Safety and Efficacy of Nivolumab in Patients With Advanced Non-Clear Cell Renal Cell Carcinoma: Results From the Phase IIIb/IV CheckMate 374 Study.

BACKGROUND: The open-label phase IIIb/IV CheckMate 374 study (NCT02596035) was conducted to validate the safety and efficacy of flat-dose nivolumab 240 mg every 2 weeks (Q2W) in previously treated advanced/metastatic renal cell carcinoma. Three cohorts included patients with predominantly clear cell histology, non-clear cell histologies, or brain metastases. We report safety and efficacy from the advanced non-clear cell RCC (nccRCC) cohort of CheckMate 374. METHODS: Eligible patients received 0 to 3 prior systemic therapies. Patients received nivolumab 240 mg Q2W for ≤24 months or until confirmed progression or unacceptable toxicity. The primary endpoint was incidence of high-grade (grade 3-5) immune-mediated adverse events (IMAEs). Exploratory endpoints included objective response rate (ORR), progression-free survival (PFS), and overall survival (OS). RESULTS: Forty-four patients had advanced nccRCC (papillary [n = 24], chromophobe [n = 7], unclassified [n = 8], other [n = 5]); 34.1% received ≥1 prior systemic regimen in the advanced/metastatic setting. With median follow-up of 11 (range, 0.4-27) months, no all-cause grade 3-5 IMAEs or treatment-related grade 5 adverse events were reported. ORR was 13.6% (95% confidence interval [CI], 5.2-27.4), with 1 complete response (chromophobe) and 5 partial responses (papillary [n = 2], chromophobe [n = 1], collecting duct [n = 1], and unclassified [n = 1] histology). Median PFS was 2.2 months (95% CI, 1.8-5.4). Median OS was 16.3 months (95% CI, 9.2-not estimable). CONCLUSIONS: Safety of flat-dose nivolumab 240 mg Q2W was consistent with previous results. Clinically meaningful efficacy was observed with responses in several histologies, supporting nivolumab as a treatment option for patients with advanced nccRCC, a patient population with high unmet need.

Time-dependent effects of prognostic biomarkers of systemic inflammation in patients with metastatic renal cell carcinoma.

The goal of this study was to examine time-dependent effects of prognostic biomarkers of systemic inflammation in patients with metastatic renal cell carcinoma. Retrospective chart reviews were conducted at the Winship Cancer Institute of Emory University and the Atlanta Veterans Administration Medical Center with authorization from the Emory University Institutional Review Board and the Veterans Administration Research and Development Committee. Inclusion criteria included age ⩾18 years, treatment with targeted therapy for clear cell or non-clear cell metastatic renal cell carcinoma and concomitant assessment of C-reactive protein and albumin levels on ⩾3 occasions that were ⩾10 days apart. Discovery, expansion, and external validation cohorts were identified. Established prognostic variables were evaluated by univariate and multivariate analyses. Intensity of systemic inflammation was assessed at all time points with C-reactive protein and albumin as prognostic covariates for overall survival in an extended Cox regression model. Intensity of systemic inflammation was assessed on 3186 occasions in 181 patients. Risk status changed in 131 patients (72%). The hazard ratio for overall survival was 21.41 (95% confidence interval = 8.26-55.50) with a type 3 p value of <0.001 for the reference cohort and 9.68 (2.07-45.31) with a type-3 p value of 0.006 for the external validation cohort when time points associated with severe systemic inflammation were compared to all other time points. The bias-corrected c-statistic was 0.839 (0.773-0.905) and 0.818 (0.691-0.946), respectively. Terminal disease progression with severe systemic inflammation was detected in 87% of the 90 patients who died. In conclusion, time-dependent effects are a prominent feature of intensity of systemic inflammation, a powerful prognostic biomarker for metastatic renal cell carcinoma.

Novel (S)-(-)- and R-(+)-seco-iso-cyclopropylfurano[e]indoline-5,6,7-trimethoxyindole-2-carboxamide (iso-CFI) analogs of duocarmycin C2: synthesis and biological evaluation.

Racemic seco-iso-CFI (cyclopropylfurano[e]indoline) analogs of the duocarmycins and CC-1065 have recently been reported by our group. These compounds covalently react with AT-rich sequences of DNA, and they exhibit potent cytotoxicity against cancer cells but are less toxic to normal bone marrow cells. This article details the synthesis of enantiomerically pure (S)-(-)- and R-(+)-seco-iso-CFI (cyclopropylfurano[e]indoline)-5,6,7-trimethoxyindole-2-carboxamide analogs, (S)-(-)-1 and (R)-(+)-1, respectively. The covalent DNA binding properties and cytotoxicity of both enantiomers against L1210 murine leukemia and B16 murine melanoma cells grown in culture are reported and compared to racemate (+/-)-1. The natural (S)-(-)-enantiomer of 1 is more reactive with DNA and more cytotoxic than its unnatural mirror image and the racemic mixture.

Sequence specific recognition of DNA by tailor-made hairpin conjugates of achiral seco-cyclopropaneindoline-2-benzofurancarboxamide and pyrrole-imidazole polyamides.

Hairpin conjugates of achiral seco-cyclopropaneindoline-2-benzofurancarboxamide (achiral seco-CI-Bf) and three diamides (ImPy 1, PyIm 2, and PyPy 3, where Py is pyrrole, and Im is imidazole), linked by a gamma-aminobutyrate group, were synthesized. The sequence-specific covalent alkylation of the achiral CI moiety with adenine-N3 in the minor groove was ascertained by thermally induced DNA cleavage experiments. The results provide evidence that hairpin conjugates of achiral seco-CI-Bf-gamma-polyamides could be tailored to target specific DNA sequences according to a set of general rules: the achiral CI moiety selectively reacts with adenine-N3, a stacked pair of imidazole/benzofuran prefers a G/C base pair, and a pyrrole/benzofuran prefers an A/T or T/A base pair. Models for the binding of hairpin conjugates 1-3 with sequences 5'-TCA(888)G-3', 5'-CAA(857)C-3', and 5'-TTA(843)C-3' are proposed.

Unexpected syntheses of seco-cyclopropyltetrahydroquinolines - from a radical 5-exo-trig cyclization reaction: analogs of CC-1065 and the duocarmycins.

Analogs of the seco-cyclopyrroloindoline (seco-CPI), the DNA alkylation pharmacophore of CC-1065 and the duocarmycins, can be prepared through a 5-exo-trig radical cyclization of a free radical and a 3-chloro-2-allylic moiety. This manuscript reports an unexpected discovery that, depending on the structure and stability of the free radical, the cyclization process leads to the production of an appreciable amount of seco- cyclopropyltetrahydroquinolines 7a-d along with the seco-cyclopropoyltetra- hydroindoline products (6a-e). For instance, free radical reaction of the bromoallylic chloride 5a produced an equal amount of 6-benzyloxy-N-t-butoxycarbonyl-3- (chloromethyl)furano[e]indoline (6a), and 7-benzyloxy-N-t-butoxycarbonyl-3-chloro- 1,2,3,4-tetrahydrofurano[f]quinoline (7a). Three other examples that produced mixtures of indoline and quinoline products are provided. In only one of the examples reported in this manuscript, the 6-benzyloxy-N-t-butoxycarbonyl-3-(chloromethyl)benzo[e]indoline, was a seco-CBI precursor 6e formed exclusively, consistent with literature precedents.

Preparation of quantum dot-biotin conjugates and their use in immunochromatography assays.

Biotinylated, highly luminescent CdSe-ZnS quantum dot (QD) conjugates were prepared and used in immunofiltration assays. Water-soluble quantum dot surfaces having a homogeneous negative charge density at basic pH were initially coated with a two-domain recombinant maltose-binding protein appended with a positively charged leucine zipper. Biotin functionalization of these electrostatically stabilized QD-protein complexes was then carried out using amine-reactive NHS biotin. These protein-coated biotin-functionalized quantum dot conjugates were incorporated into flow immunofiltration/displacement assays employing Affi-gel agarose resin for antibody immobilization, analyte capture, and immune complex formation with a second biotinylated antibody. A key component of the assay was the use of tetranitromethane-modified NeutrAvidin, used to link the biotinylated QDs to the immune complexes and facilitate their release at basic pH for subsequent quantification. This assay methodology was used to detect as little as 10 ng/mL staphylococcal enterotoxin type-B.

2,4,6-Trinitrotoluene detection using recombinant antibodies.

The ability to detect low levels of 2,4,6-trinitrotoluene (TNT) in aqueous samples is important due to the toxicity of both TNT and its breakdown products. We have been characterizing recombinant anti-TNT antibodies isolated from the Griffin library of phage displayed scFvs by selection for binders to the TNT-surrogate 2,4,6-trinitrobenzene (TNB) coupled to the protein bovine serum albumin. Two candidate antibody fragments, TNB1 and TNB2, were isolated and evaluated by ELISA for their ability to bind to TNB coupled to the protein ovalbumin. Competition ELISA was then used to demonstrate antibody fragment binding to TNT in solution and to examine cross-reactivity towards several TNT-related compounds and other explosives. Both recombinant antibody fragments were incorporated into a continuous flow assay for the detection of TNT. TNB2, the best single chain antibody, showed a limit of detection of 1 ng ml(-1), comparable to a commercially available anti-TNT antibody in the same assay format.

Analysis of aqueous 2,4,6-trinitrotoluene (TNT) using a fluorescent displacement immunoassay.

We report a rapid, simple, and sensitive assay that is potentially amenable to high throughput screening for analysis of 2,4,6-trinitrotoluene (TNT) present in aqueous solutions. The assay is based on the change in fluorescence emission intensity of a fluorescently labeled TNT analogue pre-bound to an anti-TNT antibody that occurs upon its competitive displacement by TNT. The assay can be performed in both cuvette- and 96-well plate-based formats. TNT at a level of 0.5 micro g L(-1) (0.5 ppb) was detected in phosphate buffered saline; detection improved to 0.05 micro g L(-1) (0.05 ppb) for TNT dissolved in artificial seawater.

Nine-analyte detection using an array-based biosensor.

A fluorescence-based multianalyte immunosensor has been developed for simultaneous analysis of multiple samples. While the standard 6 x 6 format of the array sensor has been used to analyze six samples for six different analytes, this same format has the potential to allow a single sample to be tested for 36 different agents. The method described herein demonstrates proof of principle that the number of analytes detectable using a single array can be increased simply by using complementary mixtures of capture and tracer antibodies. Mixtures were optimized to allow detection of closely related analytes without significant cross-reactivity. Following this facile modification of patterning and assay procedures, the following nine targets could be detected in a single 3 x 3 array: Staphylococcal enterotoxin B, ricin, cholera toxin, Bacillus anthracis Sterne, Bacillus globigii, Francisella tularensis LVS, Yersiniapestis F1 antigen, MS2 coliphage, and Salmonella typhimurium. This work maximizes the efficiency and utility of the described array technology, increasing only reagent usage and cost; production and fabrication costs are not affected.

Novel furano analogues of duocarmycin C1 and C2: design, synthesis, and biological evaluation of seco-iso-cyclopropylfurano[2,3-e]indoline (seco-iso-CFI) and seco-cyclopropyltetrahydrofurano[2,3-f]quinoline (seco-CFQ) analogues.

The design, synthesis and biological evaluation of novel seco-iso-cyclopropylfurano[2,3-e]indoline (seco-iso-CFI) and the seco-cyclopropyltetrahydrofurano[2,3-f]quinoline (seco-CFQ) analogues of the duocarmycins are described. These novel analogues (4-7) were designed on the premise that the lone pair of electrons on the furano-oxygen atom could enter into conjugation with the isocyclopropylfurano[e]indolone (iso-CFI) alkylating moiety, formed from the loss of HCl in compounds 4-7. The seco-iso-CFI DNA alkylating pharmacophore was synthesized through a well precedented approach of 5-exo-trig aryl radical cyclization with a vinyl chloride. In our studies, in addition to the formation of the seco-iso-CFI product, an equal amount of an unexpected seco-CFQ product was also generated during the radical cyclization reaction. Like CC-1065 and adozelesin, using Taq DNA polymerase stop and thermal cleavage assays, the seco-iso-CFI compounds (4 and 6) and the seco-CFQ compounds (5 and 7) were shown to preferentially alkylate the adenine-N3 position within the minor groove of long stretches of A residues. A MM2 energy optimized molecular model of a 1:1 complex of compound 6 with DNA reveals that the iso-CFI compound fits snugly within the minor groove. Using a MTT based experiment, the cytotoxicity of compounds 4-7 were determined against the growth of murine leukemia (L1210), mastocytoma (P815) and melanoma (B16) cell lines. The concentrations of compounds required to inhibit the growth of these tumor cells by 50% is in the range of 10(-8)M. These compounds were also tested against a panel of human cancer cells by the National Cancer Institute, demonstrating that the compounds exhibited a high level of activity against selected solid tumors. At a concentration of 0.0084 microM (based on the IC(50) of compound 17 (seco-CBI-TMI) against the growth L1210 cells), while compounds 4 and 17 were toxic against murine bone marrow cells as judged by a colony forming study of freshly isolated murine progenitor hematopoeitic cells, compound 5, a seco-CFQ compound, was significantly less toxic. Flow cytometric analysis of P815 cells that had been incubated for 24h with compounds 4 and 5 at their cytotoxic IC(50) concentrations indicated the induction of apoptosis in a large percentage of cells, thereby suggesting that this might be the mechanism by which the iso-CFI compounds kill cells.