CD40 Agonist Research Articles

Combining CD40 agonist mitazalimab with mFOLFIRINOX in previously untreated metastatic pancreatic ductal adenocarcinoma (OPTIMIZE-1): a single-arm, multicentre phase 1b/2 study

Current systemic therapies for metastatic pancreatic ductal adenocarcinoma are associated with poor outcomes with a 5-year overall survival rate under 5%. We aimed to assess the safety and antitumour activity of mitazalimab, a human CD40 agonistic IgG1 antibody, with modified FOLFIRINOX (mFOLFIRINOX; fluorouracil, leucovorin, oxaliplatin, and irinotecan), in chemotherapy-naive patients with metastatic pancreatic ductal adenocarcinoma. OPTIMIZE-1 was a single-arm, multicentre, phase 1b/2 study which enrolled adults with histologically-confirmed metastatic pancreatic ductal adenocarcinoma and European Cooperative Oncology Group performance status 0 or 1 in 14 university hospitals in Belgium, France, and Spain. The primary endpoint of phase 1b was to determine the recommended phase 2 dose of intravenous mitazalimab (450 μg/kg or 900 μg/kg) when combined with intravenous mFOLFIRINOX (oxaliplatin 85 mg/m2, leucovorin 400 mg/m2, irinotecan 150 mg/m2, fluorouracil 2400 mg/m2). In the first 21-day treatment cycle, mitazalimab was administered on days 1 and 10, and mFOLFIRINOX on day 8. In subsequent 14-day cycles mitazalimab was administered 2 days after mFOLFIRINOX. The phase 2 primary endpoint was objective response rate. Activity and safety analyses were conducted on the full analysis set (all patients who received the combination of mitazalimab at the recommended phase 2 dose and mFOLFIRINOX for at least two treatment cycles) and safety set (all patients who received any study treatment), respectively. Enrolment is complete, and data represents a primary analysis of the ongoing trial. The trial is registered at Clinicaltrials.gov (NCT04888312). Between Sept 29, 2021, and March 28, 2023, 88 patients were screened and 70 patients were enrolled (40 [57%] were female and 30 [43%] were male). In phase 1b, 900 μg/kg mitazalimab was determined as the recommended phase 2 dose. Overall, five patients received 450 μg/kg mitazalimab; 65 received 900 μg/kg mitazalimab. No dose-limiting toxicities were observed at 450 μg/kg, and one dose-limiting toxicity was observed at 900 μg/kg. 57 patients were evaluated for activity, and all 70 patients were included in the safety set. At data cutoff on Nov 14, 2023, median follow-up was 12·7 months (95% CI 11·1-15·7). Of the 57 patients, 29 (51%) remained on study and 18 (32%) remained on treatment. The primary endpoint (objective response rate >30%) was met (objective response rates in 23 [40%]; one-sided 90% CI ≥32 of 57 patients). The most common grade 3 or worse adverse events were neutropenia (18 [26%] of 70 patients), hypokalaemia (11 patients [16%]), and anaemia and thrombocytopenia (eight patients [11%]). Serious adverse events were reported in 29 (41%) of 70 patients, the most common being vomiting (five [7%] of 70 patients), decreased appetite (four [6%]), and diarrhoea and cholangitis (three [4%] of 70 patients for each), none considered related to mitazalimab. No treatment-related deaths were reported. Mitazalimab with mFOLFIRINOX demonstrated manageable safety and encouraging activity, warranting continued development in a phase 3, randomised, controlled trial. The results from OPTIMIZE-1 pave the way for further exploration and confirmation of a novel immunotherapy treatment regimen for metastatic pancreatic ductal adenocarcinoma, which is a complex and aggressive cancer with very low survival rates and restricted treatment options. Alligator Bioscience.

OPTIMIZE-1 primary analysis: Safety, efficacy and biomarker results of a phase 1b/2 study combining CD40 agonist mitazalimab with mFOLFIRINOX in previously untreated metastatic pancreatic ductal adenocarcinoma (mPDAC).

4133 Background: With a 5 year overall survival (OS) rate <5%, PDAC is a leading cause of cancer related mortality. Currently available systemic therapies are not curative and new therapeutic options are needed. Mitazalimab is a human CD40 agonistic IgG1antibody that reduces immune suppression, sensitizes the tumor to chemotherapy, and induces long lasting anti-tumor T cell responses. OPTIMIZE-1 (NCT04888312) is a Phase 1b/2, open label, multicenter study assessing mitazalimab's safety and efficacy combined with mFOLFIRINOX (mFFX) in chemotherapy naïve mPDAC patients (pts). Methods: In the first 21 day cycle, mitazalimab was administered on day 1 and 10 and mFFX infusion started on day 8. In subsequent cycles, treatment followed a 14 day cycle with mitazalimab given 2 days after mFFX. The primary endpoint is objective response rate (ORR) compared to 30% ORR for FFX (Conroy, 2011) (80% power; α (1-sided) =0.10). Secondary and exploratory endpoints include Duration of Response (DoR), progression free survival (PFS), OS, safety, PK and PD biomarker assessments. Results: Seventy pts with mPDAC were treated with mFFX + mitazalimab (safety set: 5 at 450 µg/kg and 65 at 900 µg/kg). 57 patients at 900 µg/kg received ≥2 treatment cycles and were efficacy evaluable. The most common grade ≥3 AEs were neutropenia (25.7%), anemia (11.4%), hypokalemia (15.7%) and thrombocytopenia (11.4%), consistent with FFX safety profile. Two pts discontinued treatment due to AEs. Confirmed ORs were observed in 23 pts (40.4%), including 1 complete responder (CR). Median OS, PFS and duration of Response (DoR) were 14.3 months (mo), 7.4 mo and 12.5 mo respectively, with a median follow up of 12.7 mo. 29 pts (51%) remain in the study (32% on treatment, 19% in survival follow up). Efficacy results including correlation with detected KRAS G12 mutations are summarized (Table). Conclusions: Mitazalimab in combination with mFFX is a feasible regimen with a manageable safety profile. The primary endpoint was met; KRAS G12 V and R mutations were associated with better efficacy. Given the promising DoR linked with survival benefit in previously untreated mPDAC, these results merit continued development of mitazalimab in a confirmatory phase 3 study. Clinical trial information: NCT04888312 . [Table: see text]

A randomized phase 2 study of combination atezolizumab and varlilumab (CDX-1127) with or without addition of cobimetinib in previously treated unresectable biliary tract cancer.

4017 Background: Combined MEK inhibition (MEKi) and PD-L1 blockade significantly improved progression-free survival (PFS) versus PD-L1 monotherapy in patients with advanced biliary tract cancer (BTC) following first-line chemotherapy (NCT03201458). Further study has shown MEKi enhances tumor immunogenicity but impairs host T cell activation/priming. The addition of immune agonists, e.g. a CD27 agonist, can rescue T cell function in the setting of systemic MEKi in vivo and may be an effective strategy to optimize MEKi’s immunomodulatory potential when used in combination with checkpoint blockade. Methods: We conducted a randomized ph 2 trial (NCT04941287) evaluating Atezolizumab in combination with the CD27 immune agonist (CDX-1127 [Varlilumab]) with or without the addition of a MEKi (Cobimetinib) in patients with unresectable, previously treated, BTC. Adults with pathologically-confirmed BTC, s/p at least 1 prior line (no more than 2) of systemic therapy, measurable disease, and ECOG PS ≤1 were enrolled. Patients who had received previous PD-[L]1 were eligible. The study was planned for 64 evaluable subjects randomized in a 1:1 ratio, stratified by BTC site, to either AV (Atezolizumab [840mg IV days 1,15])+ Varlilumab [3mg/kg IV days 1, 15]) or CAV (Cobimetinib [60mg oral daily days 1-21, off 22-28] + Atezolizumab + Varlilumab). Overall response rate (ORR) and PFS were co-primary endpoints. The primary correlative outcome was treatment-related changes in CD8+ infiltrating T cells. Results: A preplanned interim analysis based on objective response rates did not meet the threshold for continuance in either treatment arm, so the trial was closed early for futility. In total, 57 patients were enrolled (n = 29 [CAV], n = 28 [AV], 67% had intrahepatic cholangiocarcinoma, and 33% had received previous PD-[L]1. Both CAV and AV regimens were well-tolerated without new safety signals. Adding CDX-1127 did not appear to meaningful increase immunotoxicity beyond established PD-L1 toxicity profiles. ORR was 0% (CAV) and 4% (AV). With a median follow up time of 6.2 months, median PFS was 2.2 (CAV) and 1.8 (AV) months (HR 0.71 [0.40,1.25]). In PD[L]1-experienced patients, median PFS was 3.6 (CAV) and 1.7 (AV) months (HR 0.44 [0.14,1.33]). Median OS was 10.2 (CAV) and 6.1 (AV) months (HR 0.76 [0.37,1.55]). In PD[L]1-experienced patients, median OS was 6.4 (CAV) and 4.4 (AV) months for CAV and AV (HR 0.68 [0.19,2.42]). Primary correlative endpoint and updated survival will be reported at time of meeting. Conclusions: The combination of Atezolizumab and Varlilumab with or without Cobimetinib was safe but did not improve clinical outcomes in advanced stage BTC patients treated in later lines. Though not statistically significant, treatment with CAV demonstrated numerically favorable PFS/OS compared AV among immunotherapy-experienced patients. Clinical trial information: NCT03201458 .

First-in-human study (FIH) of FS222, a next-generation tetravalent PD-L1/CD137 bispecific antibody: Safety, pharmacodynamics (PD), and antitumor activity in patients (pts) with advanced solid tumors including PD-1 refractory melanoma.

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 .

Phase 1 study of CTX-471, a novel CD137 agonist antibody, in patients with progressive disease following PD-1/PD-L1 inhibitors in metastatic or locally advanced malignancies.

Phase 1 study of CTX-471, a novel CD137 agonist antibody, in patients with progressive disease following PD-1/PD-L1 inhibitors in metastatic or locally advanced malignancies.

280MO CD40 agonist mitazalimab combined with mFOLFIRINOX (mFFX) in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC): Primary analysis of the OPTIMIZE-1 phase Ib/II study

280MO CD40 agonist mitazalimab combined with mFOLFIRINOX (mFFX) in patients with metastatic pancreatic ductal adenocarcinoma (mPDAC): Primary analysis of the OPTIMIZE-1 phase Ib/II study

Reversible Chemical Modification of Antibody Effector Function Mitigates Unwanted Systemic Immune Activation.

Antibody effector functions including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP) are mediated through the interaction of the antibody Fc region with Fcγ receptors present on immune cells. Several approaches have been used to modulate antibody Fc-Fcγ interactions with the goal of driving an effective antitumor immune response, including Fc point mutations and glycan modifications. However, robust antibody-Fcγ engagement and immune cell binding of Fc-enhanced antibodies in the periphery can lead to the unwanted induction of systemic cytokine release and other dose-limiting infusion-related reactions. Creating a balance between effective engagement of Fcγ receptors that can induce antitumor activity without incurring systemic immune activation is an ongoing challenge in the field of antibody and immuno-oncology therapeutics. Herein, we describe a method for the reversible chemical modulation of antibody-Fcγ interactions using simple poly(ethylene glycol) (PEG) linkers conjugated to antibody interchain disulfides with maleimide attachments. This method enables dosing of a therapeutic with muted Fcγ engagement that is restored in vivo in a time-dependent manner. The technology was applied to an effector function enhanced agonist CD40 antibody, SEA-CD40, and experiments demonstrate significant reductions in Fc-induced immune activation in vitro and in mice and nonhuman primates despite showing retained efficacy and improved pharmacokinetics compared to the parent antibody. We foresee that this simple, modular system can be rapidly applied to antibodies that suffer from systemic immune activation due to peripheral FcγR binding immediately upon infusion.

A spatial architecture-embedding HLA signature to predict clinical response to immunotherapy in renal cell carcinoma.

An important challenge in the real-world management of patients with advanced clear-cell renal cell carcinoma (aRCC) is determining who might benefit from immune checkpoint blockade (ICB). Here we performed a comprehensive multiomics mapping of aRCC in the context of ICB treatment, involving discovery analyses in a real-world data cohort followed by validation in independent cohorts. We cross-connected bulk-tumor transcriptomes across >1,000 patients with validations at single-cell and spatial resolutions, revealing a patient-specific crosstalk between proinflammatory tumor-associated macrophages and (pre-)exhausted CD8+ T cells that was distinguished by a human leukocyte antigen repertoire with higher preference for tumoral neoantigens. A cross-omics machine learning pipeline helped derive a new tumor transcriptomic footprint of neoantigen-favoring human leukocyte antigen alleles. This machine learning signature correlated with positive outcome following ICB treatment in both real-world data and independent clinical cohorts. In experiments using the RENCA-tumor mouse model, CD40 agonism combined with PD1 blockade potentiated both proinflammatory tumor-associated macrophages and CD8+ T cells, thereby achieving maximal antitumor efficacy relative to other tested regimens. Thus, we present a new multiomics and spatial map of the immune-community architecture that drives ICB response in patients with aRCC.

Abstract PO1-19-08: Phase I trial of pegylated liposomal doxorubicin chemotherapy in combination with CD40 agonist and Flt3 ligand in metastatic triple-negative breast cancer

Abstract Background: Only a subset of patients with metastatic triple-negative breast cancers (TNBC) demonstrate response to FDA approved PD-1 immune checkpoint blockade (ICB), and few have durable responses. Data suggests that breast cancers have defects in antigen presentation and that antigen presenting cells especially the cDC1 subtype of dendritic cells (DCs) are required for response to ICB. CD40 agonists activate antigen presenting cells including DCs and B cells and repolarize macrophages to an anti-tumor phenotype. Flt3 ligand is a growth factor that increases differentiation and expansion of DCs. We recently demonstrated in pre-clinical TNBC models that the combination of liposomal doxorubicin chemotherapy, a CD40 agonist, and a Flt3 ligand improves outcomes compared to alternate combinations. Methods: This is a single arm phase I pilot study of liposomal doxorubicin, CDX-1140 (CD40 agonist monoclonal antibody), and CDX-301 (recombinant Flt3 ligand) combination therapy in patients with metastatic or unresectable locally advanced metastatic TNBC. Patients will be randomized to 3 lead-in arms (triplet therapy, doublet immunotherapy only, or liposomal doxorubicin only) for one cycle prior to receiving triplet therapy with tissue biopsies done before and after the lead-in treatment. CDX-301 will be given for only two cycles; liposomal-doxorubicin and CDX-1140 will be continued until disease progression or clinically limiting toxicities. Primary endpoint is determination of a recommended phase II dose based on treatment-related adverse events and dose-limiting toxicities. Secondary endpoints include anti-tumor immune response after triplet therapy, after immunotherapy alone, and after liposomal doxorubicin alone; median progression-free survival, overall response rate, duration of response, and clinical benefit rate. Key eligibility criteria are unresectable stage III or stage IV TNBC (ER ≤10%, PR ≤10%, HER2/neu negative), 1st to 3rd line treatment for metastatic disease (1st line patients need to be PD-L1 negative by 22C3 assay), measurable disease by RECIST 1.1 criteria, consent for pre-treatment and on-treatment biopsies of amenable soft tissue tumor lesions, no prior treatment with an anti-CD40 antibody or a Flt3 ligand, no anthracycline treatment in the metastatic setting, no prior progression while on anthracycline-based therapy or within 6 months of completing neoadjuvant chemotherapy, and no history of non-infectious pneumonitis or current pneumonitis. This trial will enroll up to 45 patients across multiple sites (NCT05029999) and is currently open at University of Texas Southwestern Medical Center, Texas Oncology, University of Chicago, University of Texas San Antonio, Sarah Cannon Research Institute, and Johns Hopkins. Citation Format: Sangeetha Reddy, Joyce O'Shaughnessy, Navid Sadeghi, Samira Syed, Cesar Santa-Maria, Virginia Kaklamani, Nan Chen, Denise Yardley, Yisheng Fang, Isaac Chan, Nisha Unni, Sarah Kashanian, Namrata Peswani, Shahbano Shakeel, Meredith Carter, Kelly Kyle, Farjana Fattah, Chul Ahn, Ina Patel, Joshua Gruber, Dawn Klemow, Glenda Delgado, Nicole Sinclair, Michael Yellin, Heather McArthur, Rita Nanda, Suzanne Conzen, Carlos Arteaga. Phase I trial of pegylated liposomal doxorubicin chemotherapy in combination with CD40 agonist and Flt3 ligand in metastatic triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO1-19-08.

Abstract LT08: Extracellular Vesicle Surface Display Enhances the Therapeutic Efficacy and Safety Profile of Cancer Immunotherapy

Abstract As our understanding of the complex interactions between the immune system and cancer has progressed, so has the identification of novel targets and ligands that can modulate effective anti-cancer immune responses. While immunotherapy has emerged as a mainstay in cancer therapy, its efficacy has been limited by the potential for off-target toxicity against normal body cells. In this study, we present a nanoparticle-based delivery system that enhances the signaling efficacy of immunomodulatory ligands while concurrently limiting systemic toxicity. We demonstrate that extracellular vesicles (EVs), lipid bilayer enclosed nanoparticles released by cells, can be efficiently engineered to display multiple immunomodulatory ligands on their surface. Display of immunomodulatory ligands such as agonistic CD137 and CD40 antibodies on the EV surface significantly enhanced their therapeutic efficacy over equivalent doses of free ligands by inducing ligand multimerization, efficient receptor crosslinking and the formation of immune synapses. EVs conjugated with a complementary combination of immune stimulatory ligands and immune checkpoint inhibitors were able to shift the tumor immune milieu towards an anti-tumorigenic phenotype in a lung metastatic melanoma allograft and suppress tumor progression to a greater extent than an equivalent dose of free ligands. We also demonstrate the ability of EVs to co-deliver intraluminally loaded TLR agonists which synergize with surface displayed agonistic CD40 antibodies to induce effective anti-tumor responses and tumor regression in a metastatic autochthonous cell-derived model of PDAC. Remarkably, we also observed that EV-associated immunotherapeutics were limited in their biodistribution to the tumor microenvironment, resulting in significantly lower off-target toxicity than treatment with free ligands. In summary, we present an EV-based delivery approach that allows the use of potent cancer immunotherapy combinations to achieve superior anti-tumor responses at significantly lower doses with less side-effects than is possible with conventional administration methods for cancer immunotherapy. Citation Format: Migara Kavishka Jayasinghe, Dawn Liu Xiao Tian, Yock Sin Lay, Chang Yu Lee, Brendon Zhi Jie Yeo, Dong Van Hoang, Rebecca Carissa Prajogo, Boya Peng, Thach Tuan Pham, Minh Thi Nguyet Le. Extracellular Vesicle Surface Display Enhances the Therapeutic Efficacy and Safety Profile of Cancer Immunotherapy [abstract]. In: Proceedings of Frontiers in Cancer Science; 2023 Nov 6-8; Singapore. Philadelphia (PA): AACR; Cancer Res 2024;84(8_Suppl):Abstract nr LT08.

CB307: A Dual Targeting Costimulatory Humabody VH Therapeutic for Treating PSMA-Positive Tumors.

CD137 is a T- and NK-cell costimulatory receptor involved in consolidating immunologic responses. The potent CD137 agonist urelumab has shown clinical promise as a cancer immunotherapeutic but development has been hampered by on-target off-tumor toxicities. A CD137 agonist targeted to the prostate-specific membrane antigen (PSMA), frequently and highly expressed on castration-resistant metastatic prostate cancer (mCRPC) tumor cells, could bring effective immunotherapy to this immunologically challenging to address disease. We designed and manufactured CB307, a novel half-life extended bispecific costimulatory Humabody VH therapeutic to elicit CD137 agonism exclusively in a PSMA-high tumor microenvironment (TME). The functional activity of CB307 was assessed in cell-based assays and in syngeneic mouse antitumor pharmacology studies. Nonclinical toxicology and toxicokinetic properties of CB307 were assessed in a good laboratory practice (GLP) compliant study in cynomolgus macaques. CB307 provides effective CD137 agonism in a PSMA-dependent manner, with antitumor activity both in vitro and in vivo, and additional activity when combined with checkpoint inhibitors. A validated novel PSMA/CD137 IHC assay demonstrated a higher prevalence of CD137-positive cells in the PSMA-expressing human mCRPC TME with respect to primary lesions. CB307 did not show substantial toxicity in nonhuman primates and exhibited a plasma half-life supporting weekly clinical administration. CB307 is a first-in-class immunotherapeutic that triggers potent PSMA-dependent T-cell activation, thereby alleviating toxicologic concerns against unrestricted CD137 agonism.

Abstract CT192: A first-in-human phase 1 study of TQB2916, a novel CD40 agonist antibody for advanced malignancies

Abstract Background: CD40 receptor is a member of the tumor necrosis factor receptor family that plays a prominent role in immune responses. TQB2916, a humanized IgG2 CD40 monoclonal antibody, has demonstrated CD40 agonistic activity and promising anti-tumor activity in preclinical studies. A first-in-human study of TQB2916 single agent in advanced solid tumors and lymphomas is ongoing (NCT05213767). Methods: Patients with an ECOG performance status of 0 or 1, adequate hematologic and organ function and refractory to standard therapies met inclusion criteria. TQB2916 was given intravenously every 3 weeks until progression or unacceptable toxicity. Bayesian optimal interval (BOIN) design was used to determine the maximum tolerated dose (MTD) and recommended Phase 2 dose (RP2D). The objective is to assess the safety, tolerability, pharmacokinetics, and preliminary anti-tumor activity of TQB2916. Results: From April 2022 to November 2023, 18 patients with solid tumors and 2 with lymphoma have been treated with TQB2916 monotherapy, and were assigned to 7 cohorts (0.5 mg n=1, 2.5 mg n=1, 12.5 mg n=3, 60 mg n=3, 200 mg n=7, 300 mg n=2, and 400 mg n=3). Three dose limiting toxicities (DLTs) were observed: one with grade 3 pneumonia and one with grade 4 lipase/amylase increase in the 400 mg cohort; one with grade 3 pancreatitis in the 300 mg cohort. All patients experienced at least one treatment-related adverse event (TRAE). The most common TRAEs were lipase increase (n=15, 75%), amylase increase (n=14, 70%), lymphocyte count decrease (n=13, 65%), alanine aminotransferase (ALT) increase (n=12, 60%), alkaline phosphatase (ALP) increase (n=12, 60%), aspartate aminotransferase (AST) increase (n=11, 55%), hypoalbuminemia (n=11, 55%), and anorexia (n=11, 55%). Serious TRAEs occurred in 5 (25%) patients. Most of TRAEs were of grade 1 or grade 2 and manageable. Sixteen patients were evaluated for efficacy, and 3 of them achieved stable disease as per immune response evaluation criteria in solid tumors (iRECIST) and lymphoma response to immunomodulatory therapy criteria (LYRIC). The pharmacokinetics were well-behaved with increasing exposure dose-proportionally and no accumulation occurred after repeated dosing. The Cmax, AUC(0-t) and T1/2 of 400 mg single dosing were 109±21.8 μg/ml, 611±228 d*µg/mL and 5.54±3.37 d. Dose-dependent occupancy of CD40 was detected of 0.5 mg and above doses. Reduction in peripheral B cells and increasing in cytokines secretion were also observed. Conclusions: Pharmacodynamics analysis suggested that TQB2916 treatment achieved CD40 occupancy and immune activation with the changes in cytokines. Considering the safety profile and efficacy, 200 mg was determined as the preliminary expansion dose as it was well tolerated. Studies of TQB2916 in combination with immune checkpoint inhibitors and/or other anti-cancer therapies are ongoing. Citation Format: Yi Ba, Huilai Zhang, Ting Deng, Tao Ning, Qian Fan. A first-in-human phase 1 study of TQB2916, a novel CD40 agonist antibody for advanced malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT192.

Nanofluidic delivery implant sustains localization and maximizes efficacy of intratumoral immunotherapy

Intratumoral immunotherapy holds promise for improving cancer treatment efficacy. However, rapid clearance from the tumor upon bolus volume injections limits efficacy and impedes assessment of dose-dependent effects on the tumor immune microenvironment (TIME). To this end, we developed a drug-agnostic nanofluidic implant to deliver immunotherapy within the tumor, providing a mechanism for sustained and controlled intratumoral dosing. Diffusive drug elution from the implant reservoir is controlled by a nanofluidic membrane, which abrogates rapid drug elimination from the tumor and maximizes immunotherapy localization. Here we first achieve in vitro sustained release of agonist CD40 monoclonal antibody (mAb), anti-programmed death ligand-1 (PDL1) mAb, and immune-cell targeted polymeric prodrugs of Resiquimod (toll-like receptor 7/8,TLR 7/8) and a small molecule STING agonist. We then demonstrated in vivo sustained intratumoral drug localization of agonist CD40 Ab and anti-PDL1 Ab in the 4T1 murine model of triple-negative breast cancer (TNBC). Further, we show highly effective anti-tumor efficacy with radiation therapy and sustained intratumoral co-delivery of agonist CD40 Ab and anti-PDL1 Ab in the EMT6 TNBC murine model. Finally, we demonstrate the versatility of this implant by showing that sustained intratumoral delivery of the STING or Resiquimod polymeric prodrugs strongly inhibits 4T1 tumor growth. Collectively, these results support our nanofluidic system as a therapeutic platform technology to investigate sustained intratumoral dosing of immunotherapy.

Abstract 2366: Balancing efficacy and toxicity of a fibroblast activation protein dependent tetravalent CD40 agonist antibody for cancer therapy

Abstract Background: CD40 agonist antibodies have been found to augment tumor antigen presentation and have shown potential as anti-tumor treatments in human clinical trials. The clinical success of CD40 agonist antibodies has been restricted due to their limited efficacy and on-target, off-tumor toxicity. We hypothesized that toxicity associated with CD40 agonist antibodies could be overcome by activating CD40 specifically through tumor-specific antigens. Additionally, limited efficacy can be improved by rational design of CD40 bispecific antibodies (BsAbs). Herein, we present FAPxCD40, a trivalent CD40 agonist BsAb targeting fibroblast activation protein (FAP) possessing an optimized therapeutic window. Method: We generated a series of FAPxCD40 bispecific antibodies with 2, 4, and 6 CD40 valences by using a high-affinity anti-FAP antibody and a crosslink-dependent CD40 agonist VHH antibody. These BsAbs were evaluated using multiple immune cell functional assays, including assays for dendritic cell (DC) maturation, B cell maturation, and CMV-reactive T cell recall. The in vivo therapeutic window of the FAPxCD40 bispecifics was compared in CD40 humanized mice with subcutaneously transplanted FAP-positive MC38 tumor xenografts. Results: We developed FAPxCD40 BsAbs with varying valences, including bivalent FAPxCD40-2, tetravalent FAPxCD40-4, and hexavalent FAPxCD40-6. FAPxCD40-2 showed strict FAP-dependent CD40 agonist activity in both DC and B cell maturation assays, while its CD40 agonist activity upon FAP-crosslinking was modest. FAPxCD40-4 exhibited efficient CD40 agonist activity upon FAP-crosslinking, but however, it also exhibited some undesired FAP-independent CD40 agonist activity. FAPxCD40-6 efficiently clustered CD40 but marginally benefited from FAP binding, making it unsuitable as a choice. In the humanized CD40 mice model, FAPxCD40-2 effectively eliminated systemic toxicity at all tested doses. Its antitumor activity, however, is modest with TGI (tumor growth inhibition) rate of 32.8 % at a dose of 6.96 mpk. Further escalation of the FAPxCD40-2 dosage to 13.92 mpk did not result in enhanced antitumor activity with TGI rate of 46.9 %, indicating that its CD40 agonist activity may have reached a plateau. In contrast, FAPxCD40-4 broke this plateau and induce a promising antitumor response at a dose of 1.3 mpk, resulting in a 92% TGI rate. Furthermore, no liver enzyme upregulation or platelet count decrease was observed under these conditions, indicating an ideal therapeutic window. In addition, during nonclinical toxicology studies conducted on cynomolgus monkeys, it was found that doses of FAPxCD40-4 up to 99 mg/kg administered weekly for a total of four doses were well-tolerated. Conclusion: Our study demonstrates that FAPxCD40-4, a tetravalent FAPxCD40 BsAb, exhibits efficient CD40 agonist activity with an optimized therapeutic window. Citation Format: Simeng Chen, Yuan Lin, Xiaoru Zhou, Xing Sun, Changyong Yang, Cheng Liao. Balancing efficacy and toxicity of a fibroblast activation protein dependent tetravalent CD40 agonist antibody for cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2366.

Abstract 5314: Improved breast cancer tumor control with addition of CD40 agonist and Flt3 ligand to pegylated liposomal doxorubicin is only partially mediated by CD8 T cells

Abstract Background: There is a need for improved immunotherapy strategies beyond current FDA approved treatments for triple negative breast cancer. Defective antigen presentation has been shown to play a contributory role in deficient anti-tumor immunity in breast cancers. Flt3 ligand (Flt3L) is a growth factor that increases differentiation into DC1 dendritic cells. CD40 agonist (CD40a) activates all 3 classes of antigen presenting cells - DCs, B cells, and macrophages. Synergy with chemotherapy and CD40a has been seen in other cancers but not explored in breast cancers. Methods: C57BL/6 mice (aged 6-8 weeks) were injected with 150,000 E0771 breast cancer cells within the mammary fat pad. Tumors were allowed to grow to a volume of ~50 mm3 before randomization into treatment groups. Mice were treated with triplet therapy of pegylated liposomal doxorubicin (PLD) via tail vein followed by Flt3L intraperitoneal (IP) daily for 5 days and CD40a IP 1 week later as well as monotherapy and doublet therapy controls. Tumor growth and survival were monitored throughout the experiment. Tumors were harvested for single cell RNA sequencing (scRNAseq) and immunohistochemistry. To further study subset specific effects on efficacy of the immunotherapy, CD8 and CD4 T-cells were systemically depleted with anti-CD8 and anti-CD4 antibodies administered IP every 4 days starting 2 days before PLD treatment. Results: Triplet therapy led to improved tumor control compared to no treatment, monotherapy, and doublet controls (p<0.0001). Increased tumor infiltration with CD8 T cells was observed with triplet therapy via immunohistochemistry. scRNAseq showed increased cDC1 subset with Flt3L treatment. Contrastingly, CD40a decreased DC subsets likely due to activation and emigration out of the tumor, remodeled macrophage populations, and decreased T regulatory cells. CD8 depletion led to faster tumor growth whereas CD4 depletion reduced tumor growth. Triplet therapy maintained efficacy over PLD with both CD8 and CD4 depletion but showed reduced magnitude of benefit with CD8 T cell depletion suggesting a partial CD8 mediated process. CD4 T cell depletion did not hinder triplet therapy efficacy compared to PLD but depletion further enhanced tumor control in combination with triplet therapy, with 20% (2/10) of animals achieving complete regression. Conclusion: Triplet therapy with PLD, Flt3L, and CD40a improved tumor control and survival in syngeneic E0771 breast cancer mouse model. Added benefit of immunotherapy to PLD is partially CD8 T cell mediated. CD4 depletion further enhances tumor control in combination with triplet therapy. Experiments to explore the interactive effect of triplet therapy with other pertinent immune subsets are ongoing. A clinical trial with this combination in metastatic triple negative breast cancer patients is open and recruiting (NCT05029999). Citation Format: Preteesh L. Mylabathula, Shruthi Nooka, Anas Alkhani, James Zhu, Ashley Vu, Riyasat Ali, Varshini Ramasamy Balasubramanian, Isaac Chan, Sangeetha M. Reddy. Improved breast cancer tumor control with addition of CD40 agonist and Flt3 ligand to pegylated liposomal doxorubicin is only partially mediated by CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5314.

Abstract 5311: KK2269, an epithelial cell adhesion molecule-targeted CD40 agonist, stimulates anti-tumor immunity resulting in sustained anti-tumor effect against mouse intrahepatic tumor without hepatotoxicity

Abstract Several anti-CD40 agonist antibodies have been evaluated in clinical trials based on their preclinical antitumor activity via generation of antitumor innate and adaptive immunity through activation of antigen-presenting cells. However, conventional monospecific anti-CD40 agonist antibodies have faced limitations in terms of safety margins and clinical efficacy. To address this problem, we developed KK2269, a novel bispecific antibody (REGULGENT™ format) that binds to both CD40 and epithelial cell adhesion molecule (EpCAM), which is highly expressed in several types of tumors. KK2269 demonstrated EpCAM-dependent activation of CD40 signals both in vitro and in vivo, confirming that it is a tumor-targeting CD40 agonist. Liver metastasis is common in EpCAM-positive cancers, including colon, stomach, and lung cancers. Intrahepatic tumors can attenuate the efficacy of immune checkpoint inhibitors due to the liver tumor-specific suppressive immune milieu. Although CD40 agonists are expected to overcome the immunosuppressive environment, non-tumor-targeting CD40 agonists cause hepatotoxicity. In the present study, we used a syngeneic intrahepatic tumor model to examine whether KK2269 exhibits antitumor efficacy without causing hepatotoxicity. Firstly, we confirmed that KK2269 administration did not increase aspartate aminotransferase (AST) or cause pathological abnormalities in the liver despite Il12b induction in the EpCAM-positive tumor. Additionally, gene expression analysis of peritumoral normal liver revealed that KK2269 did not induce inflammatory cytokines. In contrast, a conventional anti-CD40 agonist antibody increased AST, caused pathological abnormalities, and upregulated inflammatory cytokines in peritumoral normal liver, as reported in the clinical setting. Secondly, the antitumor effect of KK2269 in combination with docetaxel was evaluated. The combination therapy synergistically reduced intrahepatic tumor volume, and the effect persisted even 21 days after the administration. Gene expression analysis revealed a lower immune infiltration in the intrahepatic tumor versus subcutaneous tumor and that KK2269 in combination with docetaxel increased T-cell infiltration and activation even in the immunosuppressive intrahepatic tumor. In summary, our study demonstrated that KK2269 activated dendritic cells in an EpCAM-positive tumor in the liver but did not affect peritumoral normal liver tissues. In addition, KK2269 in combination with docetaxel showed potent antitumor effect accompanied by T-cell infiltration and activation against the intrahepatic tumor, which has a lower immune infiltration. These results suggest that the combination therapy of KK2269 and docetaxel is promising for metastatic EpCAM-positive tumors. Citation Format: Yoshiki Sumitomo, Kyoko Kawasaki, Yuta Tezuka, Masato Saito, Akari Yao, Munetoshi Ando. KK2269, an epithelial cell adhesion molecule-targeted CD40 agonist, stimulates anti-tumor immunity resulting in sustained anti-tumor effect against mouse intrahepatic tumor without hepatotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5311.

Abstract 5308: Ifngr1 signaling on tumor-specific CD4 T cells is required to mitigate Treg differentiation during CD40 agonist and anti-PD-L1 immunotherapy in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDA) is a particularly lethal malignancy that lacks effective therapeutic strategies. A promising therapeutic modality that can elicit transient antitumor responses in a subset of metastatic PDAs is chemotherapy + CD40 agonist +/- anti-PD-1. A deeper understanding of the cellular mechanisms that mediate the initial responses will inform new strategies to enhance the durability of this therapy. Our prior studies demonstrated that tumor-specific CD4 T cells are critical for mitigating tumor growth in a syngeneic KPC orthotopic tumor model of PDA that expresses a model neoantigen, click beetle red luciferase (CBR). CD4 T cells direct tumor antigen recognition by tumor-associated macrophages and host interferon-gamma receptor (Ifngr1) signaling. Here, we demonstrate that CD4 T cells are required for the antitumor effects of anti-CD40 agonist indicating that this therapy fails to compensate for a lack of CD4 T cell help. We sought to test the hypothesis that host Ifngr1 signaling acts in a CD4 T cell intrinsic manner to promote antitumor responses. As such, we develop and validate a novel MHC class II tetramer (CBR54-62:I-Ab) to track the fate of endogenous CD4 T cells during tumor growth. We demonstrate that tumor-specific CD4 T cell abundance and Th1 polarization is decreased in orthotopic tumors, particularly between day 14 and day 21 post tumor implantation. Combination agonistic anti-CD40 + anti-PD-L1 maintained tumor-specific CD4 T cell numbers in the spleen and pancreas-draining lymph nodes of tumor-bearing mice. Intriguingly, Th1 polarized tumor-infiltrating CD4 T cells were decreased by aPD-L1, but were restored by anti-CD40, a finding further corroborated by single-cell RNA sequencing. Further, agonistic anti-CD40 + anti-PD-L1 alone or in combination robustly decreased Treg polarized tumor-specific CD4 T cells. Using a mixed bone marrow chimeric approach, we show immunotherapy’s Treg-depletion effect is dependent on Ifngr1 on antigen-specific CD4 T cells. In contrast, Ifngr1 signaling on tumor-specific CD8 T cells appeared inconsequential to CD8 T cell fate following immunotherapy. To our knowledge, this is the first study to interrogate tumor-specific CD4 T cells in PDA and uncover novel interplay between CD4 T cells, CD8 T cells, and myeloid cells that are critical for immunotherapy response. Citation Format: Eduardo Cruz-Hinojoza, Adam Burrack, Zoe Schmiechen, Cara-Lin Lonetree, Ebony A. Miller, Michael Patterson, Jesse Williams, Thamotharampillai Dileepan, Ingunn Stromnes. Ifngr1 signaling on tumor-specific CD4 T cells is required to mitigate Treg differentiation during CD40 agonist and anti-PD-L1 immunotherapy in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5308.

Abstract 5316: SM2256: A bispecific conditional CD40 and 4-1BB dual agonist for enhanced solid tumor immune response

Abstract Background: Monoclonal antibodies targeting the co-stimulatory receptors CD137 (4-1BB) and CD40 have displayed encouraging activity against a wide range of solid tumors in preclinical animal models. However, their clinical development as monotherapy has been hindered by dose-related liver toxicity, leading to limited efficacy at safe doses. To address this challenge and bolster tumor-specific immunity by orchestrating the interaction between dendritic cells (DCs) and cytotoxic T cells, we introduce SM2256 - a bispecific conditional CD40 and 4-1BB dual agonist. SM2256 physically bridges DCs and T cells, enhancing T cell priming and reactivation. In preclinical animal studies, SM2256 exhibited both excellent tolerability and potent anti-tumor activity. Methods: SM2256 was engineered by fusing high-affinity, non-ligand-blocking VHH domains targeting 4-1BB and CD40 to a silenced IgG1 Fc backbone. This fusion resulted in a tetravalent bispecific antibody capable of conditionally activating CD40 and 4-1BB upon mutual receptor binding. We assessed the binding specificity and affinity of SM2256 to human CD40 and 4-1BB using ELISA and flow cytometry. The target-dependent activation of CD40 and 4-1BB was confirmed via reporter assays and co-culture systems using primary DCs and T cells. In vivo safety and anti-tumor efficacy were evaluated in a syngeneic transplantation model using the MC38 cancer cell line in B-h41BB/B-hCD40 double humanized mice. Results: SM2256 exhibited sub-nanomolar binding affinity to recombinant human and cynomolgus monkey CD40 and 4-1BB, activating 4-1BB-NFκB-luc and CD40-NFκB-luc reporter cell lines at picomolar concentrations in a strictly target-dependent manner. Furthermore, SM2256 physically facilitated the interaction between DCs and T cells, leading to increased expression of DC co-stimulatory ligands and elevated secretion of cytokines by T cells in mixed lymphocyte cultures. In a syngeneic tumor transplantation model, SM2256 significantly restrained the growth of MC38 tumors in B-h41BB/B-hCD40 double humanized mice. Conclusions: SM2256 is a novel conditional CD40 and 4-1BB dual agonist that demonstrates promising preclinical efficacy against solid tumors while boasting an improved safety profile when compared to existing monoclonal antibodies. Citation Format: Wenjing Song, Simin Yang, Yi Wei, Shihao Lu, Huiqin Geng, Xiaodan Liu, Hong Zhou, Siyao Xie, Xing Zhang, Sheila Zhou, Yanbin Liang. SM2256: A bispecific conditional CD40 and 4-1BB dual agonist for enhanced solid tumor immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5316.

Abstract 5313: CB307: A dual targeting costimulatory Humabody® VH therapeutic for treating PSMA-positive tumors

Abstract INTRODUCTION: CD137 is a T and NK cell costimulatory receptor involved in consolidating immunological responses. The potent CD137 agonist urelumab has shown clinical promise as a cancer immunotherapeutic but development has been hampered by on-target off-tumour toxicities. A CD137 agonist targeted to the prostate-specific membrane antigen (PSMA), frequently and highly expressed on castration-resistant metastatic prostate cancer (mCRPC) tumour cells, could bring effective immunotherapy to this immunologically challenging to address disease. EXPERIMENTAL PROCEDURES: We designed and manufactured CB307, a novel half-life extended bispecific costimulatory Humabody® VH therapeutic to elicit CD137 agonism exclusively in a PSMA-high tumour microenvironment (TME). The functional activity of CB307 was assessed in cell-based assays and in syngeneic mouse anti-tumour pharmacology studies. Non-clinical toxicology and toxicokinetic properties of CB307 were assessed in a good laboratory practice (GLP) compliant study in cynomolgus macaques. RESULTS: CB307 provides effective CD137 agonism in a PSMA-dependent manner, with anti-tumour activity both in vitro and in vivo, and additional activity when combined with checkpoint inhibitors. A validated novel PSMA/CD137 immunohistochemistry (IHC) assay demonstrated a higher prevalence of CD137-postive cells in the PSMA-expressing human mCRPC TME with respect to primary lesions. CB307 did not show substantial toxicity in non-human primates and exhibited a plasma half-life supporting weekly clinical administration. CONCLUSIONS: CB307 is a first-in-class immunotherapeutic that triggers potent PSMA-dependent T cell activation thereby alleviating toxicological concerns against unrestricted CD137 agonism. Citation Format: Sophie Archer, Phillip M. Brailey, Minjung Song, Phillip D. Bartlett, Ines Figueiredo, Bora Gurel, Christina Guo, Verena Brucklacher-Waldert, H. Lorraine Thompson, Jude Akinwale, Samantha E. Boyle, Christine Rossant, Neil R. Birkett, Julia Pizzey, Mark Maginn, James Legg, Richard Williams, Colette M. Johnston, Philip Bland-Ward, Johann S. de Bono, Andrew J. Pierce. CB307: A dual targeting costimulatory Humabody® VH therapeutic for treating PSMA-positive tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5313.

Abstract 5301: Tumor-targeted activation of CD137 using Bicycles: New insights into mechanism of action and discovery of BT7455, a clinical candidate for the treatment of EphA2-expressing cancers

Abstract Introduction: Clinical studies in cancer patients have validated CD137 agonism as an activator of the immune system to enable tumor rejection. We have demonstrated that small, chemically synthetic bicyclic peptides can drive tumor-localized agonism of CD137 and anti-tumor immunity in mouse models. Here, we report the next stage of our work - delving into the mechanism of action of these novel agents and extending our program to serve patients whose tumors express EphA2. Experimental Procedures: MultiOmyx™ hyperplexed immunofluorescence assay was used to evaluate the expression of CD137 and EphA2 in head and neck squamous cell carcinoma samples. Human PBMC/tumor cell co-culture assays were used to assess CD137 Bicycle TICA™ in vitro bioactivity and syngeneic mouse tumor models were used to evaluate CD137 Bicycle TICA anti-tumor activity as mono- or combination therapies. Pharmacodynamic activity was evaluated by transcriptional profiling using NanoString assays or single cell RNA sequencing. Summary of the Data: Studies utilizing a syngeneic mouse model and deep RNA sequencing of tumors from CD137 Bicycle TICA-treated mice implicated intratumoral dendritic cells in addition to T cells as early responders to localized CD137 agonism and potential contributors to the anti-tumor response. Furthermore, a mouse efficacy model demonstrated synergy with checkpoint inhibitor therapy. Multiplex imaging revealed that EphA2 and CD137 are co-expressed in human tumors of high unmet medical need and therefore, using our highly modular Bicycle platform, we discovered BT7455, a Bicycle TICA that engages EphA2 and CD137 with high affinity, resulting in potent EphA2-dependent activity in vitro and robust anti-tumor activity in vivo with intermittent dosing in mouse models. Gene expression profiling of tumors revealed that BT7455 led to increased production of cytokines and chemokines known to drive T cell infiltration, the extent of which differentiated it from both a checkpoint inhibitor and an anti-CD137 monoclonal antibody agonist. BT7455 was well tolerated in preclinical species and liver function tests indicated no evidence of hepatic toxicity. Statement of Conclusions: In summary, we have identified EphA2 as a promising target to pair with a CD137 agonist. In addition to CD8+ T cells, tumor-resident dendritic cells are a likely contributor to anti-tumor immunity following treatment with Bicycle® TICAs, supporting utility for Bicycle® tumor-targeted CD137 agonists in solid tumors beyond those that are highly T cell-infiltrated. We have advanced a clinical development candidate, BT7455, to realize this potential for patients with EphA2-expressing cancers. Citation Format: Johanna Lahdenranta, Kristen Hurov, Heather Cohen, Lia Luus, Cara Bray, Peter Brown, Anna Devlen, Carly Campbell, Matthew Gray, Mike Kelly, Gemma Mudd, Punit Upadhyaya, Sailaja Battula, Kelvin Zhang, Anne-Sophie Dugast, Kevin McDonnell, Phil Brandish, Nicholas Keen. Tumor-targeted activation of CD137 using Bicycles: New insights into mechanism of action and discovery of BT7455, a clinical candidate for the treatment of EphA2-expressing cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5301.