WO2025242842A1 - Dosage regimes for the administration of an anti-cd137/pd-l1 antigen-binding protein - Google Patents

Abstract

The present application relates to dosage regimes for the administration of an antigen-binding protein which binds CD137 and programmed death-ligand 1 (PD-L1) and their medical use in the treatment of cancer in human patients.

Description

Dosage Regimes for the Administration of an Anti-CD137/PD-L1 Antigen- Binding Protein

This application claims priority from US63/650,742 filed 22 May 2024, the contents and elements of which are herein incorporated by reference for all purposes.

Field of the Invention

The present invention relates to dosage regimes for the administration of an antigen-binding protein which binds CD137 and programmed death-ligand 1 (PD-L1) and their use in the treatment of cancer in human patients.

Background

Cancer is a complex disease for which there is a still significant unmet medical need. Evasion of the immune system is now widely recognised as a key hallmark of cancer progression and the interplay between the host immune system and the tumour has been an area of intense assessment in recent years. Clinical evidence suggests that increased tumour-infiltrating lymphocytes (TILs), which directly kill the tumour cells, have improved cancer prognosis. However, tumours modulate the local microenvironment through expression of multiple inhibitory molecules that lead to T-cell exhaustion. The clinical benefit of targeting inhibitory molecules has been highlighted by the approval of several monoclonal antibodies (mAbs) against PD-1 , PD-L1 and CTLA-4 checkpoint receptors, collectively known as immune checkpoint blockade (ICB) therapy. Monotherapy with ICB has resulted in significant clinical benefit in a subpopulation of subjects.

CD137 is a costimulatory receptor of the tumour necrosis factor receptor (TNFR) superfamily and is expressed on activated T cells, including CD4+ (Th1 and Th2 cells), CD8+ and regulatory T cells, in addition to B cells, NK cells, natural killer T cells and dendritic cells. CD137 interaction with its ligand, CD137 ligand, results in receptor clustering which triggers the activation of intracellular signalling pathways. CD137 clustering preferentially stimulates CD8+ T cells, compared to CD4+ T cells, and leads to their proliferation, survival and cytotoxic effector function via the production of inflammatory cytokines. It also contributes to the differentiation and maintenance of memory CD8+ T cells. CD137 has been shown to be highly expressed on TILs in several oncology indications including ovarian, non-small cell lung cancer (NSCLC) and triple-negative breast cancer (TNBC) and has been reported to be associated with tumour-reactive subsets of TILs. Furthermore, culturing TILs from TNBC with urelumab, a CD137 agonist mAb, enhanced their cytotoxic function ex vivo. This expression by TILs, and enhanced activity upon CD137 agonist mAb engagement, provides some of the rationale behind its agonistic engagement in vivo and its use in TIL selection for cell transfer. ICB therapy combined with CD137 agonist mAb therapy has shown enhanced effects in tumour growth reduction compared to monotherapy (Bartkowiak T, Curran MA. Front Oncol. 2015,5:117). To initiate signalling, agonist antibodies binding to CD137 typically need to be crosslinked to ensure receptor clustering, which would otherwise occur via binding to CD137 ligand. In vivo, this secondary crosslinking requires the low affinity interaction of the mAb with Fc-gamma receptors (FcyRs), which is a complex process as there are six FcyRs in humans with different expression patterns and affinities for the four human IgG isotypes. FcyRs have been shown to be required for optimal antitumour activity of agonist antibodies targeting TNFR superfamily targets in vivo. However, to date, limited clinical activity has been reported with FcyR-dependent CD137 agonist mAbs. In mouse syngeneic tumour models, CD137 agonist mAbs can eradicate established tumours as monotherapy treatments or in combination with other agents such as chemotherapy, cytokines and other checkpoint regulators (Chu DT, Bac ND, Nguyen KH, et al. IntJ Mol Sci. 2019;20:1822).

Agonist antibodies to CD137, e.g. urelumab and utomilumab, are currently in clinical trials for cancer treatment; however, clinical progress has been slowed by dose-limiting high-grade liver inflammation observed with urelumab (Sanchez-Paulete AR, Labiano S, Rodriguez-Ruiz ME, et al. Eur J Immunol. 2016;46:513-22, and Segal NG, He AR, Doi T, et al. Clin Cancer Res. 2018;24:1816-23). Additional clinical trials are underway with both antibodies, testing both as monotherapies and in combination with radiotherapy and chemotherapy and ICB therapies. Developing CD137 agonists that provide therapeutic benefit without the risk of systemic toxicities is currently a major focus of research in the IO field (Chester C, Sanmamed MF, Wang J, et al.. Blood. 2018;131:49-57).

PD-L1 is a type I transmembrane protein belonging to the B7 family of proteins. It is expressed broadly across immune cell subtypes and binds to its receptors PD-1 (CD274, B7-H1), expressed on activated T cells, or CD80 (B7.1), expressed on activated T cells and APCs. The consequence of either interaction is to inhibit cellular immune responses, limiting T-cell activation and expansion.

PD-L1 is also expressed on Kupffer cells and non-immune cells (including vascular endothelium and placenta) and can be upregulated on certain epithelial cell types in response to inflammation. It is via this mechanism that PD-L1 is believed to maintain peripheral tolerance and protect immune-privileged sites.

Tumours resist immune attack by inducing tolerance among tumour-specific T cells and by expressing ligands that engage inhibitory receptors and dampen T-cell functions within the tumour microenvironment. PD-L1 is selectively expressed on many tumours and on cells within the tumour microenvironment in response to inflammatory stimuli. Blockade of the interaction between PD-1 and PD-L1 potentiates immune responses in vitro and mediates anti-tumour activity in a preclinical model. PD-L1 is the primary PD-1 ligand that is upregulated in solid tumours, where it can inhibit cytokine production and the cytolytic activity of PD-1 + tumour-infiltrating CD4+ and CD8+ T cells. Anti-PD-L1 mAbs (atezolizumab, avelumab and durvalumab) have been developed for therapeutic use in subjects with advanced cancer and show clinical benefit across a range of tumour types. These agents have recently all received accelerated approval in bladder cancer. Additionally, avelumab has received approval in merkel cell carcinoma, while atezolizumab and durvalumab have been approved in metastatic NSCLC. However, although these agents provide dramatic responses in a small percentage of subjects, fewer than one third of unselected subjects respond to these treatments (Bustamante Alvarez JG, Gonzalez-Cao M, Karachaliou N, et al. Cancer Biol Med. 2015;12:209-22), with many showing innate and adaptive resistance to therapy.

In mouse models resistant to single agent treatment with either CD137 agonists or PD-1/PD-L1 blockade, significant synergistic effects are observed when mAbs targeting both these pathways are combined. These observations correlate with enhanced T-cell responses to tumour antigens. The mechanistic basis for this synergy, even in poorly immunogenic mouse cancer models, is that TILs co-express both PD-1 and CD137, and following dual combination treatment, CD8+ T cells can respond to recognise tumour neoantigens (Morales-Kastresana A, Sanmamed MF, Rodriguez I, et al. Clin Cancer Res. 2013;19:6151-62).

Preliminary clinical data have shown the tolerability of the CD137 agonist mAb urelumab given at 8 mg flat dose every three weeks along with the standard doses of the PD-1 mAb nivolumab. The most striking efficacy result was a high overall response rate (ORR) of up to 47% in metastatic melanoma cases, with less than 1 % of tumour cells expressing PD-L1 (Lundqvist A, van Hoef V, Zhang S, et al. J Immunother Cancer. 2016; 4(Suppl 1, part 1):1-106).

Similar clinical results following combined treatment with the CD137 agonist mAb utomilumab and ICB with the PD-1 mAb pembrolizumab have been observed in a phase 1 dose escalation study. The safety profile of the combination was consistent with the side effects expected for pembrolizumab alone.

FS222 is an immunoglobulin G1 (IgGI)-based bispecific antigen-binding protein, that has independent binding sites for the CD137 and PD-L1 proteins. Co-engagement of these targets results in CD137-receptor clustering and signalling, which activates CD4+ and CD8+ T cells and NK cells. FS222 contains two binding sites for CD137, produced by engineering amino acid mutations into the CH3 domains of a propriety anti-human PD-L1 lgG1 mAb. The resulting bispecific tetravalent molecule simultaneously binds to both CD137 and PD-L1 with subnanomolar affinity to result in potent agonism and ICB. This binding of both targets is required for optimal function in primary immune assay systems. As FcyR engagement is not required for FS222 activity, and to minimise the impact of antibody-dependent cellular cytotoxicity (ADCC), mutations to decrease FcyR binding (L234A and L235A, known as the LALA mutation) were engineered into the CH2 domains of FS222. As noted above, dose-limiting high-grade liver inflammation has been observed with anti-CD137 mAbs when used as monotherapy. Due to the expression of PD-L1 in the tumour microenvironment on both tumour cells and immune cells, it is hypothesised that FS222 will target the molecule and its activity will be maximised within this environment, thereby reducing the likelihood of liver toxicity.

Despite the proven benefits of ICB for subjects with solid malignancies, the majority of subjects do not respond or have a short duration of response (DoR). Therefore, there is a need in the art for treatments which improve the rate and durability of anti-tumour responses, induce clinical remissions and ultimately improve survival. The present invention has been devised in light of the above considerations.

Summary of the Invention

FS222 is an antigen-binding protein which binds to both CD137 and PD-L1 and thereby mediates both CD137 activation and PD-L1 checkpoint blockade via the same molecule. In view of the dual functionality and agonist element of FS222, as well as the dose-limiting high-grade liver inflammation which has been shown to be associated with CD137 agonism (see discussion of urelumab above), it was not clear that it would be possible to identify dose levels and administration schedules which provide both efficacy and acceptable safety in human patients.

Despite the difficulties associated with dosing of bispecific molecules, the invention provided herein describes the optimal dosing regimen for FS222. This optimal dosing regimen is detailed in the following embodiments.

The antigen-binding protein employed in the present invention (FS222) comprises the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 2. The antigen-binding protein may further comprise a C-terminal lysine residue (K) at the immediate C- terminus of SEQ ID NO: 1 in one or both heavy chains, such that the FS222 antibody may comprise the heavy chain sequence set forth in SEQ ID NO: 3, in addition to the light chain sequence set forth in SEQ ID NO: 2. For simplicity, SEQ ID NO: 1 is referred to as the heavy chain sequence of the antigen-binding protein herein. However, wherever SEQ ID NO: 1 is mentioned, the antigen-binding protein could instead comprise the heavy chain sequence set forth in SEQ ID NO: 3 in one or both of its heavy chains.

In one aspect, the present invention provides an antigen-binding protein which binds CD137 and programmed death-ligand 1 (PD-L1) for use in a method of treating cancer in a human patient, wherein the antigen-binding protein comprises the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 2; and wherein the antigen binding protein is administered to the patient every four weeks at a dose of 2 mg per kg of body weight of the patient.

In another aspect, the present invention provides a method of treating cancer in a human patient, wherein the method comprises administering to the patient a therapeutically effective amount of an antigen-binding protein which binds CD137 and PD-L1 , wherein the antigen-binding protein comprises the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 2; and wherein the method comprises administering the antigen-binding protein to the patient every four weeks at a dose of 2 mg per kg of body weight of the patient.

In a further aspect, the present invention provides the use of an antigen-binding protein which binds CD137 and PD-L1 in the manufacture of a medicament for treating cancer in a human patient, wherein the antigen-binding protein comprises the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 2; and wherein the antigen-binding protein is administered to the patient every four weeks at a dose of 2 mg per kg of body weight of the patient. A cancer to be treated in accordance with the present invention may be selected from the list consisting of: melanoma, ovarian cancer, soft tissue sarcoma (STS), non-small cell lung cancer (NSCLC), triple negative breast cancer (TNBC), colorectal cancer (CRC), mesothelioma, prostate cancer, head and neck cancer, squamous cell carcinoma of head and neck (SCCHN), and renal cell carcinoma (RCC).

In a preferred embodiment, a cancer to be treated in accordance with the present invention may be selected from the list consisting of: melanoma, ovarian cancer, soft tissue sarcoma (STS), and triple negative breast cancer (TNBC).

In a preferred embodiment, the cancer to be treated in accordance with the present invention is melanoma. The melanoma may be mucosal melanoma, uveal melanoma, or cutaneous melanoma, but preferably is cutaneous melanoma.

The present inventors have shown that treatment of patients with 2 mg/kg of FS222 results in a high proportion of patients achieving a partial response or stable disease in response to treatment (Figures 7 and 9). In particular, achievement of a partial response in response to treatment with FS222 has not previously been demonstrated. The high percentage of patients treated with 2 mg/kg of FS222 every four weeks who achieved a partial response in response to treatment is particularly surprising and demonstrates the high clinical benefit of FS222 treatment for patients with a wide variety of cancers.

Thus, in one embodiment, the patient achieves a complete response, a partial response, or stable disease in response to the treatment with the antigen-binding protein. In a preferred embodiment, the patient achieves a partial response or stable disease in response to the treatment with the antigenbinding protein, most preferably a partial response. The possibility of achieving a complete response in response to FS222 treatment is shown in Figures 7 and 9 with a 1 mg/kg dose.

Figures 7 and 9 also show that many patients achieved a long period of progression-free survival (PFS) in response to FS222 treatment.

Thus, in one embodiment, the antigen-binding protein is administered to the patient every four weeks at a dose of 2 mg per kg of body weight of the patient, and wherein the patient achieves at least 8 weeks, at least 10 weeks, at least 12 weeks, at least 14 weeks, at least 16 weeks, at least 18 weeks, at least 20 weeks, at least 22 weeks, at least 24 weeks, at least 26 weeks, at least 28 weeks, at least 30 weeks, at least 35 weeks, at least 40 weeks, at least 45 weeks, at least 50 weeks, at least 55 weeks, or at least 60 weeks of PFS in response to treatment with the antigen-binding protein. In one such embodiment, the patient achieves at least 24 weeks of PFS. In another such embodiment, the patient achieves at least 24 weeks of PFS including a partial response. In another such embodiment, the patient achieves at least 24 weeks of PFS including a complete response. In another such embodiment, the patient achieves at least 40 weeks of PFS. In another such embodiment, the patient achieves at least 40 weeks of PFS including a partial response. In another such embodiment, the patient achieves at least 40 weeks of PFS including a complete response. In another such embodiment, the patient achieves at least 55 weeks of PFS. In another such embodiment, the patient achieves at least 55 weeks of PFS including a partial response. In another such embodiment, the patient achieves at least 55 weeks of PFS including a complete response. In another such embodiment, the patient achieves at least 60 weeks of PFS. In another such embodiment, the patient achieves at least 60 weeks of PFS including a partial response. In another such embodiment, the patient achieves at least 60 weeks of PFS including a complete response.

In a preferred example, the cancer is selected from the list consisting of: melanoma, ovarian cancer, triple negative breast cancer, and colorectal cancer, and the method comprises administering the antigen-binding protein to the patient every four weeks at a dose of 2 mg per kg of body weight of the patient, and wherein the patient achieves at least 20 weeks, at least 25 weeks, at least 30 weeks, at least 35 weeks, or at least 40 weeks of progression-free survival in response to treatment with the antigen-binding protein.

As shown in Figure 6A, cancer patients treated with 2 mg/kg of FS222 showed an ORR of 35%, and a DCR of 53% in addition to good tolerability.

Thus, a patient population treated in accordance with the present invention may achieve a DCR of at least 40%, at least 45%, at least 50%, at least 55%, or at least 60%. In one embodiment the patient shows a DCR of at least 50%.

In addition, or alternatively, a patient population treated in accordance with the present invention may achieve an ORR of at least 25%, at least 30%, or at least 35%. In one embodiment the patient shows an ORR of at least 25%.

As can be seen in Figure 8, 100% of melanoma patients treated with 2 mg/kg of FS222 showed at least 23 weeks of PFS in response to treatment, with many patients achieving longer PFS of at least 30 weeks and up to 60 weeks.

The cancer may be resistant to a first anti-cancer treatment or may have relapsed during or following a first anti-cancer treatment. The first anti-cancer treatment may be treatment with an immune checkpoint inhibitor, such as an inhibitor of programmed cell death protein 1 (PD-1), PD-L1 , or CTLA- 4. Alternatively, the first anti-cancer treatment may be selected from chemotherapy, radiotherapy, treatment with an anti-cancer biologic therapy, or hormone therapy. Treatment of such pre-treated patients is particularly difficult in view of the advanced nature of the disease and the failure of a first- line treatment.

In one preferred embodiment, the cancer may be melanoma, such as mucosal melanoma, uveal melanoma, or cutaneous melanoma, but preferably cutaneous melanoma, wherein the melanoma is resistant to treatment with a first PD-1 inhibitor or PD-L1 inhibitor, preferably a PD-1 inhibitor, or has relapsed during or following treatment with a first PD-1 inhibitor or PD-L1 inhibitor, preferably a PD-1 inhibitor. Treatment efficacy of FS222 in patients with such resistant or recurrent melanomas has been shown to be particularly high (Figures 8 and 10).

The findings of the inventors reported herein is particularly significant because re-treatment of patients with a PD-(L)1 antibody after disease progression on a prior PD-(L)1 containing treatment regimens is not recommended and historically patients have been shown to derive little benefit from such therapy (Fujita et al. Thoracic Cancer, 2019, 1759-7706 and Martini et al., J. Immunotherapy Cancer, 2017, 5:66).

Thus, in a preferred embodiment, the cancer is melanoma, preferably cutaneous melanoma, and the melanoma is optionally resistant to treatment with a first PD-1 inhibitor or PD-L1 inhibitor, or has relapsed during or following treatment with a first PD-1 inhibitor or PD-L1 inhibitor, wherein the antigen-binding protein is administered to the patient every four weeks at a dose of 2 mg per kg of body weight of the patient, and wherein the patient achieves at least 12 weeks, at least 14 weeks, at least 16 weeks, at least 18 weeks, at least 20 weeks, at least 22 weeks, at least 23 weeks, at least 24 weeks, at least 25 weeks, at least 26 weeks, at least 28 weeks, at least 30 weeks, at least 32 weeks, at least 34 weeks, at least 36 weeks, at least 38 weeks, or at least 40 weeks of PFS. Thus, in a further preferred embodiment, the cancer is melanoma, preferably cutaneous melanoma, and the melanoma is resistant to treatment with a first PD-1 inhibitor or PD-L1 inhibitor, preferably a PD-1 inhibitor, or has relapsed during or following treatment with a first PD-1 inhibitor or PD-L1 inhibitor, preferably a PD-1 inhibitor, wherein the antigen-binding protein is administered to the patient every four weeks at a dose of 2 mg per kg of body weight of the patient, and wherein the patient achieves at least 23 weeks of PFS.

As shown in Figure 6B, melanoma patients who were resistant to treatment with a first PD-1 inhibitor or had relapsed following treatment with a first PD-1 inhibitor, and who were treated with 2 mg/kg of FS222 showed an ORR of 75%, and a DCR of 100% in addition to good tolerability.

The treatment efficacy shown in Figure 6B is particularly surprising, as the efficacy seen with existing second-line treatments for melanoma following failure of anti-PD-1/PD-L1 therapy is significantly lower. Specifically, the ORR of patients treated with ipilimumab after pembrolizumab, was reported as 13% ORR. The ORR of patients treated with ipilimumab and pembrolizumab after failure of anti-PD- 1/PD-L1 therapy was reported as 29% (Olson et al. J Clin Oncol. 2021 Aug 20,39(24) .2647 -2655. doi: 10.1200/JCO.21.00079. Epub 2021 May 4. PMID: 33945288; PMCID: PMC8376314). A similar ORR of 31.5% for patients treated with lifileucel after failure of anti-PD-1/PD-L1 therapy has been reported (Lifileucel Full Prescribing Information. Available at: https://www.fda.gov/media/176417/download and https://www.iovance.com/AMTAGVI_USPI/ (Accessed: 20 May 2024).). The ORR of nivolumab and relatlimab combination treatment after failure of anti-PD-1/PD-L1 therapy was reported as 12% ORR (Ascierto PA et al. J Clin Oncol. 2023 May 20;41(15):2724-2735. doi: 10.1200/JC0.22.02072. Epub 2023 Feb 13. PMID: 36780608; PMCID: PMC 10431305). Thus, in a preferred embodiment, the cancer is melanoma, preferably cutaneous melanoma, and the melanoma is resistant to treatment with a first PD-1 inhibitor or PD-L1 inhibitor, preferably a PD-1 inhibitor, or has relapsed during or following treatment with a first PD-1 inhibitor or PD-L1 inhibitor, preferably a PD-1 inhibitor, wherein the antigen-binding protein is administered to the patient once every four weeks at a dose of 2 mg per kg of body weight of the patient, and wherein the treatment results in a DCR of at least 80%, at least 90%, or 100%, preferably 100%.

In another preferred embodiment, the cancer is melanoma, preferably cutaneous melanoma, and the melanoma is resistant to treatment with a first PD-1 inhibitor or PD-L1 inhibitor, preferably a PD-1 inhibitor, or has relapsed during or following treatment with a first PD-1 inhibitor or PD-L1 inhibitor, preferably a PD-1 inhibitor, wherein the antigen-binding protein is administered to the patient every four weeks at a dose 2 mg per kg of body weight of the patient, and wherein the treatment results in an ORR of at least 75%.

Also provided is an antigen-binding protein which binds CD137 and PD-L1 for use in a method of treating cancer in a human patient who has been subjected to treatment with a prior immune checkpoint inhibitor therapy, selected from an anti-PD-1 , anti-PD-L1 or anti-CTLA-4 therapy, the antigen-binding protein comprising the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 2; wherein a tumour of the cancer in the patient has been determined to have an acquired resistance phenotype in respect of the prior immune checkpoint inhibitor therapy, and wherein a tumour with an acquired resistance phenotype is a tumour which showed a complete or partial response to treatment with the prior immune checkpoint inhibitor therapy, or showed stable disease whilst subjected to treatment with the prior immune checkpoint inhibitor therapy.

Further provided is a method of treating cancer in a human patient who has been subjected to treatment with a prior immune checkpoint inhibitor therapy, selected from an anti-PD-1 , anti-PD-L1 or anti-CTLA-4 therapy, the method comprising administering to the patient a therapeutically effective amount of an antigen-binding protein which binds CD137 and PD-L1 and comprises the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 2; wherein a tumour of the cancer in the patient has been determined to have an acquired resistance phenotype in respect of the prior immune checkpoint inhibitor therapy, and wherein a tumour with an acquired resistance phenotype is a tumour which showed a complete or partial response to treatment with the prior immune checkpoint inhibitor therapy, or showed stable disease whilst subjected to treatment with the prior immune checkpoint inhibitor therapy.

The immune checkpoint inhibitor is preferably a PD-1 , PD-L1 , or CTLA-4 inhibitor, more preferably an anti-PD-1 , anti-PD-L1 , or anti-CTLA-4 antibody. The prior treatment with one or more immune checkpoint inhibitors (other than FS222) may have been administered alone or in combination with one or more additional therapies (e.g., one or more chemotherapeutic agents). Anti-PD-1 or anti-PD-L1 therapy may refer to treatment with an anti-PD-1 or anti-PD-L1 antibody (other than an antibody which binds to both CD137 and PD-L1 , such as FS222), including, but not limited to, treatment with nivolumab, pembrolizumab, avelumab, durvalumab or atezolizumab.

The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

Summary of the Figures

Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

Figure 1 shows the general structure and function of the CD137/PD-L1 tetravalent bispecific antigenbinding protein molecule, FS222. A shows the location of the two PD-L1 binding sites, the two CD137 binding sites, and the LALA mutation in the CH2 domain which abrogates binding of the antigenbinding protein molecule to Fey receptors. B shows how FS222 drives conditional T-cell activation. FS222 binds to PD-L1 on tumour cells. The interaction prevents binding of tumour cell surface-bound PD-L1 to PD-1 expressed on T cells. Simultaneous avid binding of the FS222 molecule to CD137 on T cells and to PD-L1 on tumours cells results in conditional CD137 agonism in areas with PD-L1 expression (such as the tumor microenvironment). CD137 agonism is thought to be achieved through receptor clustering, which is driven by the close proximity of the two CD137 binding sites in the constant regions of FS222 (see A).

Figure 2 shows the design of a phase 1 , multi-centre, multi-part, open-label, sequential-group, first-in- human (FIH) study to evaluate the safety, tolerability, pharmacokinetics (PK), pharmacodynamics (PD), immunogenicity, and clinical activity of FS222 in adult subjects diagnosed with advanced malignancies. In an initial accelerated dose titration (ADT) design, single-subject cohorts were evaluated followed by a 3+3 dose escalation design. The dose-limiting toxicity windows were 28 days where administration took place on Day 1 of every four-week cycle (Q4W) and 42 days where administration took place on Day 1 of every three-week cycle (Q3W)-

Figure 3 shows the characteristics of 114 patients, 100 of whom received treatment on Day 1 of every four-week cycle (Q4W)- The remaining patients were dosed at Q3W. Characteristics included median lines of prior therapy, exposure to prior anti-PD-L1/anti-CTLA4 therapy and cancer types.

Figure 4 shows the maximum fold change in levels of Ki67⁺ CD8⁺ T cells, Ki67⁺ Effector Memory (EM) CD8⁺ T cells and Ki67⁺ CD4⁺ T cells compared to baseline levels across patients in a first Q4W cycle of FS222 treatment. FS222 doses ranged from 0.75 mg/kg to 4.5 mg/kg. All FS222 concentrations led to an increase in proliferation of both CD8+ and CD4+ cells. An increase in median CD8⁺ T-cell proliferation greater than or equal to (>) six-fold was observed at an FS222 concentration of > 0.75 mg/kg and no evidence of a hook effect at higher concentrations was observed. Figure 5 shows the density of CD3⁺ CD8⁺ T cells in tumours in patients during a second Q4W cycle of FS222 treatment at > 0.75 mg/kg compared to baseline levels prior to the start of the treatment. The median of all samples, paired and unpaired, is illustrated by the box plots (A). FS222 treatment increased the density of CD3⁺ CD8⁺ T cells within tumour and stromal compartments (B).

Figure 6 shows the response of cancer patients to treatment with FS222 at doses ranging from 0.75 mg/kg to 4.5 mg/kg administered every four weeks. Disease progression was assessed using iRECIST. A: Anti-tumour activity was observed in patients with different cancer types and across all tested FS222 concentrations. A partial response (PR) was observed in a substantial proportion of the patient cohorts treated with 2 mg/kg FS222. B: Response of cutaneous melanoma patients who had completed at least one treatment cycle and who received treatment with a PD-1 inhibitor prior to FS222 treatment. Similar to the data shown in A, anti-tumour activity was observed in patients across all FS222 doses investigated and at least half of the patients in each cohort treated with > 1.5 mg/kg of FS222 showed a partial response (PR). C: Treatment-related adverse events (TRAEs) observed in response to treatment with FS222 at doses ranging from 0.75 mg/kg to 4.5 mg/kg administered every four weeks. Instances of increased aspartate aminotransferase (AST), increased alanine aminotransferase (ALT), pyrexia, asthenia, thrombocyopenia, neutropenia, anaemia, and increased gamma-glutamyl transferase (GGT) are reported, as is the number and percentage of patients who experienced one or more TRAE, and a TRAE of grade 3 or higher. The dose of 4.5 mg/kg was determined to exceed the maximum tolerated dose (MTD).

Figure 7 shows the response of patients with a variety of different cancers to FS222 treatment at doses ranging from 300 pg (fixed dose) to 4.5 mg/kg of FS222 administered every four weeks (Q4W)- The data shown represent a data cut from the clinical trial obtained on 15 April 2024. A partial response was observed in a significant proportion of patients treated with > 1 .5 mg/kg of FS222, as indicated (half-filled triangles). The bars indicate the treatment duration for each patient and the best overall response (BOR) recorded. The legend for Figure 7 is shown to the right of the figure: Progressive disease (PD) (filled squares), stable disease (SD) (filled triangles), partial response (PR) (half-filled triangles), complete response (CR) (empty triangles), not evaluable (NE) (no symbol). EOT refers to end of treatment. Arrows indicate active, i.e., continuing treatment.

Figure 8 shows the response of melanoma patients to FS222 treatment at doses ranging from 1 mg/kg to 4.5 mg/kg administered every four weeks (Q4W)- The data shown represent a data cut from the clinical trial obtained on 15 April 2024. Only limited anti-tumour effects were observed in cutaneous melanoma patients treated with 1 mg/kg of FS222. Nine of 12 cutaneous melanoma patients treated with 1 .5 to 3 mg/kg of FS222 showed at least a partial response to treatment and three patients showed stable disease. The bars indicate the treatment duration for each patient and the best overall response recorded. The legend for Figure 8 is shown to the right of the figure: Progressive disease (PD) (filled squares), stable disease (SD) (filled triangles), partial response (PR) (half-filled triangles), not evaluable (NE) (no symbol). EOT refers to end of treatment. Arrows indicate active, i.e., continuing treatment. Figure 9 shows the response of patients with a variety of different cancers to FS222 treatment at doses ranging from 300 pg (fixed dose) to 4.5 mg/kg of FS222 administered every four weeks (Q4W). The data shown represent a data cut from the clinical trial obtained on 24 November 2024. A partial response was observed in a significant proportion of patients treated with > 1 .5 mg/kg of FS222, as indicated (half-filled triangles). The bars indicate the treatment duration for each patient and the best overall response (BOR) recorded. The legend for Figure 9 is shown to the right of the figure: Progressive disease (PD) (filled squares), stable disease (SD) (filled triangles), partial response (PR) (half-filled triangles), complete response (CR) (empty triangles), not evaluable (NE) (no symbol). EOT refers to end of treatment. Arrows indicate active, i.e., continuing treatment.

Figure 10 shows the response of melanoma patients to FS222 treatment at doses ranging from 1 mg/kg to 4.5 mg/kg administered every four weeks (Q4W). The data shown represent a data cut from the clinical trial obtained on 24 November 2024. Only limited anti-tumour effects were observed in cutaneous melanoma patients treated with 1 mg/kg of FS222. Nine of 12 cutaneous melanoma patients treated with 1 .5 to 3 mg/kg of FS222 showed at least a partial response to treatment and three patients showed stable disease. The bars indicate the treatment duration for each patient and the best overall response recorded. The legend for Figure 10 is shown to the right of the figure: Progressive disease (PD) (filled squares), stable disease (SD) (filled triangles), partial response (PR) (half-filled triangles), not evaluable (NE) (no symbol). EOT refers to end of treatment. Arrows indicate active, i.e., continuing treatment.

Detailed Description of the Invention

Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference.

Antigen-binding protein

The anti-CD137/PD-L1 antigen-binding protein used in the present invention (FS222) is described in WO 2020/011964 A1 , the contents of which are incorporated herein in their entirety and for all purposes. The anti-CD137/PD-L1 antigen-binding protein comprises the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 2. Alternatively, the anti- CD137/PD-L1 antigen-binding protein used in the present invention may comprise an additional lysine residue at the C-terminus of the heavy chain sequence of one or both of its heavy chains. In this case, the anti-CD137/PD-L1 antigen-binding protein used in the present invention comprises the heavy chain sequence set forth in SEQ ID NO: 3 and the light chain sequence set forth in SEQ ID NO: 2. Thus, the antigen-binding protein used in the present invention may comprise the heavy chain sequence set forth in SEQ ID NO: 1 in both of its heavy chains, and the light chain sequence set forth in SEQ ID NO: 2; the heavy chain sequence set forth in SEQ ID NO: 1 in one of its heavy chains, the heavy chain sequence set forth in SEQ ID NO: 3 in the other of its heavy chains, and the light chain sequence set forth in SEQ ID NO: 2; or the heavy chain sequence set forth in SEQ ID NO: 3 in both of its heavy chains, and the light chain sequence set forth in SEQ ID NO: 2.

The heavy chain of the antigen-binding protein used in the present invention comprises the LALA mutation, which involves substitution of the leucine residues at positions 234 and 235 (according to the Eu numbering scheme) of the lgG1 CH2 domain with alanine residues (i.e., an L234A/L235A double mutation). This and other Fc mutations are known to decrease or abrogate binding of the CH2 domain of lgG1 molecules to one or more Fey receptors and/or the complement component C1 q to diminish or nullify lgG1 effector functions, such as ADCC, antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC). Other such mutations, with reference to the Eu numbering system, include, but are not limited to, the substitution of the leucine residue at position 234 and the glycine residue at position 327 of the CH2 domain with alanine residues (i.e., an L234A/G327A double mutation), the substitution of the asparagine residue at position 297 of the CH2 domain with an alanine, glycine or glutamine residue (i.e., an N297A, N297G or N297Q mutation); and the substitution of the proline residue at position 329 of the CH2 domain with an alanine or glycine residue (i.e., a P329A or P329G mutation). These and other mutations to reduce or abolish Fc effector functions may be used as alternatives to, or combined with, the LALA mutation, as appropriate, in the antigen-binding protein used in the present invention.

The term “antigen-binding protein”, as used herein, refers to an isolated protein, an antibody or antibody molecule, one or more antibody fragments (e.g., Fabs, etc.), and other antibody-derived protein constructs, including antibody fusion proteins, such as those comprising domains (e.g., domain antibodies, etc.) which are capable of binding to the antigen. Such alternative antibody formats include triabody, tetrabody, mini-antibody, and minibody. Also included are alternative scaffolds in which the one or more CDRs of any molecules in accordance with the disclosure can be arranged onto a suitable non-immunoglobulin protein scaffold or skeleton, such as an affibody, a SpA scaffold, an LDL receptor class A domain, an avimer or an EGF domain. An antigen binding protein also includes antigen binding fragments of such antibodies or other molecules. Further, an antigen binding protein may comprise the VH regions of the invention formatted into a full length antibody, a (Fab')2 fragment, a Fab fragment, a bi-specific or biparatopic molecule or equivalent thereof (such as scFV, bi- tri- or tetra-bodies, Tandabs, etc.), when paired with an appropriate light chain. The antigenbinding protein may comprise an antibody that is an lgG1 , lgG2, lgG3, or lgG4; or IgM; IgA, IgE or IgD or a modified variant thereof. The constant domain of the antibody heavy chain may be selected accordingly. The light chain constant domain may be a kappa or lambda constant domain.

The antigen binding protein used in the present invention may be referred to as an antibody molecule, in particular a bispecific antibody, e.g., a bivalent bispecific antibody or a tetravalent bispecific antibody. Bivalent bispecific antibody formats that the antigen-binding protein may take include the DuoBody™ format and the Biclonics™ format. Tetravalent bispecific antibody formats that the antigenbinding protein may take include the mAb²™ format, the RUBY™ format, and the tetravalent bispecific tandem IgG (TBTI) format. The antigen-binding protein used in the present invention may also be referred to as a fusion protein, such as an antibody-anticalin fusion protein (e.g., a tetravalent bispecific antibody-anticalin fusion protein) or an antibody-scFv fusion protein (e.g., a tetravalent bispecific antibody-scFv fusion protein).

In a preferred embodiment the antigen binding protein is a tetravalent bispecific antibody.

The present invention provides an antigen-binding protein which binds CD137 and PD-L1 for use in a method of treating cancer in a human patient, wherein the antigen-binding protein comprises the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 2.

The present invention also provides a method of treating cancer in a human patient, wherein the method comprises administering to the patient a therapeutically effective amount of an antigenbinding protein which binds CD137 and PD-L1 , wherein the antigen-binding protein comprises the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 2.

The present invention further provides the use of an antigen-binding protein which binds CD137 and PD-L1 in the manufacture of a medicament for treating cancer in a human patient, wherein the antigen-binding protein comprises the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 2.

Formulations

For therapeutic use, the antigen-binding protein which binds CD137 and PD-L1 is formulated with a carrier that is pharmaceutically acceptable and is appropriate for delivering the antigen-binding protein by the chosen route of administration, such as intravenous administration. Suitable pharmaceutically acceptable carriers are those conventionally used for intravenous administration of antibody molecules, such as diluents and excipients and the like. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co.

Mode of administration

The anti-CD137/PD-L1 antigen-binding protein is preferably administered to the patient by intravenous injection or intravenous infusion, more preferably by intravenous infusion. For example, the anti-CD137/PD-L1 antigen-binding protein may be administered to the patient by intravenous bolus injection or by intravenous infusion, e.g., using a continuous infusion pump. Intravenous infusion may be conducted using a continuous infusion pump over 30 minutes or 60 minutes.

The anti-CD137/PD-L1 antigen-binding protein is administered to the patient at a dose calculated based on the patient’s weight in kilograms (kg). A patient receiving a dose of 2 mg/kg and weighing 70 kg, would thus receive a dose of 140 mg of the anti-CD137/PD-L1 antigen-binding protein. The antigen-binding protein is administered to the patient at a dose of 2 mg per kg of body weight of the patient.

The antigen-binding protein is administered to the patient once every four weeks (Q4W).

The antigen-binding protein is administered to the patient at a dose of 2 mg per kg of body weight of the patient.

The antigen-binding protein is administered to the patient once every four weeks (Q4W).

Where the antigen-binding protein is administered to the patient once every four weeks, the doses of the antigen-binding protein may be separated in time by 28 days. As will be appreciated in the art, the time between doses may be varied to some extent so that each and every dose is not separated by precisely the same time. This will often be directed under the discretion of the administering physician.

Cancer

A cancer to be treated in accordance with the present invention is preferably a solid cancer.

A cancer to be treated in accordance with the present invention expresses PD-L1 . Preferably, the cancer has been determined to express PD-L1 . In addition, a cancer to be treated in accordance with the present invention comprises immune cells, such as TILs, which express CD137. Preferably, the cancer has been determined to comprise CD137-expressing immune cells.

The present inventors have shown that the anti-CD137/PD-L1 antigen-binding protein employed in the present invention is capable of treating a wide-variety of solid cancers (Figures 7 and 9).

In a preferred embodiment, a cancer to be treated in accordance with the present invention may be selected from the list consisting of: melanoma, ovarian cancer, soft tissue sarcoma (STS), non-small cell lung cancer (NSCLC), triple negative breast cancer (TNBC), colorectal cancer, mesothelioma, prostate cancer, head and neck cancer, squamous cell carcinoma of head and neck (SCCHN), and renal cell carcinoma (RCC). In a further preferred embodiment, a cancer to be treated in accordance with the present invention is selected from the list consisting of: melanoma, ovarian cancer, soft tissue sarcoma, non-small cell lung cancer (NSCLC), triple negative breast cancer (TNBC), and colorectal cancer. In a yet preferred embodiment, a cancer to be treated in accordance with the present invention may be selected from the list consisting of: melanoma, ovarian cancer, soft tissue sarcoma (STS), and triple negative breast cancer (TNBC).

In one embodiment, the cancer to be treated in accordance with the present invention is ovarian cancer, such as ovarian clear cell carcinoma. In another embodiment, the cancer to be treated in accordance with the present invention is soft tissue sarcoma. In another embodiment, the cancer to be treated in accordance with the present invention is non-small cell lung cancer. In another embodiment, the cancer to be treated in accordance with the present invention is triple negative breast cancer. In another embodiment, the cancer to be treated in accordance with the present invention is colorectal cancer. In another embodiment, the cancer to be treated in accordance with the present invention is mesothelioma. In another embodiment, the cancer to be treated in accordance with the present invention is prostate cancer. In a further embodiment, the cancer to be treated in accordance with the present invention is renal cell carcinoma.

In a preferred embodiment, the cancer to be treated in accordance with the present invention is melanoma. The melanoma may be mucosal melanoma, uveal melanoma, or cutaneous melanoma, but preferably is cutaneous melanoma.

The cancer may be resistant to a first anti-cancer treatment, or may have relapsed during or following a first anti-cancer treatment. Tumours which have relapsed following a first anti-cancer treatment are considered to have an “acquired resistance phenotype” to the prior anti-cancer treatment, such as an immune checkpoint inhibitor.

The first anti-cancer treatment may be treatment with a first immune checkpoint inhibitor, such as an inhibitor of PD-1 , PD-L1 , or CTLA-4 inhibitor. In a preferred embodiment, the cancer may be resistant to treatment with a first PD-1 inhibitor or PD-L1 inhibitor, preferably a PD-1 inhibitor.

Alternatively, the first anti-cancer treatment may be selected from chemotherapy, radiotherapy, treatment with an anti-cancer biologic therapy, or hormone therapy.

Treatment of such pre-treated patients is particularly difficult in view of the advanced nature of the disease and the failure of the first-line treatment.

Where the cancer to be treated is melanoma, in particular cutaneous melanoma, the melanoma is preferably resistant to treatment with a first PD-1 inhibitor or PD-L1 inhibitor, or has relapsed during or following treatment with a first PD-1 inhibitor or PD-L1 inhibitor. In a further preferred embodiment, the melanoma is resistant to treatment with a first PD-1 inhibitor, or has relapsed during or following treatment with a first PD-1 inhibitor.

Whether a tumour shows a complete response, partial response, stable disease or progressive disease during treatment with an anti-cancer therapy, such as an immune checkpoint inhibitor, may be evaluated according to the RECIST 1.1 criteria (Eisenhauer et al., Revised RECIST guideline (version 1. 1), European Journal of Cancer, 2009, 28-247.) or the iRECIST criteria (Seymour et al., IRECIST: Lancet Oncol., 2017, 18(3):e143-e152.), preferably the RECIST 1.1 criteria. This may involve obtaining scans (e.g., MRI scans) of the patient’s tumour and measuring the size/volume of the tumour lesions. For the purposes of defining acquired resistance herein, it is assumed that, where a patient had, for example, a first scan classified as showing stable disease (or a partial response or complete response) followed by a later scan classified as showing progressive disease, the patient showed stable disease (or a partial response or complete response) for the time period until which the scan showing progressive disease was obtained. In other words, the acquired resistance phenotype may be defined as tumours that (a) had a best overall response (BOR) of a complete response or partial response to a prior immune checkpoint inhibitor, or (b) had stable disease as a best overall response (BOR). Clinical endpoints such as BOR may be defined according to the RECIST 1.1 criteria or the iRECIST criteria, preferably the RECIST 1.1 criteria.

Patient characteristics

The present inventors have shown that the anti-CD137/PD-L1 antigen-binding protein used in the present invention is capable of producing a significant anti-tumour effect in a wide variety of human cancers. The patient to be treated in accordance with the present invention is thus a human patient. The patient preferably has histologically or cytologically confirmed, locally advanced, unresectable or metastatic solid tumours. The patient has preferably not been treated with any agent directed to another stimulatory or costimulatory T-cell receptor (e.g., a CD137 agonist, 0X40 agonist, or CD40 agonist, or a GITR- or CD27-targeting therapy).

Treatment effects

The present inventors have shown that treatment with FS222 may result in the patient achieving a complete response, a partial response, or stable disease in response to the treatment with the anti- CD137/PD-L1 antigen-binding protein.

In one embodiment, the patient achieves a complete response in response to the treatment with the antigen-binding protein. In another embodiment, the patient achieves a partial response in response to the treatment with the antigen-binding protein. In a further embodiment, the patient achieves stable disease in response to the treatment with the antigen-binding protein. The achievement of a partial response to treatment with FS222 was shown for the first time by the present inventors.

The patient may achieve one or more weeks of progression-free survival (PFS) in response to treatment with the antigen-binding protein. Progression-free survival refers to the patient exhibiting a complete response, a partial response, or stable disease in response to treatment with the antigenbinding protein.

In one embodiment, the patient achieves at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 16 weeks, at least 17 weeks, at least 18 weeks, at least 19 weeks, at least 20 weeks, at least 21 weeks, at least 22 weeks, at least 23 weeks, at least 24 weeks, at least 25 weeks, at least 26 weeks, at least 27 weeks, at least 28 weeks, at least 29 weeks, at least 30 weeks, at least 31 weeks, at least 32 weeks, at least 33 weeks, at least 34 weeks, at least 35 weeks, at least 36 weeks, at least 37 weeks, at least 38 weeks, at least 39 weeks, at least 40 weeks, at least 41 weeks, at least 42 weeks, at least 43 weeks, at least 44 weeks, at least 45 weeks, at least 46 weeks, at least 47 weeks, at least 48 weeks, at least 49 weeks, at least 50 weeks, at least 51 weeks, at least 52 weeks, at least 53 weeks, at least 54 weeks, at least 55 weeks, at least 56 weeks, at least 57 weeks, at least 58 weeks, at least 59 weeks, or at least 60 weeks of PFS in response to treatment with the antigen-binding protein.

In another preferred embodiment, the patient achieves at least 12 weeks of PFS in response to treatment with the antigen-binding protein. In this case, the cancer may be selected from the group consisting of: melanoma, preferably cutaneous melanoma or mucosal melanoma, ovarian clear cell carcinoma, soft tissue sarcoma (STS), triple negative breast cancer (TNBC), colorectal cancer, and mesothelioma.

In another preferred embodiment, the patient achieves at least 23 weeks of PFS in response to treatment with the antigen-binding protein. In this case, the cancer may be selected from the group consisting of: melanoma, preferably cutaneous melanoma or mucosal melanoma, ovarian clear cell carcinoma, triple negative breast cancer (TNBC), and colorectal cancer.

In another preferred embodiment, the patient achieves at least 24 weeks of PFS in response to treatment with the antigen-binding protein. In this case, the cancer may be selected from the group consisting of: melanoma, preferably cutaneous melanoma or mucosal melanoma, ovarian clear cell carcinoma, triple negative breast cancer (TNBC), and colorectal cancer.

In another preferred embodiment, the patient achieves at least 32 weeks of PFS in response to treatment with the antigen-binding protein. In this case, the cancer may be selected from the group consisting of: cutaneous melanoma, ovarian clear cell carcinoma, triple negative breast cancer (TNBC), and colorectal cancer.

In another preferred embodiment, the patient achieves at least 40 weeks of PFS in response to treatment with the antigen-binding protein. In this case, the cancer may be selected from the group consisting of: cutaneous melanoma, ovarian clear cell carcinoma, triple negative breast cancer (TNBC), and colorectal cancer.

In another preferred embodiment, the patient achieves at least 50 weeks of PFS in response to treatment with the antigen-binding protein. In this case, the cancer may be selected from the group consisting of: cutaneous melanoma, ovarian clear cell carcinoma, and triple negative breast cancer (TNBC).

In another preferred embodiment, the patient achieves at least 55 weeks of PFS in response to treatment with the antigen-binding protein. In this case, the cancer may be selected from the group consisting of: cutaneous melanoma, ovarian clear cell carcinoma, and triple negative breast cancer (TNBC).

In another preferred embodiment, the patient achieves at least 56 weeks of PFS in response to treatment with the antigen-binding protein. In this case, the cancer may be selected from the group consisting of: cutaneous melanoma, and ovarian clear cell carcinoma. In another preferred embodiment, the patient achieves at least 50 weeks of PFS in response to treatment with the antigen-binding protein. In this case, the cancer may be cutaneous melanoma.

In another preferred embodiment, the patient achieves at least 60 weeks of PFS in response to treatment with the antigen-binding protein. In this case, the cancer may be cutaneous melanoma.

As shown in Figure 6A and B, cancer patients treated with FS222 showed high objective response rates (ORR) and high disease control rates (DCR).

Thus, a patient population treated in accordance with the present invention may achieve a DCR of at least 50%.

A patient population treated in accordance with the present invention may, in addition, or alternatively, achieve an ORR of at least 35%.

In a preferred embodiment, the cancer is melanoma, preferably cutaneous melanoma, and the melanoma is resistant to treatment with a first PD-1 inhibitor or PD-L1 inhibitor, preferably a PD-1 inhibitor, or has relapsed during or following treatment with a first PD-1 inhibitor or PD-L1 inhibitor, preferably a PD-1 inhibitor, wherein the antigen-binding protein is administered to the patient every four weeks at a dose of 2 mg per kg of body weight of the patient, and wherein the treatment results in a DCR of at least 80%, at least 90%, or 100%, preferably 100%.

In a further preferred embodiment, the cancer is melanoma, preferably cutaneous melanoma, and the melanoma is resistant to treatment with a first PD-1 inhibitor or PD-L1 inhibitor, preferably a PD-1 inhibitor, or has relapsed during or following treatment with a first PD-1 inhibitor or PD-L1 inhibitor, preferably a PD-1 inhibitor, wherein the antigen-binding protein is administered to the patient every four weeks at a dose of 2 mg per kg of body weight of the patient, and wherein the treatment results in an ORR of at least 75%.

The disease control rate, objective response rate, complete response, partial response, and stable disease may be assessed according to the iRECIST criteria (Seymour et al., 2017).

Alternatively, the disease control rate, objective response rate, complete response, partial response, and stable disease may be assessed according to the RECIST 1.1 criteria (Eisenhauer et al., 2009).

Whether a patient has achieved disease control, an objective response, a complete response, a partial response, and/or stable disease in response to treatment may be assessed according to the RECIST 1.1 criteria or the iRECIST criteria at any timepoint from six weeks from the start of treatment onwards, e.g., at week 6, at week 7, or at week 8, and may then be assessed at further timepoints, e.g. at 4 to 8 weeks, such as at 4 weeks, at 5 weeks, at 6 weeks, at 7 weeks, or at 8 weeks, after the previous assessment. For example, the disease control rate may be assessed at 24 weeks from the start of treatment. Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means, for example, a value within two standard deviations of the mean for any particular measured value.

Examples

EXAMPLE 1 - A Phase 1, Open-Label, First-in-Human (FIH) Study to Evaluate the Safety and Antitumour Activity of FS222, a CD137/PD-L1 Bispecific Antibody, in Subjects with Advanced Malignancies

1.1 Study Design

This was a Phase 1 , multi-centre, multi-part, open-label, sequential-group, first-in-human (FIH) study to evaluate the safety, tolerability, PK, PD, immunogenicity and clinical activity of FS222 in adult subjects diagnosed with advanced malignancies. The study was designed to systematically assess safety and tolerability and to identify the maximum tolerated dose (MTD) and the dose with optimal biological effect and acceptable tolerability. The study was initiated with an accelerated dose titration (ADT) design in which single-subject cohorts were evaluated followed by a classical 3+3 dose escalation design. In both the ADT design and 3+3 design, doses were planned to escalate in a sequential fashion contingent on the safety, tolerability, and PK results from the preceding dose levels in the study. The ADT and the 3+3 cohorts were open to specific tumour types known to be immunogenic. The study also consisted of optional PK/PD expansion cohorts and specific tumour types were enriched into these cohorts.

The initial five cohorts (dose levels 300 pg, 1 mg, 3 mg, 10 mg, and 30 mg) were enrolled sequentially as single-subject cohorts and followed specific rules associated with the ADT design. Subsequent cohorts were assessed according to 3+3 study design. Dosages tested in the 3+3 study design were 0.75 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, and 4.5 mg/kg (Figure 2).

Subjects received treatment until disease progression, unacceptable toxicity, withdrawal of consent by subject, discontinuation of study treatment by investigator, Sponsor decision to terminate the study, initiation of alternative anticancer therapy or death, whichever occurs earlier. The maximum treatment period for this study was 24 months. Efficacy assessments included tumour assessments. Radiographic assessments (CT scan with contrast/MRI) of chest, abdomen, and pelvis (and head for subjects with SCCHN) to evaluate response to treatment were conducted at screening and every 8 weeks (±7 days) thereafter up to Cycle 12, every 12 weeks (±7 days) Cycle 13 onwards and at EOT according to RECIST 1.1 and iRECIST 1.1.

The safety assessments included adverse events (AEs) (including dose-limiting toxicities [DLTs] and serious adverse events [SAEs]), physical examination findings including ECOG performance status, vital sign measurements, standard clinical laboratory measurements, and electrocardiogram (ECG) and echocardiogram/multigated acquisition (ECHO/MUGA) recordings. Serial blood samples were collected for evaluation of immunogenicity of FS222. Immunogenicity was evaluated by determination of the presence of anti-drug antibody (ADAs) as measured in serum by a tiered-testing approach involving screening, confirmation, and titration of confirmed positive samples. Blood samples were collected as part of safety assessments if a subject showed an infusion reaction and/or immune- related adverse event (irAE).

1.2 Study Drug, Dosage, and Route of Administration:

The FS222 Drug Product was presented as a lyophilised powder for concentrate for solution for infusion (80 mg in a 10-mL vial). The FS222 Drug Product was designed to be reconstituted with 4.4 mL of sterile Water for Injection to obtain a nominal concentration of 20.0 ± 2.0 mg/mL. To ensure that an extractable volume of 4 mL can be withdrawn from the vial, there was an overfill of 0.5 mL. The FS222 Drug Product does not contain any preservative and is intended for single use only. Prior to infusion the reconstituted FS222 Drug Product was diluted with dextrose 5% in water.

Subjects received FS222 at escalating doses as detailed in Figure 2. To ensure accurate delivery of low volume doses, the initial five cohorts (dose levels 300 pg, 1 mg, 3 mg, 10 mg, and 30 mg) were administered in a fixed-dose schedule. The subsequent dose levels were administered in a body weight-based dosing schedule.

FS222 was administered by a slow IV bolus injection to Cohort 1 and 2 and by continuous IV infusion pump over 30 minutes (to Cohorts 3-7) or 60 minutes (to Cohort 8).

1.3 Primary Objectives 1.4 Secondary Objectives

1.5 Exploratory objectives

1.6 Inclusion Criteria

Each subject must meet all of the following criteria to be enrolled in this study:

1. Age > 18 years.

2. Confirmed and documented disease progression on the last prior therapy as per investigator assessment.

3. Subjects who have received prior anti-PD-1 or anti-CTLA-4 therapy (ICB), or any concurrent chemotherapy, radiotherapy, investigational, biologic or hormonal therapy for cancer treatment may be eligible for enrolment following a washout period of these treatments totalling at least 28 days or 5 half-lives, whichever is shorter, before the first dose of FS222. NOTE 1 : Subjects must have recovered from all AEs due to previous therapies to Grade < 1 or baseline. Subjects with Grade < 2 neuropathy or endocrine-related AEs requiring hormone replacement therapy may be eligible after discussion with the Sponsor’s medical monitor. Alopecia of any grade is acceptable. Other toxicities that would not be expected to be exacerbated by FS222, e.g. hearing loss, may be approved after discussion with the medical monitor. NOTE 2: If the subject has had major surgery, the subject must have recovered adequately from the procedure and/or any complications from the surgery prior to starting study intervention.

4. Measurable disease (defined as at least 1 measurable lesion outside the CNS), as determined by the investigator using RECIST 1 .1 . A previously irradiated lesion can be considered a target lesion if the lesion is increasing in size and is well defined; is accurately measured at baseline as being > 10 mm in the longest diameter (except lymph nodes, which must have a short axis > 15 mm) using CT or MRI; and is suitable for accurate repeated measurements according to RECIST 1.1 guidelines. Subjects must consent to undergo a CT scan or MRI before enrolling in the study. Radiographic disease assessment at baseline can be performed up to 28 days prior to the first dose of study drug.

5. Eastern Cooperative Oncology Group Performance Status < 1.

6. The subject agrees to undergo a mandatory pre-treatment biopsy of the tumour during the screening period (exceptions in NOTES below). An on-treatment biopsy (ideally from the same lesion as the baseline biopsy) is also mandatory unless the biopsy procedure is judged by the investigator to be high risk. Further biopsies (e.g. on-treatment and/or at disease progression) may be collected at the investigator’s discretion where the subject agrees, and the biopsy procedure is not judged by the investigator to be high risk. Core needle (image-guided) or excisional biopsies (including punch biopsy) or surgically resected tissue is required. Fine needle aspirates, Endobronchial Ultrasound (EBUS) or cell blocks are not acceptable.

NOTE 1 : Subjects in the initial 5 single subject cohorts (ADT design) are not mandated to undergo pre-treatment or on-treatment biopsy but a biopsy is encouraged to be performed where the subject agrees, and the biopsy procedure is not judged to be high risk by the investigator.

NOTE 2: The lesion chosen to perform the biopsy should not have been previously irradiated and should be different from the measurable lesions.

NOTE 3: If the subject meets all other eligibility criteria but under-going a baseline biopsy would represent a significant risk to the subject, there should be a discussion with the medical monitor/sponsor about whether the subject can enter the study.

NOTE 4: Archival FFPE tumour material may be used as a baseline biopsy but only if there is sufficient quantity (see laboratory manual) and the biopsy was taken within 6 months prior to the date of signing the ICF. If more than one archival sample meets these criteria the most recent should be used.

7. The subject agrees to utilise highly effective contraception (i.e. methods with a failure rate of less than 1% per year) for both male and female subjects if the risk of conception exists. Highly effective contraception must be used 28 days prior to the first study drug administration, for the duration of study treatment, and for at least 60 days after stopping study treatment. 8. Willing and able to provide written informed consent.

9. A female subject is eligible to participate if she is not pregnant, not breastfeeding, and at least one of the following conditions applies: a) Not a WOCBP b) A WOCBP who agrees to follow the contraceptive guidance in inclusion criterion number 7.

10. Subjects with HIV who are healthy and, in the opinion of the investigator, have a low risk of AIDS- related outcomes may be considered if they meet the following criteria: a) Subjects on antiretroviral therapy (ART) must have a CD4+T-cell count > 350 cells/mm3at time of screening. b) Subjects on ART must have achieved and maintained virologic suppression defined as confirmed HIV RNA level below 50 copies/mL or the lower limit of qualification (below the limit of detection) using the locally available assay at the time of screening and for at least 12 weeks prior to screening. c) Subjects on ART must have been on a stable regimen, without changes in drugs or dose modification, for at least 4 weeks prior to study entry (Day 1).

1.6. 1 Inclusion Criteria for APT and 3+3 Dose Escalation:

In addition to the core inclusion criteria listed above, each subject considered for the ADT and 3+3 dose escalation cohorts must meet all the following inclusion criteria to be enrolled in this part of the study.

11 . Subjects with histologically or cytologically confirmed, locally advanced, unresectable or metastatic solid tumours where there is a regulatory approval for ICB therapy (except for hepatobiliary cancers), and for whom standard therapy has proven to be ineffective, intolerable or is considered inappropriate.

NOTE 1 : Subjects with cancers that harbour molecular alterations for which targeted therapy is standard of care should have received Health Authority-approved targeted therapy for their cancers prior to enrolment in this study.

NOTE 2: For subjects with SCCHN only cancers with origins in the oral cavity, oropharynx, larynx, or hypopharynx are eligible.

12. Prior treatment with an ICB containing regimen is acceptable but the following conditions apply: a) No more than 1 line of a prior ICB containing regimen NOTE: 2 lines of prior ICB containing regimens are permitted for melanoma. b) Only treatment regimens containing anti-PD-L1 , anti-PD-1 , and/or CTLA-4 are permitted. c) Subjects should have achieved CR, PR, or SD as the best response and have had disease progression whilst on ICB therapy. d) If prior PD-L1 tumour scoring was conducted and is available, the resulting PD-L1 score (e.g. TPS, CPS) should be documented.

1.6.2 Inclusion Criteria for PK/PD Expansion Cohorts:

In addition to the core inclusion criteria, each subject considered for the PK/PD expansion cohorts must also meet the following tumour-specific inclusion criteria to be enrolled in this part of the study.

NOTE: Subjects who meet these tumour specific criteria are also eligible for the 3+3 dose escalation cohorts.

Subjects with the following tumour types are eligible for the PK/PD expansion cohorts:

1.6.2.1 Colorectal cancer

13. Histologically confirmed advanced/metastatic RAS/RAF wild-type, MSS colon or rectal adenocarcinoma, naive to ICB therapy, who had a prior radiological response to first or -second-line treatment with FOLFIRI/FOLFOX in combination with cetuximab but have subsequently progressed on this treatment.

14. Enter screening for this study < 35 days of the date of confirmed progression (investigator assessed) on prior FOLFIRI/FOLFOX in combination with cetuximab.

1.6.2.2 Triple negative breast cancer (TNB C)

15. Metastatic or locally advanced, histologically confirmed and documented TNBC characterised by absence of HER2, ER, and progesterone receptor expression. NOTE: TNBC is defined as per the American Society of Clinical Oncology and College of American Pathologists guidelines. From the most recent tumour sample (primary or metastatic): ER negative with <1% of tumour cells positive for ER on IHC or IHC score (Allred) of <2; PR negative with <1% of tumour cells positive for PR on IHC or IHC score (Allred) of <2 or PR unknown and HER2 negative with 0 or 1+ intensity on IHC and no evidence of amplification on in situ hybridisation.

16. Prior treatment with a maximum of 3 prior systemic treatment regimens for locally advanced or metastatic disease (1 of which should have included a taxane or anthracycline based regimen. Subjects with known germline BRCA mutations should have received either platinum and/or PARP inhibitor therapy). 1.6.2.3 Non-small cell lung cancer (NSCLC)

17. Histologically or cytologically confirmed stage II I B, 11 IC and IV NSCLC (AJCC Cancer Staging Manual, eighth edition) that are not candidates for curative treatment modalities in one of the following 2 categories: a. Subjects who have received and progressed under prior SOC (such as but not limited to prior line of platinum-based chemotherapy, chemoradiation, small molecule inhibitors and/or ICB).

- Subjects should have received no more than 3 prior lines of therapy.

- Subjects who have received a prior ICB should meet inclusion criteria number 12. b. ICB naive subjects who have progressed under prior SOC (such as but not limited to prior line of platinum-based chemotherapy, chemoradiation, small molecule inhibitors) and with documented PD- L1 TPS of 0% to 49% (by local or central testing) are also eligible.

1.6.2.3 Squamous cell carcinoma of the Head and Neck (SCCHN)

19. Histologically and/or cytologically confirmed relapsed metastatic SCCHN originating from the following sites is eligible: oral cavity, oropharynx, larynx, or hypopharynx. Subjects must not have a primary tumour site of nasopharynx (any histology). The results of HPV testing should be available and documented for subjects with oropharyngeal cancer.

20. Subjects who have received a maximum of 2 prior systemic treatment regimens (1 of which must have included a platinum-based regimen with or without cetuximab) for relapsed metastatic SCCHN which may have included an ICB regimen as per the conditions listed under inclusion criteria number 12.

1.6.2.4 Soft tissue sarcoma (STS)

21 . Subjects with histologically confirmed STS (subtype must be documented e.g. dedifferentiated liposarcoma, UPS, leiomyosarcoma) who have received a minimum of 1 but no more than 4 prior lines of systemic treatment for advanced or metastatic disease and are naive to ICB therapy.

1.6.2.5 Ovarian cancer

23. Subjects with histologically confirmed, platinum resistant or refractory carcinoma of the ovary, peritoneum or fallopian tube, naive to ICB therapy, who have received a maximum of 4 prior lines of systemic therapy for advanced or metastatic disease.

1.7 Exclusion Criteria

Core Exclusion Criteria. Subjects meeting any of the following criteria will be excluded from the study: 1. During the COVID-19 pandemic, the following exclusions apply: a. Subject shows clinical signs and symptoms (e.g., fever, headache, persistent cough, alterations of smell and/or taste, fatigue) of COVID-19 and/or potential infection with SARS- CoV-2 cannot be ruled out at the time of enrolment. b. Subject, in the opinion of the treating physician, is deemed at high risk of fatal outcome in case of COVID-19 disease. c. Subject has a history of COVID-19 disease and has not provided a negative test for SARS- CoV-2 infection within 28 days of the planned first dose date with FS222.

2. Concurrent enrolment in another clinical study with the exception of non- interventional/observational studies or the follow-up period of an interventional study or has used an investigational device within the 28 days prior to the first dose of study treatment.

NOTE: Subjects who have entered the follow-up phase of an investigational study may participate as long as it has been 28 days.

3. Prior treatment with any agent directed to another stimulatory or costimulatory T cell receptor (e.g.CD137 [4-1 BB] agonist, 0X40 agonist, CD40 agonist, GITR or CD27 targeting therapy) as a single agent or in combination with other anti-cancer therapies.

4. Subjects who experienced a toxicity related to prior ICB that led to permanent discontinuation of treatment.

5. Have received prior radiotherapy within 2 weeks of start of study treatment. Subjects must have recovered from all radiation-related toxicities, not require corticosteroids, and not have had radiation pneumonitis. A 1-week washout is permitted for palliative radiation (<2 weeks of radiotherapy) to non- CNS disease.

6. Subjects with active autoimmune disease requiring treatment in the previous 2 years or subjects with a documented history of any autoimmune disease.

NOTE: This includes subjects with a history of inflammatory bowel disease, ulcerative colitis and Crohn’s Disease, diverticulitis (with the exception of diverticulosis), rheumatoid arthritis, systemic progressive sclerosis (scleroderma), systemic lupus erythematosus, autoimmune vasculitis (e.g. Wegener’s granulomatosis), CNS or motor neuropathy considered of autoimmune origin (e.g. Guillain Barre syndrome, myasthenia gravis, multiple sclerosis) and moderate or severe psoriasis. However, subjects with rheumatoid arthritis or psoriasis (in stable remission for at least 6 months and without contraindications to possible cotreatment with corticosteroids for immune-related AEs), vitiligo, Sjogren’s syndrome, interstitial cystitis, Graves’disease, Hashimoto’s disease or hypothyroidism stable on hormone replacement will be allowed with the study medical monitor’s approval. 7. Subjects with a diagnosis of immunodeficiency or who are receiving chronic systemic steroid therapy (in dosing exceeding 10 mg daily of prednisone equivalent) or any other form of immunosuppressive therapy within 7 days prior to the first dose of study drug.

8. Receipt of any live or live attenuated virus vaccine within 30 days prior to the first dose of study drug. NOTE 1 : Subjects, if enrolled, should not receive any live virus vaccines while receiving study drug nor during the 30-day period after the last dose of study drug.

NOTE 2: Administration of killed vaccines are permitted. For COVID-19 vaccine, F-star recommends that there should be a 48-hour window between subjects receiving a COVID-19 vaccine and IP administration. The decision to vaccinate trial subjects remains with treating study investigator and should be based on adequate balance of benefits/risks.

9. A WOCBP who has a positive urine pregnancy test (within 72 hours) prior to treatment. If the urine test is positive or cannot be confirmed as negative, a serum pregnancy test will be required.

10. History of uncontrolled intercurrent illness including but not limited to: a. Documented hypertension, uncontrolled by treatment with standard therapies (not stabilised to <150/90 mmHg). b. Documented uncontrolled diabetes. NOTE: Subjects with well-controlled diabetes under stable insulin replacement therapy for at ast 6 months and without contraindications to possible cotreatment with corticosteroids for irAEs may be enrolled. c. Significant cardiac abnormalities, including a history of long QTc syndrome and/or pacemaker, cerebral vascular accident/stroke (<6 months prior to enrolment), myocardial infarction (<6 months prior to enrolment), unstable angina, congestive heart failure (New York Heart Association Classification Class > II), or clinically significant and symptomatic cardiac arrhythmia that has not been controlled with medication for at least 6 months. d. History of (non-infectious) pneumonitis/interstitial lung disease that required steroids or has current pneumonitis/interstitial lung disease. e. Known history of liver disease(s) or drug-induced liver injury (DILI) e.g. alcoholic hepatitis or non-alcoholic steohepatitis, drug-related or autoimmune hepatitis, or hepatic cirrhosis. f. Known history of myositis, Guillian-Barre syndrome, or myasthenia gravis of any grade. g. Any condition that, in the opinion of the investigator or Sponsor, would interfere with evaluation of the study drug or interpretation of the study results or subject’s safety e.g. Presence of nephrostomy or other ureteral stent that may put the subject at risk of systemic infection, peritoneal carcinomatosis (these patients may be eligible but should be discussed with the medical monitor prior to enrolment), symptomatic pulmonary embolism (known asymptomatic cases of pulmonary embolism should be reviewed with the medical monitor prior to enrolment).

11 . Psychological, familial, sociological or geographical conditions that do not permit compliance with the protocol.

12. Judgment by the investigator that the subject is unsuitable to participate in the study, and the subject is unlikely to comply with study procedures, restrictions and requirements.

13. Screening laboratory values with the following criteria: a. Haemoglobin <9.0 g/dL (5.7 pmol/L)

NOTE: Criteria must be met without packed RBC infusion within the prior 2 weeks. Subjects can be on a stable dose of erythropoietin (> approximately 3 months) b. Absolute neutrophil count <1.0 x 109/L c. Absolute lymphocyte count <0.5 x 109/L d. Platelets <100 x 109/L e. Serum creatinine >1.5 x ULN and/or creatinine clearance <30 mL/min (Cockcroft-Gault formula or Modification of Diet in Renal Disease formula) f. LDH >2.5 x ULN g. Total bilirubin >1 .5 x ULN; or AST and ALT >2.5 x ULN (AST and ALT > 5 x ULN, if liver metastasis are present)

NOTE 1 : For any subject with LFTs outside the normal range there should be a discussion with the medical monitor as to whether the subject should enter the study.

NOTE 2: Criteria must be met without the need for steroids or other treatments to improve liver function either during the screening period or in the 28 days prior to the subject signing the ICF. h. Albumin <2.8 g/L.

14. Subjects with haematological malignancies (any grade).

15. Subjects with previous episodes of severe neutropenia within the last 12 months prior to enrolment in the study should be discussed with the study medical monitor prior to enrolment. 16. Subject has a known additional malignancy that is progressing or has required active treatment in the past 3 years.

NOTE: Subjects with basal cell carcinoma of the skin, squamous cell carcinoma of the skin, transitional cell carcinoma of urothelial cancer, localized prostate carcinoma that has been treated and/or on hormone deprivation therapy with a normal PSA, or carcinoma in situ (e.g. breast carcinoma, cervical cancer in situ) that has undergone potentially curative therapy are not excluded.

17. Subjects who have had a prior allogenic tissue/solid organ transplant or prior allogenic hematopoetic stem cell transplantation (HSCT) within the last 5 years (subjects who received allogenic HSCT greater than 5 years ago are eligible as long as there are no symptoms of GVHD).

18. Known infections: a. Human immunodeficiency virus, HBV (i.e. HBsAg positive), HCV (i.e. detectable HCV RNA), or EBV (i.e. IgM positive). NOTE: Subjects with a prior history of treated HBV infection who are antigen negative or subjects with a prior history of treated HCV infection in whom HCV RNA is undetectable may be enrolled after consultation with the medical monitor. Subjects with HIV who are healthy and, in the opinion of the investigator, have a low risk of AIDS-related outcomes may be considered after consultation with the SRC and medical monitor. b. Active infections (including asymptomatic infections with positive virus titres) and investigator’s judgment that worsening of condition is likely with study drug or condition would impair/prohibit a subject’s participation in the study. c. Subject has tested positive for SARS-CoV-2 infection.

19. HIV-infected subjects with a history of Kaposi sarcoma and/or Multicentric Castleman Disease.

20. Uncontrolled CNS metastases, primary CNS tumours with CNS metastases as only measurable disease. A brain MRI (CT is an acceptable alternative, especially in cases where MRI is contraindicated) is required at Screening to assess for any evidence of CNS involvement. Subjects with active but stable CNS disease may be enrolled.

NOTE: Stable CNS disease is defined as no evidence of progression for at least 4 weeks by repeat imaging (note that repeat imaging should be performed during study screening), clinically stable and without requirement for steroid treatment for at least 14 days prior to first dose of study treatment. This exception does not include carcinomatous meningitis which is excluded regardless of clinical stability.

21 . Prior history of: a. Any Grade > 3 irAE that has not improved to Grade <1 , except for endocrine deficiencies that are managed by HRT. b. Significant (Grade > 3 NCI CTCAE Version 5.0) treatment-related cytokine release syndrome. c. Systemic inflammatory response syndrome.

22. Current use of immunosuppressive agents, hypersensitivity or intolerance to mAb or their excipients, or persisting toxicity related to prior therapy of Grade >1 NCI CTCAE Version 5.0 with the following exceptions: a. Endocrine dysfunction on replacement therapy is allowed (including stable hypophysitis on HRT). b. Non-systemic steroids; topical, intraocular, intranasal, intraarticular, or inhalative steroids are allowed. c. Systemic steroid replacement treatment <10 mg/day prednisone or equivalent in subjects with adrenal insufficiency is allowed. d. Enrolment of subjects who receive systemic steroid treatment <10 mg/day prednisone or equivalent as part of their palliative treatment or symptomatic disease control should be discussed with the medical monitor and/or Sponsor.

1 .8 Assessment of Disease

RECIST v1 .1 was used as the primary measure for assessment of tumour response (investigator assessed), date of disease progression, and as a basis for all protocol guidelines related to disease status (e.g. discontinuation of study treatment). Although RECIST v1 .1 references a maximum of 5 target lesions in total and 2 per organ, this protocol allows a maximum of 10 target lesions in total and 5 per organ, if clinically relevant to enable a broader sampling of tumour burden. iRECIST is based on RECIST v1 .1 but adapted to account for the unique tumour response seen with immunotherapeutic drugs. iRECIST was used by the investigator to assess tumour response and progression and make treatment decisions. When clinically stable, subjects should not be discontinued until progression is confirmed by the investigator. This allowance to continue treatment despite initial radiologic PD takes into account the observation that some subjects can have a transient tumour flare in the first few months after the start of immunotherapy, and then experience subsequent disease response.

Clinical stability was defined as the following:

• Absence of symptoms and signs indicating clinically significant progression of disease • No decline in ECOG performance status

• No requirements for intensified management, including increased analgesia, radiation, or other palliative care

Any subject deemed clinically unstable was discontinued from study treatment at site-assessed first radiologic evidence of PD and is not required to have repeat tumour imaging for confirmation of PD by iRECIST. Lack of clinical stability was defined as:

• Unacceptable toxicity

• Clinical signs or symptoms indicating clinically significant disease progression

• Decline in performance status

• Rapid disease progression or threat to vital organs or critical anatomical sites (e.g. CNS metastasis, respiratory failure due to tumour compression) requiring urgent alternative medical intervention.

If the investigator decided to continue treatment, the subject continued to receive study treatment and the tumour assessment was repeated 4 to 8 weeks later to confirm PD by iRECIST, per investigator assessment.

If repeat imaging did not confirm PD per iRECIST, as assessed by the investigator, and the subject continued to be clinically stable, study treatment continued and followed the regular imaging schedule. If PD was confirmed, subjects were discontinued from study treatment.

If a subject had confirmed radiographic progression (iCPD) study treatment was discontinued; however, if the subject achieved a clinically meaningful benefit, an exception to continue study treatment may be considered following consultation with the Sponsor. In this case, if study treatment was continued, tumour imaging continued to be performed.

A description of the adaptations and iRECIST process is provided in below, with additional details in the iRECIST publication (Seymour et al 2017).

1.9 Analysis of Primary Endpoints

Adverse events (AEs), severe adverse events (SAEs), Abnormal Laboratory Parameters, Vital Signs, Electrocardiogram (ECG) and dose-limiting toxicitis (DLTs) were analysed as described below.

The safety profile was based on AEs (including DLTs and SAEs), physical examination findings (including ECOG performance status), vital sign measurements, standard clinical laboratory measurements, and ECG recordings. Safety analyses in general were descriptive and were presented in tabular format with the appropriate summary statistics for the Safety Analysis Set. A treatment-emergent adverse event (TEAE) were defined as an AE with a start date and time on or after the first dose of study drug. The number and percentage of subjects with TEAEs were tabulated by SOC and preferred term by dose cohort and in total.

Similarly, the number and percentage of subjects reporting treatment-emergent SAEs, TEAEs and treatment-emergent SAEs considered related to FS222 and TEAEs leading to discontinuation of study drug were tabulated in the same manner.

The number and percentage of subjects reporting TEAEs and FS222-related TEAEs were also be tabulated by the worst NCI CTCAE grade, SOC, and preferred term.

Incidence of DLTs were also be tabulated and listed by dose cohort.

A by-subject AE data listing were provided including, but not limited to, verbatim term, preferred term, SOC, NCI CTCAE grade, and relationship to study drug. Death, SAEs and other significant AEs, including those leading to permanent discontinuation from FS222, were listed.

Descriptive statistics were provided for vital signs (temperature, heart rate, respiratory rate, and blood pressure) and change from baseline in vital signs by scheduled time point. ECG measurements and change from baseline in ECG measurements will be summarised by scheduled time point. Abnormal laboratory results were graded according to NCI CTCAE Version 5.0, if applicable. Treatment- emergent abnormal laboratory results, defined as at least 1 grade increase from baseline, were summarised by scheduled time point and dose cohort.

1.10 Analysis of Secondary Endpoints

Tumour responses were assessed by the investigator according to RECIST 1.1 criteria.

All references to CR and PR were based on confirmed response.

1.10.1 Definition of Efficacy Endpoints

Objective response rate per RECIST 1.1 was defined as the proportion of subjects who achieved a best overall response (OR) of CR or PR.

Disease control rate per RECIST 1 .1 is defined was the proportion of subjects whose best OR is a CR, PR, or stable disease.

The time-to-event endpoints are defined as follows:

Duration of response per RECIST 1.1 : DoR was calculated for subjects achieving a best OR of CR or PR and is defined as the interval from the first documentation of objective response until the first date that progressive disease per RECIST 1.1 was objectively documented, initiation of alternative anticancer therapy or the date of death due to underlying cancer. For subjects who had not progressed, DoR was censored at the last recorded date that the subject was known to be progression-free as of the cutoff date for the analysis.

Progression-free survival per RECIST 1.1 : PFS was defined as the time from the first dose date of study drug to first observation of documented progressive disease per RECIST 1.1 or death due to any cause within 12 weeks of last tumour assessment, per RECIST 1 .1 . For subjects who had not progressed and were still alive at time of data cutoff for study analysis or who were lost to follow-up, PFS was censored at the last recorded date that the subject was known to be progression-free and alive as of the cutoff date for analysis.

Overall survival: OS was defined as time from the first dose date of study drug to death due to any cause. For subjects who were still alive at time of data cutoff for study analysis or who v lost to followup, OS was censored at the last recorded date that the subject is known to be alive as of the cutoff date for analysis.

1.11 Analyses of Exploratory Endpoints

Disease control rate, ORR, DoR and PFS were defined and analysed in a similar way to the secondary endpoints but will be assessed according to iRECIST 1.1 criteria instead of RECIST 1.1.

1.12 Results summary

The characteristics of the patients enrolled in the study to date are shown in Figure 3.

The treatment results are summarised in Figure 6A and 6B.

Figure 6C shows the results of the safety assessment of cancer patients treated with 1 .5 to 4.5 mg/kg of FS222. The treatment-related adverse events are reported.

Figures 7 and 9 show response data for cancer patients treated with 300 pg (fixed dose) to 4.5 mg/kg of FS222 once every four weeks. The data in Figure 7 was obtained at a later time point in the trial than the data shown in Figure 6 and so includes data for additional patients enrolled in the study in the meantime. Similarly, the data in Figure 9 was obtained at a later time point in the clinical trial than the data shown in Figure 7 (November 2024 compared with April 2024) and thus shows increased length of treatment of some of the patients in the trial as well as reflecting some data corrections.

Figures 8 and 10 show response data for melanoma patients treated with 1 mg/kg to 4.5 mg/kg of FS222 once every four weeks. This data is a subset of the data shown in Figures 7 and 9, respectively. Sequence Listing

Heavy chain amino acid sequence of anti-human CD137/PD-L1 mAb² FS222 (with LALA mutation)

(SEQ ID NO: 1)

VH domain (italics); LALA mutation (bold underlined)

EVQLVQSGAEVKRPGASVKVSCKASGYPFTSYGISWVRQAPGQGLEWMGWISAYSGGTNYAQKLQ

GRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDLFPTIFGVSYYYYWGQGTLVTVSSASTKGPSVF

PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL

GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT

CWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN

KALPAPIEKTISKAKGQPREPQVYTLPPSRDELPYIIPPYNQVSLTCLVKGFYPSDIAVEWESNGQPEN

NYKTTPPVLDSDGSFFLYSKLTVGADRWLEGNVFSCSVMHEALHNHYTQKSLSLSPG

Light chain amino acid sequence of anti-human CD137/PD-L1 mAb² FS222 (SEQ ID NO: 2)

VL domain (italics)

DIQMTQSPSTLSASVRDRVIITCRASQSIGNRLAWYQHKPGKAPKLLIYEASTSETGVPSRFSGSGSG

TDFTLT/SSLQPEDFATYYCQQSYSTPYTFGQGTKLE/KRTVAAPSVFIFPPSDEQLKSGTASVVCLLN

NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSS

PVTKSFNRGEC

Heavy chain amino acid sequence of anti-human CD137/PD-L1 mAb² FS222 (with LALA mutation and C-terminal lysine) (SEQ ID NO: 3)

VH domain (italics); LALA mutation (bold underlined); C-terminal lysine in bold and italics

EVQLVQSGAEVKRPGASVKVSCKASGYPFTSYGISWVRQAPGQGLEWMGWISAYSGGTNYAQKLQ

GRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARDLFPTIFGVSYYYYWGQGTLVTVSSASTKGPSVP

PLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSL

GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVT

CWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN

KALPAPIEKTISKAKGQPREPQVYTLPPSRDELPYIIPPYNQVSLTCLVKGFYPSDIAVEWESNGQPEN

NYKTTPPVLDSDGSFFLYSKLTVGADRWLEGNVFSCSVMHEALHNHYTQKSLSLSPGK

Claims

Claims

1. An antigen-binding protein which binds CD137 and programmed death-ligand 1 (PD-L1) for use in a method of treating cancer in a human patient, wherein the antigen-binding protein comprises the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 2; and wherein the antigen-binding protein is administered to the patient every four weeks at a dose of 2 mg per kg of body weight of the patient.

2. The antigen-binding protein for use according to claim 1 , wherein the cancer is selected from the list consisting of: melanoma, ovarian cancer, soft tissue sarcoma, non-small cell lung cancer (NSCLC), triple negative breast cancer (TNBC), and colorectal cancer.

3. The antigen-binding protein for use according to any preceding claim, wherein the patient achieves a complete response, a partial response, or stable disease in response to the treatment with the antigen-binding protein.

4. The antigen-binding protein for use according to any preceding claim, wherein the patient achieves a partial response in response to the treatment with the antigen-binding protein.

5. The antigen-binding protein for use according to any preceding claim, wherein the antigenbinding protein is administered at a dose of 2 mg per kg of body weight of the patient, and wherein the patient achieves at least 8 weeks of progression-free survival (PFS) in response to treatment with the antigen-binding protein.

6. The antigen-binding protein for use according to any preceding claim, wherein the cancer is selected from the list consisting of: melanoma, ovarian cancer, triple negative breast cancer, and colorectal cancer, and the antigen-binding protein is administered at a dose of 2 mg per kg of body weight of the patient, and wherein the patient achieves at least 30 weeks, preferably at least 40 weeks, more preferably at least 50 weeks, of PFS in response to treatment with the antigen-binding protein.

7. The antigen-binding protein for use according to any preceding claim, wherein the antigenbinding protein is administered at a dose of 2 mg per kg of body weight of the patient, and wherein the treatment results in an objective response rate of at least 35%.

8. The antigen-binding protein for use according to any preceding claim, wherein the antigenbinding protein is administered at a dose of 2 mg per kg of body weight of the patient, and wherein the treatment results in a disease control rate of at least 50%.

9. The antigen-binding protein for use according to any preceding claim, wherein the cancer is resistant to a first anti-cancer treatment or has relapsed during or following a first anti-cancer treatment.

10. The antigen-binding protein for use according to claim 9, wherein the first anti-cancer treatment is treatment with an immune checkpoint inhibitor.

11 . The antigen-binding protein for use according to claim 10, wherein the immune checkpoint inhibitor is an inhibitor of PD-1 , PD-L1 , or CTLA-4.

12. The antigen-binding protein for use according to any preceding claim, wherein the cancer is cutaneous melanoma.

13. The antigen-binding protein for use according to claim 12, wherein the cutaneous melanoma is resistant to treatment with a first PD-1 inhibitor or PD-L1 inhibitor, or has relapsed during or following treatment with a first PD-1 inhibitor or PD-L1 inhibitor.

14. The antigen-binding protein for use according to claim 13, wherein the cutaneous melanoma is resistant to treatment with a first PD-1 inhibitor, or has relapsed during or following treatment with a first PD-1 inhibitor.

15. The antigen-binding protein for use according to any one of claims 2 to 14, wherein the antigen-binding protein is administered at a dose of 2 mg per kg of body weight of the patient, and wherein the patient achieves at least 23 weeks of PFS.

16. The antigen-binding protein for use, according to claim 15, wherein the patient achieves at least 30 weeks of PFS.

17. The antigen-binding protein for use, according to claim 9, wherein the first anti-cancer treatment is selected from chemotherapy, radiotherapy, treatment with an anti-cancer biologic therapy, or hormone therapy.

18. An antigen-binding protein which binds CD137 and PD-L1 for use in a method of treating cancer in a human patient who has been subjected to treatment with a prior immune checkpoint inhibitor therapy, selected from an anti-PD-1 , anti-PD-L1 or anti-CTLA-4 therapy, the antigen-binding protein comprising the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 2; wherein a tumour of the patient has been determined to have an acquired resistance phenotype in respect of the prior immune checkpoint inhibitor therapy, and wherein a tumour with an acquired resistance phenotype is a tumour which showed a complete or partial response to treatment with the prior immune checkpoint inhibitor therapy or showed stable disease whilst subjected to treatment with the prior immune checkpoint inhibitor therapy.

19. A method of treating cancer in a human patient, wherein the method comprises administering to the patient a therapeutically effective amount of the antigen-binding protein according to any preceding claim.

20. A method of treating cancer in a human patient who has been subjected to treatment with a prior immune checkpoint inhibitor therapy, selected from an anti-PD-1 , anti-PD-L1 or anti-CTLA-4 therapy, the method comprising administering to the patient a therapeutically effective amount of an antigen-binding protein which binds CD137 and PD-L1 and comprises the heavy chain sequence set forth in SEQ ID NO: 1 and the light chain sequence set forth in SEQ ID NO: 2; wherein a tumour of the patient has been determined to have an acquired resistance phenotype in respect of the prior immune checkpoint inhibitor therapy, and wherein a tumour with an acquired resistance phenotype is a tumour which showed a complete or partial response to treatment with the prior immune checkpoint inhibitor therapy, or showed stable disease whilst subjected to treatment with the prior immune checkpoint inhibitor therapy.

21 . The antigen-binding protein for use according to any one of claims 1 to 18, or the method according to claim 19 or 20, wherein the antigen-binding protein comprises the heavy chain sequence set forth in SEQ ID NO: 3.