Bicycle Toxin Conjugate Research Articles

304 (PB292): Effects of Nectin-4 targeted Bicycle Toxin Conjugate, zelenectide pevedotin (BT8009), and enfortumab vedotin on a human corneal tissue model

304 (PB292): Effects of Nectin-4 targeted Bicycle Toxin Conjugate, zelenectide pevedotin (BT8009), and enfortumab vedotin on a human corneal tissue model

654P Peripheral neuropathy (PN) following treatment (tx) with bicycle toxin conjugates (BTCs) BT8009 or BT5528 monotherapy in patients (pts) with advanced solid tumors

654P Peripheral neuropathy (PN) following treatment (tx) with bicycle toxin conjugates (BTCs) BT8009 or BT5528 monotherapy in patients (pts) with advanced solid tumors

647P EphA2-targeting bicycle toxin conjugate (BTC) BT5528 in patients (pts) with advanced solid tumors: A phase I/II study

647P EphA2-targeting bicycle toxin conjugate (BTC) BT5528 in patients (pts) with advanced solid tumors: A phase I/II study

Breaking from the paradigm of antibody-drug conjugates: Evaluation of clinical pharmacokinetics and safety of bicycle toxin conjugates (BTCs).

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]

A phase 2/3 study of Bicycle toxin conjugate BT8009 targeting nectin-4 in patients with locally advanced or metastatic urothelial cancer (la/mUC): Duravelo-2.

TPS4619 Background: BT8009 is a Bicycle toxin conjugate (BTC), comprising a highly selective bicyclic peptide targeting nectin-4 linked to the cytotoxin monomethyl auristatin E (MMAE) via a cleavable linker. Nectin-4 is an adhesion molecule commonly expressed in many tumor types, including urothelial cancer (UC), and is a validated therapeutic target (Hoffman-Censits 2021). BT8009 has a low molecular weight and short plasma half-life, with potential to rapidly penetrate solid tumors and reduce toxicity by minimizing exposure to normal tissue (Rigby 2022). Results from the ongoing phase 1/2 clinical trial of BT8009 (NCT04561362) indicate preliminary antitumor activity and a tolerable safety profile in patients (pts) with advanced malignancies including UC (Baldini 2023). This global, open label, phase 2/3 multicenter adaptive study aims to evaluate the safety and efficacy of BT8009 as monotherapy, or combined with pembrolizumab (pembro), vs chemotherapy in pts with locally advanced or metastatic UC (la/mUC) (NCT06225596/BT8009-230; Duravelo-2). Methods: The trial will enroll up to 956 adult pts in 2 cohorts. Cohort 1 will include n≤641 previously untreated pts eligible for platinum-based chemotherapy. Cohort 2 will include n≤315 pts with ≥1 prior systemic therapy, excluding enfortumab vedotin or other MMAE-based therapy. Pts must have la/mUC of the renal pelvis, ureter, bladder, or urethra, ECOG performance status ≤2 (Cohort 1) or ≤1 (Cohort 2), and adequate organ function. Cohort 1 will be randomized 1:1:1 to receive: 1) BT8009 5 mg/m2 on days (D)1, 8, and 15 + pembro 200 mg on D1; 2) BT8009 6 mg/m2 on D1 and 8 + pembro 200 mg on D1; or 3) chemotherapy (gemcitabine + cisplatin / carboplatin, followed by avelumab maintenance). Cohort 2 will be randomized 1:1 to receive: 1) BT8009 5 mg/m2 on D1, 8, and 15 or 2) BT8009 6 mg/m2 on D1 and 8. Cycle lengths will be 21D (28D for avelumab). After 30 pts in each dose arm have 9 weeks follow-up, an interim analysis will determine the optimal dose of BT8009 + pembro (Cohort 1) or BT8009 monotherapy (Cohort 2) to be used for the rest of the study. An additional Cohort 2 arm, optimal dose of BT8009 + pembro, will open after completion of the interim analysis. Discontinuation criteria include planned completion of therapy, progressive disease, and intolerable toxicity. Primary endpoints are progression-free survival (PFS; Cohort 1) and objective response rate (ORR; Cohort 2) assessed by blinded independent central review. Secondary endpoints are ORR (Cohort 1), PFS (Cohort 2), and overall survival, duration of response, disease control rate, safety/tolerability, and health-related quality of life (Cohorts 1 and 2). Pharmacokinetics, incidence/titers of antidrug antibodies, and tumor/peripheral biomarkers are exploratory endpoints. Efficacy endpoints will be assessed per RECIST v1.1. This study is actively recruiting. Clinical trial information: NCT06225596 .

Abstract 5807: Bicycle Toxin Conjugates®for the treatment of solid tumors

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.

Revisiting Treatment of Metastatic Urothelial Cancer: Where Do Cisplatin and Platinum Ineligibility Criteria Stand?

Cisplatin-based chemotherapy has been the standard of care in metastatic urothelial cancer (mUC) for more than two decades. However, many patients with comorbidities cannot receive cisplatin or its alternative, carboplatin. 'Cisplatin-ineligible' and 'platinum-ineligible' patients lacked effective therapy options. However, the recent combination of enfortumab vedotin (EV), an antibody-drug conjugate targeting Nectin-4, with pembrolizumab (P), an antibody targeting the programmed death-1 (PD-1) immune checkpoint, is changing the status quo of frontline mUC treatment, with potential synergy seen in the EV-103 and EV-302 clinical trials. First, we review the working definitions of 'cisplatin ineligibility' and 'platinum ineligibility' in mUC clinical trials and the standard of care in both categories. Then, we review select clinical trials for frontline treatment of cisplatin- and platinum-ineligible mUC patients on ClinicalTrials.gov. We classify the investigated drugs in these trials by their therapeutic strategies. Alongside chemotherapy combinations, the field is witnessing more immunotherapy combinations with fibroblast growth factor receptor (FGFR) inhibitors, bicycle toxin conjugates, bispecific antibodies, innovative targeted therapies, and many others. Most importantly, we rethink the value of classifying patients by cisplatin or platinum ineligibility in the frontline setting in the post-EVP era. Lastly, we discuss new priority goals to tailor predictive, monitoring, and prognostic biomarkers to these emergent therapies.

Systematic review of recent advancements in antibody-drug and bicycle toxin conjugates for the treatment of urothelial cancer.

Antibody-drug conjugates and bicycle toxin conjugates represent a tremendous advance in drug delivery technology and have shown great promise in the treatment of urothelial cancer. Previously approved systemic therapies, including chemotherapy and immunotherapy, are often impractical due to comorbidities, and outcomes for patients with advanced disease remain poor, even when receiving systemic therapy. In this setting, antibody-drug and bicycle toxin conjugates have emerged as novel treatments, dramatically altering the therapeutic landscape. These drugs harness unique designs consisting of antibody or bicycle peptide, linker, and cytotoxic payload with more targeted delivery than conventional chemotherapy, thus eliminating malignant cells while reducing systemic toxicities. Potential targets investigated in urothelial cancer include Nectin-4, TROP2, HER2, and EphA2. Initial clinical trials demonstrated efficacy in treatment of refractory advanced urothelial cancer, as well as improvement in quality of life. These initial studies led to FDA approval of two antibody-drug conjugates, enfortumab vedotin and sacituzumab govitecan. Moreover, antibody-drug and bicycle toxin conjugates are being studied in ongoing clinical trials in frontline treatment of advanced disease as well as for localized cancer. These studies highlight the potential for additional future therapies with novel targets, novel antibodies, cytotoxic and immunomodulatory payloads, and unique structural designs enhancing efficacy and safety. There is increasing evidence that combinations with other cancer therapies, especially immunotherapy, improve treatment outcomes. The combination of enfortumab vedotin and pembrolizumab was recently approved for first-line treatment of advanced urothelial carcinoma. Despite the great promise of these novel drugs, robust predictive biomarkers are needed to determine the patients who would maximally benefit. This review surveys the rationale and current state of the evidence for these new drugs and describes future directions actively being explored.

Abstract LB_A17: 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 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.

Abstract B014: Characterization of Nectin-4 protein expression in non-small cell lung cancer patients

Abstract Nectin-4, a cell adhesion molecule from the Nectin-like family, is a validated tumor target overexpressed on the surface of cells in several cancers. Using Bicycles, a novel class of fully synthetic, short, constrained peptides, we are developing Bicycle Toxin Conjugates® (BTCs) and Bicycle Tumor-Targeted Immune Cell Agonists (Bicycle TICAs) that target Nectin-4 expressing tumors. BT8009 is a BTC™ which consists of a bicyclic peptide targeting Nectin-4 linked to the cytotoxin monomethyl auristatin E (MMAE) via a molecular spacer and cleavable linker. BT7480 is a Bicycle TICA™ that binds Nectin-4 on tumor cells and CD137 on immune cells to induce CD137 mediated immune agonism. Both BT8009 and BT7480 are currently under investigation in clinical trials, BT8009-100 (NCT04561362) and BT7480-100 (NCT05163041), respectively. To identify tumors expressing Nectin-4, we previously developed a proprietary Nectin-4 monoclonal antibody to measure its protein expression in an immunohistochemistry (IHC) assay. In collaboration with The Christie Hospital & The Manchester Cancer Research Centre Biobank (MCRC), we performed Nectin-4 IHC on tissue microarrays (TMAs) consisting of tumor biopsies from 59 non-small cell lung cancer (NSCLC) patients, including 4 longitudinal pairs, and 10 synchronous primaries with an average of 4 cores per tissue. A pathologist manually scored the TMAs for Nectin-4, evaluating both H-score and tumor proportion score (TPS) independently in the tumor cell membrane and tumor cytoplasm. Given the high degree of heterogeneity observed across multiple TMA cores from a single sample, Nectin-4 positivity was defined as a tumor proportion score (TPS) > 1 in either the tumor membrane or tumor cytoplasm, in at least 1 core. In samples collected around diagnosis, 69.5% of patients were positive for Nectin-4. In the 4 longitudinal pairs, all samples were Nectin-4 positive at the later timepoint. No statistically significant differences were observed in Nectin-4 positivity when analyzed by grade (grade 1 66.6% (n=6), grade 2 88.9% (n=9), grade 3 69.7% (n=33)), stage (stage I 75% (n=24), stage II 75% (n=16), stage III 64.3% (n=14), stage IV 40% (n=5)) or morphology (adenocarcinoma 65.2% (n=23), squamous cell carcinoma 77.4% (n=31)) when compared using Fisher’s exact test. A similarly high percent of Nectin-4 positivity was observed when comparing samples across demographic and mutational data. The high percent of Nectin-4 positivity observed in NSCLC supports the inclusion of this indication in Bicycle's BT8009-100 and BT7480-100 studies. Citation Format: Sean M Santos, Emma Darlington, Laura Woodhouse, Niamh Peters, Erin Peat, Assunta De Rienzo, Rob Smale, Shawn Watson, Stephen Blakemore, Louise Carter, Carly Campbell. Characterization of Nectin-4 protein expression in non-small cell lung cancer patients [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 B014.

BT8009-100: A phase I/II study of novel bicyclic peptide and MMAE conjugate BT8009 in patients (pts) with advanced malignancies associated with nectin-4 expression, including urothelial cancer (UC).

498 Background: BT8009 is a Bicycle Toxin Conjugate (BTC), a novel class of chemically synthesized molecules comprising a small (~2 kDa) bicyclic peptide targeting Nectin-4 tumor antigen linked to cytotoxin MMAE via a valine-citrulline cleavable linker. BTCs represent a unique therapeutic class, distinct from ADCs and with pharmacokinetic properties analogous to small molecules. Presented are the monotherapy dose escalation results of the ongoing Phase I/II clinical trial of BT8009 (NCT04561362). Methods: In the Part A 3+3 dose escalation portion of the study, BT8009 monotherapy was administered weekly (2.5, 5, 7.5 mg/m2) or every other week (7.5, 10 mg/m2) of a 28-day cycle, or on days 1 and 8 of a 21-day cycle (7.5 mg/m2). Primary objectives were to assess safety of the monotherapy and define maximum tolerated and recommended Phase II dose(s) (RP2D). Key secondary objectives were antitumor activity per RECIST v1.1 and PK parameters. Results: At time of the 20 Sept 2022 data cut-off, 49 pts had received BT8009 (24 pts with UC): weekly (7 pts at 2.5 mg/m2, 20 pts at 5 mg/m2, 5 pts at 7.5 mg/m2); biweekly (5 pts at 7.5 mg/m2, 7 pts at 10 mg/m2); and days 1+8 of a 21-day cycle (5 pts at 7.5 mg/m2). Two RP2Ds have been determined: 5 mg/m2 given weekly on a 28-day cycle and 7.5 mg/m2 given on days 1+8 of a 21-day cycle. Median age was 66 y, 59% pts were male, and median prior lines of therapy was 3 (range 1-15). Most common treatment-related adverse events of any grade experienced by ≥15% of pts were nausea, fatigue, diarrhea, decreased appetite, alopecia, asthenia, pyrexia, and neutrophil count decreased/neutropenia; majority were mild to moderate in severity. One DLT (G3 asthenia) was reported at the 7.5 mg/m2 weekly dose level and 1 DLT (G4 sepsis) at the 10 mg/m2 biweekly dose level. Treatment-related SAEs occurred in 5 pts. No pt discontinued treatment due to an AE. No treatment-related deaths occurred. Of 8 efficacy evaluable pts with UC treated with a RP2D of 5 mg/m2, there was 1 complete response, 3 partial responses (PR), and 3 stable diseases (SD), for an ORR of 50% and clinical benefit rate of 75% (CR+PR+SD≥16 wks). Median duration of response has not yet been reached, but as of 20 Sept 2022 was at least 11 months. Both plasma BT8009 and MMAE exhibited linear pharmacokinetics, with short half-life for BT8009 (<1 h) and longer half-life for MMAE (37-46 h). Conclusions: BT8009 was well tolerated and demonstrated promising preliminary antitumor activity. Evaluation of BT8009 as monotherapy at RP2D(s) and in combination with an immune checkpoint inhibitor will be explored during the expansion phase of this trial; cohorts will include pts with renal impairment to support further development in cis-ineligible pts, pts with previously untreated cis-ineligible metastatic UC, pts with refractory UC, and tumor-specific cohorts outside of bladder cancer. Clinical trial information: NCT04561362 .

Discovery of BT8009: A Nectin-4 Targeting Bicycle Toxin Conjugate for the Treatment of Cancer.

Bicycle toxin conjugates (BTCs) are a promising new class of molecules for targeted delivery of toxin payloads into tumors. Herein we describe the discovery of BT8009, a Nectin-4 targeting BTC currently under clinical evaluation. Nectin-4 is overexpressed in multiple tumor types and is a clinically validated target for selective delivery of cytotoxic payloads. A Nectin-4 targeting bicyclic peptide was identified by phage display, which showed highly selective binding for Nectin-4 but suffered from low plasma stability and poor physicochemical properties. Multiparameter chemical optimization involving introduction of non-natural amino acids resulted in a lead Bicycle that demonstrated high affinity for Nectin-4, good stability in biological matrices, and a much-improved physicochemical profile. The optimized Bicycle was conjugated to the cytotoxin Monomethyl auristatin E via a cleavable linker to give the targeted drug conjugate BT8009, which demonstrates potent anticancer activity in in vivo rodent models.

Comprehensive Surfaceome Profiling to Identify and Validate Novel Cell-Surface Targets in Osteosarcoma.

Immunoconjugates targeting cell-surface antigens have demonstrated clinical activity to enable regulatory approval in several solid and hematologic malignancies. We hypothesize that a rigorous and comprehensive surfaceome profiling approach to identify osteosarcoma-specific cell-surface antigens can similarly enable development of effective therapeutics in this disease. Herein, we describe an integrated proteomic and transcriptomic surfaceome profiling approach to identify cell-surface proteins that are highly expressed in osteosarcoma but minimally expressed on normal tissues. Using this approach, we identified targets that are highly expressed in osteosarcoma. Three targets, MT1-MMP, CD276, and MRC2, were validated as overexpressed in osteosarcoma. Furthermore, we tested BT1769, an MT1-MMP-targeted Bicycle toxin conjugate, in osteosarcoma patient-derived xenograft models. The results showed that BT1769 had encouraging antitumor activity, high affinity for its target, and a favorable pharmacokinetic profile. This confirms the hypothesis that our approach identifies novel targets with significant therapeutic potential in osteosarcoma.

Abstract LB008: Integrative surfaceome profiling identifies immunotherapeutic targets in osteosarcomas (OS); Preclinical testing of BT1769, an MT1-MMP-targeted Bicycle Toxin Conjugate, in OS by the Pediatric Preclinical Testing Consortium (PPTC)

Abstract Introduction: Toxin-conjugated agents targeting cell-surface antigens (e.g. antibody-drug conjugates) show promising clinical activity for some solid tumors, but none yet for OS. There is an urgent need to identify high-confidence OS cell-surface antigens and evaluate the antitumor activity of toxin conjugates that can deliver cytotoxic payloads to OS tumors expressing these targets. Methods: To identify cell-surface proteins that are highly expressed in OS but not in normal tissues, we prepared surface protein extracts from 4 standard, 4 patient-derived OS cell lines, and 10 OS PDX tissues. Quantitative mass spectrometry (MS) was used to profile the surface proteins. We included the proteomics data of OS and 39 types of normal human tissues via a public MS database (ProteomicsDB). We compared RNA-sequencing data from 101 OS patient samples via the NCI TARGET project with the RNA-sequencing data of 54 types of normal human tissue (GTEx, 17382 samples). We integrated the MS and RNA sequencing datasets to prioritize candidate surface targets by differential and absolute expression at both protein and mRNA levels. The cell-surface expression of the targets was further validated by IHC and flow cytometry with OS cell lines and tumor tissue microarray. BT1769, which consists of a bicyclic peptide targeting the tumor antigen MT1-MMP, linked to the cytotoxin monomethyl auristatin E (MMAE) via a molecular spacer and cleavable linker was tested in 6 OS PDX models using standard PPTC methods at 3 mg/kg given IV weekly x 4. Ewing sarcoma (EWS) models lacking MT1-MMP expression were also tested. Results: MS identified 1156 surface proteins as highly expressed in OS cell lines/PDX tissues. 129 proteins overexpressed in OS were identified from the public MS database. RNA sequencing data identified 195 overexpressed plasma membrane-associated genes in OS compared to normal tissues. The targets identified by both proteomics and transcriptomic data analysis were cross-referenced with existing toxin-conjugated agents. Three potential targets, MT1-MMP, CD276, and MRC2 were validated and MT1-MMP was prioritized because it had the highest absolute expression level in OS. BT1769 significantly inhibited tumor growth in 6/6 OS PDX models when compared to vehicle, with complete response maintained at 6 weeks in 3/6 models. Minimal activity was observed in 2/2 EWS models. Correlation analysis between the MT1-MMP gene expression level, IHC staining intensity and drug response are underway and will be presented. Conclusion: Integrated proteomic and transcriptomic OS surfaceome profiling identified high-confidence OS cell-surface antigens as therapeutic targets. MT1-MMP was highly expressed in most OS patient samples and PDX models, and BT1769 was highly active against select OS PDX models. Citation Format: Yifei Wang, Wendong Zhang, Zhongting Zhang, Xiangjun Tian, Rossana N. Lazcano Segura, Pooja Hingorani, Michael Roth, Jonathan Gill, Douglas Harrison, Zhaohui Xu, Jing Wang, Eric J. Earley, Stephen W. Erickson, Edward A. Kolb, Malcolm A. Smith, Peter Houghton, Raushan Kurmasheva, Tara Gelb, Philip Huxley, Johanna Lahdenranta, Gemma Mudd, Richard Gorlick. Integrative surfaceome profiling identifies immunotherapeutic targets in osteosarcomas (OS); Preclinical testing of BT1769, an MT1-MMP-targeted Bicycle Toxin Conjugate, in OS by the Pediatric Preclinical Testing Consortium (PPTC) [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 LB008.

Abstract 1319: Rapid accumulation of cytotoxic payload in tumor tissue drives BT5528 activity in tumor models

Abstract BT5528 was developed as a Bicycle® toxin conjugate to deliver monomethyl auristatin E (MMAE) -payload to EphA2 overexpressing tumors. It consists of a bicyclic peptide targeting the tumor antigen EphA2, linked to the cytotoxin MMAE via a molecular spacer and cleavable linker. Administration of BT5528 results in rapid uptake of payload into EphA2 overexpressing xenograft tumors associated with persistent toxin levels in tumor tissue and limited systemic exposure of both parent drug and payload (1). BT5528 showed a favorable preclinical profile supporting the initiation of a first-in-human Phase I/II study (NCT04180371) to investigate safety and efficacy of BT5528 in indications with evidence of EphA2 expression including non-small-cell lung cancer (NSCLC), ovarian cancer, triple-negative breast cancer (TNBC), gastric/upper gastrointestinal (GI), pancreatic and urothelial cancers (2). We have used EphA2 overexpressing tumor xenograft models in both mice and rats to elucidate the relative differences between toxin payload delivery and systemic exposure of BT5528. Comparison of payload-delivery and systemic exposure from EphA2 -targeted Bicycle toxin conjugate and antibody-drug conjugate (ADC) demonstrate the differentiation of these toxin-payload delivery platforms. ADC payload delivery depends upon a sustained plateau-like pharmacokinetic (PK) profile that exposes target and potentially nontarget organs to intact ADC for several days. In contrast, BT5528 achieves prolonged toxin delivery to tumors, following transient exposure in plasma of less than one hour. A Phase I/II study of BT5528 in patients with solid tumors is ongoing, which will investigate if this PK novel, differentiated pharmacokinetic profile, unique to Bicycle toxin conjugates, gives rise to a favorable balance of efficacy and safety.

Abstract 391: Molecular-based enrichment strategy for Nectin-4 targeted Bicycle toxin conjugate BT8009

Abstract Introduction: BT8009 consists of a bicyclic peptide targeting the tumor antigen Nectin-4, linked to the cytotoxin monomethyl auristatin E (MMAE) via a molecular spacer and cleavable linker. BT8009 is currently being investigated in a Phase 1/2 clinical trial (BT8009-100, NCT04561362) in relapsed and/or refractory solid tumor patients. Nectin-4 positive tumor tissue will be a requirement for enrolment eligibility (with the exception of bladder cancer) starting in dose escalation level 3. Nectin-4 positivity will be determined by IHC on tumor tissue, requiring either tumor membrane or tumor cytoplasmic H-score ≥ 100. Methods: Nectin-4 is not included on commercial NGS panels or routinely screened by IHC. Therefore, the work reported here aimed to identify a routinely measured molecular surrogate of tumor Nectin-4 protein expression (i.e. somatic mutation or gene amplification) that could potentially increase patient screening frequency, yield, and likelihood of response to BT8009. Here we demonstrate that Nectin-4 copy number is associated with Nectin-4 mRNA expression in 9 cancer indications in TCGA including breast, bladder, and lung cancer. In addition, across >30 cancer indications in TCGA, copy number of Nectin-4 is highly correlated with SDHC copy number (these two genes are ~225 kb apart on 1q23), which is included on multiple commercially available NGS panels. Taken together, these results suggest that the existence of an SDHC amplification could be used as an enrichment tool to identify patients with Nectin-4 positive tumors. To test this hypothesis, we obtained 100 TNBC human tumor samples and generated data to determine Nectin-4 and SDHC copy number as well as Nectin-4 protein expression status by IHC. Results: SDHC and Nectin-4 copy number were observed to be highly positively correlated (Spearman's Rho=0.93). Furthermore, all (N = 22) TNBC tumors with a Nectin-4 copy number ≥ 3 were identified as positive for Nectin-4 protein expression and were at or above the H-score threshold required for enrolment to BT8009-100. In contrast, within the subset of tumors that were diploid for Nectin-4/SDHC (n=30), there were both Nectin-4 positive and negative tumors. This suggests that the presence of SDHC/Nectin-4 amplification can be used to increase the probability of identifying Nectin-4 expressing tumors. Conclusions: We demonstrate the potential utility of using pre-existing patient NGS data with SDHC amplifications to increase the frequency of identifying patients with high Nectin-4 expressing tumors via IHC. The use of this strategy is ongoing at all current sites enrolling patients to BT8009-100. Citation Format: Tara Gelb, Sean Santos, Amy Dickson, Carly Campbell, Mike Rigby, Andrew J. Mckenzie, Dan Rozelle, Stephen J. Blakemore. Molecular-based enrichment strategy for Nectin-4 targeted Bicycle toxin conjugate BT8009 [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 391.

Association of combined phase I/II study of a novel bicyclic peptide and MMAE conjugate BT8009 in patients with advanced malignancies with Nectin-4 expression.

TPS2668 Background: BT8009 is a Bicycle Toxin Conjugate (BTC), a novel class of chemically synthesized molecules, comprising a bicyclic peptide targeting Nectin-4 tumor antigen, linked to cytotoxin (monomethyl auristatin E [MMAE]) via a valine-citrulline (val-cit) cleavable linker. Nectins (Nectin-1, -2, -3, and -4) and nectin-like molecules (Necl) are Ca2+ independent immunoglobulin-like cell adhesion molecules. Recent studies have shown the importance of Nectin-4 in several human cancers, including lung, ovarian, breast and bladder cancer; however, the precise roles and clinical relevance of Nectin-4 in tumors remain largely unknown. The Nectin-4 targeted enfortumab vedotin, linked to MMAE via a val-cit linker, is highly active in late-stage bladder cancer and demonstrates notable additional clinical activity as a single agent and in combination with pembrolizumab1. Skin toxicities, bone marrow suppression, peripheral neuropathy and diabetes have been associated with enfortumab, with some of these toxicities already noted with MMAE-bearing antibody therapies. We anticipate a similar toxicity profile for BT8009 in clinical studies. BT8009 exhibited a favorable preclinical profile and was effective in a range of cell-derived xenograph tumor models. Methods: Study BT8009-100 (NCT04561362) will evaluate safety and tolerability of weekly and every other week BT8009 administration, alone and in combination with q4w nivolumab. Determination of both a realistic phase 2 dose and a sequence will also be key to further exploration of safety and efficacy signals, along with an early examination of the role of baseline immunohistochemistry-ascertained levels of tumor Nectin-4. Patients will be recruited with advanced solid tumors associated with Nectin-4 expression after exhausting SOC options (i.e., bladder, breast, pancreatic, head and neck, gastric, esophageal and ovarian). Patients must have available tumor tissue, acceptable hematologic and other critical organ function and be willing to participate. Appropriate ethical and regulatory approvals and advice will be in place and adhered to. Exclusion criteria include uncontrolled brain metastases, uncontrolled hypertension, concomitant CYP3A4 inhibitors and significant history of autoimmune disease for the nivolumab cohorts. PK serial collections will be taken on D1 through D15. Radiologic tumor assessments for response per RECIST will be taken every two months. 1. Enfortumab Vedotin. FDA_data. 761137Orig1s000MultiDiscliplineR.pdf (fda.gov). Clinical trial information: NCT04561362.

Abstract 5300: A survey of EphA2 expression by immunohistochemistry (IHC) in tumor tissue microarrays (TMAs) to support BT5528 indication selection

Abstract Introduction: BT5528 is a Bicycle® Toxin Conjugate (BTC) which contains an EphA2 binding peptide (Bicycle) conjugated through a molecular spacer and cleavable linker to the antimitotic toxin MMAE. EphA2 is an attractive tumor target; overexpression has been reported in multiple tumor types. Preclinical activity of BT5528 has been described previously, demonstrating increased efficacy is associated with high EphA2 tumor cell expression levels in xenograft models. Enrollment to a Phase I dose escalation study with BT5528 (BT5528-100) has started in patients with advanced solid tumors associated with EphA2 expression. Here we describe the analysis of TMAs stained using an EphA2 IHC assay developed for retrospective analysis of archive tumor from the BT5528-100 clinical trial. Methods: A clinical grade EphA2 IHC assay was developed on the Dako platform using R&D Systems EphA2 primary antibody (AF3035) at 10 µg/mL and the Dako FLEX detection kit. TMAs of indications reported to have high EphA2 including pancreatic, bladder, head & neck, stomach, NSCLC, TNBC, and ovarian cancer were stained and scored for EphA2 expression. H-scores (the product of stain intensity on a scale of 0-3 and % positive tumor cells) were generated by a pathologist independently for tumor cell membrane (TM) and tumor cytoplasm (TC). H-scores ≥20 were considered positive for EphA2. Results: The pattern of EphA2 expression was different across indications tested with pancreas having the greatest frequency of EphA2 expression. In contrast to the other indications assessed, pancreas and bladder had a greater percentage of cores positive for TM over TC. These differences may be relevant for BT5528 indication selection given that the mechanism of BTCs may be enhanced in cases which are TM positive. Conclusion: Use of an IHC assay assessing expression of EphA2 across multiple TMAs and scoring TM and TC individually may help guide indication selection for the BT5528 clinical program. EphA2 expression by IHC in multiple indicationsIndicationTotal cores (N)TM Positive N (%)TC Positive N(%)TM or TC Positive N (%)Pancreatic9248 (52)14 (15)48 (52)Bladder14242 (30)37 (26)51 (36)Head & Neck6914 (20)17 (25)22 (32)Stomach5310 (19)15 (28)15 (28)NSCLC22145 (20)52 (24)60 (27)Ovarian838 (10)13 (16)17 (20)TNBC1243 (2)25 (20)25 (20) Citation Format: Carly T. Campbell, Robert Smale, James Lee, Josette William Ragheb, Michael Rigby, Tara Gelb, Lisa Mahnke, Gavin Bennett, Stephen J. Blakemore. A survey of EphA2 expression by immunohistochemistry (IHC) in tumor tissue microarrays (TMAs) to support BT5528 indication selection [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5300.

MMAE Delivery Using the Bicycle Toxin Conjugate BT5528.

The EphA2 receptor is found at high levels in tumors and low levels in normal tissue and high EphA2 expression in biopsies is a predictor of poor outcome in patients. Drug discovery groups have therefore sought to develop EphA2-based therapies using small molecule, peptide, and nanoparticle-based approaches (1-3). However, until now only EphA2-targeting antibody-drug conjugates (ADC) have entered clinical development. For example, MEDI-547 is an EphA2-targeting ADC that displayed encouraging antitumor activity in preclinical models and progressed to phase I clinical testing in man. Here we describe the development of BT5528, a bicyclic peptide ("Bicycle") conjugated to the auristatin derivative maleimidocaproyl-monomethyl auristatin E to generate the Bicycle toxin conjugate BT5528. The report compares and contrasts the Pharmacokinetics (PK) characteristics of antibody and Bicycle-based targeting systems and discusses how the PK and payload characteristics of different delivery systems impact the efficacy-toxicity trade off which is key to the development of successful cancer therapies. We show that BT5528 gives rise to rapid update into tumors and fast renal elimination followed by persistent toxin levels in tumors without prolonged exposure of parent drug in the vasculature. This fast in, fast out kinetics gave rise to more favorable toxicology findings in rats and monkeys than were observed with MEDI-547 in preclinical and clinical studies.Graphical Abstract: http://mct.aacrjournals.org/content/molcanther/19/7/1385/F1.large.jpg.

BT5528-100 phase I/II study of the safety, pharmacokinetics, and preliminary clinical activity of BT5528 in patients with advanced malignancies associated with EphA2 expression.

TPS3655 Background: BT5528 is a Bicycle Toxin Conjugate (BTC), comprising a bicyclic peptide targeting the tumor antigen EphA2, linked to a cytotoxin (monomethyl auristatin E [MMAE]) via a tumor microenvironment cleavable linker. Bicycles are a novel class of chemically synthesized constrained peptides, developed by Bicycle Therapeutics. EphA2 is reported to be overexpressed in a range of solid tumors, contributes to oncogenesis, tumor-associated angiogenesis and metastasis. Intracellular EphA2 signaling converges on pathways that are integral to cell growth, proliferation, migration and invasion. Increased EphA2 expression has been identified as a resistance mechanism to EGFR Tyrosine Kinase Inhibitor based therapy. BT5528 mechanism of action is dependent on tumor penetration, target binding and release of MMAE toxin payload. BTCs offer advantages over antibody-toxin conjugates exhibiting rapid penetration of dense tumors and decreased extra-tumor exposure. BT5528 exhibited a favorable preclinical profile supporting the initiation of a first-in-human study to investigate safety and efficacy of BT5528 in indications with evidence of EphA2 expression including non-small-cell lung cancer (NSCLC), ovarian cancer, triple-negative breast cancer (TNBC), gastric/upper gastrointestinal (GI), pancreatic and urothelial cancers. Methods: BT5528-100 (NCT04180371) is a Ph I/II study to evaluate safety and tolerability of weekly BT5528 alone and in combination with Q4W nivolumab. Each dose escalation utilizes a 3+3 design which converts to a Bayesian design to determine MTD or MAD and RP2D for BT5528 with and without nivolumab. Eligible patients must have advanced solid tumors associated with EphA2 expression which have recurred after exhausting standard treatment options. Patients must have available tumor tissue and acceptable hematologic and organ function, with exclusions for uncontrolled brain metastases, thromboembolic events, bleeding disorders, uncontrolled hypertension, CYP3A4 inhibitors/inducers or, for the nivolumab cohorts, autoimmune disease. On-study tumor and blood samples will be collected for biomarker evaluations including tumor EphA2 expression, ADA, and candidate response biomarkers for BT5528 alone and combination with nivolumab. Pharmacokinetic data will be reported for C1D1 and D15 for BT5528 and MMAE. The expansion phase will enroll specific tumor types to evaluate clinical activity of BT5528. Enrollment is ongoing. Clinical trial information: NCT04180371 .