Paula Kaplan-Lefko

c-Met inhibitors with novel binding mode show activity against several hereditary papillary renal cell carcinoma-related mutations.

c-Met is a receptor tyrosine kinase often deregulated in human cancers, thus making it an attractive drug target. One mechanism by which c-Met deregulation leads to cancer is through gain-of-function mutations. Therefore, small molecules capable of targeting these mutations could offer therapeutic benefits for affected patients. SU11274 was recently described and reported to inhibit the activity of the wild-type and some mutant forms of c-Met, whereas other mutants are resistant to inhibition. We identified a novel series of c-Met small molecule inhibitors that are active against multiple mutants previously identified in hereditary papillary renal cell carcinoma patients. AM7 is active against wild-type c-Met as well as several mutants, inhibits c-Met-mediated signaling in MKN-45 and U-87 MG cells, and inhibits tumor growth in these two models grown as xenografts. The crystal structures of AM7 and SU11274 bound to unphosphorylated c-Met have been determined. The AM7 structure reveals a novel binding mode compared with other published c-Met inhibitors and SU11274. The molecule binds the kinase linker and then extends into a new hydrophobic binding site. This binding site is created by a significant movement of the C-helix and so represents an inactive conformation of the c-Met kinase. Thus, our results demonstrate that it is possible to identify and design inhibitors that will likely be active against mutants found in different cancers.

Discovery and Optimization of Triazolopyridazines as Potent and Selective Inhibitors of the c-Met Kinase.

Tumorigenesis is a multistep process in which oncogenes play a key role in tumor formation, growth, and maintenance. MET was discovered as an oncogene that is activated by its ligand, hepatocyte growth factor. Deregulated signaling in the c-Met pathway has been observed in multiple tumor types. Herein we report the discovery of potent and selective triazolopyridazine small molecules that inhibit c-Met activity.

Adoptive Transfer of MART-1 T-Cell Receptor Transgenic Lymphocytes and Dendritic Cell Vaccination in Patients with Metastatic Melanoma

Purpose: It has been demonstrated that large numbers of tumor-specific T cells for adoptive cell transfer (ACT) can be manufactured by retroviral genetic engineering of autologous peripheral blood lymphocytes and expanding them over several weeks. In mouse models, this therapy is optimized when administered with dendritic cell (DC) vaccination. We developed a short 1-week manufacture protocol to determine the feasibility, safety, and antitumor efficacy of this double cell therapy. Experimental Design: A clinical trial (NCT00910650) adoptively transferring MART-1 T-cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide-pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma. Autologous TCR transgenic cells were manufactured in 6 to 7 days using retroviral vector gene transfer, and reinfused with (n = 10) or without (n = 3) prior cryopreservation. Results: A total of 14 patients with metastatic melanoma were enrolled and 9 of 13 treated patients (69%) showed evidence of tumor regression. Peripheral blood reconstitution with MART-1–specific T cells peaked within 2 weeks of ACT, indicating rapid in vivo expansion. Administration of freshly manufactured TCR transgenic T cells resulted in a higher persistence of MART-1–specific T cells in the blood as compared with cryopreserved. Evidence that DC vaccination could cause further in vivo expansion was only observed with ACT using noncryopreserved T cells. Conclusion: Double cell therapy with ACT of TCR-engineered T cells with a very short ex vivo manipulation and DC vaccines is feasible and results in antitumor activity, but improvements are needed to maintain tumor responses. Clin Cancer Res; 20(9); 2457–65. ©2014 AACR.

Design, synthesis, and biological evaluation of potent c-Met inhibitors.

c-Met is a receptor tyrosine kinase that plays a key role in several cellular processes but has also been found to be overexpressed and mutated in different human cancers. Consequently, targeting this enzyme has become an area of intense research in drug discovery. Our studies began with the design and synthesis of novel pyrimidone 7, which was found to be a potent c-Met inhibitor. Subsequent SAR studies identified 22 as a more potent analog, whereas an X-ray crystal structure of 7 bound to c-Met revealed an unexpected binding conformation. This latter finding led to the development of a new series that featured compounds that were more potent both in vitro and in vivo than 22 and also exhibited different binding conformations to c-Met. Novel c-Met inhibitors have been designed, developed, and found to be potent in vitro and in vivo.

Discovery of a Potent, Selective, and Orally Bioavailable c-Met Inhibitor: 1-(2-Hydroxy-2-methylpropyl)-N-(5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl)-5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-carboxamide (AMG 458)

Deregulation of the receptor tyrosine kinase c-Met has been implicated in human cancers. Pyrazolones with N-1 bearing a pendent hydroxyalkyl side chain showed selective inhibition of c-Met over VEGFR2. However, studies revealed the generation of active, nonselective metabolites. Blocking this metabolic hot spot led to the discovery of 17 (AMG 458). When dosed orally, 17 significantly inhibited tumor growth in the NIH3T3/TPR-Met and U-87 MG xenograft models with no adverse effect on body weight.

Antagonism of Ang-Tie2 and Dll4-Notch signaling has opposing effects on tumor endothelial cell proliferation, evidenced by a new flow cytometry method

Sustained angiogenesis is essential for tumor growth as it provides the tumor with a network of blood vessels that supply both oxygen and essential nutrients. Limiting tumor-associated angiogenesis is a proven strategy for the treatment of human cancer. To date, the rapid detection and quantitation of tumor-associated endothelial cell (TAEC) proliferation has been challenging, largely due to the low frequency of endothelial cells (ECs) within the tumor microenvironment. In this report, we address this problem using a new multiparametric flow cytometry method capable of rapid and precise quantitation of proliferation by measuring bromodeoxyuridine (BrdUrd) uptake in mouse TAECs from established human tumor xenografts. We determined the basal proliferation labeling index of TAECs in two human tumor xenografts representing two distinct histologies, COLO 205 (colorectal cancer) and U-87 (glioblastoma). We then investigated the effects of two large-molecule antiangiogenic agents targeting different biochemical pathways. Blocking angiopoietin-Tie2 signaling with the peptide-Fc fusion protein, trebananib (AMG 386), inhibited proliferation of TAECs, whereas blocking Dll4-Notch signaling with an anti-Dll4-specific antibody induced hyperproliferation of TAECs. These pharmacodynamic studies highlight the sensitivity and utility of this flow cytometry-based method and demonstrate the value of this assay to rapidly assess the in vivo proliferative effects of angiogenesis-targeted agents on both the tumor and the associated vasculature.

Abstract 3765: IND-Enabling GLP study to support a clinical trial of dual adoptive cell therapy combining stem cells and T cells engineered with an NY-ESO-1 TCR

T cell receptor (TCR) engineered adoptive T cell transfer (ACT) has shown remarkable antitumor efficacy in several clinical trials. However, low persistence of modified cells limits long-term clinical responses. To overcome this hurdle, we propose a clinical trial co-administering genetically modified T cells and stem cells both expressing an NY-ESO-1 TCR such that the engrafted stem cells generate a source for constant renewal of modified T cells. Here we report a pre-clinical IND-enabling study performed at UCLA under Good Laboratory Practice (GLP) compliance to assess whether co-administration impacts (I) safety; (II) engraftment and cell lineage differentiation of gene modified stem cells; and (III) persistence of adoptively transferred T cells and stem cell-derived progeny. We performed 12 optimization studies to define the optimal conditions for TCR gene modified ACT and TCR gene modified hematopoietic stem cell (HSC) bone marrow transplantation (BMT). Sixty-four HLA-A2/kb transgenic mice were myelodepleted and received syngeneic BMT with Lineage depleted bone marrow (Lin-) cells transduced with the LV-NYESO-1 TCR/sr39TK and ACT with T cells transduced with the RV-NYESO-1 TCR. Control groups were as follows: untreated mice, mice receiving mock transduced Lin- cells and T cells, mice receiving transduced Lin- cells and mock transduced T cells, and mice receiving mock transduced Lin- cells and transduced T cells (n = 16 per group). Overall survival at 3 months was 87.5%; no significant differences in survival were observed among cohorts. After BMT we observed a decrease in body weight, elevation in creatinine kinase and transaminases, and gonadal germ cell ablation in all cohorts. Three months after BMT, all blood cell lineages were reconstituted in surviving mice. Using digital droplet PCR and flow cytometry, we confirmed that transduced stem cells engrafted and their progeny persisted long term. In the bone marrow, NY-ESO-1 TCR was expressed intracellularly among progenitor cells (Lin-, LSK and HSC) as well as all hematopoietic cell lineages within the spleen (CD8+ T cells, CD4+ T cells, NKT cells, B cells and granulocytes). Co-administration with gene modified T cells and stem cells did not affect engraftment, cell lineage differentiation or persistence of the gene modified stem cells. Moreover, co-administration with stem cells did not affect persistence of adoptively transferred T cells. These data demonstrate that 1) NY-ESO-1 TCR genetically modified stem cells engraft and differentiate into all hematopoietic cell lineage progeny, which persists at 3 months; 2) adoptively transferred NY-ESO-1 TCR T cells persist at 3 months; 3) co-administration of stem cells and T cells genetically modified to express an NY-ESO-1 TCR is safe and does not negatively impact stem cell engraftment, lineage differentiation and progeny persistence or T cell persistence. Citation Format: Cristina Puig-Saus, Giulia Parisi, Paige Krystofinski, Angel Garcia-Diaz, Salemiz Sandoval, James McCabe, Ruixue Zhang, Gardenia Cheung-Lau, Nhat Truong, Justin Saco, Sara Komenan, Agustin Vega-Crespo, Mignonette H Macabali, Begona Comin-Anduix, Beata Berent-Maoz, Donald Kohn, Paula Kaplan-Lefko, Antoni Ribas. IND-Enabling GLP study to support a clinical trial of dual adoptive cell therapy combining stem cells and T cells engineered with an NY-ESO-1 TCR [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 3765. doi:10.1158/1538-7445.AM2017-3765