Abstract Background: Erythropoietin-producing hepatocellular receptor A2 (EphA2) is a receptor tyrosine kinase critical for cell development; it is highly expressed in a range of solid tumors, and its expression correlates with higher grade, later stage disease, and poor prognosis. There is high unmet need for patients with EphA2-expressing tumors, including pancreatic cancer, which has one of the highest EphA2 expression levels across solid tumors. BT5528 is a Bicycle® Drug Conjugate (BDC®), comprising a highly selective EphA2-targeting bicyclic peptide conjugated to the cytotoxin MMAE via a stable valine-citrulline cleavable linker. BT5528 has low molecular weight (4.4 kDa), enabling rapid and efficient delivery of the BDC® to the tumor and subsequent payload release, with minimal systemic exposure to the conjugate. Here we evaluate EphA2 expression and antitumor activity of BT5528 in murine PDX models of PDAC. Methods: Cohorts of female NOD SCID gamma (NSG) mice were used to generate 16 PDAC PDX models by subcutaneous implantation of patient-derived PDAC tumors into the left abdominal flank. Flash frozen paraffin embedded tumor samples from vehicle treated mice were used to assess EphA2 expression by immunohistochemistry (IHC); Tumor Proportion Score (TPS) was calculated as the number of membrane EphA2+ cells/total number of viable tumor cells x 100. Tumor-bearing mice were treated once weekly for 4 weeks with either vehicle control or BT5528 (3 mg/kg IV). Tumor growth was monitored by caliper measurements and tumor volume was calculated as [(width)2 x length]/2. Tumor growth inhibition (TGI) was estimated for each treatment group per model using the average response across replicates after 4 weeks. Results: Of 16 PDAC PDX models tested, all 16 displayed some degree of EphA2 membrane staining, with 16/16 considered positive (TPS ≥1%), including models JH029 (20%), Panc163 (30%), and Panc421 (90%). Fourteen of 16 PDAC PDX models were assessed for antitumor activity. Six of 14 models showed high sensitivity to BT5528 (TGI ≥100%), including JH029 (157%), Panc163 (114%), and Panc421 (110%), with most models showing some level of sensitivity to BT5528 (only 3/14 had TGI <50%). Anti-tumor activity of BT5528 was not affected by the extent of desmoplasia, with TGI scores roughly equivalent across mature, intermediate, and immature tumor models. No increase in response was seen with increasing membrane EphA2 TPS. Conclusions: Expression of EphA2 was found in all PDAC PDX models. Most models were sensitive to BT5528 treatment, which was not affected by the degree of desmoplasia or membrane EphA2 TPS. These results may reflect a lack of truly EphA2-negative models in this analysis. These data support the potential for BT5528 to offer a novel option for the treatment of PDAC. Citation Format: Lukas Stanczuk, Assunta De Rienzo, Gavin Bennett. Preclinical assessment of BT5528 anti-tumor activity in patient-derived xenograft (PDX) models of pancreatic ductal adenocarcinoma (PDAC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4518.

Abstract Background: Erythropoietin-producing hepatocellular receptor A2 (EphA2) is a receptor tyrosine kinase critical for cell development; it is highly expressed in a range of solid tumors, and its expression correlates with higher grade, later stage disease, and poor prognosis. There is high unmet need for patients with EphA2-expressing tumors, including HNSCC, which has one of the highest EphA2 expression levels across solid tumors. BT5528 is a Bicycle® Drug Conjugate (BDC®), comprising a highly selective EphA2-targeting bicyclic peptide conjugated to the cytotoxin MMAE via a stable valine-citrulline cleavable linker. BT5528 has low molecular weight (4.4 kDa), enabling rapid and efficient delivery of the BDC® to the tumor and subsequent payload release, with minimal systemic exposure to the conjugate. Here we evaluate anti-tumor activity of BT5528 in murine CDX models of HNSCC. Methods: Three HNSCC CDX models were generated with female NOD SCID (SCC-9 [tongue]) or Balb/c nude (Fadu [hypopharynx] and Cal27 [tongue]) mice by subcutaneous inoculation of cell-line-derived HNSCC tumor cells into the right abdominal flank. Tumor-bearing mice were treated once weekly with either vehicle or BT5528 at 1, 3, or 5 mg/kg IV (n=6 per treatment group per CDX model) once average tumor volume reached ∼160 mm3. EphA2 expression in cell lines was assessed by quantitative FACS. Tumor growth was monitored by caliper measurements and tumor volume calculated as (width2 x length)/2. Average tumor growth inhibition (TGI) was calculated twice weekly for each treatment group per model for up to 28 days. Results: Average EphA2 expression (receptors/cell) was 29,000 (Fadu, low expression), 46,000 (SCC-9, medium expression), and 135,000 (Cal27, high expression), which are comparable to expression levels observed in previous preclinical work in CDX and patient-derived xenograft models, and which correlated with BT5528 anti-tumor activity. Compared with vehicle treated mice, significant anti-tumor activity was observed for all three models. For Cal27, TGI reached 53% and 92% at 3 and 5 mg/kg IV, respectively, on Day 28 (p<0.001 and p<0.0001). For SCC-9 and Fadu, the day of measurement was the last day all vehicle-treated mice survived. TGI reached 101% and 107%, respectively, for SCC-9 on Day 21 (p<0.0001); and 95% and 107%, respectively, for Fadu on Day 25 (p<0.0001). Assessment of tumor volume over time demonstrated a clear dose response for all models tested, with Fadu and SCC-9 being the most sensitive. BT5528 was generally tolerable in line with previous studies.1 Conclusions: BT5528 has shown potent preclinical anti-tumor activity in EphA2-expressing CDX models of HNSCC at clinically relevant doses. These data support the potential to further evaluate BT5528 in HNSCC. 1. Bennett et al. Mol Cancer Ther. 2020;19(7):1385-1394. Citation Format: Lukas Stanczuk, Daniel A. Peterson, Inma Rioja, Michael Method, Michael Skynner, Gavin Bennett. Preclinical assessment of BT5528 anti-tumor activity in cell-line-derived xenograft (CDX) models of head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1325.

Therapeutic advances have improved cancer survival outcomes for an increasing number of patients, but novel approaches are still urgently needed for patients who cannot tolerate, or do not respond to current treatments. Erythropoietin-producing hepatocellular receptor A2 (EphA2) is highly expressed in a variety of solid tumors, which is associated with poor prognosis, especially in tumors considered difficult-to-treat, such as pancreatic and head and neck cancer. EphA2 has emerged as a promising therapeutic target for the treatment of solid tumors; however, efficacy and safety issues have halted clinical development of previous EphA2-targeting agents including MEDI-547, DS-8895a, MM-310, and dasatinib. Despite these setbacks, interest in targeting EphA2 in solid tumors remains, with ongoing development of investigational therapies such as antibodies, antibody drug conjugates, EphA2 antagonists, peptide drug conjugates, bicyclic peptide drug conjugates, and tyrosine kinase inhibitors. Among these, BT5528, a Bicycle® Drug Conjugate (BDC), has shown an emerging differentiated safety profile, in contrast to prior EphA2-targeting agents, and promising antitumor activity in patients with advanced solid tumors. BT5528 comprises an EphA2-targeting bicyclic (Bicycle) peptide, linked to the cytotoxin monomethyl auristatin E (MMAE) via a valine-citrulline cleavable linker. The high specificity of BT5528 to EphA2, combined with its high affinity, enables precision-guided delivery of MMAE, while its peptidic nature results in rapid distribution and retention of MMAE within the tumor, limited systemic exposure, and liver-sparing renal elimination. The preclinical and emerging clinical data for BT5528 suggest that novel approaches to targeting EphA2 can achieve efficacy without the safety issues that plagued earlier agents. Here, we review EphA2 as a target and the historical and current clinical development of EphA2-targeting therapeutic agents.

INTRODUCTION:Antibody drug conjugates (ADCs) selectively deliver cytotoxins to tumor cells using highly specific monoclonal antibodies. While ADCs are a standard treatment paradigm in oncology, safety- and efficacy-related challenges remain. Additional platforms are needed to deliver more effective targeted therapy with differentiated safety profiles. AREAS COVERED:This narrative review discusses the preclinical and clinical journey of ADCs, their shortcomings, and avenues for refinement. Development of novel antibody fragment-drug conjugates, small molecule-drug conjugates (SMDCs), peptide drug conjugates (PDCs), and Bicycle drug conjugate (BDC) molecules are discussed based on targeted literature searches for each technology described; searches were not bound by specific terms or dates in PubMed or ClinicalTrials.gov. EXPERT OPINION:Targeted delivery of cytotoxins will continue to be a powerful tool against cancer; however, we believe novel SMDCs, PDCs, and BDC molecules will help circumvent current challenges surrounding cytotoxin-containing ADCs and provide differentiated treatment options in solid tumors, including those particularly difficult to treat. We expect promising preclinical and clinical data emerging for SMDCs, PDCs, and BDC molecules will translate into approvals, such as BDC molecules that are under pivotal randomized phase II clinical investigation. These novel treatments will increase survival and quality of life for patients with solid tumors.

The membrane-type 1 matrix metalloproteinase MT1-MMP (or MMP14) is a member of the type 1 transmembrane proteinase family, highly overexpressed in several solid tumors and associated with poor prognosis (e.g. non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma, breast cancer, gastric cancer, and osteosarcoma). Its expression profile makes this target amenable to novel precision targeting with a toxin drug conjugate across a broad spectrum of solid tumors. Bicycle® drug conjugates (BDC®) intrinsically possess several advantages over other targeted cytotoxic modalities such as antibody-drug conjugates (ADCs). This Bicycle® advantage includes improved kinetics of extravasation and tumor penetration, fast renal clearance, and a lack of engagement of Fc receptors. Targeting MT1-MMP with a BDC® molecule represents an attractive potential approach for targeted tumor therapy. Bicycle® binders for MT1-MMP were identified using a proprietary phage display peptide technology consisting of highly diverse phage libraries of Bicycle® molecules, then conjugated to cleavable linkers and toxins to form Bicycle® Drug Conjugates (BDC®). Tool molecules were identified, containing an MT1-MMP binding Bicycle® conjugated to the cytotoxic auristatin derivative maleimidocaprolyl-monomethyl auristatin E (MMAE) via a proteolytically cleavable valine-citrulline (Val-Cit) linker. The properties of tool molecules were evaluated using a range of in vitro and in vivo models assessing MT1-MMP binding and specificity/selectivity, internalisation, in vitro cytotoxicity, and in vivo antitumor activity using xenograft models (cell and patient derived). Toxicology studies were performed in Han Wistar rats and Cynomologous monkeys. In vitro, tool molecules bound to MT1-MMP with high affinity (KD ∼3nM), and demonstrated both, cross-reactivity to species orthologues of MT1-MMP and selectivity over related human MMP family members by surface plasmon resonance (SPR) measurements. A fluorescent version of the MT1-MMP binding Bicycle® bound to cell lines dependent on expression levels of MT1-MMP, effectively internalised, and the tool BDC® molecule (MMAE containing) demonstrated MT1-MMP dependent cytotoxicity in an in vitro cell model.In vivo, the tool BDC® molecule demonstrated the expected PK profile in pre-clinical species of a short half life, and was effective in MT1-MMP expressing cell line (CDX) xenograft models in both mice and rats, showing complete tumor regression at 3mg/kg weekly dosing. The molecule also showed antitumor activity in a panel of NSCLC patient-derived xenograft models (PDX). Finally, in vivo toxicology (2 doses 1 week apart) demonstrated a similar tolerated range to other clinical stage BDC® molecules. Targeting MT1-MMP with a BDC® molecule shows potent antitumour activity in a range of solid tumour xenograft models, with a focus on NSCLC, and represents an attractive opportunity to target this novel tumor antigen for therapeutic benefit in this indication. Steve B. Ludbrook, Stephen Walsh, Gemma Mudd, Johanna Lahdenranta, Philip Huxley, Luca Mascheroni, Maximillian Harman, Jennifer Bré, Inma Rioja, Michael Skynner, Gavin Bennett. Targeting the novel tumor antigen MT1-MMP with Bicycle® Drug Conjugates (BDC®) for the potential treatment of NSCLC and other solid tumor indications [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr B026.

Background: The Bicycle (R) toxin conjugate BT5528 is a novel peptide therapeutic conjugated to the cytotoxic agent monomethyl auristatin E (MMAE). A bioanalytical assay was developed to quantify BT5528 and unconjugated MMAE in human plasma. Methodology: BT5528 quantitation used a protein precipitation procedure followed by LC-MS/MS detection. Quantitation of MMAE required a selective offline and online solid-phase extraction with detection via LC-MS/MS. Results: BT5528 was quantified over the assay range of 5-2500 ng/ml and free MMAE was quantified over the assay range of 0.05-50 ng/ml. Conclusion: Bioanalytical methods were used in the bioanalysis of intact BT5528 and released MMAE, in a phase I/IIa clinical trial; to date, over 2000 human patient samples have been analyzed.

Abstract Bicycle Therapeutics is developing a unique class of chemically synthesized medicines. Based on its proprietary bicycle peptide (Bicycle®) phage display platform, Bicycles are a unique class of highly constrained peptides, which have demonstrated utility in the targeted delivery of different classes of payloads (for example cytotoxic agents, radioisotopes, immune modulators) into tumors. Bicycles are currently being explored in the clinic as Bicycle Toxin Conjugates® (BTCs) for targeted delivery of cytotoxic payloads into tumors. BTCs consist of a bicyclic peptide that is conjugated to a cytotoxic payload via a cleavable linker, which allows payload release in the tumor microenvironment or within the tumor cell. BTCs were developed to address the shortcomings of antibody drug conjugates (ADCs) in several ways. First, the small size of BTCs (∼4 kDa) compared to large biologic entities such as monoclonal antibody (mAb)-based conjugates (∼150 kDa) allows rapid distribution to tissues and extensive tumor penetration, which enables rapid delivery of payload into the tumor. Second, the peptidic nature of BTCs results in relatively short, yet tunable, duration of systemic exposure and liver-sparing renal elimination. These properties limit the body’s exposure to payload and should therefore minimize damage to normal tissue. In this body of work, we used in vitro cytotoxicity and cell uptake assays and mouse and rat cell line derived xenograft models for 1) cytotoxicity and anti-tumor activity evaluation, and 2) Bicycle and toxin uptake and biodistribution evaluation. Here, we show that BTCs targeting a number of different tumor antigens can deliver toxins to tumor tissue producing durable responses in a range of preclinical in vivo models, spanning several solid tumor indications. Citation Format: Stephen J. Walsh, Johanna Lahdenranta, Philip Huxley, Gemma Mudd, Gavin Bennett, Amy Brown, Katerine an Rietschoten, Liuhong Chen, Heather Scott, Gabriella Ivanova-Berndt, Katarzyna Dzionek, Mike Rigby, Olga Burenkova, Phil Jeffrey, Paul Beswick, Michael Skynner, Nicholas Keen. Bicycle Toxin Conjugates®for the treatment of solid 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 5807.

3088 Background: Bicycle toxin conjugates (BTCs) are chemically synthesized molecules comprising a small (~4.5 kDa) bicyclic peptide linked to a cytotoxin. BT8009 and BT5528 are BTCs linked to monomethyl auristatin E (MMAE) targeting nectin-4 and EphA2, respectively, and have shown preliminary activity. BTCs are a unique therapeutic class of small size, with corresponding pharmacokinetic (PK) properties distinct from antibody-drug conjugates (ADCs). MMAE-containing ADCs demonstrate anti-tumor efficacy but substantial safety issues attributable to the slow clearance of ADCs, which increases uptake into off-target tissues and exposure to plasma proteases (ie, payload release).To evaluate BTC PK and safety relative to ADCs, we present results of the ongoing Phase I/II trials for BT8009 (NCT04561362) and BT5528 (NCT04180371). Methods: Patients with advanced solid tumors receiving IV BT8009 (n=147) or BT5528 (n=109) monotherapy were evaluated. Doses were given as 2.5–10 mg/m2 weekly (QW), every 2 weeks (Q2W), or 2 weeks on/1 week off for BT8009 and 2.2–10 mg/m2 QW or Q2W for BT5528. Population PK (PPK) models for BT8009 and BT5528 were developed. PK exposures (Cycle 1 area under the concentration curve [AUC]) for BTCs, conjugated MMAE, and unconjugated MMAE were simulated for 5 mg/m2 QW BT8009 and BT5528 and compared with those of an MMAE-containing ADC (approved regimen, using the PPK model in the literature). Treatment-related adverse events (TRAEs) were tabulated for BT8009 and BT5528. Results: BTCparent drugs were rapidly eliminated (half-life [t1/2] <1 hour) while unconjugated MMAE exhibited persistent exposures and gradual elimination (MMAE t1/2, 1.9 days [D] for both BT8009 and BT5528), in contrast with the ADC (parent drug t1/2, 3.6 D; MMAE t1/2, 2.6 D). Conjugated MMAE exposures for the ADC were higher than for BT8009 and BT5528 (75- and 73-fold, respectively). Unconjugated MMAE exposures for BT8009 and BT5528 were comparable to the ADC (25% and 8% higher, respectively). Among the BTC regimens most thoroughly evaluated (BT8009 5 mg/m2 QW and BT5528 6.5 mg/m2 Q2W), TRAEs of interest occurred with low frequency and severity (Table). Conclusions: Preliminary data show substantial differences between BTC and ADC PK, and a promising BTC safety profile, possibly resulting from the distinct PK and selectivity (and specificity) of Bicycle peptides. These data highlight the potential value of BTCs as a platform for developing therapies against advanced malignancies. Clinical trial information: NCT04561362 and NCT04180371 . [Table: see text]

PURPOSE BT5528 is a Bicycle Toxin Conjugate, a novel class of chemically synthesized molecules, comprising a bicyclic peptide targeting EphA2 tumor antigen, linked to a cytotoxin (monomethyl auristatin E [MMAE]). EphA2 is overexpressed in many solid tumors and contributes to oncogenesis, tumor-associated angiogenesis, and metastasis. MATERIALS AND METHODS The primary objectives were to investigate the safety and tolerability of BT5528 and to define the maximum-tolerated dose, if observed, and recommended phase II dose (RP2D)/expansion dose. Dose escalation exploring once every week or once every 2 weeks administration of BT5528 employed a 3 + 3 dose-escalation design for the first two dose levels, followed by a Bayesian logistic regression model. Secondary and exploratory end points included preliminary efficacy and the pharmacokinetics of BT5528 and MMAE. RESULTS Forty-five patients were enrolled and received BT5528 doses between 2.2 mg/m 2 once every week to 10.0 mg/m 2 once every 2 weeks within the dose-escalation stage of the study. The most frequent BT5528-related adverse events (AEs) were nausea (44.4%), diarrhea (35.6%), and fatigue (33.3%), and the most common grade ≥3 BT5528-related AE was neutropenia/neutrophil count decrease (22.2%). Dose level 6.5 mg/m 2 once every 2 weeks was selected as a RP2D. At 6.5 mg/m 2 once every 2 weeks, the overall response rate was 6.7%, and the disease control rate was 20.0%. BT5528 and MMAE pharmacokinetics are generally dose proportional. BT5528 has a short half-life (0.4-0.7 hours), and the half-life of MMAE is longer (35-47 hours). CONCLUSION BT5528 was well tolerated and demonstrated favorable and preliminary antitumor activity. We believe these data provide preliminary validation of a Bicycle Toxin Conjugate approach to EphA2 tumor antigen. The study is ongoing and is evaluating BT5528 as monotherapy at a RP2D of 6.5 mg/m 2 once every 2 weeks.

The file contains supplemental details related to sequence information of the bicyclic peptides referred to in the text, binding data, organ distribution data, PK parameters, mouse and human plasma stability, in vivo metabolism of BCY-B2 in mice, cell binding and internalization data, additional PET imaging data. It also contains detailed methods related to protein expression, phage selection, peptide synthesis, affinity determination by fluorescence polarization and SPR, radiolabeling, internalization, confocal microscopy, plasma protein binding, plasma stability of radiolabeled compounds, µPET imaging, autoradiography, immunohistochemistry, and 6 references related to these methods. €¢ Supplementary Tables S1 - S14: o Table S1: Affinities and plasma stabilities of BCY-B and stabilized BCY-C. o Table S2: Sequence information and binding data of bicyclic peptides referred to in text. o Table S3: Surface plasmon resonance (SPR) data. o Table S4-6: Organ distribution data, BCY-B3/B4 o Table S7: PK parameters. o Table S8: Selectivity data of BCY-C towards other metalloproteinases. o Table S9-11: Organ distribution data- BCYC2/C4 o Table S12-14: Organ distribution data- BCYD1/D2 o Table S15: Organ distribution data- MAb €¢ Supplementary Figures S1-S6: o Figure S1: Comparative HT1080 cell binding and internalization of non-stabilized BCY-B3 and stabilized BCY-C2 o Figure S2: Organ distribution time course studies with active BCY-B3, and inactive BCY-B4 in HT1080 xenograft mice. o Figure S3: Mouse and human plasma stability of BCY-B5, BCY-C3, BCY-D1; o Figure S4: Pharmacokinetic profile of BCY-B2 in mouse; o Figure S5: In vivo metabolism of BCY-B2 in mice; o Figure S6: Confocal microscopy on HT1080 cells and BCY-C2 o Figure S7: PET imaging. Time-resolved whole-body maximum intensity projections of 68Ga-BCY-C2 in HT1080 xenograft mouse. €¢ Supplementary Methods: o Protein expression, o Phage selection, o Peptide synthesis, o Affinity determination by fluorescence polarization and SPR, o Radiolabeling, o Internalization, o Confocal microscopy, o Plasma protein binding, o Plasma stability of radiolabeled compounds, o µPET imaging, o Autoradiography, o Immunohistochemistry, o 6 references related to these methods.

Supplementary Data from BT8009; A Nectin-4 Targeting Bicycle Toxin Conjugate for Treatment of Solid Tumors

Supplementary Chemical Structures provides an inventory and structural diagrams of all Bicycle conjugates used in paper

Abstract Background: Bicycles are a novel class of synthetic molecules formed by short linear peptides constrained in a stabilized bi-cyclic structure that can be conjugated to other molecules. BT5528 is a Bicycle Toxin Conjugate, comprising a bicyclic peptide targeting EphA2 linked to a cytotoxin (monomethyl auristatin E [MMAE]). EphA2 is expressed at low levels in normal tissues but overexpressed in many solid tumors; EphA2 expression correlates with malignant progression and poor patient outcome. BT5528 is ~40X smaller than antibody-drug conjugates (ADCs) and has the potential to penetrate solid tumors. Within the tumor, release of MMAE disrupts intracellular microtubule dynamics leading to inhibited proliferation and tumor cell death with bystander killing effect. The pharmacokinetic (PK) profile of BT5528 is distinct from ADCs with lack of Fc-mediated non-specific uptake, fast distribution, and renal elimination, leading to lower vascular exposure and potentially reduced toxicity in non-target tissues. The preclinical profile supported the initiation of a first-in-human study to investigate BT5528 safety and efficacy in advanced solid tumors with EphA2 expression (NCT04180371). Methods: This phase 1/2 multicenter, open-label trial evaluates BT5528 as monotherapy and in combination with nivolumab (nivo) in up to 288 patients. Phase 1 employs a 3+3 dose-escalation design for the first two dose levels followed by a Bayesian logistic regression model. Phase 2 expansion will test the selected dose(s) of BT5528 in six tumor-specific cohorts with high EphA2 expression (NSCLC, ovarian, TNBC, gastric/upper GI, head and neck, and urothelial cancer). BT5528 will be administered intravenously (IV) in ascending doses either weekly or every other week as monotherapy in Part A-1 and in combination with nivo in Part A-2 (480 mg of IV nivo every 4 weeks or 240 mg every 2 weeks), and as per the recommended phase 2 dose(s) (RP2D) in Part B. Part A patients must have a metastatic solid tumor type known to have high EphA2 tumor expression and Part B patients must have one of the specified tumor types; all patients must have failed or be ineligible for appropriate treatment options with evidence of radiographic progression on the most recent line of therapy. Additional eligibility criteria include ≥18 years of age, ECOG performance status of 0 or 1, and adequate organ function. Primary objectives for Part A are safety and tolerability and to define the maximum tolerated dose (if observed) and RP2D(s) as monotherapy and in combination with nivo. The primary objective for Part B is to assess clinical activity of BT5528 monotherapy. Secondary objectives include preliminary efficacy (Part A), safety and tolerability (Part B), correlation of EphA2 expression with efficacy (Part B), PK parameters of BT5528 and MMAE, and incidence of anti-drug antibodies. Tumor and blood samples will be collected for biomarker evaluations including tumor EphA2 expression, ADAs, and candidate response biomarkers for BT5528 alone and in combination with nivo. This study is actively recruiting. Citation Format: Elisa Fontana, Babar Bashir, Judy S. Wang, Raid Aljumaily, Jean-Pascal Machiels, Maria Vieito, Gerald Falchook, Louise Carter, Bernard Doger de Spéville, Alastair Greystoke, Sang Wun Kim, Nuria Kotecki, Alexander I. Spira, Irene Moreno Candilejo, Bristi Basu, Hans Prenen, Alberto Bessudo, Misako Nagasaka, Jordi Rodón Ahnert, Joo-Hwan Park, Min-Yuen Teo, Julia Rotow, Jie Liu, Assunta De Rienzo, Mengyao Li, Adriana Domingo, Hanna Orr, Gavin Bennett, Rajiv Sharma, Meredith McKean. Trial in progress: First-in-human phase I dose-escalation study of a novel Bicycle toxin conjugate (BT5528) targeting EphA2 in patients with advanced solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_A17.

Supplementary data are provided to further understand the pharmacokinetics and efficacy of BT5528 and EphA2 expression in tumors. Figure S1: Plasma concentration-time curves of BT5528 and MMAE following IV dosing of BT5528 1 in mouse, rat, and cynomolgus Monkey at 1 mg/kg (n=3 per species). Related to in vivo PK. Figure S2: EphA2 expression and tumour growth inhibition in CDX and PDX xenograft models are correlated. BT5528 3 mg/kg qw dosing has a range of anti-tumour activities across different cell-line derived and patient-derived xenograft models (*p<0.05, **p<0.01, ***p<0.001 2way ANOVA from D0 to last day of vehicle tumour measurement).Data used to generate Figure 3. Figure S3: EphA2 Immunohistochemistry staining of CDX and PDX xenograft models. A range of EphA2 expression is observed in stained tumour tissue from mouse xenograft models. Related to figure 3. Table T1: Ki values for BT5528, 1C1-mcMMAF and control compounds binding to human, mouse and rat Epha2 receptor. Ki values for Bicycle, BTC and DOTA analogues used to generate data in Figures 1, 4 and 5. Table T2: Binding affinities for BT5528 binding to EphA and EphB tyrosine kinases receptors determined by Surface Plasmon Resonance. Data defining BTC and Bicycle KD values for EphA2 orthologues. Table T3: In vitro ADME properties of BT5528 in mouse, rat, NHP and human: plasma protein binding, plasma stability and metabolic stability in hepatocytes. Related to in vitro ADME properties of BT5528. Table T4: Summary of experimentally determined and calculated PK Parameters for BT5528 and MMAE Following IV Dosing of 1 mg/kg BT5528 in Mouse, Rat and Cynomolgus Monkey. Related to in vivo PK Table T5a: Anti-Human EphA2 Antibody Binding Sites on CDX Cell Lines. Expression data used to generate Figure 3. Table T5b:Anti-Human EphA2 Antibody Binding Sites on PDX Cell Lines. Expression data used to generate Figure 3. Table T6: Doses of BT5528 and corresponding toxin (MMAE) used in preclinical toxicology studies compared with the clinical dose of MED-547 and corresponding toxin (MMAF)1. Related to the discussion (ADC versus BTC toxicology responses).

Supplementary information detailing methods used to generate in vitro and in vivo data presented in paper

AbstractMultiple tumor types overexpress Nectin-4 and the antibody–drug conjugate (ADC), enfortumab vedotin (EV) shows striking efficacy in clinical trials for metastatic urothelial cancer, which expresses high levels of Nectin-4, validating Nectin-4 as a clinical target for toxin delivery in this indication. Despite excellent data in urothelial cancer, little efficacy data are reported for EV in other Nectin-4 expressing tumors and EV therapy can produce significant toxicities in many patients, frequently leading to discontinuation of treatment. Thus, additional approaches to this target with the potential to extend utility and reduce toxicity are warranted. We describe the preclinical development of BT8009, a “Bicycle Toxin Conjugate” (BTC) consisting of a Nectin-4–binding bicyclic peptide, a cleavable linker system and the cell penetrant toxin mono-methylauristatin E (MMAE). BT8009 shows significant antitumor activity in preclinical tumor models, across a variety of cancer indications and is well tolerated in preclinical safety studies. In several models, it shows superior or equivalent antitumor activity to an EV analog. As a small hydrophilic peptide-based drug BT8009 rapidly diffuses from the systemic circulation, through tissues to penetrate the tumor and target tumor cells. It is renally eliminated from the circulation, with a half-life of 1–2 hours in rat and non-human primate. These physical and PK characteristics differentiate BT8009 from ADCs and may provide benefit in terms of tumor penetration and reduced systemic exposure. BT8009 is currently in a Phase 1/2 multicenter clinical trial across the US, Canada, and Europe, enrolling patients with advanced solid tumors associated with Nectin-4 expression.