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BackgroundIn 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).MethodsPhage 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 in vitro and in vivo assays to characterize their pharmacology and mechanism of action.ResultsLinking 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.ConclusionTumor 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.

Background After disappointing first clinical experiences with agonistic anti-CD137 (4-1BB) antibodies, a new generation of both systemic and targeted CD137 agonists is entering clinical development.1–3 These strategies rely on biologic agents with suboptimal properties for CD137 agonism due to their relatively large sizes and long circulating half-lives. These properties may limit their tissue penetration and cause sustained agonism resulting in overstimulation and activation-induced cell death of lymphocytes due to continuous exposure.Fully synthetic constrained bicyclic peptides (Bicycles™) with antibody-like affinities and target selectivity are uniquely suited to circumvent the above barriers to optimal targeted CD137 agonistic therapeutics. BT7480 is a tumor-targeted immune cell agonist (TICA) designed to deliver a highly potent CD137 agonist to Nectin-4 overexpressing tumor tissue with a flexible dosing schedule maximizing anti-tumor activity while circumventing the need for continuous systemic exposure. Methods BT7480 functional activity in vitro was analyzed by measuring IL-2 and IFN gamma production from primary human PBMC/tumor cell co-cultures. BT7480 in vivo activity was determined in huCD137-syngeneic tumor models using tumor immune cell and transcriptional profiling by FACS, IHC, and Nanostring as well as tumor growth kinetics as read-outs. Results BT7480 binds potently and simultaneously to Nectin-4 and CD137 as assessed biochemically and caused Nectin-4-dependent CD137 agonism in primary human PBMC co-cultured with tumor cells. Treatment of Nectin-4 expressing tumors in immunocompetent mice with BT7480 leads to profound reprogramming of the tumor immune microenvironment including increased T cell infiltration and upregulation of a cytotoxic cell gene signature. BT7480 treatment induces complete tumor regressions and subsequent resistance to tumor re-challenge. TICA-dependent anti-tumor activity and established immunologic memory are dependent on cytotoxic T cells. Importantly, BT7480 in vivo activity is not dependent on continuous plasma exposure since once weekly dosing of BT7480 provides a maximum anti-tumor activity despite minimal BT7480 plasma exposure after day 2.BT7480 demonstrates linear pharmacokinetics in non-human primates and appears well tolerated at exposures in excess of the predicted efficacious exposure in humans. Conclusions BT7480 is a highly potent Nectin-4 expression dependent CD137 agonist with optimal target binding, pharmacologic, and pharmacokinetic properties that enable intermittent dosing for curative effect through modulation of tumor immune microenvironment in syngeneic mouse tumor models. BT7480 is currently being evaluated in IND-enabling safety studies. Ethics Approval The care and use of animals were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of WuXi AppTec and conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). References Hinner, et al. Tumor-localized costimulatory t-cell engagement by the 4-1BB/HER2 Bispecific antibody-anticalin fusion PRS-343. Clin. Cancer Res 2019 Oct 1;25(19):5878–5889. Claus, et al. Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy. Sci. Transl. Med 2019 Jun 12;11(496):eaav5989. Eskiocak, et al. Differentiated agonistic antibody targeting CD137 eradicates large tumors without hepatotoxicity. JCI Insight 2020 Mar 12;5(5):e133647.

Clonal heterogeneity associated with acquired resistance presents a critical therapeutic challenge. Whole-exome sequencing of paired tumor biopsies and targeted sequencing of cell-free DNA (cfDNA) from patients with BRAFV600E colorectal cancer receiving BRAF inhibitor combinations identified 14 distinct alterations in MAPK pathway components driving acquired resistance, with as many as eight alterations in a single patient. We developed a pooled clone system to study clonal outgrowth during acquired resistance, in vitro and in vivo. In vitro, the dynamics of individual resistant clones could be monitored in real time in cfDNA isolated from culture media during therapy. Outgrowth of multiple resistant clones was observed during therapy with BRAF, EGFR, and MEK inhibitor combinations. However, ERK inhibition, particularly in combination with BRAF and EGFR inhibition, markedly abrogated clonal outgrowth in vitro and in vivo. Thus, convergent, up-front therapy may suppress outgrowth of heterogeneous clones harboring clinically observed resistance alterations, which may improve clinical outcome. Significance: We observed heterogeneous, recurrent alterations in the MAPK pathway as key drivers of acquired resistance in BRAFV600E colorectal cancer, with multiple concurrent resistance alterations detectable in individual patients. Using a novel pooled clone system, we identify convergent up-front therapeutic strategies capable of intercepting multiple resistance mechanisms as potential approaches to suppress emergence of acquired resistance. Cancer Discov; 8(4); 417–27. ©2018 AACR. See related commentary by Janku, p. 389. See related article by Corcoran et al., p. 428. This article is highlighted in the In This Issue feature, p. 371

Harnessing costimulatory molecules expressed on T and NK cells namely 4-1BB (CD137/TNFRSF9) is ideal for cancer immunotherapy. Despite promise preclinically, 4-1BB agonistic antibodies, were unable to demarcate hepatoxicity from efficacy in the clinic. Bispecific approaches promote target-mediated clustering of 4-1BB while limiting systemic and liver toxicity. Bicycles® are novel, fully synthetic, constrained bicyclic peptides with high affinity and selectivity to their targets. Their small size (~2kDa) and tunable pharmacokinetic parameters allow Bicycles to have superior tumor penetration and de-risk hepatoxicity concerns due to a renal clearance mechanism. We hypothesized that clustering and activation of 4-1BB could be attained by conjugating a 4-1BB binding Bicycle to a tumor antigen targeting Bicycle promoting a potent tumor-localized immune response. BT7480 is a tumor-targeted immune cell agonist (TICA™) targeting Nectin-4 and agonizing 4-1BB. Nectin-4 (PVRL4) is highly expressed in numerous tumor indications with unmet clinical needs. In an engineered 4-1BB reporter system, we found BT7480 induced potent 4-1BB agonism correlating to target antigen surface expression on the co-cultured tumor cells. Moreover, BT7480 induces cytokine secretion, including interleukin-2 and interferon gamma, in immune cell co-culture in the presence of tumor antigen. This activity is strictly dependent on Nectin-4 expression on tumor cells and the ability of the TICA to bind to both Nectin-4 and 4-1BB. In a MC38 (Nectin-4-expressing) syngeneic mouse model, intermittent dosing of BT7480 led to robust anti-tumor efficacy (22 out of 24 complete responders (CRs)). Importantly, a memory response was established in CR mice as resistance to a re-challenge with MC38 tumors was observed. Additionally, BT7480 led to increased intratumoral T cell infiltration without elevation of liver enzymes in a CT26 (Nectin-4-expressing) syngeneic mouse model. In non-human primates (NHPs), BT7480 exhibits dose linear exposure and is well tolerated up to 10mpk. BT7480 represents a new generation of chemically synthetic tumor antigen targeted 4-1BB agonists with potent efficacy and a favorable safety profile. Citation Format: Elizabeth M. Repash, Punit Upadhyaya, Johanna Lahdenranta, Jessica Kublin, Jun Ma, Marianna Kleyman, Liuhong Chen, Eric Haines, Sailaja Battula, Kevin McDonnell, Kristen Hurov, Philip Brandish, Nicholas Keen. BT7480, a novel fully synthetic tumor-targeted immune cell agonist (TICA™) induces tumor localized 4-1BB agonism [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2020 Oct 19-20. Philadelphia (PA): AACR; Cancer Immunol Res 2021;9(2 Suppl):Abstract nr PO077.

Costimulatory molecules expressed on activated T and NK cells such as 4-1BB (CD137/TNFRSF9) can be leveraged for cancer immunotherapy. Despite compelling preclinical data, 4-1BB agonistic antibodies have been hampered by failure to delineate hepatoxicity from efficacy in the clinic [1,2]. Next generation strategies are focused on bispecific approaches aimed at promoting target-mediated clustering of 4-1BB to limit systemic and liver effects [3,4]. Bicycles® are fully synthetic, constrained bicyclic peptides that have antibody-like affinity and selectivity to their targets. Unlike traditional biologic approaches, the small size (~2 kDa) and tunable pharmacokinetic (PK) parameters of Bicycles enable superior tumor penetration and allow exploration into the relationship between pulsatile dosing and 4-1BB activation while de-risking hepatoxicity concerns due to a differentiated renal elimination mechanism combined with a tumor-localized immune response. We hypothesized that clustering and activation of 4-1BB could be achieved by conjugating a 4-1BB binding Bicycle to a tumor antigen targeting Bicycle. BT7480 is a tumor-targeted immune cell agonist (TICATM) targeting Nectin-4 and agonizing 4-1BB. Nectin-4 (PVRL4) is highly expressed on numerous tumors with unmet medical need, including bladder, pancreatic, breast, ovarian, esophageal, and lung. BT7480 exhibits highly potent 4-1BB agonism in an engineered 4-1BB reporter system that correlates with Nectin-4 surface expression on the co-cultured tumor cells. In addition, BT7480 induces robust production of interleukin-2 (IL-2) and interferon gamma (IFNγ) in primary PBMC/tumor cell co-culture assays. This activity is strictly dependent on the tumor cells expressing Nectin-4 and on the ability of the TICA to bind to both Nectin-4 and 4-1BB. Nectin-4/4-1BB TICAs are also target-specific immune cell stimulators of patient-derived lung tumors with an intact immune microenvironment. Intermittent dosing of BT7480 led to robust anti-tumor efficacy with 22 out of 24 complete responders (CRs) in a MC38 (Nectin-4-expressing) syngeneic mouse model. Importantly, a memory response was established as the CR mice were resistant to re-challenge with MC38 tumors. Additionally, BT7480 led to increased intratumoral T cell infiltration without elevation of liver enzymes in a CT26 (Nectin-4-expressing) syngeneic mouse model. In non-human primates (NHPs), BT7480 exhibits dose linear exposure and is well tolerated up to 10mpk. Further dose-range finding and safety analysis in NHPs is currently ongoing. BT7480 represents a new generation of chemically synthetic tumor antigen targeted 4-1BB agonists. References 1. Segal NH, Logan TF, Hodi FS, et al. Results from an integrated safety analysis of urelumab, an agonist anti-CD137 monoclonal antibody. Clin Cancer Res. 2017;23(8):1929-1936. 2. Chester et al. Immunotherapy targeting 4-1BB: mechanistic rationale, clinical results, and future strategies. Blood 2018;131(1): 49-57. 3. Hinner et al. Tumor-localized costimulatory T-cell engagement by the 4-1BB/HER2 bispecific antibody-anticalin fusion PRS-343. Clin Cancer Res. 2019; 25(19): 5878-5889. 4. Claus C, Ferrara, C, Xu W, et al. Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy. Sci Transl Med. 2019; 11(496): eaav5989. Citation Format: Kristen Hurov, Punit Upadhyaya, Johanna Lahdenranta, Jessica Kublin, Jun Ma, Elizabeth Repash, Marianna Kleyman, Julia Kristensson, Liuhong Chen, Eric Haines, Sailaja Battula, Kevin McDonnell, Nicholas Keen. BT7480, a novel fully synthetic tumor-targeted immune cell agonist (TICATM) induces tumor localized 4-1BB agonism [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 5552.

4-1BB (CD137) is a member of the TNFR superfamily involved in stimulation of several immune cell types, including T cells and NK cells. CD137 is well validated pre-clinically, as agonism with anti-CD137 antibodies is effective in vivo [1], however, clinical utility to date has been limited by dose dependent hepatotoxicity. Bicycles® are a new therapeutic modality - fully synthetic, constrained bicyclic peptides with high affinity and excellent target selectivity. We hypothesized that Bicycle CD137 agonists that lack Fc domains and exhibit rapid renal elimination may induce CD137 mediated anti-tumor activity while avoiding liver toxicity. Initially, we identified and optimized CD137 specific Bicycle agonists which, when multimerized together, induced CD137 mediated anti-tumor activity [2]. We have built on this initial approach and developed second generation molecules to enable potent stimulation of immune cells exclusively at the tumor site. These tumor-targeted immune cell agonists (TICAsTM) comprise CD137 binding Bicycles coupled to tumor antigen binding Bicycles. Erythropoietin-producing hepatocellular A2 receptor (EphA2) is a tumor target overexpressed in several human cancers and its overexpression correlates with poor clinical prognosis. Here, we present new preclinical data demonstrating the potent immunomodulatory activity of dual targeting EphA2/CD137 TICAs. EphA2/CD137 TICAs engage EphA2 and CD137 with high affinity resulting in picomolar potency in co-culture assays consisting of cancer cell lines endogenously expressing EphA2, and CD137 Jurkat NF-kB/luciferase reporter cells. Moreover, EphA2/CD137 TICAs potentiate cytokine secretion (e.g. IFNγ) in immune cell co-culture experiments and promote caspase activity in T cell mediated cell killing assays. In vivo, EphA2/CD137 TICA (BCY9173), when dosed at 15 mg/kg (BID) to PBMC-humanized mice bearing HT29 xenografts, increased CD8+ T cells in tumors but not in plasma, suggesting local tumor target specific stimulation of T cells without systemic CD137 agonism. Intermittent dosing with various regimens of BCY12491, an EphA2/CD137 TICA, demonstrated robust anti-tumor activity including complete responses (CR) in 10/12 animals in a syngeneic humanized CD137 MC38 mouse model. Importantly, CR mice are resistant to re-challenge with MC38 tumor cells demonstrating a memory response, a phenomenon which has been previously reported for CD137 agonists [1]. Our EphA2/CD137 TICAs have shown the potential to precisely and potently stimulate immune cells in tumors without systemic immune activation. This provides a strong rationale to further develop first-in-class Bicycle TICAs to potentially treat EphA2 expressing cancers. References 1. Melero I, Shuford WW, Newby SA, Aruffo A, Ledbetter JA, Hellström KE, Mittler RS, Chen L. Monoclonal antibodies against the 4-1BB T-cell activation molecule eradicate established tumors. Nat Med. 1997; 3(6): 682-5 2. K. Hurov, P. Upadhyaya, J. Kublin, M. Kleyman, X. Zhou, J. Kristensson, G. Mudd, K. Rietschoten, S. Watcham, R. Lani, W. F. An, T. Stephen, E. Haines, J. Lahdenranta, L. Chen, S. Battula, K. McDonnell, P. Park, and N. Keen. Activation of 4-1BB using multivalent and tumour targeted bicyclic peptides; Poster 2019 Annual AACR meeting, Cancer Research, DOI: 10.1158/1538-7445.AM2019-3257 Published July 2019 Citation Format: Sailaja Battula, Gemma Mudd, Punit Upadhyaya, Julia Kristensson, Marianna Kleyman, Elizabeth Repash, Jessica Kublin, Jun Ma, Eric Haines, Kristen Hurov, Liuhong Chen, Johanna Lahdenranta, Paul Beswick, Kevin McDonnell, Nicholas Keen. A fully synthetic EphA2/4-1BB tumor-targeted immune cell agonist (TICATM) induces tumor localized 4-1BB agonism [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 6703.

Clonal heterogeneity associated with acquired resistance presents a critical therapeutic challenge. Whole-exome sequencing of paired tumor biopsies and targeted sequencing of cell-free DNA (cfDNA) from BRAF(V600E) colorectal cancer patients receiving BRAF inhibitor combinations identified 14 distinct alterations in MAPK pathway components driving acquired resistance, with as many as eight alterations in a single patient. We developed a novel pooled clone system to study clonal outgrowth during acquired resistance, in vitro and in vivo. In vitro, the dynamics of individual resistant clones could be monitored in real-time in cfDNA isolated from culture media during therapy. Outgrowth of multiple resistant clones was observed during therapy with BRAF, EGFR, and MEK inhibitor combinations. However, ERK inhibition, particularly in combination with BRAF and EGFR inhibition, markedly abrogated clonal outgrowth in vitro and in vivo. Thus, convergent, upfront therapy may suppress outgrowth of heterogeneous clones harboring clinically observed resistance alterations, which may improve clinical outcome.

Summary: Three recent studies, approaching the question from different angles and using different and/or overlapping models, provide compelling evidence for the involvement of tumor-infiltrating B cells in the initiation and progression of pancreatic ductal adenocarcinoma. These studies highlight the need for a better understanding of pancreatic tumor–immune system interactions and the immunologic mechanisms that promote or inhibit tumorigenesis, paving the way for better treatment strategies., Bloodwise (UK) (Visiting Fellowship Grant (14043))

Clonal heterogeneity associated with the emergence of acquired resistance to therapy presents a critical challenge for therapeutic strategies to overcome resistance. We investigated the molecular landscape of acquired resistance in BRAF-mutant colorectal patients treated with BRAF inhibitor combinations. Through whole-exome sequencing of paired pre-treatment and post-progression tumor biopsies and targeted sequencing of pre-treatment and post-progression plasma circulating tumor DNA (ctDNA), we identified 14 unique alterations in MAPK pathway components driving acquired resistance, affecting KRAS, NRAS, BRAF, MEK1 and MEK2. Analysis of ctDNA at the time of disease progression revealed profound tumor heterogeneity associated with acquired resistance, with multiple concurrent resistance alterations detectable in ctDNA in individual patients, with one patient harboring as many as 8 co-existing resistance alterations. These findings necessitate development of a strategy capable of simultaneously overcoming multiple heterogeneous resistance mechanisms. To evaluate potential strategies for convergent targeting of multiple concurrent resistance alterations, we generated individual resistant models harboring the full spectrum of clinically observed mutations driving acquired resistance. Individually, these alterations drove resistance to BRAF inhibitor combinations currently in clinical trials by maintaining MAPK signaling. However, since acquired resistance is thought to arise from pre-existing clones that emerge during treatment, we developed a novel pooled clone model system to study clonal outgrowth under the selective pressure of therapy. In this system, each resistant clone was pooled at an abundance of 1% in a background of the sensitive parental cells and exposed to an array of potential therapies, both in vitro, and in vivo as xenografts. The change in clonal abundance from baseline to the completion of therapy was assessed for each clone by digital PCR to measure the degree of clonal outgrowth. Moreover, in vitro, we were able to monitor clonal dynamics in real-time by isolating cell-free DNA (cfDNA) from the cell culture media every 3-4 days during therapy. We observed rapid outgrowth of resistant clones during BRAF-EGFR and BRAF-MEK therapy, and delayed, but robust outgrowth during BRAF-MEK-EGFR therapy, all of which are therapies that have been evaluated in recent clinical trials. However, ERK inhibitor alone, and to a greater degree BRAF-ERK and BRAF-ERK-EGFR combinations markedly abrogated the clonal outgrowth of resistant clones. Moreover, in xenograft tumors derived from clonal pools, BRAF-ERK-EGFR triple combination resulted in profound tumor regressions and completely prevented the outgrowth of all resistant clones. In conclusion, we observed the potential for profound heterogeneity of acquired resistance mechanisms in BRAF-mutant colorectal cancer patients, with multiple alterations observed in ctDNA from individual patients. Our data suggest that convergent, upfront therapy with RAF-ERK or RAF-ERK-EGFR inhibitor combinations may suppress outgrowth of clones harboring clinically observed resistant alterations, offering the potential for improved clinical outcome. Citation Format: Mehlika Hazar-Rethinam, Marianna Kleyman, G. Celine Han, David Liu, Leanne G. Ahronian, Heather A. Shahzade, Lifeng Chen, Aparna R. Parikh, Jill N. Allen, Jeffrey W. Clark, Eunice L. Kwak, Jason E. Faris, Janet E. Murphy, Theodore S. Hong, Emily E. Van Seventer, Brandon Nadres, Catriona B. Hong, Joseph M. Gurski Jr., Nicholas A. Jessop, Dora Dias-Santagata, A. John Iafrate, Eli M. Van Allen, Ryan B. Corcoran. Modeling convergent therapeutic strategies to overcome the heterogeneity of acquired resistance in BRAF-mutant colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A34.