Methods of nucleic acid amplification and sequencing

Methods for amplification and sequencing of at least one nucleic acid comprising the following steps: (1) forming at least one nucleic acid template comprising the nucleic acid(s) to be amplified or sequenced, wherein said nucleic acid(s) contains at the 5' end an oligonucleotide sequence Y and at the 3' end an oligonucleotide sequence Z and, in addition, the nucleic acid(s) carry at the 5' end a means for attaching the nucleic acid(s) to a solid support; (2) mixing said nucleic acid template(s) with one or more colony primers X, which can hybridize to the oligonucleotide sequence Z and carries at the 5' end a means for attaching the colony primers to a solid support, in the presence of a solid support so that the 5' ends of both the nucleic acid template and the colony primers bind to the solid support; (3) performing one or more nucleic acid amplification reactions on the bound template(s), so that nucleic acid colonies are generated and optionally, performing at least one step of sequence determination of one or more of the nucleic acid colonies generated. Solid supports, kits and apparatus for use in these methods are also describe

Zr-89-Lumretuzumab PET Imaging before and during HER3 Antibody Lumretuzumab Treatment in Patients with Solid Tumors

Purpose: We evaluated biodistribution and tumor targeting of Zr-89-lumretuzumab before and during treatment with lumretuzumab, a human epidermal growth factor receptor 3 (HER3)targeting monoclonal antibody. Experimental Design: Twenty patients with histologically confirmed HER3-expressing tumors received Zr-89-lumretuzumab and underwent positron emission tomography (PET). In part A, (89)-Zr-lumretuzumab was given with additional, escalating doses of unlabeled lumretuzumab, and scans were performed 2, 4, and 7 days after injection to determine optimal imaging conditions. In part B, patients were scanned following tracer injection before (baseline) and after a pharmacodynamic (PD)-active lumretuzumab dose for saturation analysis. HER3 expression was determined immunohistochemically in skin biopsies. Tracer uptake was calculated as standardized uptake value (SUV). Results: Optimal PET conditions were found to be 4 and 7 days after administration of Zr-89-lumretuzumab with 100-mg unlabeled lumretuzumab. At baseline using 100-mg unlabeled lumretuzumab, the tumor SUVmax was 3.4(+/- 1.9) at 4 days after injection. SUVmean values for normal blood, liver, lung, and brain tissues were 4.9, 6.4, 0.9 and 0.2, respectively. Saturation analysis (n = 7) showed that 4 days after lumretuzumab administration, tumor uptake decreased by 11.9% (+/- 8.2), 10.0% (+/- 16.5), and 24.6% (+/- 20.9) at PD-active doses of 400, 800, and 1,600 mg, respectively, when compared with baseline. Membranous HER3 was completely downregulated in paired skin biopsies already at and above 400-mg lumretuzumab. Conclusions: PET imaging showed biodistribution and tumor-specific Zr-89-lumretuzumab uptake. Although, PD-active lumretuzumab doses decreased Zr-89-lumretuzumab uptake, there was no clear evidence of tumor saturation by PET imaging as the tumor SUV did not plateau with increasing doses. (C) 2017 AACR

First-in-Human Phase I Study of Lumretuzumab, a Glycoengineered Humanized Anti-HER3 Monoclonal Antibody, in Patients with Metastatic or Advanced HER3-Positive Solid Tumors

PURPOSE: A first-in-human phase I study was conducted to characterize safety, efficacy, and pharmacokinetic (PK) and pharmacodynamic (PD) properties of lumretuzumab, a humanized and glycoengineered anti-HER3 monoclonal antibody, in patients with advanced cancer. EXPERIMENTAL DESIGN: Twenty-five patients with histologically confirmed HER3-expressing tumors received lumretuzumab (100, 200, 400, 800, 1,600, and 2,000 mg) every two weeks (q2w) in 3+3 dose-escalation phase. In addition, 22 patients were enrolled into an extension cohort at 2,000 mg q2w. RESULTS: There were no dose-limiting toxicities. Common adverse events (any grade) included diarrhea (22 patients, 46.8%), fatigue (21 patients, 44.7%), decreased appetite (15 patients, 31.9%), infusion-related reactions (13 patients, 27.7%), and constipation (10 patients, 21.3%). The peak concentration (Cmax) and area under the concentration-time curve up to the last measurable concentration (AUClast) of lumretuzumab increased more than dose proportionally from 100 mg up to 400 mg. Linear PK was observed with doses ≥ 400 mg q2w indicating target-mediated drug disposition saturation. Downregulation of HER3 membranous protein was observed in on-treatment tumor biopsies from 200 mg, and was maximal at and above 400 mg. An ex vivo assay demonstrated increased activation potential of peripheral NK lymphocytes with lumretuzumab compared with a non-glycoengineered anti-HER3 antibody. Ten patients (21.3%) had stable disease and remained on study at a median of 111 days (range, 80-225 days). CONCLUSIONS: Lumretuzumab was well tolerated and showed evidence of clinical activity. Linear serum PK properties and plateauing of PD effects in serial tumor biopsies indicate optimal biologically active doses of lumretuzumab from 400 mg onwards

Phase Ib Study of Lumretuzumab Plus Cetuximab or Erlotinib in Solid Tumor Patients and Evaluation of HER3 and Heregulin as Potential Biomarkers of Clinical Activity

AbstractPurpose: This study investigated the safety, clinical activity, and target-associated biomarkers of lumretuzumab, a humanized, glycoengineered, anti-HER3 monoclonal antibody (mAb), in combination with the EGFR-blocking agents erlotinib or cetuximab in patients with advanced HER3-positive carcinomas.Experimental Design: The study included two parts: dose escalation and dose extension phases with lumretuzumab in combination with either cetuximab or erlotinib, respectively. In both parts, patients received lumretuzumab doses from 400 to 2,000 mg plus cetuximab or erlotinib according to standard posology, respectively. The effect of HRG mRNA and HER3 mRNA and protein expression were investigated in a dedicated extension cohort of squamous non–small cell lung cancer (sqNSCLC) patients treated with lumretuzumab and erlotinib.Results: Altogether, 120 patients were treated. One dose-limiting toxicity (DLT) in the cetuximab part and two DLTs in the erlotinib part were reported. The most frequent adverse events were gastrointestinal and skin toxicities, which were manageable. The objective response rate (ORR) was 6.1% in the cetuximab part and 4.2% in the erlotinib part. In the sqNSCLC extension cohort of the erlotinib part, higher tumor HRG and HER3 mRNA levels were associated with a numerically higher disease control rate but not ORR.Conclusions: The toxicity profile of lumretuzumab in combination with cetuximab and erlotinib was manageable, but only modest clinical activity was observed across tumor types. In the sqNSCLC cohort, there was no evidence of meaningful clinical benefit despite enriching for tumors with higher HRG mRNA expression levels. Clin Cancer Res; 23(18); 5406–15. ©2017 AACR.</jats:p