Search

TRAIL can activate cell surface death receptors, resulting in potent tumor cell death via induction of the extrinsic apoptosis pathway. Eftozanermin alfa (ABBV-621) is a second generation TRAIL receptor agonist engineered as an IgG1-Fc mutant backbone linked to two sets of trimeric native single-chain TRAIL receptor binding domain monomers. This hexavalent agonistic fusion protein binds to the death-inducing DR4 and DR5 receptors with nanomolar affinity to drive on-target biological activity with enhanced caspase-8 aggregation and death-inducing signaling complex formation independent of FcγR-mediated cross-linking, and without clinical signs or pathologic evidence of toxicity in nonrodent species. ABBV-621 induced cell death in approximately 36% (45/126) of solid cancer cell lines in vitro at subnanomolar concentrations. An in vivo patient-derived xenograft (PDX) screen of ABBV-621 activity across 15 different tumor indications resulted in an overall response (OR) of 29% (47/162). Although DR4 (TNFSFR10A) and/or DR5 (TNFSFR10B) expression levels did not predict the level of response to ABBV-621 activity in vivo, KRAS mutations were associated with elevated TNFSFR10A and TNFSFR10B and were enriched in ABBV-621–responsive colorectal carcinoma PDX models. To build upon the OR of ABBV-621 monotherapy in colorectal cancer (45%; 10/22) and pancreatic cancer (35%; 7/20), we subsequently demonstrated that inherent resistance to ABBV-621 treatment could be overcome in combination with chemotherapeutics or with selective inhibitors of BCL-XL. In summary, these data provide a preclinical rationale for the ongoing phase 1 clinical trial (NCT03082209) evaluating the activity of ABBV-621 in patients with cancer.Significance:This study describes the activity of a hexavalent TRAIL-receptor agonistic fusion protein in preclinical models of solid tumors that mechanistically distinguishes this molecular entity from other TRAIL-based therapeutics.

Supplementary file containing all supplementary figures and tables

TRAIL can activate cell surface death receptors, resulting in potent tumor cell death via induction of the extrinsic apoptosis pathway. Eftozanermin alfa (ABBV-621) is a second generation TRAIL receptor agonist engineered as an IgG1-Fc mutant backbone linked to two sets of trimeric native single-chain TRAIL receptor binding domain monomers. This hexavalent agonistic fusion protein binds to the death-inducing DR4 and DR5 receptors with nanomolar affinity to drive on-target biological activity with enhanced caspase-8 aggregation and death-inducing signaling complex formation independent of FcγR-mediated cross-linking, and without clinical signs or pathologic evidence of toxicity in nonrodent species. ABBV-621 induced cell death in approximately 36% (45/126) of solid cancer cell lines in vitro at subnanomolar concentrations. An in vivo patient-derived xenograft (PDX) screen of ABBV-621 activity across 15 different tumor indications resulted in an overall response (OR) of 29% (47/162). Although DR4 (TNFSFR10A) and/or DR5 (TNFSFR10B) expression levels did not predict the level of response to ABBV-621 activity in vivo, KRAS mutations were associated with elevated TNFSFR10A and TNFSFR10B and were enriched in ABBV-621–responsive colorectal carcinoma PDX models. To build upon the OR of ABBV-621 monotherapy in colorectal cancer (45%; 10/22) and pancreatic cancer (35%; 7/20), we subsequently demonstrated that inherent resistance to ABBV-621 treatment could be overcome in combination with chemotherapeutics or with selective inhibitors of BCL-XL. In summary, these data provide a preclinical rationale for the ongoing phase 1 clinical trial (NCT03082209) evaluating the activity of ABBV-621 in patients with cancer. Significance: This study describes the activity of a hexavalent TRAIL-receptor agonistic fusion protein in preclinical models of solid tumors that mechanistically distinguishes this molecular entity from other TRAIL-based therapeutics.

The murine gammaherpesvirus 68 (MHV-68 or γHV-68) model provides many advantages for studying virus-host interactions involved in gammaherpesvirus replication, including the role of cellular responses to infection. We examined the effects of cellular cyclooxygenase-2 (COX-2) and its by-product prostaglandin E2(PGE2) on MHV-68 gene expression and protein production following de novo infection of cultured cells. Western blot analyses revealed an induction of COX-2 protein in MHV-68-infected cells but not in cells infected with UV-irradiated MHV-68. Luciferase reporter assays demonstrated activation of theCOX-2promoter during MHV-68 replication. Two nonsteroidal anti-inflammatory drugs, a COX-2-specific inhibitor (NS-398) and a COX-1-COX-2 inhibitor (indomethacin), substantially reduced MHV-68 protein production in infected cells. Inhibition of viral protein expression and virion production by NS-398 was reversed in the presence of exogenous PGE2. Global gene expression analysis using an MHV-68 DNA array showed that PGE2increased production of multiple viral gene products, and NS-398 inhibited production of many of the same genes. These studies suggest that COX-2 activity and PGE2production may play significant roles during MHV-68 de novo infection.

Diesel exhaust particles (DEP) and their organic constituents modulate the immune system and exacerbate allergic airway inflammation. We investigated the role of DEP extract and associated polycyclic aromatic hydrocarbons (PAHs) on prostaglandin synthesis in endotoxin-activated murine macrophages and in mitogen-stimulated fibroblasts. In both macrophages and fibroblasts, DEP extract, phenanthrene, anthracene, phenanthrenequinone, and beta-napthoflavone inhibit prostaglandin production from endogenous arachidonic acid in response to ligand stimulation. However, DEP extract and PAHs do not block ligand induction of cyclooxygenase-2 (COX-2) protein, either in mitogen-stimulated fibroblasts or endotoxin-treated macrophages. Release of total arachidonic acid and total lipid products is not reduced by DEP or PAHs following ligand stimulation of macrophages or fibroblasts. DEP extract and the PAHs inhibit the activity of purified COX-2 enzyme in vitro but do not inhibit COX-1 activity. Thus, DEP and PAHs do not affect ligand-induced COX-2 gene expression, phospholipase activation, or arachidonic acid release in macrophages and fibroblasts but exert their inhibitory effect on prostaglandin production by preferentially blocking COX-2 enzyme activity.

Based on the finding that gene expression for the actin-bundling protein L-plastin is inducible by androgen and that L-plastin is overexpressed in malignant epithelium of the prostate, we examined the functional consequences of L-plastin down-regulation in prostate carcinoma cell lines by both transfection and retroviral infection. We constructed retroviral vectors to express two different regions of the L-plastin gene, a 1713-bp 3′-coding portion and a 163-bp 5′-untranslated region, both in antisense orientation. Introduction of either constructs into prostate carcinoma cell lines, PC-3 and its isogenic but metastatic variant PC-3M cells, reduced the growth rates of both cell lines. In vitro invasion and motility of PC-3 and PC-3M cells were drastically suppressed (approximately 10-fold) by the expression of the antisense constructs. Evidence was obtained to indicate that L-plastin protein levels were indeed decreased by the antisense expression. The antisense construct for the 5′-untranslated region with the most unique sequence for the L-plastin gene was more effective in down-regulation efficiency compared with the larger antisense construct in the coding region, which maintains homology to other members of the plastin gene family. Cells infected with the 163-bp antisense virus, which were also tested in a nude mouse diaphragm invasion model, showed suppression of in vivo . invasion of both PC-3 and PC-3M cells. These results suggested that overexpression of L-plastin might be functionally involved in prostate cancer invasion and metastasis, and raised the possibility that L-plastin gene-specific antisense delivery could potentially be a useful approach to interfere with prostate cancer progression in vivo .

Previously, we described cloning of three alternatively spliced mRNA forms of human FGF8, a, b, and e, of which the b form is the major expressed species in both normal and tumor prostatic epithelial cells. In this report, we describe construction and overexpression of sense and antisense sequences of either the full length FGF8b coding region (215-amino acids or 215aa), 103aa N-terminal part or a smaller N-terminal region (34aa), each including the 23aa putative signal peptide domain, via a retrovirus system. While the morphologic transforming activities of the sense 215aa and 103aa constructs were similar in NIH3T3 cells, 103aa displayed reduced soft agar clonogenic activity. The 34aa construct was practically inert in these assays, although its expression could mimic the ability of 215aa or 103aa in conferring cell growth under reduced serum condition. Overexpression of any of the three constructs in antisense orientation, however, was similarly effective in reversing the morphology and anchorage-independent growth property of FGF8b-transfected NIH3T3 cells. The expression of the antisense 215aa construct significantly reduced the growth rate of the human prostatic carcinoma DU145 cells and inhibited their soft agar clonogenic activity and in vivo tumorigenicity in nude mice. Taken together, these results identify N-terminal portions of FGF8 protein isoform for having the domains necessary for one or more of the biologic effects examined, and suggest that low levels of FGF8 expressed in prostatic epithelial cells may contribute significantly to their growth and tumorigenic properties.

FLT3 is a member of the class III receptor tyrosine kinase family that includes C-KIT, C-FMS and platelet derived growth factor receptor (PDGFR). FLT3 is primarily expressed in early myeloid and lymphoid progenitors and plays an important role in their proliferation and differentiation. In human leukemia, FLT3 is expressed on 70-90% acute myeloid leukemia (AML) and most B-acute lymphoblastic leukemia (B-ALL). FLT3 genetic aberrations are commonly detected in patients with AML. The most common aberration is internal tandem duplication (ITD), which occurs in 25-30% of AML patients and causes constitutive activation of FLT3. Point mutation in codon D835 of the FLT3 tyrosine kinase domain is reported in 7-10% of AML patients and also causes constitutive activation of the receptor. FLT3 small molecule inhibitors targeting the kinase domain are predominantly active against FLT3 activated AML. The restricted normal tissue expression profile and higher differential in leukemic specimens makes FLT3 amenable to antibody-based therapeutics, requiring only target expression independent of kinase activation status. Therefore, development of an antibody-drug conjugate (ADC) may provide a therapeutic alternative for AML patients. Here, we report the development of the first FLT3specific ADC, AGS62P1, employing site-specific conjugation using the non-natural amino acid, p-acetyl phenylalanine (pAF). AGS62P1 comprises a human gamma one antibody including an inserted pAF residue in each of the heavy chains. The antibody was conjugated to a potent cytotoxic payload via an oxime bond at the pAF sites, thus creating a nearly homogeneous drug distribution, with approximately 2 drug molecules per antibody. Strong binding affinity (0.1-0.9 nM) and potent in vitro cytotoxic activity (IC50 = 0.2-12 nM) was achieved in AML cell lines. The anti-FLT3 ADC was highly efficacious in AML tumor xenografts, leading to statistically significant tumor growth inhibition of both FLT3 ITD and non-ITD models. Additional characterization of both the antibody and ADC was performed, including ligand receptor interaction, degradation, internalization, and apoptosis. In summary, we have developed a site-specific ADC targeting FLT3 that exhibits potent anti-tumor activity in xenograft models regardless of FLT3 activation status. This drug can potentially offer a new and more versatile approach in targeting FLT3-expressing leukemia through a mechanism independent of FLT3 genetic aberration. Citation Format: Nandini Rudra-Ganguly, Pia M. Challita-Eid, Christine Lowe, Mike Mattie, Sung-Ju Moon, Brian A. Mendelsohn, Monica Leavitt, Cyrus Virata, Alla Verlinsky, Linnette Capo, Mi Sook Chang, Deanna L. Russell, Baljinder Randhawa, Gao Liu, René Hubert, Mary Brodey, Hector Aviña, Chunying Zhang, Joseph D. Abad, Banmeet Anand, Sher Karki, Zili An, Roland Luethy, Fernando Doñate, Daniel S. Pereira, Kendall Morrison, Ingrid B.J. Joseph, David R. Stover. AGS62P1, a novel site-specific antibody drug conjugate targeting FLT3 exhibits potent anti-tumor activity regardless of FLT3 kinase activation status. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 574.

FLT3 (FMS-like tyrosine kinase 3) is a member of the class III receptor tyrosine kinase family, which is highly expressed in the blasts of both AML and ALL patients. In addition to FL ligand stimulation, FLT3 can also be activated by two distinct clusters of mutations: internal tandem duplications (FLT3/ITDs) in 20% to 25% patients and point mutations at position D835 in the tyrosine-kinase domain (FLT3/TKD) in 7% to 10% patients. FLT3 tyrosine kinase inhibitors (TKI) are mainly active against FLT3 mutant AML. An antibody drug conjugate (ADC), directed against the extracellular domain of FLT3 may only require FLT3 cell surface expression independent of mutation status. The restricted cellular distribution of FLT3 receptor and a higher expression in AML than in normal bone marrow makes FLT3 a favorable ADC target. Therefore, this ADC based strategy may offer a therapeutic alternative for AML patients independent of FLT3 status. Here, we report the preclinical assessment of a novel FLT3 targeting ADC, AGS62P1. AGS62P1 consists of a human anti-FLT3 monoclonal antibody, site specifically conjugated to a potent cytotoxic payload. FLT3 expression is confirmed in a large panel of AML and ALL tumor cells as well as in AML patient specimens via flow cytometry. The anti-leukemic activity of AGS62P1 was evaluated against AML and ALL tumor cell lines, in vitro and in vivo. AGS62P1 demonstrated strong binding affinity (0.1-0.5 nM) and potent cytotoxic activity in FLT3/ITD and Non-ITD tumor models, in vitro. Cytotoxic IC50 potency for AGS62P1 was 0.5-13 nM in FLT3/ITD and 0.2-12 nM in FLT3 non-ITD models. A fluorescence based assay confirmed that AGS62P1 is rapidly internalized in AML tumor cell lines. AGS62P1 is highly efficacious in FLT3/ITD and non-ITD tumor xenografts, leading to significant tumor growth inhibition or complete tumor regression. In primary AML patient xenograft drug treatment studies, the engraftment and outgrowth of 5/6 samples were significantly reduced when treated with AGS62P1. Taken together our data demonstrate that AGS62P1 exhibits potent antitumor activity against a broad panel of AML tumor models and primary AML samples, regardless of FLT3 status. We believe AGS62P1 may be an effective and alternative therapeutic for AML patients, which can bypass the TKI mediated resistance and deliver target specific effect through a different mode of action. Disclosures Jin: Agensys: Research Funding. Anand:Agensys: Employment. Dick:Agensys: Research Funding.

Discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family. The receptor is activated upon binding to its ligand, collagen, and plays a crucial role in many fundamental processes such as cell differentiation, adhesion, migration and invasion. Although DDR1 is expressed in many normal tissues, upregulated expression of DDR1 in a variety of human cancers such as lung, colon and brain cancers is known to be associated with poor prognosis. Using shRNA silencing, we assessed the oncogenic potential of DDR1. DDR1 knockdown impaired tumor cell proliferation and migration in vitro and tumor growth in vivo. Microarray analysis of tumor cells demonstrated upregulation of TGFBI expression upon DDR1 knockdown, which was subsequently confirmed at the protein level. TGFBI is a TGFβ-induced extracellular matrix protein secreted by the tumor cells and is known to act either as a tumor promoter or tumor suppressor, depending on the tumor environment. Here, we show that exogenous addition of recombinant TGFBI to BXPC3 tumor cells inhibited clonogenic growth and migration, thus recapitulating the phenotypic effect observed from DDR1 silencing. BXPC3 tumor xenografts demonstrated reduced growth with DDR1 knockdown, and the same xenograft tumors exhibited an increase in TGFBI expression level. Together, these data suggest that DDR1 expression level influences tumor growth in part via modulation of TGFBI expression. The reciprocal expression of DDR1 and TGFBI may help to elucidate the contribution of DDR1 in tumorigenesis and TGFBI may also be used as a biomarker for the therapeutic development of DDR1 specific inhibitors.

Multidrug resistance may play a critical role in developing cancer therapeutics for acute leukemia. The expression of the ATP binding cassette (ABC) transporter Pgp/MDR1 (also known as ABCB1) has been reported in AML patients and correlates with poor clinical outcome. Additional transporters like MRP1 and BCRP also appeared to contribute towards this resistance mechanism. We have profiled a panel of AML tumor cell lines and tumor xenografts for both expression and activity of Pgp/MDR1 via FACS and MSD;. Our profiling also includes analysis of the known SNPs associated with these transporters in key models. We have evaluated the sensitivity of the AML models to a series of standard of care therapies, including auristatins, in both in vitro and in vivo studies. These payloads alone or when conjugated to antibodies as antibody drug conjugates (ADCs) are known substrates of the ABC transporters, Using Pgp inhibitors, we could restore the sensitivity of the MDR expressing cells to the cytotoxic drugs and ADCs, which confirmed that the drugs were MDR substrates. These inhibitors could also block the activity of the Pgp pump. We have also observed limited activity in vivo with these ADCs when used in a MDR positive model. Our initial observation indicates that a threshold level of activity of MDR1 may be critical to confer resistance to these standard of care molecules and antibody drug conjugates using similar payloads. Currently, we are investigating the contribution of other efflux pumps in this mechanism of drug resistance MRP1 and BCRP, in similar manner. This can also aid in identifying cytotoxic drugs that are able to bypass the resistance mediated by these efflux pumps. Additional work is underway to characterize patient samples and identify MDR threshold level of activity as a potential predictive biomarker in developing AML therapeutics. Citation Format: Nandini Rudra-Ganguly, Christine Lowe, Mukta Shiwalkar, Claudia I. Guevara, Christopher Kemball, Min Michelle Wu, Cyrus Virata, Alla Verlinsky, Ssucheng J Hsu, Michael Mattie, William Yeh, Peng Yang, Sung-Ju Moon, Ingrid Joseph, David R. Stover, Daniel S. Pereira, Dowdy Jacksosn. MDR expression/ activity may serve as potential biomarker in developing therapeutic drugs for AML therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1976. doi:10.1158/1538-7445.AM2014-1976

A search for differentially expressed genes in a pair of nonmetastatic (PC-3) versus metastatic variant (PC-3M) human prostate carcinoma cell lines led to identification of the human heat shock factor (HSF1) as an overexpressed gene product in PC-3M cells. Analysis of primary prostate cancer specimens indicated that HSF1 is generally up-regulated in most of the malignant prostate epithelial cells relative to the normal prostate cells. Among the known effectors of HSF1 action, constitutive levels of HSP70 and HSP90 are not significantly altered by the naturally elevated expression of HSF1 as in PC-3M cells or by transduced overexpression of HSF1 in PC-3 cells. The basal levels of HSP27 in both cases are, however, consistently increased by two- to threefold. With respect to response to heat shock, high basal concentration of HSP90 is not further enhanced in these cells, and HSP70 is up-regulated irrespective of HSF1 level. Heat shock, however, causes an increase in HSP27 when HSF1 is up-regulated, except when the expression of HSF1 is already too high. These results document for the first time that HSF1 is overexpressed in human prostate cancer cells, at least one consequence of which in the prostate cancer cell lines tested is stimulation of both basal and stress-induced expression of HSP27, an important factor in cell growth, differentiation, or apoptosis.

Discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family. It is activated by binding to its ligand, collagen and plays a key role in cell survival, adhesion, migration and invasion. Although DDR1 is present in several normal tissues, it is overexpressed in various cancer types, including lung, colon, ovary and breast tumors as well as in gliomas, where it is known to be associated with poor prognosis. The significance of DDR1 in cancer was illustrated using shRNA silencing, which impaired tumor cell growth, both in vitro and in vivo. Using microarray analysis of tumor cells with DDR1 knockdown, we identified an upregulation of TGFBI expression, which was subsequently confirmed at the protein level. TGFBI is a TGFβ induced extracellular matrix protein secreted by tumor cells and has been reported to act as either a tumor promoter or suppressor, depending upon tumor type. Exogenous addition of recombinant TGFBI to tumor cells inhibited clonogenic growth, recapitulating shRNA data. When grown in vivo as xenografts, the DDR1 knockdown cell lines demonstrated a similar phenotype of reduced growth and tumors exhibited an increase in TGFBI protein levels. In summary, our data suggests that DDR1 expression levels influences tumor growth in part through modulation of TGFBI expression. Ongoing studies will help to understand how DDR1 and TGFBI are linked and contribute to tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1086. doi:1538-7445.AM2012-1086