The GDF15-GFRaL-RET signaling complex is involved in a broad range of disease states, with agonistic action of GDF15 affecting metabolism and body weight control, while inhibition is indicated in cancer and wasting disorders like cachexia. Here, we describe the discovery of the peptide inhibitors of the GDF15-GFRaL protein-protein interaction to prevent RET-induced signaling using both a structure-guided design and a phage display approach. Phage display provided bicyclic peptide hits with high affinity for GFRaL, and these were dimerized to mimic the bidentate interaction of homodimeric GDF15. Guided by structural data, the monomeric peptides were converted into tandem Bicycle molecules with picomolar affinities, similar to that of the endogenous GDF15 ligand. These dimerized protein mimetics inhibited cell signaling in a functional assay and showed improved pharmacokinetic properties compared with their monomeric counterparts. This is the first example of a homodimeric Bicycle molecule inhibiting receptor complex formation, thereby antagonizing the intracellular signaling response.

Thymic stromal lymphopoietin (TSLP) is an epithelial-derived pro-inflammatory cytokine involved in the development of asthma and other atopic diseases. We used Bicycle Therapeutics' proprietary phage display platform to identify bicyclic peptides (Bicycles) with high affinity for TSLP, a target that is difficult to drug with conventional small molecules due to the extended protein–protein interactions it forms with both receptors. The hit series was shown to bind to TSLP in a hotspot, that is also used by IL-7Rα. Guided by the first X-ray crystal structure of a small peptide binding to TSLP and the identification of key metabolites, we were able to improve the proteolytic stability of this series in lung S9 fractions without sacrificing binding affinity. This resulted in the potent Bicycle 46 with nanomolar affinity to TSLP (KD = 13 nM), low plasma clearance of 6.4 mL/min/kg, and an effective half-life of 46 min after intravenous dosing to rats.

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

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.

The tumor specific activation of natural killer (NK) cells is an area of active investigation in immune oncology, but to date has relied on complex biologic modalities (e.g., antibodies, fusion proteins, or cell therapies). NK cells are highly responsive immune cells that can detect and eliminate tumor cells and bridge innate to adaptive immune responses. Bicycles® are small (ca.1.5kDa), chemically synthetic, structurally constrained peptides discovered via phage display and optimized using structure-driven design and medicinal chemistry approaches. We have applied the Bicycle platform technology to identify Bicycles® that bind specifically to the key activating receptor, NKp46. When chemically coupled to tumor antigen binding Bicycles, this results in highly potent, antigen-dependent receptor activation and NK cell activation. We term this new class of fully synthetic molecules NK-TICAs and we will describe herein their discovery and evaluation.We demonstrate potent, selective binding of our Bicycles to receptor-expressing cells and the capability of the bifunctional molecule to induce NK cell function in vitro. With Bicycle’s novel NK-TICA™ compound, we demonstrate the engagement of NK cells, the specific activation and function of NK cells, and enhanced tumor cytotoxicity in a tumor target- and dose-dependent manner.In conclusion, NK-TICAs drive NK cell-mediated tumor cell killing and cytokine production in vitro and as such have the potential to catalyze the development of durable anti-tumor immunity in tumor types not well served by current therapies. We hypothesize that utilization of Bicycle NK-TICA™ as a multifunctional immune cell engager will promote the modulation of NK cells, as well as the infiltration and anti-tumor activity of NK cells in solid tumors. The data presented here provide initial proof of concept for the application of our Bicycle technology to drive NK cell-mediated tumor immunity. Citation Format: Fay J. Dufort, Christopher J. Leitheiser, Gemma Mudd, Julia Kristensson, Alexandra Rezvaya, Katie Gaynor, Sandra Uhlenbroich, Liudvikas Urbonas, Heather Scott, Liuhong Chen, Helen Harrison, Michael Skynner, Kevin McDonnell, Philip E. Brandish, Nicholas Keen. Generation of a Bicycle NK-TICA™, a novel NK cell engaging molecule designed to induce targeted tumor cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4233.

Natural killer (NK) cells are immune cells that can detect and eliminate tumor cells and bridge innate to adaptive immune responses. Tumor specific activation of NK cells is thus an area of active investigation in immune oncology, but to date has relied on complex biologic modalities (e.g., antibodies, fusion proteins, or cell therapies), each of which has inherent disadvantages in this application. Thus, alternative approaches are warranted. Bicycle® are small (ca. 1.5 kDa), chemically synthetic, structurally constrained peptides discovered via phage display and optimized using structure-driven design and medicinal chemistry approaches. We have now applied this technology to identify Bicycles that bind specifically to the key activating receptors, NKp46 and CD16a. When chemically coupled to tumor antigen binding Bicycles this results in highly potent, antigen-dependent receptor activation and NK cell activation. We term this new class of fully synthetic molecules Bicycle® natural killer- tumor-targeted immune cell agonists (NK-TICAs™) and we will describe their discovery and evaluation in this presentation.Using our unique phage display screening platform, we have identified high affinity, selective binders to NKp46 and CD16a. By conjugating the Bicycle® NK cell-engaging binders to a model tumor antigen EphA2-binding Bicycle®, we have developed a bifunctional Bicycle NK-TICA™ molecule. In in vitro functional assays, we evaluated the ability of the Bicycle NK-TICAs™ to induce NK cell activation as well as cell-mediated cytotoxicity and cytokine production in NK-tumor co-culture assays.We have developed a novel modular compound with high affinity and selectivity to NK cell receptors with specific tumor targeting capability. We demonstrate potent, selective binding of our Bicycles to receptor-expressing cells and the capability of the bifunctional molecule to induce NK cell function. With Bicycle's novel NK-TICA™ compound, we demonstrate engagement of NK cells, specific activation and function of NK cells, and enhanced EphA2-expressing tumor cytotoxicity, in a dose dependent manner.Bicycle NK-TICAs™ are novel therapeutic agents capable of enhancing the landscape of immune oncology. We hypothesize that utilization of Bicycle NK-TICA™ as a multifunctional immune cell engager will promote modulation of NK cells, and infiltration and anti-tumor activity of NK cells in solid tumors. The data presented here provide initial proof of concept for application of the Bicycle technology to drive NK cell-mediated tumor immunity.

Background In contrast to immune checkpoint inhibitors, the use of antibodies as agonists of immune costimulatory receptors as cancer therapeutics has largely failed. We sought to address this problem using a new class of modular synthetic drugs, termed tumor-targeted immune cell agonists (TICAs), based on constrained bicyclic peptides (Bicycles).Methods Phage libraries displaying Bicycles were panned for binders against tumor necrosis factor (TNF) superfamily receptors CD137 and OX40, and tumor antigens EphA2, Nectin-4 and programmed death ligand 1. The CD137 and OX40 Bicycles were chemically conjugated to tumor antigen Bicycles with different linkers and stoichiometric ratios of binders to obtain a library of low molecular weight TICAs (MW <8 kDa). The TICAs were evaluated in a suite of in vitro and in vivo assays to characterize their pharmacology and mechanism of action.Results Linking Bicycles against costimulatory receptors (e.g., CD137) to Bicycles against tumor antigens (e.g., EphA2) created potent agonists that activated the receptors selectively in the presence of tumor cells expressing these antigens. An EphA2/CD137 TICA (BCY12491) efficiently costimulated human peripheral blood mononuclear cells in vitro in the presence of EphA2 expressing tumor cell lines as measured by the increased secretion of interferon γ and interleukin-2. Treatment of C57/Bl6 mice transgenic for the human CD137 extracellular domain (huCD137) bearing EphA2-expressing MC38 tumors with BCY12491 resulted in the infiltration of CD8+ T cells, elimination of tumors and generation of immunological memory. BCY12491 was cleared quickly from the circulation (plasma t1/2 in mice of 1–2 hr), yet intermittent dosing proved effective.Conclusion Tumor target-dependent CD137 agonism using a novel chemical approach (TICAs) afforded elimination of tumors with only intermittent dosing suggesting potential for a wide therapeutic index in humans. This work unlocks a new path to effective cancer immunotherapy via agonism of TNF superfamily receptors.

The treatment of infection by Gram-negative bacteria is increasingly challenging as resistance to existing antibiotics spreads. Constrained peptides, selected for high target specificity and affinity via library display technologies, are an emerging therapeutic modality in many disease areas and may be a fertile source of new antibiotics. Currently, the utility of constrained peptides and other large molecules as antibiotics is limited by the outer membrane (OM) barrier of Gram-negative bacteria. However, the addition of certain moieties to large molecules can confer the ability to cross the OM; these moieties function as intramolecular trans-OM "vectors". Here, we present a method to systematically assess the carrying capacity of candidate trans-OM vectors using a real-time luminescence assay ("SLALOM", Split Luciferase Assay for Live monitoring of Outer Membrane transit), reporting on periplasmic entry. We demonstrate the usefulness of our tools by constructing a 3800 Da chimeric compound composed of a constrained bicyclic peptide (Bicycle) with a periplasmic target, linked to an intramolecular peptide vector; the resulting chimera is a broad-spectrum inhibitor of pathogenic Gram-negative bacterial growth.

BACKGROUND:Few cross-sectional studies report iron deficiency (ID) prevalence in women of different race/ethnicity and ages in US or Canada. MATERIALS AND METHODS:We evaluated screening observations on women who participated between 2001-2003 in a cross-sectional, primary care-based sample of adults ages ≥25 y whose observations were complete: race/ethnicity; age; transferrin saturation; serum ferritin; and HFE p.C282Y and p.H63D alleles. We defined ID using a stringent criterion: combined transferrin saturation <10% and serum ferritin <33.7 pmol/L (<15 μg/L). We compared ID prevalence in women of different race/ethnicity subgrouped by age and determined associations of p.C282Y and p.H63D to ID overall, and to ID in women ages 25-44 y with or without self-reported pregnancy. RESULTS:These 62,685 women included 27,079 whites, 17,272 blacks, 8,566 Hispanics, 7,615 Asians, 449 Pacific Islanders, 441 Native Americans, and 1,263 participants of other race/ethnicity. Proportions of women with ID were higher in Hispanics and blacks than whites and Asians. Prevalence of ID was significantly greater in women ages 25-54 y of all race/ethnicity groups than women ages ≥55 y of corresponding race/ethnicity. In women ages ≥55 y, ID prevalence did not differ significantly across race/ethnicity. p.C282Y and p.H63D prevalence did not differ significantly in women with or without ID, regardless of race/ethnicity, age subgroup, or pregnancy. CONCLUSIONS:ID prevalence was greater in Hispanic and black than white and Asian women ages 25-54 y. p.C282Y and p.H63D prevalence did not differ significantly in women with or without ID, regardless of race/ethnicity, age subgroup, or pregnancy.

We have identified a Nectin-4 targeting peptide for delivery of cytotoxic agents to Nectin-4 expressing tumors. Nectin-4 is a cell adhesion molecule, that is highly expressed in certain tumor types, including bladder, TNBC and NSCLC, but has a restricted distribution in normal tissue. Bicycles® are small (1.5kDa) fully synthetic, structurally constrained, peptide drugs that combine the affinity of antibodies with the pharmacokinetic properties of small molecules. The Bicycle phage display platform was utilised to rapidly identify a high affinity (18 nM) and highly selective bicyclic Nectin-4 binding peptide. Synthetic modification of the initial lead peptide improved affinity (0.3 nM), hydrophilicity and stability. The optimised peptide is conjugated through an inert sarcosine spacer chain and a cleavable linker to the toxin MMAE, to form the Bicycle Toxin Conjugate (BTC) BT8009. Once bound to cell surface Nectin-4, the linker system is cleaved by peptidases (e.g. cathepsin B) upregulated in the tumor micro environment. Full structure of BT8009 will be disclosed within the presentation. Fluorescence polarisation and surface plasmon resonance show BT8009 has low nanomolar affinity (3nM) for Nectin-4 and high selectivity (>1000 fold) over Nectins 1-3, and the 5 nectin-like, family members. Good affinity for the cognate native protein is maintained across the preclinical safety species. High content imaging of cultures of MDA-MB-468 cells, demonstrates binding of the BTC to cell membrane. In vivo, BT8009 is well tolerated in mouse and rat and shows regressions across a range of cell and patient derived xenograft models. Efficacy correlates with the expression level of Nectin-4 on the tumor cells, and the dose delivered. In the MDA-MB-468 (TNBC) CDX model full tumor regression was seen with weekly i.v. administration of 3 mg/k g i.v. (4 doses), with no tumor regrowth out to 70 days post last dose. Similar efficacy was seen in two NSCLC PDX models (adenocarcinoma and squamous cell carcinoma) with full regression attained in both. Near full regression was seen in a squamous cell esophageal cancer PDX. Larger tumors show a similar degree of sensitivity to the BTC, with rapid tumor regression from a volume of 800 mm3. As a small peptide BT8009 undergoes rapid clearance from the plasma with minimal exposure to non-targeted organs. After a single dose, MMAE has been shown to be retained within tumor tissue in excess of 60 h, at exposure levels significantly greater than corresponding plasma and other tissue levels. Bicycle Toxin Conjugates represent a novel treatment modality for nectin-4 expressing tumors with excellent efficacy in several mouse xenograft models. Citation Format: Mike Rigby, Paul Beswick, Gemma Mudd, Katerine Van Rietschoten, Liuhong Chen, Sophie M. Watcham, Heather Allen, Amy Brown, Helen Harrison, Gavin Bennett, Phil Jeffrey, Peter U. Park, Maria Koehler, Nicholas Keen. BT8009: A bicyclic peptide toxin conjugate targeting Nectin-4 (PVRL4) displays efficacy in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4479.

Abstract Small molecule toxin conjugates offer a novel approach to tumor antigen specific targeting, allowing reduced systemic exposures and efficient tumor penetration of cytotoxic payloads, compared to large antibody toxin conjugates. BT8009 is a Bicycle Toxin Conjugate (BTC®) in which a Nectin-4 binding Bicycle® (bicyclic peptide) is conjugated through an inert sarcosine spacer chain, and a cleavable linker, to the antimitotic toxin MMAE. Increased Nectin-4 expression has been reported in multiple tumor types (including bladder, breast, esophageal, colorectal, lung, ovarian and pancreatic cancers) and so represents an appropriate tumor binding target. Bicycles are small (1.5KDa) fully synthetic, structurally constrained peptides with the specificities and affinities akin to antibodies, and pharmacokinetic properties more like small molecules. These features make Bicycles very attractive novel drug modalities for therapeutic approaches in which low systemic exposures coupled with high local drug concentrations are desired. We have previously described the identification and optimization process leading to selection of BT8009 as a candidate for IND enabling studies. BT8009 has low nanomolar (3nM) affinity for Nectin-4 and high selectivity (>1000 fold) over Nectins 1-3 and the 5 nectin-like family members (Necl1-5). BT8009 is active in a broad range of Nectin-4 positive cell line and patient derived xenograft tumors in vivo, leading to stable diseases and tumor regressions with durable responses. This abstract describes recent work characterizing this molecule. Selectivity of the Bicycle has been further demonstrated by assessing the binding of the biotinylated version of the Nectin-4 Bicycle on a human, plasma membrane protein cell array expressing 5528 full length human plasma membrane and secreted proteins. In this assay the Bicycle bound only Nectin-4 itself, again demonstrating the excellent target specificity. Efficacy data for BT8009 has now been extended to demonstrate the anti-tumor activity of BT8009 in large (1000mm3) xenograft tumors, in which rapid and near complete responses are observed. We have further characterized the efficacy profile of BT8009 in a panel of patient-derived non-small cell lung cancer (NSCLC) xenograft models, as well as in a panel of patient-derived pancreatic ductal (PDAC) xenograft models demonstrating a high frequency of stable diseases and partial responses. We will also describe the ongoing work for identification and validation of biomarkers for treatment response. BT8009 shows excellent efficacy in xenograft models expressing Nectin-4 target and its pharmacokinetic profile enables it to provide a rapid attainment of high tumor exposure levels with reduced systemic exposure. IND-enabling studies are currently ongoing with BT8009. Citation Format: Michael Rigby, Gavin Bennett, Liuhong Chen, Gemma Mudd, Paul Beswick, Helen Harrison, Sophie Watcham, Heather Allen, Amy Brown, Katerine Van Rietschoten, Philip Jeffrey, Peter U Park, Eric Haines, Nicholas Keen, Johanna Lahdenranta. BT8009, a Bicycle Toxin Conjugate targeting Nectin-4, shows target selectivity, and efficacy in preclinical large and small tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C061. doi:10.1158/1535-7163.TARG-19-C061

Molecular imaging of cancers using probes specific for tumor-associated target proteins offers a powerful solution for providing information regarding selection of targeted therapy, patient stratification, and response to therapy. Here we demonstrate the power of bicyclic peptides as targeting probes, exemplified with the tumor-overexpressed matrix metalloproteinase MT1-MMP as a target. A bicyclic peptide with subnanomolar affinity towards MT1-MMP was identified, and its radioconjugate showed selective tumor uptake in an HT1080 xenograft mouse model. Proteolytic stabilization of the peptide by chemical modification significantly enhanced the in vivo tumor signal [from 2.5% ID/g to 12% ID/g at 1 hour post injection (p. i.)]. Studies using mouse xenograft models with different cell lines show a robust correlation between tumor signals and in vivo MT1MMP expression levels. Fatty acid modification of the bicy-clic peptide extended its circulating half-life, resulting in increased tumor signals (36% ID/g at 6 hours p. i.). Comparative work with an equipotent radiolabeled MT1-MMP targeting antibody demonstrated starkly differential biodistribution and tumor accumulation properties, with the tumor signal slowly increasing to 6.2% ID/g within 48 hours. The rapid tumor penetration characteristics of bicyclic peptides, coupled with high potency and chemical versatility, thus offer high-contrast imaging probes for clinical diagnostics with compelling additional potential in targeted therapy. Significance: This work demonstrates the potential of bicyclic peptides as a platform for the development of highcontrast imaging probes for potential use in clinical cancer diagnostics and molecularly targeted therapeutics.

INTRODUCTION:Venipuncture is a psychomotor skill required in many healthcare professions. E-learning could be used to overcome current barriers in face-to-face learning in healthcare education such as insufficient classroom space or qualified instructors. We sought to evaluate the effectiveness of an e-learning module on students' performance when used in addition to in-class training.METHODS:Overall, 224 health sciences students were approached to participate in this pilot study. Recruited students were divided into control and study groups. The control group received only in-class training, whereas the study group had access to the e-learning module in addition to in-class learning. Both groups were evaluated on their self-confidence using a Likert scale, academic competence using a multiple-choice questionnaire, and psychomotor competence from video skill recordings using an in-house rubric. Nonparametric, independent sample Mann-Whitney tests were performed to evaluate differences between groups.RESULTS:Overall, 114 students provided written informed consent; 84 students (control: n = 50, study: n = 34) participated in at least one component of the study. Significantly higher (p = 0.017) academic competence scores were observed in the study group. Significantly higher confidence levels were also observed postintervention for both the control (p = 0.0025) and study (p = 0.0011) groups; however, no significant differences were found between the study and control groups before (p = 0.441) or after (p = 0.883) intervention. Finally, no significant differences (p = 0.428) were observed for psychomotor skills between the study arms.CONCLUSION:Our results suggest that there is potential for e-learning to increase the academic competence of students when used in conjunction with traditional learning; however, further research is needed to determine its efficacy on psychomotor skills.

Plasma kallikrein, a member of the kallikrein-kinin system, catalyzes the release of the bioactive peptide bradykinin, which induces inflammation, vasodilation, vessel permeability, and pain. Preclinical evidence implicates the activity of plasma kallikrein in diabetic retinopathy, which is a leading cause of visual loss in patients suffering from diabetes mellitus. Employing a technology based on phage-display combined with chemical cyclization, we have identified highly selective bicyclic peptide inhibitors with nano- and picomolar potencies toward plasma kallikrein. Stability in biological matrices was either intrinsic to the peptide or engineered via the introduction of non-natural amino acids and nonpeptidic bonds. The peptides prevented bradykinin release in vitro, and in vivo efficacy was demonstrated in both a rat paw edema model and in rodent models of diabetes-induced retinal permeability. With a highly extended half-life of ∼40 h in rabbit eyes following intravitreal administration, the bicyclic peptides are promising novel agents for the treatment of diabetic retinopathy and diabetic macular edema.

Bicycles® are novel binding agents comprising small bicyclic peptides (1.5-3 KDa) constrained via a chemical scaffold, selected for high affinity and selectivity to targets of interest. MT1 (MMP14/MT1-MMP) is a membrane-associated metalloprotease overexpressed in many solid tumours and is implicated in tumor invasion and metastasis. MT1 expression positively correlates with poor prognosis. Phage libraries containing 1015 unique peptide sequences were post-translationally cyclized with thiol-reactive scaffold and used in an optimized, high-throughput selection process to identify Bicycles® to the hemopexin domain of MT1. Additional iterative rounds of directed phage based screening were used to optimize affinity and off-phage non-natural amino acids were introduced at select positions to improve plasma stability to generate the lead Bicycle binder. The lead anti-MT1 Bicycle was further modified with a sarcosyl spacer to form N241. N241 binds specifically to the hemopexin domain of MT1 with a Kd of approximately 2 nM with no binding observed to the catalytic domain of the protease nor to any of the related MMP family members tested. Importantly and in contrast to most antibodies, N241 binds with similar affinity to MT1 from multiple species including rodent, dog and non-human primate. Since the expected rapid tumor penetration and specific binding of these small peptidyl-binders makes them ideal for use in targeted delivery approaches, a series of Bicycle drug conjugates (BDCs) were prepared; N241 was conjugated to potent maytansinoid cytotoxics via linkers which varied in their cleavability. Though all the BDCs maintained high affinity for MT1, efficacy toward MT1-positive human tumor mouse xenografts varied with linker stability. BDCs with the most stable linkers were the least active suggesting that optimal tumor activation was obtained with linkers that could be cleaved more rapidly. Due to the rapid clearance and limited systemic exposure of these small-targeting BDCs, only the most labile linker showed toxicity in the mouse studies. Of the BDCs tested, BT1718, composed of N241 and DM1 conjugated via the SPP linker, demonstrated an optimal therapeutic index. Potent anti-tumor efficacy with BT1718 was observed across a panel of MT1-positive xenografts with complete tumor regressions observed in most models at doses that were well tolerated. In one example, HT-1080 fibrosarcoma subcutaneous xenografts were intraveneously treated with BT1718 when the tumor size had reached approximately 180 mm3. BT1718 given at 3 mg/kg once a week resulted in tumor stasis while BT1718 given at 10 mg/kg once a week or 3 mg/kg twice a week induced complete regression. In summary, BT1718, a highly active, targeted drug conjugate with unique pharmacological properties is a promising therapeutic candidate for the treatment of MT1-MMP-positive solid tumors. Citation Format: Helen Harrison, Gavin Bennett, Diane Blakeley, Amy Brown, Spencer Campbell, Liuhong Chen, Robert J. Lutz, Silvia Pavan, Katerine van Rietschoten, Daniel Teufel, Peter U. Park, Kevin Lee. BT1718, a novel bicyclic peptide-maytansinoid conjugate targeting MT1-MMP for the treatment of solid tumors: Design of bicyclic peptide and linker selection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5144. doi:10.1158/1538-7445.AM2017-5144

Abstract BT1718 is a Bicycle Drug Conjugate (BDC®) comprising a constrained bicyclic peptide (Bicycle®) that binds with high affinity and specificity to membrane type 1-matrix metalloprotease (MT1-MMP; MMP14) covalently linked through a hindered disulfide linker to the potent anti-tubulin agent DM1. MT1-MMP is involved in normal tissue remodeling and is also expressed in tumor associated stromal cells. However, overexpression is linked to increased tumor aggression and metastasis. Specifically, over-expression of MT1-MMP is also associated with poor clinical prognosis and shorter survival times in patients with NSCLC and a range of other solid tumors. The Bicycle MT1-MMP binding element within BT1718 was identified using a proprietary phage display peptide technology consisting of highly diverse phage libraries of linear amino acid sequences constrained into two loops by a central tri-functional chemical scaffold. These MT1-MMP binding Bicycles exhibit a profound affinity and specificity, more often associated with monoclonal antibodies, whilst their low molecular weight (1.5-3 kDa), akin to that of a small molecule, aids in rapid extravasation and tumor penetration. Together these attributes make Bicycles an ideal format for the targeted delivery of cytotoxic payloads. We evaluated the ability of BT1718 to bind to and kill tumor cells in vitro and in vivo in a panel of tumor cells. BT1718 demonstrated MT1-MMP target-specific binding and MT1-MMP-dependent cell killing of lung tumor cells in vitro as well as efficacy across a panel of lung tumor xenograft mouse models. For example, in the Met-amplified squamous NSCLC lung EBC-1 model, complete regressions were observed in all mice at doses as low as 5 mg/kg (iv) twice weekly and across a range of other dosing schedules, from daily to weekly. MT1-MMP-dependent activity was demonstrated by blocking target specific interactions through co-administration of an excess of unconjugated Bicycle binder, which inhibited tumour regression, or a non-binding Bicycle, which had no effect. Further evaluation in patient-derived lung xenograft (PDX) models indicates a similar activity to that seen in cell-line derived xenografts, with efficacy seen from 3mg/kg twice weekly and rapid full regression of tumors at higher doses. The molecular attributes of these Bicycles: rapid tumor penetration and specific binding, makes them ideal therapeutics for targeted delivery of toxins as Bicycle drug conjugates (BDCs). The small size of the BDC may offer a significant advantage to other targeted cytotoxic approaches such as antibody-drug conjugates due to rapid extravasation and improved tumor penetration. BT1718, a Bicycle Drug Conjugate, shows potent anti-tumor activity in human lung tumor xenograft models and IND-enabling studies are underway. Citation Format: Gavin Bennett, Robert Lutz, Peter Park, Helen Harrison, Kevin Lee. Development of BT1718, a novel Bicycle Drug Conjugate for the treatment of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1167. doi:10.1158/1538-7445.AM2017-1167

Abstract The Bicycle® technology is based on repertoires of short peptides displayed on the surface of bacteriophages which can be modified with homo-trifunctional organochemical scaffolds, thus creating large diverse libraries of constrained, bicyclic peptides. These large combinatorial libraries have been extensively used for iterative selections to identify high affinity binding peptides to a wide array of targets, including receptors, interleukins and proteases. Bicyclic peptides are chemically synthesized macrocyclic entities with drug-like properties that exhibit sub-nanomolar affinities and exquisite selectivity towards targets. Unlike biologics, their synthetic nature allows facile modulation of metabolic and pharmacokinetic properties, as well as site-specific conjugation to effector molecules such as fluorophores, radionuclides, and cytotoxic drugs. In the present work, novel phage display derived bicyclic peptides were identified targeting the matrix metalloproteinase 14 (also known as MT1-MMP), a tumor associated surface protein overexpressed in a variety of cancers (i.e. lung, breast). A prototype bicyclic peptide with high affinity to MT1-MMP (Kd at ~1 nM) was identified, and confocal microscopy using fluoresceinated bicyclic peptide derivatives shows target-dependent internalisation in MT1-MMP+ cells. In the in vivo mouse, selective tumor binding in an MT1-MMP+ xenograft model is demonstrated for a DOTA conjugate loaded with Ga-68 or Lu-177. Upon proteolytic optimization of the prototype bicycle peptide, a striking enhancement in tumor signal is observed in biodistribution studies. Compared to radiolabeled antibodies directed against the same target, the lead compound showed fast background clearance (< 1 %ID/g for all organs apart from kidneys) resulting in high imaging contrast in µPET studies as early as 30 minutes post injection. Importantly, most of the non-tumor associated bicyclic peptide rapidly clears into the bladder. Together, tumor targeting bicyclic peptides can, through their small size and high selectivity, facilitate efficient penetration and visualization of tumors in vivo, demonstrating their potential as diagnostic imaging agents in profiling and therapeutic management of patients. Citation Format: Daniel Teufel, Helen Harrison, Spencer Campbell, Catherine Stace, Edward Walker, Robert J. Lutz, Peter Park, Matthias Eder, Ulrike Bauder-Wüst, Ursula Schierbaum, Karin Leotta, Klaus Kopka, Uwe Haberkorn. Bicyclic peptides for PET imaging of MT1-MMP expressing tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3719. doi:10.1158/1538-7445.AM2017-3719