Combination Targeted Therapy in Relapsed Diffuse Large B-Cell Lymphoma.

BACKGROUND: The identification of oncogenic mutations in diffuse large B-cell lymphoma (DLBCL) has led to the development of drugs that target essential survival pathways, but whether targeting multiple survival pathways may be curative in DLBCL is unknown. METHODS: We performed a single-center, phase 1b-2 study of a regimen of venetoclax, ibrutinib, prednisone, obinutuzumab, and lenalidomide (ViPOR) in relapsed or refractory DLBCL. In phase 1b, which included patients with DLBCL and indolent lymphomas, four dose levels of venetoclax were evaluated to identify the recommended phase 2 dose, with fixed doses of the other four drugs. A phase 2 expansion in patients with germinal-center B-cell (GCB) and non-GCB DLBCL was performed. ViPOR was administered every 21 days for six cycles. RESULTS: In phase 1b of the study, involving 20 patients (10 with DLBCL), a single dose-limiting toxic effect of grade 3 intracranial hemorrhage occurred, a result that established venetoclax at a dose of 800 mg as the recommended phase 2 dose. Phase 2 included 40 patients with DLBCL. Toxic effects that were observed among all the patients included grade 3 or 4 neutropenia (in 24% of the cycles), thrombocytopenia (in 23%), anemia (in 7%), and febrile neutropenia (in 1%). Objective responses occurred in 54% of 48 evaluable patients with DLBCL, and complete responses occurred in 38%; complete responses were exclusively in patients with non-GCB DLBCL and high-grade B-cell lymphoma with rearrangements of MYC and BCL2 or BCL6 (or both). Circulating tumor DNA was undetectable in 33% of the patients at the end of ViPOR therapy. With a median follow-up of 40 months, 2-year progression-free survival and overall survival were 34% (95% confidence interval [CI], 21 to 47) and 36% (95% CI, 23 to 49), respectively. CONCLUSIONS: Treatment with ViPOR was associated with durable remissions in patients with specific molecular DLBCL subtypes and was associated with mainly reversible adverse events. (Funded by the Intramural Research Program of the National Cancer Institute and the National Center for Advancing Translational Sciences of the National Institutes of Health and others; ClinicalTrials.gov number, NCT03223610.).

First-in-human phase 1 study of filanesib (ARRY-520), a kinesin spindle protein inhibitor, in patients with advanced solid tumors.

Purpose Filanesib (ARRY-520) is a highly selective, targeted inhibitor of kinesin spindle protein (KSP) inhibitor that induces mitotic arrest and subsequent tumor cell death. This first-in-human Phase 1 study evaluated dose-limiting toxicities (DLTs) and determined a maximum tolerated dose (MTD) for filanesib administered as a 1-h intravenous infusion on 2 treatment schedules in patients with advanced solid tumors. The pharmacokinetics (PK), pharmacodynamics and preliminary efficacy of filanesib were also evaluated. Methods Filanesib was administered on Day 1 of each 3-week cycle (Initial Schedule) or Days 1 and 2 of each 2-week cycle (Alternate Schedule). A standard 3 + 3 dose-escalation design was employed. An expansion cohort was conducted at the MTD of the Initial Schedule. Filanesib PK was evaluated in plasma (both schedules) and urine (Initial Schedule only). Monopolar spindle formation was evaluated in biopsies taken from patients in the expansion cohort. Results Forty-one patients received filanesib. The MTD was equivalent for both the Initial and Alternate Schedules (2.50 mg/m(2)/cycle). The prevalence of neutropenia as a DLT for both schedules necessitated adding prophylactic filgrastim to another dose escalation on the Alternate Schedule (highest tolerated dose 3.20 mg/m(2)/cycle). Neurotoxicity related to filanesib was not observed. Dose-proportional increases in filanesib exposure were observed. The half-life for filanesib was ~70 h. Monopolar spindles in patient biopsy samples indicated KSP inhibition. Stable disease was the best tumor response observed in 18 % (7/39) of evaluable patients. Conclusion Filanesib provided exposures with acceptable tolerability and evidence of target-specific pharmacodynamic effects.

Identification of inhibitory scFv antibodies targeting fibroblast activation protein utilizing phage display functional screens.

Fibroblast activation protein (FAP) is a serine protease selectively expressed on tumor stromal fibroblasts in epithelial carcinomas and is important in cancer growth, adhesion, and metastases. As FAP enzymatic activity is a potent therapeutic target, we aimed to identify inhibitory antibodies. Using a competitive inhibition strategy, we used phage display techniques to identify 53 single-chain variable fragments (scFvs) after three rounds of panning against FAP. These scFvs were expressed and characterized for binding to FAP by surface plasmon resonance and flow cytometry. Functional assessment of these antibodies yielded an inhibitory scFv antibody, named E3, which could attenuate 35% of FAP cleavage of the fluorescent substrate Ala-Pro-7-amido-4-trifluoromethylcoumarin compared with nonfunctional scFv control. Furthermore, a mutant E3 scFv was identified by yeast affinity maturation. It had higher affinity (4-fold) and enhanced inhibitory effect on FAP enzyme activity (3-fold) than E3. The application of both inhibitory anti-FAP scFvs significantly affected the formation of 3-dimensional FAP-positive cell matrix, as demonstrated by reducing the fibronectin fiber orientation from 41.18% (negative antibody control) to 34.06% (E3) and 36.15% (mutant E3), respectively. Thus, we have identified and affinity-maturated the first scFv antibody capable of inhibiting FAP function. This scFv antibody has the potential to disrupt the role of FAP in tumor invasion and metastasis.

Long-Term Follow-Up of Patients With Follicular Lymphoma Using Next Generation Sequencing to Detect Minimal Residual Disease.

BACKGROUND: Follicular lymphoma (FL) is a highly treatable, indolent non-Hodgkin lymphoma. Although FL is considered incurable, a patient without progression of disease by 24 months after treatment is predicted to have a survival consistent with persons without lymphoma. Using a sensitive assessment of minimal residual disease (MRD), we tested the hypothesis that MRD monitoring can predict long term remissions. METHODS: Unselected patients who were in a clinical remission for at least 24 months after their last treatment were enrolled and monitored prospectively for MRD detectability using a sensitive next-generation sequencing assay (clonoSEQ, Adaptive Biotechnologies, Seattle, WA). RESULTS: Forty-seven consecutive patients were monitored. We evaluated the MRD thresholds 10-4, 10-5, and 10-6 for the ability to predict long-term remissions in this cohort and determined that undetectable disease at 10-6 was the best predictor with a specificity and negative predictive value (NPV) of 70% and 100%, respectively. While 3 patients exhibited clinical disease progression during the course of the study, none of the 31 patients with persistent MRD undetectability at 10-6 experienced relapse. CONCLUSIONS: A significant proportion (31/47; 66.0%) of FL patients in clinical remission after ≥24 months following last therapy were undetectable at 10-6 by a sensitive assay of MRD. The threshold of sensitivity was 100%, specificity 70%, with a PPV of 19%, but a NPV of 100%. Although longer follow-up is needed for confirmation, many of these patients may continue to have durable complete remissions.

A phase 1 dose-escalation study of ARRY-520, a kinesin spindle protein inhibitor, in patients with advanced myeloid leukemias.

BACKGROUND: ARRY-520 selectively inhibits the mitotic kinesin spindle protein (KSP), which leads to abnormal monopolar spindle formation and apoptosis. METHODS: A phase 1 trial was conducted to establish the safety and the maximum tolerated dose (MTD) of ARRY-520 given as a 1-hour infusion in either a single dose or on a day 1, 3, and 5 divided-dose schedule per cycle in patients with advanced or refractory myeloid leukemias. Additional objectives were to characterize pharmacokinetics, assess preliminary clinical activity, and explore biomarkers of KSP inhibition with ARRY-520. A total of 36 patients with acute myelogenous leukemia (n = 34) or myelodysplastic syndromes (n = 2) with a median age of 66 years (range, 21-88 years) were enrolled: 15 in the single-dose schedule (dose levels: 2.5, 3.75, 4.5, and 5.6 mg/m(2)) and 21 in the divided-dose schedule (dose levels: 0.8, 1.2, 1.5, and 1.8 mg/m(2)/day). RESULTS: The MTD was 4.5 mg/m(2) total dose per cycle for both dose schedules. Dose-limiting toxicities included mucositis, exfoliative rash, hand-foot syndrome, and hyperbilirubinemia. Grades 3 or 4 reversible drug-related myelosuppression were observed in 33 of 36 patients. Plasma pharmacokinetic analyses revealed low clearance of ARRY-520 (~3 L/hour), a volume of distribution of ~450 L, and a median terminal half-life of >90 hours. Monopolar spindles were observed in blood mononuclear cells, through use of 4',6-diamidino-2-phenylindole nucleic acid stain and antitubulin antibodies. CONCLUSIONS: On the basis of the relative lack of clinical activity, further development of ARRY-520 as an antileukemic agent was halted. (Clinicaltrials.gov identifier NCT00637052).

Adverse Effects of Virus-Specific T-Cell Therapy: An Integrative Review.

Allogeneic hematopoietic stem cell transplant (HSCT) remains the mainstay in treating many hematologic malignancies. T-cell-depleted grafts designed to reduce graft-vs.-host disease (GVHD) may be complicated by severe viral infections that increase morbidity and mortality. Despite the use of antiviral pharmacologic therapy, challenges in controlling viral infections include drug resistance and/or side-effect intolerability. Virus-specific T-cell (VST) therapy is a promising targeted therapy for treating severe or drug-refractory viral infections after HSCT. An integrative review was conducted to inform advanced practitioners of the adverse effects associated with VST. A total of 836 articles were identified using PubMed, Scopus, and CINAHL databases, with 7 included in this review. Studies reviewed indicate that the adverse effects associated with VST therapy are limited and generally treatable. These studies reported low rates of adverse events of mild to moderate severity, including acute, recurrent, chronic, and de novo GVHD; cytokine release syndrome; infusion toxicity; and other adverse events. No deaths were attributed to VSTs in these studies.

Development of engineered antibodies specific for the Müllerian inhibiting substance type II receptor: a promising candidate for targeted therapy of ovarian cancer.

The Müllerian inhibiting substance type II receptor (MISIIR) is involved in Müllerian duct regression as part of the development of the male reproductive system. In adult females, MISIIR is present on ovarian surface epithelium and is frequently expressed on human epithelial ovarian cancer cells. Müllerian inhibiting substance has been found to be capable of inhibiting the growth of primary human ovarian cancer cells derived from ascites and ovarian cancer cell lines. This suggested to us that MISIIR could be an attractive target for antibody-based tumor targeting and growth inhibition strategies. Here, we describe the production of recombinant human MISIIR extracellular domain-human immunoglobulin Fc domain fusion proteins and their use as targets for the selection of MISIIR-specific human single-chain variable fragments (scFv) molecules from a human nonimmune scFv phage display library. The binding kinetics of the resulting anti-MISIIR scFv clones were characterized and two were employed as the basis for the construction of bivalent scFv:Fc antibody-based molecules. Both bound specifically to human ovarian carcinoma cells in flow cytometry assays and cross-reacted with mouse MISIIR. These results indicate that antibody-based constructs may provide a highly specific means of targeting MISIIR on human ovarian carcinoma cells for the purpose of diagnosing and treating this disease.

A single treatment of yttrium-90-labeled CHX-A"-C6.5 diabody inhibits the growth of established human tumor xenografts in immunodeficient mice.

Antitumor diabody molecules are noncovalent single-chain Fv dimers that recapitulate the divalent binding properties of native IgG antibodies. Diabodies are capable of substantial accumulation in tumor xenografts expressing relevant antigens in immunodeficient mouse models. With a Mr of approximately 55,000, diabodies are rapidly cleared from the circulation, resulting in tumor-to-blood ratios that significantly exceed those achieved early after the administration of monoclonal antibodies. We have evaluated the therapeutic potential of the beta-emitting isotope yttrium-90 (t1/2, 64 hours) conjugated to the C6.5K-A diabody that specifically targets the HER2/neu human tumor-associated antigen. We have found that a single intravenous dose of 150 microCi (200 microg) 90Y-CHX-A"-C6.5K-A diabody substantially inhibits the growth rates of established MDA-361/DYT2 human breast tumor xenografts in athymic nude mice. In contrast, 300 microCi (300 microg) 90Y-CHX-A"-C6.5K-A diabody resulted in only a minor delay in the growth of SK-OV-3 human ovarian cancer xenografts. The maximum tolerated dose was also dependent on the tumor xenograft model used. These studies indicate that genetically engineered antitumor diabody molecules can be used as effective vehicles for radioimmunotherapy.

Identification of NOM1, a nucleolar, eIF4A binding protein encoded within the chromosome 7q36 breakpoint region targeted in cases of pediatric acute myeloid leukemia.

Proteins that contain the recently described MIF4G and/or MA3 domains function in translation, cell growth, proliferation, transformation, and apoptosis. Examples of MIF4G/MA3 containing proteins and their functions include eIF4G, which serves as a scaffold for assembly of factors required for translation initiation, programmed cell death protein 4 (Pdcd4) that inhibits translation and functions as a tumor suppressor, and NMD2, which is essential for nonsense-mediated mRNA decay. MIF4G and MA3 domains serve as binding sites for one or more isoforms of the eIF4A family of ATP-dependent DEAD-box RNA helicases that are required for translation and for nonsense-mediated decay. In this report, we describe the characterization of a novel MIF4G/MA3 family member called NOM1 (nucleolar protein with MIF4G domain 1) that was identified at the chromosome 7q36 breakpoint involved in 7;12 translocations associated with certain acute leukemias of childhood. NOM1, which includes a previously described EST called c7orf3, encodes a ubiquitously expressed transcript composed of 11 exons and an approximately 3 kb 3' UTR that contains several Alu repeats. The predicted NOM1 protein contains one MIF4G domain and one MA3 domain and, consistent with data obtained with other MIF4G/MA3 proteins, interacts with members of the eIF4A family of helicases. Database searches reveal that NOM1 homologs exist in several organisms and that at least two of these are essential genes. Finally, like its Saccharomyces cerevisiae homolog Sgd1p, NOM1 localizes predominantly to the nucleolus. These data demonstrate that NOM1 is a new member of the MIF4G/MA3 family of proteins and suggest that it may provide an essential function in metazoans.

Isolation of scFvs to in vitro produced extracellular domains of EGFR family members.

The members of the epidermal growth factor receptor (EGFR) family are over expressed in a variety of malignancies and are frequently linked to aggressive disease and a poor prognosis. Although clinically effective monoclonal antibodies (MAbs) have been developed to target HER2 and EGFR, the remaining two family members, HER3 and HER4, have not been the subject of significant efforts. In this paper, we have taken the initial steps required to generate antibodies with potential clinically utility that target the members of the EGFR family. The genes for the extracellular domains (ECDs) of all four members of the EGFR family were cloned and used to stably transfect 293 (HEK) cells. Milligram quantities of each ECD were produced and characterized. The HER3, HER4, and EGFR ECDs were then employed as targets for the selection of antibodies from naïve human scFv (single-chain Fv) phage display libraries. Six unique scFv clones were isolated that bound specifically to HER3, 13 unique clones were isolated with specificity for HER4 and 52 unique anti-EGFR clones were isolated. These scFvs provide a valuable and potentially clinically relevant panel of agents to target the members of the EGFR family.

Anti-CD70 immunocytokines for exploitation of interferon-γ-induced RIP1-dependent necrosis in renal cell carcinoma.

Metastatic renal cell carcinoma (RCC) is an incurable disease in clear need of new therapeutic interventions. In early-phase clinical trials, the cytokine IFN-γ showed promise as a biotherapeutic for advanced RCC, but subsequent trials were less promising. These trials, however, focused on the indirect immunomodulatory properties of IFN-γ, and its direct anti-tumor effects, including its ability to kill tumor cells, remains mostly unexploited. We have previously shown that IFN-γ induces RIP1 kinase-dependent necrosis in cells lacking NF-κB survival signaling. RCC cells display basally-elevated NF-κB activity, and inhibiting NF-κB in these cells, for example by using the small-molecule proteasome blocker bortezomib, sensitizes them to RIP1-dependent necrotic death following exposure to IFN-γ. While these observations suggest that IFN-γ-mediated direct tumoricidal activity will have therapeutic benefit in RCC, they cannot be effectively exploited unless IFN-γ is targeted to tumor cells in vivo. Here, we describe the generation and characterization of two novel 'immunocytokine' chimeric proteins, in which either human or murine IFN-γ is fused to an antibody targeting the putative metastatic RCC biomarker CD70. These immunocytokines display high levels of species-specific IFN-γ activity and selective binding to CD70 on human RCC cells. Importantly, the IFN-γ immunocytokines function as well as native IFN-γ in inducing RIP1-dependent necrosis in RCC cells, when deployed in the presence of bortezomib. These results provide a foundation for the in vivo exploitation of IFN-γ-driven tumoricidal activity in RCC.

Isolation of anti-MISIIR scFv molecules from a phage display library by cell sorter biopanning.

While cell surface antigens represent the most common targets for antibody-based cancer therapy, isolation of new antibodies specific for these targets from single-chain Fv phage display libraries has been hindered by limitations associated with traditional selection techniques. Solid phase panning is often associated with conformational changes to the target protein due to its immobilization on plastic tubes that can limit the ability of the isolated scFv to bind to conformational epitopes and solution panning methods require the use of secondary tags that often mask desired sequences and create unintended epitopes. Commonly utilized cell-based panning methods typically yield a panel of single-chain Fv (scFv) molecules that are specific for numerous cell surface antigens, often obscuring the desired clones. Here, we describe a novel cell sorter-based system to isolate single-chain Fv molecules specific for defined antigen targets expressed on stably-transformed mammalian cells. We employed these methods to isolate promising scFv clones that bind specifically to the Müllerian inhibiting substance type II receptor, a cell surface ovarian cancer antigen that has proven to be a difficult target for selection strategies.

Phase I pharmacokinetic and pharmacodynamic study of the oral, small-molecule mitogen-activated protein kinase kinase 1/2 inhibitor AZD6244 (ARRY-142886) in patients with advanced cancers.

PURPOSE: To assess the tolerability, pharmacokinetics (PKs), and pharmacodynamics (PDs) of the mitogen-activated protein kinase kinase (MEK) 1/2 inhibitor AZD6244 (ARRY-142886) in patients with advanced cancer. PATIENTS AND METHODS: In part A, patients received escalating doses to determine the maximum-tolerated dose (MTD). In both parts, blood samples were collected to assess PK and PD parameters. In part B, patients were stratified by cancer type (melanoma v other) and randomly assigned to receive the MTD or 50% MTD. Biopsies were collected to determine inhibition of ERK phosphorylation, Ki-67 expression, and BRAF, KRAS, and NRAS mutations. RESULTS: Fifty-seven patients were enrolled. MTD in part A was 200 mg bid, but this dose was discontinued in part B because of toxicity. The 50% MTD (100 mg bid) was well tolerated. Rash was the most frequent and dose-limiting toxicity. Most other adverse events were grade 1 or 2. The PKs were less than dose proportional, with a median half-life of approximately 8 hours and inhibition of ERK phosphorylation in peripheral-blood mononuclear cells at all dose levels. Paired tumor biopsies demonstrated reduced ERK phosphorylation (geometric mean, 79%). Five of 20 patients demonstrated >or= 50% inhibition of Ki-67 expression, and RAF or RAS mutations were detected in 10 of 26 assessable tumor samples. Nine patients had stable disease (SD) for >or= 5 months, including two patients with SD for 19 (thyroid cancer) and 22 (uveal melanoma plus renal cancer) 28-day cycles. CONCLUSION: AZD6244 was well tolerated with target inhibition demonstrated at the recommended phase II dose. PK analyses supported twice-daily dosing. Prolonged SD was seen in a variety of advanced cancers. Phase II studies are ongoing.