Human Tumor Xenograft Models Research Articles

Abstract PS12-20: A dose escalation study of the novel oral SERD-ZN-c5 in women with ER-positive, HER2-negative advanced/metastatic breast cancer

Abstract Background: ZN-c5 is an orally bioavailable selective estrogen receptor degrader (SERD) that binds potently to the estrogen receptors alpha and beta. It shows improved activity over fulvestrant in human tumor xenograft models and activity in tumor models that are resistant to tamoxifen. This is a Phase 1/2, open-label, multicenter, dose-escalation and expansion study to evaluate the safety, tolerability, pharmacokinetics (PK), and clinical activity of ZN-c5 in subjects with advanced/metastatic estrogen receptor (ER) positive/ human epidermal growth factor receptor (HER2) negative breast cancer, both as monotherapy and in combination with palbociclib. The results from the ongoing monotherapy dose escalation are reported. Methods: Single agent ZN-c5 is being evaluated at sequentially escalating doses starting at 50 mg/day, administered orally, once daily (QD). The endpoints are to determine a maximum tolerated dose (MTD) or recommended Phase 2 dose (RP2D), preliminary clinical activity and to characterize the PK profile. Subjects must be intolerant to or have breast cancer refractory to established therapies and to have received up to 2 prior lines of chemotherapy for the treatment of advanced breast cancer. Subjects must have a documented prior response to endocrine therapy for advanced/metastatic disease (SD, PR, or CR) lasting > 6 months or disease recurrence after at least 24 months of adjuvant endocrine treatment. Results: A total of 15 female subjects (median age 57 years, range 51 - 89 years) were enrolled across 5 cohorts (3 subjects/dose level). The dose levels were 50, 75, 100, 150, and 300 mg/day. The subjects had a median of 4 prior therapies for advanced/metastatic disease, with a median of 3 prior hormonal-based therapies and a median of 1 prior chemotherapy. Eleven of 15 subjects (73%) received prior fulvestrant. The cut off-date for this analysis was 30 June 2020. There was no increase in incidence or severity of TEAEs with increase in dose level. The most frequent TEAEs reported in > 1 subject were nausea (33%), arthralgia, cough, musculoskeletal pain and vomiting (20% each), alanine aminotransferase increased, anemia, back pain, blood alkaline phosphatase increased, breast pain, diarrhea, fatigue, gamma-glutamyl transferase increased, headache, hypophosphatemia, myalgia and skin mass (13% each). Grade 3 events were COVID-19, hypercalcemia, arthralgia, back pain musculoskeletal chest pain, pain in extremity and hypertension, none were deemed related to ZN-c5. Grade 4 events were not reported. No bradycardia was observed. A single subject reported a Grade 1 visual field defect, not deemed related to ZN-c5. No DLTs were reported. ZN-c5 demonstrated a best response of stable disease (SD) in 10/15 subjects (66.5%), while progression of disease (PD) was reported in 5/15 subjects (33.5%). The clinical benefit rate (CBR, SD ≥ 24 weeks) was 40%. In addition, the progression free survival (PFS) was a median of 3.8 months (95% [CI], 1.6 to 6.3). The preliminary PK was characterized by fast absorption with median Tmax values of 1 - 2 hrs. The exposures were approximately dose-proportional at the dose levels of 50 - 100 mg and less than dose-proportional between 100 - 300 mg. No ZN-c5 accumulation after 15 days of QD dosing was observed. The estimated mean elimination half-lives ranged between 11 - 18 hrs. Conclusion: This monotherapy dose escalation study demonstrates that ZN-c5 is very well-tolerated and has promising clinical activity in patients with ER+/HER2-negative advanced breast cancer who have disease that progressed on standard therapies. The trial with ZN-c5 in monotherapy and with palbociclib is ongoing and the RP2D has not been determined yet. Citation Format: Pavani Chalasani, Vandana Abramson, Joanne Mortimer, Julie R Nangia, Jose Suarez, Matt Suster, Mieke Ptaszynski, Kevin Kalinsky. A dose escalation study of the novel oral SERD-ZN-c5 in women with ER-positive, HER2-negative advanced/metastatic breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS12-20.

Quantifying ADC bystander payload penetration with cellular resolution using pharmacodynamic mapping

With the recent approval of 3 new antibody drug conjugates (ADCs) for solid tumors, this class of drugs is gaining momentum for the targeted treatment of cancer. Despite significant investment, there are still fundamental issues that are incompletely understood. Three of the recently approved ADCs contain payloads exhibiting bystander effects, where the payload can diffuse out of a targeted cell into adjacent cells. These effects are often studied using a mosaic of antigen positive and negative cells. However, the distance these payloads can diffuse in tumor tissue while maintaining a lethal concentration is unclear. Computational studies suggest bystander effects partially compensate for ADC heterogeneity in tumors in addition to targeting antigen negative cells. However, this type of study is challenging to conduct experimentally due to the low concentrations of extremely potent payloads. In this work, we use a series of 3-dimensional cell culture and primary human tumor xenograft studies to directly track fluorescently labeled ADCs and indirectly follow the payload via an established pharmacodynamic marker (γH2A. X). Using TAK-164, an anti-GCC ADC undergoing clinical evaluation, we show that the lipophilic DNA-alkylating payload, DGN549, penetrates beyond the cell targeted layer in GCC-positive tumor spheroids and primary human tumor xenograft models. The penetration distance is similar to model predictions, where the lipophilicity results in moderate tissue penetration, thereby balancing improved tissue penetration with sufficient cellular uptake to avoid significant washout. These results aid in mechanistic understanding of the interplay between antigen heterogeneity, bystander effects, and heterogeneous delivery of ADCs in the tumor microenvironment to design clinically effective therapeutics.

15580 Effect of TGF-β1/COX-2 small interfering RNA combination product (STP705) on cell viability and tumor growth in a human squamous carcinoma xenograft tumor model in nude mice

Background: TGF-β1 and COX-2 overexpression plays key role in tumorigenesis of nonmelanoma skin cancer. STP705 is a combination of TGF-β1/COX-2 siRNAs formulated in histidine-lysine copolymer nanoparticle delivery system. We studied the effect of STP705 on the growth of human squamous carcinoma (A431) xenograft tumors in nude mice and viability of human cutaneous squamous carcinoma (HSC-1) cells.

Futibatinib Is a Novel Irreversible FGFR 1-4 Inhibitor That Shows Selective Antitumor Activity against FGFR-Deregulated Tumors.

FGFR signaling is deregulated in many human cancers, and FGFR is considered a valid target in FGFR-deregulated tumors. Here, we examine the preclinical profile of futibatinib (TAS-120; 1-[(3S)-[4-amino-3-[(3,5-dimethoxyphenyl)ethynyl]-1H-pyrazolo[3, 4-d] pyrimidin-1-yl]-1-pyrrolidinyl]-2-propen-1-one), a structurally novel, irreversible FGFR1-4 inhibitor. Among a panel of 296 human kinases, futibatinib selectively inhibited FGFR1-4 with IC50 values of 1.4 to 3.7 nmol/L. Futibatinib covalently bound the FGFR kinase domain, inhibiting FGFR phosphorylation and, in turn, downstream signaling in FGFR-deregulated tumor cell lines. Futibatinib exhibited potent, selective growth inhibition of several tumor cell lines (gastric, lung, multiple myeloma, bladder, endometrial, and breast) harboring various FGFR genomic aberrations. Oral administration of futibatinib led to significant dose-dependent tumor reduction in various FGFR-driven human tumor xenograft models, and tumor reduction was associated with sustained FGFR inhibition, which was proportional to the administered dose. The frequency of appearance of drug-resistant clones was lower with futibatinib than a reversible ATP-competitive FGFR inhibitor, and futibatinib inhibited several drug-resistant FGFR2 mutants, including the FGFR2 V565I/L gatekeeper mutants, with greater potency than any reversible FGFR inhibitors tested (IC50, 1.3-50.6 nmol/L). These results indicate that futibatinib is a novel orally available, potent, selective, and irreversible inhibitor of FGFR1-4 with a broad spectrum of antitumor activity in cell lines and xenograft models. These findings provide a strong rationale for testing futibatinib in patients with tumors oncogenically driven by FGFR genomic aberrations, with phase I to III trials ongoing. SIGNIFICANCE: Preclinical characterization of futibatinib, an irreversible FGFR1-4 inhibitor, demonstrates selective and potent antitumor activity against FGFR-deregulated cancer cell lines and xenograft models, supporting clinical evaluation in patients with FGFR-driven tumors. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/22/4986/F1.large.jpg.

In Vivo Pre-Clinical Evaluation of LOXO-305 Alone and in Combination with Venetoclax, Rituximab, R-CHOP or Obinutuzumab on Human Xenograft Lymphoma Tumor Models in Mice

Introduction: Bruton's Tyrosine Kinase (BTK) is an essential component of normal and malignant B-cell receptor signaling. Covalent BTK inhibitors have transformed the treatment of B-cell malignancies. Despite the marked efficacy of covalent BTKi, treatment failure can occur through the development of resistance and discontinuation for adverse events. The activity of these covalent BTK inhibitors are markedly reduced or absent in the presence of BTK cysteine binding site (C481) mutations. Moreover, these agents share pharmacologic liabilities (e.g. low oral bioavailability, short half-life) that may sometimes lead to suboptimal BTK target coverage, for example in rapidly proliferating tumors with high BTK protein turnover, ultimately manifesting as acquired resistance for some patients. To address these limitations, LOXO-305, a highly selective, non-covalent BTKi that inhibits both WT and C481-mutated BTK with equal low nM potency was developed. Proof-of-concept Phase I results demonstrated LOXO-305's anti-tumor activity across patients with heavily pretreated B-cell malignancies (Mato et al. ASH 2019). We previously showed pre-clinical data demonstrating that LOXO-305 potently inhibited wild-type (WT) BTK and different variants of the BTK mutation C481 with nanomolar potency and caused regression in BTK-dependent lymphoma mouse xenograft models (Brandhuber et al. SOHO 2018, and Gomez et al. ASH 2019). Here we describe the activity of LOXO-305 alone or in combination with venetoclax (BCL-2 inhibitor), in TMD8 BTK WT and TMD8 BTK C481S human tumor xenograft models of diffuse large B lymphoma and a REC-1 human tumor xenograft model of mantle cell lymphoma in mice. We also report the activity of LOXO-305 alone and in combination with R-CHOP (rituximab, cyclophosphamide, doxorubicin hydrochloride, vincristine sulfate, and prednisone) or obinutuzumab (anti-CD20 antibody), in the TMD8 xenograft tumor model. Methods: In all studies, tested articles were administered alone and in combination, following different dosing regimens. Table 1 shows the tested compound(s), dosing frequency, cell line used, disease, BTK status (WT or C481S), and mouse strain used, for each study presented in this abstract. Human TMD8 BTK WT, TMD8 BTK C481S or REC-1 cells were injected subcutaneously in the right flank of mice. When tumors reached a mean volume between 150 mm3 and 400 mm3, mice were randomized based on their tumor volumes. Mice were next dosed for 17 to 23 days depending on the study design. The potencies of the compounds stand-alone or in combination on the inhibition of the tumor growth were assessed based on the tumor volume changes and weights after collection at the end of the study. Additionally, in the TMD8 studies, the plasma concentrations of tested articles were measured at multiple time points after the last dose. Results: All treatments were well tolerated without any significant body weight loss or clinical signs being observed on the mice. LOXO-305 potently inhibited the growth of BTK WT and BTK C481S driven xenograft tumors. In all combinations tested, significantly greater tumor growth inhibition was observed in groups where LOXO-305 was co-administered with clinically approved agents. Conclusion: These data suggest that the co-administration of LOXO-305 with venetoclax, R-CHOP or obinutuzumab could have an increased benefit for patients with B-cell malignancies compared to stand-alone treatments and warrants further investigation. Disclosures Gomez: Loxo Oncology, Inc, a wholly owned subsidiary of Eli Lilly and Company: Current Employment, Divested equity in a private or publicly-traded company in the past 24 months. Wu:Eli Lilly and Company: Current Employment, Current equity holder in publicly-traded company. Stephens:Eli Lilly and Company: Current Employment, Current equity holder in publicly-traded company. Rosendahl:Loxo Oncology, Inc, a wholly owned subsidiary of Eli Lilly and Company: Current Employment, Divested equity in a private or publicly-traded company in the past 24 months. Brandhuber:Loxo Oncology, Inc, a wholly owned subsidiary of Eli Lilly and Company: Current Employment, Divested equity in a private or publicly-traded company in the past 24 months.

PODO447: a novel antibody to a tumor-restricted epitope on the cancer antigen podocalyxin

BackgroundThe success of new targeted cancer therapies has been dependent on the identification of tumor-specific antigens. Podocalyxin (Podxl) is upregulated on tumors with high metastatic index and its presence is...

Characterization of a Novel Barbituric Acid and Two Thiobarbituric Acid Compounds for Lung Cancer Treatment.

Previously, we reported the identification of a cytotoxic chemotype compound CC-I (1a), a derivative of thiobarbituric acid. We also reported the anticancer activity of a series of novel thio- and seleno-barbituric acid analogs. We herein evaluated the effect of 1a and its modified compounds on in vitro and in vivo lung cancer models. The compounds 1b and 2a showed more potent cytotoxicity than 1a to lung cancer cells. Moreover, 1b did not have any cytotoxicity on normal cells, such as fibroblasts. In the human lung cancer A549 mouse tumor xenograft model, 1b and 2a showed more pronounced antitumor effects than 1a In the A549 lung cancer cells, 1a induced cell death mainly via JNK and p38 MAPK activation. However, compound 1b and 2a induced lung cancer cell death mostly through JNK activation. The results suggest that 1b and 2a can be useful therapeutic agents for lung cancer.

Thioredoxin Interacting Protein (TXNIP) Is Differentially Expressed in Human Tumor Samples but Is Absent in Human Tumor Cell Line Xenografts: Implications for Its Use as an Immunosurveillance Marker.

Simple SummaryThe metabolic protein TXNIP plays a crucial role in various cellular processes. Abnormal TXNIP levels are notable, e.g., in type II diabetes, cardiovascular diseases, and tumors. Using immunohistochemical staining for TXNIP in different tumor entities, we give new insights of TXNIP expression on the protein level. In human tumors, staining intensity inversely correlated with aggressiveness of the tumor entity. In contrast, human tumor cell lines grown in mice (xenografts), consistently revealed no staining. Hence, loss of TXNIP suggests a critical role for the development of tumors in xenografts. Furthermore, we investigated TXNIP staining of immunocompetent cells in the proximity of the xenograft tumor tissue. Our findings demonstrate that TXNIP downregulation is a common feature in human tumor xenograft models. Subsequently, TXNIP expression might be used to monitor the functional state of tumor-infiltrating leukocytes in tissue sections and may help to predict response to modern immune therapy.Thioredoxin interacting protein (TXNIP) is a metabolic protein critically involved in redox homeostasis and has been proposed as a tumor suppressor gene in a variety of malignancies. Accordingly, TXNIP is downregulated in breast, bladder, and gastric cancer and in tumor transplant models TXNIP overexpression inhibits growth and metastasis. As TXNIP protein expression has only been investigated in few malignancies, we employed immunohistochemical detection in a large multi-tumor tissue microarray consisting of 2,824 samples from 94 different tumor entities. In general, TXNIP protein was present only in a small proportion of primary tumor samples and in these cases was differently expressed depending on tumor stage and subtype (e.g., renal cell carcinoma, thyroid cancer, breast cancer, and ductal pancreatic cancer). Further, TXNIP protein expression was determined in primary mouse xenograft tumors derived from human cancer cell lines and was immunohistochemically absent in all xenograft tumors investigated. Intriguingly, TXNIP expression became gradually lower in the proximity of the primary tumor tissue and was absent in leukocytes directly adjacent to tumor tissue. In conclusion, these findings suggest that TXNIP downregulation is as a common feature in human tumor xenograft models and that intra-tumoral leukocytes down-regulate TXNIP. Hence TXNIP expression might be used to monitor the functional state of tumor-infiltrating leukocytes in tissue sections.

Preclinical Antitumor Activity and Biodistribution of a Novel Anti-GCC Antibody–Drug Conjugate in Patient-derived Xenografts

Guanylyl cyclase C (GCC) is a unique therapeutic target with expression restricted to the apical side of epithelial cell tight junctions thought to be only accessible by intravenously administered agents on malignant tissues where GCC expression is aberrant. In this study, we sought to evaluate the therapeutic potential of a second-generation investigational antibody-dug conjugate (ADC), TAK-164, comprised of a human anti-GCC mAb conjugated via a peptide linker to the highly cytotoxic DNA alkylator, DGN549. The in vitro binding, payload release, and in vitro activity of TAK-164 was characterized motivating in vivo evaluation. The efficacy of TAK-164 and the relationship to exposure, pharmacodynamic marker activation, and biodistribution was evaluated in xenograft models and primary human tumor xenograft (PHTX) models. We demonstrate TAK-164 selectively binds to, is internalized by, and has potent cytotoxic effects against GCC-expressing cells in vitro A single intravenous administration of TAK-164 (0.76 mg/kg) resulted in significant growth rate inhibition in PHTX models of metastatic colorectal cancer. Furthermore, imaging studies characterized TAK-164 uptake and activity and showed positive relationships between GCC expression and tumor uptake which correlated with antitumor activity. Collectively, our data suggest that TAK-164 is highly active in multiple GCC-positive tumors including those refractory to TAK-264, a GCC-targeted auristatin ADC. A strong relationship between uptake of 89Zr-labeled TAK-164, levels of GCC expression and, most notably, response to TAK-164 therapy in GCC-expressing xenografts and PHTX models. These data supported the clinical development of TAK-164 as part of a first-in-human clinical trial (NCT03449030).

Amivantamab (JNJ-61186372), an Fc Enhanced EGFR/cMet Bispecific Antibody, Induces Receptor Downmodulation and Antitumor Activity by Monocyte/Macrophage Trogocytosis.

Small molecule inhibitors targeting mutant EGFR are standard of care in non-small cell lung cancer (NSCLC), but acquired resistance invariably develops through mutations in EGFR or through activation of compensatory pathways such as cMet. Amivantamab (JNJ-61186372) is an anti-EGFR and anti-cMet bispecific low fucose antibody with enhanced Fc function designed to treat tumors driven by activated EGFR and/or cMet signaling. Potent in vivo antitumor efficacy is observed upon amivantamab treatment of human tumor xenograft models driven by mutant activated EGFR, and this activity is associated with receptor downregulation. Despite these robust antitumor responses in vivo, limited antiproliferative effects and EGFR/cMet receptor downregulation by amivantamab were observed in vitro Interestingly, in vitro addition of isolated human immune cells notably enhanced amivantamab-mediated EGFR and cMet downregulation, leading to antibody dose-dependent cancer cell killing. Through a comprehensive assessment of the Fc-mediated effector functions, we demonstrate that monocytes and/or macrophages, through trogocytosis, are necessary and sufficient for Fc interaction-mediated EGFR/cMet downmodulation and are required for in vivo antitumor efficacy. Collectively, our findings represent a novel Fc-dependent macrophage-mediated antitumor mechanism of amivantamab and highlight trogocytosis as an important mechanism of action to exploit in designing new antibody-based cancer therapies.

Favorable therapeutic response after anti-Mesothelin antibody\u2013drug conjugate treatment requires high expression of Mesothelin in tumor cells

PurposeThe cell surface glycoprotein Mesothelin is overexpressed in ovarian, fallopian tube, endometrial, cervical and primary peritoneal cancer and, therefore, might become a particular interesting tumor target in gynecologic oncology. However, even in malignant tumors of the same entity the level of Mesothelin expression varies between individuals, hence it can be expected that the response to Mesothelin-targeting therapies will be variable as well. In this study we explored the therapeutic potency of a novel anti-Mesothelin antibody–drug conjugate (Anetumab ravtansine) as a function of Mesothelin expression in the targeted tumor cells.MethodsAnti-tumor activity studies were performed in human uterine xenograft tumor models that express Mesothelin at high, moderate or low levels. The antibody–drug conjugate (ADC) was applied in varying doses ranging from 2 to 15 mg/kg at variable intervals in tumor bearing SCID/beige mice and the impact on tumor growth was monitored.ResultsThe therapeutic response to the anti-Mesothelin ADC correlated closely with the level of Mesothelin expression in tumor cells. Within the applied dose levels complete tumor regression was achieved only in tumors which expressed Mesothelin at particularly high levels (Hela cell tumors). The application of high anti-Mesothelin ADC doses less frequently was more efficious than giving lower doses at a higher frequency.ConclusionThe studies confirm the great therapeutic potential of Anetumab ravtansine. However, a favorable treatment outcome requires strong Mesothelin expression in tumor cells. Future clinical trials may benefit from a more rigorous selection of appropriate patients based on the level of Mesothelin expression in their tumor tissue. If, in addition, it is possible to better control side effects by introducing protective measures and by doing so to increase the maximum tolerated dose, Anetumab ravtansine has the potency to become a valuable therapeutic tool, especially in the field of gynecological oncology.

Abstract 5619: PBMC humanized NSG-(KbDb)null (IA)null mouse model to evaluate immune-oncology drug efficacy

Abstract Multiple types of humanized mouse models using immunodeficient NOD-scid IL2rgnull (NSG) mice have been developed by The Jackson Laboratory for preclinical studies of new cancer therapies. Humanized NSG mice engrafted with both human peripheral blood mononuclear cells (PBMC) and human cancers have been used for evaluating antibody-based therapeutics targeting T cells. Compared to CD34+ HSC-mediated humanization, NSG mice engrafted with PBMC are ready to use more quickly and are more cost-effective. The major limitation of PBMC engrafted NSG is the development of acute xenogeneic graft-versus-host disease (GVHD) due to human T cell activation through mouse MHC class I and class II molecules. The experimental window for drug efficacy evaluation is limited to three-four weeks post PBMC injection and strong xeno-GVHD may hinder drug-induced T cell activation. A new strain lacking murine MHC molecules, NSG-(KbDb)null (IA)null, was created to address this limitation. Here we engrafted non-irradiated adult NSG and MHC double knockout NSG-(KbDb)null (IA)null mice using the same human PBMC donors to compare their survival and engraftment level. We found that most of the MHC deficient mice can survive over 100 days and can support human T cell engraftment. We also evaluated drug efficacy response using checkpoint inhibitors anti-CTLA-4 (lpilimumab) and anti-PDL-1 (Avelumab) in human tumor xenograft models. Anti-CTLA-4 treatment resulted in reduced growth of mantle cell lymphoma Jeko-1. Anti-PDL-1 treatment reduced growth of a melanoma PDX. In consistent with their anti-tumor response, flow analysis showed increased T cell frequency in blood and tumor in the drug treated group when compared to the vehicle control. Additional cancer treatment evaluated in the PBMC engrafted NSG-(KbDb)null (IA)null mice will also be discussed. Citation Format: Li-Chin Yao, Mingshan Cheng, Leonard D. Shultz, James G. Keck. PBMC humanized NSG-(KbDb)null (IA)null mouse model to evaluate immune-oncology drug efficacy [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 5619.

Abstract 4373: Discovery of ZN-c5, a novel potent and oral selective estrogen receptor degrader

Abstract Majority of breast cancers express the estrogen receptor alpha (ERα) and the two major strategies for therapeutic targeting of ER-alpha positive breast cancer are aromatase inhibitors (which deprives estrogen) and inhibition of the estrogen receptor alpha. Although these strategies can be effective, many patients develop resistance which ultimately leads to disease progression which continues to rely on estrogen receptor signaling. One of the resistance mechanisms is the acquisition of activating mutations in the ER gene (ESR1) that allow tumors to proliferate without depending on estrogen. One approach to overcome resistance is to develop high affinity small molecules that degrade the estrogen receptor and effectively shut down ER signaling. Fulvestrant, an ER antagonist and a selective estrogen receptor degrader (SERD) is the only small molecule that is approved for the treatment of ER+/HER2− metastatic breast cancer. However, fulvestrant's limitation include low oral bioavailability and administration via intramuscular injection, which limits its exposure and leads to sub-optimal estrogen receptor degradation. The poor pharmacokinetic properties of fulvestrant has fueled the interest in developing an orally bioavailable small molecule estrogen receptor antagonist and degrader that could potentially benefit breast cancer patients. Here we describe the discovery of Zn-c5 a novel, small molecule with potent antagonism and degradative properties against the estrogen receptor both in vitro and in vivo. ZN-c5 showed a high oral bioavailability across several preclinical species as compared to other SERDs. To test if the high oral bioavailability can be translated to potent efficacy in vivo, we evaluated the anti-tumor activity of ZN-c5 in MCF-7 orthotopic tumor xenograft model. Oral ZN-c5 treatment at 5 mg/kg and 10 mg/kg resulted in 89% and 102% tumor growth inhibition respectively. Combination of ZN-c5 with cell cycle inhibitors such as CDK4/6 inhibitors or PI3K inhibitors results in enhanced antitumor activity. In addition to MCF-7 model, we evaluated the activity of ZN-c5 in ER mutant models including WHIM20, a Y537S ESR1 patient derived xenograft model. Treatment with ZN-c5 at 40 mg/kg induced 64% tumor growth inhibition while fulvestrant at 200 mg/kg (exposures 8-fold higher than that achieved in the clinic) resulted in 13% tumor growth inhibition. These data indicate that ZN-c5 has improved antitumor activity over fulvestrant in human tumor xenograft models. Zn-c5 is currently in clinical trials as a single agent and in combination studies. The PK profile of ZN-c5 in breast cancer patients indicates that Zn-c5 has greater than 5-fold exposure than fulvestrant. We believe that the high exposure of ZN-c5 coupled with its potency and degradative properties could therapeutic benefit estrogen receptor positive breast cancer patients. Citation Format: Ahmed A. Samatar, Jiali Li, Sayee Hegde, Peter Huang, Jianhui Ma, Kevin Bunker, Robert Winkler, Fernando Donate, Masha Sergeeva. Discovery of ZN-c5, a novel potent and oral selective estrogen receptor degrader [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 4373.

Abstract 4560: Conditionally active biologics (CAB): A novel class of molecules targeting solid tumors

Abstract Conditionally Active Biologics (CAB) technology is a proprietary platform that selects antibodies binding to target antigens in the context of diseased tissues, but not normal tissues, taking advantage of the unique conditions in the tumor microenvironment. Using our CAB technology, we have developed CAB ADCs to various well-established tumor associated antigens (TAA), including Nectin-4, Her2 and CD46. Each of these molecules play an important role in cancer biology and are attractive targets for the development of therapeutic antibodies. These targets, however, are expressed on many normal tissues and have been implicated in a wide range of biological processes; therefore, limiting the use of traditional ADCs as cancer therapy. Using CAB technology, we have developed conditionally active ADCs that have no or very little binding to the target antigen in healthy tissue (normal physiological conditions), but have strong binding in the context of diseased tissues (tumor microenvironment) based on the glycolytic tumor metabolism (including Warburg effect). In vitro and in vivo efficacy data demonstrating the abilities of CAB ADCs to induce cytotoxicity of human target-expressing tumor cell lines, and to inhibit tumor growth in human tumor xenograft models will be presented. In conclusion, our data is consistent with our previous work on EpCAM, our clinical stage AXL and ROR2 CAB ADCs, and suggests that conditionally active ADCs generated using the CAB technology will provide a new generation of biologics with increased safety margin and therapeutic index in the clinic. Citation Format: Jing Wang, Charles Xing, Haizhen Liu, Ana Paula G. Cugnetti, Christina Wheeler, Matthew Lucas, Gerhard Frey, Cathy Chang, William J. Boyle, Jay M. Short. Conditionally active biologics (CAB): A novel class of molecules targeting solid tumors [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 4560.

Abstract 2401: Oncogenic H-Ras is a key regulator of death receptor-induced apoptosis

Abstract Ras oncogenes, including H-Ras, K-Ras and N-Ras, are widely implicated in tumorigenesis. Oncogenic K-Ras mutations are found in about 30% of human tumors rendering constitutive activation of Ras-dependent pathways to promote cell proliferation, survival, and transformation. By contrast, H-Ras mutations are rarely detected; however, its expression levels are often elevated in cancer cells. This study aims to identify the molecular pathways by which H-Ras exerts its oncogenic activity. Our previous data drew a link between H-Ras and apoptosis via death receptors (DRs), particularly DR4 and DR5 that are known to induce apoptosis upon ligation of their cognate ligand namely Tumor Necrosis Factor-related apoptosis inducing ligand (TRAIL). We showed that wild-type H-Ras was overexpressed in several human cancer cell lines that were resistant to TRAIL (Chen et al, Oncotarget, 2014). In this study, we tested H-Ras expression and its relationship with cellular susceptibility to TRAIL in a large panel of human cancer cell lines and xenograft tumor models. This included the use of stable cell lines that were developed via transfection of doxycycline-inducible sh-RNA against H-Ras transcript. These cancer cell lines were used to detect surface expression of DR4 and DR5 as well as TRAIL-induced apoptosis. A xenograft mouse model was developed by injecting the TRAIL-resistant H-Ras mutant cells into athymic mice. The mice were fed with water containing doxycycline, which effectively induced sh-RNA mediated reduction of H-Ras protein in the xenografts, and were subjected to intratumor injection with TRAIL, and immunohistochemistry analysis. Our results show that wild-type H-Ras GTPase is upregulated in TRAIL-resistant cells compared to TRAIL-sensitive cells. The elevated H-Ras expression correlated with a loss of DR4 and DR5 receptor expression on the plasma membrane in TRAIL-resistant cells. Knockdown of H-Ras in TRAIL-resistant cells restored the surface expression of DR4 and DR5, and subsequently increased apoptosis in response to TRAIL treatment. The xenograft mice developed tumors upon injection of the H-Ras mutant cells. Knockdown of H-Ras and TRAIL injection showed an increase of apoptotic markers in the tumor. This study confirms our previous report that high expression levels of H-Ras, while is found in its wild-type state, protect cancer cells from TRAIL induced apoptosis. As a TNF-related cytokine, TRAIL has been implicated in tumor immune surveillance by inducing apoptosis in target cells. H-Ras appears to suppress surface expression of DR4 and DR5, thereby promoting cancer cell survival through evasion from the immune surveillance mechanisms. These data have potential implications in identifying novel therapeutic targets for the development of effective cancer therapy. Citation Format: Su-Ryun Kim, Ancy D. Nalli, Baolin Zhang. Oncogenic H-Ras is a key regulator of death receptor-induced apoptosis [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 2401.

Abstract 1951: FCN-159: A novel, potent and selective oral inhibitor of MEK1/2 for the treatment of solid tumors

Abstract MEK1/2 (Mitogen-activated protein kinase kinase 1/2) is a key member in the RAS/RAF/MEK/ERK signaling pathway. The MEK pathway is found ubiquitously in tissues and regulates multiple cellular processes including proliferation, differentiation, migration, survival and angiogenesis. Dysregulation of MEK pathway through mutations in BRAF, KRAS and NRAS is frequently found in many types of cancers, such as melanoma, non-small cell lung cancer (NSCLC), prostate cancer and breast cancer, acute myeloid leukemia and acute lymphoblastic leukemia (ALL). MEK inhibitors inhibit cell proliferation, induce cell cycle arrest, as well as cell apoptosis in cancer cells, making MEK inhibition an effective anti-cancer strategy. Here we introduce FCN-159, a novel, selective and orally active inhibitor of MEK1/2. FCN-159 demonstrated selective kinase activities against MEK1 and MEK2. FCN-159 exhibited remarkable potency against the cell proliferation of a panel of human cancer cell lines with RAS/RAF mutations while sparing normal or cancer cell lines expressing wild type RAS/RAF, indicating highly selective inhibition against RAS/RAF/MEK signaling pathway. In human colon cancer cells, FCN-159 dose-dependently inhibited the phosphorylation of MEK downstream effector ERK and induced cell cycle arrest as well as cell apoptosis. FCN-159 showed significant and dose-dependent anti-tumor activities in a variety of human tumor xenograft models, derived from colon cancer (HT-29 and Colo205), melanoma (A375), NSCLC (Calu-6) and AML (HL-60). In addition, FCN-159 potently inhibited tumor growth in two patient-derived xenograft (PDX) models bearing NRAS mutation. The in vivo anti-tumor activity of FCN-159 was comparable to or stronger than FDA-approved MEK inhibitor trametinib. In non-clinical studies, FCN-159 exhibited excellent pharmacokinetic (PK) and safety properties. In particular, FCN-159 had longer T1/2 and higher dose-normalized AUC in both rats and dogs, compared with trametinib. Overall, FCN-159 exhibits great potential as a clinically-useful MEK inhibitor, as seen in its marked in vitro and in vivo anti-tumor efficacy, improved PK properties and good safety profiles. FCN-159 can be a novel and effective targeted monotherapy and potentially in combination to treat patients with advanced solid tumors. A Phase I clinical trial of FCN-159 is ongoing in China to treat patients with NRAS-aberrant (Ia) and NRAS-mutant (Ib) advanced melanoma (NCT03932253). Citation Format: Shu Lin, Xingdong Zhao, Zuwen Zhou, Haohan Tan, Ling Chen, Rui Tan, Weipeng Zhang, Lihua Jiang, Li Linghu, Jing Sun, Jiashu Zhou, Te Li, Yunlong Song, Weibo Wang. FCN-159: A novel, potent and selective oral inhibitor of MEK1/2 for the treatment of solid tumors [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 1951.

Abstract 2890: IMGN151 - A next generation folate receptor alpha targeting antibody drug conjugate active against tumors with low, medium and high receptor expression

Abstract Folate Receptor alpha (FRα) is an attractive antibody drug conjugate (ADC) target due to its over expression in multiple epithelial malignancies including ovarian, endometrial, triple negative breast, and non-small cell lung cancer, with limited expression on normal tissues. IMGN853 (i.e., mirvetuximab soravtansine and M9346A-sulfo-SPDB-DM4), a FRα targeting ADC, is currently in phase III (MIRASOL) clinical evaluation as monotherapy in patients with platinum-resistant epithelial ovarian cancer with high levels of FRα expression. The MIRASOL study builds on the results from the prior randomized study, FORWARD I, which demonstrated that improved outcomes with IMGN853 correlated with FRα expression, with the strongest treatment effects for all efficacy endpoints in ovarian cancer patients with FRα-high disease (Moore, ESMO 2019). In order to address the unmet needs of additional patient populations, we sought to develop a next generation FRα-targeting ADC active against tumors with a broad range of FRα expression. Development of a new molecular entity with the desired antitumor properties included optimization of the antibody format and the linker-payload. The resulting lead ADC denoted IMGN151 comprises an asymmetric, bivalent, biparatopic antibody targeting two independent epitopes of FRα, linked to the highly potent maytansinoid derivative DM21 via a stable cleavable peptide linker. The average drug per antibody ratio is 3.5. The binding, internalization and processing of the biparatopic IMGN151 and the parent monospecific antibodies were compared using 3H-antibodies. In tumor cells with medium (JHOS4) and high (KB) FRα expression the biparatopic antibody boosted antibody binding events and processing by 100% and 170%, respectively. The plasma stability of IMGN151 was tested in a cynomolgus monkey pharmacokinetic study. The stable linker increased ADC half-life by 60 hours and conjugate exposure in vivo by 40%, as compared to IMGN853. IMGN151 activity was characterized against cell lines and xenograft models with a wide range of FRα expression and compared to IMGN853. In in vitro studies, both ADCs had similar activity against FRα-high KB cells; IMGN151 was up to 200 times more active against four FRα-medium cell lines. IMGN151 had also notably stronger bystander killing activity in a mixed culture of target-positive and negative cells. In vivo IMGN151 induced complete tumor regressions of human tumor xenograft models with high (KB, H-score of 300), medium (Igrov-1 and Ishikawa, H-score of 140 and 100, respectively) and low (Ov-90, H-score of 30) FRα expression. All tested doses were well tolerated with no body weight loss observed. With a novel biparatopic antibody and linker payload design, IMGN151 has shown potent antitumor activity against ovarian cancer models with a broad range of FRα expression, which warrants further development into the clinic for patients with tumors expressing FRα at a wide range of levels. Citation Format: Olga Ab, Laura M. Bartle, Leanne Lanieri, Jose F. Ponte, Qifeng Qiu, Surina Sikka, Juliet A. Costoplus, Wayne Deats, Nicholas C. Yoder, Wayne C. Widdison, Katherine Mucciarone, Kate Selvitelli, Ying Chen, Neeraj Kohli, Thomas Chittenden, Richard Gregory, Yulius Setiady, Eric H. Westin. IMGN151 - A next generation folate receptor alpha targeting antibody drug conjugate active against tumors with low, medium and high receptor expression [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 2890.

Relaxin-expressing oncolytic adenovirus induces remodeling of physical and immunological aspects of cold tumor to potentiate PD-1 blockade

BackgroundCurrently, several antibody (Ab)-based therapies have shown excellent therapeutic effects in the clinic. Nonetheless, Ab penetration into tumor tissues is limited due to abnormal vasculature, tumor interstitial pressure, and excessive...

Manipulating the function of tumor-associated macrophages by siRNA-loaded lipid nanoparticles for cancer immunotherapy

The tumor-microenvironment contains large numbers of tumor-associated macrophages (TAMs) which are largely M2 phenotypes and are involved in pro-tumorous functions. Targeting TAMs so as to manipulate them and to modify their functions could be a novel immunotherapy for the treatment of cancer. Such a strategy would involve targeting TAMs with short interfering RNA (siRNA) to modify their functions by silencing certain genes that are responsible for their M2 polarization. In this study, a lipid nanoparticle (LNP) formulation was used to target and deliver siRNA to TAMs. The LNP was mainly composed of a novel, pH-sensitive cationic lipid, referred to as the CL4H6 lipid, which had previously been optimized to target hepatocytes. The optimized siRNA-loaded CL4H6-LNPs were selectively and efficiently taken up and showed strong gene silencing activity in TAMs in a human tumor xenograft model in nude mice. Furthermore, an anti-tumor therapeutic response in the same tumor model was obtained by targeting TAMs using the optimized siRNA-loaded CL4H6-LNPs. The anti-tumor therapeutic response was obtained through the silencing of the signal transducer and activator of transcription 3 (STAT3) and hypoxia inducible factor 1 α (HIF-1α), which resulted in an increase in the level of infiltrated macrophage (CD11b+ cells) into the tumor-microenvironment (TME) as well as a tendency to increase the concentration of M1 macrophages (CD169+ cells). The treatment also resulted in reversing the pro-tumorous functions of TAMs -mainly angiogenesis and tumor cell activation-, as evidenced by a decrease in the related gene expression at the mRNA level. This research has promising clinical and pharmaceutical applications for achieving novel macrophage-based cancer immunotherapy.

Anti-Cancer Activity of Acriflavine as Metabolic Inhibitor of OXPHOS in Pancreas Cancer Xenografts.

PurposeAll currently available therapies for the treatment of pancreatic ductal adenocarcinoma (PDAC) show limited success. PDACs fast progression depends on the tumor characteristics and can be influenced by the microenvironment. The antibacterial drug acriflavine (ACF) has been shown to have anti-cancer activities in cell lines.Materials and MethodsTo understand the working mechanism of ACF on cancer progression and tumor-stromal interplay, we evaluated pancreatic cancer in cell culture (Panc-1) (morphology, cell invasion and RNA expression) and the macrophage cell line THP1 (representing innate immune stromal cells). In the translational arm, the activity of ACF on xenograft models of human PDAC tumors representing different clinical subclasses was investigated (tumor growth, morphology and immunofluorescence, RNA expression and pathway analysis).ResultsIn vitro, ACF reduces epithelial-to-mesenchymal transition (EMT) and invasion of Panc-1 cells and shifts macrophage polarization to a M1-like anti-tumoral phenotype. At non-toxic concentrations, ACF downregulates cell metabolism. In xenografts, effect on tumor growth and metabolism could be confirmed; however, the innate immune stromal cells did not respond. Importantly, only in the moderately differentiated PDAC model, ACF could significantly suppress tumor growth and not in the fast-growing EMT-high model. Pathway analysis shows that ACF highly significantly downregulates metabolic pathways, especially OXPHOS and MYC/cell proliferation pathways in xenografts.ConclusionACF, with known pleiotropic effects on cancer cells, is in this study shown to be an attractive therapeutic based on its novel observed metabolic activity. Repurposing this compound for cancer treatment should be in the setting with other targeting agents, which offers reduced chance of resistance development in PDAC. Further evaluation should best be done in biological complex models such as human xenografts or syngeneic cancer models.