NOVEL DOSAGES OF ANTI-CD137 ANTIBODY

Field of the Invention

The present invention relates to antibody-based polypeptides with binding specificity for CD137, which have utility in the treatment of diseases such as cancer at particularly advantageous dosages.

Background to the Invention

Cancer is a leading cause of premature deaths in the developed world. The aim of immunotherapy in cancer is to mount an effective immune response by the body against a tumour, particularly a solid tumour. This may be achieved by, for example, breaking tolerance against tumour antigen, augmenting anti-tumor immune responses, and stimulating local cytokine responses at the tumor site. The key effector cell of a long-lasting anti-tumor immune response is the activated tumor specific effector T cell. Potent expansion of activated effector T cells can redirect the immune response towards the tumour. In this context, regulatory T cells (Treg) play a role in inhibiting the anti-tumour immunity. Depleting, inhibiting, reverting or inactivating Tregs may therefore provide anti-tumour effects and revert the immune suppression in the tumour microenvironment. Further, incomplete activation of effector T cells by, for example, dendritic cells can cause T-cell anergy, which results in an inefficient anti- tumor response, whereas adequate induction by dendritic cells can generate a potent expansion of activated effector T cells, redirecting the immune response towards the tumor. In addition, Natural killer (NK) cells play an important role in tumour immunology by attacking tumour cells with down-regulated human leukocyte antigen (NLA) expression and by inducing antibody dependent cellular cytotoxicity (ADCC). Stimulation of NK cells may thus also reduce tumour growth.

CD137 (4-1BB, TNFRSF9) is a TNF receptor (TNFR) superfamily member and is expressed on activated CD4 ⁺ and CD8 ⁺ T cells, Treg, DC, monocytes, mast cells and eosinophils. CD137 activation plays an important role in CD8+ T cell activation and survival (Lee et al., 2002; Pulle et al., 2006). It sustains and augments, rather than initiates, effector functions and preferentially supports Th1 cytokine production (Shuford et al., 1997). In CD4 ⁺ T cells, CD137 stimulation initially results in activation and later in activation-induced cell death, explaining why CD137 agonistic antibodies have shown therapeutic effect in tumour immunity as well as in autoimmunity (Zhang, JCI, 2007, Sun, Trends Mol Med, 2003). CD137 also suppresses Treg function (So, Cytokine Growth Factor Rev, 2008). Activation of CD137 is dependent on receptor oligomerization (Rabu et al., 2005; Wyzgol et al., 2009).

CD137 agonistic antibody has been shown to activate endothelial cells in the tumour environment, leading to upregulation of ICAM-1 and VCAM-1 and improved T cell recruitment (Palazon, Cancer Res, 2011).

CD137 is upregulated on NK cells activated by cytokines or CD16, in mice or humans, respectively (see Melero, CCR 19 (5)1044-53, 2013 and references cited therein). CD137 has been shown to activate NK cells in mice as well as humans, potentiating ADCC (Kohrt et al., 2014), though there are reports suggesting opposite effects on NK cells in mice and humans, leading to NK cell activation in mice and inhibition in humans (Baessler, Blood, 2010).

Several studies have demonstrated induction of tumour immunity by treatment with agonistic CD137 antibody (Dubrot et al., 2010; Gauttier et al. , 2014; Kim et al., 2001; McMillin et al. , 2006; Melero et al. , 1997; Miller et al. , 2002; Sallin et al. , 2014; Taraban et al., 2002; Uno et al., 2006; Vinay and Kwon, 2012; Wilcox et al., 2002). In addition, it synergizes with several immunomodulators, including CpG, TRAIL, CD40, OX-40, DR5, PD-1/PD-L1, CTLA-4 Tim-3, IL-2, IL-12(Curran et al., 2011; Gray et al., 2008; Guo et al., 2013; Kwong et al., 2013; Lee et al., 2004; Morales-Kastresana et al., 2013; Pan et al., 2002; St Rose et al., 2013; Uno et al., 2006; Wei et al., 2013; Westwood et al., 2010; Westwood et al., 2014a; Westwood et al., 2014b) in pre-clinical models.

Two CD137 antibodies are in clinical development. Urelumab (BMS-66513) is a fully human IgG4 antibody developed by Bristol-Myers Squibb. Urelumab is a strong 4-1BB agonist that has demonstrated limited clinical efficacy (Chester et al. 2017; Chin et al. 2018). Development of urelumab was however hampered by hepatotoxicity at doses ≥0.3 mg/kg (including 2 fatal events at doses ≥lmg/kg) (Segal et al. 2017). The maximum tolerated dose was therefore set to 0.1 mg/kg (or a flat dose of 8 mg). In the subsequent studies, no clear objective responses was observed for urelumab as monotherapy (Chester et al. 2017). The mechanism behind the hepatotoxicity is not fully understood. Utomilumab, on the other hand, is regarded as a weaker agonist than urelumab and has also shown limited clinical efficacy (Chin et al. 2018; Segal et al. 2018; Tolcher et al. 2017). Utomilumab showed a tolerable clinical safety profile up to 10 mg/kg with no dose limiting toxicity (DLT). Utomilumab, but not urelumab, is dependent on FcR-crosslinking to execute its agonistic effect. As FcγRs in the blood are saturated by endogenous circulating human

IgG, at approximately 10 g/L, FcγR-crosslinking dependent antibodies such as utomilumab need to compete with IgG to bind to FcγRs (Jolliff 1982). Endogenous IgG of 10 g/L is more than 60-fold higher than the maximum serum concentration (Cmax) reached with the highest clinical dose of utomilumab (155 μg/mL at 10 mg/kg) (Segal et al. 2018). The liver is a highly vascularized organ, and endogenous IgG concentrations in the liver have been shown to be similar to circulating levels (Eigenmann et al. 2017). Therefore, it can be expected that FcγR- crosslinking dependent 4-1BB activation is also reduced in the liver, due to competition with endogenous IgG. This reduced possibility for FcγR-crosslinking of utomilumab in the liver may explain the absence of liver toxicity with utomilumab that was detected with urelumab.

Nine additional monospecific 4-1BB antibodies: ADG106, administered at doses of 0.03 to 10 mg/kg, currently in phase I/II (Liu et al. 2017), and CTX-471, AGEN2373, LVGN6051 ATOR-1017, EU101 (IND/CTA), PE0116, STA551 and HOT1030 have entered clinical development during 2018-2021 and are currently being evaluated for safety in phase I studies.

The agonistic effect of CD137 antibodies is affected by the isotype of the Fc region. The antibodies tested in the clinic are either IgG2 or IgG4. Like most TNFR family members, CD137 depends on cross linking for activation (Wilson 2011, Cancer Cell). The CD137L expressed on the membrane of an ARC may induce significant multiple cross linking of the receptor. An antibody can by itself only cross link two CD137 receptors, and to induce a strong signal, further cross linking via FcγRs expressed on other cells (in trans) may be necessary for induction of a strong CD137 mediated signal. An exception to this may be IgG2 antibodies, which induce a cross linking independent signaling by an unknown mechanism (White et al, 2015 Cancer Cell). T cells do not express FcγRs, and the FcγR mediated cross linking in vivo is thought to be mediated by monocytes, macrophages, DCs and potentially B cells and other cell types.

Another factor to take into account is that engagement of FcγR receptors may also induce ADCC, antibody-dependent cellular phagocytosis (ADCP) and complement- dependent cytotoxicity (CDC) on cells coated with antibodies (for simplicity ADCC below includes ADCP and CDC). Typically, human IgG1 is a strong inducer of NK/Macrophage dependent ADCC, depending on the nature of the target, the cell type and the receptor density. IgG4 antibodies may also induce ADCC but to a lower extent than IgG1 (Wang 2015, Front Imm; Vidarson 2014 Front Imm).

The effect of a CD137 agonistic antibody with different isotypes may thus be affected by the balance between 1) inducing cross linking, which results in a stronger immune activation, and 2) inducing ADCC, which may lead to killing of both effector T cells (predominantly CD8 T cells) and Tregs. The net effect of 1) and 2) will likely depend on the distribution of CD137 expressing cells, the possibility of the target cells to engage with FcγR expressing immune cells, the receptor density and affinity and the sensitivity of Teff vs Treg to ADCC. The CD137 expression is high both on CD8 and Tregs in melanoma tumours (Quezada, presentation SITC 2015). The IgG4 format would allow for FcγRI mediated cross linking by macrophages and monocytes, yet minimizing NK mediated ADCC of effector CD8 T cells.

However, as outlined above, it is difficult to translate comparison of different human Fc in mouse models due to differences in expression and affinity between murine and human FcRs. Further, the functional consequence in vivo of antibodies blocking the binding of the CD137L to CD137 is currently debated, but it may be speculated that CD137 agonists that blocks the CD137L, and thus not allow for simultaneous activation via C137L and the CD137 agonistic antibody, have a reduced risk of inducing exaggerated activation and systemic toxicity.

Several studies have demonstrated induction of tumour immunity by treatment with agonistic CD137 mAb (Dubrot et al., 2010; Gauttier et al., 2014; Kim et al., 2001; McMillin et al., 2006; Melero et al., 1997; Miller et al., 2002; Sallin et al., 2014; Taraban et al., 2002; Uno et al., 2006; Vinay and Kwon, 2012; Wilcox et al., 2002). Two different antibodies are commonly used for in vivo studies in mice, Lobl2.3 and 3H3 (Shuford 1997 J Exp Med).

The toxicity seen in mouse models has been detected following repeated dosing in a time dependent but not dose dependent manner (Ascierto 2010 Semin One, Dubrot 2010 Can Imm, Niu 2007 JI). The toxicity includes skin toxicity and liver toxicity: aspartate amino transferase/alanine amino transferase ratio (ASAT/ALAT) and cytokine release. This suggests that either the toxicity requires CD137 mediated pre- activation of immune cell populations (likely T cells) or it depends on secondary effects caused by antidrug-antibodies (ADA) response, potentially forming aggregations of CD137 antibodies that may lead to enhanced cross-linking. The toxicities seen in mice are reversible and seems to depend on TNFa/CD8 cell dependent manner (Ascierto 2010 Sem One). Toxicology studies in monkeys showed that both single and repeated dosing of up to 100mg/kg once weekly for four weeks was tolerable with no skin or liver toxicity detected (Ascierto 2010, Semin One).

Prolonged and continuous activation through TNF receptor family members may lead to immune exhaustion. Therefore, it may be of advantage to administer such antibodies in a manner allowing resting periods for the cells expressing the receptors. One approach to increase the resting period in a specific dosing protocol is to reduce the half-life of an antibody by for example decreasing the binding to the neonatal Fc receptor (FcRn). This could, depending on the administration route, also reduce the toxicity associated with the treatment.

Summary of Invention

There remains a need for improved anti-tumour therapies, particularly anti-CD137 antibodies suitable for clinical use and with improved properties, such as reduced toxicity.

The inventors have surprisingly found that an antibody or antigen-binding fragment thereof that specifically binds to CD137 when administered to a cancer patient at a high dosage of about 500 mg or more per administration has an impressive safety profile, and a good efficacy.

Accordingly, a first aspect of the invention provides an antibody or antigen-binding fragment thereof that specifically binds to CD137, for use in the treatment of cancer in a patient; wherein a dosage of about 500 mg or more of the antibody or antigen-binding fragment thereof is administered to the patient per administration.

A second aspect of the invention provides a use of an antibody or antigen-binding fragment that specifically binds to CD137, in the manufacture of a medicament for the treatment of cancer in a patient; wherein a dosage of about 500 mg or more of the antibody or antigen-binding fragment thereof is administered to the patient per administration.

A third aspect of the invention provides a method for treating a patient with cancer, the method comprising the step of administering to the patient in need thereof an antibody or antigen-binding fragment thereof that specifically binds to CD137; wherein a dosage of about 500 mg or more of the antibody or antigen-binding fragment thereof is administered to the patient per administration.

A fourth aspect of the invention provides a kit for treating cancer, as described in the first to third aspects of the invention, wherein the kit comprises an antibody or antigen- binding fragment thereof that specifically binds to CD137, as described herein; and wherein the kit comprises an antibody or antigen-binding fragment thereof at a dosage of about 500 mg or more, to be administered to the patient per administration.

Detailed description of the invention

As outlined above, a first aspect of the invention provides an antibody or antigen- binding fragment thereof that specifically binds to CD137, for use in the treatment of cancer in a patient; wherein a dosage of about 500 mg or more of the antibody or antigen-binding fragment thereof is administered to the patient per administration.

A second aspect of the invention provides a use of an antibody or antigen-binding fragment that specifically binds to CD137, in the manufacture of a medicament for the treatment of cancer in a patient; wherein a dosage of about 500 mg or more of the antibody or antigen-binding fragment thereof is administered to the patient per administration.

A third aspect of the invention provides a method for treating a patient with cancer, the method comprising the step of administering to the patient in need thereof an antibody or antigen-binding fragment thereof that specifically binds to CD137; wherein a dosage of about 500 mg or more of the antibody or antigen-binding fragment thereof is administered to the patient per administration.

The embodiments described herein relate to, and applicable to, each of these first to third aspects of the invention, as well as the kit of the fourth aspect of the invention described below.

In one embodiment, the dosage is about 500 mg to about 1500 mg. In a preferred embodiment, the dosage is about 600 mg to about 900 mg.

In a particularly preferred embodiment, the dosage is about 600 mg. In an alternative particularly preferred embodiment, the dosage is about 900 mg. In one embodiment, the antibody or antigen-binding fragment thereof is administered intravenously.

In one embodiment, the dosage of the antibody or antigen-binding fragment thereof is administered about every 21 days.

In one embodiment, the cancer is a tumour, preferably a solid tumour.

In one embodiment, the cancer is a relapsed cancer and/or a refractory cancer.

In a preferred embodiment, the cancer is a cancer selected from the list consisting of: a gynaecological cancer; a cancer of the digestive system; melanoma; and breast cancer.

In a particularly preferred embodiment, the cancer is a gynaecological cancer, and that gynaecological cancer is ovarian cancer.

In a preferred embodiment, the treatment is palliative treatment.

Antibodies and antigen-binding fragments thereof that specifically binds to CD137

In one embodiment, the antibody or an antigen-binding fragment thereof ('antibody polypeptides') that specifically binds to CD137: a) has binding specificity for domain 2 of CD137, preferably domain 2 of human CD137; b) is a CD137 agonist; and/or c) is capable of inhibiting the binding of reference antibody '1630/1631' to human CD137.

In one embodiment, the antibody or an antigen-binding fragment thereof ('antibody polypeptides') that specifically binds to CD137: a) has binding specificity for domain 2 of CD137, preferably domain 2 of human CD137; b) is a CD137 agonist; and/or c) is capable of inhibiting the binding of reference antibody '2674/2675' to human CD137. In one embodiment, the antibody or antigen binding fragment that specifically binds to CD137 is capable of inhibiting the binding of reference antibody '1630/1631' and/or '2674/2675' to human CD137. In a particular embodiment, the antibody is the reference antibody '1630/1631', or an antigen binding fragment thereof. In an alternative, and preferred, embodiment the antibody is the reference antibody '2674/2675', or an antigen binding fragment thereof.

According to the invention, antibody or an antigen-binding fragment thereof that specifically binds to CD137 are provided which are capable of inhibiting the binding of one or more reference antibodies to human CD137, for example is capable of inhibiting the binding of reference antibody '1630/1631' and/or '2674/2675' to human CD137. Exemplary anti-CD137 antibodies are disclosed in WO 2018/091740 to Alligator Bioscience AB (the disclosures of which are incorporated herein by reference). For example, such anti-CD-137 antibodies are explicitly disclosed on pages 7-8, 11-12, 16-17 and 19 of WO 2018/091740, the disclosures of which are incorporated herein by reference.

By"CD137" we specifically include the human CD137 protein, for example as described in GenBank Accession No. AAH06196.1 (the sequence of which is set out in SEQ ID NO: 11, below). CD137 is also known in the scientific literature as 4-1BB and TNFRSF9.

Human CD137, amino acid sequence: >gi| 571321 |gb|AAA53133.1 | 4-1BB [Homo sapiens]

[SEQ ID NO: 11]

"Domain 2", as referred to above, corresponds to amino acids 66 to 107 of human CD137 (see bold, underlined region in SEQ ID NO: 11 above).

Thus, the antibody and antigen-binding fragments thereof of the invention have specificity for CD137. By "specificity" we mean that the antibody polypeptide is capable of binding to CD137 in vivo, i.e. under the physiological conditions in which CD137 exists within the human body. Preferably, the antibody polypeptide does not bind to any other protein in vivo. Such binding specificity may be determined by methods well known in the art, such as ELISA, immunohistochemistry, immunoprecipitation, Western blots and flow cytometry using transfected cells expressing CD137.

The antibody or antigen-binding fragment thereof preferably binds to human CD137 with a Kd value which is less than 10x10 ^-9 M or less than 7x10 ^-9 M, more preferably less than 4, or 2x10 ^-9 M, most preferably less than 1.2x10 ^-9 M. Advantageously, the antibody polypeptide is capable of binding selectively to CD137, i.e. it bind at least 10-fold more strongly to CD137 than to any other proteins. The anti-CD137 antibody preferably specifically binds to CD137, i.e. it binds to CD137 but does not bind, or binds at a lower affinity (e.g. a 10-fold lower affinity), to other molecules (such as 0X40 and/or CD40) - it therefore binds to CD137 with greater binding affinity than that at which it binds another molecule. Therefore, typically, the Kd for the antibody with respect to human CD137 will be 2-fold, preferably 5-fold, more preferably 10-fold less than Kd with respect to the other, non-target molecule, such as murine CD137, other TNFR superfamily members, or any other unrelated material or accompanying material in the environment. More preferably, the Kd will be 50-fold less, even more preferably 100-fold less, and yet more preferably 200-fold less.

Methods for measuring the overall affinity (KD) and on-rate (ka) and off-rate (kd) of an interaction (such as an interaction between an antibody and a ligand) are well known in the art. Exemplary in vitro methods are described in the accompanying Examples. It is also conceivable to use flow cytometry-based methods (Sklar et al., Annu Rev Biophys Biomol Struct, (31), 97-119, 2002).

The term CD137 as used herein typically refers to human CD137. The antibody may have some binding affinity for CD137 from other mammals, such as CD137 from a non-human primate, for example Macaca fascicularis (cynomolgus monkey). The antibody preferably does not bind to murine CD137 and/or does not bind to other human TNFR superfamily members, for example human 0X40 or CD40.

In an embodiment, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may have affinity for CD137 in its native state, and in particular for CD137 localised on the surface of a cell.

By "localised on the surface of a cell" it is meant that CD137 is associated with the cell such that one or more region of CD137 is present on the outer face of the cell surface. For example, CD137 may be inserted into the cell plasma membrane (i.e. orientated as a transmembrane protein) with one or more regions presented on the extracellular surface. This may occur in the course of expression of CD137 by the cell. Thus, in one embodiment, "localised on the surface of a cell" may mean "expressed on the surface of a cell." Alternatively, CD137 may be outside the cell with covalent and/or ionic interactions localising it to a specific region or regions of the cell surface.

In a preferred embodiment, the antibodies and antigen-binding fragments thereof that specifically binds to CD137 as defined herein are CD137 agonists. For example, they may be capable of inducing the release of interferon-gamma from CD8+ T cells. Agonistic activity of anti-CD137 antibodies may be evaluated in a T cell assay based on primary CD8+ T cells (see the Examples of WO 2018/091740).

Thus, the antibody or antigen binding fragment thereof that specifically binds to CD137 may modulate the activity of a cell expressing CD137, wherein said modulation is an increase or decrease in the activity of said cell. The cell is typically a T cell. The antibody may increase the activity of a CD4+ or CD8+ effector cell, or may decrease the activity of, or deplete, a regulatory T cell (T reg). In either case, the net effect of the antibody will be an increase in the activity of effector T cells, particularly CD4+, CD8+ or NK effector T cells. Methods for determining a change in the activity of effector T cells are well known and are as described earlier.

The antibody or antigen binding fragment thereof that specifically binds to CD137 preferably causes an increase in activity in a CD8+ T cell in vitro, optionally wherein said increase in activity is an increase in proliferation, IFN-y production and/or IL-2 production by the T cell. The increase is preferably at least 2-fold, more preferably at least 10-fold and even more preferably at least 25-fold higher than the change in activity caused by an isotype control antibody measured in the same assay.

Reference antibody 1630/1631 and reference antibody 2674/2675

As outlined above, antibody or antigen binding fragment thereof that specifically binds to CD137 which are capable of inhibiting the binding of one or more reference antibodies to human CD137 are provided. The reference antibodies described herein are reference antibody 1630/1631 and reference antibody 2674/2675. By reference antibody "1630/1631" we mean an intact IgG antibody comprising heavy and light chains having the amino acid sequences of SEQ ID NOS: 17 and 18, respectively.

1630/1631- Full sequence Heavy chain [SEQ ID NO: 17]

1630/1631 - Full sequence Light chain

[SEQ ID NO: 18]

By reference antibody "2674/2675" we mean an intact IgG antibody comprising heavy and light chains having the amino acid sequences of SEQ ID NOS: 29 and 30, respectively. Antibody 2674/2675 is also known as ATOR-1017 and these terms are fully interchangeable.

2674/2675 - Full sequence heavy chain

[SEQ ID NO: 29] 2674/2675 - Full sequence light chain

[SEQ ID NO: 30]

The identification of 1630/1631 and 2674/2675, the binding characteristics of those antibodies and their therapeutic efficacy was described in WO 2018/091740, which is incorporated by reference herein in its entirety.

As discussed below, the reference antibody '1630/1631' binds to domain 2 of CD137. Reference antibody 2674/2675 also binds to domain 2 of CD137. Thus, it will be appreciated that the antibody or an antigen-binding fragment of the invention also binds to domain 2 of CD137.

By "capable of inhibiting the binding of reference antibody '1630/1631' to human CD137" we mean that the presence of the antibody polypeptides of the invention inhibits, in whole or in part, the binding of '1630/1631' to human CD137. Similarly, by "capable of inhibiting the binding of reference antibody '2674/2675' to human CD137" we mean that the presence of the antibody polypeptides of the invention inhibits, in whole or in part, the binding of '2674/2675" to human CD137. The anti- CD137 antibodies or antigen-binding fragments thereof of the invention may therefore compete for binding to human CD137 with 'reference antibody' 1630/1631 and/or with 'reference antibody' 2674/2675. Such competitive binding inhibition can be determined using assays and methods well known in the art, for example using BIAcore chips with immobilised CD137 and incubating in the presence of the reference antibody '1630/1631' or '2674/2675' with and without an antibody polypeptide to be tested. Alternatively, a pair-wise mapping approach can be used, in which the reference antibody '1630/1631' or '2674/2675' is immobilised to the surface of the BIAcore chip, CD137 antigen is bound to the immobilised antibody, and then a second antibody is tested for simultaneous CD137-binding ability (see 'BIAcore Assay Handbook', GE Healthcare Life Sciences, 29-0194-00 AA 05/2012; the disclosures of which are incorporated herein by reference). In a further alternative, competitive binding inhibition can be determined using flow cytometry. For example, to test whether a test antibody is able to inhibit the binding of the 1630/1631 or 2674/2675 reference antibody to a cell surface antigen, cells expressing the antigen can be pre-incubated with the test antibody for 20 min before cells are washed and incubated with the reference 1630/1631 or 2674/2675 antibody conjugated to a fluorophore, which can be detected by flow cytometry. If the pre- incubation with the test antibody reduces the detection of the reference 1630/1631 or 2674/2675 antibody in flow cytometry, the test antibody inhibits the binding of the reference antibody to the cell surface antigen. If the antibody to be tested exhibits high affinity for CD137, then a reduced pre-incubation period may be used (or even no pre-incubation at all).

In a further alternative, competitive binding inhibition can be determined using an ELISA (as would be well known to one skilled in molecular biology).

In some embodiments, the antibodies and antigen binding fragments thereof that specifically bind CD137 are defined by reference to the variable regions of reference antibodies 1630/1631 and 2674/2675.

The reference antibody designated '1630/1631' comprises:

(a) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 1:

[SEQ ID NO:1] and

(b) a light chain variable region having the amino acid sequence of SEQ ID NO: 2:

[SEQ ID NO:2]

The reference antibody designated '2674/2675' comprises: (a) a heavy chain variable region having the amino acid sequence of SEQ ID NO: 19:

[SEQ ID NO: 19] and

(b) a light chain variable region having the amino acid sequence of SEQ ID NO: 20:

[SEQ ID NO:20]

The term "amino acid" as used herein includes the standard twenty genetically- encoded amino acids and their corresponding stereoisomers in the 'D' form (as compared to the natural 'L' form), omega-amino acids and other naturally-occurring amino acids, unconventional amino acids (e.g. o,o-disubstituted amino acids, N-alkyl amino acids, etc.) and chemically derivatised amino acids (see below).

When an amino acid is being specifically enumerated, such as "alanine" or "Ala" or "A", the term refers to both L-alanine and D-alanine unless explicitly stated otherwise. Other unconventional amino acids may also be suitable components for polypeptides of the present invention, as long as the desired functional property is retained by the polypeptide. For the peptides shown, each encoded amino acid residue, where appropriate, is represented by a single letter designation, corresponding to the trivial name of the conventional amino acid.

In one embodiment, the antibody polypeptides as defined herein comprise or consist of L-amino acids.

A "polypeptide" is used herein in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. The term "polypeptide" thus includes short peptide sequences and also longer polypeptides and proteins. As used herein, the term "amino acid" refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.

It will be appreciated by persons skilled in the art that the binding specificity of an antibody or antigen-binding fragment thereof is conferred by the presence of complementarity determining regions (CDRs) within the variable regions of the constituent heavy and light chains, such as those CDRs described herein.

It will be further appreciated by persons skilled in the art that any intact IgG antibody comprising the above variable regions may be used as the reference antibody to identify antibody polypeptides of the invention that competitively inhibit 1630/1631 or 2674/2675 binding to CD137. Preferably however, reference antibody 1630/1631 consists of heavy and light chains as defined in SEQ ID NOs:17 and 18, respectively, and reference antibody 2674/2675 consists of heavy and light chains as defined in SEQ ID NOs:29 and 30, respectively

Competitive binding typically arises because the test antibody binds at, or at least very close to, the epitope on the antigen to which binds the reference antibody (in this case, 1630/1631 or 2674/2675). However, it will be appreciated by persons skilled in the art that competitive binding may also arise by virtue of steric interference; thus, the test antibody may bind at an epitope different from that to which the reference antibody binds but may still be of sufficient size or configuration to hinder the binding of the reference antibody to the antigen.

The antibodies and antigen-binding fragments thereof that bind to CD137 were identified after screening of anti-CD137 antibodies, on the basis of exhibiting properties that make them particularly suitable as diagnostic and therapeutic agents for cancer (as discussed in WO 2018/091740).

Thus, in one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to CD137 exhibits one or more of the following properties: a) the ability to stimulate CD137 and activate T cells and other immune cells via a cross-linking dependent mechanism (e.g. to induce release of interferon-gamma from CD8+ T cells; see Examples); and/or b) cross-reactivity with cynomolgus CD137 (see Examples). For example, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may exhibit both of the above properties. The antibody or antigen-binding fragment thereof that specifically binds to CD137 may be or may comprise a variant or a fragment of one of the specific anti-CD137 antibodies disclosed herein, provided that said variant or fragment retains specificity for CD137, and in some embodiments retains at least one of functional characteristics (a) to (b) above.

As described above, the antibodies and antigen binding fragments thereof that specifically bind to CD137 may have a cross linking dependent mechanism. By "cross linking dependent mechanism", we include an Fc cross linking dependent mechanism wherein the antibody has to bind both CD137 and an Fc receptor in order to stimulate CD137. As such, in some embodiments, the antibody has to be capable of binding both CD137 and an Fc receptor.

In an embodiment, the antibody or antigen binding fragment thereof that specifically binds to CD137 is capable of binding an Fc receptor. In one embodiment, the antibody or antigen binding domain is capable of simultaneous binding to CD137 and a Fc receptor. In a preferred embodiment, the ability of the antibody and/or antigen binding domain thereof that specifically binds to CD137 to activate T cells is dependent upon binding to both CD137 and Fc receptors.

In a preferred embodiment, the Fc receptor that is targeted is an FcγR. Examples of FcγRs include, FcγRI, FcγRIIA and FcγRIIB Thus, in one embodiment, the FcγR may be FcγRIIA. By FcγRIIA, we include both the R131 and H131 allotypes of FcγRIIA. Thus, in one embodiment, the FcγR to be targeted is the R131 allotype of FcγRIIA.

In an alternative embodiment, the antibody or antigen binding fragment thereof that specifically binds to CD137 could be Fc crosslinking independent, such that it can stimulate CD137 in the absence of binding to an Fc receptor.

Thus, exemplary antibodies 2674/2675 and 1630/1631 are FcγR-crosslinking dependent agonistic antibodies targeting the co-stimulatory CD137 receptor. They are therefore only active in tissues or tumours containing cells expressing CD137 and FcγR. By "tumours containing cells expressing CD137 and FcγR" we include tumours or tumour draining lymph nodes comprising tumour cells and/or tumour infiltrating immune cells (such as monocytes, macrophages, dendritic cells, NK cells, T cells, B cells and granulocytes) expressing CD137 and FcγR. It will be appreciated that CD137 and FcγR may be expressed on separate cells within the tumour and/or co-expressed in the same cells. Reference antibodies 2674/2675 and 1630/1631 will thus provide a tumour directed immune activation in indications associated with cells that express both CD137 and FcγR in the tumour microenvironment; this contrasts with FcγR independent CD137 agonists (e.g. Urelumab), which capable of inducing systemic immune activation. The tumour localizing effect of antibodies 2674/2675 and 1630/1631 will primarily depend oonn the number of tumour infiltrating macrophages/myeloid cells expressing different FcγRs.

It is known that IgG4 binds with high affinity to FcγRI and with moderate/low affinity to FcγRIIa and FcγRIIb. FcγRI and FcγRIIa are expressed on monocytes and FcγRIIb is expressed with a high density on B cells. Crosslinking of antibodies 2674/2675 and 1630/1631 will preferentially occur intratumorally as well as in adjacent draining lymph nodes. Systemically in the blood, where serum IgG levels are high, the availability of free non-blocked FcγRs are believed to be too low for an effective crosslinking to occur. Therefore, the risk for a systemic immune activation of is believed to be low which improves the risk-benefit profile compared to other CD137 mAbs.

Patient selection and a biomarker rationale for treatment with antibodies or antigen binding fragments thereof that specifically binds to CD137 of the invention, such as 2674/2675 and 1630/1631, may be guided by tumour types that have infiltrating cells expressing CD137 and FcγRs. Thus, the antibodies of the invention may be for use in patients selected on the basis of having a tumour containing cells expressing CD137 and FcγRs (7.e. a as companion diagnostic test).

By "infiltrating cells" we include tumour infiltrating immune cells such as monocytes, macrophages, dendritic cells, NK cells, T cells, B cells and granulocytes

Advantageously, the antibody or antigen-binding fragment thereof that specifically binds to CD137 is capable of inducing tumour immunity. Tumour immunity can be demonstrated using methods well known in the art, for example by re-challenging mice that have been cured from a given tumour by CD317 antibody treatment with the same tumour and/or by re-challenging mice that have been cured from a given tumour by the antibody or antigen-binding fragment thereof that specifically binds to CD137 of the present invention with the same tumour. If tumour immunity has been induced by the antibody therapy, then the tumour is rejected upon re-challenge. In one embodiment, the antibody or antigen binding fragment thereof that specifically binds to CD137 is substantially incapable of inducing the following upon binding to cells expressing CD137: a) antibody-dependent cellular cytotoxicity (ADCC); b) antibody-dependent cellular phagocytosis (ADCP); and/or c) complement-dependent cytotoxicity (CDC).

The antibody may be or may comprise a variant or a fragment of one of the specific anti-CD137 antibodies disclosed herein, provided that said variant or fragment retains specificity for CD137 and is incapable of inducing one or more of (a) to (c) upon binding to cells expressing CD137.

Methods for determining the level of ADCC-mediated lysis or apoptosis in a sample of cells are well known in the art. For example, a chromium-51 release assay, europium release assay or sulphur-35 release assay may be used. In such assays, a previously labelled target cell line expressing the antigen is incubated with an antibody to be tested. After washing, effector cells (typically expressing Fc receptor CD16) are co-incubated with the antibody-labelled target cells. Target cell lysis is subsequently measured by release of intracellular label by a scintillation counter or spectrophotometry. As an alternative to the labelling with radioisotopes required in such assays, methods may be used in which lysis is detected by measuring the release of enzymes naturally present in the target cells. This may be achieved by detection (for example bioluminescent detection) of the products of an enzyme- catalysed reaction. No previous labelling of the cells is required in such an assay. A typical cellular enzyme detected with such an assay is GAPDH.

Methods for determining the level of ADCP in a sample of cells are well known in the art. For example, the tumor antigen-expressing cancer cells may be incubated in the presence of a titration of mAb and the human leukemia monocytic cell line THP-1. Both effector and target cells may be fluorescently labelled and cell engulfment may be measured by flow cytometry. Phagocytosis may also be confirmed using microscopy or imaging cytometry.

Methods for determining the level of CDC in a sample of cells are well known in the art. For example, serum comprising the components of the complement system (typically human serum) may be mixed with target cells bound by the antibody being detected, and then cell death may be determined by a suitable method. Cell death may be determined via pre-loading the target cells with a radioactive compound. As cells die, the radioactive compound is released from them. Hence, the efficacy of the antibody to mediate cell death is may be determined by the radioactivity level. Non- radioactive CDC assays may also be used, which may determine the release of abundant cell components, such as GAPDH, with fluorescent or luminescent determination.

In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to CD137 is capable of binding to an epitope on the extracellular domain of CD137 which overlaps, at least in part, with the epitope on CD137 to which reference antibody 1630/1631 and/or 2674/2675 is capable of binding. Thus, the antibody or antigen- binding fragment thereof that specifically binds to CD137 may be capable of binding to an epitope located at/within domain 2 of CD137 (i.e. amino acids 66 to 107 of human CD137).

In one embodiment, the antibody polypeptide of the invention comprises or consists of an intact antibody (for example, an IgG1, IgG2, IgG3 or IgG4 antibody). In a preferred embodiment, the antibody is an IgG4 antibody.

In an alternative embodiment, the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises or consists of an antigen-binding fragment selected from the group consisting of: Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab' fragments and F(ab)2 fragments) and domain antibodies (e.g. single V _H variable domains or V _L variable domains). In particular, the antibody polypeptide may be a scFv.

In a further embodiment, as discussed above, the antibody or antigen-binding fragment thereof that specifically binds to CD137 of the invention comprises or consists of an antibody mimic selected from the group comprising or consisting of affibodies, tetranectins (CTLDs), adnectins (monobodies), anticalins, DARPins (ankyrins), avimers, iMabs, microbodies, peptide aptamers, Kunitz domains and affilins.

In one embodiment, the antibody or antigen binding fragment thereof that specifically binds to CD137 comprises: a) a heavy chain CDR1 sequence with the consensus sequence G, F, T/N, F, G, Y, S, Y; b) a heavy chain CDR2 sequence with the consensus sequence I, G, S, G/T, S, S, Y/H, T; and c) a heavy chain CDR3 sequence with the sequence ARVYSSPGIDY.

In one embodiment, the antibody or antigen binding fragment thereof that specifically binds to CD137 comprises: a) a light chain CDR1 sequence with the consensus sequence Q, S, I, S/G, S, Y/T; b) a light chain CDR2 sequence with the consensus sequence A/G, A, S; and c) a light chain CDR3 sequence with the sequence QQYYTWVPFT.

In a preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to CD137 of the invention comprises a heavy chain variable region comprising the following CDRs: a) GFTFGYSY [SEQ ID NO: 3] or an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 3, for example 1, 2 or 3 mutations; b) IGSGSSYT [SEQ ID NO: 4] or an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 4, for example 1, 2 or 3 mutations; and c) ARVYSSPGIDY [SEQ ID NO: 5] or an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 5, for example 1, 2 or 3 mutations.

Thus, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a heavy chain variable region comprising one, two or all three of the CDRs of SEQ ID NOs 3, 4 and 5. Preferably, the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises a heavy chain variable region comprising all three of the CDRs of SEQ ID NOs 3, 4 and 5.

For example, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a heavy chain variable region having the amino acid sequence of the corresponding region of the 1630/1631 reference antibody, i.e. SEQ ID NO:1.

In some embodiments the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO 1: or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

Percent sequence identity can be determined by, for example, the LALIGN program (Huang and Miller, Adv. Appl. Math. (1991) 12:337-357, the disclosures of which are incorporated herein by reference) at the Expasy facility site (http://www.ch.embnet.org/software/LALIGN_form.html) using as parameters the global alignment option, scoring matrix BLOSUM62, opening gap penalty -14, extending gap penalty -4. Alternatively, the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percentage sequence identity is calculated in relation to polypeptides whose sequence has been aligned optimally.

The alignment may alternatively be carried out using the Clustal W program (as described in Thompson et al. , 1994, Nucl. Acid Res. 22:4673-4680, which is incorporated herein by reference). The parameters used may be as follows:

Fast pair-wise alignment parameters: K-tuple(word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent.

Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05.

Scoring matrix: BLOSUM.

Alternatively, the BESTFIT program may be used to determine local sequence alignments.

In an alternative preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises a heavy chain variable region comprising the following CDRs: a) GFNFGYSY [SEQ ID NO: 21] or an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 21, for example 1, 2 or 3 mutations; b) IGSTSSHT [SEQ ID NO: 22] or an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 22, for example 1, 2 or 3 mutations; and c) ARVYSSPGIDY [SEQ ID NO: 23] or an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 23, for example 1, 2 or 3 mutations.

Thus, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a heavy chain variable region comprising one, two or all three of the CDRs of SEQ ID NOs 21, 22 and 23. Preferably, the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises a heavy chain variable region comprising all three of the CDRs of SEQ ID NOs 21, 22 and 23.

For example, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a heavy chain variable region having the amino acid sequence of the corresponding region of the 2674/2675 reference antibody, i.e. SEQ ID NO:19.

In some embodiments the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises a heavy chain variable region having the amino acid sequence of SEQ ID NO 19: or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

However, it will be appreciated (in relation to either embodiment, 1630/1631 or 2674/2675) that a low level of mutation (typically, just one, two or three amino acids) within a CDR sequence may be tolerated without loss of the specificity of the antibody or antigen-binding fragment for CD137.

For example, in an alternative embodiment, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a heavy chain variable region comprising the CDRs as defined above, wherein the H1 and H2 CDRs are mutated versions of SEQ ID NO: 3 and 4, respectively, and wherein the H3 CDR is SEQ ID NO: 5.

In a further alternative embodiment, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a heavy chain variable region comprising the CDRs as defined above, wherein the H1 and H2 CDRs are mutated versions of SEQ ID NO: 21 and 22, respectively, and wherein the H3 CDR is SEQ ID NO: 23. In a further preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises a light chain variable region comprising the following CDRs: a) QSISSY [SEQ ID NO: 6] or an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 6, for example 1 , 2 or 3 mutations; b) AAS [SEQ ID NO: 7] or an amino acid sequence containing up to 2 amino acid mutations compared to SEQ ID NO: 7; for example 1 or 2 mutations and

C) QQYYTWVPFT [SEQ ID NO: 8] or an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 8, for example 1, 2 or 3 mutations.

Thus, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a light chain variable region comprising the one, two or all three of CDRs of SEQ ID NOs 6, 7 and 8. Preferably, the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises a light chain variable region comprising all three of the CDRs of SEQ ID NOs 6, 7 and 8.

For example, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a light chain variable region having the amino acid sequence of the corresponding region of the 1630/1631 reference antibody, i.e. SEQ ID NO: 2.

In some embodiments the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises a light chain variable region having the amino acid sequence of SEQ ID NO 2: or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

In an alternative embodiment, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a light chain variable region comprising the CDRs as defined above, wherein the L1 and L2 CDRs are mutated versions of SEQ ID NO: 6 and 7, respectively, and wherein the L3 CDR is SEQ ID NO:8.

In a further preferred embodiment, the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises a light chain variable region comprising the following CDRs: a) QSIGST [SEQ ID NO: 24] or an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 24, for example 1, 2 or 3 mutations; b) GAS [SEQ ID NO: 25] or an amino acid sequence containing up to 2 amino acid mutations compared to SEQ ID NO: 25; for example 1 or 2 mutations and c) QQYYTWVPFT [SEQ ID NO: 26] or an amino acid sequence containing up to 3 amino acid mutations compared to SEQ ID NO: 26, for example 1, 2 or 3 mutations.

Thus, the antibody or antigen-binding fragment thereof that specifically binds to CD137may comprise a light chain variable region comprising one, two or all three of the CDRs of SEQ ID NOs 24, 25 and 26. Preferably, the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises a light chain variable region comprising all three of the CDRs of SEQ ID NOs 24, 25 and 26.

For example, the antibody or antigen-binding fragment thereof may comprise a light chain variable region having the amino acid sequence of the corresponding region of the 2674/2675 reference antibody, i.e. SEQ ID NO: 20.

In some embodiments the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises a light chain variable region having the amino acid sequence of SEQ ID NO 20: or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

In an alternative embodiment, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a light chain variable region comprising the CDRs as defined above, wherein the L1 and L2 CDRs are mutated versions of SEQ ID NO: 24 and 25, respectively, and wherein the L3 CDR is SEQ ID NO: 26.

The antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise one, two or all three of the CDR sequences of SEQ ID NOs: 3 to 5 and/or one, two, or all three of the CDR sequences of SEQ ID NOs: 6 to 8. The antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise all six CDR sequences of SEQ ID NOs: 3 to 8. The antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise or consist of the light chain variable region sequence of SEQ ID NO: 2 and/or the heavy chain variable region sequence of SEQ ID NO: 1, or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 and/or SEQ ID NO:2.

The antibody or antigen-binding fragment thereof that specifically binds to CD137 may be, or may bind to the same epitope as, an antibody comprising the light chain variable region sequence of SEQ ID NO: 2 and the heavy chain variable region sequence of SEQ ID NO: 1. In addition, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise the light chain constant region sequence of SEQ ID NO: 16 and/or the heavy chain constant region sequence of SEQ ID NO: 13, or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 16 and/or SEQ ID NO: 13.

The antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise one, two or all three of the CDR sequences of SEQ ID NOs: 21 to 23 and/or one, two, or all three of the CDR sequences of SEQ ID NOs: 24 to 26. The antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise all six CDR sequences of SEQ ID NOs: 21 to 26.

The antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise or consist of the light chain variable region sequence of SEQ ID NO: 20 and/or the heavy chain variable region sequence of SEQ ID NO: 19 or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 20 and/or 19.

The antibody or antigen-binding fragment thereof that specifically binds to CD137 may be, or may bind to the same epitope as, an antibody comprising the light chain variable region sequence of SEQ ID NO: 20 and the heavy chain variable region sequence of SEQ ID NO: 19. In addition, the antibody may comprise the light chain constant region sequence of SEQ ID NO: 16 and/or the heavy chain constant region sequence of SEQ ID NO: 13 or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 16 and/or 13. It will be appreciated by persons skilled in the art that for human therapy, human or humanised antibodies are preferably used. Humanised forms of non-human (e.g. murine) antibodies are genetically engineered chimeric antibodies or antibody fragments having preferably minimal-portions derived from non-human antibodies. Humanised antibodies include antibodies in which complementary determining regions of a human antibody (recipient antibody) are replaced by residues from a complementary determining region of a non-human species (donor antibody) such as mouse, rat of rabbit having the desired functionality. In some instances, Fv framework residues of the human antibody are replaced by corresponding non-human residues. Humanised antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported complementarity determining region or framework sequences. In general, the humanised antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the complementarity determining regions correspond to those of a non-human antibody and all, or substantially all, of the framework regions correspond to those of a relevant human consensus sequence. Humanised antibodies optimally also include at least a portion of an antibody constant region, such as an Fc region, typically derived from a human antibody (see, for example, Jones et al., 1986. Nature 321:522-525; Riechmann et al., 1988, Nature 332:323-329; Presta, 1992, Curr. Op. Struct. Biol. 2:593-596, the disclosures of which are incorporated herein by reference).

Methods for humanising non-human antibodies are well known in the art. Generally, the humanised antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues, often referred to as imported residues, are typically taken from an imported variable domain. Humanisation can be essentially performed as described (see, for example, Jones et al., 1986, Nature 321:522-525; Reichmann et al. , 1988. Nature 332:323-327; Verhoeyen et al., 1988, Science 239:1534-15361; US 4,816,567, the disclosures of which are incorporated herein by reference) by substituting human complementarity determining regions with corresponding rodent complementarity determining regions. Accordingly, such humanised antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanised antibodies may be typically human antibodies in which some complementarity determining region residues and possibly some framework residues are substituted by residues from analogous sites in rodent antibodies. Chimeric antibodies are discussed by Neuberger et al (1998, 8 ^th International Biotechnology Symposium Part 2, 792-799). Human antibodies can also be identified using various techniques known in the art, including phage display libraries (see, for example, Hoogenboom & Winter, 1991, J. Mol. Biol. 227:381; Marks et al., 1991, J. Mol. Biol. 222:581; Cole et al., 1985, In: Monoclonal antibodies and Cancer Therapy, Alan R. Liss, pp. 77; Boerner et al., 1991.

J. Immunol. 147:86-95, the disclosures of which are incorporated herein by reference).

It will be appreciated by persons skilled in the art that humanised antibodies or antigen-binding fragments thereof that specifically bind to CD137 may further comprise a heavy chain constant region, or part thereof (see below).

Constant domains

In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to CD137 comprises a CHI, CH2 and/or CH3 region of an IgG heavy chain (such as an IgG1, IgG2, IgG3 or IgG4 heavy chain). Thus, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise part or all of the constant regions from an IgG4 heavy chain. For example, the antibody or antigen- binding fragment thereof that specifically binds to CD137 may be a Fab fragment comprising CH1 and CL constant regions, combined with any of the above-defined heavy and light variable regions respectively.

Likewise, the above-defined antibodies or antigen-binding fragments thereof that specifically binds to CD137 may further comprise a light chain constant region, or part thereof (see below). For example, the antibody or antigen-binding fragments thereof that specifically binds to CD137 may comprise a CL region from a kappa or lambda light chain.

In one embodiment, the antibodies or antigen-binding fragments thereof that specifically bind to CD137 comprise an antibody Fc-region. It will be appreciated by a skilled person that the Fc portion may be from an IgG antibody, or from a different class of antibody (such as IgM, IgA, IgD or IgE). In one embodiment, the Fc region is from an IgG1, IgG2, IgG3 or IgG4 antibody. Advantageously, however, the Fc region is from an IgG4 antibody.

The Fc region may be naturally-occurring (e.g. part of an endogenously produced antibody) or may be artificial (e.g. comprising one or more point mutations relative to a naturally-occurring Fc region). A variant of an Fc region typically binds to Fc receptors, such as FcγR and/or neonatal Fc receptor (FcRn) with altered affinity providing for improved function and/or half-life of the polypeptide. The biological function and/ or the half-life may be either increased or a decreased relative to the half-life of a polypeptide comprising a native Fc region. Examples of such biological functions which may be modulated by the presence of a variant Fc region include antibody dependent cell cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or apoptosis.

Thus, the Fc region may be naturally-occurring (e.g. part of an endogenously produced human antibody) or may be artificial (e.g. comprising one or more point mutations relative to a naturally-occurring human Fc region).

As is well documented in the art, the Fc region of an antibody mediates its serum half- life and effector functions, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP).

Engineering the Fc region of a therapeutic monoclonal antibody or Fc fusion protein allows the generation of molecules that are better suited to the pharmacology activity required of them (Strohl, 2009, Curr Opin Biotechnol 2O(6):685-91, the disclosures of which are incorporated herein by reference).

(a) Engineered Fc regions for increased half-life

One approach to improve the efficacy of a therapeutic antibody is to increase its serum persistence, thereby allowing higher circulating levels, less frequent administration and reduced doses.

The half-life of an IgG depends on its pH-dependent binding to the neonatal receptor FcRn. FcRn, which is expressed on the surface of endothelial cells, binds the IgG in a pH-dependent manner and protects it from degradation.

Some antibodies that selectively bind the FcRn at pH 6.0, but not pH 7.4, exhibit a higher half-life in a variety of animal models.

Several mutations located at the interface between the CH2 and CH3 domains, such as T250Q/M428L (Hinton et al., 2004, J Biol Chem. 279(8) :6213-6, the disclosures of which are incorporated herein by reference) and M252Y/S254T/T256E + H433K/N434F (Vaccaro et al., 2005, Nat. Biotechnol. 23(10): 1283-8, the disclosures of which are incorporated herein by reference), have been shown to increase the binding affinity to FcRn and the half-life of IgG1 in vivo.

(b) Engineered Fc regions for altered effector function

Depending on the therapeutic antibody or Fc fusion protein application, it may be desired to either reduce or increase the effector function (such as ADCC).

For antibodies that target cell-surface molecules, especially those on immune cells, abrogating effector functions may be required for certain clinical indications.

The four human IgG isotypes bind the activating Fey receptors (FcγRI, FcγRIIa, FcγRIIIa), the inhibitory FcγRIIb receptor, and the first component of complement (C1q) with different affinities, yielding very different effector functions (Bruhns et al., 2009, Blood. 113(16) :3716-25, the disclosures of which are incorporated herein by reference). FcγRI binding affinity of IgG4 vs IgG2

Bruhns et al performed a series of experiments that evaluated the specificity and affinity of the known human FcγRs, and their polymorphic variants, for the different human IgG subclasses (Bruhnset al. , 2009, Blood. 113(16) :3716-25, the disclosures of which are incorporated herein by reference). In this study, it was clearly demonstrated that while IgG2 had no detectable affinity for FcγRI, IgG1, IgG3 and IgG4 all displayed a binding affinity for FcγRI in the nanomolar range (Bruhns et al., 2009, Blood. 113(16):3716-25, Lu et al., 2015, Proc Natl Acad Sci U S A. 112(3):833- 8, the disclosures of which are incorporated herein by reference). A summary of the relative binding affinities between the major human FcγRs and their variants and IgG isotypes is summarized in Table 1. (Stewart et al. 2014, J Immunother. 2(29), the disclosures of which are incorporated herein by reference)

Table 1. Binding affinity between human FcγRs and IgG isotypes.

However, cellular activation influences the affinity of FcγRI for IgG immune complexes and the data generated by surface plasmon resonance in the Bruhns paper may not correctly reproduce what occurs at an inflammatory site. A review paper by Hogarth et al (Hogarth et al. 2012, Nat Rev Drug Discov 11(4):311-31, the disclosures of which are incorporated herein by reference) summarizes this as well as other studies focusing on FcγR binding for IgG.

FcvRI expression on myeloid cell subsets

Human FcγRs are primarily expressed by cells of the myeloid lineage, which has been demonstrated in numerous studies for circulating myeloid cell subsets. Classical monocytes, generally identified as CD14 ⁺ CD16- display high levels of FcγRII (CD32), intermediate levels of FcγRI and low levels of FcγRIII (CD16) (Almeida et al. 2001, 100(3) :325-38, Cheeseman et al. 2016, PLoS One ll(5):e0154656, the disclosures of which are incorporated herein by reference). CD14- CD16 ⁺ non-classical monocytes, however, display high levels of FcγRIII, intermediate levels of FcγRII and low levels of FcγRI (Almeida et al. 2001). A summary and compilation of several published microarray data sets showing the expression of human FcγR genes on different myeloid cell subsets confirms these observations (Guilliams et al. 2014, Nat Rev Immunol. 14(2):94-108, the disclosures of which are incorporated herein by reference).

Once within tissues, monocytes differentiate towards macrophages and, depending on environmental cues, these macrophages obtain specific phenotypes. In a study by Roussel et al (Roussel et al. 2017, J Leukoc Biol. 102(2) :437-447, the disclosures of which are incorporated herein by reference), peripheral blood monocytes were polarized towards different macrophage lineages by using various inflammatory stimuli and the expression profile of these cells evaluated. Here, IFN-y stimulated monocytes resulted in a highly elevated expression specifically of CD64. A similar observation was made in SLE patients where increased CD64 expression was detected on circulating CD14 ⁺ monocytes, which correlated with expression of interferon-stimulated genes (Li et al. 2010, Arthritis Res Ther 12(3): R90, the disclosures of which are incorporated herein by reference). Myeloid cell infiltration within various human tumors

Various myeloid cell subsets such as inflammatory monocytes, monocytic myeloid- derived suppressor cells (MDSC) and macrophages have, in numerous studies, been shown to accumulate in cancer patients (Solito et al. 2014, Ann N Y Acad Sci 1319:47- 65., Hu et al. 2016, Clin Transl Oncol.18(3) :251-8, the disclosures of which are incorporated herein by reference). Although recent attempts have aimed at proposing strategies to standardize the characterization of these cells (Bronte et al. 2016, Nat Commun. 7:12150, the disclosures of which are incorporated herein by reference), many phenotypic definitions of these cell populations can still be found throughout the literature (Elliott et al. 2017, Front Immunol. 8:86, the disclosures of which are incorporated herein by reference). Most commonly, these cells are defined by the expression of the markers CD11b, CD14, CD33 and the low expression of HLA-DR (monocytic MDSC) (Bronte et al. 2016). Additionally, tumor-associated macrophages (TAM) are commonly identified by the expression of CD64 and CD68 (M1-polarized, anti-tumorigenic), or CD163 and CD206 (M2-polarized, pro-tumorigenic) (Elliott et al. 2017).

A recent review by Elliott et al., 2017 summarizes the numerous phenotypes used to identify myeloid cell subsets in cancer patients. Most of these studies have focused their analyses on circulating cells and increased frequencies of myeloid CD11b ⁺ cells have been observed in the blood of patients with e.g. bladder, breast, colorectal, hepatocellular, pancreatic, prostate and renal cell carcinoma (Solito et al. 2014, Elliott et al. 2017). Other studies have also attempted to characterize the level of infiltration of these cells into tumor tissue. In colorectal tumors, a high frequency of CD14 ⁺ CD169 ⁺ cells was observed. These cells also expressed CD163 and CD206 and were thus suggested to be M2-polarized TAM (Li et al. 2015, PLoS One 10(10):e0141817, the disclosures of which are incorporated herein by reference). Another study in colorectal cancer patients also detected increased numbers of CD11b ⁺ CD33+ HLA-DR- cells, compared to healthy individuals (Zhang et al. 2013, PLoS One 8(2):e57114, the disclosures of which are incorporated herein by reference).

Similarly, CD11b ⁺ myeloid cells were also identified in bladder tumors, where they accounted for 10-20% of all nucleated cells (Eruslanov et al. 2012, Int J Cancer 130(5): 1109-19, the disclosures of which are incorporated herein by reference). An even higher frequency of CD11b ⁺ cells was observed in pancreatic cancer where over 60% of the CD45+ cells were CD11b ⁺ CD15 ⁺ CD33 ⁺ (Porembka et al. 2012, Cancer Immunol Immunother 61(9) : 1373-85, the disclosures of which are incorporated herein by reference). Also, one study concluded that the major myeloid cell population within non-small cell lung carcinoma is a CD11b ⁺ CD15 ⁺ CD66b ⁺ neutrophil-like population. Interestingly, once these cells migrate from blood to the tumor tissue, these cells display an altered expression profile, including upregulated FcγRI (Eruslanov et al. 2014, J Clin Invest. 124(12):5466-80, the disclosures of which are incorporated herein by reference). FcγRI expression on tumor-infiltrating cells

Although numerous studies have identified a high infiltration of myeloid cells within human tumors, no study has thoroughly explored the expression of FcγRs on these cells in detail. Several publications have, however, demonstrated the presence of FcγRI-expressing cells within tumor tissue.

A study by Morimura et al (Morimura et al. 1990, Acta Neuropathol. 80(3):287-94, the disclosures of which are incorporated herein by reference) evaluated gliomas from 12 human samples by immunocytochemistry and compared these to peritumoral control tissue. This study demonstrated a high presence of macrophages (using the marker CD163, RM3/1) in gliomas, compared to peritumoral tissue, as well as an increase in FcγRI and FcγRII (CD32). A more recent study by Griesinger et al (Griesinger et al. 2013, J Immunol. 191(9) :4880-8, the disclosures of which are incorporated herein by reference) confirmed these observations by performing flow cytometric analyses of various pediatric brain tumor types. Here, a high frequency of CD45 ⁺ CD11b ⁺ myeloid cells was observed for tissues from pilocytic astrocytoma and ependymoma patients. These cells also expressed high levels of FcγRI.

In addition to brain tumors, FcγRI expression has also been shown for other types of tumors. Grugan et al (Grugan et al. 2012, J Immunol. 189(ll):5457-66, the disclosures of which are incorporated herein by reference) demonstrated the presence of CD11b ⁺ CD14 ⁺ cells within human breast tumor tissue. These cells were shown to express high levels of FcγRI and FcγRIIa, as well as FcγRIIb and FcγRIII. Also, CD45 ⁺ CD11b ⁺ CD14 ⁺ CD68 ⁺ TAM were identified in gastrointestinal stromal tumors displaying expression of FcγRI (Cavnar et al. 2013, J Exp Med. 210(13):2873-86, the disclosures of which are incorporated herein by reference). CD45 ⁺ CD11b ⁺ FcγRI ⁺ cells were also identified in colorectal cancer patients and these cells displayed a higher expression of FcγRI in tumor tissue, compared to healthy control tissue (Norton et al. 2016, Clin Transl Immunology. 5(4):e76, the disclosures of which are incorporated herein by reference). FcγRI expression has also been demonstrated for melanoma metastases (Hansen et al. 2006, Acta Oncol 45(4):400-5, the disclosures of which are incorporated herein by reference).

Binding of IgG to the FcγRs or C1q depends on residues located in the hinge region and the CH2 domain. Two regions of the CH2 domain are critical for FcγRs and C1q binding, and have unique sequences in IgG2 and IgG4. Substitutions into human IgG1 of IgG2 residues at positions 233-236 and IgG4 residues at positions 327, 330 and 331 were shown to greatly reduce ADCC and CDC (Armour et al., 1999, Eur J Immunol. 29(8):2613-24; Shields et al., 2001, J Biol Chem. 276(9):6591-604, the disclosures of which are incorporated herein by reference). Furthermore, Idusogie et al. demonstrated that alanine substitution at different positions, including K322, significantly reduced complement activation (Idusogie et al. , 2000, J Immunol. 164(8) :4178-84, the disclosures of which are incorporated herein by reference). Similarly, mutations in the CH2 domain of murine IgG2A were shown to reduce the binding to FcγRI, and C1q (Steurer. et al., 1995. J Immunol. 155(3):1165- 74, the disclosures of which are incorporated herein by reference).

Numerous mutations have been made in the CH2 domain of human IgG1 and their effect on ADCC and CDC tested in vitro (see references cited above). Notably, alanine substitution at position 333 was reported to increase both ADCC and CDC (Shields et al., 2001, supra; Steurer et al., 1995, supra). Lazar et al. described a triple mutant (S239D/I332E/A330L) with a higher affinity for FcγRIIIa and a lower affinity for FcγRIIb resulting in enhanced ADCC (Lazar et al. , 2006, PNAS 103(11):4005-4010, the disclosures of which are incorporated herein by reference). The same mutations were used to generate an antibody with increased ADCC (Ryan et al., 2007, Mol. Cancer Ther. 6:3009-3018, the disclosures of which are incorporated herein by reference). Richards et al. studied a slightly different triple mutant (S239D/I332E/G236A) with improved FcγRIIIa affinity and FcγRIIa/FcγRIIb ratio that mediates enhanced phagocytosis of target cells by macrophages (Richards et al., 2008. Mol Cancer Ther. 7(8):2517-27, the disclosures of which are incorporated herein by reference).

Due to their lack of effector functions, IgG4 antibodies represent a preferred IgG subclass for receptor modulation without cell depletion. IgG4 molecules can exchange half-molecules in a dynamic process termed Fab-arm exchange. This phenomenon can also occur in vivo between therapeutic antibodies and endogenous IgG4.

The S228P mutation has been shown to prevent this recombination process allowing the design of less unpredictable therapeutic IgG4 antibodies (Labrijn et al., 2009, Nat Biotechnol. 27(8) :767-71, the disclosures of which are incorporated herein by reference).

In a further embodiment, the effector function of the Fc region may be altered through modification of the carbohydrate moieties within the CH2 domain therein, for example by modifying the relative levels of fucose, galactose, bisecting N-acetylglucosamine and/or sialic acid during production (see Jefferis, 2009, Nat Rev Drug Discov. 8(3) :226- 34 and Raju, 2008, Curr Opin Immunol., 20(4) :471-8; the disclosures of which are incorporated herein by reference)

Thus, it is known that therapeutic antibodies lacking or low in fucose residues in the Fc region may exhibit enhanced ADCC activity in humans (for example, see Peipp et al., 2008, Blood 112(6):2390-9, Yamane-Ohnuki & Satoh, 2009, MAbs 1(3) :230-26, lida et al., 2009, BMC Cancer 9;58 (the disclosures of which are incorporated herein by reference). Low fucose antibody polypeptides may be produced by expression in cells cultured in a medium containing an inhibitor of mannosidase, such as kinfunensine (see Example I below).

Other methods to modify glycosylation of an antibody into a low fucose format include the use of the bacterial enzyme GDP-6-deoxy-D-lyxo-4-hexulose reductase in cells not able to metabolise rhamnose (e.g. using the GlymaxX® technology of ProBioGen AG, Berlin, Germany).

Another method to create low fucose antibodies is by inhibition or depletion of alpha- (1,6)-fucosyltransferase in the antibody-producing cells (e.g. using the Potelligent® CHOK1SV technology of Lonza Ltd, Basel, Switzerland).

An exemplary heavy chain constant region amino acid sequence which may be combined with any VH region sequence disclosed herein (to form a complete heavy chain) is the IgG1 heavy chain constant region sequence reproduced here:

[SEQ ID NO: 12] Other heavy chain constant region sequences are known in the art and could also be combined with any VH region disclosed herein. For example, as indicated above, a preferred constant region is a modified IgG4 constant region such as that reproduced here:

[SEQ ID NO: 13]

This modified IgG4 sequence results in stabilization of the core hinge of IgG4 making the IgG4 more stable, preventing Fab arm exchange.

Another preferred constant region is a modified IgG4 constant region such as that reproduced here :

[SEQ ID NO: 14]

This modified IgG4 sequence exhibits reduced FcRn binding and hence results in a reduced serum half-life relative to wild type IgG4. In addition, it exhibits stabilization of the core hinge of IgG4 making the IgG4 more stable, preventing Fab arm exchange.

Also suitable for use in the polypeptides of the invention is a wild type IgG4 constant region such as that reproduced here :

[SEQ ID NO: 15]

An exemplary light chain constant region amino acid sequence which may be combined with any VL region sequence disclosed herein (to form a complete light chain) is the kappa chain constant region sequence reproduced here:

[SEQ ID NO: 16]

Other light chain constant region sequences are known in the art and could also be combined with any VL region disclosed herein.

In an exemplary embodiment of the invention, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise the IgG4 constant regions of SEQ ID NOs: 13 and 16, respectively.

Thus, exemplary antibody or antigen-binding fragment thereof that specifically binds to CD137 of the invention comprise:

(a) a heavy chain comprising a variable region of SEQ ID NO: 1 together with a constant region of SEQ ID NO: 13, or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1 and/or 13; and/or

(b) a light chain comprising a variable region of SEQ ID NO: 2 together with a constant region of SEQ ID NO: 16, or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2 and/or 16.

For example, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may be an intact IgG4 molecule comprising or consisting of two heavy chains having an amino acid sequence of SEQ ID NO: 17 and two light chains having an amino acid sequence of SEQ ID NO: 18, or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 17 and/or 18.

Alternative antibody or antigen-binding fragment thereof that specifically binds to CD137 of the invention comprise:

(a) a heavy chain comprising a variable region of SEQ ID NO: 19 together with a constant region of SEQ ID NO: 13 or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 19 or 13; and/or

(b)a light chain comprising a variable region of SEQ ID NO: 20 together with a constant region of SEQ ID NO: 16 or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 20 or 16.

For example, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may be an intact IgG4 molecule comprising or consisting of two heavy chains having an amino acid sequence of SEQ ID NO: 29 and two light chains having an amino acid sequence of SEQ ID NO: 30 or an amino acid sequence having at least 60% sequence identity therewith, for example at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 29 and/or 30.

Modified antibodies or antigen-binding fragments thereof

In one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to CD137 is or comprises a "fusion" polypeptide.

In addition to being fused to a moiety in order to improve pharmacokinetic properties, it will be appreciated that the antibody or antigen-binding fragment thereof that specifically binds to CD137 may also be fused to a polypeptide such as glutathione-S- transferase (GST) or protein A in order to facilitate purification of said antibody or antigen-binding fragment thereof. Examples of such fusions are well known to those skilled in the art. Similarly, the said antibody or antigen-binding fragment thereof that specifically binds to CD137 may be fused to an oligo-histidine tag, such as His6, or to an epitope recognised by an antibody such as the well-known Myc tag epitope. Fusions to any variant or derivative of said antibody or antigen-binding fragment thereof that specifically binds to CD137 are also included in the scope of the invention. It will be appreciated that fusions (or variants, derivatives or fusions thereof) which retain or improve desirable properties, such as IL-1R binding properties or in vivo half-life are preferred.

Thus, the fusion may comprise an amino acid sequence as detailed above together with a further portion which confers a desirable feature on the said antibody or antigen- binding fragment thereof that specifically binds to CD137 of the invention; for example, the portion may useful in detecting or isolating the antibody or antigen-binding fragment thereof that specifically binds to CD137, or promoting cellular uptake of the antibody or antigen-binding fragment thereof that specifically binds to CD137. The portion may be, for example, a biotin moiety, a radioactive moiety, a fluorescent moiety, for example a small fluorophore or a green fluorescent protein (GFP) fluorophore, as well known to those skilled in the art. The moiety may be an immunogenic tag, for example a Myc tag, as known to those skilled in the art or may be a lipophilic molecule or polypeptide domain that is capable of promoting cellular uptake of the antibody or antigen-binding fragment thereof that specifically binds to CD137, as known to those skilled in the art.

It will be appreciated by persons skilled in the art that the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise or consist of one or more amino acids which have been modified or derivatised.

Chemical derivatives of one or more amino acids may be achieved by reaction with a functional side group. Such derivatised molecules include, for example, those molecules in which free amino groups have been derivatised to form amine hydrochlorides, p-toluene sulphonyl groups, carboxy be nzoxy groups, t- butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatised to form salts, methyl and ethyl esters or other types of esters and hydrazides. Free hydroxyl groups may be derivatised to form O-acyl or O-alkyl derivatives. Also included as chemical derivatives are those peptides which contain naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline may be substituted for proline; 5-hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine and ornithine for lysine. Derivatives also include peptides containing one or more additions or deletions as long as the requisite activity is maintained. Other included modifications are amidation, amino terminal acylation (e.g. acetylation or thioglycolic acid amidation), terminal carboxylamidation (e.g. with ammonia or methylamine), and the like terminal modifications. It will be further appreciated by persons skilled in the art that peptidomimetic compounds may also be useful. The term 'peptidomimetic' refers to a compound that mimics the conformation and desirable features of a particular peptide as a therapeutic agent.

For example, the said antibody or antigen-binding fragment thereof that specifically binds to CD137 may include not only molecules in which amino acid residues are joined by peptide (-CO-NH-) linkages but also molecules in which the peptide bond is reversed. Such retro-inverso peptidomimetics may be made using methods known in the art, for example such as those described in Meziere et al. (1997) J. Immunol. 159, 3230-3237, which is incorporated herein by reference. This approach involves making pseudo-peptides containing changes involving the backbone, and not the orientation of side chains. Retro-inverse peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis. Alternatively, the said polypeptide may be a peptidomimetic compound wherein one or more of the amino acid residues are linked by a -y(CH ₂ NH)- bond in place of the conventional amide linkage.

In a further alternative, the peptide bond may be dispensed with altogether provided that an appropriate linker moiety which retains the spacing between the carbon atoms of the amino acid residues is used; it may be advantageous for the linker moiety to have substantially the same charge distribution and substantially the same planarity as a peptide bond.

It will also be appreciated that the said antibody or antigen-binding fragment thereof that specifically binds to CD137 may conveniently be blocked at its N- or C-terminus so as to help reduce susceptibility to exo-proteolytic digestion.

A variety of un-coded or modified amino acids such as D-amino acids and N-methyl amino acids have also been used to modify mammalian peptides. In addition, a presumed bioactive conformation may be stabilised by a covalent modification, such as cyclisation or by incorporation of lactam or other types of bridges, for example see Veber et al. , 1978, Proc. Natl. Acad. Sci. USA 75:2636 and Thursell et al. , 1983, Biochem. Biophys. Res. Comm. 111:166, which are incorporated herein by reference.

Typically, the antibody or antigen-binding fragment thereof that specifically binds to CD137 will be a 'naked' antibody polypeptide, i.e. without any additional functional moieties such as cytotoxic or detectable moieties. For example, where the therapeutic effect is mediated by a direct effect of the antibody or antigen-binding fragment thereof that specifically binds to CD137 on immune cells, e.g. to reduce inflammation, it may be advantageous for the antibody to lack any cytotoxic activity.

However, in alternative embodiment, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may be augmented with a functional moiety to facilitate their intended use, for example as a diagnostic (e.g. in vivo imaging) agent or therapeutic agent. Thus, in one embodiment, the antibody or antigen-binding fragment thereof that specifically binds to CD137 is linked, directly or indirectly, to a therapeutic moiety. A suitable therapeutic moiety is one that is capable of reducing or inhibiting the growth, or in particular killing, a cancer cell (or associated stem cells or progenitor cells). For example, the therapeutic agent may be a cytotoxic moiety, such as a radioisotope (e.g. ⁹⁰ Y, ¹⁷⁷ Lu, ⁹⁹ Tc ^m , etc) or cytotoxic drug (e.g. antimetabolites, toxins, cytostatic drugs, etc).

Alternatively, the cytotoxic moiety may comprise or consist of one or more moieties suitable for use in activation therapy, such as photon activation therapy, neutron activation therapy, neutron-induced Auger electron therapy, synchrotron irradiation therapy or low energy X-ray photon activation therapy.

Optionally, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may further comprise a detectable moiety. For example, a detectable moiety may comprise or consist of a radioisotope, such as a radioisotope selected from the group consisting of ^99m Tc, ^m In, ⁶⁷ Ga, ⁶⁸ Ga, ⁷² As, ⁸⁹ Zr, ¹²³ I and ²⁰¹ TI Optionally, the agent may comprise a pair of detectable and cytotoxic radionuclides, such as ⁸⁶ Y/ ⁹⁰ Y or ¹²⁴ I/ ²¹¹ At. Alternatively, the antibody or antigen-binding fragment thereof that specifically binds to CD137 may comprise a radioisotope that is capable of simultaneously acting in a multi-modal manner as a detectable moiety and also as a cytotoxic moiety to provide so-called "Multimodality theragnostics". The binding moieties may thus be coupled to nanoparticles that have the capability of multi-imaging (for example, SPECT, PET, MRI, Optical, or Ultrasound) together with therapeutic capability using cytotoxic drugs, such as radionuclides or chemotherapy agents.

Therapeutic and/or detectable moieties (such as a radioisotope, cytotoxic moiety or the like) may be linked directly, or indirectly, to the antibody or fragment thereof. Suitable linkers are known in the art and include, for example, prosthetic groups, non- phenolic linkers (derivatives of N-succimidyl- benzoates; dodecaborate), chelating moieties of both macrocyclics and acyclic chelators, such as derivatives of 1,4,7,10- tetraazacyclododecane-1, 4, 7, 10, tetraacetic acid (DOTA), deferoxamine (DFO), derivatives of diethylenetriaminepentaacetic avid (DTPA), derivatives of S-2-(4- Isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triaceti c acid (NOTA) and derivatives of 1,4,8,ll-tetraazacyclodocedan-1,4,8,ll-tetraacetic acid (TETA), derivatives of 3,6,9,15-Tetraazabicyclo[9.3.1]-pentadeca-l(15),ll,13-triene -4-(S)- (4-isothiocyanato-benzyl)-3,6,9-triacetic acid (PCTA), derivatives of 5-S-(4- Aminobenzyl)-l-oxa-4,7,10- triazacyclododecane-4,7,10-tris(acetic acid) (DO3A) and other chelating moieties.

One preferred linker is DTPA, for example as used in ¹⁷⁷ Lu-DTPA-[antibody or antigen- binding fragment thereof that specifically binds to CD137]. A further preferred linker is deferoxamine, DFO, for example as used in ⁸⁹ Zr-DFO-[antibody or antigen-binding fragment thereof that specifically binds to CD137].

However, it will be appreciated by persons skilled in the art that many medical uses of the antibody or antigen-binding fragment thereof that specifically binds to CD137 will not require the presence of a cytotoxic or diagnostic moiety.

As discussed above, methods for the production of antibody or antigen-binding fragment thereof that specifically binds to CD137 are well known in the art.

Conveniently, the antibody or antigen-binding fragment thereof that specifically binds to CD137 is or comprises a recombinant polypeptide. Suitable methods for the production of such recombinant polypeptides are well known in the art, such as expression in prokaryotic or eukaryotic hosts cells (for example, see Green & Sambrook, 2012, Molecular Cloning, A Laboratory Manual, Fourth Edition, Cold Spring Harbor, New York, the relevant disclosures in which document are hereby incorporated by reference).

Although the antibody or antigen-binding fragment thereof that specifically binds to CD137 may be a polyclonal, it is preferred if it is a monoclonal antibody, or that the antigen-binding fragment, variant, fusion or derivative thereof, is derived from a monoclonal antibody.

Suitable monoclonal antibodies may be prepared by known techniques, for example those disclosed in "Monoclonal Antibodies; A manual of techniques", H Zola (CRC Press, 1988) and in "Monoclonal Hybridoma Antibodies: Techniques and Application", SGR Murrell (CRC Press, 1982). Polyclonal antibodies may be produced which are poly- specific or mono-specific. It is preferred that they are mono-specific.

The antibody or antigen-binding fragment thereof that specifically binds to CD137 can also be produced using a commercially available in vitro translation system, such as rabbit reticulocyte lysate or wheatgerm lysate (available from Promega). Preferably, the translation system is rabbit reticulocyte lysate. Conveniently, the translation system may be coupled to a transcription system, such as the TNT transcription- translation system (Promega). This system has the advantage of producing suitable mRNA transcript from an encoding DNA polynucleotide in the same reaction as the translation.

It will be appreciated by persons skilled in the art that antibody or antigen-binding fragment thereof that specifically binds to CD137 may alternatively be synthesised artificially, for example using well known liquid-phase or solid phase synthesis techniques (such as t-Boc or Fmoc solid-phase peptide synthesis).

The Fc region may be naturally-occurring (e.g. part of an endogenously produced antibody) or may be artificial (e.g. comprising one or more point mutations relative to a naturally-occurring Fc region). A variant of an Fc region typically binds to Fc receptors, such as FcγR and/or neonatal Fc receptor (FcRn) with altered affinity providing for improved function and/or half-life of the polypeptide. The biological function and/ or the half-life may be either increased or a decreased relative to the half-life of a polypeptide comprising a native Fc region. Examples of such biological functions which may be modulated by the presence of a variant Fc region include antibody dependent cell cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/or apoptosis.

Thus, the Fc region may be naturally-occurring (e.g. part of an endogenously produced human antibody) or may be artificial (e.g. comprising one or more point mutations relative to a naturally-occurring human Fc region).

As is well documented in the art, the Fc region of an antibody mediates its serum half- life and effector functions, such as complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell phagocytosis (ADCP). Fc regions may be engineered as described above in relation to the CD137 antibodies and antigen-binding fragments thereof.

The term "antibody" as referred to herein includes whole antibodies and any antigen binding fragment (i.e., "antigen-binding portion") or single chains thereof. An antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

By "an antibody or an antigen-binding fragment thereof" we include substantially intact antibody molecules, as well as chimeric antibodies, humanised antibodies, isolated human antibodies, single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and/or light chains, and antigen-binding fragments and derivatives of the same. Suitable antigen-binding fragments and derivatives include, but are not necessarily limited to, Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g. Fab fragments, Fab' fragments and F(ab)2 fragments), single variable domains (e.g. V _H and V _L domains) and domain antibodies (dAbs, including single and dual formats [i.e., dAb-linker-dAb]). The potential advantages of using antibody fragments, rather than whole antibodies, are several-fold. The smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue. Moreover, antigen-binding fragments such as Fab, Fv, ScFv and dAb antibody fragments can be expressed in and secreted from E. coli, thus allowing the facile production of large amounts of the said fragments.

For example, the antigen-binding fragment may comprise an scFv molecule, i.e. wherein the V _H and V _L partner domains are linked via a flexible oligopeptide. Heavy chains can be of any isotype, including IgG (IgG1, IgG2, IgG3 and IgG4 subtypes), IgA (IgA1 and IgA2 subtypes), IgM and IgE.

Light chains include kappa chains and lambda chains.

Antibodies include, but are not limited to, synthetic antibodies, monoclonal antibodies, single domain antibodies, single chain antibodies, recombinantly produced antibodies, multi-specific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, intrabodies, scFvs (e.g. including mono-specific and bi- specific, etc.), Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies, and epitope-binding fragments of any of the above.

Of particular relevance are antibodies and their antigen-binding fragments thereof that have been "isolated" so as to exist in a physical milieu distinct from that in which it may occur in nature or that have been modified so as to differ from a naturally occurring antibody in amino acid sequence.

The phrase "an antibody or an antigen-binding fragment thereof" is also intended to encompass antibody mimics (for example, non-antibody scaffold structures that have a high degree of stability yet allow variability to be introduced at certain positions). Those skilled in the art of biochemistry will be familiar with many such molecules, as discussed in Gebauer & Skerra, 2009, Curr Opin Chem Biol 13(3): 245-255 (the disclosures of which are incorporated herein by reference). Exemplary antibody mimics include: affibodies (also called Trinectins; Nygren, 2008, FEBS J, 275, 2668-2676); CTLDs (also called Tetranectins; Innovations Pharmac. Technol. (2006), 27-30); adnectins (also called monobodies; Meth. Mol. Biol., 352 (2007), 95-109); anticalins (Drug Discovery Today (2005), 10, 23-33); DARPins (ankyrins; Nat. Biotechnol. (2004), 22, 575-582); avimers (Nat. Biotechnol. (2005), 23, 1556-1561); microbodies (FEBS J, (2007), 274, 86-95); peptide aptamers (Expert. Opin. Biol. Ther. (2005), 5, 783-797); Kunitz domains (J. Pharmacol. Exp. Ther. (2006) 318, 803-809); affilins (Trends. Biotechnol. (2005), 23, 514-522); affimers (Avacta Life Sciences, Wetherby, UK).

Persons skilled in the art will further appreciate that the invention also encompasses modified versions of antibodies and antigen-binding fragments thereof, whether existing now or in the future, e.g. modified by the covalent attachment of polyethylene glycol or another suitable polymer (see below). An antibody may be a polyclonal antibody or a monoclonal antibody. The antibody may be produced by any suitable method.

Methods of generating antibodies and antibody fragments are well known in the art. For example, antibodies may be generated via any one of several methods which employ induction of in vivo production of antibody molecules, screening of immunoglobulin libraries (Orlandi. et al, 1989. Proc. Natl. Acad. Sci. U.S.A. 86:3833- 3837; Winter et al. , 1991, Nature 349:293-299, the disclosures of which are incorporated herein by reference) or generation of monoclonal antibody molecules by cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Barr virus (EBV)-hybridoma technique (Kohler et al., 1975. Nature 256:4950497; Kozbor et al., 1985. 3. Immunol. Methods 81:31-42; Cote et al., 1983. Proc. Natl. Acad. Sci. USA 80:2026-2030; Cole et al., 1984. Mol. Cell. Biol. 62:109-120, the disclosures of which are incorporated herein by reference).

Suitable methods for the production of monoclonal antibodies are also disclosed in "Monoclonal Antibodies: A manual of techniques", H Zola (CRC Press, 1988, the disclosures of which are incorporated herein by reference) and in "Monoclonal Hybridoma Antibodies: Techniques and Applications", J G R Murrell (CRC Press, 1982, the disclosures of which are incorporated herein by reference).

Likewise, antibody fragments can be obtained using methods well known in the art (see, for example, Harlow & Lane, 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, New York, the disclosures of which are incorporated herein by reference). For example, antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coll or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Alternatively, antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.

The term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen, such as CD137. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include a Fab fragment, a F(ab')2 fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a dAb fragment and an isolated complementarity determining region (CDR). Single chain antibodies such as scFv and heavy chain antibodies such as VHH and camel antibodies are also intended to be encompassed within the term "antigen- binding portion" of an antibody. These antibody fragments may be obtained using conventional techniques known to those of skill in the art, and the fragments may be screened for utility in the same manner as intact antibodies.

The terms "binding activity" and "binding affinity" are intended to refer to the tendency of a molecule (e.g. an antibody molecule antigen binding fragment thereof) to bind or not to bind to a target. Binding affinity may be quantified by determining the dissociation constant (Kd) for an antibody and its target. Similarly, the specificity of binding of an antibody to its target may be defined in terms of the comparative dissociation constants (Kd) of the antibody for its target as compared to the dissociation constant with respect to the antibody and another, non-target molecule.

Typically, the Kd for the antibody with respect to the target will be 2-fold, preferably 5-fold, more preferably 10-fold less than Kd with respect to the other, non-target molecule such as unrelated material or accompanying material in the environment. More preferably, the Kd will be 50- fold less, even more preferably 100-fold less, and yet more preferably 200-fold less.

The value of this dissociation constant can be determined directly by well-known methods, and can be computed even for complex mixtures by methods such as those, for example, set forth in Caceci et al. (Byte 9:340-362, 1984). For example, the Kd may be established using a double-filter nitrocellulose filter binding assay such as that disclosed by Wong & Lohman (Proc. Natl. Acad. Sci. USA 90, 5428-5432, 1993). Other standard assays to evaluate the binding ability of ligands such as antibodies towards targets are known in the art, including for example, ELISAs, Western blots, RIAs, and flow cytometry analysis. The binding kinetics (e.g., binding affinity) of the antibody also can be assessed by standard assays known in the art, such as by Biacore™ system analysis.

A competitive binding assay can be conducted in which the binding of the antibody to the target is compared to the binding of the target by another, known ligand of that target, such as another antibody. The concentration at which 50% inhibition occurs is known as the Ki. Under ideal conditions, the Ki is equivalent to Kd. The Ki value will never be less than the Kd, so measurement of Ki can conveniently be substituted to provide an upper limit for Kd. An anti-CD137 antibody or antigen binding fragment thereof is preferably capable of binding to its target with an affinity that is at least two-fold, 10-fold, 50-fold, 100-fold or greater than its affinity for binding to another non-target molecule.

An antibody or antigen binding fragment thereof that specifically binds to CD137 for use in the methods of the invention may be a human antibody. The term "human antibody", as used herein, is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences - such antibodies are typically referred to as chimeric or humanised.

A human antibody or antigen binding fragment thereof that specifically binds to CD137 for use the methods of the invention is typically a human monoclonal antibody, or antigen binding fragment thereof. Such a human monoclonal antibody may be produced by a hybridoma which includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell. Human antibodies may also be prepared by in vitro immunisation of human lymphocytes followed by transformation of the lymphocytes with Epstein-Barr virus. The term "human antibody derivatives" refers to any modified form of the human antibody, e.g., a conjugate of the antibody and another agent or antibody.

An antibody or antigen binding fragment thereof that specifically binds to CD137 for use in the methods of the invention may alternatively be a humanised antibody, or antigen binding fragment thereof.

The term "humanised" refers to an antibody molecule, generally prepared using recombinant techniques, having aann antigen binding site derived from an immunoglobulin from a non-human species and a remaining immunoglobulin structure based upon the structure and /or sequence of a human immunoglobulin. The antigen- binding site may comprise either complete non-human antibody variable domains fused to human constant domains, or only the complementarity determining regions (CDRs) of such variable domains grafted to appropriate human framework regions of human variable domains. The framework residues of such humanised molecules may be wild type (e.g., fully human) or they may be modified to contain one or more amino acid substitutions not found in the human antibody whose sequence has served as the basis for humanization. Humanization lessens or eliminates the likelihood that a constant region of the molecule will act as an immunogen in human individuals, but the possibility of an immune response to the foreign variable region remains (LoBuglio, A.F. et al. (1989) "Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics And Immune Response," Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224). Another approach focuses not only on providing human-derived constant regions, but modifying the variable regions as well so as to reshape them as closely as possible to human form. It is known that the variable regions of both heavy and light chains contain three complementarity- determining regions (CDRs) which vary in response to the antigens in question and determine binding capability, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide a scaffolding for the CDRs. When nonhuman antibodies are prepared with respect to a particular antigen, the variable regions can be "reshaped" or "humanised" by grafting CDRs derived from nonhuman antibody on the FRs present in the human antibody to be modified. Application of this approach to various antibodies has been reported by Sato, K. et al. (1993) Cancer Res 53:851-856. Riechmann, L. et al. (1988) "Reshaping Human Antibodies for Therapy," Nature 332:323-327; Verhoeyen, M. et al. (1988) "Reshaping Human Antibodies: Grafting An Antilysozyme Activity," Science 239:1534- 1536; Kettleborough, C. A. et al. (1991) "Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting: The Importance Of Framework Residues On Loop Conformation," Protein Engineering 4:773-3783; Maeda, H. et al. (1991) "Construction Of Reshaped Human Antibodies With HIV-Neutralizing Activity," Human Antibodies Hybridoma 2:124-134; Gorman, S. D. et al. (1991) "Reshaping A Therapeutic CD4 Antibody," Proc. Natl. Acad. Sci. (U.S.A.) 88:4181-4185; Tempest, P.R. et al. (1991) "Reshaping A Human Monoclonal Antibody To Inhibit Human Respiratory Syncytial Virus Infection in vivo," Bio/Technology 9:266-271; Co, M. S. et al. (1991) "Humanized Antibodies For Antiviral Therapy," Proc. Natl. Acad. Sci. (U.S.A.) 88:2869-2873; Carter, P. et al. (1992) "Humanization Of An Anti-pl85her2 Antibody For Human Cancer Therapy," Proc. Natl. Acad. Sci. (U.S.A.) 89:4285-4289; and Co, M.S. et al. (1992) "Chimeric And Humanized Antibodies With Specificity For The CD33 Antigen," J. Immunol. 148:1149-1154. In some embodiments, humanised antibodies preserve all CDR sequences (for example, a humanized mouse antibody which contains all six CDRs from the mouse antibodies). In other embodiments, humanised antibodies have one or more CDRs (one, two, three, four, five, six) which are altered with respect to the original antibody, which are also termed one or more CDRs "derived from" one or more CDRs from the original antibody. The ability to humanise an antigen is well known (see, e.g., US Patents No. 5,225,539; 5,530,101; 5,585,089; 5,859,205; 6,407,213; 6,881,557).

In one embodiment, the antibodies and antigen binding fragments thereof that specifically bind CD137 are defined by reference to the variable regions of reference antibodies 1630/1631 and 2674/2675.

The antibody may be or may comprise a variant or a fragment of one of the specific antibodies disclosed herein, provided that said variant or fragment retains specificity for its target. For example, the antibody may be or may comprise a variant or a fragment of one of the specific anti-CD137 antibodies disclosed herein, provided that said variant or fragment retains specificity for CD137.

A fragment is preferably an antigen binding portion of a said antibody. A fragment may be made by truncation, e.g. by removal of one or more amino acids from the N and/or C-terminal ends of a polypeptide. Up to 10, up to 20, up to 30, up to 40 or more amino acids may be removed from the N and/or C terminal in this way. Fragments may also be generated by one or more internal deletions.

A variant may comprise one or more substitutions, deletions or additions with respect to the sequences of a specific anti-CD137 antibody. A variant may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and/or deletions from the specific sequences disclosed herein. "Deletion" variants may comprise the deletion of individual amino acids, deletion of small groups of amino acids such as 2, 3, 4 or 5 amino acids, or deletion of larger amino acid regions, such as the deletion of specific amino acid domains or other features. "Substitution" variants preferably involve the replacement of one or more amino acids with the same number of amino acids and making conservative amino acid substitutions. For example, an amino acid may be substituted with an alternative amino acid having similar properties, for example, another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid or another aliphatic amino acid. Some properties of the 20 main amino acids which can be used to select suitable substituents are as follows:

Preferred "variants" include those in which instead of the naturally occurring amino acid the amino acid which appears in the sequence is a structural analog thereof. Amino acids used in the sequences may also be derivatized or modified, e.g. labelled, providing the function of the antibody is not significantly adversely affected.

Variants may be prepared during synthesis of the antibody or by post- production modification, or when the antibody is in recombinant form using the known techniques of site- directed mutagenesis, random mutagenesis, or enzymatic cleavage and/or ligation of nucleic acids.

Preferably variant antibodies have an amino acid sequence which has more than 60%, or more than 70%, e.g. 75 or 80%, preferably more than 85%, e.g. more than 90 or 95% amino acid identity to the VL or VH domain of an antibody disclosed herein. This level of amino acid identity may be seen across the full length of the relevant SEQ ID NO sequence or over a part of the sequence, such as across 20, 30, 50, 75, 100, 150, 200 or more amino acids, depending on the size of the full length polypeptide.

In connection with amino acid sequences, "sequence identity" refers to sequences which have the stated value when assessed using ClustalW (Thompson et al., 1994, supra) with the following parameters: Pairwise alignment parameters -Method: accurate, Matrix: PAM, Gap open penalty: 10.00, Gap extension penalty: 0.10;

Multiple alignment parameters -Matrix: PAM, Gap open penalty: 10.00, % identity for delay: 30, Penalize end gaps: on, Gap separation distance: 0, Negative matrix: no, Gap extension penalty: 0.20, Residue-specific gap penalties: on, Hydrophilic gap penalties: on, Hydrophilic residues: GPSNDQEKR. Sequence identity at a particular residue is intended to include identical residues which have simply been derivatized.

An anti-CD137 antibody or antigen-binding fragment thereof that specifically binds to CD137 of the invention may bind to the same epitope as a specific antibody as disclosed herein (e.g. an anti-CD137 antibody may bind domain 2 of CD137), since such an antibody is likely to mimic the action of the disclosed antibody. Whether or not an antibody binds to the same epitope as another antibody may be determined by routine methods. For example, the binding of each antibody to a target may be using a competitive binding assay. Methods for carrying out competitive binding assays are well known in the art. For example they may involve contacting together an antibody and a target molecule under conditions under which the antibody can bind to the target molecule. The antibody/target complex may then be contacted with a second (test) antibody and the extent to which the test antibody is able to displace the first antibody from antibody/target complexes may be assessed. Such assessment may use any suitable technique, including, for example, Surface Plasmon Resonance, ELISA, or flow cytometry. The ability of a test antibody to inhibit the binding of a first antibody to the target demonstrates that the test antibody can compete with said first antibody for binding to the target and thus that the test antibody binds to the same epitope or region on the target as the first antibody, and may therefore mimic the action of the first antibody.

Any antibody or antigen-binding fragment thereof referred to herein may be provided in isolated form or may optionally be provided linked (directly or indirectly) to another moiety. The other moiety may be a therapeutic molecule such as a cytotoxic moiety or a drug.

The therapeutic molecule may be directly attached, for example by chemical conjugation, to an antibody of the invention. Methods for conjugating molecules to an antibody are known in the art. For example, carbodiimide conjugation (Bauminger & Wilchek (1980) Methods Enzymol. 70, 151-159) may be used to conjugate a variety of agents, including doxorubicin, to antibodies or peptides. The water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) is particularly useful for conjugating a functional moiety to a binding moiety.

Other methods for conjugating a moiety to antibodies can also be used. For example, sodium periodate oxidation followed by reductive alkylation of appropriate reactants can be used, as can glutaraldehyde cross-linking. However, it is recognised that, regardless of which method of producing a conjugate of the invention is selected, a determination must be made that the antibody maintains its targeting ability and that the functional moiety maintains its relevant function.

A cytotoxic moiety may be directly and/or indirectly cytotoxic. By "directly cytotoxic" it is meant that the moiety is one which on its own is cytotoxic. By "indirectly cytotoxic" it is meant that the moiety is one which, although is not itself cytotoxic, can induce cytotoxicity, for example by its action on a further molecule or by further action on it. The cytotoxic moiety may be cytotoxic only when intracellular and is preferably not cytotoxic when extracellular.

The antibody or antigen-binding fragment is linked to a cytotoxic moiety which is a directly cytotoxic chemotherapeutic agent. Optionally, the cytotoxic moiety is a directly cytotoxic polypeptide. Cytotoxic chemotherapeutic agents are well known in the art.

Cytotoxic chemotherapeutic agents, such as anticancer agents, include: alkylating agents including nitrogen mustards such aass mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin) aanndd chlorambucil; ethylenimines and methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulphonates such as busulfane; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU) and streptozocin (streptozotocin); and triazenes such as decarbazine (DTIC; dimethyltriazenoimidazole-carboxamide); Antimetabolites including folic acid analogues such as methotrexate (amethopterin); pyrimidine analogues such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) and cytarabine (cytosine arabinoside); and purine analogues and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6- thioguanine; TG) and pentostatin (2'-deoxycoformycin). Natural Products including vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C); enzymes such as L-asparaginase; and biological response modifiers such as interferon alphenomes. Miscellaneous agents including platinum coordination complexes such as cisplatin (cis-DDP) and carboplatin; anthracenedione such as mitoxantrone and anthracycline; substituted urea such as hydroxyurea; methyl hydrazine derivative such as procarbazine (N-methylhydrazine, MIH); and adrenocortical suppressant such aass mitotane (o,p'-DDD) and aminoglutethimide; taxol aanndd analogues/derivatives; and hormone agonists/antagonists such as flutamide and tamoxifen.

The cytotoxic moiety may be a cytotoxic peptide or polypeptide moiety which leads to cell death. Cytotoxic peptide and polypeptide moieties are well known in the art and include, for example, ricin, abrin, Pseudomonas exotoxin, tissue factor and the like. Methods for linking them to targeting moieties such as antibodies are also known in the art. Other ribosome inactivating proteins are described as cytotoxic agents in WO 96/06641. Pseudomonas exotoxin may also be used as the cytotoxic polypeptide. Certain cytokines, such as TNFo and IL-2, may also be useful as cytotoxic agents.

Certain radioactive atoms may also be cytotoxic if delivered in sufficient doses. Thus, the cytotoxic moiety may comprise a radioactive atom which, in use, delivers a sufficient quantity of radioactivity to the target site so as to be cytotoxic. Suitable radioactive atoms include phosphorus-32, iodine-125, iodine-131, indium-ill, rhenium-186, rhenium-188 or yttrium-90, or any other isotope which emits enough energy to destroy neighbouring cells, organelles or nucleic acid. Preferably, the isotopes and density of radioactive atoms in the agents of the invention are such that a dose of more than 4000 cGy (preferably at least 6000, 8000 or 10000 cGy) is delivered to the target site and, preferably, to the cells at the target site and their organelles, particularly the nucleus.

The radioactive atom may be attached to the antibody, antigen-binding fragment, variant, fusion or derivative thereof in known ways. For example, EDTA or another chelating agent may be attached to the binding moiety and used to attach 111ln or 90Y. Tyrosine residues may be directly labelled with 1251 or 131I.

The cytotoxic moiety may be a suitable indirectly-cytotoxic polypeptide. The indirectly cytotoxic polypeptide may be a polypeptide which has enzymatic activity and can convert a non-toxic and/or relatively non-toxic prodrug into a cytotoxic drug. With antibodies, this type of system is often referred to as ADEPT (Antibody-Directed Enzyme Prodrug Therapy). The system requires that the antibody locates the enzymatic portion to the desired site in the body of the patient and after allowing time for the enzyme to localise at the site, administering a prodrug which is a substrate for the enzyme, the end product of the catalysis being a cytotoxic compound. The object of the approach is to maximise the concentration of drug at the desired site and to minimise the concentration of drug in normal tissues. The cytotoxic moiety may be capable of converting a non-cytotoxic prodrug into a cytotoxic drug.

The enzyme and prodrug of the system using a targeted enzyme as described herein may be any of those previously proposed. The cytotoxic substance may be any existing anti-cancer drug such as an alkylating agent; an agent which intercalates in DNA; an agent which inhibits any key enzymes such as dihydrofolate reductase, thymidine synthetase, ribonucleotide reductase, nucleoside kinases or topoisomerase; or an agent which effects cell death by interacting with any other cellular constituent. Etoposide is an example of a topoisomerase inhibitor.

Reported prodrug systems include those listed in Table 2, below.

Suitable enzymes for forming part of an enzymatic portion include: exopeptidases, such as carboxypeptidases G, G1 and G2 (for glutamylated mustard prodrugs), carboxypeptidases A and B (for MTX-based prodrugs) and aminopeptidases (for 2-a- aminocyl MTC prodrugs); endopeptidases, such as e.g. thrombolysin (for thrombin prodrugs); hydrolases, such as phosphatases (e.g. alkaline phosphatase) or sulphatases (e.g. aryl sulphatases) (for phosphylated or sulphated prodrugs); amidases, such as penicillin amidases and arylacyl amidase; lactamases, such as β- lactamases; glycosidases, such as β-glucuronidase (for β-glucuronomide anthracyclines), a-galactosidase (for amygdalin) and β-galactosidase (for β-galactose anthracycline); deaminases, such as cytosine deaminase (for 5FC); kinases, such as urokinase and thymidine kinase (for gancyclovir); reductases, such as nitroreductase (for CB1954 and analogues), azoreductase (for azobenzene mustards) and DT- diaphorase (for CB1954); oxidases, such as glucose oxidase (for glucose), xanthine oxidase (for xanthine) and lactoperoxidase; DL-racemases, catalytic antibodies and cyclodextrins.

Preferably, the prodrug is relatively non-toxic compared to the cytotoxic drug. Typically, it has less than 10% of the toxicity, preferably less than 1% of the toxicity as measured in a suitable in vitro cytotoxicity test.

It is likely that the moiety which is able to convert a prodrug to a cytotoxic drug will be active in isolation from the rest of the agent of the invention but it is necessary only for it to be active when (a) it is in combination with the rest of the anti-CD137 antibody or antigen-binding fragment thereof of the invention and (b) the anti-CD137 antibody or antigen-binding fragment thereof of the invention is attached to, adjacent to or internalised in target cells. When each moiety is a polypeptide, the two portions may be linked together by any of the conventional ways of cross-linking polypeptides. For example, the antibody or antigen-binding fragment may be enriched with thiol groups and the further moiety reacted with a bifunctional agent capable of reacting with those thiol groups, for example the N-hydroxysuccinimide ester of iodoacetic acid (NHIA) or N-succinimidyl- 3-(2-pyridyldithio)propionate (SPDP). Amide and thioether bonds, for example achieved with m-maleimidobenzoyl-N-hydroxysuccinimide ester, are generally more stable in vivo than disulphide bonds.

The cytotoxic moiety may be a radiosensitizer. Radiosensitizers include fluoropyrimidines, thymidine analogues, hydroxyurea, gemcitabine, fludarabine, nicotinamide, halogenated pyrimidines, 3-aminobenzamide, 3-aminobenzodiamide, etanixadole, pimonidazole and misonidazole. Also, delivery of genes into cells can radiosensitise them, for example delivery of the p53 gene or cyclin D. The further moiety may be one which becomes cytotoxic, or releases a cytotoxic moiety, upon irradiation. For example, the boron-10 isotope, when appropriately irradiated, releases α pprticles which are cytotoxic. Similarly, the cytotoxic moiety may be one which is useful in photodynamic therapy such as photofrin.

Dosages, and dosage regimes

In the context of the present invention, the dosage of about 500 mg or more of the antibody or antigen-binding fragment thereof that specifically binds to CD137 is the dosage administered to the patient per administration. This definition of dosage per administration may be referred to as a 'unit dosage' or a 'single dosage'. As described further herein, the defined dosage can be, and often will be, administered once or more; to put it another way, the patient of the present invention can receive one or more of the about 500 mg or more unit dosages of the antibody or antigen-binding fragment thereof that specifically binds to CD137.

As would be known to one skilled in medicine, the dosages described herein without reference to the weight of the patient are known as a 'flat dosage' or a 'fixed dosage'. It would be appreciated by one skilled in medicine that dosages that are equivalent to those described herein could be expressed using other metrics, such as a dosage calculated based on the weight of the patient.

For the avoidance of doubt, the dosage as expressed in mg relates to the amount of the antibody or antigen-binding fragment thereof that specifically binds to CD137, and not to any other components and/or ingredients of any pharmaceutical composition in which the antibody or antigen-binding fragment thereof that specifically binds to CD137 is formulated.

As shown in the Examples, the dosages described here are therapeutically effective, so are an 'therapeutically effective amount', which might be referred to as an 'effective amount' or as being 'therapeutically effective'.

In one embodiment, the dosage is about 600 mg or more, for example: about 650 mg or more; about 700 mg or more; about 750 mg or more; about 800 mg or more; about 850 mg or more; about 900 mg or more; about 950 mg or more; about 1000 mg or more; about 1050 mg or more; about 1100 mg or more; about 1150 mg or more; about 1200 mg or more; about 1250 mg or more; about 1300 mg or more; about 1350 mg or more; about 1400 mg or more; about 1450 mg or more; about 1500 mg or more; about 1600 mg or more; about 1700 mg or more; about 1800 mg or more; about 1900 mg or more; or about 2000 mg or more.

In one embodiment, the dosage is about 500 mg to about 2000 mg, for example: about 500 mg to about 1900 mg; about 500 mg to about 1800 mg; about 500 mg to about 1700 mg; about 500 mg to about 1600 mg; about 500 mg to about 1500 mg; about 500 mg to about 1400 mg; about 500 mg to about 1300 mg; about 500 mg to about 1200 mg; about 500 mg to about 1100 mg; about 500 mg to about 1000 mg; about 500 mg to about 900 mg; about 600 mg to about 2000 mg; 600 mg to about 1900 mg; about 600 mg to about 1800 mg; about 600 mg to about 1700 mg; about 600 mg to about 1600 mg; about 600 mg to about 1500 mg; about 600 mg to about 1400 mg; about 600 mg to about 1300 mg; about 600 mg to about 1200 mg; about 600 mg to about 1100 mg; about 600 mg to about 1000 mg; or about 600 mg to about 900 mg.

In a preferred embodiment, the dosage is about 600 mg to about 1500 mg. In an alternative preferred embodiment, the dosage is about 600 mg to about 900 mg.

As described in the Examples, a dosage of about 600 mg to about 900 mg is particularly preferred because those high dosages have an impressive safety profile, and a good efficacy.

In a preferred embodiment, the dosage is about 600 mg. In an alternative preferred embodiment, the dosage is about 900 mg. In one embodiment, the dosage of the antibody or antigen-binding fragment thereof that specifically binds to CD137 is administered for about 30 minutes or more, for example: about 45 minutes or more; about 1 hour or more; about 1 hour 30 minutes or more; about 2 hours or more; about 2 hours 30 minutes or more; about 3 hours or more; about 3 hours 30 minutes or more; about 4 hours or more; about 4 hours 30 minutes or more; or about 5 hours or more.

In one embodiment, the dosage of the antibody or antigen-binding fragment thereof that specifically binds to CD137 is administered for about 30 minutes to about five hours; for example, about one hour to about five hours; about two hours to about five hours; about one hour to about four hours; or about one hour to about three hours.

In a preferred embodiment, the dosage of the antibody or antigen-binding fragment thereof that specifically binds to CD137 is administered for about one hour to about three hours, preferably the antibody or antigen-binding fragment thereof is administered for about two hours.

In one embodiment, the dosage of the antibody or antigen-binding fragment thereof that specifically binds to CD137 is administered twice or more, for example: about three or more times; about four or more times; about five or more times; about six or more times; about seven or more times; about eight or more times; about nine or more times; or about ten or more times.

In one embodiment, the dosage of the antibody or antigen-binding fragment thereof that specifically binds to CD137 is administered every about 7 or more days, for example: every about 14 or more days; every about 21 or more days; every about 28 or more days; every about 35 or more days; or every about 42 or more days. A period of time expressed as such relates to the time between dosages of the antibody or antigen-binding fragment thereof being administered to the patient. This can be referred to as a 'treatment cycle'; to put it another way, the dosage of the antibody or antigen-binding fragment thereof being administered every 21 days can be referred to as a treatment cycle of 21 days.

As will be appreciated by one skilled in medicine, the overall period of time that the antibody or antigen-binding fragment thereof that specifically binds to CD137 is administered can be referred to as a course of treatment; for example, if the antibody or antigen-binding fragment thereof that specifically binds to CD137 is administered every 21 days four times then the course of treatment could be referred to as 84 days. A course of treatment can be finished for a number of reasons; for example: a planned treatment regime; the curing of the cancer; and/or a sufficient reduction in cancer symptoms without cure.

As will also be appreciated, a patient might receive one or more course of treatment, separated by any period of time; for example, separated by about 1 or more months, such as: about two or more months; about three or more months; about four or more months; about six or more months; about seven or more months; about eight or more months; about nine or more months; about ten or more months; about 11 or more months; about one or more year; about two or more years; about three or more years; about four or more years; or about five or more years.

In a preferred embodiment, the dosage of the antibody or antigen-binding fragment thereof that specifically binds to CD137 is administered about every 14 days to about every 28 days. In a more preferred embodiment, the dosage of the antibody or antigen-binding fragment thereof that specifically binds to CD137 is administered about every 21 days.

In a preferred embodiment, the dosage of the antibody or antigen-binding fragment thereof that specifically binds to CD137 is administered once daily.

As disclosed further below, the antibody or antigen-binding fragment thereof that specifically binds to CD137 can be formulated into various pharmaceutical compositions, and/or can be administered via numerous administration routes. In a particularly preferred embodiment, the antibody or antigen-binding fragment thereof is administered intravenously, preferably the antibody or antigen-binding fragment thereof is administered via an intravenous infusion.

It will be appreciated that based on the disclosure of the present invention, any particular combination of the above features of the dosage could be combined into a particular dosage regime.

For example, in one embodiment, the dosage of the antibody or antigen-binding fragment thereof that specifically binds to CD137 is about 600 mg to about 900 mg, administered for about two hours via an intravenous infusion, once daily, and about every 21 days. In a preferred embodiment, the dosage of the antibody or antigen- binding fragment thereof that specifically binds to CD137 is about 600 mg, administered for about two hours via an intravenous infusion, once daily, and about every 21 days. In an alternative preferred embodiment, the dosage of the antibody or antigen-binding fragment thereof that specifically binds to CD137 is about 900 mg, administered for about two hours via an intravenous infusion, once daily, and about every 21 days.

In some embodiments, prior to one or more (preferably each) of the antibody or antigen-binding fragment thereof administrations, the patient is also administered one or more (preferably all) of the following:

• an acetaminophen (such as paracetamol); for example, at an amount of 650-1000 mg, preferably by oral administration;

• an antihistamine (such as diphenhydramine); for example, at an amount of 50 mg, preferably by oral administration or intravenously;

• a glucocorticoid (such as prednisolone); for example, at an amount of 100 mg, preferably by oral administration;

• a H2 antagonist (such as ranitidine); for example, at an amount of 50 mg; and

• an antiemetic (such as ondansetron); for example, at an amount of 16- 24 mg, optionally wherein those are administered about 30 to about 120 minutes prior to the administration of the antibody or antigen-binding fragment thereof.

Patients and cancers to be treated

It will be further appreciated by persons skilled in the art that the antibodies or antigen- binding fragments thereof that specifically bind to CD137 have utility in both the medical and veterinary fields. Thus, the invention may be used in the treatment of both human and non-human animals (such as horses, dogs and cats). Preferably, however, the patient is human.

By 'treatment' we include 'prophylactic treatment', 'therapeutic treatment', and 'palliative treatment' of the patient.

The term 'prophylactic treatment' is used to encompass the use of the antibodies or antigen-binding fragments thereof that specifically bind to CD137, as described herein, which either prevents or reduces the likelihood of a cancer, or the spread, dissemination, or metastasis of the cancer in the patient. The term 'prophylactic' also encompasses the use of the antibodies or antigen-binding fragments thereof that specifically bind to CD137, as described herein, to prevent recurrence of a cancer in a patient who has previously been treated for the cancer.

The term 'therapeutic treatment' is used to encompass the use of the antibodies or antigen-binding fragments thereof that specifically bind to CD137, as described herein, with the ultimate aim or goal of clearing the cancer from the patient, so the patient is cured of the cancer.

The term 'palliative treatment' is used to encompass the use of the antibodies or antigen-binding fragments thereof that specifically bind to CD137, as described herein, to treat a patient who it is accepted will not be cured of the cancer. However, palliative treatment can still bring a great benefit to the patient by improving their quality of life and/or extending their lifetime. For example, palliative treatment can result in a cancer characterised as being progressive and/or aggressive being, following treatment, characterised as a stable cancer. By 'stable cancer' we include that the cancer is neither growing nor shrinking, as would be appreciated by one skilled in medicine and/or oncology. Palliative treatment can also result in a reduction in the severity and/or number of cancer symptoms. The effects of the palliative treatment may be exhibited during the course of treatment, or beyond the end of the course of treatment.

In a preferred embodiment, the treatment is palliative treatment. In a particularly preferred embodiment, the palliative treatment results in the cancer being characterised as a stable cancer, after the administration of the antibody or antigen- binding fragment thereof that specifically binds to CD137. As described in the Examples, the antibody or antigen-binding fragment thereof that specifically binds to CD137 of the invention particularly demonstrates characteristics of a palliative treatment, specifically in stabilising cancer.

In one embodiment, the cancer is a relapsed cancer. 'Relapsed cancer' is a term that would be known to one skilled in medicine; herein it encompasses a cancer that a patient has been cured of, or partially cured of, but which has returned and/or worsened.

In one embodiment, the cancer is a refractory cancer. 'Refractory cancer' is a term that would be known to one skilled in medicine; herein it encompasses a cancer that is, or has become, resistant to a previous cancer treatment administered to the patient, such that that previous cancer treatment is no longer effective. In one embodiment, the cancer is a progressive cancer. 'Progressive cancer' is a term that would be known to one skilled in medicine; herein it encompasses a cancer which is developing rapidly, for example: if the cancer is a tumour, then that tumour is growing rapidly in size; that the cancer is metastatic; and/or that the health of the patient is rapidly deteriorating.

In one embodiment, the cancer is an advanced cancer. 'Advanced cancer' is a term that would be known to one skilled in medicine; herein it encompasses that the cancer will not be possible to cure, so any treatment is likely to be palliative treatment.

As will be appreciated by one skilled in medicine, a cancer can be characterised as being one or more of: a relapsed cancer; a refractory cancer; a progressive cancer; and an advanced cancer. For example, the cancer can be a relapsed and refractory cancer; or a relapsed, refractory, progressive, and advanced cancer. As described in the Examples, the antibody or antigen-binding fragment thereof that specifically binds to CD137 of the invention is particularly effective at treating cancer that can be described as relapsed, refractory, progressive, and/or advanced cancer

Cancer can be characterised using the 'TNM Staging System' (also referred to as the 'TNM System'), which would be known to one skilled in medicine and/or oncology.

In the TNM System:

• T refers to the size and extent of the main tumor (also referred to as a primary tumor).

• The N refers to the number of nearby lymph nodes that have cancer.

• The M refers to whether the cancer has metastasized.

Numbers used after each letter that give more details about the cancer— for example, T1N0MX or T3N1M0, as explained below

Primary tumor (T):

• TX: Main tumor cannot be measured. • T0: Main tumor cannot be found.

• T,, T2, T3, T4: Refers to the size and/or extent of the main tumor. The higher the number after the T, the larger the tumor or the more it has grown into nearby tissues. T's may be further divided to provide more detail, such as T3a and T3b.

Regional lymph nodes (N):

NX: Cancer in nearby lymph nodes cannot be measured.

N0: There is no cancer in nearby lymph nodes.

N1, N2, N3: Refers to the number and location of lymph nodes that contain cancer. The higher the number after the N, the more lymph nodes that contain cancer.

Distant metastasis (M):

MX: Metastasis cannot be measured.

M0: Cancer has not spread to other parts of the body.

M1: Cancer has spread to other parts of the body.

Whilst the TNM System can be used to describe a cancer in great detail, the TNM combinations can grouped into five less-detailed Stages 0-IV, which are used within the Examples and are as follows:

Stage 0 -Abnormal cells are present but have not spread to nearby tissue. Also called carcinoma in situ, or CIS. CIS is not cancer, but it may become cancer. This can be referred to as N0, M0.

Stage I, Stage II, and Stage III - Cancer is present. The higher the number, the larger the cancer tumor and the more it has spread into nearby tissues. Stage I - Localized cancer. T1-T2, N0, M0. Stage II - Locally advanced cancer, early stages. T2-T4, N0, M0. Stage III - Locally advanced cancer, late stages. T1-T4, N1-N3, M0.

Stage IV - The cancer has spread to distant parts of the body. Metastatic cancer. T1-T4, N1-N3, M1.

In some select cancers, there can be a Stage V, in which specific pathology is involved.

In one embodiment, the cancer is a Stage II, Stage III or Stage IV cancer, preferably a Stage III or Stage IV cancer, mor preferably a Stage IV cancer. In a further embodiment, the cancer is a Stage V cancer. In one embodiment, the cancer is a high grade cancer or a low grade cancer.

The terms 'high grade' and 'low grade' in relation to cancer severity would be known to one skilled in medicine and/or oncology. Here, we include that a high grade cancer grows and spreads more quickly than a low grade cancer. Accordingly, generally speaking, describing a cancer as being high grade indicates that it is more aggressive and acute than a low grade cancer, which is a more chronic condition.

The cancer may be associated with formation of solid tumours or may be a haematologic cancer. Cancer types that may be treated include carcinomas, sarcomas, lymphomas, leukemias, blastomas and germ cell tumours.

In a preferred embodiment, the cancer is a tumour, preferably a solid tumour. In a further preferred embodiment, the cancer referred to in the first to third aspects of the invention is a squamous cell cancer. In another further preferred embodiment, the cancer referred to in the first to third aspects of the invention is a carcinoma or adenocarcinoma, preferably a mucinous carcinoma or mucinous adenocarcinoma.

In one embodiment, the patient has previously received surgery to treat the cancer. In an alternative embodiment, the patient has not previously received surgery to treat the cancer.

In one embodiment, the patient has previously received radiotherapy to treat the cancer. In an alternative embodiment, the patient has not previously received radiotherapy to treat the cancer.

In a preferred embodiment, the cancer (in particular, wherein the cancer is a tumour) is unresectable. By 'unresectable' we include that the cancer cannot be surgically removed .

In one embodiment, the cancer may be selected from the list of cancers in Table 3 or Table 4 below (taken from WO 2018/091740).

Table 3: Mean expression values of solid human tumors with an above average expression (mean expression level ≥10) of both Fey receptor and CD137 (TNFRSF9). The ten tumors with the highest expression of the six Fey receptors are shown.

Table 4: Mean expression values of hematological malignancies with an above average expression (mean expression level ≥10) of both Fey receptor and CD137. The ten malignancies with the highest expression of the six Fey receptors are shown.

For example, the cancer can be selected from the group consisting of: prostate cancer; breast cancer; colorectal cancer; kidney cancer; pancreatic cancer; ovarian cancer; lung cancer; cervical cancer; rhabdomyosarcoma; neuroblastoma; bone cancer; multiple myeloma; leukemia (such as acute lymphoblastic leukemia [ALL] and acute myeloid leukemia [AML]), skin cancer (e.g. melanoma), bladder cancer and glioblastoma.

In one embodiment, the cancer is a cancer selected from the list consisting of: a gynaecological cancer; a cancer of the digestive system; melanoma; and breast cancer.

In a preferred embodiment, the gynaecological cancer is a gynaecological cancer selected from the list consisting of: ovarian cancer; endometrial cancer; and cervical cancer, preferably ovarian cancer.

In a preferred embodiment, the ovarian cancer is an adenocarcinoma of the ovary. In a preferred embodiment, the cancer of the digestive system is a cancer of the digestive system selected from the list consisting of: gastric cancer; sigmodal cancer; bile duct cancer; liver cancer; appendix cancer; mandibular cancer; an adenoneuroendocine carcinoma; an adenoid cystic cancer; pancreatic cancer; stomach cancer; rectal cancer; and anal cancer.

In a preferred embodiment, the stomach cancer is a stromal sarcoma of stomach antrum (GIST).

In a preferred embodiment, the bile duct cancer is a cholangiocarcinoma (CCA).

In a preferred embodiment, the mandibular cancer is an adenoid cystic cancer.

In a preferred embodiment, the anal cancer is a squamous cell anal cancer.

In a preferred embodiment, the appendix cancer is a mucinous adenocarcinoma of the appendix.

In a preferred embodiment, the pancreatic cancer is an adenoneuroendocine carcinoma of the pancreas.

In a preferred embodiment, the melanoma is a choroidal melanoma.

In a preferred embodiment, the breast cancer is a triple negative breast cancer. As would be known by one skilled in medicine, a triple negative breast cancer does not have receptors for oestrogen, progesterone and Her2.

In one embodiment, prior to the antibody or antigen-binding fragment thereof being administered, the patient is characterised by one or more (preferably all) of the "inclusion criteria" described in Example 1. However, as will be appreciated by one skilled in medicine, those inclusion criteria are of most relevance to a clinical trial setting, and some/all will (or may) not be relevant to a clinical setting. Therefore, it is not necessarily inappropriate to treat a patient that is not characterised by one or more (or all) of the inclusion criteria. In a preferred embodiment, prior to the antibody or antigen-binding fragment thereof being administered, the patient is characterised by one or more (preferably all) of the following : • a neutrophil number of about 1 x 10 ⁸ or more/Litre of blood, preferably a neutrophil number of about 1.5 x 10 ⁹ or more/Litre of blood; • a platelet number of about 100 x 10 ⁸ or more/Litre of blood, preferably a platelet number of about 100 x 10 ⁹ or more/Litre of blood; • a hemoglobin concentration of about 4 mmol or more/Litre of blood, preferably a hemoglobin concentration of about 5.9 mmol or more/Litre of blood; • an albumin amount of about 15g or more/Litre of blood, preferably an albumin amount of about 24g or more/Litre of blood; • a glomerular filtration rate (GFR) of about 30mL or more/minute, preferably a glomerular filtration rate (GFR) of about 45mL or more/minute; • a level of creatinine of about 2x or less of the upper limit of normal (ULN) for the patient, preferably a level of creatinine of about 1.5x or less of the upper limit of normal (ULN) for the patient; • a level of alanine transaminase (ALT) of about 4x or less of the upper limit of normal (ULN) for the patient, preferably a level of alanine transaminase (ALT) of about 3x or less of the upper limit of normal (ULN) for the patient; • a level of aspartate aminotransferase (AST) of about 4x or less of the upper limit of normal (ULN) for the patient, preferably a level of aminotransferase (AST) of about 3x or less of the upper limit of normal (ULN) for the patient; and • a level of bilirubin of about 2x or less of the upper limit of normal (ULN) for the patient, preferably a level of bilirubin of about 1.5x or less of the upper limit of normal (ULN) for the patient. It would be known to one skilled in medicine how to measure the above patient characteristics, with further relevant details explained in the Examples.

In one embodiment, prior to the antibody or antigen-binding fragment thereof being administered, the patient is not characterised by one or more (preferably all) of the "exclusion criteria" described in Example 1. However, as will be appreciated by one skilled in medicine, those exclusion criteria are of most relevance to a clinical trial setting, and some/all will (or may) not be relevant to a clinical setting. Therefore, it is not necessarily inappropriate to treat a patient that is characterised by one or more (or all) of the exclusion criteria.

In a preferred embodiment, following the antibody or antigen-binding fragment thereof being administered, and/or during the course of treatment (as described herein), the patient is characterised by one or more (preferably all) of the following: a neutrophil number of about 0.5 x 10 ⁸ or more/Litre of blood, preferably a • neutrophil number of about 0.5 x 10 ⁹ or more/Litre of blood; • a platelet number of about 50 x 10 ⁸ or more/Litre of blood, preferably a platelet number of about 50 x 10 ⁹ or more/Litre of blood; • a level of alanine transaminase (ALT) of about 6x or less of the upper limit of normal (ULN) for the patient, preferably a level of alanine transaminase (ALT) of about 5x or less of the upper limit of normal (ULN) for the patient; and • a level of aspartate aminotransferase (AST) of about 6x or less of the upper limit of normal (ULN) for the patient, preferably a level of aminotransferase (AST) of about 5x or less of the upper limit of normal (ULN) for the patient.

In one embodiment, following administration of the antibody or antigen-binding fragment thereof, the patient is characterised by an increase in the number and/or activation of immune cells, such as T cells, B cells, Natural Killer (NK) cells and/or dendritic cells, preferably NK cells and/or T cells (most preferably, cytotoxic CD8+ T cells). In one embodiment, CD54 and/or CD38 are markers of T cell activation and/or Natural Killer (NK) cell activation, so an increase of those markers can show T cell activation and/or NK cell activation. In a preferred embodiment, the increase in the number and/or activation of immune cells is compared to a base line, which is the number of immune cells prior to treatment with the antibody or antigen-binding fragment thereof. This base line can be calculated based on the patient to be treated with the antibody or antigen-binding fragment thereof, but prior to said treatment; or based on a subject that has not, and will not necessarily, be treated with the antibody or antigen-binding fragment thereof.

How to measure the number of immune cells would be known to one skilled in cell biology and/or medicine; for example, as discussed in the Examples.

In one embodiment, following administration of the antibody or antigen-binding fragment thereof, the patient is negative for anti-drug antibodies with specificity for the antibody or antigen-binding fragment thereof.

By "negative" in the context of anti-drug antibodies (ADA), we include that the patient does not have detectable ADA. How to measure ADA would be known to one skilled in cell biology and/or medicine; for example, as discussed in the Examples.

ADA are generated to a therapeutic antibody - the 'drug' - by a patient's immune system recognising it as foreign material. ADA can be detrimental for antibody treatments, because they ccaann alter the antibody's pharmacokinetic and/or pharmacodynamic properties, which can lead to a loss of efficacy. Furthermore, ADA can also have a detrimental impact on the health of the patient due to the immune response generated against the therapeutic antibody. Accordingly, it is preferable if a therapeutic antibody does not lead to the generation of ADA in a patient. As shown in Example 3, the inventors have surprisingly found that at high dosages of 500 mg or more (preferably 600 mg, more preferably 900 mg) the antibodies or antigen-binding fragments thereof that specifically bind to CD137 do not generate, or do not generate detectable, ADA.

ADAs are detected using highly specific and sensitive immunoassays to detect antibodies that: a) bind the drug; and b) can potentially neutralize the activity of the drug (a so-called "neutralizing antibody (nAb) assay"). These methods would be known to one skilled in the art.

In one embodiment, following administration of the antibody or antigen-binding fragment thereof, the patient is characterised by an increase in soluble CD137. In a preferred embodiment, the increase in soluble CD137 is compared to a base line, which is a level of soluble CD137 prior to treatment with the antibody or antigen-binding fragment thereof. This base line can be calculated based on the patient to be treated with the antibody or antigen-binding fragment thereof, but prior to said treatment; or based on a subject that has not, and will not necessarily, be treated with the antibody or antigen-binding fragment thereof.

How to measure soluble CD137 would be known to one skilled in cell biology and/or medicine; for example, as discussed in the Examples.

In a particular embodiment, the soluble CD137 is circulating soluble CD137. By 'circulating soluble CD137', we include soluble CD137 that is detectable and/or present in the blood of the patient.

During treatment with an antibody or an antigen-binding fragment thereof that specifically binds to CD137, CD137 can be inducibly expressed as a transmembrane protein or as a soluble protein (sometimes referred to as sCD137). It has been found that soluble CD137 can be used as a quantitative parameter reflecting therapeutic costimulatory activity, so can be used as a biomarker for the efficacy of the antibody or antigen-binding fragment thereof that specifically binds to CD137 (Glez-Vaz et al., 2022). The inventors have surprisingly found that high dosages (preferably 600 mg, more preferably 900 mg) of the antibody or an antigen-binding fragment thereof that specifically binds to CD137 of the invention induces high levels of soluble CD137, showing a good efficacy using that biological marker.

In a particular embodiment, the increase in soluble CD137 is characterised by a fold change of about ten or more, for example: about 11 or more; about 12 or more; about 13 or more; about 14 or more; about 15 or more; about 16 or more; about 17 or more; about 18 or more; about 19 or more; about 20 or more; about 21 or more; about 22 or more; about 23 or more; about 24 or more; about 25 or more; about 26 or more; about 27 or more; about 28 or more; about 29 or more; about 30 or more; about 31 or more; about 32 or more; about 33 or more; about 34 or more; about 35 or more; about 36 or more; about 37 or more; about 38 or more; about 39 or more; about 40 or more; about 41 or more; about 42 or more; about 43 or more; about 44 or more; about 45 or more; about 46 or more; about 47 or more; about 48 or more; about 49 or more; or about 50 or more. In a preferred embodiment, the fold change is compared to a base line, as discussed herein.

In a particularly preferred embodiment, the increase in soluble CD137 is characterised by a fold change of about 15 or more. In an alternative particularly preferred embodiment, the increase in soluble CD137 is characterised by a fold change of about 20 or more.

In a preferred embodiment, the fold change of the increase in soluble CD137 is over a

5 period of about three or more days, for example: about four or more days; about five or more days; about six or more days; about seven or more days; about eight or more days; about nine or more days; about ten or more days; about 11 or more days; about 12 or more days; about 13 or more days; about 14 or more days; about 15 or more days; about 16 or more days; about 17 or more days; about 18 or more days; about

10 19 or more days; about 20 or more days; about three or more weeks; about four or more weeks; about five or more weeks; about six or more weeks; about seven or more weeks; about eight or more weeks; about nine or more weeks; or about ten more weeks. In a particular embodiment, the period of time of which fold change is considered can be the course of treatment, and/or beyond the end of the course of

15 treatment.

In a preferred embodiment, the soluble CD137 is at a concentration of about 500 pg or more/ml, for example: about 550 pg or more/ml; about 600 pg or more/ml; about 650 pg or more/ml; about 700 pg or more/ml; about 750 pg or more/ml; about 800

20 pg or more/ml; about 850 pg or more/ml; about 900 pg or more/ml; about 950 pg or more/ml; about 1000 pg or more/ml; about 1100 pg or more/ml; about 1200 pg or more/ml; about 1300 pg or more/ml; about 1400 pg or more/ml; about 1500 pg or more/ml; about 1600 pg or more/ml; about 1700 pg or more/ml; about 1800 pg or more/ml; about 1900 pg or more/ml; about 2000 pg or more/ml; about 2100 or

25 more/ml; about 2200 or more/ml; about 2300 or more/ml; about 2400 or more/ml; about 2500 or more/ml; about 2600 or more/ml; about 2700 or more/ml; about 2800 or more/ml; about 2900 or more/ml; or about 3000 or more/ml.

In a particularly preferred embodiment, the soluble CD137 is at a concentration of

30 about 750 pg or more/ml. In an alternative particularly preferred embodiment, the soluble CD137 is at a concentration of about 1000 pg or more/ml.

In one embodiment, following administration of the antibody or antigen-binding fragment thereof, the patient is characterised by an increase in one of more (preferably

35 all) of the cytokines from the list consisting of: TNF-o, IFN-y, IL 10, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70 and IL 13. In a preferred embodiment, the increase those cytokines is compared to a base line, which is a level of those cytokines prior to treatment with the antibody or antigen-binding fragment thereof. This base line can

75 be calculated based on the patient to be treated with the antibody or antigen-binding fragment thereof, but prior to said treatment; or based on a subject that has not, and will not necessarily, be treated with the antibody or antigen-binding fragment thereof.

In one embodiment, the patient to be treated has been pre-screened and identified as having a tumour with cells expressing CD137 and FcγR, such as FcγRI, FcγRIIA, FcγRIIB or combinations thereof.

In one embodiment, the patient has been identified as being suitable for treatment with the antibodies or antigen-binding fragments thereof that specifically bind to CD137 of the invention, based on the presence of one or more relevant biomarkers.

It will be further appreciated that the antibodies or antigen-binding fragments thereof that specifically bind to CD137 of the invention may be used as a sole treatment for cancer in a patient or as part of a combination treatment (which further treatment may be a pharmaceutical agent, radiotherapy and/or surgery).

Thus, the patient may also receive one or more further treatments for cancer, for example pharmaceutical agents (such as chemotherapeutic agents), radiotherapy and/or surgery.

For example, the antibodies or antigen-binding fragments thereof that specifically bind to CD137 of the invention may be administered in combination with other therapeutic agents used in the treatment of cancers, such as antimetabolites, alkylating agents, anthracyclines and other cytotoxic antibiotics, vinca alkyloids, etoposide, platinum compounds, taxanes, topoisomerase I inhibitors, antiproliferative immunosuppressants, corticosteroids, sex hormones and hormone antagonists, and other therapeutic antibodies (such as trastuzumab).

In one embodiment, the one or more further treatments are selected from the group consisting of conventional chemotherapeutic agents (such as alkylating agents, anti- metabolites, plant alkaloids and terpenoids, topoisomerase inhibitors and antineoplastics), radiotherapeutic agents, antibody-based therapeutic agents (such as gemtuzumab, alemtuzumab, rituximab, trastuzumab, nimotuzumab, cetuximab, bevacizumab), and steroids.

Kits, pharmaceutical compositions, and administration routes In a fourth aspect, the invention also provides a kit for treating a cancer, as described in the first to third aspects of the invention, wherein the kit comprises an antibody or antigen-binding fragment thereof that specifically bind to CD137, as described herein; and wherein the kit comprises an antibody or antigen-binding fragment thereof at a dosage of about 500 mg or more, to be administered to the patient per administration.

In one embodiment, the kit comprises instructions for treating the patient in line with the disclosures of any one of the first to third aspects of the invention.

The antibody or antigen-binding fragment thereof that specifically bind to CD137 is preferably provided in a form suitable for local administration to a tumour site.

The kits of the invention may additionally comprise one or more other reagents or instruments which enable any of the embodiments mentioned above to be carried out. Such reagents or instruments include one or more of the following: suitable buffer(s) (aqueous solutions) and means to administer the anti-CD137 antibody antigen-binding fragment thereof (such as a vessel or an instrument comprising a needle).

The anti-CD137 antibody or antigen-binding fragment thereof used in the methods of the invention, or provided in the kits of the invention, may each be provided as a separate pharmaceutical composition formulated together with a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible and are also compatible with the required routes of administration.

The person skilled in medicine would know how to formulate the anti-CD137 antibody or antigen-binding fragment thereof for use in the treatments described herein.

Thus, the carrier for the anti-CD137 antibody or antigen-binding fragment thereof may be suitable for systemic administration, which as defined above means administration into the circulatory system of the subject, including the vascular and/or lymphatic system. Such administration may be by any suitable route, but is typically parenteral. The phrase "parenteral administration" as used herein means modes of administration other than enteral and topical administration, and is typically achieved by injection, infusion or implantation. Suitable routes include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral routes of administration, preferably intravenous.

However, the carrier for the anti-CD137 antibody or antigen-binding fragment thereof is preferably suitable for local administration, which as defined above includes peritumoral, juxtatumoral, intratumoral, intralesional, perilesional, intracranial and intravesicle administration by any suitable means, such as injection, Local administration may also include intra cavity infusion and inhalation, depending on the site of the tumour.

Depending on the route of administration, the anti-CD137 antibody or antigen-binding fragment thereof may be coated in a material to protect the antibody from the action of acids and other natural conditions that may inactivate or denature the antibody and/or agent. Preferred pharmaceutically acceptable carriers comprise aqueous carriers or diluents. Examples of suitable aqueous carriers that may be employed in the pharmaceutical compositions of the invention include water, buffered water and saline. Examples of other carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.

It will be appreciated by persons skilled in the art that the anti-CD137 antibody or antigen-binding fragment thereof components of the present invention are typically provided in the form of one or more pharmaceutical compositions, each containing a therapeutically-effective amount of the antibody component(s) together with a pharmaceutically-acceptable buffer, excipient, diluent or carrier.

It will be appreciated by persons skilled in the art that additional compounds may also be included in the pharmaceutical compositions, including, chelating agents such as EDTA, citrate, EGTA or glutathione.

By "pharmaceutically acceptable" we mean a non-toxic material that does not decrease the effectiveness of the CD137-binding activity of the antibody polypeptide of the invention. Such pharmaceutically acceptable buffers, carriers or excipients are well- known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed ., Pharmaceutical Press (2000), the disclosures of which are incorporated herein by reference).

A pharmaceutical composition may include a pharmaceutically acceptable anti-oxidant. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of presence of microorganisms may be ensured both by sterilization procedures, supra, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminium monostearate and gelatin.

Pharmaceutical compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active agent (e.g. antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration. Generally, dispersions are prepared by incorporating the active agent into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active agent plus any additional desired ingredient from a previously sterile-filtered solution thereof. Pharmaceutical compositions may comprise additional active ingredients as well as those mentioned above.

Suitable pharmaceutically acceptable buffers, diluents, carriers and excipients are well- known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A.R Gennaro, Ed., Mack Publishing Company (1990) and handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000), the disclosures of which are incorporated herein by reference).

The term "buffer" is intended to include an aqueous solution containing an acid-base mixture with the purpose of stabilising pH. Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES.

The term "diluent" is intended to include an aqueous or non-aqueous solution with the purpose of diluting the agent in the pharmaceutical preparation. The diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil).

The term "adjuvant" is intended to include any compound added to the formulation to increase the biological effect of the agent of the invention. The adjuvant may be one or more of zinc, copper or silver salts with different anions, for example, but not limited to fluoride, chloride, bromide, iodide, tiocyanate, sulfite, hydroxide, phosphate, carbonate, lactate, glycolate, citrate, borate, tartrate, and acetates of different acyl composition. The adjuvant may also be cationic polymers such as cationic cellulose ethers, cationic cellulose esters, deacetylated hyaluronic acid, chitosan, cationic dendrimers, cationic synthetic polymers such as poly(vinyl imidazole), and cationic polypeptides such as polyhistidine, polylysine, polyarginine, and peptides containing these amino acids.

The excipient may be one or more of carbohydrates, polymers, lipids and minerals. Examples of carbohydrates include lactose, glucose, sucrose, mannitol, and cyclodextrines, which are added to the composition, e.g., for facilitating lyophilisation. Examples of polymers are starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polysulphonate, polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone, all of different molecular weight, which are added to the composition, e.g., for viscosity control, for achieving bioadhesion, or for protecting the lipid from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di-, and triglycerides, ceramides, sphingolipids and glycolipids, all of different acyl chain length and saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are added to the composition for reasons similar to those for polymers. Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduction of liquid accumulation or advantageous pigment properties.

The anti-CD137 antibody or antigen-binding fragment thereof of the invention may be formulated into any type of pharmaceutical composition known in the art to be suitable for the delivery thereof.

In one embodiment, the pharmaceutical compositions of the invention may be in the form of a liposome, in which the anti-CD137 antibody or antigen-binding fragment thereof is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids, which exist in aggregated forms as micelles, insoluble monolayers and liquid crystals. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Suitable lipids also include the lipids above modified by polyethylene glycol) in the polar headgroup for prolonging bloodstream circulation time. Preparation of such liposomal formulations is can be found in for example US 4,235,871 and in EP 0 213 303, the disclosures of which are incorporated herein by reference.

The pharmaceutical compositions of the invention may also be in the form of biodegradable microspheres. Aliphatic polyesters, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), copolymers of PLA and PGA (PLGA) or poly(carprolactone) (PCL), and polyanhydrides have been widely used as biodegradable polymers in the production of microspheres. Preparations of such microspheres can be found in US 5,851,451 and in EP 0 213 303, the disclosures of which are incorporated herein by reference.

In a further embodiment, the pharmaceutical compositions of the invention are provided in the form of nanoparticles, for example based on poly-gamma glutamic acid. Details of the preparation and use of such nanoparticles can be found in WO 2011/128642, the disclosures of which are incorporated herein by reference. It will be appreciated by persons skilled in the art that one or more of the active components of the combination therapies of the present invention may be formulated in separate nanoparticles, or both active components may be formulated in the same nanoparticles.

In a further embodiment, the pharmaceutical compositions of the invention are provided in the form of polymer gels, where polymers such as starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polyvinyl imidazole, polysulphonate, polyethyleng lycol/polyethylene oxide, polyethyleneoxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone are used for thickening of the solution containing the agent. The polymers may also comprise gelatin or collagen.

Alternatively, the anti-CD137 antibody or antigen-binding fragment thereof may simply be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil), tragacanth gum, and/or various buffers.

It will be appreciated that the pharmaceutical compositions of the invention may include ions and a defined pH for potentiation of action of the anti-CD137 antibody or antigen-binding fragment thereof. Additionally, the compositions may be subjected to conventional pharmaceutical operations such as sterilisation and/or may contain conventional adjuvants such as preservatives, stabilisers, wetting agents, emulsifiers, buffers, fillers, etc.

The pharmaceutical compositions, antibodies, or antigen-binding fragments thereof according to the invention may be administered via any suitable route known to those skilled in the art. Thus, possible routes of administration include parenteral (intravenous, subcutaneous, and intramuscular), topical, ocular, nasal, pulmonar, buccal, oral, parenteral, vaginal and rectal. Also administration from implants is possible. Most preferably, the route of administration pharmaceutical compositions, antibodies, or antigen-binding fragments thereof according to the invention is an intravenous administration, more preferably the antibody or antigen-binding fragment thereof is administered via an intravenous infusion.

Advantageously, the pharmaceutical composition is suitable for administration at or near the site of a tumour, e.g. intra-tumourally or peri-tumourally.

It is preferred that the pharmaceutical composition is suitable for parenteral administration, for example the pharmaceutical composition is preferably suitable for administration intravenously, intracerebroventricularly, intraarticularly, intra- arterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intramuscularly or subcutaneously, or by infusion techniques. Methods for formulating an antibody into a pharmaceutical composition, such as a pharmaceutical composition suitable for parenteral administration, will be well-known to those skilled in the arts of medicine and pharmacy.

Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

The pharmaceutical compositions, antibodies, or antigen-binding fragments thereof according to the invention of the invention may be delivered using an injectable sustained-release drug delivery system. These are designed specifically to reduce the frequency of injections. An example of such a system is Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period. Preferably, delivery is performed intra-muscularly (i.m.) and/or sub-cutaneously (s.c.) and/or intravenously (i.v.).

The pharmaceutical compositions, antibodies, or antigen-binding fragments thereof according to the invention can be administered by a surgically implanted device that releases the drug directly to the required site. For example, Vitrasert releases ganciclovir directly into the eye to treat CMV retinitis. The direct application of this toxic agent to the site of disease achieves effective therapy without the drug's significant systemic side-effects.

Electroporation therapy (EPT) systems can also be employed for the administration of the pharmaceutical compositions, antibodies, or antigen-binding fragments thereof according to the invention. A device which delivers a pulsed electric field to cells increases the permeability of the cell membranes to the drug, resulting in a significant enhancement of intracellular drug delivery. The pharmaceutical compositions, antibodies, or antigen-binding fragments thereof according to the invention can also be delivered by electro-incorporation (El). El occurs when small particles of up to 30 microns in diameter on the surface of the skin experience electrical pulses identical or similar to those used in electroporation. In El, these particles are driven through the stratum corneum and into deeper layers of the skin. The particles can be loaded or coated with drugs or genes or can simply act as "bullets" that generate pores in the skin through which the drugs can enter.

An alternative pharmaceutical composition, antibody, or antigen-binding fragment thereof according to the invention is the ReGel injectable system that is thermo- sensitive. Below body temperature, ReGel is an injectable liquid while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active substance is delivered over time as the biopolymers dissolve.

The pharmaceutical compositions, antibodies, or antigen-binding fragments thereof according to the invention can also be delivered orally. The process employs a natural process for oral uptake of vitamin B12 and/or vitamin D in the body to co-deliver proteins and peptides. By riding the vitamin B12 and/or vitamin D uptake system, the agents, medicaments and pharmaceutical compositions of the invention can move through the intestinal wall. Complexes are synthesised between vitamin B12 analogues and/or vitamin D analogues and the drug that retain both significant affinity for intrinsic factor (IF) in the vitamin B12 portion/vitamin D portion of the complex and significant bioactivity of the active substance of the complex.

The pharmaceutical compositions, antibodies, or antigen-binding fragments thereof according to the invention can be introduced to cells by "Trojan peptides". These are a class of polypeptides called penetratins which have translocating properties and are capable of carrying hydrophilic compounds across the plasma membrane. This system allows direct targeting of oligopeptides to the cytoplasm and nucleus, and may be non- cell type specific and highly efficient. See Derossi et al. (1998), Trends Cell Biol. 8, 84- 87.

The antibodies or antigen-binding fragments thereof according to the invention will normally be administered orally or by any parenteral route, in the form of a pharmaceutical composition comprising the active ingredient, optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated, as well as the route of administration, the compositions may be administered at varying doses.

In human therapy, the antibodies or antigen-binding fragments thereof according to the invention can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.

For example, the pharmaceutical compositions, antibodies, or antigen-binding fragments thereof according to the invention can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. The agents, medicaments and pharmaceutical compositions of the invention may also be administered via intracavernosal injection.

Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the pharmaceutical compositions, antibodies, or antigen-binding fragments thereof according to the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

The pharmaceutical compositions, antibodies, or antigen-binding fragments thereof according to the invention ccaann be administered parenterally, for example, intravenously, intra-arterially, intraperitoneally, intra-thecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. Preferably, the pharmaceutical compositions, antibodies, or antigen-binding fragments thereof according to the invention are administered intravenously. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

Medicaments and pharmaceutical compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti- oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The medicaments and pharmaceutical compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

Thus, the pharmaceutical compositions of the invention are particularly suitable for parenteral, e.g. intravenous, administration.

The pharmaceutical compositions, antibodies, or antigen-binding fragments thereof can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1,1,1,2-tetrafluoroethane (HFA 134A3 or 1, 1,1, 2, 3,3,3- heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas. In the case of a pressurised aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active agent, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of an agent of the invention and a suitable powder base such as lactose or starch. Aerosol or dry powder formulations are preferably arranged so that each metered dose or 'puff' contains at least 1 mg of a compound of the invention for delivery to the patient. It will be appreciated that the overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.

Alternatively, the pharmaceutical compositions, antibodies, or antigen-binding fragments thereof can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, gel, ointment or dusting powder. The agents, medicaments and pharmaceutical compositions of the invention may also be transdermally administered, for example, by the use of a skin patch. They may also be administered by the ocular route, particularly for treating diseases of the eye.

For ophthalmic use, the pharmaceutical compositions, antibodies, or antigen-binding fragments thereof can be formulated as micronised suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.

For application topically to the skin, the pharmaceutical compositions, antibodies, or antigen-binding fragments thereof can be formulated as a suitable ointment containing the active agent suspended or dissolved in, for example, a mixture with one or more of the following : mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene agent, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier. Generally, in humans, local administration of the pharmaceutical compositions, antibodies, or antigen-binding fragments thereof at or near the site of a tumour is the preferred route, in particular intra-tumoural or peri-tumoural administration.

For veterinary use, the antibodies, antigen-binding fragments thereof that specifically bind to CD137, and pharmaceutical compositions of the invention are administered as a suitably acceptable formulation in accordance with normal veterinary practice and the veterinary surgeon will determine the dosing regimen and route of administration which will be most appropriate for a particular animal.

Nucleic acids, vectors and hosts

Also encompassed by the present invention are isolated nucleic acid molecules comprising or consisting of nucleic acid sequences encoding the antibodies and antigen- binding fragments thereof that specifically bind to CD137, described herein.

By "nucleic acid molecule" we include DNA (e.g. genomic DNA or complementary DNA) and mRNA molecules, which may be single- or double-stranded. By "isolated" we mean that the nucleic acid molecule is not located or otherwise provided within a cell.

In one embodiment, the nucleic acid molecule(s) is/are cDNA molecule(s).

In an embodiment, the nucleic acid molecules encode an antibody heavy chain or variable region thereof and/or encode an antibody light chain or variable region thereof.

In one embodiment, the present invention relates to a nucleic acid sequence comprising or consisting of one of the following nucleic acid sequences: SEQ ID NO: 9; SEQ ID NO: 10; SEQ ID NO: 27; and SEQ ID NO: 28.

In a preferred embodiment, the nucleic acid sequence comprises or consists of SEQ ID NO: 9 and/or SEQ ID NO: 10. In an alternative preferred embodiment, the nucleic acid sequence comprises or consists of SEQ ID NO: 27 and/or SEQ ID NO: 28.

Nucleotide sequence encoding VH region of "1630"

Nucleotide sequence encoding VL region of "1631"

Nucleotide sequence encoding VH region of "2674"

[SEQ ID NO: 27] Nucleotide sequence encoding VL region of "2675"

[SEQ ID NO: 28]

It will be appreciated by persons skilled in the art that the first nucleic acid molecule may be codon-optimised for expression of the antibody polypeptide in a particular host cell, e.g. for expression in human cells (for example, see Angov, 2011, Biotechnol. J. 6(6):650-659, the disclosures of which are incorporated herein by reference).

Also encompassed by the present invention are vectors (such as an expression vector) comprising the nucleic acid sequences, described herein.

Also encompassed by the present invention are host cells (such as a mammalian cell, e.g. human cell, or Chinese hamster ovary cell, e.g. CHOK1SV cells) comprising the nucleic acid sequences and/or the vectors, described herein.

Brief Description of the Sequence Listing

SEQ ID NO: 1 is the amino acid sequence of VH region of "1630".

SEQ ID NO: 2 is the amino acid sequence of VL region of "1631".

SEQ ID NO: 3 is the amino acid sequence of HCDR 1 of "1630".

SEQ ID NO: 4 is the amino acid sequence of HCDR 2 of "1630".

SEQ ID NO: 5 is the amino acid sequence of HCDR 3 of "1630".

SEQ ID NO: 6 is the amino acid sequence of LCDR 1 of "1631".

SEQ ID NO: 7 is the amino acid sequence of LCDR 2 of "1631".

SEQ ID NO: 8 is the amino acid sequence of LCDR 3 of "1631".

SEQ ID NO: 9 is the nucleotide sequence encoding VH region of "1630".

SEQ ID NO: 10 is the nucleotide sequence encoding VL region of "1631". SEQ ID NO: 11 is the amino acid sequence of human CD137 sequence (amino acids 66 to 107 correspond to domain 2 of human CD137).

SEQ ID NO: 12 is the amino acid sequence of IgG1 heavy chain constant region.

SEQ ID NO: 13 is the amino acid sequence of modified IgG4 constant region.

SEQ ID NO: 14 is the amino acid sequence of modified IgG4 constant region.

SEQ ID NO: 15 is the amino acid sequence of wild-type IgG4 constant region.

SEQ ID NO: 16 is the amino acid sequence of kappa chain constant region.

SEQ ID NO: 17 is the full amino acid sequence of heavy chain of "1630".

SEQ ID NO: 18 is the full amino acid sequence of the light chain of "1631".

SEQ ID NO: 19 is the amino acid sequence of the VH region of "2674".

SEQ ID NO: 20 is the amino acid sequence of the VL region of "2675".

SEQ ID NO: 21 is the amino acid sequence of HCDR 1 of "2674".

SEQ ID NO: 22 is the amino acid sequence of HCDR 2 of "2674".

SEQ ID NO: 23 is the amino acid sequence of HCDR 3 of "2674".

SEQ ID NO: 24 is the amino acid sequence of LCDR 1 of "2675".

SEQ ID NO: 25 is the amino acid sequence of LCDR 2 of "2675".

SEQ ID NO: 26 is the amino acid sequence of LCDR 3 of "2675".

SEQ ID NO: 27 is the nucleotide sequence encoding VH region of "2674".

SEQ ID NO: 28 is the nucleotide sequence encoding VL region of "2675".

SEQ ID NO: 29 is the full amino acid sequence of the heavy chain "2674".

SEQ ID NO: 30 is the full amino acid sequence of the light chain of "2675".

It is to be understood that different applications of the disclosed pharmaceutical compositions, antibodies, or antigen-binding fragments thereof, and medical uses and methods, may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting. Where a feature is described with reference to a specific aspect, it will be appreciated by the skilled person that said feature may also apply to other related aspects.

In addition as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "an antibody" includes "antibodies", reference to "an antigen" includes two or more such antigens, reference to "a subject" includes two or more such subjects, and the like.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

The present invention is further illustrated by the following examples which should not be construed as further limiting. The contents of all figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

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Brief Description of the Figures

Preferred, non-limiting examples which embody certain aspects of the invention will now be described, with reference to the following figures:

Figure 1: Dose escalation.

Figure 2: Design of the dose escalation.

Figure 3: Dosage schedule and key assessments.

Figure 4A: Dosages and time over which the patients remained in the study, and the patients' cancer type.

Figure 4B: Dosages and time over which the patients remained in the study, and the patients' cancer type updated to include additional data points (cut-off date 29 March 2023).

Figure 5: Information regarding patients that received a dosage of 0.38 mg, 1.5 mg, 5 mg, 15 mg, 40 mg, 100 mg, and 200mg.

Figure 6: Information regarding patients that received a dosage of 360 mg, 600 mg, and 900mg. Figure 7A: Pharmacokinetics (PK) data showing log concentration of ATOR-1017 over time.

Figure 7B: Pharmacokinetics (PK) data showing log concentration of ATOR-1017 over time adjusted to exclude the influence of anti-drug antibody (ADA). Only the first cycle is plotted and nominal time is shown on the x-axis.

Figure 8: PK data showing dose proportionality expressed as C _max .

Figure 9: PK data showing dose proportionality expressed as AUCtau.

Figure 10: Anti-drug antibodies detected in patients at the of the dosages (with corrected values).

Figure 11A and 11B (corrected): Individual soluble CD137 concentration per dosage. The units for the y-axis in all graphs is pg/ml. The units for the x-axis of the 40 mg, 100 mg, 200 mg and 360 mg graphs is days, and for the 600 mg and 900 mg graphs it is cycle.

Figure 12A: Fold change of soluble CD137 when compared to pre-dose.

Figure 12B: Continuous fold change of soluble CD137 when compared to pre-dose. Fold change is different from Figure 12A due to lower detection limit. Continuous as long as patient is receiving the initial dose level.

Figure 13A and 13B (900mg dose corrected): Concentration of soluble CD137 for cycle 1.

Examples

Example 1 - study design

This Example describes the first-in-human, multicenter, open-label, phase 1 study in patients with advanced solid malignancies to evaluate the safety of intravenously administered ATOR-1017 - described elsewhere herein as antibody 2674/2675.

ATOR-1017 is a human monoclonal antibody targeting 4-1BB (CD137) developed for immunotherapy of cancer. Repeated doses of ATOR-1017 will be administered intravenously at intervals of 3 weeks.

Ten flat dose levels were used - 0.38 mg; 1.5 mg; 5 mg; 15 mg; 40 mg; 100 mg; 200 mg; 360 mg; 600 mg; 900 mg. As show in Figure 1.

Executive summary

Background: ATOR-1017 is a human Fcy-receptor cross-linking dependent IgG4 4- 1BB (CD137) agonist antibody. ATOR-1017 activates T cells and natural killer cells in the tumor environment, leading to immune-mediated tumor cell killing.

Methods: In this first-in-human, dose escalation, multicenter, phase 1 study, adult patients with solid tumors refractory to standard therapy were enrolled in single patient cohorts for doses up to 40 mg, and thereafter in cohorts of 3-6 patients. Intra-patient dose escalation is allowed. ATOR-1017 is administered intravenously as monotherapy (flat doses) every three weeks until disease progression. The primary objectives are assessment of safety (maximum tolerated dose (MTD), adverse events (AEs), dose- limiting toxicities (DLT)), and determination of recommended phase 2 dose. Secondary and exploratory objectives include pharmacokinetics (PK), immunogenicity, efficacy (by iRECIST), and Pharmacodynamic (PD) biomarkers.

Results: At cut off date 14 June 2022, 25 patients (20 females/ 5 males), with median age 57 years (34-76), median of 3 (1-9) prior lines of chemotherapy and/or median 1 (1-3) lines of immunotherapy, 21 (84%) at disease stage IV had been treated. Ten dose levels were evaluated; 0.38mg, 1.5mg, 5mg, 15mg, 40mg, 100mg, 200mg, 360mg, 600mg, and 900mg. Treatment-related AEs (TRAEs) were reported in 13 patients (52%); most common (≥10%) were fatigue (16%) and neutropenia (12%). Five patients experienced a grade 3-4 TRAE; neutropenia (n = 2), febrile neutropenia (n = 1), non-cardiac chest pain (n = 1), increased liver enzymes (n = 1) and leukopenia/thrombocytopenia (n = 1). No patients discontinued due to TRAEs, no DLTs were observed, and MTD has not been reached. Three patients remained on treatment and 22 had discontinued treatment [(confirmed disease progression (n = 12), clinical deterioration (n = 6), withdrawal of consent (n = 1), death due to disease progression (n = 2), investigator's decision (n=l)]. The median time on treatment was 12.1 weeks (range 5.3-67.3). A dose-proportional pharmacokinetics was observed. RD biomarkers demonstrated activation of peripheral CD8 T cells and a dose-dependent increase in soluble 4-1BB confirming biological activity and proof-of-mechanism. Stable disease was observed in 13 patients (52%), which lasted longer than 6 months for 6 (24%) patients (of which 2 had ovarian cancer).

Conclusions: ATOR-1017 demonstrated excellent safety at doses up to 900 mg, together with a favorable PK and confirmation of biologic activity. These data warrant further development of ATOR-1017, a 4-1BB agonistic antibody, in combination with other therapeutic approaches in solid tumors. Clinical trial ID: NCT04144842.

Eligibility criteria

A patient is eligible to be included in the study if all the following criteria apply - 'inclusion criteria' as described herein:

1. Has provided written informed consent

2. Is ≥18 years of age at the time of signing the informed consent form (ICF)

3. Has a body weight ≥40 kg

4. Has a diagnosis of advanced and/or refractory solid malignancy (histologically or cytologically documented) that is metastatic or unresectable and has received standard of care therapy and the remaining therapeutic options are participation in a clinical study or best supportive care

5. Has an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1

6. Has a minimum of one measurable tumor lesion (≥10 mm in diameter) or nodal lesion (≥15 mm in the short axis) in a non-irradiated area (CT scan thickness no greater than 5 mm)

7. Has a life expectancy of at least 3 months

8. Has acceptable hematologic laboratory values defined as: a. Neutrophils ≥1.5 x 109/L, without growth factor stimulation within 3 weeks prior to the blood test b. Platelets ≥100 x 109/L c. Hemoglobin ≥5.9 mmol/L (~95 g/L), without transfusion or erythropoietin therapy within 4 weeks prior to the blood test

9. Has acceptable clinical chemistry laboratory values defined as: a. Albumin ≥24 g/L b. Creatinine ≤1.5 x upper limit of normal (ULN) or glomerular filtration rate (GFR) of ≥45 mL/min c. AST ≤3 x ULN with or without hepatic metastases d. ALT ≤3 x ULN with or without hepatic metastases e. Total bilirubin ≤1.5 x ULN

10. For women of childbearing potential: Has a negative highly sensitive serum (β-human chorionic gonadotropin [β-hCG]) pregnancy test at screening

11. Is willing to comply with all study procedures

Table 5: ECOG performance status

A patient is excluded if any of the following criteria apply - 'exclusion criteria' as described herein:

1. Has received anti-cancer medications within 4 weeks prior to first dose (6 weeks required for nitrosurea or mitomycin) except for medications with half-lives <5.5 days. Bisphosphonates, denosumab and androgen deprivation therapies such as LHRH (GnRH) agonists are allowed if patients are stabilized on treatment for at least 4 weeks prior to screening 2. Has not recovered from AEs to at least grade 1 by CTCAE version 5.0 due to prior anti-cancer medications (except for alopecia, grade 2 neuropathy or adequately controlled grade 2 endocrinopathies) prior to signing the IGF

3. Has received radiotherapy within 14 days before first dose (palliative radiotherapy for pain allowed)

4. Has symptomatic, steroid-dependent oorr progressive brain metastasis/metastases within 4 weeks prior to signing the IGF

5. Has clinically significant cardiac disease, including : a. Has known congestive heart failure grade III or IV by the New York Heart Failure Association b. Has a myocardial infarction within 6 months prior to signing the IGF c. Has an onset of unstable angina within 6 months prior to signing the IGF

6. Has a history of another primary malignancy, except for: a. Malignancy treated with curative intent and with no known active disease within 2 years prior to first dose of ATOR-1017 b. Adequately treated non-invasive basal skin cancer or squamous cell skin carcinoma c. Adequately treated uterine cervical cancer stage IB or less

7. Has an autoimmune disorder requiring immune modulating treatment during the last 2 years prior to first dose of ATOR-1017. Patients with vitiligo, resolved atopy, limited psoriasis, hypothyroidism stable on hormone replacement, type I diabetes, Graves' or Hashimoto's disease that are under treatment and stable, are allowed.

8. Receives treatment with systemic immunosuppressant medication (except for inhaled and low dose systemic corticosteroids, i.e. ≤10 mg prednisolone or equivalent per day) within 4 weeks prior to first dose of ATOR-1017

9. Has a known positive serology for HIV

10. Has a positive serology for hepatitis B (anti-HBc) or known prior hepatitis B

11. Has a positive serology for hepatitis C (anti-HCV) (unless undetectable virus load by polymerase chain reaction (PCR) after HCV treatment or due to immunoglobulin therapy)

12. Has been exposed to live or live attenuated vaccine within 4 weeks prior to signing the ICF

13. Participate, or have participated within the previous 4 weeks prior to signing the IGF, in an investigational drug or device study with any intervention

14. Is a female patient who is pregnant or nursing 15. Is a female patient of childbearing potential, not willing to use a highly effective form of contraception during treatment and for at least 6 months after the last dose of ATOR-1017

Highly effective forms of contraception include (if using hormonal contraception this method must be supplemented with a barrier method, preferably male condom): o Combined (estrogen and progestogen containing) hormonal contraception associated with inhibition of ovulation: o oral o intravaginal o transdermal o Progestogen-only hormonal contraception associated with inhibition of ovulation: o oral o injectable o implantable o Intrauterine device (IUD) o Intrauterine hormone-releasing system (IUS) o Bilateral tubal occlusion o Vasectomized partner o True heterosexual abstinence defined as when this is in line with the preferred and usual lifestyle of the patient. Periodic abstinence (e.g. calendar, ovulation, symptothermal, post-ovulation methods), declaration of abstinence for the duration of a study, and withdrawal are not acceptable methods of contraception

16. Is a sexually active male patient with a female partner not practicing effective double barrier contraceptive methods or not willing to abstain from sperm donation during the study and for 6 months after last dose of ATOR- 1017

17. Any condition that, in the opinion of the Investigator, would place the patient at increased risk or preclude the patient's compliance with the study

Methodology:

This is a phase 1, multicenter, open-label dose escalation study in which patients will be administered intravenous (iv) doses of ATOR-1017. The study starts with a screening period of up to 21 days which is followed by a treatment period with treatment cycles of 21 days. ATOR 1017 will be administered every 21 days (Day 1 of each cycle) and a tumor response evaluation (by Computed tomography [CT]) will be performed approximately every 6 weeks during the first 4 cycles, thereafter approximately every 12 weeks. Patients that do not progress can continue in the study with dosing every 3 weeks (see Duration of treatment below). A Treatment Follow-up Visit will be performed 28-56 days after last dose.

All patients will be monitored for at least 8 hours after the first infusion of ATOR 1017, and for at least 4 hours after the second, third and the fourth infusions of ATOR-1017. If an infusion-related reaction grade >1 has not been observed at the latest infusion (fourth or later), the monitoring of the patient can be reduced to 1 hour for subsequent infusions. If an infusion-related reaction grade >1 has been observed at the latest infusion, the monitoring should be maintained at 4 hours after the infusion. Staggered dosage of at least 2 days between the first dosing of the first patient and the second patient at each dose level with 3 or more patients planned will be applied.

Dose escalation will be determined by a DRC following review of safety data, including clinical laboratory tests and AEs, obtained during the DLT evaluation period. The DLT evaluation period is defined as the time from the first dose of ATOR-1017 (Day 1) until Day 21.

Initially the study will have an accelerated dose escalation design, with single-patient cohorts for dose levels <40 mg. However, if a patient in a single-patient cohort experiences one grade ≥2 toxicity lasting for more than 72 hours or two grade ≥2 toxicities during the DLT evaluation period, an additional 2 patients (at least) will be included at this dose level and the study will shift to a modified 3+3 design. For dose levels ≥40 mg, the modified 3+3 design will be applied with at least 3 patients enrolled at each dose level.

Intrapatient dose escalation is allowed after the first 2 treatment cycles according to the judgement of the treating Investigator in agreement with Sponsor up to a dose level declared safe by the DRC.

Duration of treatment:

Patients may continue study treatment until iCPD, or clear clinical deterioration, according to Investigator's judgment, as long as the patients are tolerating the treatment and agree to continue. The patients may receive treatment for a maximum of 2 years after the last patient's first dose in the study.

Study assessments:

Assessments include medical history, previous anti-cancer treatments, height and weight, vital signs (blood pressure, pulse rate, oxygen saturation and body temperature), physical examination, ECOG performance status, ECG and clinical laboratory tests (clinical chemistry, hematology, urinalysis), concomitant medication and collection of AEs.

Blood samples will be taken for PK and pharmacodynamic analyses, and for immunogenicity testing.

Anti-tumor activity will be evaluated by assessing CT scans according to iRECIST.

Statistical methods:

All analyses will be descriptive. Categorical variables will be presented with numbers and, if meaningful, percentages. Continuous variables will be presented by n, mean, median, standard deviation and range (minimum and maximum) as appropriate.

Introduction to the Investigational Drug ATOR-1017

ATOR-1017 is a fully human agonistic IgG4 antibody targeting the co-stimulatory receptor 4 IBB (CD137). The IgG4 is stabilized, containing a well characterized and clinically evaluated mutation (S228P), that will inhibit Fab arm exchange ("half- molecule exchange" usually seen with IgG4 antibodies) [1]. The mode of action for ATOR-1017 is to activate effector T cells (Teffs) and natural killer (NK) cells in the tumor environment, leading to immune-mediated tumor cell killing. ATOR-1017 is dependent on Fc gamma receptor (FcγR) crosslinking for its effect. The FcγR crosslinking-dependency of ATOR-1017 is expected to direct the agonistic effect to the tumor environment and tumor draining lymph nodes and reduce the systemic immune activation due to the high abundance of endogenous circulating IgG which will compete with ATOR-1017 for binding to FcγRs. ATOR-1017 has been developed to reduce tumor burden, and thus prolong progression-free survival and overall survival in patients with cancer and will be administered intravenously.

Background to study

Immunotherapy using monoclonal antibodies has been a great advancement in cancer therapy. Immunomodulatory approaches include both the approved checkpoint inhibitors targeting T-lymphocyte associated protein 4 (CTLA-4), programmed cell death protein 1 (PD 1) and programmed cell death protein 1 ligand (PD-L1), as well as immunostimulatory agonistic antibodies, targeting co-stimulatory receptors such as 4- 1BB, 0X40 and CD40 within the tumor necrosis factor (TNF) receptor superfamily. A multitude of clinical trials are ongoing with both inhibitory and stimulating antibodies given as single agents or in combinations to treat cancer.

T-cell receptor (TCR) engagement by antigen is the main signal for the activation of naive T cells. This signal is however not sufficient to transform resting T cells into Teffs. Full activation of T cells requires additional signals via so called co-stimulatory receptors on the T cells. 4 IBB is a co-stimulatory receptor of the TNF receptor superfamily, which is transiently expressed and upregulated on Teffs, regulatory T cells (Tregs), natural killer (NK) cells and dendritic cells upon activation [2]. It has also been shown to be highly expressed on intratumoral tumor reactive CD8+ T cells, while expression of 4-1BB on Teffs in the circulation is low [3].

The only confirmed natural ligand binding to 4-1BB is 4-1BB ligand (4-1BBL), which is expressed on antigen-presenting cells (APCs) including macrophages, B cells and dendritic cells. Activation of 4-1BB on T cells supports proliferation, cytokine production, cytolytic effector functions and survival [4]. 4-1BB has also been shown to be important for the induction of long-lived memory T cells [2]. On NK cells, 4-1BB ligation increases cytokine release and cytolytic responses such as antibody-dependent cellular cytotoxicity (ADCC) [5].

Several studies in experimental tumor models have demonstrated a potent induction of tumor immunity by treatment with agonistic 4-1BB antibodies [6, 7]. In addition, 4- 1BB antibody treatment has been shown to be synergistic with other immunomodulatory antibodies such as anti-CTLA-4 and anti-PD-l/PD-Ll, as well as with standard of care treatments such as radiotherapy and chemotherapy [6, 8]. Summary of Non-Clinical Data

ATOR-1017 binds to the 4-1BB receptor with high affinity and activates cytotoxic effector CD8+ T cells and NK cells at nanomolar concentrations in primary human T and NK cell assays. The agonistic effect of ATOR-1017 is FcγR crosslinking-dependent, which means that T and NK cells will only be activated if ATOR-1017 binds to 4-1BB and FcγR simultaneously. The effect of ATOR-1017 is dose-dependently reduced in presence of IgG. These IgG competition assays indicate that ATOR-1017 will not induce systemic immune activation due to the high abundance of endogenous IgG in the circulation, and that the immune activating effect will be directed to the tumor tissue where IgG concentration is lower [9-11].

Due to lack of cross-reactivity (binding) of ATOR-1017 to mouse 4-1BB, the anti-tumor effect of ATOR-1017 in vivo was tested in a human 4-1BB Knock-In transgenic mouse model. ATOR 1017 was found to reduce tumor growth and improve survival in a dose- dependent manner, and to induce immunological memory.

A full non-clinical safety package of ATOR-1017 has been performed, including toxicology studies in cynomolgus monkeys, tissue cross-reactivity studies and cytokine release assays. Cynomolgus monkey was considered the most relevant species for toxicology studies. This was based on high sequence homology, similar binding affinities, expression profiles as well as potency in a functional assay with primary CD8+ T cells. Overall, ATOR-1017 was well tolerated at all dose levels and no significant safety concerns were identified. The cross reactivity studies did not show any unexpected results, and ATOR-1017 did not increase cytokine levels in the cytokine release assays.

Summary of Clinical Data

This is a first-in-human study, and hence there is no clinical data available for ATOR 1017. There are currently four monoclonal 4-1BB antibodies that are or have been in clinical development, urelumab, utomilumab, ADG106 and CTX-471. ADG106 and CTX- 471 are still in early clinical exploration (entered clinical phase 1 in 2018 and 2019 respectively) and clinical data have not yet been disclosed. Clinical data have been published for urelumab and utomilumab, and key clinical information from each of these is summarized in the next sections.

Urelumab Urelumab is an IgG4 monoclonal antibody targeting domain 1 of the 4-1BB receptor, it is FcγR crosslinking-independent, and has similar in vitro potency compared to ATOR- 1017 [12]. It has been assessed in the clinic at doses ranging from 0.1 mg/kg to 15 mg/kg every 3 weeks [13]. Grade 3-4 neutropenia was observed in 8 out of 346 patients (2.3%) and elevation of transaminases were observed in 41 out of 346 patients (11.8%) [9]. Elevation in transaminases was mainly seen at doses of 1 mg/kg and higher. Of the 229 patients receiving ≥1 mg/kg urelumab, 2 cases of fatal hepatotoxicity (0.9%) were reported. It was concluded that urelumab was associated with hepatotoxicity at doses ≥ 0.3 mg/kg [9]. A flat dose of 8 mg (approximately 0.1 mg/kg) was subsequently chosen for further clinical development. No clear objective responses were observed for urelumab as a monotherapy [14]. The mechanism behind the hepatic toxicity induced by urelumab is not well understood.

Utomilumab

Utomilumab is a FcγR crosslinking-dependent, IgG2 monoclonal antibody targeting domain 3 4 of the 4-1BB receptor [12], and has similar in vitro potency compared to ATOR-1017 [12, 15]. It has been tested at doses ranging from 0.006 mg/kg to 10 mg/kg every 4 weeks in several advanced malignancies [10]. Utomilumab was well tolerated at doses up to 10 mg/kg. None of the patients experienced a dose-limiting toxicity (DLT), and AEs were generally of grade 1 2. No significant liver toxicity was reported.

Other Compounds in Clinical Development

In addition to the monospecific 4-1BB antibodies described above, there are 4 bispecific 4 IBB antibodies in clinical development. RG7827, an anti-41BB x anti-FAP antibody entered clinical phase 1 in 2018, and PRS343, an anti-4-lBB x anti-HER2 antibody entered clinical phase 1 as monotherapy in 2017 and started a combination trial with PD-L1 in 2018. Initiation of the combination therapy study indicates a good safety profile of PRS343, though no data are yet publicly available. Two additional bispecific antibodies targeting 4-1BB x PD-L1 have entered clinical phase in 2019, INBRX-105 and MCLA-145.

Several other 4-1BB targeting antibodies are in preclinical development.

Patient Population Patients, at least 18 years of age, diagnosed with advanced and/or refractory solid malignancies and who have progressive disease (RD) and/or intolerable adverse effects with established therapy, can be enrolled in the study. The patients eligible for the study, are patients who have received standard of care therapy and the remaining therapeutic options are participation in a clinical study or best supportive care.

The patients to be enrolled must meet all inclusion and exclusion criteria specified above.

Scientific Rationale

ATOR-1017 is a fully human agonistic IgG4 antibody targeting the co-stimulatory receptor 4 IBB. 4-1BB has been shown to be highly expressed on tumor infiltrating CD8+ T effector cells (Teffs) in several cancer indications, while expression on Teffs in the circulation is low [3, 16, 17]. By binding to 4-1BB, ATOR-1017 is expected to enhance the activity of tumor reactive Teffs and NK cells within the tumor, and thereby induce a more powerful anti-tumor attack. ATOR-1017 is an IgG4 antibody, and the IgG4 subclass was chosen to allow efficient immune activation and tumor cell killing by crosslinking with FcγRs without the undesired killing of 4 IBB expressing effector cells by ADCC. ATOR-1017 is dependent on FcγR crosslinking for its agonistic effect. The FcγR crosslinking-dependency of ATOR-1017 is expected to direct the agonistic effect to the tumor environment and tumor draining lymph nodes and reduce the systemic immune activation due to the high abundance of endogenous circulating IgG which will compete with ATOR-1017 for binding to FcγRs [9-11].

Potential liver impairment

The clinical study will include close monitoring of liver function through repeated assessment of standard laboratory tests. Patients must also have normal or maximum grade 1 increase of liver function tests (ALT, AST and bilirubin) for enrolment, and patients with prior hepatitis B and/or prior untreated hepatitis C infection are excluded from participating in the study to reduce the risk of inducing clinically important hepatotoxicity. In case of signs of hepatic toxicity related to treatment with ATOR- 1017, the Protocol includes instructions for repeated laboratory testing of ALT, AST, and bilirubin (total and direct). The clinical study will include close monitoring of liver function through repeated assessment of standard laboratory tests. Patients must also have normal or maximum grade 1 increase of liver function tests (ALT, AST and bilirubin) for enrolment, and patients with prior hepatitis B and/or prior untreated hepatitis C infection are excluded from participating in the study to reduce the risk of inducing clinically important hepatotoxicity. In case of signs of hepatic toxicity related to treatment with ATOR- 1017, the Protocol includes instructions for repeated laboratory testing of ALT, AST, and bilirubin (total and direct).

Potential Neutropenia

Neutropenia may increase the risk for infections, especially grade 4 neutropenia that lasts longer than 7 days [19]. Grade 3-4 neutropenia was observed with urelumab [13, 20]. Neutropenia was not observed for patients treated with utomilumab [18]. To mitigate the risk for induction of clinically important neutropenia, patients must have neutrophils ≥1500/μL (≥1.5 x 109/L) to be enrolled in the study, and ≥500/μL (≥0.5 x 109/L) before each dosing of ATOR 1017.

Potential Renal Impairment

In the toxicology in cynomolgus monkeys, an increase in urine volume was observed in two animals following one month of repeated dosing with ATOR-1017. Furthermore, in tissue cross reactivity studies, membrane staining was observed on the mesangial cells of the kidney glomeruli, but not in the tubuli where fluid absorption occurs.

To mitigate risk of renal impairment, patients must have a creatinine ≤1.5 x ULN or glomerular filtration rate (GFR) of ≥45 mL/min, to be eligible for the study. In addition, measurements of creatinine and electrolytes are carried out at multiple time points throughout the study.

Glomerular filtration rate (GFR) may be estimated based on commonly used and accepted formulae, i.e. one of the below formula.

Cockroft Gault formula:

Units: GFR [ml/ min], age [years], weight [kg], serum creatinine [mg/dl], FS is a correction Factor for Sex: in males FS = 1, in females FS = 0.85 Modification of Diet in Renal Disease (MDRD) formula:

Units: GFR [ml/min], age [years], serum creatinine [mg/dl], FS is a correction Factor for Sex: in males FS = 1, in females FS = 0.762

Variations of the MDRD formula:

Units: GFR [ml/min], age [years], serum creatinine [mg/dl], Fs is a correction Factor for Sex: in males Fs = 1, in females Fs = 0.742

Potential Adrenal Insufficiency

Binding of ATOR-1017 to the membrane of cells in the adrenals was observed in tissue cross reactivity studies. One case of adrenal insufficiency has been reported in a clinical trial combining utomilumab and pembrolizumab [21]. However, adrenal insufficiency has not been reported in clinical studies of monotherapy with co-stimulatory receptor 4-1BB antibodies [13, 18]. Adrenal insuffiency, most often secondary to hypophysitis, does occur with treatment with checkpoint inhibitors [22].

Potential Immunogenicity

Introduction of a foreign protein may provoke an immune response in humans. Though ATOR 1017 is a human antibody, it may still be immunogenic and induce an immune response. Immunogenicity against ATOR-1017 will be evaluated during the clinical study at regular intervals. The samples for immunogenicity testing will be used for anti-drug antibody (ADA) analysis (i.e. antibodies against ATOR-1017) and confirmed positive ADA samples will be tested for neutralizing antibodies.

If the infusion of ATOR-1017 is interrupted due to an AE, a sample for immunogenicity should be collected at the time of interruption (except during the first infusion) together with a PK sample.

ADA was measured as follows:

Equipment The following equipment was found to be suitable for use during the validation study.

Equivalent equipment may be employed provided adequate selectivity and sensitivity are achieved.

Positive control

Goat anti-ATOR-1017 antibody (Batch No. 356.40.76FT) was supplied by the Sponsor with a stated concentration of 1.08 mg/mL. The material was stored in a freezer set to maintain a temperature of -80°C.

Upon receipt into the Department of Immunobiology, goat anti-ATOR-1017 was divided into aliquots each with sufficient volume to prepare positive control samples for one analytical batch. Aliquots were stored in a freezer set to maintain -80°C and were considered stable for a maximum of 12 months following the initial thawing and aliquoting procedure. Following the use of each aliquot any residual material was discarded.

Critical reagent

ATOR-1017 (Batch No. ATOR-1017-DP-01) was supplied as slightly yellow liquid with a stated concentration of 20.0 mg/mL.

ATOR-1017 was divided into aliquots each with sufficient volume to prepare immunodepleted samples for one analytical batch. Aliquots were stored in a refrigerator set to maintain 2-8°C. Following the use of each aliquot any residual material was discarded.

Biotin Labelling of ATOR-1017 ATOR-1017 (Batch No. ATOR-1017-DP-01) was labelled using an EZ-Link™ Sulfo-NHS- LC-Biotinylation Kit (Thermo Scientific Cat. No. 21435) following the instructions provided by the manufacturer. ATOR-1017 was diluted in BupH PBS to obtain a solution of 2 mg/mL. This solution was treated with Sulfo-NHS-LC-Biotin and incubated for 45 min. Following buffer exchange and removal of excess labelling material using a desalting column, the biotinylated material (with an assumed concentration of 2 mg/mL) was aliquoted and stored in a refrigerator set to maintain a temperature of 2- 8°C. A date identical to the expiry date of the unlabelled ATOR-1017 material was assigned to the labelled material.

Following the use of each aliquot of ATOR-1017-Biotin, any residual material was discarded.

SULFO-TAG™ Labelling of ATOR-1017

ATOR-1017 (Batch No. ATOR-1017-DP-01) was labelled using an MSD GOLD SULFO-TAG NHS-Ester Conjugation Pack (MSD Cat. No. R31AA) following the instructions provided by the manufacturer. ATOR-1017 was diluted in conjugation buffer to obtain a solution of 2 mg/mL. This solution was treated with MSD GOLD SULFO-TAG NHS-Ester and incubated for 2 hours (± 12 min). Following buffer exchange and removal of excess labelling material using a desalting column, the SULFO-TAG™ material (with an assumed concentration of 2 mg/mL) was aliquoted and stored in a refrigerator set to maintain a temperature of 2-8°C. A date identical to the expiry date of the unlabelled ATOR-1017 material was assigned to the labelled material.

Following the use of each aliquot of ATOR-1017-SULFO-TAG™, any residual material was discarded.

Control Matrix

Control human serum was obtained from BioIVT. Upon arrival serum was divided into volumes of 3 mL. All serum was stored in a freezer set to maintain a temperature of - 20°C when not in use and subjected to a maximum of 3 freeze thaw cycles (following arrival). All control human serum was used within the stated supplier provided expiry date.

Other Materials Chemicals were of analytical grade where available:

Benefit-Risk Assessment

Monoclonal antibodies given intravenously may be associated with infusion-related reactions, especially for the first infusion. Close monitoring of the patients during and after the infusions allows rapid detection and mitigation of infusion-related reactions. There is no clinical experience with ATOR-1017 in humans; therefore, ATOR-1017 dosing will start at a dose level based on a minimal anticipated biological effect level (MABEL) calculation. This dose level is well below the highest dose tested with no adverse events in cynomolgus monkeys, the pharmacodynamically active dose (PAD), and the clinically well tolerated doses of other agonistic 4-1BB antibodies.

The observation period for DLTs will be 21 days from first dose of ATOR-1017, i.e. corresponding to the first treatment cycle. This is considered sufficient based on the predicted half-life of ATOR-1017 (14-21 days) and the fact that adverse reactions to immune activating antibodies usually are observed within a few days of drug administration.

The purpose of the safety precautions and close monitoring of the patients is to secure a positive benefit-risk ratio for the patients. ATOR-1017 has not previously been tested in humans and there is a risk that unknown side effects that can be mild or severe in intensity may occur. While there is a potential risk that ATOR-1017 may be associated with systemic reactions such as cytokine release syndrome, immune-related adverse events, elevations in liver transaminases or severe hepatotoxicity, it can potentially reduce tumor burden as well as prolong survival of patients with advanced solid tumors.

It is considered that patients who are eligible for this study, may benefit from treatment with ATOR-1017, in terms of reduced tumor burden, tumor stabilization or improvement of tumor related symptoms, as well as prolonged survival. The current study enrolls adult patients diagnosed with advanced and/or refractory solid malignancies who have progressive disease. This patient population has a very poor prognosis, and a need for new therapeutic options.

The potential benefit of ATOR-1017 treatment is expected to outweigh the treatment- related risks, and overall, ATOR-1017 is believed to have an acceptable risk/benefit profile.

Objective and endpoints

Study Overview

This first-in-human study is an open-label, multicenter, phase 1 dose escalation study to determine the safety and tolerability of ATOR-1017 administered intravenously.

The study has an accelerated dose escalation design followed by a modified 3+3 design as illustrated in Figure 2. The accelerated part consists of single-patient cohorts for dose levels below 40 mg. However, when a patient in a single-patient cohort experiences one grade ≥2 toxicity lasting for more than 72 hours or two grade ≥2 toxicities (regardless of duration) during the DLT evaluation period the cohort will be mandatorily expanded with additional 2 (at least) patients at this dose level and this marks the start of the modified 3+3 design part of the study.

The study starts with a screening period which is followed by a treatment period consisting of treatment cycles of 21 days. The schedule of ATOR-1017 administration is every 21 days (Day 1 of each cycle). A tumor response evaluation will be performed by assessing CT scans using iRECIST, at screening, approximately after 6 and 12 weeks (at the end of Cycle 2 and Cycle4) and thereafter approximately every 12th week (in Cycles 8, 12, 16 etc.) during treatment until progression of disease. The overview of the study is illustrated in Figure 3. Flat Dose

ATOR-1017 will be administered as flat (fixed) doses without adjustment of body weight. This will simplify the preparation for administration as no dose calculations based on body weight of the individual patient will be needed. The flat doses are justified by:

• Distribution volume of monoclonal antibodies is generally the blood plasma and extracellular fluids that vary much less than body weight or body surface area [27]

• Monoclonal antibodies usually have a wide therapeutic window [27]

• Elimination of monoclonal antibodies are mainly by non-specific IgG pathways and/or target mediated clearance

• The number of target cells, i.e. T cells, including subsets, is not considered to correlate to body weight or body surface area

Dosage Schedule

The dosage schedule with iv administration every 3 weeks is based on animal data and the expected half-life of ATOR-1017. The half-life of ATOR-1017 in cynomolgus monkeys was in the range of 6-8.5 days after a single iv infusion. The half-life of IgG monoclonal antibodies is usually around 3 weeks [28]. The objective of the dosing regimen is to achieve intermittent high exposure, with periods of lower exposure in between. The reason for this is that high intensity prolonged stimulus of co-stimulatory pathways, such as 4-1BB, may be associated with exhaustion of signaling and a diminished response (also known as tachyphylaxia or desensitization). It is expected that dosing every 3 weeks will result in significant exposure but with reduced risk of desensitization, as compared to a more frequent dosing schedule. PK data collected in this study will be used to select the dosage schedule in further clinical development of ATOR-1017.

Administration

The following guidance for iv administration of ATOR-1017 will apply: • The infusion will be administrated at a constant rate over a 2-hour period. • Administration via a peripheral vein is preferred, but administration via a central venous catheter or infusion port is acceptable for doses ≥1 mg. • For medical reasons, the duration of the infusion may be extended (provided that the ATOR-1017 solution for infusion are used within 24 hours of preparation).

For a patient to continue ATOR-1017 treatment, all the following criteria must be met: • Neutrophils ≥500/μL (0.5 x 109/L) and platelets ≥50.000/μL (50 x 109/L), with or without growth factor support or transfusions • AST and ALT ≤5 x ULN • The patient has tolerated previous doses of ATOR-1017, in the opinion of the Investigator

Premedication

Premedication, as often used together with monoclonal antibodies, is not mandatory in this study.

If a patient has an infusion-related reaction during the infusion or during the post- infusion monitoring period, premedication for subsequent infusions must be considered by the Investigator. The premedication can include one or more of the following medications given 30-120 minutes prior to the infusion:

• Acetaminophen (paracetamol), e.g. 650-1000 mg per os (i.e., oral administration)

• Antihistamine, e.g. diphenhydramine 50 mg iv or per os (i.e., oral administration) (or equivalent)

• Glucocorticoid, e.g. prednisolone 100 mg iv

• H2 antagonist, e.g. ranitidine (50 mg) or equivalent

• Antiemetic, e.g. ondansetron (16-24 mg) or equivalent

Prohibited Medication

The following medications are prohibited during the study: • Anti-cancer medication other than ATOR-1017 • Any other investigational therapy • Systemic corticosteroids >10 mg prednisolone or equivalent per day, except if used to mitigate and/or relieve AEs related to ATOR 1017 treatment such as infusion-related reactions • Systemic immunosuppressants, except if used to mitigate and/or relieve

AEs related to ATOR-1017 treatment • Dietary supplements, except for multivitamins, vitamin D and calcium, and supplements for prevention of weight loss • Traditional medicines (natural or herbal medicines and products) • The patient should consult with the Investigator before taking any medications, including over the-counter products.

Permitted Concomitant Medications and Therapies • Palliative radiotherapy for local pain control provided that the patient does not have PD in the opinion of the Investigator, that no more than 10% of the patient's bone marrow is irradiated and that the radiation field does not encompass a target lesion • Concurrent bisphosphonates, denosumab and androgen deprivation therapies such as LHRH (GnRH) agonists (as long as the patient has been on stable doses for at least 4 weeks prior to screening) • Hormonal therapy for non-cancer related conditions • Allopurinol and/or rasburicase as well as hydration according to medical practice for patients at risk of developing tumor lysis syndrome are recommended • Red cell transfusion if clinically indicated • G-CSF and other hematopoietic growth factors in the management of acute toxicity, such as febrile neutropenia at the Investigator's discretion and as clinically indicated • Prophylactic antibiotics, antiviral and antifungal therapy • Multivitamins, vitamin D and calcium, and supplements for prevention of weight loss • Inhaled and low dose systemic corticosteroids of ≤10 mg prednisolone or equivalent per day • Systemic corticosteroids >10 mg prednisolone or equivalent per day for the treatment of AEs related to ATOR-1017 treatment, e.g. infusion-related reactions.

Clinical Laboratory Tests

The clinical laboratory tests to be performed are listed in Table 6. Table 6: Clinical Laboratory Tests Pharmacokinetics (PK)

The samples for PK analysis must be taken from a peripheral vein contralateral to the arm into which ATOR-1017 is infused.

The following PK parameters will be derived :

• C _max • T _max

• AUC _(0-T)

Other PK parameters may be derived if data allows such as:

• AUC _0-∞

• AUC _[

• Elimination half-life

• Total serum clearance (CL)

• Volume of distribution (V _d )

Immunogenicity

The samples for immunogenicity testing will be used for ADA analysis (i.e. antibodies to ATOR 1017). Confirmed positive ADA samples will be tested for neutralizing antibodies.

Assessment of Anti-Tumor Activity

Computed Tomography (CT) Scan

CT scans of chest/abdomen/pelvis should be taken according to local practice. Other body areas may also be CT scanned if needed to assess the tumor(s) (e.g. a CT scan of neck would be needed for a patient having cervical nodes or a head and neck tumor). Additional CT scans may be taken based on Investigator's judgement at regular visits or at additional (unscheduled) visits.

The use of iv contrast is at the discretion of the radiologist performing the scanning, but imaging must be consistent per patient throughout the study. If a CT scan is considered not feasible, as judged by the Investigator, a Magnetic Resonance Imaging (MRI) may be performed. The same scanning modality must be used throughout the study.

The Investigator and/or radiologist will identify the tumors to be followed throughout the study. These will be recorded on the relevant eCRF page(s).

Participants in this study will undergo CT scans for evaluation of disease once during screening, after approximately 6 and 12 weeks (at the end of Cycle 2 and Cycle 4) and thereafter approximately every 12th week (in Cycles 8, 12, 16 etc.) during treatment until progression of disease. The number of additional scans is dependent on how long the patient remains in the study.

The CT scans will be evaluated according to iRECIST. Patients with response (iPR or ICR) must have a confirmatory CT scan at least 4 weeks later to confirm the response. Patients with an unconfirmed iPD (iUPD) can continue treatment with ATOR-1017, but they must have a confirmatory CT scan performed at least 4 weeks, and no later than 8 weeks, after the last CT scan. If the iPD is confirmed (iCPD), the patient should discontinue study treatment and be withdrawn from the study.

Tumor Response Evaluation

The evaluation of tumor response will be done according to iRECIST. iRECIST Guideline

Response Evaluation Criteria in Solid Tumors for immune-based therapeutics ( iRECIST) [30] is a consensus guideline which is built on the RECIST version 1.1 [31] and takes into account the tumor response observed with immunotherapies. The major change from RECIST version 1.1 to iRECIST is the requirement of confirmation of PD. This allows for increases in tumor size that can be seen with immunotherapeutics before a shrinkage is observed.

The target lesions should be selected based on their size (lesions with the longest diameter), be representative of all involved organs, but in addition should be those that lend themselves to reproducible repeated measurements. It may be the case that the largest lesion does not lend itself to reproducible measurement in which circumstance the next largest lesion which can be measured reproducibly should be selected.

A sum of the diameters (longest for non-nodal lesions, short axis for nodal lesions) for all target lesions will be calculated and reported as the baseline sum diameters.

All other lesions (or sites of disease) including pathological lymph nodes should be identified as non-target lesions and should also be recorded at baseline. Measurements are not required, and these lesions should be followed as 'present', 'absent', or in rare cases 'unequivocal progression'.

Please see Table 7 below for definitions according to iRECIST.

Table 7: Definitions according to iRECIST

iUPD but not ICPD, can precede ICR, IPR or ISD.

The clinical status of the patient must be taken into consideration after iUPD for the decision of continued ATOR-1017 treatment.

Pharmacodynamics

Blood Blood samples will be taken and the following pharmacodynamic biomarkers will be evaluated : • Serum samples will be analyzed for levels of the following cytokines: TNF- α, IFN-γ, IL 1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70 and IL 13 • Serum sample will be analyzed for the levels of soluble 4-1BB (S4-1BB) • Whole blood samples will be used for immunophenotyping, including : ■ Quantification of immune cell populations (such as T cells, B cells, NK cells and dendritic cells) ■ Markers for specific T cell populations (such as CD4+, CD8+, effector memory T cells, central memory T cells and Tregs) ■ Markers for T cell activation (such as Ki67, ICOS and EOMES)

Cytokine analysis

Cytokine levels in serum from patients were analyzed at Cerba Research using a pre- validated 10-plex. Pro-inflammatory kit from MSD (Meso Scale Diagnostics, 1601 Research Boulevard, Rockville, Maryland, USA) The following cytokines were included : IL-ip, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, TNF-α and IFN-γ.

Immunophenotyping

INSTRUMENT & ANALYSIS SOFTWARE INSTRUMENTS

3 laser (405 nm, 488 nm, 633 nm) / 10 color flowcytometer; Beckton Dickinson FACSCanto™ equipped with FacsDiva software version 8.0.1. (S/N : V657338000177 (referred to as F6; CC-EQ-738-CHIM), V657338000025 (referred to as F7; = CC-EQ- 760-CHIM)).

Analysis software: FacsDiva software version 8.0.1.

GENERAL EQUIPMENT AND MATERIAL Heraeus Multifuge Centrifuge ThermoFisher Vortex VWR international 444-1372

Sysmex XN-3000 Hematology analyzer 3 laser (405 nm, 488 nm, 633 nm) / 10 color BD FACSCanto flow cytometer equipped with FacsDiva version 8.0.1. Instrument codes: CC-EQ-738-CHIM and CC-EQ-760 CHIM Distilled PBS (DPBS)

FACS Shutdown Solution BD Biosciences 334224

FACS Flow Solution BD Biosciences 342003 FACS Clean Solution BD Biosciences 340345

Falcon 12x75mm, 5mL polystyrene round bottom tubes VWR 734-0000

Stain Buffer BD Biosciences 554656

FACS Lysing Solution BD Biosciences 349202

CompBeads BD Biosciences 552843

CS81T Beads BD Biosciences 641319

BD OneFlow setup Beads BD Biosciences 658620

ANTIBODY LIST

CCR7 (CD197) BV421 150503 BD Biosciences 562555

CD25 PE 2A3 BD Biosciences 341011

CD4 V500 RPA-T4 BD Biosciences 560768

CD45RA FITC LEU-18 BD Biosciences 335039 CD8 PerCP-Cy5.5 RPA- T8 BD Biosciences 560662

CD127 AF647 HIL-7R-M21 BD Biosciences 558598

CD3 APC-H7 SK7 BD Biosciences 560176

ICOS PE-Cy7 C.38.A4 BioLegend 313519

Eomes PE-Cy7 WD1928 eBioscience 25-4877-42

Ki-67 BV605 Ki-67 BioLegend 350521

OTHER ASSAY-SPECIFIC REAGENTS

Viability Stain 700 BD Biosciences 564997 Perm/Fix Solution2 BD Biosciences 555899

Sample preparation - Intracellular staining (Eomes and Ki67 tube)

1. Determine the leukocyte concentration (cells/μL) with a hematology analyzer. Forthe panel below a leukocyte concentration of 10 x 10 ³ cells/μL (between 5 - 15 x10 ³ cells/μL) is recommended. If the leukocyte concentration exceeds 15 x 10 ³ cells/μL, dilute the sample first with Stain Buffer to the recommended leukocyte concentration.

2. Pipette the below listed antibodies in their respective volumes in 12x75 mm polystyrene tubes.

SUBSTITUTE SHEET (RULE 26)

3. Add 150 μL whole blood to each tube.

4. Vortex.

5. Incubate 30 minutes at room temperature in the dark.

6. Add 1.5 mL FACS Lysing Solution (first diluted 1/10 with distilled water).

7. Vortex.

8. Incubate 15 minutes at room temperature in the dark.

9. Centrifuge during 5 minutes at 470 g.

10. Decant the supernatant.

11. Add 2 mL of Staining Buffer.

12. Vortex.

13. Centrifuge during 5 minutes at 470 g.

14. Decant the supernatant.

15. Add 0.5 mL 1x BD FACS PermSolution 2.

16. Vortex.

17. Incubate 10 minutes at room temperature. (No longer than 10 minutes!!)

18. Add immediately 2 mL Staining Buffer Vortex.

20. Centrifuge during 5 minutes at 470g.

21. Decant the supernatant.

22. Add 100 μL Staining Buffer.

23. Pipet the antibodies in their respective concentrations.

24. Vortex.

25. Incubate 30 minutes in the dark at room temperature.

26. Add 2 mL Staining Buffer.

27. Centrifuge 5 minutes at 470 g.

28. Decant supernatant.

29. Resuspend in 300 μL Staining Buffer

Reportable parameters

Determination of the absolute cell counts is based on the dual platform methodology. In this methodology, the absolute number of CD3 T cells is calculated based on the absolute lymphocyte count of the hematology analyzer (Sysmex XN-9000). As the hematology analyzer cannot distinguish between living and dead lymphocytes, an extra step is implemented to exclude the number of dead lymphocytes. Therefore, the absolute number of CD3 T cells is calculated as follows:

CD3 T cells (cells/μL) - (CD3 T cells (% of Lympho)/100 * ((Viable Lympho #Events/ Lympho #Events)* Lymphocytes (10 ³ cells/μL)) *1000.

Calculation of the absolute number of cell populations downstream from CD3 T cells has been calculated as follows:

CD4 T cells (cells/μL ) = [CD4 T cells (% CD3 T)xCD3 T cells (cells/μL )] / 100

Test principle

The panel is designed to identify and enumerate T cell populations including their activation status in terms of their expression of activation and proliferation markers ICOS, Eomes and Ki67. First, viable CD4 and CD8 T cells are defined based on CD4, CD8 and CD3 expression. Further, naive CD4 and CD8 T cells and TEM and TCM CD4 and CD8 T cells are defined based on memory markers CCR.7 and CD45RA. Finally, CD4 Treg cells are define based on their CD25high and CD127low expression profile. The presence of ICOS, Eomes and Ki67 is reported on all of the above-listed subsets

Soluble 4-1BB

Soluble 4-1BB (TNFRSF9) was measured in serum samples obtained from patients using the commercial Kit: Human TNFRSF9 ELISA Kit Catalogue Number: EHTNFRSF9.

Adverse events

Adverse Event (AE)

An AE is any untoward medical occurrence in a patient or clinical investigation subject administered a medicinal product and which does not necessarily have a causal relationship with this treatment. An AE can be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom or disease temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product.

Adverse Reaction (AR) All untoward and unintended responses to an investigational medicinal product related to any dose administered.

The phrase "responses to a medicinal product" means that a causal relationship between a medicinal product and an AE is at least a reasonable possibility, i.e. the relationship cannot be ruled out.

Serious Adverse Event (SAE) or Serious Adverse Reaction (SAR)

A serious AE (SAE) or serious AR (SAR) is any untoward medical occurrence or effect that at any dose: • Results in death • Is life-threatening (Note: The term "life-threatening" in the definition of "serious" refers to an event in which the patient was at risk of death at the time of the event; it does not refer to an event which hypothetically might have caused death if it were more severe) • Requires in-patient hospitalization oorr prolongation of existing hospitalization, unless the hospitalization is for:

■ Routine treatment or monitoring of the disease under study, including hospitalization due to study-related procedures (e.g. administration of medicinal product) or to manage AEs related to signs or symptoms of disease under study

■ Elective treatment (planned before signing the ICF) for a pre-existing condition that is unrelated to the disease under study and has not worsened since signing the ICF

■ Treatment on an emergency outpatient basis for an event not fulfilling any of the definitions for an SAE

■ Social reasons, respite care in the absence of a medical condition

Results in persistent or significant disability/incapacity or substantial • disruption of the ability to conduct normal life functions • Is or results in a congenital abnormality or birth defect

Medical and scientific judgement should be exercised in deciding whether expedited reporting is appropriate in other situations, such as important medical events that may not be immediately life-threatening or result in death or hospitalization but may jeopardize the patient or may require intervention to prevent one of the other outcomes listed in the definition above. These AEs should also usually be considered serious. Examples of such events are intensive treatment in an emergency room or at home for allergic bronchospasm; blood dyscrasias or convulsions that do not result in hospitalization; or development of drug dependency or drug abuse.

To ensure no confusion or misunderstanding of the difference between the terms "serious" and "severe," which are not synonymous, the following note of clarification is provided:

The term "severe" is often used to describe the intensity (severity) of a specific event (as in mild, moderate, or severe myocardial infarction); the event itself, however, may be of relatively minor medical significance (such as severe headache). This is not the same as "serious," which is based on patient/event outcome or action criteria usually associated with events that pose a threat to a patient's life or functioning. Seriousness (not severity) serves as a guide for defining regulatory reporting obligations.

Adverse Event of Special Interest (AESI)

An AESI is any AE, serious or non-serious, irrespective of its relationship to the IMP, that is of scientific and medical concern to the Sponsor's product or program, for which ongoing monitoring and rapid communication by the Investigator to the Sponsor can be propagated.

The following AEs are considered as AESIs for this Clinical Study Protocol:

• Grade ≥2 infusion-related reaction • Grade ≥2 cytokine release syndrome • Grade ≥3 liver enzyme elevation of AST and/or ALT • Grade ≥2 bilirubin elevation

Unexpected Adverse Reaction

An unexpected AR is an AR where the nature or severity of which is not consistent with the applicable product information (e.g. the RSI in the Investigator's Brochure for an unapproved investigational medicinal product).

Suspected Unexpected Serious Adverse Reaction (SUSAR)

A Suspected Unexpected Serious Adverse Reaction (SUSAR) is a SAR that is also unexpected according to the definition above. Disease Progression

Disease progression can be considered as a worsening of a patient's condition attributable to the disease for which the investigational product is being studied. It may be an increase in the severity of the disease under study and/or increases in the symptoms of the disease.

Deterioration of the disease under study and associated symptoms or findings, including the development of new, or the progression of existing, metastases, should not be regarded as an AE, unless the study medication is considered to have contributed to the progression.

New Cancers

New cancers are those that are not the primary reason for the administration of the study treatment and have been identified after the patient's inclusion in this study. They do not include metastases of the original cancer. The development of a new cancer should be regarded as an AE and will generally meet at least one of the serious criteria.

References for Example 1

1. Labrijn, A.F., et al., Therapeutic IgG4 antibodies engage in Fab-arm exchange with endogenous human IgG4 in vivo. Nat Biotechnol, 2009. 27(8): p. 767-71.

2. Sanmamed, M.F., et al., Agonists of Co-stimulation in Cancer Immunotherapy

Directed Against CD137, 0X40, GITR, CD27, CD28, and ICOS. Semin Oncol, 2015. 42(4): p. 640-55.

3. Ye, Q., et al., CD137 accurately identifies and enriches for naturally occurring tumor-reactive T cells in tumor. Clin Cancer Res, 2014. 20(1): p. 44-55.

4. Vinay, D.S. and B.S. Kwon, Therapeutic potential of anti-CD137 (4- 1BB) monoclonal antibodies. Expert Opin Ther Targets, 2015. 20(3): p. 361-73.

5. Kohrt, H.E., et al., CD137 stimulation enhances the antilymphoma activity of anti-CD20 antibodies. Blood, 2011. 117(8): p. 2423-32.

6. Bartkowiak, T. and M.A. Curran, 4-1BB Agonists: Multi-Potent Potentiators of Tumor Immunity. Front Oncol, 2015. 5: p. 117.

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Infiltrating T-Cell Growth into a Distinct Repertoire Capable of Tumor Recognition in Pancreatic Cancer. Clin Cancer Res, 2017. 23(23): p. 7263-7275.

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Example 2 - patients treated, and discussion of general treatment profile

Progress of the patients through the study, and related dosages

Further information about the design of the study and the dosages administered is included in Figures 1-3.

The study included 25 patients with various cancers, as shown in Figures 4A and 4B. That figure also shows the dosages and time over which the patients remained in the study. Further information regarding the patients is included in Figure 5 and 6. Stable disease (SD) was observed in 13 patients (52%), which lasted >6 months for 6 (24%) patients (of whom 2 had ovarian cancer).

Patient profiles

Below are available profiles for patients treated in the study.

Patient 001

• Ovarian Cancer, Stage IV, diagnosed in 2011 (stage IV at study entry)

• ATOR 1017 treatment started in Dec 2019

• PDL1 status UNK, no known mutations

• Number of prior regimens - 8 (including pembrolizumab in 2018- 29 cycles- Disease Progression)

• Had modified radical hysterectomy - invasive ovarian cancer seropapillary type Grade 4.

• No radiotherapy

• 1017 Dose Cohort- 5 Dose escalations (0.38- 40mg)

• Number of cycles- 14

• Best response - ISD (immune stable disease) first recorded in Jan 2020 (EoC2)

• EoT (end of treatment) Oct 2020; iCPD (confirmed progressive disease as per iRECIST); FU (follow-up) visit on Nov 2020; new anti-cancer therapy (UNK refers to "unknown" here, and below) started in Dec 2020

• No grade 3 or higher AEs/SAEs related to ATOR 1017. Only one G3 lymphopenia; unrelated. No SAEs.

Patient 001- Prior Therapy

Patient 003

• Squamous Cell Anal Cancer, Stage IV diagnosed in 2016 (stage IV at study entry)

• PDL1 status UNK, no known mutations

• Number of prior regimens- 8

• Had curative liver resection twice in 2017

• Radiotherapy for locally advanced metastatic disease (twice in 2017); CR and palliative radiotherapy to right arcus (twice in 2019)

• 1017 Dose Cohort- 3 Dose escalations (5mg- 40mg)

• Number of cycles- 12

• Best response- iSD first recorded on 01-Apr-2022 at EoC2

• No Grade 3 AEs related to ATOR 1017. No reported SAEs

• EoT in Nov 2020 due to clinical deterioration; iUPD (Unconfirmed progressive disease) on iRECIST assessment (Oct 2020)

• Follow up visit on Nov 2020; no info on new anti-cancer therapy/ radiation therapy

> Number of prior regimens- 6

> Prior surgeries

> Palliative radiotherapy to liver (twice in 2014) and lower lobe of right lung (twice in 2016); PR (partial response)

> 1017 Dose Cohort- Dose escalated from 40 mg to 100 mg at C7

> Number of cycles- 7

> Best response- iSD first recorded in Aug 2020 at EoC2.

> EoT in Dec 2020 due to clinical progression; I RECIST assessment in Dec 2020 was iUPD.

> No Grade ≥3 AEs related to ATOR 1017; No SAEs reported

Patient 010

• Adenoid Cystic Cancer - left sub-mandibular gland diagnosed in 2004 at Stage I; stage IVC at study entry

• PDL1 status UNK; no known mutations

• Number of prior regimens- No prior systemic therapy

• Curative surgeries; removal of left sub-mandibular gland (2004); neck dissection left side (13-Jul-2004); upper lobectomy right side (2017)

• Adj radiotherapy to left neck (twice in 2004)

• 1017 Dose Cohort- Dose escalated from 100 mg to 200 mg at CIO

• Number of cycles- 12

• Best response- ISD first recorded in Oct 2020 at EoC2

• EoT in Jun 2021 due to investigator's decision; iUPD per iRECIST in May 2021; Follow up visit in Jun 2021; no info on new cancer treatment

• No Grade 3 AEs related to ATOR 1017. No reported SAEs.

Patient 016 • Mucinous adenocarcinoma of the appendix diagnosed in 2016 at stage IIB; stage IV at study entry

• PDL1 status UNK; no known mutations

• Number of prior regimens- 19

• Prior surgery

• Prior radiotherapy to both lungs (twice in 2018) • 1017 Dose Cohort- 360 mg

• Number of cycles- 2

• Best response- iSD first recorded in Jul 2021 at EoC2

• EoT in Aug 2021 due to clinical progression in the opinion of the investigator; iSD on iRECIST; no FU assessment on patient status or commencement of any therapy.

• Grade 3 AST and Grade 3 ALT elevation deemed related to ATOR 1017; No SAEs

Patient 002

• Choroidal melanoma, Stage IIB diagnosed in 2017; stage IVB at study entry

• PDL1 status UNK; no known mutations • Number of prior regimens- 3 (including pembrolizumab in 2018, 13 cycles- progressive disease) • Prior radiation therapy to right eye (2017) • Prior surgery: Enucleation of the right eye (2017) • 1017 Dose Cohort- 5 Dose escalations (1.5-100mg) • Number of cycles- 19 • Best response- iSD first recorded on Apr 2020 at EoC4. EoC2 assessment was iUPD • EoT in May 2021 due to iCPD per iRECIST evaluation in Apr 2021. Follow up visit not completed - patient started treatment in another trial. • No Grade ≥3 AEs related to ATOR 1017; no SAEs

Patient 009

• Mixed adenoneuroendocine carcinoma of the pancreas, diagnosed in 2016 at stage IIIB; Stage IV at study entry

• PDL1 status UNK; no known mutations

• Number of prior regimens- 3

• Prior surgery; no prior radiotherapy

• 1017 Dose Cohort- 100mg

• Number of cycles- 7

• Best response- iSD first recorded in Oct 2020 at EoC2

• EoT in Feb 2021 as iCPD by iRECIST evaluation in Feb 2021; No FU visit (patient went to his homeland for treatment)

• No Grade ≥3 AEs related to ATOR 1017; No SAEs

Patient 009 - Prior Therapy

Patient 014

• Cholangiocarcinoma, diagnosed in 2019 at Stage IIIA; Stage IIIA at study entry also.

• PDLI status UNK; mutations 2; other biomarkers CK7+, CK19+

• Number of prior regimens- 4

• No prior surgery; only diagnostic biopsy in 2019; No prior radiotherapy

• 1017 Dose Cohort- 360mg

• Number of cycles- 8

• Best response- iUPD in Jul 2021 at EoC2 and in Aug 2021 at EoC4; Never achieved iSD;

• EoT in Nov 2021 due to ICPD per iRECIST in Nov 2021; FU not performed; patient started new treatment

• One Grade 3 AE of febrile neutropenia related to ATOR 1017 (Jun 2021); No SAEs

Patient 014 - Prior Therapy

Patient 015

• Low grade serous ovarian cancer, diagnosed in 1995 at Stage IIIC, and stage

IIIC at study entry • PDL1 status UNK; no known mutations

• Number of prior regimens- 14

• Prior surgery: hysterosalpingo-oophorectomy in 1995 and diagnostic biopsy in

1995; No radiotherapy • 1017 Dose Cohort- 360 mg; dose escalated to 600 mg at C12

• Number of cycles- 17

• Best response- ISD first recorded in Jul 2021 at EoC2

• Patient still on treatment. Latest iRECIST assessment was iSD in May 2022 at

EoC16 o SPD at screening- 40 mm o SPD at EoC2 (44 mm), EoC4 (43mm), EoC8 (47mm) and EoC16 (40mm) • No Grade ≥3 AEs related to ATOR 1017; No SAEs

Patient 011

• Melanoma in-situ, diagnosed in 2015; Stage IVA at study entry

• PDL1 status UNK; No known mutations

• Number of prior regimens- 2 (including Nivolumab in 2016-18, 45 cycles, Progressive disease)

• No major cancer related surgeries other than diagnostic procedures (skin excision, LN excision and FN biopsy) in 2015; No radiotherapy

• 1017 Dose Cohort- 200mg

• Number of cycles- 10

• Best response- iSD first recorded in Jan 2021 at EoC2

• EoT in Jul 2021 due to iCPD on iRECIST (Jun 2021); no FU visit performed as patient started new anti-cancer treatment

• No Grade ≥3 AEs related to ATOR 1017; No SAEs

• Number of prior regimens- 7

• Prior surgery: hysterectomy and salpingo-oophorectomy, omentum resection and appendicectomy in 1993 and pelvic tumour surgery in 2014.

• Radiotherapy to right supraclavicular fossa in 2018

• 1017 Dose Cohort- 600 mg

• Number of cycles- 4

• Best response- iUPD first recorded in Jan 2022 at EoC2

• Off treatment due to iCPD on iRECIST assessment at EoC4 (Mar 2022).

• Related to ATOR 1017: Grade 4 neutropenia (SAE; Dec 2021 and Jan 2022)

• Other Grade 3 non-serious AEs: Leukopenia (Dec 2021); Grade 3 thrombocytopenia (Dec 2021 and Jan 2022)

Patient 027

• High grade serous ovarian cancer (left ovary), diagnosed in 2017 at Stage IVB, and was stage IVB at study entry

• PDL1 status UNK; no known mutations

• Number of prior regimens- 9

• Prior surgery: hysterectomy and bilateral salpingo-oophorectomy in 2017; and diagnostic lymph node biopsy in 2019; No radiotherapy

• 1017 Dose Cohort- 900 mg

• Number of cycles- 4

• Best response- iUPD first recorded in Jun 2022 at EoC2.

• Patient still on treatment (per the most current information in the eCRF)

• No Grade ≥3 AEs related to ATOR 1017; No SAEs

o 1017 Dose Cohort- 900 mg - No reported serious or non-serious adverse event(s)

• Prior therapy o No radiotherapy o Curative surgery: Auto-transplant (2019) o Diagnostic procedure: Excision of malignant melanoma R lower leg and

LN exploartion (2019) o Prior chemotherapy/Immunotherapy/Other:

Nivolumab (2019 and 2020) adjuvant;

- LOAd703 virus (2020 and 2021) palliative;

- Atezulizumab (B/w 2020 and 2021); palliative

Patient 026

• PS: ECOG 1

• Cholangiocarcinoma o Stage IB at diagnosis - (2014) o Stage IV at screening

• Prior therapy o No radiotherapy o Curative surgery: Hemi-hepatectomy (2014) o Prior chemotherapy/Immunotherapy/Other:

- Adjuvant Gemcitabine (2014 and 2015)

- GEMOX (Gemcitabine + Oxaliplatin) (2017) palliative

- GEMOX (2021) palliative

- Panetumumab ( 2021); palliative

Conclusions of Example 2

The study considered the use of ATOR-1017 in patients with a number of different cancers, with ATOR-1017 being shown to be well tolerated and safe, with no serious adverse reactions being reported at any dosage.

This is significant when compared to Urelumab, which in clinical trials was shown to cause fatal hepatotoxicity limiting its use to a flat dosage of 8 mg. No clear objective responses were observed for urelumab as a monotherapy, at that dosage. Therefore, the results show that ATOR-1017 is well-tolerated and safe in patients up to a dosage of 900 mg, which allows it to be safely used at higher dosages that other clinical anti-CD137 antibodies.

Example 3 - results of study

Through the course of the study described in Example 1, the inventors surprisingly identified that a dosage of about 500 mg or more per administration of ATOR-1017 did not result in the generation of anti-drug antibodies (ADA), and showed a good efficacy.

Pharmacokinetics (PK) data

The Pharmacokinetics (PK) data collected during the study is shown in Figures 7-9.

These data shows that the half-life of ATOR-1017 is approximately one week - Figures 7A and 7B.

These data also show a proportional increase in C _max up to a dosage of 900 mg - Figure 8 - and AUG - see Figure 9.

Anti-drua antibodies (ADA)

Data regarding anti-drug antibodies (ADA) is shown in Figure 10, for 21 patients in the study, in response to treatment with ATOR-1017. These data were collected via blood serum samples at the following post-infusion timepoints 5 minutes (min); 1h±10 min; 4h±30 min; and 8h±30 min.

Of the 25 patients in the study, two patients were positive for ADA at pre-treatment (8%).

These data show that six patients of the 21 were positive for ADA, in the 0.38 mg to 360 mg dosages. Of these six ADA positive patients, three patients had transient soluble CD137.

No ADA were detected in patient administered the 600 mg and 900 mg dosages. This data show that the potentially problematic event of ADA was surprisingly not detected at the high dosages of 600 mg and 900 mg. Soluble CD137

Data regarding soluble CD137 is shown in Figures 11-13.

These data shows that there is an increase in soluble CD137 following administration of ATOR-1017. The increase in soluble CD137 was most prominent at the 600 mg and 900 mg dosages. This shows that using this measure, in a patient the biological activity of ATOR-1017 is highest in the high dosages of 600 mg and 900 mg.

Conclusions of Example 3

These data show that there is a proportional increase in the C _max and AUC up to a dosage of 900 mg, for ATOR-1017.

Patients with the highest dosages, of 600 mg and 900 mg, were negative to ADA. This show that those dosages seem to provide the lowest risk of that particular negative even often associated with antibody therapeutics.

Additionally, the highest dosages also showed the highest levels of soluble CD137 which is a marker for therapeutic efficacy.

Therefore, surprisingly, the inventors have identified that ATOR-1017 administered at a dosage of about 500 mg and above, per administration, is particularly safe and has a good efficacy.