Antibody molecules that bind to CD137 and OX40

Abstract

To solve the problem in which: there is a potential risk of eliminating the very cells that the agonist antibodies are intended to activate.SOLUTION: The present application relates to antibody molecules that bind and are able to agonise both CD137 and OX40. The antibody molecules comprise a CDR-based binding site for CD137, and an OX40 antigen-binding site that is located in a constant domain of the antibody molecule. The antibody molecules of the invention find application, for example, in the treatment of diseases, such as cancer and infectious diseases.SELECTED DRAWING: None

Description

Technical Field

[0001] Technical Field The present invention relates to antibody molecules that can bind to and agonize both CD137 and OX40. The antibody molecule comprises a CDR-based binding site for CD137 and an OX40 antigen-binding site located in the constant domain of the antibody molecule. The antibody molecules of the present invention find use, for example, in the treatment of diseases such as cancer and infectious diseases.

Background Art

[0002] Background of the Invention The mammalian immune system is a balanced system that can be disrupted by diseases such as cancer. Checkpoint receptors play an important role in the immune system's response to disease by exerting either a costimulatory or coinhibitory effect, and their balance determines the fate of the immune response (Pardoll, 2012). Coinhibitors inhibit T cell proliferation and induce the release of anti-inflammatory cytokines. They attenuate inflammation and prevent organ / tissue damage due to excessive immune responses. On the other hand, costimulatory factors promote T cell clonal expansion, effector differentiation, and survival to promote the development of a defensive immune response.

[0003] One proven cancer immunotherapy approach is to target these checkpoint receptors with antibodies that block the function of co-inhibitory receptors or induce the activation of co-stimulatory receptors, thereby inducing the immune system to recognize and kill tumor cells (Pardoll, 2012). Antibodies that block the activity of co-inhibitory receptors have shown good clinical activity and are currently approved for the treatment of cancer (Larkin et al., 2015). Antibodies that induce the activation of co-stimulatory receptors have shown great promise in preclinical model systems (Moran et al., 2013; Schaer et al., 2014), and several agents are currently in clinical trials (Mayes et al., 2018; Melero et al., 2013). These antibodies are also called agonist antibodies because they are designed to mimic the ligands of these co-stimulatory receptors.

[0004] Some T cell co - stimulatory receptors are members of the TNF superfamily of receptors, a large family of proteins involved in both immune cell functions and non - immune cell functions expressed at the cell surface (Bremer, 2013). Structural analysis of the complexes formed between TNF family receptors and their cognate ligands has mostly shown a trimer - to - trimer stoichiometry, indicating that TNFR family ligands are typically expressed as trimers at the cell surface (Wajant, 2015). The model proposed for TNFR activation is that interaction with the trimeric ligand induces trimerization of the monomeric receptor and initiates signaling. This assumes that TNFR family members are expressed as monomers and that ligand interaction alone induces formation of the receptor trimer. This model has recently been questioned (Vanamee & Faustman, 2018), and the association of these monomers into higher - order structures in the absence of ligand interaction remains controversial. The existence of pre - assembled receptor dimers or even inactive trimers that can form ligand superclusters and induce TNF receptor superclusters, thereby inducing higher levels of receptor activation and requiring additional clustering of multiple receptor complexes compared to membrane - bound forms, explains the low activity of some soluble trimer - only TNF ligands (Mueller et al., 2008). This theory is also consistent with the observation that TNFR - specific antibodies typically have no or low agonist activity and require secondary cross - linking of the antibody - TNF receptor complex to induce sufficient receptor clustering and activation, thereby mimicking the TNF ligand supercluster (Wajant, 2015).

[0005] Secondary crosslinking of the antibody-TNF receptor complex can be achieved in vitro by a crosslinking agent such as protein A or G or a secondary antibody targeting the constant domain of a TNF receptor-specific agonist antibody (Vanamee & Faustman, 2018; Wajant, 2015). However, in vivo, this secondary crosslinking requires interaction with Fc gamma receptors present on the surface of immune cells such as macrophages, NK cells, or B cells. The interaction of antibodies with Fc gamma receptors is complex. This is because in humans, there are six Fc gamma receptors with different expression patterns and affinities for the four human IgG isotypes (Bruhns et al., 2009). Fc gamma receptors have been shown to be required for optimal antitumor activity of agonist antibodies targeting TNF receptor superfamily targets in vivo (Bulliard et al., 2013; Bulliard et al., 2014). However, the dependence of TNFR agonist antibodies on Fc gamma receptor-mediated crosslinking to induce strong receptor activation may limit their overall activity in vivo for several reasons. 1) Antibody-bound cells need to interact in trans with Fc gamma receptor-expressing cells, and the frequency of this interaction limits the activation of TNFR-expressing cells. 2) The affinity of Fc gamma receptors for human IgG is usually much lower compared to the affinity of typical therapeutic antibodies for their targets (micromolar concentration range vs. nanomolar concentration range, respectively). 3) Since Fc gamma receptors mediate effector functions of antibodies such as ADCC (antibody-dependent cell-mediated cytotoxicity) and ADCP (antibody-dependent cell phagocytosis), there is a possibility that the agonist antibody may eliminate the cells themselves that it is intended to activate (Mayes et al., 2018).

[0006] A bivalent bispecific antibody that crosslinks a TNF receptor agonist using one of the cognate antigens is an alternative to Fc gamma receptor-mediated crosslinking. The effect of antibody crosslinking can arise from binding to TNF receptor family members and another cell expressing the receptor, either on the same cell (cis) or another cell (trans). Here, this mechanism of antibody crosslinking can result in superclusterization of TNF receptors when the second target is highly expressed and mimics the TNF ligand supercluster. The bispecific antibody approach to TNFR agonist antibody development has several theoretical advantages over monospecific agonist antibodies. 1) TNFR agonist activity can be directed to specific immune cells in the tumor microenvironment and periphery by targeting a second antigen, such as a checkpoint receptor or tumor-associated antigen, as the second specificity of the bispecific antibody. 2) The affinity of the crosslinking binding domain of the bispecific antibody can be designed to be higher than the affinity of the antibody for the Fc gamma receptor, thereby making crosslinking more effective. 3) Antibody effector functions can be selectively inactivated using mutations, thereby ensuring that there is no depletion of cells intended to be activated. 4) The agonist activity of two distinct TNF receptors can be achieved with a single bispecific agonist molecule, combining the activation of different immune cells to provide a more potent stimulation of the immune response. 5) By targeting co-expressed receptors, a single cell in cis can be activated without the need for two cells to interact.

[0007] Some members of the TNF receptor family have overlapping expression patterns in immune cells. Specifically, OX40, CD137, GITR, and CD27 are expressed on activated T cells, and co-expression of OX40 and CD137 has been experimentally verified (Ma et al., 2005).

[0008] OX40 is expressed primarily on activated T cells, including CD4+ T cells, CD8+ T cells, type 1 and type 2 T helper (Th1 and Th2) cells, and regulatory T (Treg) cells, and also on activated natural killer (NK) cells. The interaction between OX40 and its ligand, OX40 ligand (OX40L), which is expressed on antigen-presenting cells (APCs), increases T cell clonal expansion, differentiation, and survival, and enhances the generation of memory T cells (Croft et al., 2009). OX40 stimulation can have a direct effect on T cells, promoting T cell proliferation and survival, or an indirect effect by promoting the production of inflammatory cytokines such as IL2 and IFNγ. OX40 signaling can also regulate the function of Tregs, but in these cells, it suppresses their suppressive activity (Takeda et al., 2004). In cancer, OX40 is expressed on tumor-infiltrating T cells from patients with head and neck cancer, melanoma, and colorectal cancer, and a high level of OX40-positive lymphocytes has been found to correlate with improved survival (Petty et al., 2002; Vetto et al., 1997). In preclinical studies of OX40 agonist antibodies in mice, therapeutic effects have been demonstrated in several syngeneic tumor models, but the effects of targeting OX40 as a single agent therapy are variable and appear to correlate with tumor immunogenicity (Kjaergaard et al., 2000). This is consistent with the view that OX40 expression in tumor-specific T cells requires sufficient priming, which may not be provided by poorly immunogenic tumors. In certain syngeneic models, the antitumor activity of the OX40 antibody OX86 has been determined to be due to its ability to deplete intratumoral Tregs that express high levels of OX40 in an Fc gamma receptor-dependent manner (Bulliard et al., 2014).

[0009] Agonist antibodies against OX40 are currently in clinical trials for cancer and most have shown a favorable safety profile but limited clinical activity (Curti et al., 2013). The isotypes selected for these antibodies vary, but some investigational drugs are human IgG1 antibodies with Fc gamma receptor engagement and are aimed at depleting Tregs as a mechanism of action. The lack of clear clinical activity of these antibodies has prompted combination trials with OX40 agonist antibodies and several other therapies including PD1 / PD-L1 or CTLA4 inhibition, anti-VEGF therapy, and the tyrosine kinase inhibitor axitinib.

[0010] This Treg depletion mechanism of action has been demonstrated to be highly effective in preclinical models and can target several receptors to eliminate Tregs such as GITR (Bulliard et al., 2014) and CTLA4 (Simpson et al., 2013). However, antibodies targeting equivalent receptors in humans have not been shown to have the same level of antitumor effect in the clinic (Glisson et al., 2016; Tran et al., 2017). The reason for this is not clear, but the lower levels of Fc gamma receptor-expressing cells such as macrophages in human tumors compared to mouse syngeneic tumor models (Milas et al., 1987) may be part of the explanation for the lack of clinical translatability of the mechanism of action of these antibodies. Another reason may be that the expression levels of these markers in human Tregs differ from those in mouse Tregs (Aspeslagh et al., 2016).

[0011] CD137 is also expressed on activated T cells, including CD4+, CD8+, Th1, Th2, and Treg, but its expression profile also includes B cells, natural killer (NK) cells, natural killer T (NKT) cells, and dendritic cells (DCs) (Bartkowiak & Curran, 2015). Similar to OX40, the interaction of CD137 with its ligand induces the activation of intracellular signaling pathways that result in the induction of T cell survival, proliferation, and cytotoxic activity. CD137 stimulation preferentially stimulates CD8+ T cells compared to CD4+ T cells, leading to their proliferation, survival, and cytotoxic effector functions via the production of inflammatory cytokines and contributing to the differentiation and maintenance of memory CD8+ T cells. CD137 has also been demonstrated to be specifically expressed in the tumor-responsive subset of tumor-infiltrating lymphocytes (TILs) (Weigelin et al., 2016), providing part of the rationale behind its use in the in vivo agonist engagement and adoptive transfer for TIL selection. Monotherapy with CD137 is effective in several preclinical immunogenic tumor models, such as MC38, CT26, and B cell lymphoma. However, for more effective treatment of established tumors, the engagement of CD137 in combination with other agents, such as chemotherapy, cytokines, and other checkpoint regulators, has shown enhanced beneficial effects in reducing tumor growth (Bartkowiak & Curran, 2015). Targeting CD137 in preclinical models using agonist antibodies is also associated with liver inflammation and hypertransaminasemia due to increased accumulation of CD8+ T cells that depend on IL27 production by myeloid cells (Bartkowiak et al., 2018).

[0012] Agonist antibodies against CD137 are currently in clinical trials for cancer, but clinical progress is slowed by high-grade liver inflammation that limits dosing, similar to observations made in mice (Sanchez-Paulete et al., 2016). Urelumab (BMS-663513) was the first CD137 agonist antibody to enter clinical trials and showed signs of clinical activity before the trial was halted due to lethal hepatotoxicity at doses above 1 mg / kg (Segal et al., 2017). This is a human IgG4 antibody that can activate CD137 in the absence of crosslinking (U.S. Patent No. 8,137,667B2), but its activity increases upon crosslinking, as predicted by the theory of complete receptor activation mediated by superclusterization. In contrast, no dose-limiting toxicity has been observed with utomilumab (PF-05082566) when tested up to 10 mg / kg (Tolcher et al., 2017). This is a human IgG2 antibody that can activate CD137 only upon crosslinking (U.S. Patent No. 8,337,850B2). Additional clinical trials are ongoing with both antibodies, testing both monotherapy and combinations with radiotherapy and chemotherapy as well as existing targeted and immuno-oncology therapies. Due to the hepatotoxicity seen with urelumab, this antibody has to be administered at very low levels, and initial signs of clinical activity have not yet been observed at these levels.

[0013] Several bispecific molecules targeting either CD137 or OX40 are in the early stages of development by multiple companies. Tumor targeting of CD137 stimulation is being tested by Macrogenics using HER2- and EphA2-targeted CD137 agonist DART molecules, Roche using FAPalpha- or CD20-targeted CD137 ligand fusion proteins, and Pieris Pharmaceuticals using HER2-targeted CD137 agonist anticalin molecules. Dual targeting of OX40 and CTLA4 is being tested by Aligator Biosciences to specifically deplete intratumoral Tregs, which are expected to express both targets at high levels.

[0014] Costimulation of OX40 and CD137 in vivo has been shown to stimulate both CD4+ and CD8+ T cells and induce the cytotoxic functions of both antigen-experienced bystander CD4+ T cells and naive bystander CD4+ T cells (Qui et al., 2011). Interestingly, dual costimulation can induce transplanted CD4+ T cells to reduce tumor growth in immunodeficient mice inoculated with a melanoma syngeneic tumor model (B16-F10), highlighting the ability of this therapy to induce the antitumor activity of CD4+ T cells (Qui et al., 2011). A phase I dose-escalation clinical trial examining the effect of combining the OX40 agonist (PF-04518600) with the CD137 agonist (utomilumab - PF-05082566) is currently underway to evaluate the safety of this combination (NCT02315066), and a phase I b / II clinical trial combining the same TNFR agonists with PD-1 blockade via avelumab is also currently underway (NCT02554812). These studies examine combinations of simple single-specific agonist antibodies that require cross-linking of the Fc gamma receptor for agonist action, and thus may underestimate the clinical activity of targeting these receptors in combination.

[0015] Dual co-stimulation of OX40 and CD137 has also recently been tested in mice using a bispecific antibody approach by chemically conjugating two existing antibodies against OX40 and CD137 (Ryan et al., 2018). A molecule called OrthomAb was able to induce the proliferation of CD4+ and CD8+ T cells and the production of the inflammatory cytokines IL-2 and IFNγ in vitro. In vivo, OrthomAb was also able to reduce tumor growth in a melanoma syngeneic tumor model (B16-F10). The bivalent bispecificity of OrthomAb is predicted to allow for efficient cross-linking of the molecule upon engagement of both targets, resulting in clustering of the OX40 and CD137 receptors and subsequent T cell activation. These results validate the bispecific antibody approach targeting OX40 and CD137 within a single molecule. In the manufacturing process of the OrthomAb molecule, dimers of the desired antibody that need to be further purified in multiple higher-order species and several rounds of size exclusion steps are generated. This manufacturing process is unlikely to enable this approach for purposes other than as a research tool to validate specific combinations of targets. Furthermore, the structure of this bispecific antibody, in which two large macromolecules are held together by a small chemical linker, is likely to be unstable in vivo, and pharmacokinetic data addressing this are not shown. Unfortunately, the in vivo anti-tumor effect of OrthomAb has only been compared to the activity of either an OX40 or CD137 agonist antibody and not to their combination, so it is unclear whether the molecule is effective due to its bispecificity or due to OrthomAb behaving as a combination of single-agent agonist antibodies against OX40 and CD137.

[0016] Accordingly, a theoretical basis is established for combining the agonistic activities of TNF receptor family members OX40 and CD137 in a single bivalent, bispecific, and stable molecule, which may effect Fc gamma receptor-independent super-clustering of OX40 and CD137, thereby activating both CD4+ and CD8+ T cells to initiate an effective anti-tumor immune response. Based on preclinical combination data generated with monoclonal antibodies targeting either the OX40 or CD137 pathway, this molecule also has the potential as a combination partner to enhance the effects of standard cancer therapies and provide benefits to patients. Summary of the Invention Means for Solving the Problems

[0017] Description of the Invention The inventors recognized that an antibody molecule that binds to both CD137 and OX40 and can induce clustering and signaling of OX40 and / or CD137 when bound to both targets is highly effective, for example, in activating immune cells in the tumor microenvironment. Further, the inventors recognized that restricting the activation of CD137 to sites where CD137 and OX40 are co-expressed is highly effective in activating immune cells without inducing the toxicity associated with known anti-CD137 agonist molecules. This is expected to be useful, for example, in immunotherapy for the treatment of cancer and other diseases.

[0018] As explained in the background section above, the initial ligation of either OX40 ligand or CD137 ligand to OX40 or CD137 is thought to initiate a series of events leading to trimerization of the receptor, subsequent clustering, activation of the receptor, and the initiation of potent anti-tumor T cell activity. Thus, it is expected that for a therapeutic agent to efficiently achieve the activation of OX40 or CD137, several receptor monomers need to be cross-linked together in a way that mimics cross-linking by the trimeric ligand.

[0019] The inventors isolated an antibody molecule comprising a complementarity-determining region (CDR)-based antigen-binding site for CD137 and an OX40 antigen-binding site located in the constant domain of the antibody molecule. The inventors showed that such an antibody molecule can bind to both targets simultaneously when both targets are co-expressed. Co-expression in this sense encompasses situations where CD137 and OX40 are expressed in the same cell, such as immune cells, as well as situations where CD137 and OX40 are expressed in two different immune cells that are adjacent to each other in the tumor microenvironment, for example. Thus, the antibody molecules of the present invention are thought to be able to bind in cis to both targets expressed on a single cell and also in trans to two targets expressed on different cells.

[0020] The inventors further showed that an antibody molecule comprising a CDR-based antigen-binding site for CD137 and an OX40 antigen-binding site located in the constant domain of the antibody molecule can bind bivalently to both targets. Specifically, the inventors showed that when such an antibody molecule was bound to OX40 and CD137, the resulting complex was cross-linked and subjected to mass spectrometry, 19% of the complex was shown to contain two OX40 moieties and two CD137 moieties. This indicates that the antibody molecule bound bivalently to both targets.

[0021] Furthermore, the inventors showed that when these antibody molecules bind to both targets, they can induce clustering and signaling of OX40 and CD137 in vitro. By acting in this way, such antibody molecules are called "dual agonists", i.e., the antibody molecules can induce receptor-mediated signaling as a result of cross-linking by dual binding to both OX40 and CD137.

[0022] As shown in the examples, OX40 is preferentially expressed in CD4+ T cells, and CD137 is preferentially expressed in CD8+ T cells. The inventors have demonstrated that antibody molecules can induce an agonistic effect of OX40 on CD4+ T cells. In these cases, the antibody molecule binds to CD137 via a CDR-based antigen-binding domain, cross-links the antibody molecules, and at the same time, the OX40 antigen-binding domain can bind to OX40 expressed on CD4+ T cells, cluster it, and activate it. Similarly, the inventors have demonstrated that antibody molecules can induce an agonistic effect of CD137 on CD8+ T cells. In these cases, the antibody molecule binds to OX40 via an OX40 antigen-binding domain, cross-links the antibody molecules, and at the same time, the OX40 antigen-binding domain can bind to CD137 expressed on CD8+ T cells, cluster it, and activate it.

[0023] Furthermore, the inventors have shown that an antibody molecule containing two antigen-binding sites as detailed above and modified to reduce or suppress binding to Fcγ receptors can induce signal transduction via receptors when CD137 and OX40 are co-expressed. This indicates that the agonistic effect occurred without the need for cross-linking by Fcγ receptors. Since Fcγ receptor-mediated cross-linking is not required for the activity of the antibody molecules of the present invention, signal transduction via the OX40 or CD137 receptor is expected to be localized at sites where both targets are present, such as the tumor microenvironment. Therefore, the antibody molecules can drive an agonistic effect autonomously without the need for additional cross-linking agents based on the expression of both specific targets.

[0024] Furthermore, since Fcγ receptor binding is required for ADCC, this decrease in binding to the Fcγ receptor is expected to also result in a decrease in ADCC such that target immune cells are not depleted by the antibody molecules of the present invention. The inventors considered this to be important because the antibody molecules were designed to activate immune cells expressing CD137 and / or OX40 in order to promote an immune response. Thus, depletion of these immune cells is undesirable. The inventors have demonstrated that antibody molecules having the properties defined herein can activate and induce immune cells, particularly T cells expressing CD137 and / or OX40.

[0025] The inventors further showed that antibody molecules comprising CD137 and OX40 antigen-binding sites as detailed above can inhibit tumor growth in vivo in mice. Furthermore, more effective tumor growth inhibition was observed with the bispecific antibody molecules compared to a combination of two monospecific antibody molecules, one comprising a CDR-based antigen-binding site for CD137 and the other comprising a CDR-based antigen-binding site for OX40. This indicates that the simultaneous engagement and agonistic action of OX40 and CD137 improve anti-tumor efficacy. Additionally, the antibody molecules were shown to be able to induce complete tumor regression and the establishment of protective immune memory against tumor cell rechallenge in a CT26 mouse tumor model. Thus, the antibody molecules of the present invention are expected to demonstrate efficacy in the treatment of cancer in human patients. Since these antibody molecules suppress ADCC activity, they are expected to inhibit tumor growth by agonizing target immune cells without significantly depleting these beneficial T cells (memory and effector cells).

[0026] As observed in in vivo studies in mice, the activation and proliferation of T cells induced by the antibody molecules described herein were systemic effects rather than effects localized to the tumor. Furthermore, increased proliferation and activation of peripheral central memory and effector memory CD4+ and CD8+ T cells were observed in a preliminary dose range finding study in cynomolgus monkeys administered the antibody molecules of the invention. Thus, similar to the targeting of T cells in the tumor microenvironment, peripheral memory T cells expressing OX40 and CD137 are expected to be targeted by the antibody molecules to drive the proliferation of tumor-responsive T cells and provide their anti-tumor effects.

[0027] Thus, in addition to the site of the actual tumor itself, anatomical locations affected by the tumor can be considered to include other locations in the body, such as peripheral lymph nodes where a tumor-specific immune response is generated.

[0028] As described in the Background section above, the clinical development of CD137 agonist molecules has been at least partially hampered due to treatment-related either dose-limiting high-grade liver inflammation (urelumab) or low clinical efficacy (utomilumab).

[0029] Although not wishing to be bound by theory, it is thought that T cells present in the liver can be activated by anti-CD137 agonist molecules and have the potential to cause 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 result in CD137-dependent T cell infiltration into the liver (Dubrot J et al., 2010). Taken together, the results of these studies indicate that highly active anti-CD137 agonist antibodies such as urelumab can cause infiltration of activated CD8+ T cells into the liver, thereby causing liver inflammation. The activity of utomilumab may have been too low to observe this effect. Instead, the dose-limiting hepatotoxicity observed with urelumab treatment may be due to the specific epitope to which this antibody binds.

[0030] The inventors have conducted an extensive selection program to isolate antibody molecules that bind with high affinity to dimeric human CD137, i.e., are expected to bind to CD137 with high binding strength. Considering the selection protocol used, the antibody molecules are expected to bind to monomeric CD137 with an affinity lower than that observed with dimeric CD137.

[0031] As used herein, "affinity" can refer to the strength of the binding interaction between an antibody molecule and its cognate antigen, as measured by K D As will be readily apparent to those skilled in the art, when an antibody molecule can form multiple binding interactions with an antigen (e.g., when the antibody molecule can bind the antigen bivalently and optionally the antigen is dimeric), the affinity measured by K D can also be affected by the binding strength, where the binding strength refers to the overall strength of the antibody-antigen complex.

[0032] The expression of CD137 by immune cells such as T cells is upregulated upon activation. Without wishing to be bound by theory, due to the high expression of CD137 on activated immune cells, CD137 is thought to exist in the form of dimers, trimers, and higher-order multimers on the surface of such cells. In contrast, naive immune cells such as naive T cells express low or negligible levels of CD137 on the cell surface, so any CD137 present is likely to be in monomeric form. Thus, antibody molecules that bind to CD137 with high affinity are expected to preferentially bind to activated immune cells such as activated T cells over naive immune cells.

[0033] Accordingly, in light of the above, it is expected that the antibody molecules of the present invention are almost impossible to activate CD137 in the absence of crosslinking via engagement with OX40. Furthermore, as described above, the inventors developed antibody molecules in which Fcγ receptor-mediated crosslinking is reduced or suppressed, expecting to avoid activation of CD137 at positions where there is little or no co-expression of OX40. Inactivation of Fcγ receptor binding has been shown not to affect the antitumor activity of the antibody molecules. Without wishing to be bound by theory, such antibody molecules are thought to exhibit reduced toxicity when administered to patients. This is thought to be because activation of CD137 is highly restricted to positions where OX40 and CD137 are co-expressed at levels sufficient to drive clustering and activation of CD137. The inventors have shown that the dose of the antibody molecules of the present invention is well tolerated up to 30 mg / kg in preliminary dose range finding studies in cynomolgus monkeys.

[0034] The inventors have shown that the antibody molecules of the present invention can induce low levels of OX40 clustering and activation even in the absence of crosslinking. Unlike CD137 agonist antibodies, OX40 agonist antibodies do not exhibit dose-limiting toxicity (DLT) in the clinic, and thus, OX40 agonist activity in the absence of crosslinking is not expected to represent a problem for clinical treatment. On the contrary, depending on the conditions being treated, low levels of OX40 agonist activity by the antibody molecules in the absence of crosslinking may be advantageous. Without wishing to be bound by theory, antibody molecules comprising an OX40 antigen-binding site with this property may induce limited activation and expansion of tumor-reactive T cells in the absence of crosslinking, resulting in a larger pool of tumor-reactive T cells, and may be useful in the context of cancer treatment by being further activated by crosslinked Fcab molecules within the tumor microenvironment.

[0035] A further advantage of the antibody molecules of the present invention modified to reduce or inhibit binding to Fcγ receptors is that these antibody molecules may have anti-tumor activity that is independent of depletion of OX40-expressing regulatory T cells (Tregs). Tregs are located peripherally and are potentially protective, and can reduce the effects of autoimmunity that can be caused by overstimulating the immune system (Vignali DA et al., 2008). Thus, depletion of Tregs is hypothesized to have a significant effect on reducing tumor growth in mouse models (Bulliard et al., 2014; Simpson et al., 2013). However, the evidence that Treg depletion can be achieved by ADCC in human tumors is limited, and when Treg depletion occurs in humans, this does not appear to result in dramatic anti-tumor activity as observed in mouse models (Powell et al., 2007; Nizar S et al., 2009; Glisson BS et al., 2016; Tran B et al., 2017). Thus, if the antibody molecules do not significantly deplete Tregs but still have anti-tumor activity, this may indicate that the antibody molecules have anti-tumor activity independent of Fcγ receptor-mediated Treg depletion.

[0036] The antibody molecule has further been shown to be able to bind with high affinity to both human and cynomolgus CD137 as well as human and cynomolgus OX40. This cross-reactivity is advantageous as it allows for the administration and safety testing of the antibody molecule in cynomolgus monkeys during preclinical development.

[0037] A further feature of the antibody molecules identified by the inventors is that both the antigen-binding site for CD137 and the antigen-binding site for OX40 are contained within the antibody structure itself. In particular, the antibody molecule does not require the fusion of other proteins to the antibody molecule via a linker or other means to result in a molecule that can bind bivalently to both of its targets. This has several advantages. Specifically, the antibody molecules identified by the inventors can be produced using methods similar to those used for the production of standard antibodies as they do not contain additional fusion moieties. This structure is also expected to lead to an improvement in the stability of the antibody as the linker can degrade over time and result in a heterogeneous population of antibody molecules. Antibodies within a population with only one protein fused may act as dual agonists as a result of cross-linking by binding to both OX40 and CD137 and may not be able to signal via the receptor. Cleavage or degradation of the linker can occur before or after administration of the therapeutic agent to an individual (e.g., via enzymatic cleavage or the in vivo pH of the individual), thereby resulting in a decrease in its efficacy while circulating within the individual. Since the antibody molecules identified by the inventors do not have a linker, the antibody molecules are expected to retain the same number of binding sites both before and after administration. Furthermore, the structure of the antibody molecules identified by the inventors is also preferred from the perspective of the immunogenicity of the molecule. This is because the introduction of a fusion protein or linker or both can induce immunogenicity when the molecule is administered to an individual, resulting in a decrease in the efficacy of the therapeutic agent.

[0038] Accordingly, the present invention provides the following.

[0039] [1]An antibody molecule that binds to CD137 and OX40, (a) a complementarity-determining region (CDR)-based antigen-binding site for CD137; and (b) an OX40 antigen-binding site located in the CH3 domain of the antibody molecule comprising, the CDR-based antigen-binding site (i) having CDR1-6 as set forth in SEQ ID NOs: 1, 2, 3, 4, 5 and 6 [FS30-10-16]; (ii) having CDR1-6 as set forth in SEQ ID NOs: 1, 2, 16, 4, 5 and 6 [FS30-10-3]; (iii) having CDR1-6 as set forth in SEQ ID NOs: 1, 2, 21, 4, 5 and 6 [FS30-10-12]; (iv) having CDR1-6 as set forth in SEQ ID NOs: 25, 26, 27, 4, 5 and 28 [FS30-35-14]; or (v) having CDR1-6 as set forth in SEQ ID NOs: 33, 34, 35, 4, 5 and 36 [FS30-5-37] and the OX40 antigen-binding site comprises a first sequence, a second sequence and a third sequence located in the AB, CD and EF structural loops of the CH3 domain, respectively, and the first, second and third sequences have the sequences set forth in SEQ ID NOs: 51, 52 and 53 [FS20-22-49], respectively, an antibody molecule.

[0040] [2]An antibody molecule that binds to CD137 and OX40, (a) a complementarity-determining region (CDR)-based antigen-binding site for CD137; and (b) an OX40 antigen-binding site located in the CH3 domain of the antibody molecule comprising, the CDR-based antigen-binding site (i) having CDR1-6 as set forth in SEQ ID NOs: 7, 8, 9, 10, 11 and 6 [FS30-10-16]; (ii) having CDR1-6 as set forth in SEQ ID NOs: 7, 8, 17, 10, 11 and 6 [FS30-10-3]; (iii) having CDR1-6 as set forth in SEQ ID NOs: 7, 8, 22, 10, 11 and 6 [FS30-10-12]; (iv) having CDR1-6 as set forth in SEQ ID NOs: 29, 30, 31, 10, 11 and 28 [FS30-35-14]; or ​(v) Each of SEQ ID NO: 37, 38, 39, 10, 11 and 36 [FS30-5-37] comprising CDR1-6 described in The OX40 antigen-binding site comprises a first sequence, a second sequence and a third sequence located in the AB, CD and EF structural loops of the CH3 domain of the antibody molecule, respectively, and the first, second and third sequences have the sequences described in SEQ ID NO: 51, 52 and 53 [FS20-22-49], respectively, an antibody molecule.

[0041] [3](i) The first sequence is located at positions 14 to 18 of the CH3 domain of the antibody molecule; (ii) The second sequence is located at positions 45.1 to 77 of the CH3 domain of the antibody molecule; and / or (iii) The third sequence is located at positions 93 to 101 of the CH3 domain of the antibody molecule; The numbering of amino acid residues follows the IMGT numbering scheme, the antibody molecule described in [1] or [2].

[0042] [4] An antibody molecule according to any one of [1] to [3], comprising the CH3 domain sequence described in SEQ ID NO: 54 [FS20-22-49].

[0043] [5] An antibody molecule according to any one of [1] to [4], comprising CDR1-6 described in any one of (i) to (iv) of [1] or [2].

[0044] [6] An antibody molecule according to any one of [1] to [5], comprising CDR1-6 described in any one of (i) to (iii) of [1] or [2].

[0045] [7] An antibody molecule according to any one of [1] to [6], comprising CDR1-6 described in (i) of [1] or [2].

[0046] [8] An antibody molecule according to any one of [1] to [7], comprising a heavy chain variable (VH) domain and / or a light chain variable (VL) domain, preferably a VH domain and a VH domain.

[0047] [9]An antibody molecule according to any one of [1] to [8], comprising an immunoglobulin heavy chain and / or an immunoglobulin light chain, preferably an immunoglobulin heavy chain and an immunoglobulin light chain.

[0048]

[10] Comprising a VH domain and / or a VL domain, preferably, the VH domain and the VH domain are, (i) SEQ ID NO: 12 and 14 respectively [FS30-10-16]; (ii) SEQ ID NO: 18 and 14 respectively [FS30-10-3]; (iii) SEQ ID NO: 23 and 14 respectively [FS30-10-12]; (iv) SEQ ID NO: 170 and 172 respectively [FS30-35-14]; or (v) SEQ ID NO: 40 and 42 respectively [FS30-5-37] The antibody molecule according to [8] or [9], as described therein.

[0049]

[11] An antibody molecule according to

[10] , comprising a VH domain and a VL domain as described in any one of (i) to (iv) of

[10] .

[0050]

[12] An antibody molecule according to

[10] or

[11] , comprising a VH and a VL domain as described in any one of (i) to (iii) of

[10] .

[0051]

[13] An antibody molecule according to any one of

[10] to

[12] , comprising a VH domain and a VL domain as described in (i) of

[10] .

[0052]

[14] A human IgG1 molecule, the antibody molecule according to any one of [1] to

[13] .

[0053]

[15] Antibody: (i) FS20-22-49AA / FS30-10-16 described in SEQ ID NO: 95 and 97 respectively; (ii) FS20-22-49AA / FS30-10-3 described in SEQ ID NO: 99 and 97 respectively; (iii) the heavy and light chains of FS20-22-49AA / FS30-10-12 described in SEQ ID NOs: 103 and 97, respectively; (iv) the heavy and light chains of FS20-22-49AA / FS30-35-14 described in SEQ ID NOs: 105 and 107, respectively; or (v) the heavy and light chains of FS20-22-49AA / FS30-5-37 described in SEQ ID NOs: 109 and 111, respectively An antibody molecule according to any one of [1] to

[14] , comprising the heavy and light chains thereof.

[0054] An antibody molecule according to

[15] , comprising the light and heavy chains described in any one of (i) to (iv) of

[16]

[15] .

[0055] An antibody molecule according to

[15] , comprising the light and heavy chains described in any one of (i) to (iii) of

[17]

[15] .

[0056] An antibody molecule according to

[15] , comprising the light and heavy chains described in (i) of

[18]

[15] .

[0057] An antibody molecule according to any one of [1] to

[18] , which binds to human CD137 and human OX40.

[0058] An antibody molecule according to

[19] , wherein human CD137 consists of or comprises the sequence described in SEQ ID NO: 127.

[0059] An antibody molecule according to

[19] or

[20] , wherein human OX40 consists of or comprises the sequence described in SEQ ID NO: 130.

[0060] An antibody molecule according to any one of [1] to

[21] , which binds to cynomolgus monkey CD137 and cynomolgus monkey OX40.

[0061] An antibody molecule according to

[22] , wherein cynomolgus monkey CD137 consists of or comprises the sequence described in SEQ ID NO: 129.

[0062]

[24] The cynomolgus monkey OX40 antibody molecule as described in

[23] or

[24] , which consists of or contains the sequence described in SEQ ID NO: 131.

[0063]

[25] An antibody molecule as described in any one of [5] to [7],

[11] to

[13] , and

[16] to

[18] , which binds to human CD137 and human OX40, and the affinity (K D ) of the antibody molecule for binding to human CD137 is within 2-fold of the affinity (K D ) of the antibody molecule for binding to human OX40.

[0064]

[26] An antibody molecule as described in any one of

[19] to

[25] , which can bind to human CD137 and human OX40 simultaneously.

[0065]

[27] An antibody molecule as described in any one of [1] to

[26] , which can activate OX40 on immune cells in the presence of CD137 expressed on the cell surface.

[0066]

[28] The binding of the antibody molecule to OX40 and CD137 on immune cells, as described in any one of [1] to

[27] , causes clustering of OX40 on immune cells.

[0067]

[29] An antibody molecule as described in any one of [1] to

[28] , which can activate CD137 on immune cells in the presence of OX40 expressed on the cell surface.

[0068]

[30] The binding of the antibody molecule to CD137 and OX40 on immune cells, as described in any one of [1] to

[29] , causes clustering of CD137 on immune cells, and OX40 is expressed on the same immune cell or a separate cell.

[0069]

[31] The antibody molecule as described in any one of claims

[27] to

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

[0070] The antibody molecule according to any one of [1] to

[31] , which is modified so as to reduce or suppress the binding of the CH2 domain of the antibody molecule to one or more Fcγ receptors.

[0071]

[33] The antibody molecule according to any one of [1] to

[32] , which does not bind to one or more Fcγ receptors.

[0072]

[34] The Fcγ receptor is selected from the group consisting of FcγRI, FcγRIIa, FcγRIIb, and FcγRIII, and the antibody molecule according to

[32] or

[33] .

[0073]

[35] The antibody molecule according to any one of [1] to

[34] , which is capable of inducing the proliferation of T cells.

[0074]

[36] A conjugate comprising the antibody molecule according to any one of [1] to

[35] and a bioactive molecule.

[0075]

[37] A conjugate comprising the antibody molecule according to any one of [1] to

[36] and a detectable label.

[0076]

[38] One or more nucleic acid molecules encoding the antibody molecule according to any one of [1] to

[35] .

[0077]

[39] One or more nucleic acid molecules encoding the antibody molecule according to any one of [1] to [4], [8] to

[10] ,

[14] to

[15] , and

[19] to

[35] , wherein (i) FS20-22-49AA / FS30-10-16 described in SEQ ID NOs: 96 and 98, respectively; (ii) FS20-22-49AA / FS30-10-3 described in SEQ ID NOs: 100 and 102, respectively; (iii) FS20-22-49AA / FS30-10-12 described in SEQ ID NOs: 104 and 102, respectively; (iv) FS20-22-49AA / FS30-35-14 described in SEQ ID NOs: 106 and 108, respectively; or (v) one or more nucleic acid molecules comprising the heavy-chain nucleic acid sequence and / or the light-chain nucleic acid sequence of FS20-22-49AA / FS30-5-37 set forth in SEQ ID NOs: 110 and 112, respectively

[0078] One or more vectors comprising one or more nucleic acid molecules according to any one of

[40] to

[39] .

[0079] A recombinant host cell comprising the nucleic acid molecule according to any one of

[41] to

[39] or the vector according to

[40] .

[0080] A method for producing an antibody molecule according to any one of

[42] to [1] to

[35] , comprising culturing the recombinant host cell of

[41] under conditions for the production of the antibody molecule.

[0081] The method according to

[42] , further comprising isolating and / or purifying the antibody molecule.

[0082] A pharmaceutical composition comprising the antibody molecule or conjugate according to any one of

[44] to [1] to

[37] and a pharmaceutically acceptable excipient.

[0083] An antibody molecule or conjugate according to any one of

[45] to [1] to

[37] for use in the treatment of the human or animal body.

[0084] A method for treating a disease or disorder in an individual, comprising administering to the individual a therapeutically effective amount of an antibody molecule or conjugate according to any one of

[46] to [1] to

[37] .

[0085] The antibody molecule or conjugate for use according to

[45] , wherein the antibody molecule or conjugate is for use in the treatment of cancer or an infectious disease in an individual.

[0086]

[48] The disease or disorder is the cancer or infectious disease of an individual, the method of

[46] .

[0087]

[49] Use of the antibody molecule or conjugate according to any one of [1] to

[37] in the preparation of a medicament for the treatment of cancer or infectious disease.

[0088]

[50] The cancer is a solid cancer, and optionally, the solid cancer is selected from the group consisting of melanoma, bladder cancer, brain cancer, breast cancer, ovarian cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, pancreatic cancer, kidney cancer, and gastric cancer, the antibody molecule or conjugate for the use according to

[47] , the method of

[48] or the use of the antibody molecule or conjugate according to

[49] .

[0089]

[51] The infectious disease is a persistent viral infection, and optionally, the persistent viral infection is selected from the group consisting of human immunodeficiency virus (HIV), Epstein - Barr virus, cytomegalovirus, hepatitis B virus, hepatitis C virus, varicella - zoster virus, the antibody molecule or conjugate for the use according to

[47] , the method of

[48] or the use of the antibody molecule or conjugate according to

[49] .

[0090]

[52] The infectious disease is a persistent bacterial infection, and optionally, the persistent bacterial infection is a persistent infection of Staphylococcus aureus, Hemophilus influenza, Mycobacterium tuberculosis, Mycobacterium leprae, Helicobacter pylori, Treponema pallidum, Enterococcus faecalis or Streptococcus pneumoniae, the antibody molecule or conjugate for the use according to

[47] , the method of

[48] or the use of the antibody molecule or conjugate according to

[49] .

[0091]

[53] The infectious disease is a persistent fungal infection, and optionally, the persistent fungal infection is a persistent infection of Candida, such as Candida albicans, Cryptococcus (gattii and neoformans), Talaromyces (Penicillium) marneffe, Microsporum, such as Microsporum audouinii and Trichophyton tonsurans, the antibody molecule or conjugate for use as described in

[47] , the method of

[48] or the use of the antibody molecule or conjugate as described in

[49] .

[0092]

[54] The infectious disease is a persistent parasitic infection, and optionally, the persistent parasitic infection is a persistent infection of Plasmodium, such as Plasmodium falciparum, or Leishmania, such as Leishmania donovani, the antibody molecule or conjugate for use as described in

[47] , the method of

[48] or the use of the antibody molecule or conjugate as described in

[49] .

[0093]

[55] The treatment comprises administering to the individual an antibody molecule or conjugate in combination with a second therapeutic agent, the antibody molecule or conjugate for use as described in any one of

[45] ,

[47] and

[50] to

[54] .

[0094]

[56] The method as described in

[46] ,

[48] and

[50] to

[54] further comprises administering to the individual a therapeutically effective amount of the second therapeutic agent.

[0095] An antibody molecule or conjugate for use in a method of treating cancer in an individual as described in

[47] to

[50] , the method comprising administering to the individual an antibody molecule or conjugate in combination with an antibody that binds to PD-1 or PD-L1. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1

Figure 2A

Figure 2B

Figure 2C

Figure 2D

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 4A

Figure 4B

Figure 4C

Figure 4D

Figure 5A

Figure 5B

Figure 5C

Figure 5D

Figure 5E

Figure 5F

Figure 6

Figure 7A

Figure 7B

Figure 7C

Figure 7D

Figure 8

Figure 9

Figure 10A

Figure 10B

Figure 10C

Figure 10D

Figure 11

Figure 12A

Figure 12B

Figure 13A

Figure 13B

Figure 13C

Figure 13D

Figure 13E

Figure 14A

Figure 14B

Figure 15A

Figure 15B

BEST MODE FOR CARRYING OUT THE INVENTION

[0097] DETAILED DESCRIPTION Here, aspects and embodiments of the invention are discussed with reference to the accompanying drawings. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

[0098] The present invention relates to antibody molecules that bind to both CD137 and OX40. Specifically, the antibody molecules of the present invention include a CDR-based antigen-binding site for CD137 and an OX40 antigen-binding site located in the constant domain of the antibody molecule. The terms "CD137" and "OX40" refer to human CD137 and human OX40, mouse CD137 and mouse OX40, and / or cynomolgus monkey CD137 and cynomolgus monkey OX40, unless the context requires otherwise. Preferably, the terms "CD137" and "OX40" refer to human CD137 and human OX40, unless the context requires otherwise.

[0099] The term "antibody molecule" describes immunoglobulins, whether natural or produced partially or wholly synthetically. Antibody molecules can be human or humanized, preferably human. Antibody molecules are preferably monoclonal antibody molecules. Examples of antibodies are immunoglobulin isotypes such as immunoglobulin G, and their isotype subclasses such as IgG1, IgG2, IgG3 and IgG4, and fragments thereof. Antibody molecules can be isolated in the sense that they are free of contaminants such as antibodies that can bind to other polypeptides and / or serum components.

[0100] Accordingly, the term "antibody molecule" as used herein includes antibody fragments, provided that said fragments include a CDR-based antigen-binding site for CD137 and an OX40 antigen-binding site located in the constant domain. Accordingly, unless the context requires otherwise, the term "antibody molecule" as used herein is equivalent to "antibody molecule or fragment thereof".

[0101] Monoclonal antibodies and other antibodies can be employed and techniques of recombinant DNA technology can be used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques can include introducing CDRs or variable regions and / or constant domain sequences that provide the OX40 antigen-binding site into different immunoglobulins. The introduction of the CDRs of one immunoglobulin into another is described, for example, in European Patent Application Publication No. A-184187, British Patent Application Publication No. 2188638A or European Patent Application Publication No. A-239400. Similar techniques can be used for the relevant constant domain sequences. Alternatively, hybridomas or other cells producing antibody molecules can be subject to genetic mutations or other changes, which may or may not alter the binding specificity of the antibodies produced.

[0102] Antibodies can be modified in many ways, so the term "antibody molecule" should be construed to include antibody fragments, derivatives, functional equivalents, and homologs of antibodies, which includes any polypeptide containing an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Thus, chimeric molecules containing an immunoglobulin binding domain or equivalent fused to another polypeptide are included. The cloning and expression of chimeric antibodies are described in European Patent Application Publication Nos. A-0120694 and A-0125023.

[0103] An example of an antibody fragment containing both CDR sequences and a CH3 domain is a minibody containing an scFv linked to a CH3 domain (Hu et al., 1996).

[0104] The antibody molecules of the present invention bind to CD137 and OX40. In this regard, binding can refer to specific binding. The term "specific" can refer to a situation where the antibody molecule does not exhibit significant binding to molecules other than its specific binding partners (here CD137 and OX40). The term "specific" is also applicable when the antibody molecule is specific for a particular epitope carried by some antigens, such as an epitope on CD137 and OX40, in which case the antibody molecule can bind to the various antigens carrying the epitope. In a preferred embodiment, the antibody molecules of the present invention do not bind to, or do not exhibit significant binding to, TNFRSF1A, TNFRSF1B, GITR, NGFR, CD40, and / or DR6.

[0105] Antibodies and methods for their construction and use are well known in the art and are described, for example, in Holliger and Hudson 2005. Monoclonal antibodies and other antibodies can be employed and techniques of recombinant DNA technology can be used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques can include introducing the CDR or variable region of one antibody molecule into a different antibody molecule (European Patent Application Publication No. A-184187, British Patent Application Publication No. 2188638A, and European Patent Application Publication No. A-239400).

[0106] A CDR-based antigen-binding site is the antigen-binding site of an antibody variable region. A CDR-based antigen-binding site can be formed by three CDRs such as three light-chain variable domain (VL) CDRs or three heavy-chain variable domain (VH) CDRs. Preferably, a CDR-based antigen-binding site is formed by six CDRs, three VL CDRs and three VH CDRs. The contribution of different CDRs to antigen binding can vary depending on the different antigen-binding sites.

[0107] The three VH domain CDRs of the antigen-binding site 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 in the antibody variable region.

[0108] An antibody molecule can have one or preferably two or more, for example two, CDR-based antigen-binding sites for a first antigen. Thus, an antibody molecule can contain one VH and one VL domain, but preferably contains two VH and two VL domains, i.e., two VH / VL domain pairs as in the case of a naturally occurring IgG molecule, for example.

[0109] The CDR-based antigen-binding site may comprise the three VH CDRs or three VL CDRs of antibody FS30-10-16, FS30-10-3, FS30-10-12, or FS30-35-14, or FS30-5-37, preferably the three VH CDRs and three VL CDRs of antibody FS30-10-16.

[0110] The VH and VL domain sequences of these antibodies are described as follows. (i) The VH and VL domain sequences of SEQ ID NO: FS30-10-16 are shown in SEQ ID NOs: 12 and 14, respectively; (ii) The VH and VL domain sequences of SEQ ID NO: FS30-10-3 are shown in SEQ ID NOs: 18 and 14, respectively; (iii) The VH and VL domain sequences of SEQ ID NO: FS30-10-12 are shown in SEQ ID NOs: 23 and 14, respectively; (iv) The VH and VL domain sequences of SEQ ID NO: FS30-35-14 are shown in SEQ ID NOs: 170 and 172, respectively; (v) The VH and VL domain sequences of SEQ ID NO: FS30-5-37 are shown in SEQ ID NOs: 40 and 42, respectively.

[0111] Those skilled in the art will have no difficulty in determining the CDR sequences from the VH and VL domain sequences of the above antibodies. The CDR sequences can be determined, for example, according to Kabat (Kabat et al., 1991) or the international ImMunoGeneTics information system (IMGT) (Lefranc et al., 2015).

[0112] The CDR1, CDR2, and CDR3 sequences of the VH domain of the antibody molecule by IMGT numbering can be the sequences located at positions 27-38, 56-65, and 105-117 of the VH domain of the antibody molecule, respectively.

[0113] The CDR1, CDR2, and CDR3 sequences of the VH domain of the antibody molecule by Kabat numbering can be the sequences located at positions 31-35, 50-65, and 95-102 of the VH domain, respectively.

[0114] The CDR1, CDR2, and CDR3 sequences of the VL domain of an antibody molecule by IMGT numbering can be sequences located at positions 27-38, 56-65, and 105-117 of the VL domain, respectively.

[0115] The CDR1, CDR2, and CDR3 sequences of the VL domain of an antibody molecule by Kabat numbering can be sequences located at positions 24-34, 50-56, and 89-97 of the VL domain, respectively.

[0116] For example, the antibody molecule may (i) have the sequences of CDR1, CDR2, and CDR3 of the VH domain as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively [FS30-10-16]; (ii) have the sequences of CDR1, CDR2, and CDR3 of the VH domain as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 16, respectively [FS30-10-3]; (iii) have the sequences of CDR1, CDR2, and CDR3 of the VH domain as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 21, respectively [FS30-10-12]; (iv) have the sequences of CDR1, CDR2, and CDR3 of the VH domain as SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, respectively [FS30-35-14]; or (v) have the sequences of CDR1, CDR2, and CDR3 of the VH domain as SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively [FS30-5-37] and may include the sequences of CDR1, CDR2, and CDR3 of the VH domain, where the CDR sequences are defined according to the numbering scheme of ImMunoGeneTics (IMGT).

[0117] The antibody molecule may (i) have the sequences of CDR1, CDR2, and CDR3 of the VH domain as SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively [FS30-10-16]; (ii) have the sequences of CDR1, CDR2, and CDR3 of the VH domain as SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 17, respectively [FS30-10-3]; (iii) have the sequences of CDR1, CDR2, and CDR3 of the VH domain as SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 22, respectively [FS30-10-12]; (iv) have the sequences of CDR1, CDR2, and CDR3 of the VH domain as SEQ ID NO: 29, SEQ ID NO: 30, and SEQ ID NO: 31, respectively [FS30-35-14]; or (v) have the sequences of CDR1, CDR2, and CDR3 of the VH domain as SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, respectively [FS30-5-37] and may include the sequences of CDR1, CDR2, and CDR3 of the VH domain, where the CDR sequences are defined according to the Kabat numbering scheme.

[0118] For example, the antibody molecule may (i) SEQ ID NOs: 4, 5, and 6, respectively [FS30-10-16]; (ii) SEQ ID NOs: 4, 5, and 6, respectively [FS30-10-3]; (iii) SEQ ID NOs: 4, 5, and 6, respectively [FS30-10-12]; (iv) SEQ ID NOs: 4, 5, and 28, respectively [FS30-35-14]; or (v) SEQ ID NOs: 4, 5, and 36, respectively [FS30-5-37] and may include the sequences of VL domain CDR1, CDR2, and CDR3 of , wherein the CDR sequences are defined according to the numbering scheme of ImMunoGeneTics (IMGT).

[0119] For example, an antibody molecule (i) SEQ ID NOs: 10, 11, and 6, respectively [FS30-10-16], (ii) SEQ ID NOs: 10, 11, and 6, respectively [FS30-10-3]; (iii) SEQ ID NOs: 10, 11, and 6, respectively [FS30-10-12], (iv) SEQ ID NOs: 10, 11, and 28, respectively [FS30-35-14]; or (v) SEQ ID NOs: 10, 11, and 36, respectively [FS30-5-37] and may include the sequences of VL domain CDR1, CDR2, and CDR3 of , wherein the CDR sequences are defined according to the Kabat numbering scheme.

[0120] The VH and VL sequences of antibodies FS30-10-16, FS30-10-3, and FS30-10-12 are identical except for the residue at position 109 of VH (residue 97 of VH according to the Kabat numbering scheme) according to the IMGT numbering scheme. Thus, the antibody molecule may comprise the VH domain CDR1, CDR2, and CDR3 sequences and / or the VL domain CDR1, CDR2, and CDR3 sequences, the VH domain sequence, and / or the VL domain sequence of antibody FS30-10-16, wherein the antibody molecule optionally comprises an amino acid substitution at position 109 of the heavy chain (residue 97 of the heavy chain according to the Kabat numbering scheme) according to the IMGT numbering scheme, and the residue at said position is preferably selected from the group consisting of asparagine (N), threonine (T), and leucine (L).

[0121] The CDR-based antigen-binding site may comprise the VH or VL domain, preferably the VH and VH domains, of antibody FS30-10-16, FS30-10-3, FS30-10-12, FS30-35-14, or FS30-5-37, preferably antibody FS30-10-16, FS30-10-3, FS30-10-12, or FS30-35-14, more preferably antibody FS30-10-16, FS30-10-3, or FS30-10-12, and most preferably antibody FS30-10-16.

[0122] The VH domains of antibodies FS30-10-16, FS30-10-3, FS30-10-12, FS30-35-14, and FS30-5-37 may have the sequences set forth in SEQ ID NOs: 12, 18, 23, 170, and 40, respectively. The VL domains of antibodies FS30-10-16, FS30-10-3, FS30-10-12, FS30-35-14, and FS30-5-37 may have the sequences set forth in SEQ ID NOs: 14, 14, 14, 172, and 42, respectively.

[0123] The antibody molecule of the present invention includes an OX40 antigen-binding site located in the constant domain of the antibody molecule. The constant domain can be a CL, CH1, CH2, CH3, or CH4 domain, preferably the constant domain is a CH1, CH2, or CH3 domain, more preferably a CH2 or CH3 domain, and most preferably a CH3 domain.

[0124] Unless otherwise specified, the positions of the amino acid residues in the constant domain are numbered herein according to the numbering scheme of ImMunoGeneTics (IMGT). The IMGT numbering scheme is described in Lefranc et al., Dev.Comp.Immunol., 29, 185-203 (2005).

[0125] The OX40 antigen-binding site may include first, second, and third sequences located in the first, second, and third structural loops of the constant domain, respectively. Manipulation of antibody constant domain structural loops to create antigen-binding sites for target antigens is known in the art and is described, for example, in Wozniak-Knopp et al., 2010 and International Publication Nos. 2006 / 072620 and 2009 / 132876. Preferably, the first, second, and third structural loops are the AB, CD, and EF structural loops of the CH3 domain of the antibody molecule, respectively. In the CH3 domain, the AB, CD, and EF structural loops are located at residues 11-18, 43-78, and 92-101 of the CH3 domain, respectively. Modification of the structural loop sequences of the antibody constant domain to create new antigen-binding sites is described, for example, in International Publication Nos. 2006 / 072620 and 2009 / 132876.

[0126] In a preferred embodiment, the OX40 antigen-binding site of the antibody molecule is (i) FS20-22-49 set forth in SEQ ID NOs: 51, 52, and 53, respectively; (ii) FS20-22-38 set forth in SEQ ID NOs: 51, 59, and 60, respectively; (iii) FS20-22-41 set forth in SEQ ID NOs: 51, 52, and 60, respectively; (iv) FS20-22-47 as set forth in SEQ ID NOs: 51, 52, and 65, respectively; or (v) FS20-22-85 as set forth in SEQ ID NOs: 51, 52, and 68, respectively comprises first, second, and third sequences.

[0127] The OX40 antigen-binding site may comprise the AB, CD, and EF structural loop sequences of FS20-22-49, FS20-22-38, FS20-22-41, FS20-22-47, or FS20-22-85, wherein the AB, CD, and EF structural loops are sequences located at residues 11-18, 43-78, and 92-101 of the CH3 domain, respectively, and the CH3 domains of FS20-22-49, FS20-22-38, FS20-22-41, FS20-22-47, or FS20-22-85 are set forth in SEQ ID NOs: 54, 61, 63, 66, and 69, respectively.

[0128] In a more preferred embodiment, the OX40 antigen-binding site of the antibody molecule comprises the first, second, and third sequences of FS20-22-49 as set forth in SEQ ID NOs: 51, 52, and 53, respectively. For example, the OX40 antigen-binding site may comprise the AB, CD, and EF structural loop sequences of FS20-22-49 as shown in SEQ ID NOs: 56, 57, and 58, respectively.

[0129] When the OX40 antigen-binding site of the antibody molecule comprises the first, second, and third sequences of FS20-22-38, FS20-22-41, FS20-22-47, FS20-22-49, or FS20-22-85, the first, second, and third sequences are preferably located at positions 14 to 18, 45.1 to 77, and 93 to 101, respectively, of the CH3 domain of the antibody molecule.

[0130] When the OX40 antigen-binding site comprises the AB, CD, and EF structural loop sequences of FS20-22-38, FS20-22-41, FS20-22-47, FS20-22-49, or FS20-22-85, the AB, CD, and EF structural loops are preferably located at positions 11 to 18, 43 to 78, and 92 to 101, respectively, of the CH3 domain of the antibody molecule.

[0131] The antibody molecule may further comprise leucine (L) at position 91 of the CH3 domain of the antibody molecule. In particular, an antibody molecule comprising an OX40 antigen-binding site comprising the first, second, and third sequences of FS20-22-85 may comprise leucine at position 91 of the CH3 domain of the antibody molecule.

[0132] In an alternative embodiment, the OX40 antigen-binding site of the antibody molecule is (i) FS20-31-58 set forth in SEQ ID NOs: 71, 72, and 73, respectively; (ii) FS20-31-66 set forth in SEQ ID NOs: 71, 72, and 76, respectively; (iii) FS20-31-94 set forth in SEQ ID NOs: 79, 80, and 81, respectively; (iv) FS20-31-102 set forth in SEQ ID NOs: 84, 85, and 76, respectively; (v) FS20-31-108 set forth in SEQ ID NOs: 84, 88, and 89, respectively; or (vi) FS20-31-115 set forth in SEQ ID NOs: 84, 92, and 89, respectively and comprises the first, second, and third sequences thereof.

[0133] The OX40 antigen-binding site may include the AB, CD, and EF structural loop sequences of FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, FS20-31-108, or FS20-31-115, where the AB, CD, and EF structural loops are sequences located at residues 11-18, 43-78, and 92-101 of the CH3 domain, respectively, and the CH3 domains of FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, FS20-31-108, or FS20-31-115 are set forth in SEQ ID NOs: 54, 61, 63, 66, and 69, respectively.

[0134] When the OX40 antigen-binding site of an antibody molecule includes the first, second, and third sequences of FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, FS20-31-108, or FS20-31-115, the first, second, and third sequences are preferably located at positions 14 to 18, 45.1 to 77, and 92 to 101, respectively, of the CH3 domain of the antibody molecule.

[0135] When the OX40 antigen-binding site includes the AB, CD, and EF structural loop sequences of FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, FS20-31-108, or FS20-31-115, the AB, CD, and EF structural loops are preferably located at positions 11 to 18, 43 to 78, and 92 to 101, respectively, of the CH3 domain of the antibody molecule.

[0136] As an alternative to IMGT numbering, the positions of amino acid residues in the constant domain, including the amino acid sequences, substitutions, deletions, and insertion positions described herein, may be numbered according to IMGT exon numbering (also referred to as continuous numbering), EU numbering, or Kabat numbering. The correspondence between IMGT numbering, IMGT exon numbering, EU numbering, and Kabat numbering of the residue positions in the CH3 domain is shown in FIG. 1.

[0137] Thus, for example, if the present application refers to the first, second, and third sequences located at positions 14 to 18, 45.1 to 77, and 93 to 101, respectively, of the CH3 domain of an antibody molecule, and the positions of the residues are numbered according to the IMGT numbering scheme, the first, second, and third sequences are located at positions 18 to 22, 46 to 50, and 74 to 82 of the CH3 domain, where the positions of the residues are numbered according to the IMGT exon numbering scheme as shown in FIG. 1.

[0138] In one embodiment, the antibody molecule comprises, has, or consists of a CH3 domain comprising the CH3 domain sequence of FS20-22-38, FS20-22-41, FS20-22-47, FS20-22-49, FS20-22-85, FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, FS20-31-108, or FS20-31-115, where the CH3 domain sequences of FS20-22-38, FS20-22-41, FS20-22-47, FS20-22-49, FS20-22-85, FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, FS20-31-108, and FS20-31-115 are set forth in SEQ ID NOs: 54, 61, 63, 66, 69, 74, 77, 82, 86, 90, and 93, respectively.

[0139] In a preferred embodiment, the antibody molecule comprises, has, or consists of a CH3 domain comprising the CH3 domain sequence of FS20-22-49 set forth in SEQ ID NO: 54.

[0140] The CH3 domain of the antibody molecule may optionally contain an additional lysine residue (K) immediately adjacent to the C-terminus of the CH3 domain sequence.

[0141] Furthermore, the antibody molecule of the present invention may include the CH2 domain of an immunoglobulin G molecule such as the CH2 domain of an IgG1, IgG2, IgG3, or IgG4 molecule. Preferably, the antibody molecule of the present invention includes the CH2 domain of an IgG1 molecule. The CH2 domain may have the sequence set forth in SEQ ID NO: 48.

[0142] The CH2 domain of the antibody molecule may include one or more mutations that reduce or suppress the binding of the CH2 domain to one or more Fcγ receptors such as FcγRI, FcγIIla, FcγRIIb, FcγRIII, and / or complement. The inventors hypothesize that by reducing or suppressing the binding to the Fcγ receptor, ADCC mediated by the antibody molecule is reduced or eliminated. Similarly, reducing or suppressing the binding to complement is expected to reduce or eliminate CDC mediated by the antibody molecule. Mutations that reduce or suppress the binding of the CH2 domain to one or more Fcγ receptors and / or complement are known in the art (Wang et al., 2018). These mutations include the "LALA mutation" described in Bruhns et al., 2009 and Hezareh et al., 2001, which involves substituting the leucine residues at positions 1.3 and 1.2 of the CH2 domain with alanine (L1.3A and L1.2A). Alternatively, generation of α-glycosylated antibodies through mutation of the conserved N-linked glycosylation site by mutating the asparagine (N) at position 84.4 of the CH2 domain to alanine, glycine, or glutamine (N84.4A, N84.4G, or N84.4Q) is also known to reduce IgG1 effector function (Wang et al., 2018). As a further alternative, it is known that complement activation (C1q binding) and ADCC are reduced by mutating the proline at position 114 of the CH2 domain to alanine or glycine (P114A or P114G) (Idusogie et al., 2000; Klein et al., 2016). These mutations can also be combined to generate antibody molecules with further reduced or no ADCC or CDC activity.

[0143] Thus, the antibody molecule may comprise a CH2 domain, where the CH2 domain (i) alanine residues at positions 1.3 and 1.2; and / or (ii) alanine or glycine at position 114; and / or (iii) alanine, glutamine or glycine at position 84.4 wherein the amino acid residue numbering follows the IMGT numbering scheme.

[0144] In a preferred embodiment, the antibody molecule comprises a CH2 domain, where the CH2 domain (i) alanine residue at position 1.3; and (ii) alanine residue at position 1.2 wherein the amino acid residue numbering follows the IMGT numbering scheme.

[0145] For example, the CH2 domain may have the sequence set forth in SEQ ID NO: 49.

[0146] In an alternative preferred embodiment, the antibody molecule comprises a CH2 domain, where the CH2 domain (i) alanine residue at position 1.3; (ii) alanine residue at position 1.2; and (iii) alanine at position 114 wherein the amino acid residue numbering follows the IMGT numbering scheme.

[0147] For example, the CH2 domain may have the sequence set forth in SEQ ID NO: 50.

[0148] In a preferred embodiment, an antibody molecule that binds to CD137 and OX40 (a) a CDR-based antigen-binding site for CD137; and (b) an OX40 antigen-binding site located in the CH3 domain of the antibody molecule comprising a CDR-based antigen-binding site that comprises three VH CDRs and three VL CDRs (CDR1-6) of antibody FS30-10-16, FS30-10-3, FS30-10-12, FS30-35-14 or FS30-5-37, preferably FS30-10-16, FS30-10-3 or FS30-10-12, more preferably FS30-10-16 or FS30-10-3, and most preferably FS30-10-16, The OX40 antigen-binding site comprises a first sequence, a second sequence and a third sequence located in the AB, CD and EF structural loops of the CH3 domain, respectively, and the first, second and third sequences have the sequence of FS20-22-49 set forth in SEQ ID NO: 51, 52 and 53, respectively.

[0149] In a more preferred embodiment, the antibody molecule that binds to CD137 and OX40 (a) a CDR-based antigen-binding site for CD137; and (b) a CH3 domain comprising, having or consisting of the sequence [FS20-22-49] set forth in SEQ ID NO: 54 comprising a CDR-based antigen-binding site that comprises three VH CDRs and three VL CDRs (CDR1-6) of antibody FS30-10-16, FS30-10-3, FS30-10-12, FS30-35-14 or FS30-5-37, preferably FS30-10-16, FS30-10-3 or FS30-10-12, more preferably FS30-10-16 or FS30-10-3, and most preferably FS30-10-16.

[0150] In an even more preferred embodiment, the antibody molecule that binds to CD137 and OX40 (a) a VH domain and a VL domain comprising a CDR-based antigen-binding site for CD137; and (b) a CH3 domain comprising, having or consisting of the sequence [FS20-22-49] set forth in SEQ ID NO: 54 comprising, wherein the VH and VL domains comprise, have or consist of the VH and VL of antibody FS30-10-16, FS30-10-3, FS30-10-12, FS30-35-14 or FS30-5-37, preferably FS30-10-16, FS30-10-3 or FS30-10-12, more preferably FS30-10-16 or FS30-10-3, and most preferably FS30-10-16.

[0151] In a further preferred embodiment, the antibody molecule that binds to CD137 and OX40 is the antibody: (i) FS20-22-49AA / FS30-10-16 set forth in SEQ ID NOs: 95 and 97, respectively; (ii) FS20-22-49AA / FS30-10-3 set forth in SEQ ID NOs: 99 and 97, respectively; (iii) FS20-22-49AA / FS30-10-12 set forth in SEQ ID NOs: 103 and 97, respectively; (iv) FS20-22-49AA / FS30-35-14 set forth in SEQ ID NOs: 105 and 107, respectively; or (v) FS20-22-49AA / FS30-5-37 set forth in SEQ ID NOs: 109 and 111, respectively comprising, having or consisting of the heavy and light chains thereof, wherein the antibody molecule preferably comprises the light and heavy chains described in (i) to (iv), more preferably the light and heavy chains described in (i) to (iii), and most preferably the light and heavy chains described in (i).

[0152] The antibody molecules of the present invention may also include variants of the first, second, or third sequences, AB, CD, or EF structural loop sequences, CH3 domain, CH2 domain, CH2 and CH3 domains, CDR, VH domain, VL domain, light chain, and / or heavy chain sequences disclosed herein. Suitable variants can be obtained by methods of sequence alteration or mutation and screening. In a preferred embodiment, an antibody molecule comprising one or more variant sequences retains one or more functional characteristics of the parental antibody molecule, such as binding specificity and / or binding affinity for CD137 and OX40. For example, an antibody molecule comprising one or more variant sequences preferably binds to CD137 and / or OX40 with the same or higher affinity as the (parental) antibody molecule. The parental antibody molecule is an antibody molecule that does not contain the amino acid substitutions, deletions, and / or insertions incorporated into the variant antibody molecule.

[0153] For example, the antibody molecules of the present invention may have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% sequence identity to the first, second, or third sequences, AB, CD, or EF structural loop sequences, CH3 domain, CH2 domain, CH2 and CH3 domains, CDR, VH domain, VL domain, light chain, and / or heavy chain sequences described herein.

[0154] In a preferred embodiment, the antibody molecule of the present invention comprises a CH3 domain sequence having at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the CH3 domain sequence [FS20-22-49] set forth in SEQ ID NO: 54.

[0155] In a more preferred embodiment, the antibody molecule has or comprises a CH2 domain sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8% or at least 99.9% sequence identity to the CH2 domain sequence set forth in SEQ ID NO: 48 or 49.

[0156] Sequence identity is generally defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego, USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximizing the number of matches and minimizing the number of gaps. Generally, default parameters where the gap generation penalty is equal to 12 and the gap extension penalty is equal to 4 are used. Although the use of GAP may be preferred, other algorithms such as BLAST (using the method of Altschul et al., 1990), FASTA (using the method of Pearson and Lipman, 1988), or the Smith-Waterman algorithm (Smith and Waterman, 1981), or the TBLASTN program of Altschul et al. 1990 supra may also generally be used with default parameters. In particular, the psi-Blast algorithm (Altschul et al., 1997) may be used.

[0157] The antibody molecule of the present invention has one or more amino acid sequence alterations (addition, deletion, substitution, and / or insertion of amino acid residues), 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, compared to the first, second, or third sequences, AB, CD, or EF structural loop sequences, CH3 domain, CH2 domain, CH2 and CH3 domains, Fcab, CDR, VH domain, VL domain, light chain, or heavy chain sequences described herein. The first, second, or third sequences, AB, CD, or EF structural loop sequences, CH3 domain, CH2 domain, CH2 and CH3 domains, CDR, VH domain, VL domain, light chain, and / or heavy chain may also be included. In particular, the alterations may be made in one or more framework regions of the antibody molecule outside the VH and VL domain sequences and / or one or more framework regions of the CH3 domain. For example, the alterations may be present in the CH3 domain outside the sequences described herein as the first, second, and third sequences or as the AB, CD, or EF structural loop sequences.

[0158] In a preferred embodiment, the antibody molecule of the present invention has one or more amino acid sequence alterations (addition, deletion, substitution, and / or insertion of amino acid residues), 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, compared to the CH3 domain sequence set forth in SEQ ID NO: 54, 61, 63, 66, 69, 74, 77, 82, 86, 90, or 93, and may include a CH3 domain sequence.

[0159] In a more preferred embodiment, the antibody molecule has one or more amino acid sequence alterations (addition, deletion, substitution, and / or insertion of amino acid residues), 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, compared to the CH2 domain sequence set forth in SEQ ID NO: 48 or 49, and includes a CH2 domain sequence.

[0160] In a preferred embodiment where one or more amino acids are substituted with another amino acid, the substitution can be, for example, a conservative substitution according to the following table. In some embodiments, amino acids in the same category in the central column are substituted with each other, i.e., nonpolar amino acids are substituted with, for example, another nonpolar amino acid. In some embodiments, amino acids in the same row in the rightmost column are substituted with each other.

[0161]

Table 1

[0162] In some embodiments, the substitution can be functionally conservative. That is, in some embodiments, the substitution may not (or may not substantially) affect one or more functional properties (e.g., binding affinity) of the antibody molecule containing the substitution as compared to an equivalent unsubstituted antibody molecule.

[0163] The antibody molecule preferably binds to human CD137 and human OX40. Preferably, the antibody molecule can bind to human CD137 and human OX40 simultaneously, where human CD137 and human OX40 are co-expressed. Co-expression in this sense includes situations where CD137 and OX40 are expressed in the same cell, such as an immune cell like a T cell, and situations where CD137 and OX40 are expressed in two different immune cells that are adjacent to each other in the tumor microenvironment. Thus, the antibody molecule of the present invention is considered to be able to bind to both targets on a single cis cell and also to two targets expressed on different trans cells.

[0164] The antibody molecule preferably binds to dimeric human CD137 with an affinity (K D ) of 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.4 nM or 0.3 nM or higher. Preferably, the antibody molecule has an affinity (K D) binds to human CD137 with an affinity equal to or higher than that. The antibody molecule can bind to dimeric CD137 with an affinity higher than that of monomeric CD137. Human CD137 can have, for example, the sequence shown in SEQ ID NO: 127.

[0165] The antibody molecule preferably has an affinity (K D ) of 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.4 nM or 0.3 nM or higher and binds to dimeric human OX40. Preferably, the antibody molecule has an affinity (K D ) of 0.3 nM or higher and binds to human OX40. The antibody molecule can bind to dimeric OX40 with an affinity higher than that of monomeric OX40. Human OX40 can have, for example, the sequence described in SEQ ID NO: 130.

[0166] The antibody molecule preferably binds to cynomolgus CD137 and cynomolgus OX40. Binding to cynomolgus CD137 and OX40 as well as human CD137 and OX40 is beneficial because it allows testing of the antibody molecule in cynomolgus for efficacy and toxicity prior to administration to humans. Preferably, the antibody molecule can bind to cynomolgus CD137 and cynomolgus OX40 simultaneously, where cynomolgus CD137 and cynomolgus OX40 are co-expressed.

[0167] The antibody molecule preferably has an affinity (K D ) of 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.4 nM or 0.3 nM or higher and binds to dimeric cynomolgus CD137. Preferably, the antibody molecule has an affinity (K D ) of 0.3 nM or higher and binds to dimeric cynomolgus CD137. Cynomolgus CD137 can have, for example, the sequence described in SEQ ID NO: 129.

[0168] The antibody molecule preferably binds to dimeric cynomolgus OX40 with an affinity (K D ) of 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2.5 nM, 2 nM, 1.5 nM or 1 nM or higher. Preferably, the antibody molecule binds to cynomolgus OX40 with an affinity (K D ) of 1 nM or higher. Cynomolgus OX40 can have, for example, the sequence set forth in SEQ ID NO: 131.

[0169] The antibody molecule preferably binds to dimeric cynomolgus OX40 with an affinity (K D ) that is within 10-fold, 9-fold, 8-fold, 7-fold, 6-fold or 5-fold of the affinity (K D ) with which the antibody molecule binds to dimeric human OX40. Preferably, the antibody molecule binds to dimeric cynomolgus OX40 with an affinity (K D ) that is within 5-fold of the affinity (K D ) with which the antibody molecule binds to dimeric human OX40.

[0170] The antibody molecule preferably binds to dimeric cynomolgus CD137 with an affinity (K D ) that is within 30-fold, 20-fold, 10-fold, 5-fold, 4-fold, 3-fold or 2-fold of the affinity (K D ) with which the antibody molecule binds to dimeric human CD137. Preferably, the antibody molecule binds to dimeric cynomolgus CD137 with an affinity (K D ) that is within 2-fold of the affinity (K D ) with which the antibody molecule binds to dimeric human CD137.

[0171] As described in this example, since it is expected that the behavior of mAb 2 in cynomolgus studies can be extrapolated to humans, the similarity in binding to human and cynomolgus antigens can be considered advantageous. This is considered beneficial for conducting efficacy and toxicity studies performed with antibody molecules in cynomolgus, which can predict the antibody molecule and efficacy and toxicity in humans.

[0172] The antibody molecule preferably binds to dimeric human CD137 with an affinity (K D ) that is within 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, or 5-fold of the affinity (K D ) with which the antibody molecule binds to dimeric human OX40. Preferably, the antibody molecule binds to dimeric human CD137 with an affinity (K D ) that is within 2-fold of the affinity (K D ) with which the antibody molecule binds to dimeric human OX40.

[0173] The antibody molecule preferably binds to dimeric cynomolgus CD137 with an affinity (K D ) that is within 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, or 5-fold of the affinity (K D ) with which the antibody molecule binds to dimeric cynomolgus OX40.

[0174] As described in this example, since the antibody molecule is more likely to bind to cells expressing both targets, it is considered that an antibody molecule having a similar affinity for binding to both targets, namely CD137 and OX40, may be advantageous.

[0175] The binding affinity of the antibody molecule for homologous antigens such as human OX40, human CD137, cynomolgus OX40, or cynomolgus CD137 can be determined, for example, by surface plasmon resonance (SPR) such as Biacore. The binding affinity of the antibody molecule for OX40 or CD137 expressed on the cell surface can be determined by flow cytometry.

[0176] The antibody molecule has been shown to have various activities with respect to ligand binding. For example, the antibody molecule may be able to block, not able to block, or may be able to partially block the binding of CD137L to CD137.

[0177] Preferably, the antibody molecule can block, not block, or possibly partially block the binding of CD137L to CD137. More preferably, the antibody molecule can partially block the binding of CD137L to CD137.

[0178] Preferably, when the two targets are co-expressed, the antibody molecule can induce signaling of OX40 and / or CD137 as a result of cross-linking by double binding to both OX40 and CD137. By acting in this way, such an antibody molecule is called a "dual agonist", that is, the antibody molecule can induce receptor-mediated signaling as a result of cross-linking by double binding to both OX40 and CD137. Thus, preferably, the antibody molecule can induce dual agonist activity when both OX40 and CD137 are co-expressed. As described herein, such dual agonists are expected to be advantageous. For example, such dual agonists can combine the activation of different immune cells, for example, by binding to both targets on different cells in trans, the activities of CD8+ and CD4+ T cells can be combined, so it is considered that a stronger stimulation of the immune response can be induced. As a further example, such dual agonists can, by binding to both targets in cis, result in the activation of a single cell co-expressing both targets without the need for two cells that interact with each other.

[0179] More preferably, the dual agonist needs to be able to drive agonist activity autonomously by simultaneous engagement with its specific targets (OX40 and CD137) without the need for additional cross-linking, such as a cross-linking agent or Fcγ receptor. As described herein, such autonomous activity is expected to be advantageous because it is restricted to the location where both targets are co-expressed, thus reducing the potential toxicity associated with the activation of CD137 at locations where there is little or no co-expression of OX40.

[0180] The ability of an antibody molecule to activate T cells can be measured using a T cell activation assay. T cells, when activated, release IL-2. Thus, a T cell activation assay can measure IL-2 release to determine the level of T cell activation induced by an antibody molecule.

[0181] For example, the ability of an antibody molecule to activate T cells is determined by measuring the concentration of the antibody molecule required to achieve half-maximal release of IL-2 by T cells in a T cell activation assay. This is referred to below as the EC 50 and is so called.

[0182] In a preferred embodiment, the antibody molecule has an EC 50 within 50-fold, 40-fold, 30-fold, 20-fold, 10-fold or 5-fold of the EC 50 of FS20-22-49AA / FS30-10-16 in a T cell activation assay, where FS20-22-49AA / FS30-10-16 consists of the heavy chain of SEQ ID NO: 95 and the light chain of SEQ ID NO: 97.

[0183] For example, the antibody molecule has an EC 50 of 30 nM or less, 25 nM or less, 20 nM or less, 14 nM or less, 10 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1.5 nM, 1 nM or 0.5 nM or less, preferably 1.5 nM or less, more preferably 1 nM or less when cross-linked, in a T cell activation assay.

[0184] Additionally or alternatively, the ability of an antibody molecule to activate T cells can be determined by measuring the maximum concentration of IL-2 released by T cells in a T cell activation assay in the presence of the antibody molecule.

[0185] In a preferred embodiment, in a T cell activation assay in the presence of an antibody molecule, the maximum concentration of IL-2 released by T cells is within 20% or 10% of the maximum concentration of IL-2 released by T cells in the presence of FS20-22-49AA / FS30-10-16 in the same assay, where FS20-22-49AA / FS30-10-16 consists of the heavy chain of SEQ ID NO: 95 and the light chain of SEQ ID NO: 97.

[0186] The T cell activation assay preferably includes T cells that co-express OX40 and CD137. In a preferred embodiment, the T cell activation assay does not include any agent capable of cross-linking antibody molecules other than CD137 and OX40.

[0187] The T cell activation assay can be a T cell assay as described herein, such as a pan-T cell assay, a CD4+ T cell assay, or a CD8+ T cell assay as described in this example.

[0188] For example, the T cell activation assay can be an IL-2 release assay based on T cells isolated from human peripheral blood mononuclear cells (PBMC). The CD4+ T cell activation assay or the CD8+ T cell activation assay can be an IL-2 release assay based on CD4+ T cells or CD8+ T cells isolated from human PBMC, respectively. As described in this example, antibody molecules capable of activating T cells in both CD4+ and CD8+ T cell assays can activate both OX40 and CD137 (also referred to as a "dual agonist"). For example, the T cell activation assay can include separating human PBMC from a leukocyte-depleted cone. Methods for isolating PBMC are known in the art and are described in this example. Next, T cells can be isolated from PBMC. Methods for isolating T cells (all T cells, CD4+ T cells, or CD8+ T cells) from PBMC are also known in the art and are described in this example.

[0189] The activation assay may include preparing the required number of T cells in an experimental medium such as a T cell medium. The required number of T cells can be prepared at a concentration of 1.0×10 6 cells / ml. Next, the T cells can be stimulated using an appropriate T cell activation reagent that provides the signals necessary for T cell activation. For example, the T cell activation reagent can be a reagent containing CD3 and CD28, such as beads containing CD3 and CD28. The isolated T cells can be incubated overnight with the T cell activation reagent to activate the T cells. Subsequently, the activated T cells are washed to separate the T cells from the T cell activation reagent and resuspended in a T cell medium at an appropriate concentration such as 2.0×10 6 cells / ml. Next, the activated T cells can be added to plates coated with anti-human CD3 antibody.

[0190] An appropriate dilution of each test antibody molecule can be prepared and added to the wells. Next, the T cells can be incubated with the test antibody at 37°C and 5% CO2 for 24 hours. The supernatant is collected and assayed to measure the concentration of IL-2 in the supernatant. Methods for determining the concentration of IL-2 in a solution are known in the art and are described in this example. The concentration of human IL-2 can be plotted against the logarithmic concentration of the antibody molecule. The resulting curve can be fitted using a log(agonist) vs response equation.

[0191] The antibody molecule can be conjugated to a bioactive molecule or a detectable label. In this case, the antibody molecule can be referred to as a conjugate. Such conjugates find use in the treatment of diseases as described herein.

[0192] For example, the bioactive molecule can be an immunomodulator such as a cytokine, preferably a human cytokine. For example, the cytokine can be a cytokine that stimulates the activation and / or proliferation of T cells. Examples of cytokines for conjugating to antibody molecules include IL-2, IL-10, IL-12, IL-15, IL-21, GM-CSF, and IFN-gamma.

[0193] Alternatively, the bioactive molecule can be a ligand trap such as a cytokine, e.g., a ligand trap of TGF-beta or IL-6.

[0194] Alternatively, the bioactive molecule can be a therapeutic radioisotope.

[0195] Radioimmunotherapy is used, for example, in the treatment of cancer. Suitable therapeutic radioisotopes for radioimmunotherapy are known in the art and include yttrium-90, iodine-131, bismuth-213, astatine-211, lutetium 177, rhenium-188, copper-67, actinium-225, iodine-125, and terbium-161.

[0196] Suitable detectable labels that can be conjugated to antibody molecules are known in the art and include radioisotopes such as iodine 125, iodine 131, yttrium 90, indium 111, technetium 99; fluorescent dyes such as fluorescein, rhodamine, phycoerythrin, Texas red, and cyanine dye derivatives (e.g., Cy7 and Alexa750); chromogenic dyes such as diaminobenzidine; latex beads; enzyme labels such as horseradish peroxidase; phosphors or laser dyes with spectrally separated absorption or emission characteristics; and chemical moieties such as biotin that can be detected via binding to a specific cognate detectable moiety (e.g., labeled avidin).

[0197] Antibody molecules can be conjugated to bioactive molecules or detectable labels by any suitable covalent or non-covalent bond, such as a disulfide or peptide bond. If the bioactive molecule is a cytokine, the cytokine can be bound to the antibody molecule by a peptide linker. Suitable peptide linkers are known in the art and can be 5 to 25 amino acids, 5 to 20 amino acids, 5 to 15 amino acids, 10 to 25 amino acids, 10 to 20 amino acids, or 10 to 15 amino acids in length.

[0198] In some embodiments, the bioactive molecule can be conjugated to the antibody molecule by a cleavable linker. The linker can enable the release of the bioactive molecule from the antibody molecule at the site of treatment. The linker can include an amide bond (e.g., a peptide linker), a disulfide bond, or a hydrazone. For example, the peptide linker can be cleaved by a site-specific protease, the disulfide bond can be cleaved by the reducing environment of the cytosol, and the hydrazone can be cleaved by acid-mediated hydrolysis.

[0199] The present invention also provides one or more isolated nucleic acid molecules encoding the antibody molecules of the present invention. Those skilled in the art will have no difficulty in preparing such nucleic acid molecules using methods well known in the art.

[0200] The one or more nucleic acid molecules can include, for example, the sequences set forth in SEQ ID NO: 55 or 113, 62, 64, 67, 70, 75, 78, 83, 87, 91, or 94, which encode the CH3 domains of FS20-22-49, FS20-22-38, FS20-22-41, FS20-22-47, FS20-22-85, FS20-31-58, FS20-31-66, FS20-31-94, FS20-31-102, FS20-31-108, and FS20-31-115, respectively. For example, the one or more nucleic acid molecules can include the sequences set forth in SEQ ID NO: 55 or 113, both of which encode the CH3 domain of FS20-22-49. In some embodiments, the one or more nucleic acid molecules include the sequence set forth in SEQ ID NO: 113, which encodes the CH3 domain of FS20-22-49. Preferably, the one or more nucleic acid molecules include the sequence set forth in SEQ ID NO: 55, which encodes the CH3 domain of FS20-22-49.

[0201] One or more nucleic acid molecules may encode the VH domain and / or VL domain of antibody FS30-10-16, FS30-10-3, FS30-10-12, FS30-35-14 or FS30-5-37, preferably antibody FS30-10-16, FS30-10-3, FS30-10-12 or FS30-35-14, more preferably antibody FS30-10-16, FS30-10-3 or FS30-10-12, and most preferably antibody FS30-10-16. The VH and VL domain sequences of these antibodies are described herein.

[0202] For example, the nucleic acid molecule may be (i) the VH domain nucleic acid sequence of antibody FS30-10-16 described in SEQ ID NO: 13 and / or the VL domain nucleic acid sequence of antibody FS30-10-16 described in SEQ ID NO: 15; or (ii) the VH domain nucleic acid sequence of antibody FS30-10-3 described in SEQ ID NO: 19 and / or the VL domain nucleic acid sequence of antibody FS30-10-3 described in SEQ ID NO: 20; (iii) the VH domain nucleic acid sequence of antibody FS30-10-12 described in SEQ ID NO: 24 and / or the VL domain nucleic acid sequence of antibody FS30-10-12 described in SEQ ID NO: 20; (iv) the VH domain nucleic acid sequence of antibody FS30-35-14 described in SEQ ID NO: 171 and / or the VL domain nucleic acid sequence of antibody FS30-35-14 described in SEQ ID NO: 32; or (v) the VH domain nucleic acid sequence of antibody FS30-5-37 described in SEQ ID NO: 41 and / or the VL domain nucleic acid sequence of antibody FS30-5-37 described in SEQ ID NO: 43 and may include.

[0203] One or more nucleic acid molecules may encode a heavy chain and / or a light chain, preferably the heavy and light chains of antibody FS20-22-49AA / FS30-10-16, FS20-22-49AA / FS30-10-3, FS20-22-49AA / FS30-10-12, FS20-22-49AA / FS30-35-14 or FS20-22-49AA / FS30-5-37, preferably the heavy and light chains of antibody FS20-22-49AA / FS30-10-16, FS20-22-49AA / FS30-10-3, FS20-22-49AA / FS30-10-12 or FS20-22-49AA / FS30-35-14, more preferably the heavy and light chains of antibody FS20-22-49AA / FS30-10-16, FS20-22-49AA / FS30-10-3 or FS20-22-49AA / FS30-10-12, most preferably FS20-22-49AA / FS30-10-16. The VH and VL domain sequences of these antibodies are described herein.

[0204] For example, the nucleic acid molecule may be (i) the heavy chain nucleic acid sequence of antibody FS20-22-49AA / FS30-10-16 described in SEQ ID NO: 96 and / or the light chain nucleic acid sequence of antibody FS20-22-49AA / FS30-10-16 described in SEQ ID NO: 98; or (ii) the heavy chain nucleic acid sequence of antibody FS20-22-49AA / FS30-10-3 described in SEQ ID NO: 100 and / or the light chain nucleic acid sequence of antibody FS20-22-49AA / FS30-10-3 described in SEQ ID NO: 102; (iii) the heavy chain nucleic acid sequence of antibody FS20-22-49AA / FS30-10-12 described in SEQ ID NO: 104 and / or the light chain nucleic acid sequence of antibody FS20-22-49AA / FS30-10-12 described in SEQ ID NO: 102; (iv) the heavy chain nucleic acid sequence of antibody FS20-22-49AA / FS30-35-14 described in SEQ ID NO: 106 and / or the light chain nucleic acid sequence of antibody FS20-22-49AA / FS30-35-14 described in SEQ ID NO: 108; or (v) The heavy-chain nucleic acid sequence of the antibody FS20-22-49AA / FS30-5-37 described in SEQ ID NO: 110 and / or the light-chain nucleic acid sequence of the antibody FS20-22-49AA / FS30-5-37 described in SEQ ID NO: 112 may be included.

[0205] When the nucleic acid encodes the VH and VL domains or the heavy and light chains of the antibody molecule of the present invention, the two domains or chains may be encoded on two separate nucleic acid molecules.

[0206] The isolated nucleic acid molecule can be used to express the antibody molecule of the present invention. The nucleic acid is generally provided in the form of a recombinant vector for expression. Thus, another aspect of the present invention provides a vector containing a nucleic acid as described above. An appropriate vector containing appropriate regulatory sequences, including promoter sequences, transcription termination fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as required, can be selected or constructed. Preferably, the vector contains appropriate regulatory sequences for driving the expression of the nucleic acid in the host cell. The vector can be, if necessary, a plasmid, viral, such as phage or phagemid.

[0207] The nucleic acid molecule or vector described herein can be introduced into a host cell. Techniques for introducing the nucleic acid or vector into a host cell are well established in the art and any appropriate technique can be used. Various host cells suitable for the production of recombinant antibody molecules are known in the art and include bacterial, yeast, insect or mammalian host cells. Preferred host cells are mammalian cells such as CHO, NS0 or HEK cells, such as HEK293 cells.

[0208] Another aspect of the invention provides a method of producing an antibody molecule of the invention, the method comprising expressing a nucleic acid encoding the antibody molecule in a host cell and optionally isolating and / or purifying the antibody molecule so produced. Methods for culturing host cells are well known in the art. The method may further comprise isolating and / or purifying the antibody molecule. Techniques for purifying recombinant antibody molecules are well known in the art and include, for example, HPLC, FPLC or affinity chromatography (e.g., using Protein A or Protein L). In some embodiments, purification may be carried out using an affinity tag on the antibody molecule. The method may also comprise formulating the antibody molecule into a pharmaceutical composition, optionally with a pharmaceutically acceptable excipient or other substances described below.

[0209] As described above, both CD137 and OX40 are expressed on cells of the immune system including T cells. For example, OX40 is expressed on activated T cells, particularly CD4+ T cells, CD8+ T cells, type 1 T helper (Th1) cells, type 2 T helper (Th2) cells and regulatory T (Treg) cells, as well as tumor infiltrating T cells, and on cells of the immune system including activated natural killer (NK) cells. CD137 is expressed on cells of the immune system including T cells, particularly CD8+ T cells, B cells, NK cells and tumor infiltrating lymphocytes (TIL). CD137 is expressed at lower levels on CD4+ T cells than on CD8+ T cells (see Example 14 and Figure 6), but has also been shown to be involved in inducing the proliferation and activation of some subsets of CD4+ T cells (Wen et al., 2002).

[0210] Activation of OX40 has been shown to play a role in enhancing T cell activation, T cell clonal expansion, T cell differentiation and survival, and the generation of memory T cells. Activation of CD137 has been shown to play a role in enhancing CD8+ T cell proliferation, survival and cytotoxic effector function, as well as CD8+ T cell differentiation and the maintenance of memory CD8+ T cells. Activation of CD137 has also been demonstrated to enhance NK cell-mediated ADCC and B cell proliferation, survival and cytokine production.

[0211] In light of the immune response enhancing activities of OX40 and CD137, OX40 and CD137 agonist molecules have been studied in relation to cancer treatment and are also expected to find use in the treatment of infectious diseases.

[0212] Accordingly, the antibody molecules described herein may be useful for therapeutic applications, particularly in the treatment of cancer and infectious diseases.

[0213] The antibody molecules described herein may be used in methods of treating the human or animal body. Related aspects of the invention are (i) the antibody molecules described herein for use as a medicament, (ii) the antibody molecules described herein for use in a method of treating a disease or disorder, (iii) the use of the antibody molecules described herein in the manufacture of a medicament for use in the treatment of a disease or disorder; and (iv) a method of treating a disease or disorder in an individual, the method comprising administering to the individual a therapeutically effective amount of the antibody molecules described herein are provided.

[0214] The individual can be a patient, preferably a human patient.

[0215] Treatment can be any treatment or therapy by which some desired therapeutic effect, such as inhibition or delay of the progression of a condition, is achieved, including a decrease in the rate of progression, a halt in the rate of progression, an improvement in the condition, a cure or remission (partial or total) of the condition, prevention, improvement, delay, alleviation or arrest of one or more symptoms and / or signs of the condition, or extension of the survival period of an individual or patient beyond that expected in the absence of treatment.

[0216] Treatment as a prophylactic measure (i.e., prophylaxis) is also included. For example, an individual who is susceptible to or at risk of the development or recurrence of a disease such as cancer can be treated as described herein. Such treatment can prevent or delay the development or recurrence of the disease in the individual.

[0217] The treatment methods as described can include, in addition to the antibody molecule, administering to the individual at least one further treatment. Thus, the antibody molecules described herein can be administered to an individual alone or in combination with one or more other treatments. When the antibody molecule is administered to an individual in combination with another treatment, the additional treatment can be administered to the individual simultaneously with, sequentially or separately from the administration of the antibody molecule. When the additional treatment is administered simultaneously with the antibody molecule, the antibody molecule and the additional treatment can be administered to the individual as a combined preparation. For example, the additional treatment can be a known treatment or therapeutic agent for the disease being treated.

[0218] The antibody molecule can be administered alone, but the antibody molecule is usually administered in the form of a pharmaceutical composition that can usually contain at least one component in addition to the antibody molecule. Thus, another aspect of the invention provides a pharmaceutical composition comprising the antibody molecules described herein. A method is also provided that includes formulating the antibody molecule into a pharmaceutical composition.

[0219] In addition to the antibody molecule, the pharmaceutical composition may include pharmaceutically acceptable excipients, carriers, buffers, stabilizers or other materials well known to those skilled in the art. As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with the tissues of a subject (e.g., a human) without undue toxicity, irritation, allergic reaction or other problems or complications, within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. The exact nature of the carrier or other material will depend on the route of administration, which may be by infusion, injection or other suitable route, as discussed below.

[0220] In the case of parenteral, such as subcutaneous or intravenous administration (e.g., by injection), the pharmaceutical composition containing the antibody molecule can be in the form of an aqueous solution that is pyrogen-free and has an appropriate pH, isotonicity, and stability, and is parenterally acceptable. Those skilled in the art with relevant technology can adequately prepare an appropriate solution using, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, and lactated Ringer's injection. Buffering agents such as phosphoric acid, citric acid, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3'-pentanol; and m-cresol); low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, and dextrose; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or preservatives, stabilizers, buffering agents, antioxidants, and / or other additives including nonionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG) can be used as needed.

[0221] In some embodiments, the antibody molecule can be provided in lyophilized form for reconstitution prior to administration. For example, the lyophilized antibody molecule can be reconstituted with sterile water and mixed with physiological saline prior to administration to an individual.

[0222] Administration can be at a "therapeutically effective amount", which is sufficient to demonstrate benefit to the individual. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of the subject being treated, the particular individual being treated, the clinical state of the individual, the cause of the disorder, the site to which the composition is to be delivered, the type of antibody molecule, the method of administration, the dosing schedule, and other factors known to the physician. Prescription of treatment, such as determination of dosage, etc., is within the responsibility of the general practitioner and other physicians and may depend on the severity of the symptoms and / or the progression of the disease being treated. Appropriate dosages of antibody molecules are well known in the art (Ledermann et al., 1991; Bagshawe et al., 1991). Specific dosages shown in this specification or in the Physician’s Desk Reference (2003) that are appropriate for the antibody molecule being administered may be used. The therapeutically effective amount or appropriate dosage of an antibody molecule can be determined by comparing its in vitro and in vivo activities in animal models. Methods for extrapolating effective dosage levels in mice and other test animals to humans are known. The exact dosage will depend on a number of factors including the size and location of the area being treated and the exact nature of the antibody molecule.

[0223] Typical antibody dosages range from 100 μg to 1 g for systemic administration and from 1 μg to 1 mg for local administration. An initial higher loading dose, followed by one or more lower doses, may be administered. This is the dosage for a single treatment of an adult individual and can be proportionally adjusted for pediatric and infant individuals and can also be adjusted for other antibody formats in proportion to the molecular weight.

[0224] Treatment can be repeated daily, twice weekly, once weekly, or once monthly at the discretion of the physician. The treatment schedule for an individual may depend on the pharmacokinetic and pharmacodynamic properties of the antibody composition, the route of administration, and the nature of the condition being treated.

[0225] The treatment can be periodic, and the period between administrations can be about 2 weeks or more, for example about 3 weeks or more, about 4 weeks or more, about once a month or more, about 5 weeks or more, or about 6 weeks or more. For example, the treatment can be every 2 to 4 weeks or every 4 to 8 weeks. Suitable formulations and routes of administration are described above.

[0226] In a preferred embodiment, the antibody molecules described herein can be for use in a method of treating cancer.

[0227] Cancer can be characterized by the abnormal proliferation of malignant cancer cells. When referring to a particular type of cancer such as breast cancer, this refers to the abnormal proliferation of malignant cells in related tissues such as breast tissue. A secondary cancer that is located in the breast but is the result of the abnormal proliferation of malignant cells in another tissue such as ovarian tissue is not breast cancer as referred to herein, but ovarian cancer.

[0228] Cancer can be primary or secondary cancer. Thus, the antibody molecules described herein can be for use in a method of treating cancer in an individual where the cancer is a primary tumor and / or tumor metastasis.

[0229] The tumors of cancer treated using the antibody molecules described herein can, for example, contain TILs that express OX40 and / or CD137 on their cell surface. In one embodiment, the tumor can be determined to contain TILs that express one or both of OX40 and / or CD137. Methods for determining the expression of antigens on the cell surface are known in the art and include, for example, flow cytometry.

[0230] For example, cancers treated using the antibody molecules described herein can be selected from the group consisting of leukemias such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia (CLL); lymphomas such as Hodgkin lymphoma, non-Hodgkin lymphoma, and multiple myeloma; and solid cancers such as sarcoma (e.g., soft tissue sarcoma), skin cancer (e.g., Merkel cell carcinoma), melanoma, bladder cancer (e.g., urothelial carcinoma of the bladder), brain tumor (e.g., glioblastoma multiforme), breast cancer, uterine / endometrial cancer, ovarian cancer (e.g., serous cystadenoma of the ovary), prostate cancer, lung cancer such as squamous cell carcinoma of the lung (e.g., non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC)), colorectal cancer (e.g., colorectal adenocarcinoma), cervical cancer (e.g., squamous cell carcinoma of the cervix and endocervical adenocarcinoma), liver cancer (e.g., hepatocellular carcinoma), head and neck cancer (e.g., squamous cell carcinoma of the head and neck), oesophageal cancer (e.g., oesophageal carcinoma), pancreatic cancer, kidney cancer (e.g., renal cell carcinoma), adrenal cancer, gastric cancer (e.g., gastric adenocarcinoma), testicular cancer (e.g., testicular germ cell tumor), cancer of the gallbladder and biliary tract (e.g., cholangiocarcinoma), thyroid cancer, thymic cancer, bone cancer, and brain cancer.

[0231] In a preferred embodiment, the cancer treated using the antibody molecules described herein is a solid cancer.

[0232] More preferably, the cancer treated using the antibody molecules described herein is a solid cancer selected from the group consisting of melanoma, bladder cancer, brain cancer, breast cancer, ovarian cancer, lung cancer, colorectal cancer, cervical cancer, liver cancer, head and neck cancer, pancreatic cancer, kidney cancer, and gastric cancer.

[0233] In a further preferred embodiment, the cancer treated using the antibody molecules described herein can be a cancer that responds to treatment with one or more checkpoint inhibitors such as antibodies that bind to PD-1, PD-L1, or CTLA4. Such tumors are thought to have higher TIL levels and / or higher tumor mutation burdens than tumors that are not sensitive to checkpoint inhibitor therapy. Such tumors are also referred to as warm tumors or hot tumors.

[0234] Examples of such tumors include head and neck squamous cell carcinoma (HNSCC), melanoma, lung cancer (such as squamous cell lung cancer, lung adenocarcinoma, non-small cell lung cancer [NSCLC] or small cell lung cancer [SCLC]), prostate cancer, cervical cancer, bladder cancer, breast cancer, thyroid cancer, renal cancer, colorectal cancer (MSI or MSS, such as colorectal adenocarcinoma), esophageal cancer, non-Hodgkin lymphoma (NHL), gastric cancer, endometrial cancer, pancreatic cancer, ovarian cancer, hepatocellular carcinoma, mesothelioma and urothelial cancer. In a preferred embodiment, the cancer is gastric cancer. The cancer can further be a cancer that has not been previously treated with a chemotherapeutic agent or a radiation therapy agent. That is, the individual to be treated can be a cancer patient who has not been treated with a chemotherapeutic agent or a radiation therapy agent for the subject cancer. In a preferred embodiment, the antibody molecules described herein are for use in a method of treating cancer responsive to one or more immune checkpoint inhibitors in an individual, where the method comprises treating the patient with an antibody molecule in combination with an agent that inhibits the interaction between PD-1 and PD-L1.

[0235] Alternatively, the cancer treated using the antibody molecules described herein can be a cancer such as pancreatic cancer or prostate cancer that does not respond to treatment with one or more checkpoint inhibitors such as an antibody that binds to PD-1, PD-L1 or CTLA4. Such tumors are also referred to as cold tumors.

[0236] The inventors have shown that tumors that did not respond to treatment with anti-PD-1 or anti-PD-L1 antibodies alone respond to treatment with anti-PD-1 or anti-PD-L1 antibodies in combination with the antibody molecules described herein. Thus, the antibody molecules of the present invention can be for use in a method of treating cancer in an individual, wherein the cancer does not respond or is resistant to treatment with one or more checkpoint inhibitors alone, and the method comprises administering to the individual the antibody molecule in combination with an agent that inhibits the interaction between PD-1 and PD-L1. Also contemplated is a method of treating cancer in an individual, wherein the cancer does not respond or is resistant to treatment with one or more checkpoint inhibitors alone, and the method comprises administering to the individual the antibody molecule in combination with an agent that inhibits the interaction between PD-1 and PD-L1.

[0237] Without wishing to be bound by theory, treatment of cancer that does not respond to treatment with one or more checkpoint inhibitors alone with chemotherapy, radiotherapy, immunotherapeutic agents such as immunostimulants or anti-tumor vaccines causes cancer cell death, resulting in an increase in TIL within the tumor and an increase in the expression of immunosuppressive receptors, whereby the cancer becomes responsive to treatment with checkpoint inhibitors, i.e., a cold tumor changes to a warm tumor. Thus, the antibody molecules of the present invention can be for use in a method of treating cancer in an individual, wherein the cancer does not respond or is resistant to treatment with one or more checkpoint inhibitors alone, and the method comprises administering to the individual the antibody molecule in combination with a chemotherapeutic agent, a radiotherapeutic agent or an immunostimulant or an anti-cancer vaccine and optionally an agent that inhibits the interaction between PD-1 and PD-L1. Also contemplated is a method of treating cancer in an individual, wherein the cancer does not respond or is resistant to treatment with one or more checkpoint inhibitors alone, and the method comprises administering to the individual the antibody molecule in combination with a chemotherapeutic agent, a radiotherapeutic agent or an immunostimulant or an anti-cancer vaccine and optionally an agent that inhibits the interaction between PD-1 and PD-L1. In a preferred embodiment, the agent that inhibits the interaction between PD-1 and PD-L1 is an antibody that binds to PD-1 or PD-L1.

[0238] In relation to cancer, treatment may include inhibition of cancer growth, including complete cancer remission, and / or inhibition of cancer metastasis and / or inhibition of cancer recurrence. Cancer proliferation generally refers to any of a number of indicators that show a change to a more developed form within the cancer. Thus, indicators for measuring inhibition of cancer proliferation include a decrease in cancer cell survival, a decrease in tumor volume or form (determined, for example, using computed tomography (CT), ultrasound, or other imaging methods), a delay in tumor growth, disruption of the tumor vasculature, improvement in the performance of a delayed hypersensitivity skin test, an increase in the activity of anti-cancer immune cells or other anti-cancer immune responses, and a decrease in the level of tumor-specific antigens. By activating or enhancing the immune response against an individual's cancerous tumor, it is possible to improve the individual's ability to resist cancer growth, particularly the growth of cancer already present in the subject, and / or to reduce the individual's tendency to cancer growth.

[0239] In relation to cancer treatment, the antibody molecules described herein can be administered to an individual in combination with another anti-cancer therapy or therapeutic agent, such as an anti-cancer therapy or therapeutic agent that has been shown to be suitable or potentially suitable for the treatment of the subject's cancer. For example, the antibody molecule can be administered to an individual in combination with a chemotherapeutic agent, radiation therapy, radionuclide, immunotherapy agent, anti-tumor vaccine, oncolytic virus, adoptive cell transfer (ACT) therapy, such as adoptive NK cell therapy or chimeric antigen receptor (CAR), autologous TIL or gamma / delta T cell therapy, or an agent for hormone therapy. The antibody molecules described herein can also be administered to an individual in combination with an adjuvant or neo-adjuvant (such as neo-adjuvant hormone therapy), an anti-angiogenic agent (such as an anti-VEGF or anti-VEGFR2 antibody), or a cytotoxic agent.

[0240] Without wishing to be bound by theory, it is believed that the antibody molecules described herein may act as an adjuvant in anti-cancer treatment. Specifically, for example, administration of the antibody molecule to an individual in combination with chemotherapy or radiation therapy is believed to induce a greater immune response against the cancer than can be achieved by chemotherapy or radiation therapy alone.

[0241] One or more chemotherapeutic agents for administration in combination with an antibody molecule as described herein may be selected from the group consisting of taxanes, cytotoxic antibiotics, tyrosine kinase inhibitors, PARP inhibitors, B-Raf enzyme inhibitors, MEK inhibitors, c-MET inhibitors, VEGFR inhibitors, PDGFR inhibitors, alkylating agents, platinum analogs, nucleoside analogs, folic acid antagonists, thalidomide derivatives, anti-cancer chemotherapeutic agents, and the like. 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, cobimetinib, and trametinib; PARP inhibitors include olaparib; 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 folic acid antagonists include methotrexate and pemetrexed. Other chemotherapeutic agents suitable for use in the present invention include defactinib, enzastaurin, eribulin, irinotecan, and vinblastine. A chemotherapeutic agent for administration in combination with an antibody molecule as described herein may be a fluoropyrimidine. For example, if the cancer to be treated is HER2-negative, such as HER2-negative gastric cancer, the antibody molecule described herein may be administered in combination with a platinum, platinum analog, and fluoropyrimidine. If the cancer to be treated is HER2-positive, such as HER2-positive gastric cancer, the antibody molecule described herein may be administered in combination with a platinum or platinum analog, a fluoropyrimidine, and trastuzumab.

[0242] Preferred therapeutic agents for administration together with the antibody molecule described herein are doxorubicin, mitoxantrone, cyclophosphamide, cisplatin, and oxaliplatin.

[0243] Radiation therapy for administration in combination with an antibody molecule as described herein can be external beam radiation therapy or brachytherapy.

[0244] Radionuclides for administration together with the antibody molecules described herein can be selected from the group consisting of yttrium-90, iodine-131, bismuth-213, astatine-211, lutetium 177, rhenium-188, copper-67, actinium-225, iodine-125, and terbium-161.

[0245] Immunotherapeutic agents for administration in combination with an antibody molecule as described herein can be therapeutic antibody molecules, nucleotides, cytokines, or cytokine-based therapeutic agents. For example, a therapeutic antibody molecule can bind to an immunomodulatory molecule, such as an inhibitory checkpoint molecule or a costimulatory molecule, a receptor of the innate immune system, or a tumor antigen, such as a cell surface tumor antigen or a soluble tumor antigen. Examples of immunomodulatory molecules to which a therapeutic antibody molecule can bind include CTLA-4, LAG-3, TIGIT, TIM-3, VISTA, programmed death ligand 1 (PD-L1), programmed cell death protein 1 (PD-1), CD47, CD73, CSF-1R, KIR, CD40, HVEM, IL-10, and CSF-1. Examples of receptors of the innate immune system to which a therapeutic antibody molecule can bind include TLR1, TLR2, TLR4, TLR5, TLR7, TLR9, RIG-I-like receptors (e.g., RIG-I and MDA-5), and STING. Examples of tumor antigens to which a therapeutic antibody molecule can bind include HER2, EGFR, CD20, and TGF-beta.

[0246] The inventors have shown that administration of the antibody molecule of the present invention in combination with an anti-PD-1 or anti-PD-L1 antibody results in enhanced T cell activation and tumor regression in a mouse tumor model compared to treatment with either the antibody molecule of the present invention or an anti-PD-1 or anti-PD-L1 antibody alone. Without wishing to be bound by theory, these results suggest that administering the antibody molecule of the present invention in combination with an agent capable of inhibiting the interaction between PD-1 and PD-L1 enhances the anti-tumor effect and that such combination administration may be suitable for treating tumors that are refractory or resistant or have recurred after monotherapy with a PD-1 or PD-L1 antibody.

[0247] Accordingly, the antibody molecule of the present invention can be for use in a method of treating cancer in an individual, wherein the method comprises administering an antibody molecule in combination with an agent capable of inhibiting the interaction between PD-1 and PD-L1. Also provided are agents capable of inhibiting the interaction between PD1 and PD-L1, such as antibody molecules that bind to PD-1 or PD-L1 for use in a method of treating cancer in an individual, wherein the method comprises administering an agent capable of inhibiting the interaction between PD-1 and PD-L1 in combination with an antibody of the present invention. A method of treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the antibody molecule of the present invention and a therapeutically effective amount of an agent capable of inhibiting the interaction between PD-1 and PD-L1.

[0248] In a preferred embodiment, the agent capable of inhibiting the interaction of PD-1 and PD-L1 is an antibody molecule that binds to PD-1 or PD-L1. Antibodies that bind to PD-1 are known in the art and include nivolumab (5C4) and pembrolizumab. Known antibodies that bind to PD-L1 include YW243.55.S1, durvalumab, atezolizumab, and avelumab. The antibody molecules of the present invention may be for administration together with one of these known anti-PD-1 or PD-L1 antibodies or another anti-PD-1 or PD-L1 antibody. The preparation of alternative antibodies that bind to PD-1 or PD-L1 is within the ability of those skilled in the art using conventional methods.

[0249] The nucleic acid for administration in combination with an antibody molecule as described herein may be siRNA.

[0250] Cytokine or cytokine-based therapy may be selected from the group consisting of IL2, a prodrug of conjugated IL-2, GM-CSF, IL-7, IL-12, IL-9, IL-15, IL-18, IL-21, and type I interferon.

[0251] Anti-tumor vaccines for the treatment of cancer are carried out clinically and discussed in detail in the scientific literature (such as Rosenberg, 2000). This mainly includes strategies to stimulate the immune system to respond to various cell markers expressed by autologous or allogeneic cancer cells by using these cells as vaccination methods, both with and without granulocyte macrophage colony-stimulating factor (GM-CSF). GM-CSF causes a strong response in antigen presentation and functions particularly well when used in the said strategy.

[0252] The antibody molecules described herein may also be administered in combination with ramucirumab and / or paclitaxel; irinotecan and docetaxel or paclitaxel; or pembrolizumab to an individual having cancer, particularly an individual having gastric cancer. In the treatment of MSI-H and / or dMMR gastric cancer, treatment with the antibody molecules described herein in combination with pembrolizumab is preferred.

[0253] In light of the immune response enhancing activities of OX40 and CD137, OX40 and CD137 dual agonist molecules are expected to find use in the treatment of infectious diseases. Thus, in another preferred embodiment, the antibody molecules described herein may be for use in a method of treating an infectious disease such as an acute or persistent infectious disease.

[0254] Without wishing to be bound by theory, OX40 and CD137 agonist molecules may be able to enhance the immune response against acute infectious diseases caused by pathogens by inducing rapid infiltration and activation of innate immune cells such as neutrophils and monocytes, thereby promoting clearance of the pathogens causing the acute infectious disease. Thus, in a further embodiment, the antibody molecules described herein may be for use in a method of treating an acute infectious disease such as an acute bacterial disease. In a preferred embodiment, the acute infectious disease is an acute bacterial disease caused by an infection with a gram-positive bacterium such as a bacterium of the genus Listeria, Streptococcus pneumoniae or Staphylococcus aureus.

[0255] Infectious diseases are usually eliminated by the immune system, but some infectious diseases persist over a long period such as several months or years and are not effectively countered by the immune system. Such infectious diseases are also referred to as persistent or chronic infectious diseases.

[0256] Preferably, the antibody molecules described herein are used to treat persistent infections, such as persistent viral, bacterial, fungal or parasitic infections, preferably persistent viral or bacterial infections.

[0257] In a preferred embodiment, the persistent viral infections treated using the antibody molecules described herein are persistent infections with human immunodeficiency virus (HIV), Epstein-Barr virus, cytomegalovirus, hepatitis B virus, hepatitis C virus, varicella-zoster virus.

[0258] In a preferred embodiment, the persistent bacterial infections treated using the antibody molecules described herein are persistent infections with Staphylococcus aureus, Hemophilus influenza, Mycobacterium tuberculosis, Mycobacterium leprae, Helicobacter pylori, Treponema pallidum, Enterococcus faecalis or Streptococcus pneumoniae.

[0259] CD137 agonist activity has been described as beneficial in the treatment of infections by Gram-positive bacteria. Thus, in a preferred embodiment, the persistent bacterial infections treated using the antibody molecules described herein are persistent infections by Gram-positive bacteria. In a more preferred embodiment, the persistent bacterial infection is a persistent infection by a Gram-positive bacterium selected from the group consisting of Staphylococcus aureus, Mycobacterium leprae, Enterococcus faecalis and Streptococcus pneumoniae.

[0260] In a preferred embodiment, the persistent fungal infections treated using the antibody molecules described herein are persistent infections of Candida, such as Candida albicans, Cryptococcus (gattii and neoformans), Talaromyces (Penicillium) marneffe, Microsporum, such as Microsporum audouinii and Trichophyton tonsurans.

[0261] In a preferred embodiment, the persistent parasitic infections treated using the antibody molecules described herein are persistent infections of Plasmodium, such as Plasmodium falciparum, or Leishmania, such as Leishmania donovani.

[0262] In connection with the treatment of persistent infections, the antibody molecules can be administered to an individual alone or in combination with a second treatment or therapeutic agent that has been shown or is expected to be suitable for the treatment of the pathogen in the subject. For example, the antibody molecules can be administered to an individual in combination with an immunotherapeutic agent. The immunotherapeutic agent for administration in combination with the antibody molecules as 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 to which the therapeutic antibody molecule can bind include TLR1, TLR2, TLR4, TLR5, TLR7, TLR9, RIG-I-like receptors (e.g., RIG-I and MDA-5), and STING.

[0263] When an antibody molecule is used to prevent an infectious disease, the antibody molecule can be administered in combination with a vaccine against the pathogen of interest. Without wishing to be bound by theory, it is believed that the antibody molecules described herein may act as adjuvants in vaccination. Specifically, administration of an antibody molecule to an individual in combination with a vaccine is believed to elicit a greater immune response against the pathogen than can be achieved with the vaccine alone.

[0264] In connection with the treatment of a persistent infection, the treatment may include eliminating the infection, reducing the pathogenic load of the individual, and preventing recurrence of the infection. For example, the treatment may include preventing, ameliorating, delaying, reducing, or arresting one or more symptoms and / or signs of the persistent infection. Alternatively, the treatment may include preventing an infectious disease.

[0265] Features appropriately expressed in the foregoing description, the following claims or the accompanying drawings, from the perspective of the specific forms in which they are disclosed or the means for performing the disclosed functions or the methods or processes for obtaining the disclosed results, may be utilized to implement the present invention in its various forms either separately or in any combination of such features.

[0266] Although the present invention has been described in conjunction with the above exemplary embodiments, many equivalent modifications and variations will be apparent to those skilled in the art given the present disclosure. Accordingly, the above exemplary embodiments of the present invention are to be considered illustrative and not limiting. Various modifications to the described embodiments may be made without departing from the spirit and scope of the present invention.

[0267] To avoid doubt, any theoretical explanations provided herein are provided solely for the purpose of improving the reader's understanding. The inventors do not wish to be bound by any of these theoretical explanations.

[0268] The headings of the sections used in this specification are for organizing purposes only and should not be construed as limiting the subject matter being described.

[0269] Throughout this specification, including the following claims, unless the context requires otherwise, the words "comprise", "comprising", and variations such as "comprises", "containing", and "including" are to be understood as including the recited integer or step or group of integers or steps but not excluding any other integer or step or group of integers or steps.

[0270] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. In this specification, ranges may be expressed as from "about" a particular value and / or to "about" another particular value. When such a range is expressed, another embodiment includes from a particular value and / or to another particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about", it will be understood that the particular value forms another embodiment. The term "about" associated with a numerical value is optional and means, for example, + / - 10%.

Examples

[0271] Examples The inventors aimed to generate an mAb that can agonize both OX40 and CD137 in the absence of an artificial cross-linker or Fcγ receptor-mediated cross-linking and can produce an enhanced immune response against diseases such as cancer. 2 In this regard, the mAb 2 is an antibody molecule comprising a CDR-based antigen-binding site that binds to CD137 and an OX40 antigen-binding site located in the CH3 domain of the antibody molecule.

[0272] To achieve this objective, the inventors first identified Fcabs that could bind to OX40 and induce T cell activation in humans and mice, respectively, using selection and affinity maturation methods (see Examples 2 and 3). Subsequently, the inventors introduced the OX40 antigen-binding sites from these Fcabs into the mAb 2 format, and showed that some of these anti-human OX40 "mock" mAbs 2 could bind to human and cynomolgus monkey OX40 with high affinity and activate T cells upon crosslinking (see Example 4). Among these, clone FS20-22-49 showed the greatest increase in agonist activity upon crosslinking and had the lowest EC 50 for agonist activity in the presence of crosslinking, and was thus advanced as the OX40 antigen-binding site for the development of the subject mAb 2 .

[0273] To develop CDR-based antigen-binding sites that can bind to CD137 and agonize, the inventors used a selection method to identify monoclonal antibodies (mAbs) that can bind to human CD137 and can only activate T cells when crosslinked (see Example 5). The CDRs from these identified mAbs were then cloned into the mAb 2 containing the FS20-22-49 OX40 antigen-binding site. The CDRs of these mAbs 2 were sequences optimized to produce the following mAbs 2 : FS20-22-49AA / FS30-10-3, FS20-22-49AA / FS30-10-12, FS20-22-49AA / FS30-10-16, FS20-22-49AA / FS30-35-14, and FS20-22-49AA / FS30-5-37 (see Example 6). All of these mAbs 2 were shown to have a high level of specificity for human CD137 and were able to activate CD137 when crosslinked in a T cell activation assay (see Example 7). None of the mAbs 2 showed a significant ability to activate CD137 in the absence of crosslinking.

[0274] selected mAb 2 The FS20-22-49AA OX40 antigen-binding site in bound to OX40 upon cross-linking and was able to activate it, and separately, the FS30-10-3, FS30-10-12, FS30-10-16, FS30-35-14, and FS30-5-37 CD137 CDR-based antigen-binding sites were able to bind to CD137 upon cross-linking and activate it, establishing that the mAbs containing these antigen-binding domains 2 sought to demonstrate the ability to activate both OX40 and CD137 (also referred to as "dual agonistic activity"). Such dual agonists bind to i) OX40 and cross-link the mAb 2 bind to CD137, cluster CD137, and activate (agonize) it, and ii) bind to CD137 and bind to the mAb 2 bind to OX40, cluster OX40, and activate (agonize) it. Importantly, the dual agonist should be able to drive agonistic activity autonomously without the need for additional cross-linking agents, based on the expression of specific targets (OX40 and CD137).

[0275] The inventors have shown that the tested mAb 2 molecule can bind to human CD137, human OX40, cynomolgus monkey CD137, and cynomolgus monkey OX40 (see Example 8), and that the tested mAb 2 molecule can bind to human CD137 and human OX40 simultaneously (see Example 9). The inventors have shown that the "LALA" mutation in the CH2 domain of the mAb 2 reduces their binding to Fcγ receptors, and that the mAb 2 clone FS20-22-49AA / FS30-10-16 was unable to induce ADCC activation in an ADCC bioassay (see Example 10).

[0276] The inventors have shown that the tested OX40 / CD137 mAb 2The molecule also showed binding to cell-expressed human and cynomolgus monkey OX40 and CD137, with no non-specific binding observed (see Example 11).

[0277] Next, the inventors tested the mAbs containing this LALA mutation in a T cell activation assay using staphylococcal enterotoxin A (SEA; see Example 12). 2 The molecule was demonstrated to be able to induce T cell activation in the absence of an artificial crosslinker. The inventors tested the mAbs 2 The molecule was demonstrated to be able to induce T cell activation in the absence of an artificial crosslinker in a pan-T cell activation assay and that this activity depends on an mAb 2 that simultaneously engages both OX40 and CD137 (see Examples 13 and 16). The inventors further confirmed that the FS20-22-49AA / FS30-10-16 mAb 2 is able to activate these receptors in CD4+ and CD8+ T cells, respectively, in the absence of crosslinking (see Example 14).

[0278] The anti-human OX40 / CD137 mAb 2 did not bind to mouse proteins. To test the possibility that the OX40 / CD137 mAb 2 might illicit a T cell-mediated anti-tumor response, parallel mAbs targeting mouse OX40 and mouse CD137 were generated in both the presence and absence of the LALA mutation (labeled FS20m-232-91AA / Lob12.3 and FS20m-232-91 / Lob12.3, respectively). The inventors showed that the FS20m-232-91AA / Lob12.3 mAb 2 is able to induce T cell activation without an additional crosslinker and that this activity depends on an mAb 2 that simultaneously engages both OX40 and CD137 (see Examples 15 and 16). 2

[0279] ​The inventors have demonstrated that FS20m-232-91AA / Lob12.3 and FS20m-232-91 / Lob12.3 mAb 2 have an anti-tumor effect in vivo in a CT26 syngeneic tumor model (see Example 17). The inventors have further demonstrated that FS20m-232-91AA / Lob12.3 mAb 2 affects circulating T cells and increases the frequency of activated and proliferating T cells (see Examples 18 and 19). The inventors have demonstrated that FS20m-232-91AA / Lob12.3 mAb 2 has an anti-tumor effect in vivo in a B16-F10 syngeneic tumor model (see Example 20).

[0280] The inventors have performed an analytical characterization and a preliminary stability assessment of the mAb 2 (see Example 21). All five mAbs tested 2 showed favorable analytical properties and favorable stability good stability.

[0281] The inventors have demonstrated that in a T cell activation assay using SEA, the combination of FS20-22-49AA / FS30-10-16 mAb 2 with an anti-PD-L1 or anti-PD-1 antibody can result in an increase in the maximum in vitro T cell activation seen with OX40 / CD137 mAb 2 alone. The inventors have further shown that treatment in vivo in a CT26 mouse tumor model with the combination of FS20m-232-91AA / Lob12.3 mAb 2 and an anti-PD-1 antibody results in an increase in anti-tumor activity, provides a life-prolonging effect, and can enhance the pharmacodynamic modulation of proliferating T cells and NK cells compared to treatment with either single agent (see Example 22).

[0282] The inventors have demonstrated that FS20m-232-91AA / Lob12.3 mAb 2has demonstrated in vivo dose-dependent anti-tumor activity in a CT26 syngeneic tumor model up to a specific dose level, and that this activity is maintained at higher dose levels. The inventors have shown that the FS20m-232-91AA / Lob12.3 mAb 2 also induces the establishment of protective immune memory in "complete responder" mice and can protect from re-challenge with CT26 cells (see Example 23). The inventors have shown that the FS20m-232-91AA / Lob12.3 mAb 2 affects circulating T cells and significantly increases the frequency of proliferating (Ki67+) CD4+ and CD8+ T cells at various dose levels (see Example 24). The inventors have shown that the FS20m-232-91AA / Lob12.3 mAb 2 can increase the frequency of activated (CD69+), proliferating (Ki67+) CD8 T cells and that CD4 T cell depletion has a detrimental effect on this peripheral pharmacodynamic response mediated by FS20m-232-91AA / Lob12.3 mAb 2 (see Example 25). The inventors have shown that the FS20-22-49AA / FS30-10-16 mAb 2 had similar functional activity in a primary cynomolgus PBMC assay compared to an equivalent human assay, that the mAb 2 was well tolerated in cynomolgus monkeys up to a dose of 30 mg / kg, and that in cynomolgus monkeys, it was able to induce a drug-related increase in the proliferation and activation of central memory and effector memory CD4+ and CD8+ T cells and NK cells (see Example 26).

[0283] The inventors have shown in studies in BALB / c mice that the FS20m-232-91AA / Lob12.3 mAb 2However, compared with the crosslinking-independent CD137 agonist, it also showed a moderate and transient increase in the levels of T cell infiltration and proliferation in the liver, which induced an increase and persistence in the infiltration, proliferation and activation of liver T cells (see Example 27). Finally, in the CT26 syngeneic mouse tumor model, the inventors showed that, despite the ability of the crosslinking-dependent Fab clone to induce increased T cell levels and proliferation compared to the crosslinking-dependent anti-CD137 Lob12.3 clone, the FS20m-232-91AA / Lob12.3 mAb 2 containing the same OX40 Fcab that pairs with the crosslinking-independent anti-CD137 Fab clone, induced no difference in tumor growth or survival among mice treated with either the FS20m-232-91AA / Lob12.3 mAb 2 or the OX40 / CD137 mAb 2 (see Example 28).

[0284] These experiments are described in more detail in the following examples.

[0285] Example 1 - Antigen Selection and Characterization The selection and screening methods used to identify mAbs 2 that bind to and agonize both OX40 and CD137 require the use of various OX40 and CD137 antigens. The production of these antigens is described in more detail below.

[0286] 1.1 OX40 Antigen The OX40 antigens used for the selection of Fcabs specific for human and mouse OX40 and the testing of the cross-reactivity of the selected Fcabs with cynomolgus monkey OX40 were either prepared in-house or obtained from commercial suppliers described below.

[0287] 1.1.1 Preparation of Recombinant Soluble Human, Cynomolgus Monkey and Mouse OX40 Antigens To prepare the recombinant soluble dimeric OX40 antigen, the extracellular domain of OX40 was fused to murine Fc to improve the solubility and stability of the antigen. Specifically, the extracellular domain of the relevant OX40 (human, cynomolgus monkey or mouse) was cloned into the pFUSE-mIgG2aFc2 vector (Invivogen catalog number pfuse-mg2afc2) using the EcoRI-HF and BglII restriction enzymes, and the antigen was produced with the murine IgG2aFc domain at the C-terminus. Next, the recombinant OX40 antigen was produced by transient expression in HEK293-6E cells (National Research Council Canada), and purified by size exclusion chromatography (SEC) using an mAb Select SuRe protein A column (GE Healthcare, 11003494), followed by ensuring that the resulting antigen was a single species and did not contain aggregates.

[0288] To prepare the biotinylated version of the recombinant OX40 antigen, the EZ-Link™ Sulfo-NHS-SS-Biotin kit (Thermo Fisher Scientific, catalog number 21331) was used according to the manufacturer's protocol to biotinylate the antigen. The biotinylated OX40 antigen was used in the selection experiments described below but not in the measurement of binding affinity. Purification of the biotinylated OX40 antigen was performed in two steps using a PD-10 desalting column (GE Healthcare, 17-0851-01) followed by an Amicon 30k spin column (Millipore, UFC903024) according to the manufacturer's instructions. The biophysical properties of the recombinant antigen were characterized by SE-HPLC analysis to ensure the absence of aggregates and PAGE to demonstrate the molecular size. Size determination by PAGE showed that the soluble antigen was a dimer as their estimated molecular weights were twice that of the predicted molecular weight of the monomer. The recombinant antigen was also analyzed by gel shift analysis which showed that the degree of biotinylation exceeded 90%. ELISA and surface plasmon resonance (SPR) were used to confirm that the biotinylated recombinant human (hOX40-mFc), mouse (mOX40-mFc) and cynomolgus (cOX40-mFc) OX40 antigens could be bound by OX40-specific antibodies for human and cynomolgus OX40 (antibody 11D4 [European Patent No. 2242771]); polyclonal sheep anti-human OX40 antibody against cynomolgus OX40 [R&D Systems catalog number AF3388]; antibody ACT35 against human OX40 [Biolegend catalog number 35002] and antibody OX86 against mouse OX40 [Biolegend catalog number 119408]). These antigens are listed in Table 2 below.

[0289] 1.1.2 Preparation of cell lines expressing human, cynomolgus and mouse OX40 Human, cynomolgus monkey, and mouse OX40 (for sequences, see Table 1) were cloned into the vector pLVX-EF1a-IRES-puro (Clontech, catalog number 631253) using SpeI-HF and NotI-HF restriction enzymes. Next, the vector was co-transformed with Lenti-X HTX packaging mix (Clontech, catalog number 631249) into the Lenti-X 293T cell line (Clontech, catalog number 632180) to generate lentivirus. Next, DO11.10 cells were transduced with the lentivirus (National Jewish Health). Cells overexpressing OX40 were selected by cloning the cell line by serial dilution after incubating the cells with 5 μg / ml puromycin (Life Technologies catalog number A11113803) for about 2 weeks. Expression of OX40 by the cell line was tested by flow cytometry using a fluorescently labeled OX40-specific antibody (OX86; ACT35; and polyclonal sheep anti-human OX40 as described in Example 1.1.1 and Table 2). Cell lines expressing human (DO11.10-hOX40), mouse (DO11.10-mOX40), or cynomolgus monkey (DO11.10-cOX40) OX40 were selected and showed fluorescence values at least 10-fold higher than those of non-transduced cells in flow cytometry analysis. These cell lines are listed in Table 2 below.

[0290]

Table 2

[0291] 1.1.3 Commercially available OX40 antigen Several commercially available OX40 antigens were tested.

[0292] The recombinant His-tagged human OX40 extracellular domain was obtained from SinoBiologicals (Catalog No. 10481-H08H-50). However, SE-HPLC analysis of this antigen showed that less than 50% of the antigen was in the monomeric non-aggregated form. Therefore, this antigen was not used in subsequent analyses.

[0293] Recombinant human OX40 / human Fc (hOX40-hFc) and recombinant mouse OX40 / human Fc (mOX40-hFc) containing the human IgG1 Fc domain at the C-terminus were obtained from R&D Systems (hOX40-hFc: Catalog No. 3388-OX-050; mOX40-hFc: Catalog No. 1256-OX-050) and by biotinylation in-house. The biophysical properties of these soluble antigens were characterized by SE-HPLC analysis to ensure the absence of aggregates and by PAGE to demonstrate the molecular size. Size determination by PAGE showed that the soluble antigens were dimers as their estimated molecular weights were twice that expected for the monomeric antigen. The soluble antigens were also analyzed by gel shift analysis which showed that the degree of biotinylation exceeded 90%. Using ELISA and SPR, it was confirmed that the biotinylated recombinant human (hOX40-hFc) and mouse (mOX40-hFc) OX40 antigens were able to bind to the OX40-specific antibodies (11D4; ACT35; and OX86) as described in Example 1.1.1 and Table 2 below.

[0294]

Table 3

[0295] 1.2 CD137 Antigen The CD137 antigen used for the selection of mAb specificity for human CD137 and testing the cross-reactivity of the selected Fcab with cynomolgus OX40 was either prepared in-house or obtained from the commercial suppliers described below.

[0296] 1.2.1 Preparation of Recombinant Soluble Human and Cynomolgus CD137 Antigens Some commercially available recombinant antigens have been found to be unsuitable for use because, for example, they contain unacceptable levels of aggregates at the time of testing. Therefore, the following recombinant dimeric and monomeric antigens (Table 3) were produced in-house for use in the selection, screening, and further characterization of anti-CD137 mAbs.

[0297] [Table 4]

[0298] The monomeric antigen was produced by cloning DNA encoding the extracellular domain of human CD137 with the Avi sequence and six C-terminal histidine residues into a modified pFUSE vector (Invivogen, catalog number pfuse-mg2afc2) using EcoRI-HF and BamHI-HF restriction enzymes. The vector was transfected into HEK293-6E cells, and the expressed CD137 was purified using a HisTrap™ excel nickel column (GE Healthcare, 17-3712-06) and size exclusion chromatography (SEC) to ensure that the antigen was a single species and did not contain aggregates.

[0299] To produce the dimeric antigen, a DNA construct encoding the extracellular domain of human or cynomolgus CD137 fused to the mIgG2a Fc domain with the Avi sequence was cloned into a modified pFUSE vector and transfected into HEK293-6E cells. The recombinant CD137 was purified using a MabSelect SuRe™ protein A column (GE Healthcare, 11003494) and size exclusion chromatography (SEC) to ensure that the antigen was a single species and did not contain aggregates.

[0300] The biotinylated versions of the dimer and monomer CD137 antigens were prepared using the BirA biotin-biotin protein ligase reaction kit (Avidity LLC, BirA500), generating monomer CD137 antigen labeled with a single biotin molecule and dimer CD137 antigen labeled with two biotin molecules, one on each of the two monomers. Specifically, 3 mg of CD137 antigen was mixed with 7.8 μl of BirA enzyme mixture at an enzyme-to-substrate molar ratio of 1:50. Next, additives were added according to the manufacturer's recommendations (142 μl of Biomix A, 142 μl of Biomix B, 142 μl of biotin), and the reaction mixture was incubated at room temperature for 2 hours. To maintain the integrity of the biotinylated antigen, the reaction mixture was immediately buffer-exchanged into DPBS using an Amicon 30 μm filter.

[0301] The CD137 antigen was further purified by SEC to ensure removal of the BirA enzyme and generate a final high-quality monodisperse protein preparation free of high-molecular-weight aggregates. Specifically, antigens from the same manufacturing lot were mixed and analyzed for stability and purity by size-exclusion high-performance liquid chromatography (SE-HPLC), SDS polyacrylamide gel electrophoresis (SDS-PAGE), and size-exclusion chromatography with multi-angle light scattering detector (SEC-MALS). Complete biotinylation of the protein was confirmed by streptavidin-shift SDS-PAGE gels. The recombinant human CD137 antigen was confirmed to bind to 20H4.9, an anti-human CD137 positive control antibody (U.S. Patent No. 7,288,638), and to human CD137 ligand-expressing DO11.10 cells by surface plasmon resonance (SPR) in vitro and flow cytometry. The recombinant cynomolgus CD137 antigen was confirmed to bind to cynomolgus CD137 ligand-expressing DO11.10 cells by flow cytometry. To ensure the highest possible purity of the CD137 antigen used in the selected protocol, a thorough protein characterization of the antigen was performed to ensure that the percentage of protein aggregates present did not exceed 2%.

[0302] 1.2.2 Preparation of cell lines expressing human, cynomolgus monkey, and mouse CD137 DO11.10 cells (National Jewish Health) expressing full-length human or cynomolgus monkey CD137, named "DO11.10-hCD137" and "DO11.10-cCD137" respectively (see Table 4), were generated to present the antigen in its most natural conformation during the selection of the selected anti-CD137 mAb and further characterization.

[0303] Anti-mouse OX40 / CD137 mAb that binds to cell-expressed mouse CD137 2 To determine the binding, DO11.10 cells expressing full-length mouse CD137, named "DO11.10-mCD137", were also generated (see Example 11.2).

[0304] Lentiviral transduction was used to generate DO11.10 cells overexpressing human, cynomolgus monkey, or mouse CD137 receptor using the Lenti-X HTX Packaging System (Clontech, 631249). The Lenti-X expression vector (pLVX) (Clontech, 631253) containing the cDNA encoding human CD137 (SEQ ID NO: 126), cynomolgus monkey CD137 (SEQ ID NO: 128), or mouse CD137 (SEQ ID NO: 164) was co-transfected into the Lenti-X 293T Cell Line (Clontech, 632180) with the Lenti-X HTX Packaging Mix to generate the virus. Next, these lentiviral vectors were transduced into the DO11.10 cell line.

[0305] The expression of human, cynomolgus monkey or mouse CD137 in these cells was confirmed by the binding of anti-CD137 positive control antibodies (20H4.9, MOR7480.1 (each of US Patent Application Publication No. 2012 / 0237498 A1) and Lob12.3 (University of Southampton)) to the cells using flow cytometry.

[0306]

Table 5

[0307] Example 2 - Selection and Characterization of Anti-Human OX40 Fcab 2.1 Naive Selection of Anti-Human OX40 Fcab To select Fcab specific for human OX40 from a naive phage library, both recombinant biotinylated soluble dimer human OX40 (hOX40-mFc; see Table 2) and cell-expressed human OX40 (DO11.10-hOX40) were used as antigens. In some selection protocols, in addition to recombinant biotinylated soluble dimer human OX40, cells expressing human OX40 were used to ensure that the selected Fcab could bind to OX40 in its natural conformation on the cell surface.

[0308] Six naive phage libraries were constructed that display a CH3 domain (IMGT number 1.4 - 130) containing an AB loop (residues 14 to 18 according to the IMGT numbering scheme) and an EF loop (residues 92 to 101 according to the IMGT numbering scheme) that are partially randomized in CH3. One of the six libraries additionally contained clones in which either two or four amino acids (encoded by two or four NNK codons) were inserted at position 101 of the EF loop of the CH3 domain (the inserted residues are numbered 101.1 to 101.4 according to the IMGT numbering scheme).

[0309] All six libraries were subjected to three rounds of selection using recombinant biotinylated soluble dimer human OX40 (hOX40-mFc; see Table 2). All six libraries were subjected to further selection campaigns using hOX40-mFc in the first selection round, followed by cell-expressed human OX40 (DO11.10-hOX40 was used in two additional selection rounds; see Table 2).

[0310] The 2133 clones identified after the third round of selection from the six libraries were screened by ELISA for binding to human OX40. This identified 32 unique positive binders, which were subcloned and expressed as soluble Fcab (consisting of the truncated hinge [SEQ ID NO: 101], CH2, and CH3 domains) in HEK Expi293 cells (Fcab cloned into the pTT5 vector [National Research Council of Canada] transfected into Expi293F cells [Life technologies, A14527] using the ExpiFectamine 293 Transfection kit [Life Technologies, A14524]).

[0311] The 32 unique Fcabs were tested for their ability to bind to cell-expressed human OX40 (DO11.10-hOX40). Fifteen of the 32 Fcabs screened had cell binding to DO11.10-hOX40 and the EC of these interactions 50showed a range of 0.1 to 62 nM. Fifteen Fcabs that showed binding to DO11.10-hOX40 were tested using an in-house human NF-κB reporter assay to test for activation of the NF-κB signaling pathway. Six of the fifteen Fcabs showed an increase in activity when cross-linked with anti-human Fc antibody in the human NF-κB reporter assay, suggesting that these Fcabs can activate OX40 signaling. Fcabs named FS20-22 and FS20-31 showed high levels of activity in this assay, and their activity increased when the Fcabs were cross-linked with anti-human CH2mAb (clone MK1A6 (Jefferis et al, 1985 and Jefferis et al, 1992), in-house production). These were selected for affinity maturation.

[0312] 2.2 Affinity Maturation of Anti-Human OX40 Fcab Using a randomization primer from ELLA Biotech with an equimolar distribution of amino acids excluding cysteine, five residues (residues 14 to 18) within the AB loop of the CH3 domain or five residues (residues 45.1 to 77) within the CD loop or a portion of the EF loop (residues 92 to 94 and residues 97 to 101 of the CH3 domain (numbering of all residue numbers according to the IMGT numbering scheme)) were randomized to generate an affinity maturation library for FS20-22 and FS20-31.

[0313] 1410 Fcabs from the output of affinity maturation were screened for binding to human OX40 by ELISA, and 204 unique positive conjugates were identified, subcloned, and expressed as soluble Fcabs in HEK Expi293 cells as described in Example 2.1 above.

[0314] The off-rates of soluble Fcab when bound to hOX40-mFc were measured using Biacore 3000 (GE Healthcare) in the absence and presence of anti-CH2 crosslinking using anti-human CH2 mAb clone MK1A6 (see Example 2.1). Fcabs with improved off-rates compared to the relevant parental Fcab were further screened for binding to cell-expressed human OX40 and activity in an in-house human T cell activation assay. All Fcabs bound to cell-expressed human OX40. Ten Fcabs from the FS20-22 line and 18 Fcabs from the FS20-31 line that showed high levels of activity in the human T cell activation assay were selected for loop shuffling as described below.

[0315] For the FS20-22 line, two loop-shuffled libraries were generated by shuffling three CD loops, six EF loops, and either the parental AB loop or the affinity-matured AB loop. For the FS20-31 line, one loop-shuffled library containing four AB loops, seven CD loops, and seven EF loops was generated.

[0316] The shuffled sequences were expressed as soluble Fcab in HEK Expi293 cells as described in Example 2.1 above and screened for binding to biotinylated hOX40-mFc antigen using the Dip and Read™ streptavidin biosensor (Pall ForteBio, 18-5050) on an OctetQK e System (Pall ForteBio). Sequences of Fcabs with improved off-rates when bound to hOX40-mFc were determined compared to the parental Fcab, and 35 unique Fcabs from the FS20-22 line and 62 unique Fcabs from the FS20-31 line were obtained. The identified unique Fcabs were tested for binding to hOX40-mFc antigen using the Biacore 3000 instrument (GE Healthcare) in the presence and absence of CH2 crosslinking using anti-human CH2 mAb clone MK1A6.

[0317] In the FS20-22 system, 18 Fcabs were the mock (4420 LALA) mAb 2 selected for further characterization based on the slowest off-rate with CH2 bridging upon expression in the format and binding to hOX40-mFc, the largest difference between the unbridged and CH2-bridged off-rates upon binding to hOX40-mFc, and as described above, the strength of binding to hOX40-mFc. In the FS20-31 system, 9 Fcabs with the slowest off-rate with CH2 bridging upon binding to hOX40-mFc and 9 Fcabs with the slowest off-rate without CH2 bridging upon binding to hOX40-mFc were expressed and the mock (4420 LALA) mAb 2 selected for further characterization in the format. Since the number of Fcabs was common to both of these groups of 9 Fcabs, additional Fcabs showing a slow off-rate upon binding to hOX40-mFc in the absence of CH2 bridging were selected from the FS20-31 system and the mock mAb 2 formatted such that the total number of Fcabs from this system was 18 for expression and further characterization. Using data from the T cell activation assay, an additional 6 Fcabs from the FS20-22 system and 8 Fcabs from the FS20-31 system were identified that showed high activity in this assay and thus the mock (4420 LALA) mAb 2 was also expressed in the format and further characterized (see Example 4).

[0318] Example 3 - Selection and Characterization of Anti-Mouse OX40 Fcab 3.1 Naive Selection of Anti-Mouse OX40 Fcab A naive yeast library displaying the CH1 to CH3 domains of human IgG1, including randomization of the AB loop (residues 11 to 18 according to the IMGT numbering scheme) and the EF loop of the CH3 domain (residues 92 to 101 according to the IMGT numbering scheme), and a 5-residue randomized insertion between residues 16 and 17 of the AB loop (according to the IMGT numbering scheme), was used for selection. Yeast was incubated with biotinylated recombinant mouse OX40 (mOX40-hFc; Table 2) fused to the human IgGFc domain and sorted by MACS using streptavidin-coated beads. Next, the concentration of biotinylated mOX40-hFc was decreased in the presence of a 5-fold molar excess of hFc, and 3 rounds of FACS selection were performed. Cells were stained with streptavidin-allophycocyanin (APC) (BD Bioscience, 349024) or anti-biotin-APC (Miltenyi Biotec, 130-090-856) and sorted using a FACSAria (BD Bioscience) cell sorter. 182 individual Fcabs from the enriched population were screened for antigen binding, and 2 unique positive binders were subcloned and expressed as soluble Fcabs as described above in Example 2.1. The Fcabs were characterized for binding to mOX40-hFc by ELISA and activity in our in-house mouse NF-κB reporter assay. In the NF-κB reporter assay, only one Fcab, FS20m-232, was active and showed binding to cells expressing mouse OX40, so this Fcab was selected for affinity maturation.

[0319] 3.2 Affinity Maturation of mOX40 Fcab Using a randomization primer manufactured by ELLA Biotech with an equimolar distribution of amino acids excluding cysteine, 7 residues (residues 15 to 16.5 according to the IMGT numbering scheme) within the AB loop of FS20m-232 Fcab (library 1), 6 residues (residues 45.1 to 78 according to the IMGT numbering scheme) within the CD loop (library 2), or 5 residues (residues 92 to 94 and residues 97 to 98 according to the IMGT numbering scheme) within the EF loop (library 3) were randomized to construct three phage display affinity maturation libraries.

[0320] Three rounds of selection were performed on the affinity maturation library using recombinant biotinylated mOX40-mFc that alternately captured streptavidin-coated (ThermoFisher Scientific, 11205D) and neutravidin-coated (ThermoFisher Scientific, 14203 and A2666) Dynabeads. The antigen concentration was decreased from 50 nM (first round) to 10 nM (second round) and 1 nM (third round) for the identification of high-affinity conjugates. 1655 individual phages from the third round of selection were screened for binding to mOX40-mFc by phage ELISA, and 98 unique positive conjugates were identified, subcloned, and expressed as soluble Fcab in HEK Expi293 cells as described in Example 2.1. The Fcab was further screened for cell binding and activity in a mouse NF-κB reporter assay. The most active Fcab was selected for loop shuffling.

[0321] A loop shuffling library containing 27 CD loops (all 26 unique sequences identified from affinity maturation and WT sequences) shuffled with 37 EF loops (the ones that bind best to mouse OX40 in phage ELISA and WT sequences) was generated, and the AB loop of FS20m-232Fcab was contained in all the shuffled clones. 750 shuffled sequences were expressed as soluble Fcab (shortened hinge-containing) in HEK Expi293 cells as described above. OctetQK e To improve the off-rate, HEK supernatants containing Fcab were screened by measuring the binding of Fcab to biotinylated mOX40-mFc (Table 2) using a Dip and Read™ streptavidin biosensor (Pall ForteBio, 18-5050) on an OctetQK

[0322] Example 4 - mAb 2 Construction, expression and characterization of anti-OX40 Fcab in the 4.1 Mock mAb 2 Construction and expression "Mock" mAbs containing anti-human OX40 and the anti-mouse OX40 Fcab identified above 2 were prepared to enable the characterization of these Fcabs in the mAb 2 format. These mock mAbs 2was prepared by replacing the CH3 domain of anti-OX40 Fcab, anti-FITC antibody, and anti-HEL antibody in the XhoI and BamHI sites present in the sequence of the unmodified CH3 domain of human IgG1 with the variable regions of anti-OX40 Fcab and anti-FITC antibody 4420 (Bedzyk et al., 1989 and Bedzyk et al., 1990) or anti-hen egg white lysozyme (HEL) antibody D1.3 (Braden et al., 1996) in the human IgG1 backbone (see SEQ ID NO: 114, SEQ ID NO: 115, and SEQ ID NO: 116 for details) or the variable regions of anti-HEL antibody D1.3 (Braden et al., 1996) in the human IgG1 backbone (see SEQ ID NO: 117 and SEQ ID NO: 118 for details). Mock mAb 2 contained the light chain of anti-FITC mAb4420 (SEQ ID NO: 116) or anti-HEL mAbD1.3 (SEQ ID NO: 118), respectively, and also contained the LALA mutation in the CH2 domain of the heavy chain to reduce Fc-gamma receptor interaction and potential Fc-gamma receptor-induced cross-linking. Mock mAb 2 and mAb 2 The presence of the LALA mutation in is mentioned in these examples and is represented by the suffix "AA" at the end of the Fcab part of their clone names.

[0323] Mock mAb 2 was produced by transient expression in HEK293-6E cells and purified using an mAb Select SuRe protein A column.

[0324] 4.2 Cell Expression of Mock mAbs against Human and Cynomolgus Macaque OX40 2 Binding Affinity of Anti-Human OX40 Fcab in the Format Regarding the binding to cells expressing human or cynomolgus macaque OX40 (DO11.10 cells expressing either human [DO11.10-hOX40] or cynomolgus macaque OX40 [DO11.10-cOX40]; see Table 2), mock (4420 LALA) mAb 2The affinity of anti-human OX40 Fcab in the form was measured using flow cytometry. Nonspecific binding was also evaluated by testing the binding to HEK cells that do not express OX40 by flow cytometry.

[0325] Mock (4420 LALA) mAb 2 And control mAb diluent (2× final concentration) was prepared three times with 1× DPBS (Gibco, 14190-094). DO11.10-hOX40 or DO11.10-cOX40 or HEK cell suspension was prepared in PBS + 2% BSA (Sigma, A7906) and seeded at 4×10 6 cells / ml at 50 μl / well in a V-bottom 96-well plate (Costar, 3897). 50 μl of mock (4420 LALA) mAb 2 or control mAb (anti-human OX40 mAb, 11D4) diluent was added to the cell-containing wells (final volume 100 μl) and incubated at 4°C for 1 hour. The plate was washed, and 100 μl / well of secondary antibody (anti-human Fc-488 antibody, Jackson ImmunoResearch, 109-546-098) diluted 1:1000 with PBS + 2% BSA was added and incubated in the dark at 4°C for 30 minutes. The plate was washed and resuspended in 100 μl of PBS containing 1 μg / ml of DAPI (Biotium, catalog number 40043). The plate was read using a CantoII flow cytometer (BD Bioscience). Dead cells were excluded and the fluorescence in the FITC channel (488 nm / 530 / 30) was measured. The data was fitted using the logarithm (agonist) vs. response of GraphPad Prism software.

[0326] In the human IgG1 backbone, Fcab containing the LALA mutation in the CH2 domain of the heavy chain (G1AA / 11D4; SEQ ID NOs: 173 and 175) (all mock [4420 LALA] mAb 2(Tested in form) and the positive control anti-human OX40 mAb, 11D4, bound to human OX40 with a range of affinities. Five clones from the FS20-22 line and six clones from the FS20-31 line were tested for their ability to bind to cell-expressed human and cynomolgus monkey OX40, and the binding affinities of these clones are shown in Table 5.

[0327]

Table 6

[0328] 4.3 Mock mAb 2 In vitro activation of OX40 by anti-OX40 Fcab in form Activated T cells express OX40 on the cell surface. Binding of trimeric OX40 ligand to OX40 results in trimerization of the receptor. Since OX40 ligand is expressed as a cluster on the cell surface of antigen-presenting cells, it is known that the interaction between OX40 ligand and OX40 results in clustering of OX40, which is essential for OX40 signaling and further activation of T cells. Antibodies that agonize OX40 need to mimic this clustering activity of OX40 ligand. In the case of monospecific anti-OX40 antibodies, Fc gamma receptors bind to the Fc domain of the antibody and crosslink them, resulting in OX40 clustering.

[0329] The above mock (4420) mAb containing the LALA mutation 2 Anti-human OX40 and anti-mouse OX40 Fcab in form were tested in a T cell activation assay for their ability to activate OX40 expressed on T cells upon crosslinking of the Fcab in the presence of a crosslinking agent. Mock (4420 LALA) mAb 2 The human T cell activation assay for testing anti-human OX40 Fcab in form included the isolation of T cells from human peripheral blood mononuclear cells (PBMC) and testing for the release of IL-2, a marker of T cell activation. The assay was performed in a method similar to the method described later in Example 13, with a positive control (11D4) or mock (4420LALA) mAb2 Involved in the use of anti-human CH2 mAb clone MK1A6 or FITC-dextran (Sigma) to crosslink Fcab in each form.

[0330] Anti-human OX40 Fcab (4420 LALA) mAb in the case of crosslinking with Fab target (FITC-dextran) 2 The form showed a certain range of activity in the T cell activation assay. All Fcab had the ability to costimulate T cells in the presence of anti-CD3 antibody and induce the production of human IL2. Fcab from FS20-22 line and FS20-31 line showed activity both with and without crosslinking. Specifically, Fcab from these lines showed activity in the absence of crosslinking agent, which increased significantly upon crosslinking. Since these Fcab retain high cross-reactivity to cynomolgus OX40 (compared to binding of human OX40), toxicology studies may be possible in this species. Among the clones of FS20-22 line, clones FS20-22-41, FS20-22-47, FS20-22-49 and FS20-22-85 had the lowest EC 50 values for agonist activity when crosslinked, and thus are preferred clones from this line. Among these, clone FS20-22-49 showed the largest increase in agonist activity upon crosslinking and the lowest EC 50 for agonist activity in the presence of crosslinking, and thus is a preferred clone.

[0331] As described above, the inventors aimed to generate mAbs that can agonize both OX40 and CD137 in the absence of additional crosslinking agent. The above experiments demonstrate that FS20-22-49 Fcab can activate OX40 in the presence of a crosslinking agent for the pair. To generate a dual agonist that does not require an additional crosslinking agent, the inventors selected to generate anti-CD137 antibodies with the intention of using the CDRs from these antibodies in the final OX40- and CD137-targeting mAb 2 molecule. 2

[0332] Example 5 - Selection and Characterization of Anti-Human CD137 Antibodies A synthetic naive phage mid library displaying Fab domains of the human germline randomized at CDR1, CDR2, and CDR3 (MSM Technologies) was used for the naive selection of anti-human CD137 mAbs using the recombinant and cell surface-expressed CD137 antigen described in Example 1.2.

[0333] The Fab library was selected in 3 rounds using streptavidin dynabeads (Thermo Fisher Scientific, 11205D) and neutravidin-binding protein conjugated to dynabeads (Thermo Fisher Scientific, 31000), and phages bound to biotinylated human CD137-mFc-Avi or human CD137-Avi-His were isolated. To ensure binding of the Fab to CD137 expressed on the cell surface, the output of the first round from the selection using the recombinant CD137 antigen also underwent two additional rounds of selection using DO11.10-hCD137 cells and a fourth round of selection using DO11.10-cCD137 cells.

[0334] Approximately 2200 clones from the outputs of rounds 3 and 4 were screened for binding to human and cynomolgus monkey CD137-mFc-Avi by phage ELISA. Biotinylated mFc was included as a negative control. The sequences of the variable regions of the positive clones (clones with a CD137 binding signal at least 4-fold higher than the binding signal to mFc) were determined, and 36 unique VH / VL sequence combinations were identified. The identified sequences were derived from both selections using recombinant CD137 antigen and cell surface-expressed CD137 antigen and had multiple clones isolated using both selection strategies. Based on phage ELISA, 22 of the 36 clones were cynomolgus monkey cross-reactive, but since the sensitivity of phage ELISA may not have been sufficient to detect weak cynomolgus monkey cross-reactive conjugates, all 36 clones were advanced to reformulation into IgG1 molecules. For each clone, the VH and VL domains were individually cloned into the pTT5 expression vector (National Research Council of Canada) containing either CH1, CH2 (each with the LALA mutation or the CL domain in the CH2 and CH3 domains). The resulting pTT5-FS30 VH and pTT5-FS30 VL vectors with the LALA mutation (AA) were transiently co-transfected into HEK293-6E cells. Twenty-eight clones were expressed as soluble IgG1 molecules. These were purified on an mAb Select SuRe protein A column and subjected to further testing.

[0335] The binding of the anti-CD137 mAb was analyzed by ELISA using human and cynomolgus CD137-mFc-Avi. Of the 28 clones tested, 10 showed dose-dependent binding to human CD137-mFc-Avi and no binding to human OX40-mFc-Avi, mFc, or streptavidin. Within this group, four clones, FS30-5, FS30-10, FS30-15, and FS30-16, cross-reacted with cynomolgus CD137-mFc-Avi. Due to the low number of cynomolgus cross-reactive clones obtained, additional clones were screened and expressed as described above. This led to the isolation of one additional cynomolgus cross-reactive conjugate, FS30-35.

[0336] The anti-human CD137 mAbs FS30-5, FS30-10, FS30-15, and FS30-16 were tested for binding to cells expressing human or cynomolgus CD137 (DO11.10-hCD137 or DO11.10-cCD137) using flow cytometry. Non-specific binding was also evaluated by testing binding to DO11.10 cells and HEK293 cells lacking CD137 expression. The binding affinity was compared to the binding affinity of two positive control mAbs, MOR7480.1 (U.S. Patent Application Publication No. 2012 / 0237498) and 20H4.9 (U.S. Patent No. 7288638), and their variable domains were cloned and expressed in a human IgG1 format containing the LALA mutation in the CH2 domain (G1AA format).

[0337] The FS30-5, FS30-10, FS30-15, and FS30-16 clones were found to bind to human and cynomolgus CD137 receptors expressed on the cell surface, EC 50The values were in the range of 0.15 - 0.57 nM, comparable to the positive control mAb. Binding to parental DO11.10 or HEK293 cells was not observed, indicating the specificity of binding. In these cells, binding of the 20H4.9 positive control anti - CD137 antibody to cynomolgus CD137 was not observed. Published data (U.S. Patent No. 7288638) show that 20H4.9 in IgG1 format binds to cynomolgus CD137 on PMA (phorbol myristate acetate) - induced cynomolgus PMBC. Under the control of the present inventors, 20H4.9 in G1AA format bound to recombinant cynomolgus CD137, but the affinity was much lower than that for human CD137 (data not shown), which could explain the lack of binding observed with the antibody to DO11.10 - cCD137 cells.

[0338] To determine the biophysical properties of the FS30 mAb, size - exclusion chromatography (SEC) was performed and the proportion of the monomer fraction was analyzed. All four FS30 mAbs tested showed a single - peak profile and were more than 97% monomer. This high level of monomeric protein enabled the initiation of functional activity tests.

[0339] Next, the functional activity of the anti - CD137 mAb was analyzed in a primary T - cell activation assay. In vivo, anti - CD137 mAbs induce an agonistic effect by mobilizing Fcy receptors, resulting in clustering of the mAb and the CD137 receptor. To mimic the maximal ability of an mAb to cluster surface CD137 receptor molecules, the FS30 mAb was cross - linked using an anti - human CH2 antibody (clone MK1A6, in - house production) prior to the assay. T - cell activation was compared with non - cross - linked mAb. An anti - hen egg white lysozyme (HEL) antibody D1.3 in a human IgG1 backbone with the LALA mutation (G1AA / HelD1.3) was included as a negative control.

[0340] When cross - linked, the FS30 - 5, FS30 - 10, FS30 - 15, and FS30 - 16 mAbs showed strong activity in the T - cell activation assay, with an EC 50The value is less than 10 nM, and the maximum level of IL-2( Emax ) was similar to that of the positive control anti-CD137 mAbs (anti-CD137 MOR7480.1 mAb, 5637 hIL-2 pg / ml; and anti-CD137 20H4.9 mAb, 10232 hIL-2 pg / ml). The E max of FS30-6 mAb (1512 hIL-2 pg / ml) was significantly lower than that of the positive control and other FS30 mAbs, indicating a low overall level of T cell activation. Unlike the positive control anti-CD137 20H4.9 mAb (3174 pg / ml of hIL-2 production) that showed activity in the absence of crosslinking, the FS30 mAbs did not show activity (when not crosslinked as indicated by the background response level of the measured IL-2 measurement).

[0341] Example 6 - mAbs Targeting Human OX40 and Human CD137 2 Constructs and Expression mAbs containing anti-human OX40 Fcab paired with anti-human CD137 Fab 2 were prepared. Fcab FS20-22-49 targeting human OX40 was selected for pairing with Fab targeting CD137 because of its high activity in the T cell assay (see Example 4.3).

[0342] 6.1 mAbs 2 Expression and Characterization of mAbs in Formats mAbs consisting of IgG1 molecules containing the CDR of any of the FS30-5, FS30-10, FS30-15, FS30-16 or FS30-35 clones, the LALA mutation in the CH2 domain and the FS20-22-49 human OX40 receptor binding site in the CH3 domain 2 molecules were prepared. These mAb 2 molecules are anti-human OX40 mAbs 2, generated by replacing the VH domain of FS20-22-49AA / HelD1.3 with the corresponding VH domain of the FS30 clone and co-transfecting the resulting VH with the corresponding light chain of the FS30 mAb. The LALA mutation in the CH2 domain of the IgG1 molecule was retained in the resulting mAb 2 molecule. These mAb 2 molecules were named FS20-22-49AA / FS30-5, FS20-22-49AA / FS30-10, FS20-22-49AA / FS30-15, FS20-22-49AA / FS30-16 and FS20-22-49AA / FS30-35. The mAb 2 was produced by transient expression in HEK293-6E cells and purified using an mAb Select SuRe protein A column.

[0343] CD137 belongs to the tumor necrosis factor superfamily (TNFRSF) of cytokine receptors (Moran et al., 2013). To analyze the specificity of the anti-CD137 Fab binding sites of the five mAb 2 molecules, binding to human CD137 and mAb 2 to five closely related human TNFRSF members (TNFRSF1A, TNFRSF1B, GITR, NGFR and CD40) was tested using SPR. The aim was to show 1000-fold specificity by showing no binding of the mAb 2 to closely related antigens at a concentration of 1 μM, but binding to the CD137 receptor at a concentration of 1 nM.

[0344] The FS20-22-49AA / FS30-5, FS20-22-49AA / FS30-10, FS20-22-49AA / FS30-16 and FS20-22-49AA / FS30-35 mAb 2 showed a high level of specificity (nearly 1000-fold), whereas the FS20-22-49AA / FS30-15 mAb 2showed non-specific binding to all five closely related TNFRSF members tested. The non-specific binding shown by this clone was on average about 5-10 times lower than binding to the CD137 receptor at the same concentration, and when tested for binding to the same five TNFRSF members closely related to CD137, the FS30-15 mAb showed the same binding profile, so the mAb 2 was concluded to be due to the Fab binding site of the molecule. Based on this data, the FS30-15 clone was excluded from subsequent selection campaigns.

[0345] 6.2 Sequence optimization of anti-CD137 mAb The FS30-5, FS30-10, FS30-16 and FS30-35 anti-CD137 mAbs showed high affinity and specificity for CD137 and were active in T cell activation assays, while they contained one or more potential post-translational modification (PTM) sites within the CDR loops. In an attempt to identify amino acid residues that could be substituted at these sites while maintaining or improving binding and activity, it was decided to further engineer these clones. The potential PTM sites identified were methionine residues in VH CDR3 (Kabat positions M100D and M100H in FS30-5, M97 in FS30-10, M100A in FS30-16 and M100F in FS30-35), a potential asparagine isomerization motif in VH CDR2 (Kabat positions D54G55 in FS30-16) and a potential deamidation site in VL CDR3 (Kabat positions Q90G91 in FS30-16).

[0346] Site-directed mutagenesis was performed on five FS20-22-49AA / FS30 mAbs 2Clones were used as templates, and primers containing the degenerate codon NNK at sites encoding methionine, aspartic acid, or glycine residues were used to enable all possible amino acid substitutions. Cysteine residues and amino acids capable of generating novel potential PTM motifs were excluded. The clones were expressed and screened for binding to DO11.10-hCD137 cells. Parent mAb 2 Clones having binding at 10 nM similar (within 2-fold) or improved compared to the clone were selected for expression at 30 - 50 ml scale, purified on a protein A column using DO11.10-hCD137 cells and anti-human CH2 antibody MK1A6 as a cross-linking agent, and screened in a T cell activation assay.

[0347] DO11.10-hCD137 cells were washed once with PBS and resuspended at a concentration of 1.0×10 6 cells / mL in DO11.10 cell medium (RPMI medium (Life Technologies)) containing 10% FBS (Life Technologies) and 5 μg / ml puromycin (Life Technologies, A11113803). A 96-well flat-bottom plate was coated with anti-mouse CD3 antibody (Thermo Fisher Scientific, clone 17A2) by incubating at 37°C, 5% CO2 for 2 hours with 0.1 μg / ml anti-mouse CD3 antibody diluted with PBS, and then washed twice with PBS. DO11.10-hCD137 cells were added at 1×10 5Cells / wells were added to the plate. A 2 μM dilution of each test antibody was prepared in DPBS (Gibco) and further diluted 1:10 in DO11.10 cell medium (30 μl + 270 μl) to obtain a 200 nM dilution. The MK1A6 crosslinker was added to the wells at a 1:1 molar ratio with the test antibody sample for crosslinking. Serial dilutions of each antibody or antibody / crosslinker mixture were prepared in 96-well plates. 100 μl of the diluted antibody or antibody / crosslinker mixture was added to the DO11.10-hCD137 cells on the plate. The cells were incubated at 37 °C and 5% CO2 for 72 hours. The supernatant was collected and assayed using a mouse IL-2 ELISA kit (eBioscience or R&D Systems) according to the manufacturer's instructions. The plate was read at 450 nm using a plate reader equipped with Gen5 software, BioTek. The absorbance value at 630 nm was subtracted from the absorbance value at 450 nm (corrected). The standard curve for calculating cytokine concentration was based on a four-parameter logistic curve fit (Gen5 software, BioTek). The concentration of mouse IL-2 (mIL-2) was plotted against the log concentration of the antibody and the resulting curve was fitted using the log(agonist) vs. response equation of GraphPad Prism.

[0348] For each clone, a limited number of amino acids that retained or improved binding to cell surface CD137 were identified for substitution of the methionine residue in the heavy chain CDR3. FS20-22-49AA / FS30-16 mAb 2 The clone contained three potential PTM sites and each mutation slightly decreased the binding affinity. When these were combined in one molecule, the decreased binding was additive (data not shown) and as a result, this clone was not pursued further. Few mutations were found that improved binding to CD137 and functional activity compared to the related parental clone. Three mutant mAbs 2 The clones were all found to have improved binding affinity and functional activity. FS20-22-49AA / FS30-10 mAb 2 were derived from the clone. These mAbs 2is one asparagine, one threonine or parental FS20-22-49AA / FS30-10 mAb 2 contains either one leucine residue in place of the methionine residue at position 97 of 2 , and is designated as FS20-22-49AA / FS30-10-3, FS20-22-49AA / FS30-10-12 and FS20-22-49AA / FS30-10-16, respectively. FS20-22-49AA / FS30-35 parental mAb 2 The EC of the variant clone derived from the clone 2 50 values did not show an improvement in functional activity compared to the parental clone, but one variant clone designated as FS20-22-49AA / FS30-35-14, which contains an alanine residue substituted for the methionine residue at position 100F of the parental clone, showed an improvement in binding. FS20-22-49AA / FS30-5 parental mAb 2 For the clone 2 , both the methionine residue at position 100D and the methionine residue at position 100H were changed to an isoleucine residue and a leucine residue, respectively, within the same molecule, to obtain a variant mAb designated as FS20-22-49AA / FS30-5-37 2 clone. The FS20-22-49AA / FS30-10-3, FS20-22-49AA / FS30-10-12, FS20-22-49AA / FS30-10-16, FS20-22-49AA / FS30-35-14 and FS20-22-49AA / FS30-5-37 clones were selected for further characterization.

[0349] 6.3 Human CD137 Ligand Blocking Assay The interaction of CD137 - CD137L is necessary for the activation of the CD137 receptor. Agonistic anti - CD137 antibodies can promote the activation of CD137 by mimicking the ligand interaction, thereby potentially blocking ligand binding or promoting receptor clustering and activation without interfering with ligand binding. If the antibody has the potential to mimic CD137L, it can block the interaction between the receptor and the ligand. Although it is known in the art that MOR7480.1 blocks the ligand / receptor interaction (US 2012 / 0237498), it has previously been reported that the 20H4.9 antibody does not block the interaction between CD137 and its ligand (U.S. Patent No. 7288638).

[0350] Anti - human CD137 mAb 2 The clones, FS20 - 22 - 49AA / FS30 - 5 - 37, FS20 - 22 - 49AA / FS30 - 10 - 3, FS20 - 22 - 49AA / FS30 - 10 - 12, FS20 - 22 - 49AA / FS30 - 10 - 16 and FS20 - 22 - 49AA / FS30 - 35 - 14, were tested for their ability to block the CD137 - CD137L interaction using an ELISA - based method. Anti - OX40 mAb 11D4 (European Patent No. 2242771) (G1 / 11D4; SEQ ID NOs: 174 and 175) in IgG1 format was used as an isotype / negative control and is an mAb containing the Fab regions of anti - OX40 Fcab clone FS20 - 22 - 49AA and anti - FITC antibody 4420 2 FS20 - 22 - 49AA / 4420 was used as a negative control mAb for OX40 binding 2 ; anti - CD137 mAbs G1 / MOR7480.1 (SEQ ID NOs: 119 and 120) and G1 / 20H4.9 (SEQ ID NOs: 121 and 122) were used as positive controls for CD137 binding and ligand blocking activity.

[0351] Specifically, the recombinant human CD137-mFc-Avi antigen was coated overnight at 4°C on a Maxisorp 96-well plate at a concentration of 1 μg / ml in PBS. The next day, the plate was washed with PBST (PBS + 0.05% Tween 20 (trademark)), and blocked with PBS + 1% BSA (Sigma, A3059-500G) with stirring for 1 hour at room temperature. After blocking, the plate was washed again with PBST. A 100 nM dilution of each test antibody was prepared in PBS + 1% BSA, added to the plate coated with CD137, and incubated with stirring for 1 hour at room temperature. After this incubation, the plate was washed with PBST, and then incubated with 20 ng / ml CD137L-His (R&D Systems, 2295-4L-025 / CF) in PBS with stirring for 1 hour at room temperature. Next, the plate was washed with PBST, and then incubated with an anti-his secondary antibody (R&D Systems, MAB050H) at a 1:1000 dilution in PBS with stirring for 1 hour at room temperature. Then, the plate was washed with PBST and incubated with TMB detection reagent (Thermo Fisher Scientific, 002023) until the positive control wells turned blue, and then the reaction was stopped by the addition of 2N H2SO4. The plate was read at 450 nm using a plate reader equipped with Gen5 software, BioTek. The absorbance value at 630 nm was subtracted from the absorbance value at 450 nm (corrected). The subtracted absorbance values were plotted against the logarithmic concentration of the antibody, and the resulting curve was fitted using the logarithmic (inhibitor) vs. response equation of GraphPad Prism. The values were normalized by setting the G1 / 11D4 and G1 / MOR7480.1 control mAbs as 0 and 100% blocking values, respectively. The data were analyzed using one-way ANOVA and the Holm-Sidak multiple comparison test using GraphPad Prism.

[0352] Five anti-human CD137 mAbs tested 2Blocking activity in a certain range was observed for the clones. FS20-22-49AA / FS30-5-37 showed complete inhibition of receptor-ligand interaction, similar to the positive control antibody. All mAbs 2 containing the Fab region of the anti-CD137 mAb of the FS30-10 line (i.e., FS20-22-49AA / FS30-10-3, FS20-22-49AA / FS30-10-12, and FS20-22-49AA / FS30-10-16) inhibited the interaction between CD137 and CD137L by 49 - 54% and were thus considered partial blockers. By only partially blocking the interaction between CD137 and CD137L, these mAbs may not be able to completely inhibit the natural interaction between CD137L and its receptor, such that even if one of these antibodies binds, some CD137 signaling may still occur via this mechanism. The FS20-22-49AA / FS30-35-14 clone was considered a non-blocker as it lacked the ability to significantly inhibit receptor-ligand interaction, similar to the negative control FS20-22-49AA / 4420 mAb 2 molecule.

[0353] In summary, the results of this ELISA-based assay showed that the panel of anti-CD137 mAbs tested exhibited a range of ligand-blocking capabilities, including complete, partial, and no blocking activity. Each of the clones FS20-22-49AA / FS30-10-3, FS20-22-49AA / FS30-10-12, FS20-22-49AA / FS30-10-16, and FS20-22-49AA / FS30-35-14 showed different blocking activities compared to that of the positive control anti-CD137 mAb. Since the range of ligand-blocking activity was identified, the functional activity of each antibody was tested.

[0354] Clones FS20-22-49AA / FS30-5-37, FS20-22-49AA / FS30-10-3, FS20-22-49AA / FS30-10-12, and FS20-22-49AA / FS30-10-16 were further tested for their ability to block the CD137-CD137L interaction using a cell-based method. A certain range of blocking activity was observed, and FS20-22-49AA / FS30-5-37 showed complete inhibition of receptor-ligand interaction, similar to the positive control antibody (G1 / MOR7480.1) used in this assay. All three mAbs 2 containing the Fab region of the anti-CD137 mAb of the FS30-10 line (i.e., FS20-22-49AA / FS30-10-3, FS20-22-49AA / FS30-10-12, and FS20-22-49AA / FS30-10-16) inhibited the interaction between CD137 and CD137L by 46 - 76% and were thus considered partial blockers. Therefore, the results of this assay were similar to those of the ELISA-based blocking assay, indicating that the panel of anti-CD137 mAbs tested exhibited a range of ligand-blocking capabilities from complete blocking activity to partial blocking activity. Each of clones FS20-22-49AA / FS30-10-3, FS20-22-49AA / FS30-10-12, and FS20-22-49AA / FS30-10-16 showed blocking activity different from that of the positive control antibody.

[0355] Example 7 - Binding Specificity and Functional Activity of mAb and mAb 2 Clones in the Human CD137 T Cell Activation Assay 7.1 mAb 2 Binding Specificity of Clones CD137 and OX40 belong to the tumor necrosis factor superfamily (TNFRSF) of cytokine receptors (Moran et al., 2013). Anti-CD137 Fab as well as five mAbs 2To analyze the specificity of the OX40 Fcab binding site of the molecule, surface plasmon resonance (SPR) was used to test the binding of FS20-22-49AA / FS30-10-3, FS20-22-49AA / FS30-10-12, FS20-22-49AA / FS30-10-16, FS20-22-49AA / FS30-35-14 and FS20-22-49AA / FS30-5-37 mAbs to human CD137, human OX40 and six closely related human TNFRSF members. 2 The purpose was to show 1000-fold specificity by showing no binding of the mAb 2 to closely related antigens at a concentration of 1 μM, but binding to the CD137 and OX40 receptors at a concentration of 1 nM. Anti-CD137 mAb MOR7480.1 and anti-OX40 mAb 11D4 were used as positive controls.

[0356] Briefly, the flow cell on the CM5 chip was immobilized with approximately 1000 RU of either human CD137-mFc-Avi (Table 3), OX40-mFc (Table 2), recombinant human TNFRSF1A-Fc, recombinant human TNFRSF1B-Fc, recombinant human GITR-Fc, recombinant human NGFR-Fc, recombinant human CD40-Fc or recombinant human DR6-Fc. Flow cell 1 was left blank for immobilization. The five mAbs 2 were diluted to 1 μM and 1 nM (GE Healthcare, product code BR100188) in 1×HBS-EP buffer, flowed over the chip for 3 minutes and then dissociated for 4 minutes. For regeneration, a 30-second injection of 10 mM glycine pH 1.5 was used. To demonstrate the coating of each antigen, positive control mAbs were injected at 50 - 100 nM. Binding levels were determined and compared at the end of the association phase.

[0357] All of the selected mAbs 2 showed high levels of specificity for the human CD137 and OX40 receptors that were similar to or higher than those of the MOR7480.1 and 11D4 positive controls, respectively.

[0358] 7.2 Functional Activity of CD137 Agonist Antibodies in Human CD137 T Cell Activation Assays To understand the activity of different anti-CD137 agonist antibodies, a T cell activation assay using DO11.10-hCD137 cells was used. The anti-CD137 agonist antibodies G1AA / MOR7480.1 (SEQ ID NOs: 125 and 120), G1AA / 20H4.9 (SEQ ID NOs: 165 and 122) and G1AA / FS30-10-16 (SEQ ID NOs: 154 and 97) and, as an isotype negative control, the anti-FITC antibody 4420 in IgG1 format (G1 / 4420; SEQ ID NOs: 115 and 116) were tested. Antibody molecules were tested both in the presence and absence of the cross-linking anti-human CH2 antibody MK1A6 (see Example 2.1). Mouse IL-2 production was used as a measure of T cell activation.

[0359] The DO11.10-hCD137 cells were washed once with PBS and resuspended in DO11.10 cell medium (RPMI medium (Life Technologies)) containing 10% FBS (Life Technologies) and 5 μg / ml puromycin (Life Technologies, A11113803) at a concentration of 1.0 × 10 6 cells / mL. A 96-well flat-bottom plate was coated with anti-mouse CD3 antibody (Thermo Fisher Scientific, clone 17A2) by incubating with 0.1 μg / ml anti-mouse CD3 antibody diluted in PBS at 37 °C, 5% CO2 for 2 hours and then washed twice with PBS. The DO11.10-hCD137 cells were at 1 × 10 5Cells / wells were added to the plates. A 2 μM dilution of each test antibody was prepared in DPBS (Gibco) and further diluted 1:10 in DO11.10 cell medium (30 μl + 270 μl) to obtain a 200 nM dilution. The MK1A6 crosslinker was added to the wells at a 1:1 molar ratio with the test antibody as needed. Serial dilutions of the antibody or antibody / crosslinked antibody mixture were prepared in 96-well plates. 100 μl of the diluted antibody or antibody / crosslinked antibody mixture was added to the DO11.10-hCD137 cells on the plates. The cells were incubated at 37 °C and 5% CO2 for 72 hours. The supernatants were collected and assayed using a mouse IL-2 ELISA kit (eBioscience or R&D Systems) according to the manufacturer's instructions. The plates were read at 450 nm using a plate reader equipped with Gen5 software, BioTek. The absorbance value at 630 nm was subtracted from the absorbance value at 450 nm (corrected). The standard curve for calculating cytokine concentration was based on a four-parameter logistic curve fit (Gen5 software, BioTek). The concentration of mouse IL-2 (mIL-2) was plotted against the log concentration of the antibody, and the resulting curve was fitted using the log(agonist) vs. response equation of GraphPad Prism.

[0360] The results of the assay are shown in FIGS. 2C and D. The anti-CD137 antibodies differed in their requirements for crosslinking antibodies for their activity. All three anti-CD137 antibodies showed an increase in IL-2 production in a concentration-dependent manner in the presence of the crosslinking antibody, but only the G1AA / 20H4.9 antibody showed activity in the absence of the crosslinking antibody. Thus, G1AA / MOR7480.1 and G1AA / FS30-10-16 required the addition of the crosslinking antibody, i.e., their activity was "crosslinking-dependent". On the other hand, G1AA / 20H4.9 showed activity both in the presence and absence of the crosslinking antibody, i.e., its activity was "crosslinking-independent".

[0361] 7.3 Function activity of mAb in human CD137 T cell activation assay 2 of Selected FS20-22-49AA / FS30-5-37, FS20-22-49AA / FS30-10-3, FS20-22-49AA / FS30-10-12 and FS20-22-49AA / FS30-10-16 mAbs 2 The functional activity of the clones was tested in a T cell activation assay using DO11.10-hCD137 cells. Anti-FITC antibody 4420 (G1 / 4420; SEQ ID NOs: 115 and 116) in IgG1 format was used as an isotype negative control, anti-OX40 mAb G1 / 11D4 (SEQ ID NOs: 174 and 175) and mAb 2 Clone FS20-22-49AA / 4420 (SEQ ID NOs: 123 and 116) was used as a negative control, and anti-CD137 antibody MOR7480.1 in both IgG1 (G1 / MOR7480.1; SEQ ID NOs: 119 and 120) and IgG2 (G2 / MOR7480.1; SEQ ID NOs: 124 and 120) formats, the IgG2 format of the antibody being tested in clinical trials (Gopal et al., 2017; Tolcher et al., 2017), was used as a positive control. The mAb and mAb 2 molecules were cross-linked using anti-human CH2 antibody, MK1A6 (see Example 2.1), and in some experiments, the activity of non-cross-linked mAb and mAb 2 molecules was investigated. Mouse IL-2 production was used as a measure of T cell activation. The experiments were conducted as described in Example 7.2.

[0362] When cross-linked, all five selected mAbs 2 clones showed strong activity in the T cell activation assay, with the average EC 50 value being less than 15 nM and the average Emax value being in the range of about 16,000 - 20,000 pg / ml IL-2 (Table 6 and Figure 2A). The activity of the mAb 2 clones was not observed in the absence of cross-linking (Figure 2B). The MOR7480.1 positive control antibody was observed to show activity only when cross-linked (for G1 / MOR7480.1, an EC of 3.3 nM 50 and an E of 12575 pg / ml maxFor G2 / MOR7480.1, an EC of 2.4 nM 50 and an E of 8547 pg / ml max ). The combination of the lack of activity of the cross-linked anti-OX40 mAb (G1 / 11D4) and the low background signal observed for the non-cross-linked anti-OX40 Fcab-containing mAb 2 molecules indicates that the results of this assay are a readout of CD137 activity only and are likely due to the high level of CD137 receptor expression and the non-detectable level of OX40 receptor expression by DO11.10 cells (data not shown).

[0363]

Table 7

[0364] Thus, mAbs containing the CDRs of the anti-human CD137 monoclonal antibodies FS30-5-37, FS30-10-3, FS30-10-12, FS30-10-16, and FS30-35-14 2 showed strong activity capable of activating CD137 in the DO11.10-hCD137T cell activation assay when cross-linked. No significant activity was observed in the absence of cross-linking. These mAbs 2 contain the CH3 domain from the anti-human OX40 Fcab FS20-22-49, which also showed high activity when cross-linked in a T cell assay (see Example 4.3). This mAb prepared with the LALA mutation 2 was designated as FS20-22-49AA / FS30-5-37, FS20-22-49AA / FS30-10-3, FS20-22-49AA / FS30-10-12, FS20-22-49AA / FS30-10-16.

[0365] These mAbs 2 were selected for further analysis to determine whether they can act as dual agonists capable of autonomously agonizing both OX40 and CD137 based on their ability to act without the need for specific target expression and additional cross-linking agents.

[0366] Example 8 - mAbs against Human and Cynomolgus Macaque OX40 and CD137 2 Binding affinity To determine the affinity for CD137, a Biacore CM5 chip (GE Healthcare) was coated with anti-human Fc using the Human Antibody Capture Kit (GE Healthcare) according to the manufacturer's conditions to a surface density of approximately 4000 RU. Samples of the test antibodies (mAb 2 FS20-22-49AA / FS30-5-37, FS20-22-49AA / FS30-10-3, FS20-22-49AA / FS30-10-12, and FS20-22-49AA / FS30-10-16, anti-CD137 positive control G1 / MOR7480.1, and anti-hOX40 negative control G1 / 11D4) were captured up to approximately 80 RU. Human or cynomolgus macaque CD137 (hCD137-mFc-Avi or cCD137-mFc-Avi) was flowed at a flow rate of 70 μl / min over a concentration range of a three-fold dilution series starting from 200 nM. The association time was 2 minutes and the dissociation time was 8 minutes. The running buffer was HBS-EP (GE Healthcare BR100188). The flow cell was regenerated by injecting 3M magnesium chloride at a flow rate of 30 μl / min for 30 seconds.

[0367] To determine the affinity for OX40, a Biacore CM5 chip was coated with anti-human Fab using the Human Fab Capture Kit (GE Healthcare 28958325) according to the manufacturer's conditions to a surface density of approximately 8000 RU. Samples of the test antibodies (FS20-22-49AA / FS30-5-37, FS20-22-49AA / FS30-10-3, FS20-22-49AA / FS30-10-12, and FS20-22-49AA / FS30-10-16 mAb 2, G1 / MOR7480.1 (negative control) and G1 / 11D4 (positive control)) were captured up to about 80 RU, and then human or cynomolgus monkey OX40 antigen (hOX40-mFc or cOX40-mFc) was flowed at a flow rate of 70 μl / min in a concentration range of a three-fold dilution series starting from 200 nM. The association time was 2 minutes and the dissociation time was 8 minutes. The running buffer was HBS-EP. The flow cell was regenerated by injecting glycine chloride at pH 2.1 at a flow rate of 30 μl / min for 30 seconds.

[0368] The data were analyzed by double referencing against a flow cell that was intentionally left blank (no antibody binding). The binding kinetics were fitted to a 1:1 Langmuir model to generate the association (k a ) and dissociation (k d ) rates. The equilibrium binding constant (K D ) was calculated by dividing the dissociation rate by the association rate for each sample. The data analysis was performed using BiaEvaluation software version 3.2. The results are shown in Table 7.

[0369]

Table 8

[0370] The binding affinity of OX40 / CD137 mAb 2 indicates that these molecules bind to both receptors with high affinity. The affinity of these molecules for human OX40 is similar, which is expected since these molecules all share OX40Fcab. The affinity for cynomolgus monkey OX40 is within 5-fold of that for human OX40. The affinity for human CD137 ranges from 4 to 0.2 nM, and the anti-CD137 Fab varies for each molecule, so the cross-reactivity with cynomolgus monkey CD137 also varies. FS20-22-49AA / FS30-10-16 has a higher affinity for human CD137 and a similar affinity for cynomolgus monkey CD137. mAb in the cynomolgus monkey study 2Since the behavior of [the subject] is expected to be extrapolatable to humans, the similarity in binding to human and cynomolgus monkey antigens can be advantageous.

[0371] Also, FS20-22-49AA / FS30-10-16 has similar affinities for human OX40 and human CD137, and when these are co-expressed, the mAb 2 is expected to bind equally well to both targets.

[0372] An mAb that binds to OX40 and CD137 and simultaneously promotes clustering and activation of both targets 2 is expected to function as a dual agonist. Both OX40 and CD137 are known to be present on T cells (Ma, et al., 2005). Without being bound by theory, an mAb with similar affinities for binding to both targets 2 is likely to bind to cells expressing both targets, and thus may be advantageous as a dual agonist. An mAb with a significantly higher affinity for one target than the other and preferentially binds 2 may preferentially bind to cells that do not express both targets, and thus may not have to act as a dual agonist. 2 2

[0373] Example 9 - Simultaneous Binding of mAbs to OX40 and CD137 2 Simultaneous Binding 9.1 Simultaneous Binding of mAbs to Human OX40 and CD137 2 Simultaneous Binding OX40 / CD137 mAb 2The ability of FS20-22-49AA / FS30-5-37, FS20-22-49AA / FS30-10-3, and FS20-22-49AA / FS30-10-16 to simultaneously bind to OX40 and CD137 was tested by SPR using a Biacore 3000. G1 / MOR7480.1 was used as a control. According to the manufacturer's instructions, biotinylated human CD137 (hCD137-mFc-Avi-Bio) was diluted to 100 nM in HBS-EP buffer and immobilized on a streptavidin (SA) chip (GE Healthcare BR100032) to a surface density of approximately 1000 RU, and the flow cell was activated and deactivated without any immobilized protein for background removal. Antibodies diluted to 100 nM in HBS-EP buffer were co-injected at a flow rate of 30 μl / min with either 100 nM human OX40 (hOX40-mFc) or HBS-EP buffer. For each binding step, dissociation was followed for 3 minutes. The sensor chip was regenerated by injecting 15 μl of 2.5 glycine (GE Healthcare) at a flow rate of 30 μl / min after each cycle. All mAbs tested 2 were able to simultaneously bind to OX40 and CD137. The control mAb, G1 / MOR7480.1, binds only to CD137.

[0374] 9.2 Mouse receptor-targeting mAbs against mouse OX40 and mouse CD137 2 Simultaneous binding mAbs containing anti-mouse OX40 Fcab with anti-mouse CD137 Fab 2It was prepared to test its ability to bind simultaneously to mouse OX40 and mouse CD137. Mouse OX40-targeted Fcab FS20m-232-91 was selected because of its higher activity in T cell assays, and the Fab of anti-mouse CD137 antibody Lob12.3 in human IgG1 isotype format (G1 / Lob12.3; University of Southampton) (Taraban et al., 2002) showed good cell binding to mouse CD137-expressing cells and was widely used in the literature as an agonistic CD137 antibody with in vitro and in vivo activities, so it was selected for pairing with FS20m-232-91 Fcab. The mAb containing the anti-mouse CD137 antibody Lob12.3 and the LALA-mutated FS20m-232-91 CH3 domain and Fab 2 is designated "FS20m-232-91AA / Lob12.3", while the mAb containing the anti-mouse CD137 antibody Lob12.3 without LALA mutation and the FS20m-232-91 CH3 domain and Fab 2 was designated "FS20m-232-91 / Lob12.3".

[0375] The ability of FS20m-232-91AA / Lob12.3 mAb 2 to bind simultaneously to the two targets was tested by SPR using a BIAcore 3000 instrument (GE Healthcare). G1 / Lob12.3 was used as a positive control. Recombinant mouse CD137 (mCD137-hFc; R&D Systems, catalog number 937-4B-050) was diluted to 200 nM with sodium acetate pH 5.0 (GE Healthcare) and immobilized on a Biacore CM5 chip at a surface density of approximately 1000 RU, and the flow cell was activated and deactivated without immobilized protein for background removal. The mAb diluted to 100 nM in HBS-EP buffer 2And the positive control was co-injected at 100 nM of either human OX40 (mOX40-MFC) or HBS-EP buffer at a flow rate of 30 μl / min. For each binding step, dissociation was followed for 3 minutes. The sensor chip was regenerated after each cycle by injecting an aqueous glycine-HCl solution at pH 1.7 at a flow rate of 20 μl / min for 30 seconds. mAb 2 was able to bind simultaneously to OX40 and CD137. The G1 / Lob12.3 mAb binds only to CD137.

[0376] Example 10 - Binding of mAb to Fcγ Receptor 2 to Agonistic antibodies targeting TNFR family members are known from the literature to require Fcγ receptor-mediated cross-linking to promote clustering and activation of the target of in vivo activity (Wajant, 2015). However, this may not be desirable for antibodies that are intended to be dual agonists. Therefore, it was determined to reduce the ability of mAb 2 to bind to Fcγ receptors by insertion of the LALA mutation.

[0377] Human IgG1 isotype antibodies are capable of binding to Fcγ receptors. Thereby, when they bind to Fcγ receptors, effector functions such as antibody-dependent cell cytotoxicity (ADCC) of cells expressing the target are induced, and cell lysis may occur. The intended mechanism of the OX40 / CD137 mAb 2 is to activate cells expressing OX40 and CD137 without killing them, so a decrease in ADCC induced by mAb 2 is desirable.

[0378] Also, the OX40 / CD137 mAb 2Since they are intended to function as dual agonists, their intended mechanism of action is to signal through the receptor as a result of cross-linking by double binding to both OX40 and CD137, whether co-expressed on the same cell or expressed on different cells, and the ability to cross-link via the Fcγ receptor is not a requirement for function.

[0379] Furthermore, CD137-targeted antibodies are known to exhibit liver toxicity clinically (Segal et al., 2017), and the toxicity mechanism is unknown but may depend on FcγR-mediated cross-linking of anti-CD137 antibodies or activation of anti-CD137-expressing cells in the liver or periphery. Prevention of CD137 agonist activity via FcγR-mediated cross-linking, since these molecules cross-link only via double binding to OX40 and CD137, the OX40 / CD137 mAb of the present invention 2 can reduce the toxicity risk.

[0380] Using binding by SPR, the presence of the LALA mutation in mAb 2 FS20-22-49AA / FS30-10-16 was confirmed to have reduced binding affinity for Fcγ receptors, particularly hFcγR1 (R&D Systems, catalog number 1257-FC-050 / CF), hFcγR2a (R&D Systems, catalog number 1330-CD-050 / CF), hFcγR2b (R&D Systems, catalog number 1460-CD-050 / CF), and hFcγR3a (R&D Systems, catalog number 4325-FC-050 / CF). Anti-hOX40 mAbs G1AA / 11D4 and G1 / 11D4 (with and without the LALA mutation, respectively) and anti-CD137 mAbs G1AA / 20H4.9 and G1 / 20H4.9 (with and without the LALA mutation, respectively) were all in the hIgG1 isotype format, and anti-hCD137 mAb G4 / 20H4.9 was in the hIgG4 isotype format and was used as a control antibody.

[0381] The binding was tested using a Biacore 3000 instrument (GE Healthcare). Human OX40 (BPS Bioscience catalog number 71310) and human CD137 (produced in-house) biotinylated his-tagged antigens were coated on an SA chip (GE Healthcare catalog number BR100398) at a concentration of 2 μM. Human OX40 and human CD137 were coated on separate flow cells, and another flow cell was left blank for background removal. The regeneration condition was determined to be 12 μl of 10 mM glycine-HCl aqueous solution at pH 2.0 at a flow rate of 20 μl / min. Antibodies (see Table 8) and human FcγRs (see Table 8) were diluted in HBS-P (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.005% weight / vol surfactant P20, GE Healthcare, BR-1003-68) to 100 nM (antibodies) or 500 nM (human FcγRs) and co-injected at a flow rate of 20 μl / min, and dissociation was followed for 5 minutes.

[0382] Data analysis was performed using BiaEvaluation software version 3.2 RC1, referencing against the blank flow cell and aligning the curves after antibody association. The value of the binding response at the end of the association phase was generated by subtracting the absolute response at the end of the association phase of FcγR from the absolute response at the end of the association phase of the antibody in order to normalize the effect of antibody binding to the OX40 and CD137 receptors.

[0383] Measurements for the binding response at the end of the dissociation phase of FcγRI were made to demonstrate the effect of the LALA mutation, which increases the off-rate of FcγRI binding, if the binding to this FcγR was not completely removed. These were generated by subtracting the absolute response at the end of the association phase of FcγR from the absolute response at the end of the dissociation phase of FcγR. Values for the anti-CD137 antibody were taken from the flow cell coated with CD137-His antigen, and values for the anti-OX40 antibody were from the flow cell coated with OX40-His antigen and OX40 / CD137 mAb 2For [specific details not provided], it was collected from both the flow cell coated with OX40-his antigen and the flow cell coated with CD137-his antigen. The results are shown in Table 8.

[0384]

Table 9

[0385] mAb without LALA mutation 2 and the control antibodies were all bound to each of the Fcγ receptors as expected in both IgG1 and IgG4 formats. The mAb of IgG1 format containing the LALA mutation 2 and the control antibodies showed significantly reduced binding at the end of the association phase (on-rate) to each of the tested Fcγ receptors, except for FcγRI, compared to the control antibodies of IgG1 format without the LALA mutation and the control antibodies of IgG4 format. The binding rate of the high-affinity Fcγ-binding receptor, FcγRI, to the hIgG1LALA-containing antibody was only slightly reduced compared to the IgG1 antibody without LALA, and was not significantly changed by the introduction of such a mutation. However, the off-rate of FcγRI was faster for the LALA mutation-containing antibody than for the antibody without LALA, as shown by a more significant decrease in the binding response at the end of the dissociation phase of FcγRI (more than 200 RU for each of the LALA-containing antibodies, compared to less than 60 RU for the antibody without LALA).

[0386] Overall, when compared to wild-type human IgG1, the LALA mutation-containing OX40 / CD137 mAb 2 showed a decreased binding to Fcγ receptors at a lower level than other LALA-containing hIgG1 antibodies and the IgG4 control antibody. Since Fcγ receptor binding is required for ADCC activity, this decrease in binding to Fcγ receptors caused by the LALA mutation is expected to also result in a decrease in ADCC such that target cells are not depleted by mAb 2 binding. The OX40 / CD137 mAb 2 is an agonist antibody, and the mAb2 Since it is a cell aimed at stimulating, depletion of target cells is not desirable, so this is considered important.

[0387] FcγRIIIa is expressed on immune effector cells such as natural killer (NK) cells and has been shown to be important for mediating ADCC (Chan et al., 2015). As confirmed by SPR data, the FS20-22-49AA / FS30-10-16 mAb against FcγRIIIa 2 To determine whether the reduced binding is translated into low activation or negligible of the ADCC pathway, an ADCC bioassay was performed using engineered Jurkat cells expressing FcγRIIIa as effector cells and large cells overexpressing either OX40 or human CD137 as target cells. By comparing the responses observed for negative or positive control antibodies used in the assay, the mAb 2 was observed not to induce ADCC activation in either OX40-expressing or CD137-expressing large cells.

[0388] Other agonist antibodies are known to rely on Fcγ receptor cross-linking of antibodies that create higher-order structures (Stewart et al., 2014; Wajant, 2015), and to bring about clustering and activation of cell surface receptors in order to exert their agonist activity. Fcγ receptor-mediated cross-linking is not required for the activity of the mAb of the present invention, and the agonist action of the cell will localize to the site where both targets are present. The LALA mutation of the OX40 / CD137 mAb 2 results in a decrease in binding to Fcγ receptors, so it is not expected that Fcγ receptor cross-linking-driven activity via only CD137 binding is possible. As a result, the mAb 2 Since the LALA mutation of results in a decrease in binding to Fcγ receptors, it is not expected that Fcγ receptor cross-linking-driven activity via only CD137 binding is possible. As a result, the mAb 2In the absence of OX40 expression, it is unlikely to activate CD137-expressing cells. In humans, there is a known liver toxicity risk associated with targeting CD137 (e.g., as seen with treatment with urelumab (BMS-663513)) (Segal et al., 2017), and the reduced possibility of Fcγ receptor-induced cross-linking of the LALA mutant-containing mAb 2 is expected to reduce the likelihood of such liver toxicity occurring during treatment with the mAb 2 since CD137 is only activated where OX40 is also expressed. The current theory of CD137-induced liver toxicity indicates that CD137-expressing myeloid cells are the cell type that causes liver inflammation seen in mice treated with CD137 agonists (Bartkowiak, et al., 2018).

[0389] Macrophages are known to express FcγRI, which can potentially mediate the cross-linking of CD137-targeted antibodies, but these cells are not known to express OX40. Therefore, the LALA mutant-containing mAb of the present invention 2 should theoretically not be able to activate liver macrophages that express CD137 but not OX40. This is the OX40 / CD137 mAb of the present invention compared to either a CD137 agonist that requires Fcγ receptor cross-linking for activity or a CD137 agonist that does not require cross-linking for activity 2 is thought to reduce the liver toxicity risk. In the case of OX40, some residual activity of Fcab has been observed in the absence of CD137 binding and in the absence of cross-linking that can lead to some activation of OX40, but since dose-limiting toxicity has not been reported in clinical trials using OX40 agonists to date, this is not considered a risk.

[0390] Example 11 - Binding of mAb 2 to cells expressing OX40 and CD137 11.1. Binding of mAb 2 to cells expressing human or cynomolgus monkey OX40 or CD137 mAbs against cell-expressed human or cynomolgus monkey OX40 and CD137 2 The binding affinities of FS20-22-49AA / FS30-5-37, FS20-22-49AA / FS30-10-3, FS20-22-49AA / FS30-10-12, and FS20-22-49AA / FS30-10-16 were determined using flow cytometry. These mAbs 2 antibodies and control antibodies G1 / 4420 (FITC), G1 / 11D4 (OX40), G1 / MOR7480.1 (CD137), and FS20-22-49AA / 4420 (OX40 / FITC mock mAb 2 )(all in IgG1 isotype format) dilutions (2X final concentration) were prepared in 1X DPBS (Gibco, 14190-094). DO11.10-hOX40, DO11.10-cOX40, DO11.10-hCD137, DO11.10-cCD137, or HEK cell suspensions were prepared in PBS + 2% BSA (Sigma, A7906) and seeded at 50 μl / well at 4 × 10 6 cells / ml in V-bottom 96-well plates (Costar, 3897). 50 μl of antibody dilutions were added to the cell-containing wells (final volume 100 μl) and incubated at 4°C for 1 hour. The plates were washed and 100 μl / well of secondary antibody (anti-human Fc-488 antibody, Jackson ImmunoResearch, 109-546-098) diluted 1:1000 in PBS + 2% BSA was added and incubated in the dark at 4°C for 30 minutes. The plates were washed and analyzed using a CantoII flow cytometer (BD Bioscience) resuspended in 100 μl of PBS containing DAPI (Biotium, catalog number 40043) at 1 μg / ml, and the data were analyzed using FlowJo. Dead cells were identified by higher fluorescence in the UV (405 nm / 450 / 50) channel and excluded from the analysis. The geometric mean fluorescence intensity (GMFI) in the FITC channel (488 nm / 530 / 30) was used as a measure of antibody binding. The GMFI data were fit using log (agonist) vs. response (three parameters) in GraphPad Prism software, and the EC 50Values were generated.

[0391]

Table 10

[0392] This result confirms that the tested OX40 / CD137 mAb 2 binds to human and cynomolgus monkey OX40 and CD137 expressed on DO11.10 cells. The mAb 2 and the positive controls (anti-human OX40 mAb, G1 / 11D4 and anti-human CD137 mAb G1 / MOR7480.1 in the human IgG1 backbone) bound to both human and cynomolgus monkey OX40 and CD137 with a certain range of affinity (see Table 9). No cross-reactivity with other proteins expressed on the surface of the HEK cell line was observed, because for none of the tested antibodies could binding to this cell line be detected. Therefore, the OX40 / CD137 mAb 2 specifically binds to human OX40 and human CD137, and no non-specific binding was confirmed.

[0393] 11.2 FS20-22-49AA / FS30-10-16 mAb 2 and binding of its components to cells expressing human or cynomolgus monkey OX40 or CD137 Cell-expressed mAb against human or cynomolgus monkey OX40 and CD137 2 To compare the affinity of FS20-22-49AA / FS30-10-16 and its components, namely OX40 Fcab (OX40 / FITC mock mAb 2 format; FS20-22-49AA / 4420) and CD137 Fab (IgG1 format; FS30-010-016), the same method as described in Example 11.1 was used. However, in this experiment, non-transduced DO11.10 cells were used instead of HEK cells to analyze non-specific binding. G1 / 4420 anti-FITC antibody was used as a control. The experiment was performed with the calculated EC 50It was repeated three times to enhance the reliability of the values. The average average EC for the tested molecules 50 values are shown in Table 10.

[0394] [Table 11]

[0395] The results show that the OX40 / CD137 mAb 2 (FS20-22-49AA / FS30-10-16) binds to human and cynomolgus monkey OX40 and CD137 expressed on DO 11.10 cells with an affinity below nanomolar concentration, and that the OX40 Fcab component of the mAb 2 binds to human and cynomolgus monkey OX40 with an affinity equivalent to that of the OX40 / CD137 mAb 2 and that the CD137 Fab component of the mAb 2 binds to human and cynomolgus monkey CD137 with an affinity equivalent to that of the OX40 / CD137 mAb 2 were confirmed. No non-specific binding was observed for either the components or isotype control antibody (G1 / 4420) of the OX40 / CD137 mAb 2 to non-transduced DO11.10 cells. The results show that the affinity of FS20-22-49AA / FS30-10-16 OX40 / CD137 mAb 2 and FS20-22-49AA OX40 Fcab for cell-expressed cynomolgus monkey OX40 was higher than previously observed (Example 11.1 and Table 9) (as indicated by lower EC 50 values) and was similar to the affinity results determined by SPR (Example 8 and Table 7). The average EC 50 values detailed in Table 10 are the results of three independent experiments and thus better represent the affinity of the molecules tested for human and cynomolgus monkey OX40 and CD137 expressed on DO11.10 cells.

[0396] 11.3 Binding of mAb to cells expressing mouse OX40 or CD137 2 Binding Binding affinity of FS20m-232-91AA / Lob12.3 mAb to cell-expressed mouse OX40 and CD137 2 was determined using flow cytometry. Dilutions (2-fold of the final concentration) of FS20m-232-91AA / Lob12.3 and control antibodies G1 / 4420 (FITC), G1 / Lob12.3 (CD137), G1 / OX86 (OX40), and FS20m-232-91AA / HELD1.3 (OX40 / HEL mock mAb 2 ) were prepared in 1X DPBS (Gibco, 14190-094). DO11.10-mOX40, DO11.10-mCD137, or HEK cell suspensions were prepared in PBS + 2% BSA (Sigma, A7906) and seeded at 4 × 10 6 cells / ml at 50 μl / well in a V-bottom 96-well plate (Costar, 3897). 50 μl of antibody dilutions were added to the cell-containing wells (final volume 100 μl) and incubated at 4°C for 1 hour. The plates were washed and 100 μl / well of secondary antibody (anti-human Fc-488 antibody, Jackson ImmunoResearch, 109-546-098) diluted 1:1000 in PBS + 2% BSA was added and incubated in the dark at 4°C for 30 minutes. The plates were washed and analyzed using a CantoII flow cytometer (BD Bioscience) resuspended in 100 μl of PBS containing DAPI (Biotium, catalog number 40043) at 1 μg / ml, and the data were analyzed using FlowJo. Dead cells were identified by higher fluorescence in the UV (405 nm / 450 / 50) channel and excluded from the analysis. Geometric mean fluorescence intensity (GMFI) in the FITC channel (488 nm / 530 / 30) was used as a measure of antibody binding. GMFI data were fitted using logarithm (agonist) vs. response (three parameters) in GraphPad Prism software to generate EC 50 values. The results are shown in Table 11.

[0397]

Table 12

[0398] The results confirm that FS20m-232-91AA / Lob12.3 mAb binds to mouse OX40 and CD137 expressed on DO11.10 cells. 2 The mAb 2 and the positive controls (anti-mouse OX40 mAb OX86 in a human IgG1 backbone and anti-mouse CD137 mAb Lob12.3 in a human IgG1 backbone) bound to both mouse OX40 and / or CD137 with a range of affinities (see Table 11). No cross-reactivity with other proteins expressed on the surface of the HEK cell line was observed, as binding to this cell line could not be detected for any of the antibodies tested.

[0399] Therefore, the anti-mouse OX40 / CD137 mAb 2 specifically bound to mouse OX40 and mouse CD137, and no non-specific binding was confirmed.

[0400] Example 12 - Activity of OX40 / CD137 mAb targeting co-expressed receptors in a staphylococcal enterotoxin A (SEA) assay 2 of OX40 expression in tumor-infiltrating lymphocytes is likely to be associated with CD137 expression because these two molecules are often co-expressed in activated T cells (Ma et al., 2005). Agonizing OX40 and CD137 with mAbs 2 targeting these two co-expressed receptors can induce the proliferation and production of inflammatory cytokines by pre-activated T cells.

[0401] To be fully activated, T cells require two signals. The first signal is antigen-specific and is provided via the T cell receptor that interacts with MHC (major histocompatibility complex) molecules presenting peptide antigens on the membrane of antigen-presenting cells (APCs). The second signal is a co-stimulatory signal, which is antigen-nonspecific and is provided by the interaction between co-stimulatory molecules expressed on the membranes of APCs and T cells.

[0402] OX40 / CD137 mAb 2 To test the activity of 2 , a T cell activation assay using staphylococcal enterotoxin A (SEA) superantigen as the first signal was established. SEA crosslinks MHC class II molecules on the surface of APCs and the TCR of T cells, thereby providing the first signal for T cell activation. For their complete activation, T cells must receive a second co-stimulatory signal by the crosslinking mAb 2 as appropriate or control molecules. This assay was performed using PBMCs isolated from blood and needs to more accurately represent what is expected to occur in vivo compared to assays performed using isolated T cells.

[0403] The SEA stimulation assay was used to establish the activity of different OX40 and CD137 agonist antibodies and OX40 / CD137 mAb 2 antibodies in the presence or absence of an artificial crosslinker, and to compare different OX40 / CD137 mAb 2 clones. Representative EC 2 values for the OX40 / CD137 mAb 50 clone FS20-22-49AA / FS30-10-16 were established in a group of 10 PBMC donors.

[0404] 12.1 Activity of OX40 and CD137 agonist antibodies on SEA-stimulated PBMCs To establish the sensitivity of the SEA assay to different OX40 and CD137 agonist antibodies, the mAb 2 antibodies (FS22-20-49AA / FS30-10-16) and control antibodies listed in Table 12 were tested for their activity in the assay. G1 / 4420 (anti-FITC), G1AA / MOR7480.1 (anti-CD137), G1AA / FS30-10-16 (anti-CD137), G1AA / 20H4.9 (anti-CD137), G1AA / 11D4 (anti-OX40) and FS20-22-49AA / 4420 (OX40 / FITC mock mAb 2) was used as a control. IL-2 production was used as a measure of T cell activation.

[0405]

Table 13

[0406] Peripheral blood mononuclear cells (PBMCs) were isolated from leukoreduced cones (NHS Blood and Transplant service), which are a by-product of platelet donation. Briefly, the contents of the leukocyte cone were washed away with PBS and layered on a Ficoll gradient (GE Lifesciences catalog number 17144002). PBMCs were separated by centrifugation and recovery of cells that did not pass through the Ficoll gradient. The PBMCs were further washed with PBS, and the remaining red blood cells were lysed by the addition of 10 ml of red blood cell lysis buffer (eBioscience) according to the manufacturer's instructions. The PBMCs were counted and resuspended at 2.0×10 6 cells / ml in T cell medium (RPMI medium (Life Technologies)) containing 10% FBS (Life Technologies), 1× penicillin streptomycin (Life Technologies), sodium pyruvate (Gibco), 10 mM HEPES (Gibco), 2 mM L-glutamine (Gibco), and 50 μM 2-mercaptoethanol (Gibco). Next, SEA (Sigma catalog number S9399) was added to the PBMCs at 200 ng / ml, and the cells were added to plates at 2×10 5 cells / well (100 μl / well).

[0407] Prepare a 2 μM dilution of each test antibody (see Table 12 for details) with DPBS (Gibco), and further dilute it 1:10 with T cell medium (30 μl + 270 μl) to obtain a 200 nM dilution. An artificial crosslinker (anti-human CH2 antibody (clone MK1A6, in-house production) or FITC-dextran (Sigma) (see Table 12) was added to the wells at a 1:1 molar ratio with the test antibody as needed. In a 96-well plate, prepare a serial dilution of the test antibody and add 100 μl of the diluted antibody mixture to the activated T cells on the plate.

[0408] The cells were incubated at 37 °C in 5% CO2 for 120 hours. The supernatant was collected and the release of IL-2 was measured using a human IL-2 ELISA kit (eBioscience or R&D Systems) according to the manufacturer's instructions. The plate was read at 450 nm using a plate reader equipped with Gen5 software, BioTek. Subtract the absorbance value at 630 nm from the absorbance value at 450 nm (corrected). The standard curve for calculating cytokine concentration was based on a four-parameter logistic curve fit (Gen5 software, BioTek). The concentration of human IL-2 (hIL-2) was plotted against the log concentration of the test antibody, and the resulting curve was fitted using the log(agonist) vs. response equation of GraphPad Prism. Table 13 shows the EC 50 values and the maximum response of IL-2 release observed in the SEA assay in the presence or absence of an artificial crosslinker. Figure 3A shows the level of IL-2 release induced by antibodies tested at a single concentration (3.7 nM) in the SEA assay. The concentration at which these antibodies induced the highest level of IL-2 production was selected for this analysis. Statistical analysis was performed by two-way ANOVA and Tukey's multiple comparison test. Asterisks on the error bars represent significant differences compared to samples treated with the isotype control (G1 / 4420) (*p < 0.032, **p < 0.0021, ***p < 0.0002, ****p < 0.0001). Figure 3B shows the OX40 / CD137 mAb in the presence or absence of an artificial crosslinker in the SEA assay 2(FS20-22-49AA / FS30-10-16)-induced IL-2 release plots are shown.

[0409]

Table 14

[0410] These results show that only OX40 / CD137 mAb 2 (FS20-22-49AA / FS30-10-16) can increase IL-2 levels in the absence of an artificial cross-linking agent, and the addition of an artificial cross-linking agent did not increase the activity of OX40 / CD137 mAb 50 from either the EC 2 or maximum response perspective. The activities of the OX40-targeting antibodies G1AA / 11D4 and FS20-22-49AA / 4420 and the anti-CD137 antibody G1AA / 20H4.9 were only observed in the presence of an artificial cross-linking agent. For the anti-CD137 antibodies G1AA / MOR7480.1 and G1AA / FS30-10-16, no statistically significant activity was detected in the presence of an artificial cross-linking agent compared to the isotype control. The anti-OX40 antibody G1AA / 11D4 induced higher IL-2 levels than the anti-CD137 antibodies G1AA / MOR7480.1 and G1AA / FS30-10-16 and equivalent IL-2 levels to the anti-CD137 antibody G1AA / 20H4.9. However, the G1AA / 11D4 antibody was observed to have a higher ability than the G1AA / 20H4.9 antibody, as indicated by its significantly lower EC 50 value. These results indicate that this SEA assay is more sensitive to OX40 agonistic activity than CD137 agonistic activity. This may be related to the fact that OX40 is preferentially expressed on CD4+ T cells and CD137 is preferentially expressed on CD8+ T cells (Croft, 2014 and internal data shown in Figure 6), due to the generally higher presence of CD8+ T cells than CD4+ T cells in human PBMC.

[0411] 12.2 Different OX40 / CD137 mAbs in SEA-stimulated PBMC 2Activity of Clones Five different OX40 / CD137 mAbs 2 The clones were tested for their activity in the SEA assay. The mAbs used in the assay 2 and details of the control antibodies are provided in Table 14. G1 / 4420 (anti-FITC), G1 / 11D4 (anti-OX40), G2 / MOR7480.1 (anti-CD137), the combination of G1 / 11D4 plus G2 / MOR7480.1, and FS20-22-49AA / 4420 (OX40 / FITC mock mAb 2 ) were used as controls. This assay was performed as described in Example 12.1.

[0412]

Table 15

[0413] Table 15 shows the EC 50 values and maximum responses of IL-2 release observed in the SEA assay in the presence or absence of cross-linking by an artificial cross-linker. Figures 3C and D show plots of IL-2 release for the SEA assay.

[0414]

Table 16

[0415] Figures 3C and D and Table 15 show that no IL-2 production was observed, as expected, with non-cross-linked or cross-linked anti-FITC antibody G1 / 4420 or non-cross-linked anti-OX40 antibody (G1 / 11D4 alone or in combination with G2 / MOR7480.1). When OX40 was activated by anti-OX40 positive control antibody binding, IL-2 was produced by T cells, but only in the presence of an artificial cross-linker (0.13 nM EC 50 for G1 / 11D4 alone and 0.11 nM EC 50 ) when combined with G2 / MOR7480.1). Mock mAb 2The OX40-targeted Fcab of format (4420 LALA) FS20-22-49AA / 4420 showed some agonist activity in this assay in the absence of crosslinking (8.53 nM EC 50 ), but when crosslinked by the binding of the Fab arm to FITC-dextran, it showed increased activity as demonstrated by a decrease in EC 50 (0.31 nM) and an increase in the maximum amount of IL-2 production (maximum response), as shown by the increase in IL-2 production.

[0416] The crosslinked CD137-targeted antibody G2 / MOR7480.1 alone showed no activity, and the activity of the combination of the OX40-targeted antibody G1 / 11D4 and the CD137-targeted antibody G2 / MOR7480.1, when crosslinked, was similar to the activity of the crosslinked OX40-targeted antibody G1 / 11D4 alone.

[0417] In this SEA T cell activation assay, the activities of the five OX40 / CD137 mAb 2 clones (see Table 15) were equivalent regardless of the presence of an artificial crosslinker. In the presence of an artificial crosslinker, the activities of the OX40 / CD137 mAb 2 were also equivalent to those of the crosslinked FS20-22-49AA / 4420 mock mAb 2 . These results of this SEA assay indicate that the OX40 / CD137 mAb 2 can signal through OX40 without the need for an artificial crosslinker as a result of crosslinking provided by the engagement of the anti-CD137 Fab arm of the mAb 2 .

[0418] In this assay, no activity was detected for the crosslinked CD137-targeted antibody G2 / MOR7480.1, but CD137 is expected to be expressed at levels on T cells that allow crosslinking of the mAb 2 . This expression, when bound to CD137, allows each of the five mAb 2 clones to also bind to OX40 and the non-crosslinked FS20-22-49AA / 4420 mock mAb2 It is hypothesized that it was at a level far higher than the low level of activity induced by

[0419] and could drive its activation in the absence of an artificial cross-linking agent. 2 T cell activation observed with OX40 / CD137 mAb also suggests that these molecules can activate T cells in which both OX40 and CD137 are expressed in vivo.

[0420] 12.3 Activity of OX40 / CD137 mAb in SEA-stimulated PBMC from 10 PBMC donors 2 of clone FS20-22-49AA / FS30-10-16 OX40 / CD137 mAb 2 Clone FS20-22-49AA / FS30-10-16 was tested on PBMC from 10 different donors in a SEA assay and accurate EC 20 values, EC 30 values and EC 50 values were established. This assay was performed as described in Example 12.1 in the absence of an artificial cross-linking agent. Mean values plus or minus standard deviation (SD) were calculated from the raw data of each donor. To calculate the EC 50 values, the raw data was fitted to a logistic function (4 parameters: Top, Bottom, Hill slope and EC 50 ).

Number

[0421] The y-axis shows the measured response (IL-2 level) as a function of log 10 (c), where c represents the concentration of the test article.

[0422] Each parameter estimate from the fit has a standard error that indicates the precision of that estimate. Different donors and / or technical replicates of a given experiment give different parameter estimates and different levels of precision (e.g., depending on the quality of the data for each case), so the parameters from each donor and / or technical replicate were included in a weighted average. The weights were defined as the reciprocal of the square of the standard error of the parameter, assuming the normality of the parameter.

[0423] Furthermore, log 10 (EC 20 ) values and log 10 (EC 30 ) values were calculated by fitting the data to a similar equation. [Number]

[0424] All logistic fits were performed using GraphPad Prism, and the weighted average was performed using Microsoft Excel. The calculation formulas used for the weighted average and the standard error of the weighted average are shown below. [Number] Here, the weighted standard deviation is estimated as follows. [Number]

[0425] In the SEA assay, the EC 2 values, EC 20 values, and EC 30 values for the IL-2 release observed for OX40 / CD137 mAb 50 are shown in Table 16.

[0426] [Table 17]

[0427] These results indicate that the OX40 / CD137 mAb 2 has activity comparable to PBMC from different donors.

[0428] Example 13 - Activity of Human OX40 / CD137 mAb in a Pan T-Cell Activation Assay 2 activity The SEA T-cell activation assay described in Example 12 used PBMS and superantigen to stimulate T cells. To evaluate the effects of OX40 and CD137 agonists on isolated T cells, a T-cell activation assay was performed. In this assay, anti-CD3 antibody immobilized on the plastic surface was used to isolate and stimulate T cells. The immobilized anti-CD3 antibody can cluster the TCR of T cells, providing the first signal necessary for T-cell activation, and the test molecule provides the second signal.

[0429] The T-cell stimulation assay was used to establish the activity of different OX40 and CD137 agonist antibodies and OX40 / CD137 mAb 2 antibodies in the presence or absence of a crosslinking agent, and to compare different OX40 / CD137 mAb 2 clones. To compare 9 PBMC donor groups of OX40 / CD137 mAb 2 clone FS20-22-49AA / FS30-10-16, representative EC 50 values were established.

[0430] 13.1 Activity of OX40 and CD137 Agonist Antibodies in a Pan T-Cell Activation Assay To determine the sensitivity of the T-cell activation assay to different OX40 and CD137 agonist antibodies, the mAbs listed in Table 17 2Antibodies (FS20-22-49AA / FS30-10-16) and control antibodies were assayed for their activities. G1 / 4420 (anti-FITC), G1AA / MOR7480.1 (anti-CD137), G1AA / FS30-10-16 (anti-CD137), G1AA / 20H4.9 (anti-CD137), G1AA / 11D4 (anti-OX40), and FS20-22-49AA / 4420 (OX40 / FITC mock mAb 2 ) were used as controls. IL-2 production was used as a measure of T cell activation.

[0431]

Table 18

[0432] As described in Example 12.1, human PBMCs were isolated. Next, T cells were isolated from PBMCs using the Pan T Cell Isolation Kit II (Miltenyi Biotec Ltd) according to the manufacturer's instructions.

[0433] Human T-Activator CD3 / CD28 Dynabeads (Life Technologies, 11452D) were resuspended by vortexing. The beads were washed twice with T cell medium (RPMI medium (Life Technologies)) containing 10% FBS (Life Technologies), 1× penicillin streptomycin (Life Technologies), sodium pyruvate (Gibco), 10 mM HEPES (Gibco), 2 mM L-glutamine (Gibco), and 50 μM 2-mercaptoethanol (Gibco).

[0434] The required number of T cells at a concentration of 1.0×10 6 cells / ml were stimulated with human T activator CD3 / CD28 dynabeads washed at a cell-to-bead ratio of 2:1 in a T-25 flask (Sigma) and incubated overnight at 37°C, 5% CO2 to activate the T cells. The activated T cells were washed from the dynabeads and 2.0×106 They were resuspended in the medium of T cells at a concentration of cells / ml. A 96-well flat-bottom plate was coated with anti-human CD3 antibody by incubating it with 2.5 μg / ml anti-human CD3 antibody (R&D Systems clone UHCT1) diluted with PBS at 37 °C and 5% CO2 for 2 hours, and then washed twice with PBS. Activated T cells were added to the plate at 2×10 5 cells / well.

[0435] A 2 μM dilution of each test antibody (see Table 17 for details) was prepared and, if necessary, added to the wells at a 1:1 molar ratio with a cross-linking agent (anti-human CH2 antibody (clone MK1A6, in-house production) or FITC-dextran (Sigma) (see Table 17)). Serial dilutions of the test antibodies were prepared in a 96-well plate, and 100 μl of the diluted antibody mixture was added to the activated T cells on the plate.

[0436] The T cells were incubated at 37 °C and 5% CO2 for 72 hours. Next, the supernatant was collected, IL-2 release was measured, and the data were prepared as described in Example 12.1. Table 18 shows the EC 50 values and maximum responses of IL-2 release observed in the T cell activation assay. Figure 4A shows the levels of IL-2 release induced by antibodies tested at a single concentration (3.7 nM) in the T cell activation assay. The concentration at which these antibodies induced the highest level of IL-2 production was selected for this analysis. Statistical analysis was performed by two-way ANOVA and Tukey's multiple comparison test. Asterisks on the error bars represent significant differences compared to samples treated with the isotype control (G1 / 4420) (*p < 0.032, **p < 0.0021, ***p < 0.0002, ****p < 0.0001). Figure 4B shows the plot of IL-2 release induced by OX40 / CD137 mAb 2 (FS20-22-49AA / FS30-10-16) in the presence or absence of a cross-linking agent in the T cell activation assay.

[0437]

Table 19

[0438] This result shows that only OX40 / CD137 mAb 2 (FS20-22-49AA / FS30-10-16) can increase the IL-2 level in the absence of an artificial cross-linking agent, and the addition of the artificial cross-linking agent did not increase the activity of OX40 / CD137 mAb 50 from either the EC 2 or maximum response perspective. The activities of the OX40-targeting antibodies G1AA / 11D4 and FS20-22-49AA / 4420 and the anti-CD137 antibody G1AA / 20H4.9 were observed only in the presence of an artificial cross-linking agent. For the anti-CD137 antibodies G1AA / MOR7480.1 and G1AA / FS30-10-16, no activity was detected even in the presence of an artificial cross-linking agent. The OX40 agonist antibody FS20-22-49AA / 4420 induced a higher IL-2 level than all three CD137 agonist antibodies. The anti-OX40 antibody G1AA / 11D4 induced a higher IL-2 level than the anti-CD137 antibodies G1AA / MOR7480.1 and G1AA / FS30-10-16 and an IL-2 level equivalent to that of the anti-CD137 antibody G1AA / 20H4.9, but the G1AA / 11D4 antibody was observed to have a higher ability than the G1AA / 20H4.9 antibody, as indicated by its lower EC 50 value. These results indicate that this T cell activation assay is more sensitive to OX40 agonist action than to CD137 agonist action. As speculated in Example 12.1, this may be related to the fact that OX40 is preferentially expressed on CD4+ T cells and CD137 is preferentially expressed on CD8+ T cells (Croft, 2014 and internal data shown in Figure 6), which is due to the fact that in human PBMCs, CD4+ T cells are generally more abundant than CD8+ T cells.

[0439] 13.2 Cytokine Analysis of the Activity of OX40 and CD137 Agonist Antibodies in a Pan-T Cell Activation Assay To better understand the effect of OX40 and CD137 stimulation on the T cell activation assay, multiple cytokine levels were analyzed. The antibodies and mAbs listed in Table 19 2 The antibody (FS20-22-49AA / FS30-10-16) and control antibodies were used. Control antibodies G1 / 4420 (anti-FITC), G1AA / FS30-10-16 (anti-CD137), and FS20-22-49AA / 4420 (OX40 / FITC mock mAb 2 ) were tested in the presence of an artificial cross-linking agent, and the OX40 / CD137 mAb 2 was tested in the absence of an artificial cross-linking agent. All antibodies were used at a single concentration (10 nM). This assay was performed as described in Example 13.1.

[0440]

Table 20

[0441] The cytokines IL-2, IL-6, IL12p70, IL-13, TNFα, IFNγ, and IL-10 in the supernatant recovered after incubation were then measured using the Pro-inflammatory V-plex kit (MSD, K15049D-1) according to the manufacturer's instructions. The results showed that the OX40 / CD137 mAb 2 (FS20-22-49AA / FS30-10-16) and the cross-linked OX40-targeting antibody (FS20-22-49AA / 4420) increased the release of IL-2, IL-6, IL-12p70, IL-13, and TNFα cytokines by T cells and decreased the release of IL-10. No activity was detected for the anti-CD137 antibody (G1AA / FS30-10-16).

[0442] 13.3 Activity of Different OX40 / CD137 mAb 2 Clones in a Pan-T Cell Activation Assay The details of the molecules tested in this assay and their respective crosslinkers are shown, where applicable, in Table 20 below. G1 / 4420 (anti-FITC), G1 / 11D4 (anti-OX40), G2 / MOR7480.1 (anti-CD137), the combination of G1 / 11D4 plus G2 / MOR7480.1, and FS20-22-49AA / 4420 (OX40 / FITC mock mAb 2 ) were used as controls. All molecules were tested in the absence of an artificial crosslinker. The single-agent controls G1 / 4420, G1 / 11D4, G2 / MOR7480.1, and FS20-22-49AA / 4420 were further tested in the presence of an artificial crosslinker. This assay was performed as described in Example 13.1.

[0443]

Table 21

[0444] Table 21 shows the EC 50 values and maximum responses of IL-2 release observed for all molecules tested in the T cell activation assay in the absence of crosslinking. Table 22 shows the EC 50 values and maximum responses of IL-2 release observed for the single-agent controls G1 / 4420, G1 / 11D4, G2 / MOR7480.1, and FS20-22-49AA / 4420 that were additionally tested in the presence of a crosslinker. Figures 4C and D show plots of IL-2 release for the T cell activation assay.

[0445]

Table 22

[0446]

Table 23

[0447] Table 21 and Figure 4C show that uncrosslinked OX40 / CD137 mAb 2 is active (EC 50 in the range of 0.2019 nM to 1.201 nM)) and thus can bind to both targets, resulting in clustering on one or both of them, indicating that T cell activation was induced. As expected, no IL-2 production was observed with non-crosslinked or crosslinked anti-FITC antibody G1 / 4420 or non-crosslinked anti-OX40 antibody (G1 / 11D4 alone or in combination with G2 / MOR7480.1). In the presence of a crosslinking agent, when the OX40 receptor was targeted with an anti-OX40 positive control antibody, IL-2 was produced by T cells (EC 50 of 0.05 nM with G1 / 11D4 alone and EC 50 ) when combined with G2 / MOR7480.1.

[0448] Mock mAb 2 The OX40-targeted Fcab in format / 4420 (4420 LALA) FS20-22-49AA / 4420 had some agonist activity in the absence of crosslinking (EC 50 of 5.02 nM and a maximum response of 1508 pg / ml hIL-2) as seen in the SEA assay, and this activity was further enhanced when the mock mAb 2 was crosslinked by binding FITC-dextran to its Fab arms.

[0449] No activity was observed with non-crosslinked anti-CD137 antibody G2 / MOR7480.1 alone, but when crosslinked, it could induce T cell activation. Unlike the SEA T cell activation assay (Example 12), this assay can measure CD137 signaling by this anti-CD137 clone, similar to the OX40 signaling confirmed above. The difference in activity observed for this crosslinked antibody compared to the same anti-CD137 clone (G1AA / MOR7480.1) in the IgG1 format of Example 13.1, where no activity was detected either in the absence or presence of an artificial crosslinking agent, can be explained by the variability of T cell donors, where some donors may respond better to CD137 stimulation than others.

[0450] In a human CD137 T cell activation assay using the DO11.10-hCD137 cells described in Example 7.1, test OX40 / CD137 mAbs 2 (FS20-22-49AA / FS30-5-37, FS20-22-49AA / FS30-10-3, FS20-22-49AA / FS30-10-12, FS20-22-49AA / FS30-10-16, and FS20-22-49AA / FS30-35-14) and the G2 / MOR7480.1 control strongly induced IL-2 production. Thus, the anti-CD137 Fab arm of the OX40 / CD137 mAb 2 is assumed to be able to agonize T cell-expressed CD137 in order to generate a detectable IL-2 signal in the primary T cell activation assay of this example.

[0451] 13.4 In a pan-T cell activation assay with T cells from 9 PBMC donors, OX40 / CD137 mAb 2 activity of clone FS20-22-49AA / FS30-10-16 OX40 / CD137 mAb 2 Clone FS20-22-49AA / FS30-10-16 was tested with PBMC from 9 different donors in a T cell activation assay, and accurate EC 20 values, EC 30 values, and EC 50 values were established. This assay was performed as described in Example 13.1 in the absence of an artificial cross-linking agent.

[0452] For each donor, the mean plus or minus the standard deviation (SD) was calculated from the raw data as described in Example 12.3. In the T cell assay, the EC 2 values, EC 20 values, and EC 30 values for the IL-2 release observed for OX40 / CD137 mAb 50 (FS20-22-49AA / FS30-10-16) were also calculated as described in Example 12.3 and are shown in Table 23.

[0453]

Table 24

[0454] These results indicate that OX40 / CD137 mAb 2 has comparable activity on T cells from different donors.

[0455] Example 14 - Activity of human OX40 / CD137 mAb in CD4+ and CD8+ T cell activation assays 2 activity T cells can be differentiated into CD4+ and CD8+ T cells according to their functions in the immune system. CD4+ T cells are called T helper cells and produce cytokines that regulate the immune response, while CD8+ T cells are called T killer cells and directly eliminate target cells. The expression of OX40 has been observed to be higher than that of CD137 on CD4+ T cells, and conversely, the expression of CD137 has been observed to be higher than that of OX40 on CD8+ T cells (see Croft, 2014 and Figure 6). Despite this difference in expression levels, both CD4+ and CD8+ T cells co-express the two receptors (Ma et al., 2005).

[0456] To further investigate the activity of OX40 / CD137 mAb in these two T cell populations, CD4+ and CD8+ T cells were separated and the ability of the molecules listed in Table 24 below was tested to activate each T cell population in separate CD4+ and CD8+ T cell activation assays. In this assay, the co-expression of OX40 and CD137 was utilized to measure the cross-linking of OX40 / CD137 mAb 2 FS20-22-49AA / FS30-10-16. G1 / 4420 (anti-FITC), G1AA / 11D4 (anti-OX40), G1AA / MOR7480.1 (anti-CD137) G1AA / FS30-10-16 (anti-CD137), FS20-22-49AA / 4420 (OX40 / FITC mock mAb 2 2 ​)And the combination of FS20-22-49AA / 4420 plus G1AA / FS30-10-16 was used as a control. IL-2 production was used as a measure of T cell activation.

[0457]

Table 25

[0458] To isolate human CD4+ and CD8+ T cells, PBMCs were first isolated as described in Example 13.1. Next, CD4+ and CD8+ T cells were separately isolated from PBMCs using a CD4+ T cell isolation kit (human) (Miltenyi Biotec, 130-096-533) and a CD8+ T cell isolation kit (human) (Miltenyi Biotec, 130-096-495), respectively, according to the manufacturer's instructions.

[0459] As described in Example 13.1, CD4+ or CD8+ T cells were activated overnight with human T activator CD3 / CD28 Dynabeads at a concentration of 1.0×10 6 cells / ml in the required amount in T cell medium.

[0460] The activated CD4+ or CD8+ T cells were washed from the Dynabeads and resuspended in T cell medium at a concentration of 2.0×10 6 cells / ml. A 96-well flat-bottom plate was coated with an anti-human CD3 antibody (R&D Systems, clone UHCT1) at either 2.5 μg / ml (for CD4+ T cell activation assay) or 10 μg / ml (for CD8+ T cell activation assay) diluted in PBS by incubating at 37°C, 5% CO2 for 2 hours, and then washed twice with PBS. Next, the activated CD4+ or CD8+ T cells were added to each plate at 2×10 5 cells / well.

[0461] Prepare a 2 μM dilution of each test antibody (see Table 24 for details), and add it to the wells at a 1:1 molar ratio with a crosslinking agent (anti-human CH2 antibody or FITC-dextran (Sigma) (see Table 24)) as described in Example 6, if necessary. Prepare serial dilutions of the test antibodies in a 96-well plate and add 100 μl of the diluted antibody mixture to the activated CD4+ or CD8+ T cells on each plate.

[0462] Incubate the T cells at 37 °C and 5% CO2 for 72 hours. Collect the supernatant and prepare the IL-2 release and data measured as described in Example 12.1. Table 25 shows the EC 50 values and maximum responses of IL-2 release observed in separate T cell activation assays in the presence or absence of crosslinking by the crosslinking agent. Figures 5A to C show plots of IL-2 release for the CD4+ or CD8+ T cell activation assays, respectively.

[0463] After collecting the supernatant, wash the T cells with PBS and stain them with Alexa Fluor 488-labeled anti-human Fc secondary antibody (Jackson Immunoresearch, catalog number 109-546-098) diluted 1:1000 in PBS at 4 °C for 1 hour. Next, wash the cells once with PBS and resuspend them in 100 μl / well of PBS containing DAPI (Biotium, catalog number 89139-054) at 1 μg / ml. Then, analyze the cells on a BD FACS CantoII flow cytometer (BD Biosciences). Figure 6 shows the geometric mean fluorescence intensity in the 488 channel of either CD4+ or CD8+ T cells treated with G1AA / MOR7480.1 or G1AA / 11D4.

[0464]

Table 26

[0465] Table 25 and FIG. 5B show that CD4+ T cells can be activated by cross-linked anti-OX40 controls G1AA / 11D4 and FS20-22-49AA / 4420 (both alone and in combination with G1AA / FS30-10-16), but not by the single-agent anti-CD137 controls G1AA / MOR7480.1 and G1AA / FS30-10-16. On the other hand, FIG. 5C shows that CD8+ T cells were activated by both the cross-linked anti-CD137 controls G1AA / MOR7480.1 and G1AA / FS30-10-16 when cross-linked, similar to the cross-linked anti-OX40 controls G1AA / 11D4 and FS20-22-49AA / 4420, but the level of response to the single-agent anti-CD137 control G1AA / FS30-10-16 was greater than that of the two single-agent anti-OX40 controls. As observed in the SEA assay (Example 12.2) and the human pan T cell activation assay (Example 13.3), the mock mAb 2 The OX40 Fcab in the form (FS20-22-49AA / 4420) showed some activity in the absence of cross-linking and in the presence of CD4+ T cells, and this activity increased when the antibody was cross-linked. The OX40 / CD137 mAb 2 (FS20-22-49AA / FS30-10-16) showed activity in the absence of cross-linking and in the presence of both CD4+ and CD8+ T cells, as expected from previous results (see Examples 12 and 13).

[0466] FIG. 6 shows that CD4+ T cells express lower levels of CD137 and higher levels of OX40 than CD8+ T cells. The binding of G1AA / MOR7480.1 to CD137 is a measure of CD137 expression, and the binding of G1AA / 11D4 to OX40 is a measure of OX40 expression.

[0467] This T cell assay with isolated CD4+ and CD8+ T cells was repeated according to the same protocol as above, using T cells isolated from different PBMC donors and adding anti-CD137 antibody G1AA / 20H4.9 (see Table 24). Consistent with the results shown in FIGS. 5A-D, FIGS. 5E and 5F show that CD8+ T cells respond to CD137 agonist action and CD4+ T cells respond to OX40 agonist action. When activated CD4+ T cells were cross-linked but CD8+ T cells were not, anti-OX40 antibodies (G1AA / 11D4 and mock mAb 2 anti-OX40 Fcab in the form FS20-22-49AA / 4420) and when activated CD8+ T cells were cross-linked but CD4+ T cells were not, CD137 antibodies (G1AA / 20H4.9 and G1AA / FS30-10-16). Similar to the results obtained in the DO11.10-hCD137 cell assay described in Example 7.1, the G1AA / 20H4.9 antibody also activated CD8+ T cells in the absence of a cross-linking antibody. In this repeated experiment, the G1AA / MOR7480.1 antibody did not activate CD8+ T cells when cross-linked. Some PBMC donors are more susceptible to the effects of CD137 co-stimulation than other donors, and the different results obtained in this experiment may be the result of this natural variation.

[0468] These data indicate that CD4+ T cells are more sensitive to activation via OX40 agonist action than CD8+ T cells, and conversely, CD8+ T cells are more sensitive to activation via CD137 agonist action than CD4+ T cells. This correlates with the reported differences in the expression levels of OX40 and CD137 receptors in CD4+ and CD8+ T cells, with the former expressing higher levels of OX40 than CD137 and the latter expressing higher levels of CD137 than OX40. The activity of the cross-linked anti-CD137 control antibody G1AA / FS30-10-16 in the presence of CD8+ T cells is such that its Fab arm is present in OX40 / CD137 mAb 2 FS20-22-49AA / FS30-10-16 and mAb 2demonstrates the ability to activate the CD137 receptor when its Fcab is crosslinked by binding to OX40. Furthermore, the mock mAb 2 activity of crosslinked anti-OX40 Fcab in the presence of CD4+ T cells in the format (FS20-22-49AA / 4420) is also present in OX40 / CD137 m...

Claims

1. An antibody molecule that binds to CD137 and OX40, comprising: (a) a complementarity-determining region (CDR)-based antigen-binding site for CD137; and (b) an OX40 antigen-binding site located in the CH3 domain of said antibody molecule , wherein said CDR-based antigen-binding site (i) comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 defined according to the ImMunoGeneTics (IMGT) numbering scheme as set forth in SEQ ID NOs: 1, 2, 3, 4, 5, and 6, respectively; (ii) comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 defined according to the ImMunoGeneTics (IMGT) numbering scheme as set forth in SEQ ID NOs: 1, 2, 16, 4, 5, and 6, respectively; (iii) comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 defined according to the ImMunoGeneTics (IMGT) numbering scheme as set forth in SEQ ID NOs: 1, 2, 21, 4, 5, and 6, respectively; (iv) comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 defined according to the ImMunoGeneTics (IMGT) numbering scheme as set forth in SEQ ID NOs: 25, 26, 27, 4, 5, and 28, respectively; or (v) comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 defined according to the ImMunoGeneTics (IMGT) numbering scheme as set forth in SEQ ID NOs: 33, 34, 35, 4, 5, and 36, respectively , or wherein said CDR-based antigen-binding site (vi) comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 defined according to the Kabat numbering scheme as set forth in SEQ ID NOs: 7, 8, 9, 10, 11, and 6, respectively; (vii) comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 defined according to the Kabat numbering scheme as set forth in SEQ ID NOs: 7, 8, 17, 10, 11, and 6, respectively; (viii) comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 defined according to the Kabat numbering scheme as set forth in SEQ ID NOs: 7, 8, 22, 10, 11, and 6, respectively; (ix) comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 defined according to the Kabat numbering scheme as set forth in SEQ ID NOs: 29, 30, 31, 10, 11, and 28, respectively; or (x) comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 defined according to the Kabat numbering scheme as set forth in SEQ ID NOs: 37, 38, 39, 10, 11, and 36, respectively , and wherein said OX40 antigen-binding site comprises a first sequence, a second sequence, and a third sequence located in the AB, CD, and EF structural loops of said CH3 domain, respectively, and said first, second, and third sequences are (i) SEQ ID NOs: 51, 59, and 60, respectively; (ii) SEQ ID NOs: 51, 52, and 60, respectively; (iii) SEQ ID NOs: 51, 52, and 65, respectively; (iv) SEQ ID NOs: 51, 52, and 68, respectively; (v) SEQ ID NOs: 71, 72, and 73, respectively; (vi) SEQ ID NOs: 71, 72, and 76, respectively; (vii) SEQ ID NOs: 79, 80, and 81, respectively; (viii) SEQ ID NOs: 84, 85, and 76, respectively; (ix) SEQ ID NOs: 84, 88, and 89, respectively; or (x) SEQ ID NOs: 84, 92, and 89, respectively of said first, second, and third sequences, an antibody molecule.

2. (i) said first sequence is located at positions 14 to 18 of said CH3 domain of said antibody molecule; (ii) the second array is located at positions 45.1 to 77 of the CH3 domain of the antibody molecule; and / or (iii) the third array is located at positions 93 to 101 of the CH3 domain of the antibody molecule; The antibody molecule according to claim 1, wherein the numbering of amino acid residues follows the IMGT numbering scheme.

3. The antibody molecule according to claim 1 or 2, comprising a CH3 domain sequence set forth in SEQ ID NO: 61, 63, 66, 69, 74, 77, 82, 86, 90 or 93.

4. The antibody molecule according to claim 3, wherein the CH3 domain contains an additional lysine residue (K) immediately adjacent to the C-terminus of the CH3 domain sequence.

5. (i) SEQ ID NOs: 12 and 14 respectively; (ii) SEQ ID NOs: 18 and 14 respectively; (iii) SEQ ID NOs: 23 and 14 respectively; (iv) SEQ ID NOs: 170 and 172 respectively; or (v) SEQ ID NOs: 40 and 42 respectively The antibody molecule according to any one of claims 1 to 4, comprising a VH domain and a VL domain as set forth therein.

6. (i) VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 defined according to the IMGT numbering scheme, set forth in SEQ ID NOs: 1, 2, 3, 4, 5 and 6 respectively; (ii) VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 defined according to the Kabat numbering scheme, set forth in SEQ ID NOs: 7, 8, 9, 10, 11 and 6 respectively; and / or (iii) a VH domain and a VL domain as set forth in SEQ ID NOs: 12 and 14 respectively The antibody molecule according to any one of claims 1 to 5, comprising the same.

7. The antibody molecule according to any one of claims 1 to 6, which binds to human CD137 and human OX40.

8. The antibody molecule according to claim 7, which is capable of binding to human CD137 and human OX40 simultaneously.

9. (i) capable of activating the OX40 on immune cells in the presence of CD137 expressed on the cell surface, and / or capable of activating CD137 on immune cells in the presence of OX40 expressed on the cell surface; (ii) binding of the antibody molecule to OX40 and CD137 on the immune cell causes clustering of OX40 on the immune cell, and / or binding of the antibody molecule to CD137 and OX40 on the immune cell causes clustering of CD137 on the immune cell; and / or (iii) modified to reduce or inhibit binding of the CH2 domain of the antibody molecule to one or more Fcγ receptors, The antibody molecule according to any one of claims 1 to 8.

10. One or more nucleic acid molecules encoding the antibody molecule according to any one of claims 1 to 9.

11. One or more vectors comprising one or more nucleic acid molecules according to claim 10.

12. A recombinant host cell comprising one or more nucleic acid molecules according to claim 10 or one or more vectors according to claim 11.

13. A method for producing the antibody molecule according to any one of claims 1 to 9, comprising culturing the recombinant host cell according to claim 12 under conditions for production of the antibody molecule.

14. The method according to claim 13, further comprising isolating and / or purifying the antibody molecule.

15. A pharmaceutical composition comprising the antibody molecule according to any one of claims 1 to 9 and a pharmaceutically acceptable excipient.

16. A pharmaceutical composition for the treatment of cancer or an infectious disease in an individual, comprising the antibody molecule according to any one of claims 1 to 9.

17. The pharmaceutical composition for the treatment of cancer according to claim 16, further comprising a second therapeutic agent.

18. The pharmaceutical composition according to claim 17, wherein the second therapeutic agent is an antibody that binds to PD-1 or PD-L1.

19. The pharmaceutical composition according to claim 18, wherein the second therapeutic agent is pembrolizumab or nivolumab.

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