Integrin-αvß6 targeted peptide-toxin therapy in a novel αvß6-expressing immunocompetent model of pancreatic cancer.

Previously we reported that a novel αvß6-specific peptide-drug conjugate (SG3299) could eliminate established human pancreatic ductal adenocarcinoma (PDAC) xenografts. However the development of effective therapies for PDAC, which is an essential need, must show efficacy in relevant immunocompetent animals. Previously we reported that the KPC mouse transgenic PDAC model that closely recapitulates most stages of development of human PDAC, unlike in humans, failed to express αvß6 on their tumours or metastases. In this study we have taken the KPC-derived PDAC line TB32043 and engineered a variant line (TB32043mb6S2) that expresses mouse integrin αvß6. We report that orthotopic implantation of the αvß6 over-expressing TB32043mb6S2 cells promotes shorter overall survival and increase in metastases. Moreover, systemic treatment of mice with established TB32043mb6S2 tumours in the pancreas with SG2399 lived significantly longer (p < 0.001; mean OS 48d) compared with PBS or control SG3511 (mean OS 25.5d and 26d, respectively). Thus SG3299 is confirmed as a promising candidate therapeutic for the therapy of PDAC.

ADCT-602, a Novel PBD Dimer-containing Antibody-Drug Conjugate for Treating CD22-positive Hematologic Malignancies.

Relapsed or refractory B-cell acute lymphoblastic leukemia (R/R B-ALL) and lymphomas have poor patient outcomes; novel therapies are needed. CD22 is an attractive target for antibody-drug conjugates (ADCs), being highly expressed in R/R B-ALL with rapid internalization kinetics. ADCT-602 is a novel CD22-targeting ADC, consisting of humanized mAb hLL2-C220, site specifically conjugated to the pyrrolobenzodiazepine dimer-based payload tesirine. In preclinical studies, ADCT-602 demonstrated potent, specific cytotoxicity in CD22-positive lymphomas and leukemias. ADCT-602 was specifically bound, internalized, and trafficked to lysosomes in CD22-positive tumor cells; after cytotoxin release, DNA interstrand crosslink formation persisted for 48 hours. In the presence of CD22-positive tumor cells, ADCT-602 caused bystander killing of CD22-negative tumor cells. A single ADCT-602 dose led to potent, dose-dependent, in vivo antitumor activity in subcutaneous and disseminated human lymphoma/leukemia models. Pharmacokinetic analyses (rat and cynomolgus monkey) showed excellent stability and tolerability of ADCT-602. Cynomolgus monkey B cells were efficiently depleted from circulation after one dose. Gene signature association analysis revealed IRAK1 as a potential marker for ADCT-602 resistance. Combining ADCT-602 + pacritinib was beneficial in ADCT-602-resistant cells. Chidamide increased CD22 expression on B-cell tumor surfaces, increasing ADCT-602 activity. These data support clinical testing of ADCT-602 in R/R B-ALL (NCT03698552) and CD22-positive hematologic cancers.

Comparison of Pyrrolobenzodiazepine Dimer Bis-imine versus Mono-imine: DNA Interstrand Cross-linking, Cytotoxicity, Antibody-Drug Conjugate Efficacy and Toxicity.

Antibody-drug conjugates (ADC) delivering pyrrolobenzodiazepine (PBD) DNA cross-linkers are currently being evaluated in clinical trials, with encouraging results in Hodgkin and non-Hodgkin lymphomas. The first example of an ADC delivering a PBD DNA cross-linker (loncastuximab tesirine) has been recently approved by the FDA for the treatment of relapsed and refractory diffuse large B-cell lymphoma. There has also been considerable interest in mono-alkylating PBD analogs. We conducted a head-to-head comparison of a conventional PBD bis-imine and a novel PBD mono-imine. Key Mitsunobu chemistry allowed clean and convenient access to the mono-imine class. Extensive DNA-binding studies revealed that the mono-imine mediated a type of DNA interaction that is described as "pseudo cross-linking," as well as alkylation. The PBD mono-imine ADC demonstrated robust antitumor activity in mice bearing human tumor xenografts at doses 3-fold higher than those that were efficacious for the PBD bis-imine ADC. A single-dose toxicology study in rats demonstrated that the MTD of the PBD mono-alkylator ADC was approximately 3-fold higher than that of the ADC bearing a bis-imine payload, suggesting a comparable therapeutic index for this molecule. However, although both ADCs caused myelosuppression, renal toxicity was observed only for the bis-imine, indicating possible differences in toxicologic profiles that could influence tolerability and therapeutic index. These data show that mono-amine PBDs have physicochemical and pharmacotoxicologic properties distinct from their cross-linking analogs and support their potential utility as a novel class of ADC payload.

PANTHER: AZD8931, inhibitor of EGFR, ERBB2 and ERBB3 signalling, combined with FOLFIRI: a Phase I/II study to determine the importance of schedule and activity in colorectal cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) is a therapeutic target to which HER2/HER3 activation may contribute resistance. This Phase I/II study examined the toxicity and efficacy of high-dose pulsed AZD8931, an EGFR/HER2/HER3 inhibitor, combined with chemotherapy, in metastatic colorectal cancer (CRC). METHODS: Treatment-naive patients received 4-day pulses of AZD8931 with irinotecan/5-FU (FOLFIRI) in a Phase I/II single-arm trial. Primary endpoint for Phase I was dose limiting toxicity (DLT); for Phase II best overall response. Samples were analysed for pharmacokinetics, EGFR dimers in circulating exosomes and Comet assay quantitating DNA damage. RESULTS: Eighteen patients received FOLFIRI and AZD8931. At 160 mg bd, 1 patient experienced G3 DLT; 160 mg bd was used for cohort expansion. No grade 5 adverse events (AE) reported. Seven (39%) and 1 (6%) patients experienced grade 3 and grade 4 AEs, respectively. Of 12 patients receiving 160 mg bd, best overall response rate was 25%, median PFS and OS were 8.7 and 21.2 months, respectively. A reduction in circulating HER2/3 dimer in the two responding patients after 12 weeks treatment was observed. CONCLUSIONS: The combination of pulsed high-dose AZD8931 with FOLFIRI has acceptable toxicity. Further studies of TKI sequencing may establish a role for pulsed use of such agents rather than continuous exposure. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov number: NCT01862003.

Efficacy, Tolerability, and Pharmacokinetic Studies of Antibody-Drug Conjugates Containing a Low-Potency Pyrrolobenzodiazepine Dimer.

Antibody-drug conjugate (ADC) research has typically focused on the release of highly potent cytotoxic agents to achieve antitumor efficacy. However, recently approved ADCs trastuzumab deruxtecan and sacituzumab govitecan release lower-potency topoisomerase inhibitors. This has prompted interest in ADCs that release lower-potency cytotoxic drugs to potentially enhance therapeutic index and reduce unwanted toxicity. Pyrrolobenzodiazepine (PBD) dimer ADCs have been widely investigated in human clinical trials, which have focused on high-potency PBDs. In this study, we evaluated five ADCs that release the low-potency PBD dimer SG3650. The relatively low clogD for this agent facilitated higher drug-to-antibody ratio (DAR) conjugation without the need for antibody engineering or functionalization of the drug. The rank order of potency for DAR 2 site-specific ADCs (conjugated at the C239i position) matched the order for the corresponding free drugs in vitro. Despite free drug SG3650 being inactive in vivo, the DAR 2 ADCs derived from the corresponding drug-linker SG3584 showed antitumor efficacy in solid (anti-HER2) and hematologic (anti-CD22) xenograft models. Antitumor activity could be enhanced by conjugating SG3584 to trastuzumab at higher DARs of 4 and 8 and by adjusting dosing and schedule. Higher-DAR conjugates were stable and displayed good rat pharmacokinetic profiles as measured by ELISA and LC/MS-MS. A single intravenous dose of isotype control SG3584 DAR 2 ADC resulted in no mortality in rats or monkeys at doses of up to 25 and 30 mg/kg, respectively. These findings suggest that further investigations of low-potency PBD dimers in ADCs that target hematologic and solid tumors are warranted.

Phase 0 Study of Vandetanib-Eluting Radiopaque Embolics as a Preoperative Embolization Treatment in Patients with Resectable Liver Malignancies.

PURPOSE: To assess the safety and tolerability of a vandetanib-eluting radiopaque embolic (BTG-002814) for transarterial chemoembolization (TACE) in patients with resectable liver malignancies. MATERIALS AND METHODS: The VEROnA clinical trial was a first-in-human, phase 0, single-arm, window-of-opportunity study. Eligible patients were aged ≥18 years and had resectable hepatocellular carcinoma (HCC) (Child-Pugh A) or metastatic colorectal cancer (mCRC). Patients received 1 mL of BTG-002814 transarterially (containing 100 mg of vandetanib) 7-21 days prior to surgery. The primary objectives were to establish the safety and tolerability of BTG-002814 and determine the concentrations of vandetanib and the N-desmethyl vandetanib metabolite in the plasma and resected liver after treatment. Biomarker studies included circulating proangiogenic factors, perfusion computed tomography, and dynamic contrast-enhanced magnetic resonance imaging. RESULTS: Eight patients were enrolled: 2 with HCC and 6 with mCRC. There was 1 grade 3 adverse event (AE) before surgery and 18 after surgery; 6 AEs were deemed to be related to BTG-002814. Surgical resection was not delayed. Vandetanib was present in the plasma of all patients 12 days after treatment, with a mean maximum concentration of 24.3 ng/mL (standard deviation ± 13.94 ng/mL), and in resected liver tissue up to 32 days after treatment (441-404,000 ng/g). The median percentage of tumor necrosis was 92.5% (range, 5%-100%). There were no significant changes in perfusion imaging parameters after TACE. CONCLUSIONS: BTG-002814 has an acceptable safety profile in patients before surgery. The presence of vandetanib in the tumor specimens up to 32 days after treatment suggests sustained anticancer activity, while the low vandetanib levels in the plasma suggest minimal release into the systemic circulation. Further evaluation of this TACE combination is warranted in dose-finding and efficacy studies.

Preclinical Development of ADCT-601, a Novel Pyrrolobenzodiazepine Dimer-based Antibody-drug Conjugate Targeting AXL-expressing Cancers.

AXL, a tyrosine kinase receptor that is overexpressed in many solid and hematologic malignancies, facilitates cancer progression and is associated with poor clinical outcomes. Importantly, drug-induced expression of AXL results in resistance to conventional chemotherapy and targeted therapies. Together with its presence on multiple cell types in the tumor immune microenvironment, these features make it an attractive therapeutic target for AXL-expressing malignancies. ADCT-601 (mipasetamab uzoptirine) is an AXL-targeted antibody-drug conjugate (ADC) comprising a humanized anti-AXL antibody site specifically conjugated using GlycoConnect technology to PL1601, which contains HydraSpace, a Val-Ala cleavable linker and the potent pyrrolobenzodiazepine (PBD) dimer cytotoxin SG3199. This study aimed to validate the ADCT-601 mode of action and evaluate its efficacy in vitro and in vivo, as well as its tolerability and pharmacokinetics. ADCT-601 bound to both soluble and membranous AXL, and was rapidly internalized by AXL-expressing tumor cells, allowing release of PBD dimer, DNA interstrand cross-linking, and subsequent cell killing. In vivo, ADCT-601 had potent and durable antitumor activity in a wide variety of human cancer xenograft mouse models, including patient-derived xenograft models with heterogeneous AXL expression where ADCT-601 antitumor activity was markedly superior to an auristatin-based comparator ADC. Notably, ADCT-601 had antitumor activity in a monomethyl auristatin E-resistant lung-cancer model and synergized with the PARP inhibitor olaparib in a BRCA1-mutated ovarian cancer model. ADCT-601 was well tolerated at doses of up to 6 mg/kg and showed excellent stability in vivo. These preclinical results warrant further evaluation of ADCT-601 in the clinic.

hMOB2 deficiency impairs homologous recombination-mediated DNA repair and sensitises cancer cells to PARP inhibitors.

Monopolar spindle-one binder (MOBs) proteins are evolutionarily conserved and contribute to various cellular signalling pathways. Recently, we reported that hMOB2 functions in preventing the accumulation of endogenous DNA damage and a subsequent p53/p21-dependent G1/S cell cycle arrest in untransformed cells. However, the question of how hMOB2 protects cells from endogenous DNA damage accumulation remained enigmatic. Here, we uncover hMOB2 as a regulator of double-strand break (DSB) repair by homologous recombination (HR). hMOB2 supports the phosphorylation and accumulation of the RAD51 recombinase on resected single-strand DNA (ssDNA) overhangs. Physiologically, hMOB2 expression supports cancer cell survival in response to DSB-inducing anti-cancer compounds. Specifically, loss of hMOB2 renders ovarian and other cancer cells more vulnerable to FDA-approved PARP inhibitors. Reduced MOB2 expression correlates with increased overall survival in patients suffering from ovarian carcinoma. Taken together, our findings suggest that hMOB2 expression may serve as a candidate stratification biomarker of patients for HR-deficiency targeted cancer therapies, such as PARP inhibitor treatments.

Durable Responses and Low Toxicity After Fast Off-Rate CD19 Chimeric Antigen Receptor-T Therapy in Adults With Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia.

PURPOSE: Prognosis for adult B-cell acute lymphoblastic leukemia (B-ALL) is poor, and there are currently no licensed CD19 chimeric antigen receptor (CAR) therapeutics. We developed a novel second-generation CD19-CAR (CAT19-41BB-Z) with a fast off rate, designed for more physiologic T-cell activation to reduce toxicity and improve engraftment. We describe the multicenter phase I ALLCAR19 (NCT02935257) study of autologous CAT19-41BB-Z CAR T cells (AUTO1) in relapsed or refractory (r/r) adult B-ALL. METHODS: Patients age ≥ 16 years with r/r B-ALL were eligible. Primary outcomes were toxicity and manufacturing feasibility. Secondary outcomes were depth of response at 1 and 3 months, persistence of CAR-T, incidence and duration of hypogammaglobulinemia and B-cell aplasia, and event-free survival and overall survival at 1 and 2 years. RESULTS: Twenty-five patients were leukapheresed, 24 products were manufactured, and 20 patients were infused with AUTO1. The median age was 41.5 years; 25% had prior blinatumomab, 50% prior inotuzumab ozogamicin, and 65% prior allogeneic stem-cell transplantation. At the time of preconditioning, 45% had ≥ 50% bone marrow blasts. No patients experienced ≥ grade 3 cytokine release syndrome; 3 of 20 (15%) experienced grade 3 neurotoxicity that resolved to ≤ grade 1 within 72 hours with steroids. Seventeen of 20 (85%) achieved minimal residual disease-negative complete response at month 1, and 3 of 17 underwent allogeneic stem-cell transplantation while in remission. The event-free survival at 6 and 12 months was 68.3% (42.4%-84.4%) and 48.3% (23.1%-69.7%), respectively. High-level expansion (Cmax 127,152 copies/µg genomic DNA) and durable CAR-T persistence were observed with B-cell aplasia ongoing in 15 of 20 patients at last follow-up. CONCLUSION: AUTO1 demonstrates a tolerable safety profile, high remission rates, and excellent persistence in r/r adult B-ALL. Preliminary data support further development of AUTO1 as a stand-alone treatment for r/r adult B-ALL.

Atomic force microscopy-A tool for structural and translational DNA research.

Atomic force microscopy (AFM) is a powerful imaging technique that allows for structural characterization of single biomolecules with nanoscale resolution. AFM has a unique capability to image biological molecules in their native states under physiological conditions without the need for labeling or averaging. DNA has been extensively imaged with AFM from early single-molecule studies of conformational diversity in plasmids, to recent examinations of intramolecular variation between groove depths within an individual DNA molecule. The ability to image dynamic biological interactions in situ has also allowed for the interaction of various proteins and therapeutic ligands with DNA to be evaluated-providing insights into structural assembly, flexibility, and movement. This review provides an overview of how innovation and optimization in AFM imaging have advanced our understanding of DNA structure, mechanics, and interactions. These include studies of the secondary and tertiary structure of DNA, including how these are affected by its interactions with proteins. The broader role of AFM as a tool in translational cancer research is also explored through its use in imaging DNA with key chemotherapeutic ligands, including those currently employed in clinical practice.

Whole-genome sequencing of single circulating tumor cells from neuroendocrine neoplasms.

Single-cell profiling of circulating tumor cells (CTCs) as part of a minimally invasive liquid biopsy presents an opportunity to characterize and monitor tumor heterogeneity and evolution in individual patients. In this study, we aimed to compare single-cell copy number variation (CNV) data with tissue and define the degree of intra- and inter-patient genomic heterogeneity. We performed next-generation sequencing (NGS) whole-genome CNV analysis of 125 single CTCs derived from seven patients with neuroendocrine neoplasms (NEN) alongside matched white blood cells (WBC), formalin-fixed paraffin-embedded (FFPE), and fresh frozen (FF) samples. CTC CNV profiling demonstrated recurrent chromosomal alterations in previously reported NEN copy number hotspots, including the prognostically relevant loss of chromosome 18. Unsupervised hierarchical clustering revealed CTCs with distinct clonal lineages as well as significant intra- and inter-patient genomic heterogeneity, including subclonal alterations not detectable by bulk analysis and previously unreported in NEN. Notably, we also demonstrated the presence of genomically distinct CTCs according to the enrichment strategy utilized (EpCAM-dependent vs size-based). This work has significant implications for the identification of therapeutic targets, tracking of evolutionary change, and the implementation of CTC-biomarkers in cancer.

Effects of N-terminus modified Hx-amides on DNA binding affinity, sequence specificity, cellular uptake, and gene expression.

Five X-HxIP (Hx-amides) 6a-e, in which the N-terminus p-anisyl moiety is modified, were designed and synthesised with the purpose of optimising DNA binding, improving cellular uptake/nuclear penetration, and enhancing the modulation of the topoisomerase IIα (TOP2A) gene expression. The modifications include a fluorophenyl group and other heterocycles bearing different molecular shapes, size, and polarity. Like their parent compound HxIP 3, all five X-HxIP analogues bind preferentially to their cognate sequence 5'-TACGAT-3', which is found embedded on the 5' flank of the inverted CCAAT box-2 (ICB2) site in the TOP2A gene promoter, and inhibit protein complex binding. Interestingly, the 4-pyridyl analog 6a exhibits greater binding affinity for the target DNA sequence and abolishes the protein:ICB2 interaction in vitro, at a lower concentration, compared to the prototypical compound HxIP 3. Analogues 6b-e, display improved DNA sequence specificity, but reduced binding affinity for the cognate sequence, relative to the unmodified HxIP 3, with polyamides 6b and 6e being the most sequence selective. However, unlike 3 and 6b, 6a was unable to enter cells, access the nucleus and thereby affect TOP2A gene expression in confluent human lung cancer cells. These results show that while DNA binding affinity and sequence selectivity are important, consideration of cellular uptake and concentration in the nucleus are critical when exerting biological activity is the desired outcome. By characterising the DNA binding, cellular uptake and gene regulatory properties of these small molecules, we can elucidate the determinants of the elicited biological activity, which can be impacted by even small structural modifications in the polyamide molecular design.

Antibody-drug conjugates (ADCs) delivering pyrrolobenzodiazepine (PBD) dimers for cancer therapy.

INTRODUCTION: The rationally designed pyrrolobenzodiazepine (PBD) dimers emerged around ten years ago as a new class of drug component for antibody-drug conjugates (ADC). They produce highly cytotoxic DNA cross-links, exploiting a completely different cellular target to the auristatin and maytansinoid tubulin inhibitor classes and a different mode of DNA damage to other DNA interacting warheads such as calicheamicin. AREAS COVERED: The properties which make the PBD dimers suitable warheads for ADCs, and the development of the two main payload structures talirine and tesirine, are discussed. The clinical experience with the twenty PBD dimer-containing ADCs to enter the clinic is reviewed, with a focus on vadastuximab talirine and rovalpituzumab tesirine, both of which were discontinued following pivotal studies, and loncastuximab tesirine and camidanlumab tesirine which are progressing towards approval. EXPERT OPINION: Reviewing the clinical efficacy and safety data from almost forty clinical trials of PBD dimer-containing ADCs highlights the complexities and challenges of ADC early clinical development. It enables some conclusions to be made about reasons for failure and suggests strategies to optimise the future clinical development of this promising class of ADCs in a rapidly expanding field.

Prognostic Threshold for Circulating Tumor Cells in Patients With Pancreatic and Midgut Neuroendocrine Tumors.

BACKGROUND: Circulating tumor cells (CTCs) are detectable in patients with neuroendocrine tumors (NETs) and are accurate prognostic markers although the optimum threshold has not been defined. OBJECTIVE: This work aims to define optimal prognostic CTC thresholds in PanNET and midgut NETs. PATIENTS AND METHODS: CellSearch was used to enumerate CTCs in 199 patients with metastatic pancreatic (PanNET) (90) or midgut NETs (109). Patients were followed for progression-free survival (PFS) and overall survival (OS) for a minimum of 3 years or until death. RESULTS: The area under the receiver operating characteristic curve (AUROC) for progression at 12 months in PanNETs and midgut NETs identified the optimal CTC threshold as 1 or greater and 2 or greater, respectively. In multivariate logistic regression analysis, these thresholds were predictive for 12-month progression with an odds ratio (OR) of 6.69 (P < .01) for PanNETs and 5.88 (P < .003) for midgut NETs. The same thresholds were found to be optimal for predicting death at 36 months, with an OR of 2.87 (P < .03) and 5.09 (P < .005) for PanNETs and midgut NETs, respectively. In multivariate Cox hazard regression analysis for PFS in PanNETs, 1 or greater CTC had a hazard ratio (HR) of 2.6 (P < .01), whereas 2 or greater CTCs had an HR of 2.25 (P < .01) in midgut NETs. In multivariate analysis OS in PanNETs, 1 or greater CTCs had an HR of 3.16 (P < .01) and in midgut NETs, 2 or greater CTCs had an HR of 1.73 (P < .06). CONCLUSIONS: The optimal CTC threshold to predict PFS and OS in metastatic PanNETs and midgut NETs is 1 and 2, respectively. These thresholds can be used to stratify patients in clinical practice and clinical trials.

The Role of Specific ATP-Binding Cassette Transporters in the Acquired Resistance to Pyrrolobenzodiazepine Dimer-Containing Antibody-Drug Conjugates.

Antibody-drug conjugates (ADC) containing pyrrolobenzodiazepine (PBD) dimers are being evaluated clinically in both hematologic and solid tumors. These include ADCT-301 (camidanlumab tesirine) and ADCT-402 (loncastuximab tesirine) in pivotal phase II trials that contain the payload tesirine, which releases the PBD dimer warhead SG3199. An important consideration in future clinical development is acquired resistance. The aim was to generate and characterize PBD acquired resistant cell lines in both hematologic and solid tumor settings. Human Karpas-299 (ALCL) and NCI-N87 (gastric cancer) cells were incubated with increasing IC50 doses of ADC (targeting CD25 and HER2, respectively) or SG3199 in a pulsed manner until stable acquired resistance was established. The level of resistance achieved was approximately 3,000-fold for ADCT-301 and 3-fold for SG3199 in Karpas-299, and 8-fold for ADCT-502 and 4-fold for SG3199 in NCI-N87. Cross-resistance between ADC and SG3199, and with an alternative PBD-containing ADC or PBD dimer was observed. The acquired resistant lines produced fewer DNA interstrand cross-links, indicating an upstream mechanism of resistance. Loss of antibody binding or internalization was not observed. A human drug transporter PCR Array revealed several genes upregulated in all the resistant cell lines, including ABCG2 and ABCC2, but not ABCB1(MDR1). These findings were confirmed by RT-PCR and Western blot, and inhibitors and siRNA knockdown of ABCG2 and ABCC2 recovered drug sensitivity. These data show that acquired resistance to PBD-ADCs and SG3199 can involve specific ATP-binding cassette drug transporters. This has clinical implications as potential biomarkers of resistance and for the rational design of drug combinations.

Integrin αvß6-specific therapy for pancreatic cancer developed from foot-and-mouth-disease virus.

Goals of investigation: The 5-year survival rate for pancreatic ductal adenocarcinoma (PDAC) has remained at <5% for decades because no effective therapies have been identified. Integrin αvß6 is overexpressed in most PDAC and represents a promising therapeutic target. Thus, we attempted to develop an αvß6-specific peptide-drug conjugate (PDC) for therapy of PDAC. Methodology: We conjugated the DNA-binding pyrrolobenzodiazepine (PBD)-based payload SG3249 (tesirine) to an αvß6-specific 20mer peptide from the VP1 coat protein of foot-and-mouth-disease virus (FMDV) (forming conjugate SG3299) or to a non-targeting peptide (forming conjugate SG3511). PDCs were tested for specificity and toxicity on αvß6-negative versus-positive PDAC cells, patient-derived cell lines from tumor xenografts, and on two different in vivo models of PDAC. Immunohistochemical analyses were performed to establish therapeutic mechanism. Results: The αvß6-targeted PDC SG3299 was significantly more toxic (up to 78-fold) for αvß6-expressing versus αvß6-negative PDAC cell lines in vitro, and achieved significantly higher toxicity at equal dose than the non-targeted PDC SG3511 (up to 15-fold better). Moreover, SG3299 eliminated established (100mm3) Capan-1 PDAC human xenografts, extending the lifespan of mice significantly (P=0.005). Immunohistochemistry revealed SG3299 induced DNA damage and apoptosis (increased γH2AX and cleaved caspase 3, respectively) associated with significant reductions in proliferation (Ki67), ß6 expression and PDAC tumour growth. Conclusions: The FMDV-peptide drug conjugate SG3299 showed αvß6-selectivity in vitro and in vivo and can specifically eliminate αvß6-positive cancers, providing a promising new molecular- specific therapy for pancreatic cancer.

Targeting of EGFR by a combination of antibodies mediates unconventional EGFR trafficking and degradation.

Antibody combinations targeting cell surface receptors are a new modality of cancer therapy. The trafficking and signalling mechanisms regulated by such therapeutics are not fully understood but could underlie differential tumour responses. We explored EGFR trafficking upon treatment with the antibody combination Sym004 which has shown promise clinically. Sym004 promoted EGFR endocytosis distinctly from EGF: it was asynchronous, not accompanied by canonical signalling events and involved EGFR clustering within detergent-insoluble plasma mebrane-associated tubules. Sym004 induced lysosomal degradation independently of EGFR ubiquitylation but dependent upon Hrs/Tsg101 that are required for the formation of intraluminal vesicles (ILVs) within late endosomes. We propose Sym004 cross-links EGFR physically triggering EGFR endocytosis and incorporation onto ILVs and so Sym004 sensitivity correlates with EGFR numbers available for binding, rather than specific signalling events. Consistently Sym004 efficacy and potentiation of cisplatin responses correlated with EGFR surface expression in head and neck cancer cells. These findings will have implications in understanding the mode of action of this new class of cancer therapeutics.

TR1801-ADC: a highly potent cMet antibody-drug conjugate with high activity in patient-derived xenograft models of solid tumors.

cMet is a well-characterized oncogene that is the target of many drugs including small molecule and biologic pathway inhibitors, and, more recently, antibody-drug conjugates (ADCs). However, the clinical benefit from cMet-targeted therapy has been limited. We developed a novel cMet-targeted 'third-generation' ADC, TR1801-ADC, that was optimized at different levels including specificity, stability, toxin-linker, conjugation site, and in vivo efficacy. Our nonagonistic cMet antibody was site-specifically conjugated to the pyrrolobenzodiazepine (PBD) toxin-linker tesirine and has picomolar activity in cancer cell lines derived from different solid tumors including lung, colorectal, and gastric cancers. The potency of our cMet ADC is independent of MET gene copy number, and its antitumor activity was high not only in high cMet-expressing cell lines but also in medium-to-low cMet cell lines (40 000-90 000 cMet/cell) in which a cMet ADC with tubulin inhibitor payload was considerably less potent. In vivo xenografts with low-medium cMet expression were also very responsive to TR1801-ADC at a single dose, while a cMet ADC using a tubulin inhibitor showed a substantially reduced efficacy. Furthermore, TR1801-ADC had excellent efficacy with significant antitumor activity in 90% of tested patient-derived xenograft models of gastric, colorectal, and head and neck cancers: 7 of 10 gastric models, 4 of 10 colorectal cancer models, and 3 of 10 head and neck cancer models showed complete tumor regression after a single-dose administration. Altogether, TR1801-ADC is a new generation cMet ADC with best-in-class preclinical efficacy and good tolerability in rats.

VEROnA Protocol: A Pilot, Open-Label, Single-Arm, Phase 0, Window-of-Opportunity Study of Vandetanib-Eluting Radiopaque Embolic Beads (BTG-002814) in Patients With Resectable Liver Malignancies.

BACKGROUND: Transarterial chemoembolization (TACE) is the current standard of care for patients with intermediate-stage hepatocellular carcinoma (HCC) and is also a treatment option for patients with liver metastases from colorectal cancer. However, TACE is not a curative treatment, and tumor progression occurs in more than half of the patients treated. Despite advances and technical refinements of TACE, including the introduction of drug-eluting beads-TACE, the clinical efficacy of TACE has not been optimized, and improved arterial therapies are required. OBJECTIVE: The primary objectives of the VEROnA study are to evaluate the safety and tolerability of vandetanib-eluting radiopaque embolic beads (BTG-002814) in patients with resectable liver malignancies and to determine concentrations of vandetanib and the N-desmethyl metabolite in plasma and resected liver following treatment with BTG-002814. METHODS: The VEROnA study is a first-in-human, open-label, single-arm, phase 0, window-of-opportunity study of BTG-002814 (containing 100 mg vandetanib) delivered transarterially, 7 to 21 days before surgery in patients with resectable liver malignancies. Eligible patients have a diagnosis of colorectal liver metastases, or HCC (Childs Pugh A), diagnosed histologically or radiologically, and are candidates for liver surgery. All patients are followed up for 28 days following surgery. Secondary objectives of this study are to evaluate the anatomical distribution of BTG-002814 on noncontrast-enhanced imaging, to evaluate histopathological features in the surgical specimen, and to assess changes in blood flow on dynamic contrast-enhanced magnetic resonance imaging following treatment with BTG-002814. Exploratory objectives of this study are to study blood biomarkers with the potential to identify patients likely to respond to treatment and to correlate the distribution of BTG-002814 on imaging with pathology by 3-dimensional modeling. RESULTS: Enrollment for the study was completed in February 2019. Results of a planned interim analysis were reviewed by a safety committee after the first 3 patients completed follow-up. The recommendation of the committee was to continue the study without any changes to the dose or trial design, as there were no significant unexpected toxicities related to BTG-002814. CONCLUSIONS: The VEROnA study is studying the feasibility of administering BTG-002814 to optimize the use of this novel technology as liver-directed therapy for patients with primary and secondary liver cancer. TRIAL REGISTRATION: ClinicalTrial.gov NCT03291379; https://clinicaltrials.gov/ct2/show/NCT03291379. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/13696.