Introduction Agonists of CD137, a co-stimulatory receptor expressed on activated lymphocytes, are under investigation, including their use in combination with immune checkpoint blockade. FS222 is a tetravalent bispecific antibody targeting both CD137 and programmed death-ligand 1 (PD-L1).Methods Fragment crystallizable regions with antigen-binding (Fcab (TM)) domains with high avidity to CD137 were engineered and matured. A resulting Fcab was introduced into a human high-affinity PD-L1 monoclonal antibody, generating FS222. Valency variants of FS222 and its mouse surrogate (FS222m) were generated. Antibody properties were assessed in vitro, while antitumor efficacy and T-cell proliferation were evaluated in vivo.Results FS222 could bind to two copies of each antigen concurrently ('2+2' binding). In T-cell activation assays, FS222 and a variant that is bivalent for CD137 and monovalent for PD-L1 activated T cells, whereas monovalent variants for CD137 did not. FS222 had no hook effect up to 300 nM and was superior to a heterodimeric '1+1' CD137/PD-L1 bispecific antibody. In mixed lymphocyte reaction assays, FS222 and another bivalently linked antibody for PD-L1 were more potent than monovalent antibodies for PD-L1. In syngeneic mouse models, FS222m was associated with more peripheral T-cell proliferation and antitumor efficacy than all MC38 or most CT26.WT valency variants.Discussion FS222 could potently target and activate high CD137-expressing tumor-infiltrating lymphocytes due to its high avidity for CD137. FS222 demonstrates activity when PD-L1 levels are limited, highlighting its potential activity in tumors insensitive to PD-1 blockade, and in situations requiring both PD-L1 and CD137 binding. This may have clinical relevance.Conclusion In this study, it was found that the '2+2' structure of FS222 is superior to that of a '1+1' CD137/PD-L1 antibody.

2505 Background: Most pts receiving immune checkpoint inhibitors do not respond to treatment or relapse. FS222 is a novel affinity optimized, tetravalent bispecific PD-L1/CD137 antibody. FS222’s structure allows for potent, PD-L1-dependent, CD137 activation across a wide range of dose levels, and is designed to provide selective CD137 agonism in the tumor. We present data from the ongoing, FIH, open-label, phase I trial of FS222 in advanced solid tumors. Methods: Pts with pretreated advanced solid tumors received increasing doses of FS222 in an accelerated dose titration and 3+3 design intravenously every 3 or 4 weeks (Q4W) until disease progression or unacceptable toxicity. The primary endpoint was safety. Secondary and exploratory endpoints included pharmacokinetics, PD and antitumor activity (by Response Evaluation Criteria in Solid Tumors [RECIST] 1.1). Results: As of data cut off (DCO) on 05Dec2023, a total of 104 pts had been treated across a range of doses and schedules in the FIH study (NCT04740424). We report interim results from the Q4W cohorts (N=90). Patients had a median age of 61 years (31-88 years) and had received a median of 2 (1-7) regimens of prior treatments. The median duration of exposure to FS222 was 82.5 days (24 – 529 days). The most common treatment-related AEs (TRAEs; >20% of pts) were increased aspartate aminotransferase (AST) and alanine aminotransferase (ALT), pyrexia, thrombocytopenia, asthenia, and neutropenia. The most common TRAEs grade ≥3 (≥10% of pts) were increased AST (13.3%) and ALT (11.1%). The most common treatment related serious AEs (≥3 pts) were febrile neutropenia (5 pts, 5.6%); and pyrexia, cytokine release syndrome, increased ALT, increased AST (all in 3 pts, 3.3%). On-target FS222 pharmacology was confirmed by the presence of dose-dependent target engagement, significant peripheral CD8 + T cell modulation and increased tumor CD3 + CD8 + T cells at multiple dose levels. At the DCO, 20 (22.2%) pts remained on treatment. Objective responses (CR, PR) were observed in pts with melanoma, NSCLC, ovarian cancer, TNBC, liposarcoma and colon cancer, for an ORR of 15.7% with evidence of further enrichment by dose. In post-PD-1 treated metastatic/advanced cutaneous melanoma the ORR was 60% (9/15, all PRs - 7 confirmed) and the disease control rate was 86.7% (13/15). Conclusions: The novel PD-L1/CD137 bispecific antibody FS222 demonstrated PD activity across a broad range of doses. The safety profile was acceptable and manageable. Encouraging anti-tumor activity was observed, including in patients with PD-1 refractory cutaneous melanoma. Next steps include further dose optimization and further evaluation of FS222 in patients with melanoma and other tumor types. Clinical trial information: NCT04740424 .

FS222 is a novel tetravalent bispecific antibody targeting PD-L1 and CD137 that aims to overcome the limitations of current PD-(L)1 and CD137 (4-1BB) monotherapies via a unique mechanism of action that elicits PD-L1-dependent CD137 activation. This has the potential to deliver strong antitumor clinical activity, even in PD-L1 low conditions as seen in preclinical models, with good tolerability.

Abstract Background: CD137 (4-1BB, TNFRSF9) is expressed on activated lymphocytes, and its clustering leads to agonism of the receptor resulting in lymphocyte proliferation and pro-inflammatory cytokine release. First-generation CD137 antibodies for cancer therapy were high affinity and enabled for FcγR engagement with either severe toxicity or weak activity limiting their therapeutic benefit. However, CD137 remains a promising target for bispecific antibodies designed to re-direct T cell activity to the tumor whilst limiting unwanted toxicities. We have rationally designed and developed a unique tetravalent bispecific antibody targeting CD137 and PD-L1 with reduced FcγR binding for safe and efficacious cancer therapy (Lakins MA et al. 2020). Methods: A CD137/PD-L1 bispecific mAb2 antibody (FS222) was generated by introducing a bivalent affinity-optimized CD137-binding Fcab into a human IgG1 bivalent PD-L1 mAb. LALA mutations were introduced to abrogate FcγR activity. To elucidate its mechanism of action, FS222's binding valency was assessed by chemically-crosslinked mass spectrometry mapping (XL/MS). Immune pharmacology models were also used to evaluate PD-L1 dependent FS222 agonism against variants with differing valency for each target. Results: FS222 was specifically designed to bind CD137 with moderate affinity, and without PD-L1-mediated crosslinking it does not cluster or activate the receptor on immune cells. When PD-L1 was bound by FS222, this lead to crosslinking and subsequent clustering and activation of CD137. This clustering was enabled by avid bivalent binding as demonstrated by tetravalent FS222 being biologically more active than variants with reduced valency. Moreover, XL/MS demonstrated all four binding sites were able to concurrently bind target antigens. FS222 was well tolerated in a GLP toxicity study up to the maximal dose (30mg/kg QW). It was pharmacologically active in peripheral blood and lymphoid tissues and had a predicted t½ of 286 hours in humans. A surrogate anti-mouse CD137/PD-L1 mAb2 significantly reduced tumor growth in a murine syngeneic model and this correlated with a significant survival benefit and T cell activation. Conclusions: FS222 was designed to be a potent anti-human CD137/PD-L1 tetravalent conditional agonist with a unique combination of high affinity PD-L1 binding and moderate monovalent affinity, and highly avid binding, to CD137 on activated T cells. A favorable safety profile and immunopharmacology was observed with FS222 in a primate GLP toxicity study. Tetravalent binding by FS222 was required for optimal activity in pharmacology studies. Preclinically, FS222 was well-tolerated with an effective mechanism of action. Citation Format: Matthew A. Lakins, Jose Munoz-Olaya, Christel Veyssier, Daniel Jones, Emma Goodman, Quincy Kaka, Jennifer Ofoedu, Robert Hughes, Daniel Gliddon, Michelle Morrow, Neil Brewis. FS222, a tetravalent bispecific antibody targeting CD137 and PD-L1, is designed for optimal CD137 interactions resulting in potent T cell activation without toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1864.

BackgroundCheckpoint Inhibitor (CPI) therapy is standard of care in a range of tumor types but many patients fail to gain long term clinical benefit. Agonist antibodies targeting the T cell costimulatory receptor CD137 (4-1BB) have shown immune-stimulatory effects, but hepatotoxicity significantly hindered development. FS222, a novel tetravalent bispecific antibody, incorporates two CD137 binding sites in the Fc-region (Fcab) and anti-PD-L1 Fabs in a natural human IgG1 antibody with silenced FcyR activity. FS222’s affinity has been tuned specifically for each target; high affinity to PD-L1 and moderate affinity to CD137. This elicited PD-L1 dependent CD137 activation for a robust immune stimulation in the absence of toxicity, which resulted in strong antitumor activity in syngeneic models (Lakins et al). FS222’s unique format has the potential to deliver strong antitumor clinical activity with good tolerability.Trial DesignThis phase 1 trial is a multi-part (A and B) adaptive, dose escalation and expansion study in subjects with advanced malignancies (NCT04740424). FS222 is administered once every 4 weeks intravenously in 28-day treatment cycles. Primary objectives are to determine safety and tolerability and the recommended phase 2 dose (RP2D). Secondary objectives include determining pharmacokinetic (PK) and pharmacodynamic (PD) profiles, immunogenicity, and clinical response. Subjects with specific solid tumor types will be enrolled who have progressed on standard of care therapy. One line of prior CPI therapy is permitted. Part A consists of an accelerated dose titration (ADT) involving single-subject cohorts followed by a classical 3+3 design and dose expansion cohorts in selected tumor types to further characterize safety, PK/PD and clinical efficacy. Part B consists of tumor-specific efficacy expansion cohorts with Bayesian adaptive statistics. Translational studies will assess peripheral immune cell proliferation and activation, soluble peripheral PD biomarker levels and the correlation of FS222 exposure with selected PD markers of target engagement and response. Other exploratory translational endpoints are also included in the study. Study enrolment began December 2020.Legal entity responsible for the studyF-star Therapeutics.FundingF-star Therapeutics.DisclosureE. Garralda: Financial Interests, Personal, Advisory Board: Genentech; Financial Interests, Personal, Advisory Board: F.Hoffmann/La Roche; Financial Interests, Personal, Invited Speaker: Ellipses Pharma; Financial Interests, Personal, Advisory Board: Neomed Therapeutics1 Inc; Financial Interests, Personal, Advisory Board: Boehringer Ingelheim; Financial Interests, Personal, Advisory Board: Janssen Global Services; Financial Interests, Personal, Invited Speaker: Seattle Genetics; Financial Interests, Personal, Expert Testimony: TFS; Financial Interests, Personal, Advisory Board: Alkermes; Financial Interests, Personal, Advisory Board: Thermo Fisher; Financial Interests, Personal, Invited Speaker: Bristol Myers Squibb; Financial Interests, Personal, Advisory Board: MabDiscovery; Financial Interests, Personal, Advisory Board: Anaveon; Financial Interests, Personal, Invited Speaker: MSD; Financial Interests, Personal, Advisory Board: Lilly; Financial Interests, Institutional, Funding: Novartis; Financial Interests, Institutional, Funding: Roche; Financial Interests, Institutional, Funding: Thermo Fisher; Financial Interests, Institutional, Funding: AstraZeneca; Financial Interests, Institutional, Funding: Taiho; Other, Personal, Other, Institutional Travel Grant: Bristol Myers Squibb; Other, Personal, Other, Institutional Travel Grant: MSD; Other, Personal, Other, Institutional Travel Grant: Menarini; Other, Personal, Other, Institutional Travel Grant: Glycotope. I. Melero: Financial Interests, Personal, Research Grant: Roche; Financial Interests, Personal, Research Grant: GenMab; Financial Interests, Personal, Research Grant: Bioncotech; Financial Interests, Personal, Research Grant: AstraZeneca; Financial Interests, Personal, Speaker’s Bureau: F-Star; Financial Interests, Personal, Speaker’s Bureau: Bristol Myers Squibb; Financial Interests, Personal, Speaker’s Bureau: Roche; Financial Interests, Personal, Speaker’s Bureau: Alligator; Financial Interests, Personal, Speaker’s Bureau: Numab; Financial Interests, Personal, Speaker’s Bureau: AstraZeneca; Financial Interests, Personal, Speaker’s Bureau: Amunix; Financial Interests, Personal, Speaker’s Bureau: Boehringer; Financial Interests, Personal, Speaker’s Bureau: Hokipa; Financial Interests, Personal, Speaker’s Bureau: Merck Serono. G. de Velasco: Financial Interests, Personal, Speaker’s Bureau: Pfizer; Financial Interests, Personal, Speaker’s Bureau: Roche; Financial Interests, Personal, Speaker’s Bureau: Ipsen; Financial Interests, Personal, Speaker’s Bureau: Merck; Financial Interests, Personal, Speaker’s Bureau: MSD; Financial Interests, Personal, Speaker’s Bureau: Bristol Myers Squibb; Financial Interests, Personal, Speaker’s Bureau: Astellas; Financial Interests, Personal, Speaker’s Bureau: Bayer; Financial Interests, Personal, Speaker’s Bureau: EUSA Pharma. V. Moreno: Financial Interests, Personal, Speaker’s Bureau: Roche; Financial Interests, Personal, Speaker’s Bureau: Bayer; Financial Interests, Personal, Speaker’s Bureau: Bristol Myers Squibb; Financial Interests, Personal, Speaker’s Bureau: Janssen; Financial Interests, Personal, Speaker’s Bureau: Basilea; Financial Interests, Institutional, Other, Principal Investigator: AbbVie, AceaBio, Adaptimmune, ADC Therapeutics, Aduro, Agenus, Amcure, Amgen, Astellas, AstraZeneca; Financial Interests, Institutional, Other, Principal Investigator: Bayer Beigene BioInvent International AB, Bristol Myers Squibb, Boehringer, Boheringer, Boston; Financial Interests, Institutional, Other, Principal Investigator: Celgene, Daiichi Sankyo, DEBIOPHARM ,Eisai, e-Terapeutics, Exelisis, Forma Therapeutics, Genmab, GSK; Financial Interests, Institutional, Other, Principal Investigator: Harpoon, Hutchison, Immutep, Incyte, Inovio, Iovance, Janssen, Kyowa Kirin, Lilly, Loxo, MedSir, Menarini, Merck, Merus, Millennium, MSD; Financial Interests, Institutional, Other, Principal Investigator: Nanobiotix, Nektar, Novartis, Odonate Therapeutics, Pfizer, Pharma Mar, PharmaMar, Principia, PsiOxus, Puma, Regeneron, Rigontec, Roche, Sanofi; Financial Interests, Institutional, Other, Principal Investigator: Sierra Oncology, Sponsor, Synthon, Taiho, Takeda, Tesaro, Transgene, Turning Point Therapeutics, Upshersmith. C. Shepherd, D. Jones, M.A. Lakins, P. Hurley, S. Khambhatwala, D. Long, L. Kayitalire: Financial Interests, Personal, Other, Employee and shareholder in F-star Therapeutics Inc.: F-Star. All other authors have declared no conflicts of interest. BackgroundCheckpoint Inhibitor (CPI) therapy is standard of care in a range of tumor types but many patients fail to gain long term clinical benefit. Agonist antibodies targeting the T cell costimulatory receptor CD137 (4-1BB) have shown immune-stimulatory effects, but hepatotoxicity significantly hindered development. FS222, a novel tetravalent bispecific antibody, incorporates two CD137 binding sites in the Fc-region (Fcab) and anti-PD-L1 Fabs in a natural human IgG1 antibody with silenced FcyR activity. FS222’s affinity has been tuned specifically for each target; high affinity to PD-L1 and moderate affinity to CD137. This elicited PD-L1 dependent CD137 activation for a robust immune stimulation in the absence of toxicity, which resulted in strong antitumor activity in syngeneic models (Lakins et al). FS222’s unique format has the potential to deliver strong antitumor clinical activity with good tolerability. Checkpoint Inhibitor (CPI) therapy is standard of care in a range of tumor types but many patients fail to gain long term clinical benefit. Agonist antibodies targeting the T cell costimulatory receptor CD137 (4-1BB) have shown immune-stimulatory effects, but hepatotoxicity significantly hindered development. FS222, a novel tetravalent bispecific antibody, incorporates two CD137 binding sites in the Fc-region (Fcab) and anti-PD-L1 Fabs in a natural human IgG1 antibody with silenced FcyR activity. FS222’s affinity has been tuned specifically for each target; high affinity to PD-L1 and moderate affinity to CD137. This elicited PD-L1 dependent CD137 activation for a robust immune stimulation in the absence of toxicity, which resulted in strong antitumor activity in syngeneic models (Lakins et al). FS222’s unique format has the potential to deliver strong antitumor clinical activity with good tolerability. Trial DesignThis phase 1 trial is a multi-part (A and B) adaptive, dose escalation and expansion study in subjects with advanced malignancies (NCT04740424). FS222 is administered once every 4 weeks intravenously in 28-day treatment cycles. Primary objectives are to determine safety and tolerability and the recommended phase 2 dose (RP2D). Secondary objectives include determining pharmacokinetic (PK) and pharmacodynamic (PD) profiles, immunogenicity, and clinical response. Subjects with specific solid tumor types will be enrolled who have progressed on standard of care therapy. One line of prior CPI therapy is permitted. Part A consists of an accelerated dose titration (ADT) involving single-subject cohorts followed by a classical 3+3 design and dose expansion cohorts in selected tumor types to further characterize safety, PK/PD and clinical efficacy. Part B consists of tumor-specific efficacy expansion cohorts with Bayesian adaptive statistics. Translational studies will assess peripheral immune cell proliferation and activation, soluble peripheral PD biomarker levels and the correlation of FS222 exposure with selected PD markers of target engagement and response. Other exploratory translational endpoints are also included in the study. Study enrolment began December 2020. This phase 1 trial is a multi-part (A and B) adaptive, dose escalation and expansion study in subjects with advanced malignancies (NCT04740424). FS222 is administered once every 4 weeks intravenously in 28-day treatment cycles. Primary objectives are to determine safety and tolerability and the recommended phase 2 dose (RP2D). Secondary objectives include determining pharmacokinetic (PK) and pharmacodynamic (PD) profiles, immunogenicity, and clinical response. Subjects with specific solid tumor types will be enrolled who have progressed on standard of care therapy. One line of prior CPI therapy is permitted. Part A consists of an accelerated dose titration (ADT) involving single-subject cohorts followed by a classical 3+3 design and dose expansion cohorts in selected tumor types to further characterize safety, PK/PD and clinical efficacy. Part B consists of tumor-specific efficacy expansion cohorts with Bayesian adaptive statistics. Translational studies will assess peripheral immune cell proliferation and activation, soluble peripheral PD biomarker levels and the correlation of FS222 exposure with selected PD markers of target engagement and response. Other exploratory translational endpoints are also included in the study. Study enrolment began December 2020. Legal entity responsible for the studyF-star Therapeutics. F-star Therapeutics. FundingF-star Therapeutics. F-star Therapeutics.

PD-1/L1 immune checkpoint blockade shows durable responses and extends overall survival in a subset of cancer patients. Tumour Necrosis Factor Receptor (TNFR) activation is being tested clinically to improve patient responses. However, low affinity FcΓR-mediated crosslinking often limits monoclonal antibody (mAb) clinical efficacy, which is further restricted by adverse safety effects. The generation of a bispecific agonist of CD137, where potent agonist activity is conditional upon PD-L1 crosslinking, allows a greater therapeutic window. FS222, an anti-CD137/PD-L1 mAb2, was generated by introducing a CD137-binding specificity into the Fc-region of a human IgG1 mAb targeting PD-L1. A LALA mutation significantly reduces FcΓR binding. Binding characterisation was assessed by surface plasmon resonance (SPR) and cell binding, and in vitro activity measured in human primary T cell assays. A dose-range finding study in cynomolgus monkey was performed to investigate toxicity, pharmacokinetics (PK) and pharmacodynamics (PD). A murine surrogate molecule was generated and its anti-tumor activity and PK/PD was tested in multiple syngeneic mouse tumor models. FS222 binds to human PD-L1 with subnanomolar affinity. This PD-L1 binding is a prerequisite to subsequently enable highly potent CD137 agonism (low nM EC50 values in primary cell in vitro assays). We term this mechanism conditional agonism. In cynomolgus monkey FS222 has a half-life of ~150h and single and repeat dosing resulted in observable PD changes in lymphocytes and soluble receptor levels at low doses. Furthermore, no hepatotoxicity as defined by changes in clinical chemistry and histopathology was detected. A surrogate anti-mouse-mAb2 significantly reduced tumor growth in multiple syngeneic mouse tumor models. The observed dose-dependent tumor growth inhibition resulted in a significant survival benefit and was concomitant with increases in tumor and peripheral activated CD8+ T cells. FS222 is a conditional CD137/PD-L1 bispecific agonist antibody and has superior activity to control mAbs and relevant combinations in vitro assays. A favorable safety profile and immunopharmacology was observed in a cynomolgus dose-range finding study and with the surrogate molecule in multiple syngeneic mouse tumor models. Anti-CD137 agonistic mAbs in clinical development have so far shown limitations due to dose limiting toxicity or poor clinical activity. In preclinical tumor models anti-CD137/PD-L1 surrogate mAb2 treatment resulted in intra-tumoral and peripheral PD changes leading to an increase in CD8+ T cell proliferation. These changes were dose dependent and coincident with tumor growth inhibition. FS222 is a highly active bispecific molecule with a favorable safety profile with the potential to serve a significant unmet need in the immunotherapy of solid tumors.Citation Format: Matthew A. Lakins, Alexander Koers, Jose Munoz Olaya, Raffaella Giambalvo, Daniel Jones, Sarka Pechouckova, Emma Goodman, Sylwia Marshall, Mateusz Wydro, Cristian Gradinaru, Francisca Wollerton, Sarah Batey, Daniel Gliddon, Michael Davies, Michelle Morrow, Mihriban Tuna, Neil Brewis. FS222 mAb2, a bispecific conditional agonist antibody targeting CD137 and PD-L1, induces potent lymphocyte activation and has a favorable safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1540.

Background: PD-1/L1 axis blockade shows durable responses and extended overall survival across cancer types in a subset of patients. Tumour Necrosis Factor Receptor (TNFR) superfamily activation is also being tested clinically to improve patient responses. Current interventions using therapeutic CD137 agonists to activate T cells are limited by adverse safety effects and poor efficacy as monotherapies. The generation of a bispecific agonist of CD137 following PD-L1 crosslinking allows a greater therapeutic window with improved safety and efficacy. Methods: An anti-CD137/PD-L1 mAb2 was generated by introducing a CD137-binding specificity into the Fc-region of a human IgG1 targeting PD-L1 mAb. FcgR binding was decreased by introducing a LALA mutation. Binding characterisation was assessed and in vitro activity measured in a mouse primary OT-1 CD8+ T cell assay. The anti-tumour activity and PK/PD of anti-CD137/PD-L1 mAb2 was tested in mouse tumour models. Results: An anti-CD137/PD-L1 mAb2 was developed, which binds to mouse PD-L1 enabling CD137 agonism (in vitro EC50: 3 pM in primary antigen-specific OT-1 assay). The mAb2 significantly reduced tumour growth in 3 syngeneic mouse tumour models (MC38, CT26 and B16-F10) with dose-dependent inhibition seen in CT26, resulting in a significant survival benefit at concentrations of 0.3 mg/kg or above. This growth inhibition was coincident with increases in tumour and peripheral activated CD8 T cells. Liver pharmacology was minimal as defined by histopathology. Conclusions: We report intra-tumoural and peripheral PD changes leading to an increase in the proliferative CD8+ T cell response following dosing with an anti-CD137/PD-L1 bispecific mAb2. These changes were dose dependent and coincident with tumour growth inhibition. In in vitro T cell activation assays the bispecific was superior to control antibodies and relevant combinations. Minimal liver pharmacology and no toxicity was observed with the anti-CD137/PD-L1 mAb2 unlike with other monoclonal antibodies targeting CD137. This warrants the development of a first-in-class anti-human CD137/PD-L1 bispecific antibody with a novel mode of action and improved therapeutic index for the treatment of human cancer. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.