Search

Purpose: To explore the role of TWEAK in tumor growth and antitumor immune response and the activity and mechanism of RG7212, an antagonistic anti-TWEAK antibody, in tumor models.Experimental Design: TWEAK-induced signaling and gene expression were explored in tumor cell lines and inhibition of these effects and antitumor efficacy with RG7212 treatment was assessed in human tumor xenograft-, patient-derived xenograft, and syngeneic tumor models and phase I patients. Genetic features correlated with antitumor activity were characterized.Results: In tumor cell lines, TWEAK induces proliferation, survival, and NF-κB signaling and gene expression that promote tumor growth and suppress antitumor immune responses. TWEAK-inducible CD274, CCL2, CXCL-10 and -11 modulate T-cell and monocyte recruitment, T-cell activation, and macrophage differentiation. These factors and TWEAK-induced signaling were decreased, and tumor, blood, and spleen immune cell composition was altered with RG7212 treatment in mice. RG7212 inhibits tumor growth in vivo in models with TWEAK receptor, Fn14, expression, and markers of pathway activation. In phase I testing, signs of tumor shrinkage and stable disease were observed without dose-limiting toxicity. In a patient with advanced, Fn14-positive, malignant melanoma with evidence of tumor regression, proliferation markers were dramatically reduced, tumor T-cell infiltration increased, and tumor macrophage content decreased. Antitumor activity, a lack of toxicity in humans and animals and no evidence of antagonism with standard of care or targeted agents in mice, suggests that RG7212 is a promising agent for use in combination therapies in patients with Fn14-positive tumors. Clin Cancer Res; 19(20); 5686–98. ©2013 AACR.

XLS file, 33K, Summary of RG7212 monotherapy and combination antitumor efficacy.

PDF file, 306K, RG7212 antitumor efficacy and characterization of tumor models used to assess RG7212 antitumor efficacy.

PDF file, 771K, TWEAK promotes proliferation and survival signaling with minimal effect on cell viability.

PDF file, 132K.

XLS file, 193K, Sorted Affymetrix gene array data.

PDF file, 29K, Lead antibodies have identical binding properties and neutralizing activities.

PDF file, 176K, Assessment of baseline gene expression in RG7212 responders and non-responders in qPCR array.

Purpose: To assess the potential for CUE-101, a novel therapeutic fusion protein, to selectively activate and expand HPV16 E711-20-specific CD8+ T cells as an off-the shelf therapy for the treatment of HPV16-driven tumors, including head and neck squamous cell carcinoma (HNSCC), cervical, and anal cancers. Experimental Design: CUE-101 is an Fc fusion protein composed of a human leukocyte antigen (HLA) complex, an HPV16 E7 peptide epitope, reduced affinity human IL2 molecules, and an effector attenuated human IgG1 Fc domain. Human E7-specific T cells and human peripheral blood mononuclear cells (PBMC) were tested to demonstrate cellular activity and specificity of CUE-101, whereas in vivo activity of CUE-101 was assessed in HLA-A2 transgenic mice. Antitumor efficacy with a murine surrogate (mCUE-101) was tested in the TC-1 syngeneic tumor model. Results: CUE-101 demonstrates selective binding, activation, and expansion of HPV16 E711-20-specific CD8+ T cells from PBMCs relative to nontarget cells. Intravenous administration of CUE-101 induced selective expansion of HPV16 E711-20-specific CD8+ T cells in HLA-A2 (AAD) transgenic mice, and anticancer efficacy and immunologic memory was demonstrated in TC-1 tumor-bearing mice treated with mCUE-101. Combination therapy with anti-PD-1 checkpoint blockade further enhanced the observed efficacy. Conclusions: Consistent with its design, CUE-101 demonstrates selective expansion of an HPV16 E711-20-specific population of cytotoxic CD8+ T cells, a favorable safety profile, and in vitro and in vivo evidence supporting its potential for clinical efficacy in an ongoing phase I trial (NCT03978689).

Background Immuno-STATsTM are novel, modular fusion proteins designed to selectively activate tumor-antigen-specific CD8+ T cells. Human papillomavirus (HPV) associated cancers serve as a model system to assess the safety and efficacy of the Immuno-STAT platform. CUE-101 is comprised of human leukocyte antigen (HLA) complex, HLA A*0201, a peptide epitope derived from the HPV type 16 E7 protein, and 4 molecules of a reduced affinity human interleukin-2 (IL2) designed to bind and activate HPV-specific T cells for eradication of HPV16-driven cancers. In preclinical studies CUE-101 demonstrated selective binding, activation, and expansion of HPV16 E7-specific CD8+ T cells, which translated into anti-tumor activity.1 Methods CUE-101-01 is a first-in-human (FIH) phase 1 study in patients diagnosed with HPV16+ recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC) refractory to one or more lines of therapy. Trial eligibility includes MHC class I type HLA-A*0201 and a diagnosis of an HPV16+ HNSCC, as assessed by p16 IHC and confirmed by HPV16 mRNA ISH. CUE-101 is administered intravenously over 60 minutes every 21 days. Objectives include determination of safety, pharmacodynamics (PD), pharmacokinetics (PK), recommended phase 2 dose (RP2D), and preliminary anti-tumor activity. The safety results from treated participants will be presented. Results 19 participants have received CUE-101 monotherapy as of August 7, 2020. Doses ranging from 0.06 to 1 mg/kg were determined to be safe and well-tolerated, enabling dose escalation to 2 mg/kg. Preliminary PK data demonstrate dose-dependent increases in drug exposure which are sustained upon repeat dosing, and low inter-subject variability. Preliminary data from systemic blood analyses show early signals of expansion of HPV-16 E711-20-specific CD8+ T cells. Stable disease (SD), as determined by RECIST 1.1, was observed in several participants in these early dose cohorts, with one subject maintaining SD up to 19 weeks. The maximum tolerated dose (MTD) has not yet been reached. As of May 14, 2020 (the development safety update report (DSUR) data-lock date), no dose limiting toxicities and the following adverse events were observed in the first 12 patients treated with CUE-101: fatigue (n=3), decreased appetite (n=1), arthralgia (n=1), muscular weakness (n=1), parasthesia (n=1), bullous pemphigoid (n=1), and infusion-related reactions (n=1). Conclusions CUE-101 is a novel agent that is demonstrating acceptable tolerability, favorable PK, and preliminary PD signals that support selective activation of tumor-specific T cells. Neither the MTD nor the monotherapy RP2D have been established. PD and PK analyses are ongoing as dose escalation continues. Acknowledgements The authors would like to thank all the patients who are participating in this study. The study is sponsored by Cue Biopharma. Trial Registration ClinicalTrials. gov NCT03978689 Ethics Approval This study was approved by Ethics and Institutional Review Boards (IRBs) at all study sites; IRB reference numbers: DF/HCC IRB# 19-374 (Massachusetts General Hospital), HRPO# 201905108 (Washington University School of Medicine), IRB 191714 (Vanderbilt University Medical Center Vanderbilt-Ingram Cancer Center), Advarra Pro00037736 (Moffitt Cancer Center), IRB(IRBMED) HUM00165746 (University of Michigan Comprehensive Cancer Center), 2019-087 (Karmanos Cancer Institute), WIRB IRB00112341(Winship Cancer Institute/Emory University), WIRB 2000026098 (Yale Cancer Center), WIRB STUDY00008948 (University of Washington, Seattle ), WIRB 1908869642 (University of Arizona Cancer Center, IRB 20-073 (Memorial Sloan Kettering Cancer Center), 2019-0578 (The University of Texas MD Anderson Cancer Center), IRB 52744 (Stanford University School of Medicine). Reference Quayle SN, Girgis N, Thapa DR, et al. CUE-101, a Novel HPV16 E7-pHLA-IL-2-Fc Fusion Protein, Enhances Tumor Antigen Specific T Cell Activation for the Treatment of HPV16-Driven Malignancies. Clin Cancer Res 2020;26:1953–64.

Purpose: To explore the role of TWEAK in tumor growth and antitumor immune response and the activity and mechanism of RG7212, an antagonistic anti-TWEAK antibody, in tumor models. Experimental Design: TWEAK-induced signaling and gene expression were explored in tumor cell lines and inhibition of these effects and antitumor efficacy with RG7212 treatment was assessed in human tumor xenograft-, patient-derived xenograft, and syngeneic tumor models and phase I patients. Genetic features correlated with antitumor activity were characterized. Results: In tumor cell lines, TWEAK induces proliferation, survival, and NF-κB signaling and gene expression that promote tumor growth and suppress antitumor immune responses. TWEAK-inducible CD274, CCL2, CXCL-10 and -11 modulate T-cell and monocyte recruitment, T-cell activation, and macrophage differentiation. These factors and TWEAK-induced signaling were decreased, and tumor, blood, and spleen immune cell composition was altered with RG7212 treatment in mice. RG7212 inhibits tumor growth in vivo in models with TWEAK receptor, Fn14, expression, and markers of pathway activation. In phase I testing, signs of tumor shrinkage and stable disease were observed without dose-limiting toxicity. In a patient with advanced, Fn14-positive, malignant melanoma with evidence of tumor regression, proliferation markers were dramatically reduced, tumor T-cell infiltration increased, and tumor macrophage content decreased. Antitumor activity, a lack of toxicity in humans and animals and no evidence of antagonism with standard of care or targeted agents in mice, suggests that RG7212 is a promising agent for use in combination therapies in patients with Fn14-positive tumors. Clin Cancer Res; 19(20); 5686–98. ©2013 AACR.

A high percentage of patients with BRAFV600E mutant melanomas respond to the selective RAF inhibitor vemurafenib (RG7204, PLX4032) but resistance eventually emerges. To better understand the mechanisms of resistance, we used chronic selection to establish BRAFV600E melanoma clones with acquired resistance to vemurafenib. These clones retained the V600E mutation and no second-site mutations were identified in the BRAF coding sequence. Further characterization showed that vemurafenib was not able to inhibit extracellular signal-regulated kinase phosphorylation, suggesting pathway reactivation. Importantly, resistance also correlated with increased levels of RAS-GTP, and sequencing of RAS genes revealed a rare activating mutation in KRAS, resulting in a K117N change in the KRAS protein. Elevated levels of CRAF and phosphorylated AKT were also observed. In addition, combination treatment with vemurafenib and either a MAP/ERK kinase (MEK) inhibitor or an AKT inhibitor synergistically inhibited proliferation of resistant cells. These findings suggest that resistance to BRAFV600E inhibition could occur through several mechanisms, including elevated RAS-GTP levels and increased levels of AKT phosphorylation. Together, our data implicate reactivation of the RAS/RAF pathway by upstream signaling activation as a key mechanism of acquired resistance to vemurafenib, in support of clinical studies in which combination therapy with other targeted agents are being strategized to combat resistance. Cancer Res; 72(4); 969–78. ©2011 AACR.

The protein kinase BRAF is a key component of the RAS–RAF signaling pathway which plays an important role in regulating cell proliferation, differentiation, and survival. Mutations in BRAF at codon 600 promote catalytic activity and are associated with 8% of all human (solid) tumors, including 8% to 10% of colorectal cancers (CRC). Here, we report the preclinical characterization of vemurafenib (RG7204; PLX4032; RO5185426), a first-in-class, specific small molecule inhibitor of BRAFV600E in BRAF-mutated CRC cell lines and tumor xenograft models. As a single agent, vemurafenib shows dose-dependent inhibition of ERK and MEK phosphorylation, thereby arresting cell proliferation in BRAFV600-expressing cell lines and inhibiting tumor growth in BRAFV600E bearing xenograft models. Because vemurafenib has shown limited single-agent clinical activity in BRAFV600E-mutant metastatic CRC, we therefore explored a range of combination therapies, with both standard agents and targeted inhibitors in preclinical xenograft models. In a BRAF-mutant CRC xenograft model with de novo resistance to vemurafenib (RKO), tumor growth inhibition by vemurafenib was enhanced by combining with an AKT inhibitor (MK-2206). The addition of vemurafenib to capecitabine and/or bevacizumab, cetuximab and/or irinotecan, or erlotinib resulted in increased antitumor activity and improved survival in xenograft models. Together, our findings suggest that the administration of vemurafenib in combination with standard-of-care or novel targeted therapies may lead to enhanced and sustained clinical antitumor efficacy in CRCs harboring the BRAFV600E mutation. Cancer Res; 72(3); 779–89. ©2011 AACR.

RO4396686 is a small molecule KDR, FGFR, and PDGFR inhibitor with good pharmacokinetic properties in rodents. In a mouse corneal neovascularization assay, this compound inhibited VEGF-induced angiogenesis. Tested in a H460a xenograft tumor model this agent effected significant tumor growth inhibition at doses as low as 50 mg/kg.

(±)-1-(anti-3-Hydroxy-cyclopentyl)-3-(4-methoxy-phenyl)-7-phenylamino-3,4-dihydro-1H-pyrimido[4,5-d]pyrimidin-2-one (RO4383596) is a potent and selective inhibitor of the pro-angiogenic receptor tyrosine kinases KDR, FGFR, and PDGFR. This agent has an excellent pharmacokinetic profile and is highly efficacious in rodent models of angiogenesis upon oral administration.

A novel class of 3,5,6-trisubstituted naphthostyril analogues was designed and synthesized to study the structure-activity relationship for inhibition of cyclin-dependent kinase 2 (CDK2). These compounds, particularly molecules with side-chain modifications providing additional hydrogen bonding capability, were demonstrated to be potent CDK2 inhibitors with cellular activities consistent with CDK2 inhibition. These molecules inhibited tumor cell proliferation and G1-S and G2-M cell-cycle progression in vitro. The X-ray crystal structure of a 2-aminoethyleneamine derivative bound to CDK2, refined to 2.5A resolution, is presented.

Here we describe the discovery and the structure of PEN-221, a somatostatin receptor 2 (SSTR2) targeting peptide conjugated to DM1. PEN-221 is the first clinical compound from Tarveda’s Pentarin platform, which utilizes miniaturized drug conjugates that diffuse rapidly and deeply into solid tumors. Antibody drug conjugates (ADCs) have garnered a significant amount of attention in their ability to direct cytotoxic drugs to cancer cells; however, the efficacy of ADCs in solid tumors is limited by the slow diffusion of such large molecules through solid tumor tissue. Pentarins are designed to improve the efficacy of targeted therapies through effective tumor cell targeting and enhanced tumor penetration. SSTR2, a GPCR overexpressed in multiple types of neuroendocrine tumors, including small cell lung cancers, internalizes rapidly upon agonist stimulation, making it an ideal vector for delivering cytotoxic payloads. Examination of a variety of SSTR2 targeting ligands, as well as several potential conjugation sites, led to the identification of the C-terminal side chain of [Tyr3]-octreotate amide as the best conjugation site for a lipophilic payload. The use of DM1 as a payload afforded superior receptor affinity and receptor internalization when compared to other similarly potent microtubule-targeting agents. In vitro studies show that PEN-221 has receptor-dependent cytotoxic effects, and preclinical studies demonstrate PEN-221 induces tumor regression in several SSTR2 expressing xenograft models. Citation Format: Brian H. White, Patrick Bazinet, Kerry Whalen, Michelle DuPont, James M. Quinn, Rossitza Alargova, Tsun Au Yeung, Adam Brockman, James Gifford, Haley Oller, Kristina Kriksciukaite, Charles-Andre Lemelin, Patrick Lim Soo, Benoit Moreau, Samantha Perino, Gitanjali Sharma, Rajesh Shinde, Beata Sweryda-Krawiec, Mary Simcox, Richard Wooster, Mark T. Bilodeau. Discovery of PEN-221, an SSTR2-targeting maytansinoid conjugate with potent activity in vitro and in vivo [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 39. doi:10.1158/1538-7445.AM2017-39

The identification of new biomarkers is essential in the implementation of personalized health care strategies that offer new therapeutic approaches with optimized and individualized treatment. In support of hypothesis generation and testing in the course of our biomarker research an online portal and respective function-tested reverse transcription quantitative real-time PCR assays (RT–qPCR) facilitated the selection of relevant biomarker genes. We have established workflows applicable for convenient high throughput gene expression analysis in biomarker research with cell lines (in vitro studies) and xenograft mouse models (in vivo studies) as well as formalin-fixed paraffin-embedded tissue (FFPET) sections from various human research and clinical tumor samples. Out of 92 putative biomarker candidate genes selected in silico, 35 were shown to exhibit differential expression in various tumor cell lines. These were further analysed by in vivo xenograft mouse models, which identified 13 candidate genes including potential response prediction biomarkers and a potential pharmacodynamic biomarker. Six of these candidate genes were selected for further evaluation in FFPET samples, where optimized RNA isolation, reverse transcription and qPCR assays provided reliable determination of relative expression levels as precondition for differential gene expression analysis of FFPET samples derived from projected clinical studies. Thus, we successfully applied function tested RT–qPCR assays in our biomarker research for hypothesis generation with in vitro and in vivo models as well as for hypothesis testing with human FFPET samples. Hence, appropriate function-tested RT–qPCR assays are available in biomarker research accompanying the different stages of drug development, starting from target identification up to early clinical development. The workflow presented here supports the identification and validation of new biomarkers and may lead to advances in efforts to achieve the goal of personalized health care.

The BRAFV600E mutation is common in several human cancers, especially melanoma. RG7204 (PLX4032) is a small-molecule inhibitor of BRAFV600E kinase activity that is in phase II and phase III clinical testing. Here, we report a preclinical characterization of the antitumor activity of RG7204 using established in vitro and in vivo models of malignant melanoma. RG7204 potently inhibited proliferation and mitogen-activated protein/extracellular signal-regulated kinase (ERK) kinase and ERK phosphorylation in a panel of tumor cell lines, including melanoma cell lines expressing BRAFV600E or other mutant BRAF proteins altered at codon 600. In contrast, RG7204 lacked activity in cell lines that express wild-type BRAF or non-V600 mutations. In several tumor xenograft models of BRAFV600E-expressing melanoma, we found that RG7204 treatment caused partial or complete tumor regressions and improved animal survival, in a dose-dependent manner. There was no toxicity observed in any dose group in any of the in vivo models tested. Our findings offer evidence of the potent antitumor activity of RG7204 against melanomas harboring the mutant BRAFV600E gene. Cancer Res; 70(13); 5518–27. ©2010 AACR.

Solid tumor growth requires new blood vessel formation or angiogenesis. Inhibition of angiogenesis as a therapeutic strategy in oncology has been validated by treatments that block vascular endothelial growth factor (VEGF) or its receptors, such as bevacizumab (Avastin®), which is an anti-VEGF antibody that prolongs survival in colorectal, lung and other cancer patients in combination with chemotherapy. However, since not all tumors are sensitive to VEGF blockade and resistance mechanisms to VEGF therapies can develop, inhibition of additional targets may be necessary in order to control tumor growth and achieve better clinical effects. PlGF is a member of the VEGF family that is found only in very low levels under normal physiological conditions, but is up-regulated in almost all major malignant diseases. PlGF expression has shown to correlate with tumor stages and patient survival in breast cancer, CRC and gastric cancer [1-3]. Pre-clinical data support a role for PlGF in tumor angiogenesis, and demonstrate that blocking PlGF can inhibit tumor growth [4]. RO5323441, a humanized IgG1 monoclonal antibody directed against PlGF, has demonstrated anti-tumor activity in human tumor xenograft models in mice, has a benign preclinical toxicology profile and is being developed for the treatment of multiple advanced cancer indications. RO5323441 binds to both PlGF-1 and PlGF-2 in a dose dependent manner, and is not cross-reactive with murine PlGF or human VEGF. RO5323441 inhibits the binding of human PlGF-1 or PlGF-2 to VEGFR −1 with IC50 values of 0.1 and 0.2 nM, respectively. RO5323441 blocks PlGF-induced VEGFR-1 phosphorylation in Flt-1-transfected HEK293 cells. Antitumor activity has been demonstrated in mutliple tumor models including ACHN and Caki-1 renal cell carcinoma and Huh-7, hepatocellular carcinoma xenografts. Inhibition of established tumors ranged from 43-97% with twice weekly dosing. A refractory model of non-small cell lung cancer has also been identified. Studies to understand the mechanism of action and to identify pharmacodynamic markers and have been carried out in ACHN-tumor bearing mice. References 1. Wei SC, Tsao PN, Yu SC, et al. Placenta growth factor expression is correlated with survival of patients with colorectal cancer. Gut. 2005 May; 54(5):666-72. 2. Parr C, Watkins G, Boulton M et al. Placenta growth factor is over-expressed and has prognostic value in human breast cancer. Eur J Cancer. 2005 Dec;41(18):2819-27. 3. Chen CN, Hsieh FJ, Cheng YM, et al. The significance of placenta growth factor in angiogenesis and clinical outcome of human gastric cancer. Cancer Lett. 2004 Sep 15;213(1):73-82. 4. Fischer C, Jonckx B, Mazzone M, et al. Anti-PlGF inhibits growth of VEGF(R)-inhibitor-resistant tumors without affecting healthy vessels. Cell. 2007. 131 : 463-75. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1370.

e14629 Background: R1530 is a multikinase inhibitor currently in clinical phase I testing. Its inhibitory profile includes several kinases that play critical roles in cancer cell growth and division as well as tumor angiogenesis. These properties translate into a potent cytotoxicity in a wide range of cancer cell lines in vitro and tumor growth inhibition in human tumor xenografts. Preclinical studies were conducted to evaluate the effects of R alone and in combination with B and P in the Caki-1 RCC xenograft model. Methods: We initially evaluated the antitumor activity of optimal dose (OD) and 1/2 OD R alone and with OD B. This was followed up with testing of OD & 1/2 OD P ± OD B, along with triplets of 1/2 OD P + OD B + 1/2 OD R and OD P + OD B + 1/2 OD R. A final study compared 1/2 OD R to OD R triplets to attempt to increase tumor growth inhibition (TGI) and increase life span (ILS). Results: No doublets or triplets tested showed antagonism or enhanced toxicity. Antitumor activity and survival results are listed below (Table). Conclusions: 1/2 OD R + OD B or OD R + OD B doublets gave better TGI and ILS than monotherapy. Comparing these two doublets, TGI is better in the high dose combination but ILS is equivalent. All TGI and ILS are better in doublet P + B combinations over respective single agent arms except for TGI (but not ILS) for 1/2 OD P vs its correlative doublet with B. The OD P + B doublet gave better TGI and ILS than 1/2 OD P + B doublet. TGI and ILS do not differ between triplets containing OD R + 1/2 OD or OD P or for triplets containing the OD P + 1/2 OD or OD R. Therefore, either agent can be alternatively dose reduced without a loss of tumor response or detriment to survival in this preclinical model of RCC. [Table: see text] [Table: see text]