Apostle 技术在临床试验领域的应用案例

Individualized, heterologous chimpanzee adenovirus and self-amplifying mRNA neoantigen vaccine for advanced metastatic solid tumors: phase 1 trial interim results.

 Christine D. Palmer, Amy R. Rappaport, Matthew J. Davis, et al. Nature Medicine  volume 28, pages 1619–1629 (2022)

Abstract Checkpoint inhibitor (CPI) therapies provide limited benefit to patients with tumors of low immune reactivity. T cell-inducing vaccines hold promise to exert long-lasting disease control in combination with CPI therapy. Safety, tolerability and recommended phase 2 dose (RP2D) of an individualized, heterologous chimpanzee adenovirus (ChAd68) and self-amplifying mRNA (samRNA)-based neoantigen vaccine in combination with nivolumab and ipilimumab were assessed as primary endpoints in an ongoing phase 1/2 study in patients with advanced metastatic solid tumors (NCT03639714). The individualized vaccine regimen was safe and well tolerated, with no dose-limiting toxicities. Treatment-related adverse events (TRAEs) >10% included pyrexia, fatigue, musculoskeletal and injection site pain and diarrhea. Serious TRAEs included one count each of pyrexia, duodenitis, increased transaminases and hyperthyroidism. The RP2D was 1012 viral particles (VP) ChAd68 and 30 µg samRNA. Secondary endpoints included immunogenicity, feasibility of manufacturing and overall survival (OS). Vaccine manufacturing was feasible, with vaccination inducing long-lasting neoantigen-specific CD8 T cell responses. Several patients with microsatellite-stable colorectal cancer (MSS-CRC) had improved OS. Exploratory biomarker analyses showed decreased circulating tumor DNA (ctDNA) in patients with prolonged OS. Although small study size limits statistical and translational analyses, the increased OS observed in MSS-CRC warrants further exploration in larger randomized studies.

(Methods section)  cfDNA was extracted from the entire plasma volume of a single draw using the Apostle MiniMax cfDNA Isolation kit (ApostleBio) 

(Methods section) Enriched libraries were sequenced on an Illumina NovaSeq to a minimum mean raw depth of 80,000... Variant allele frequency was converted to mutated haploid genomic equivalents per milliliter plasma (hGE ml1 ) using the extracted plasma volume and total cfDNA yield from the extraction. Percent change in ctDNA was calculated as the change of the median mutated hGE ml1 from the baseline sample.

Safety and tolerability of AAV8 delivery of a broadly neutralizing antibody in adults living with HIV: a phase 1, dose-escalation trial.

Casazza et al. Nature Medicine. April 11, 2022 

Adeno-associated viral vector-mediated transfer of DNA coding for broadly neutralizing anti-HIV antibodies (bnAbs) offers an alternative to attempting to induce protection by vaccination or by repeated infusions of bnAbs. In this study, we administered a recombinant bicistronic adeno-associated virus (AAV8) vector coding for both the light and heavy chains of the potent broadly neutralizing HIV-1 antibody VRC07 (AAV8-VRC07) to eight adults living with HIV. All participants remained on effective anti-retroviral therapy (viral load (VL) <50 copies per milliliter) throughout this phase 1, dose-escalation clinical trial (NCT03374202). AAV8-VRC07 was given at doses of 5 × 1010, 5 × 1011 and 2.5 × 1012 vector genomes per kilogram by intramuscular (IM) injection. Primary endpoints of this study were to assess the safety and tolerability of AAV8-VRC07; to determine the pharmacokinetics and immunogenicity of in vivo VRC07 production; and to describe the immune response directed against AAV8-VRC07 vector and its products. Secondary endpoints were to assess the clinical effects of AAV8-VRC07 on CD4 T cell count and VL and to assess the persistence of VRC07 produced in participants. In this cohort, IM injection of AAV8-VRC07 was safe and well tolerated. No clinically significant change in CD4 T cell count or VL occurred during the 1–3 years of follow-up reported here. In participants who received AAV8-VRC07, concentrations of VRC07 were increased 6 weeks (P = 0.008) and 52 weeks (P = 0.016) after IM injection of the product. All eight individuals produced measurable amounts of serum VRC07, with maximal VRC07 concentrations >1 µg ml−1 in three individuals. In four individuals, VRC07 serum concentrations remained stable near maximal concentration for up to 3 years of follow-up. In exploratory analyses, neutralizing activity of in vivo produced VRC07 was similar to that of in vitro produced VRC07. Three of eight participants showed a non-idiotypic anti-drug antibody (ADA) response directed against the Fab portion of VRC07. This ADA response appeared to decrease the production of serum VRC07 in two of these three participants. These data represent a proof of concept that adeno-associated viral vectors can durably produce biologically active, difficult-to-induce bnAbs in vivo, which could add valuable new tools to the fight against infectious diseases.

...

AAV8-VRC07 vector DNA quantitation. Plasma AAV8-VRC07 plasmid DNA was measured by extracting DNA from plasma, concentrating and then using a real-time PCR assay to measure a 103 base sequence spanning the junction of the IgG heavy chain sequence and F2A insert. DNA was extracted from serum using an Apostle MiniMax High Efficiency cfDNA Isolation Kit, following the manufacturer’s protocol with slight modification.

Dynamics of Plasma EGFR T790M Mutation in Advanced NSCLC: A Multicenter Study. 

Yang et al. Targeted Oncology. 2019;14:719-728. Published: 06 November 2019. 

Background  Droplet digital polymerase chain reaction (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, a ddPCR assay for the epidermal growth factor receptor (EGFR) p.Thr790Met (T790M) mutation suitable for clinical use remains to be established with analytical and clinical validations.  

Objective  We aimed to develop and validate a new ddPCR assay to quantify the T790M mutation in plasma for monitoring and predicting the progression of advanced non-small-cell lung cancer (NSCLC).  Methods  Specificity of the ddPCR assay was evaluated with genomic DNA samples from healthy individuals. The inter- and intraday variations of the assay were evaluated using mixtures of plasmid DNA containing wild-type EGFR and T790M mutation sequences. We assessed the clinical utility of the T790M assay in a multicenter prospective study in patients with advanced NSCLC receiving tyrosine kinase inhibitor (TKI) treatment by analyzing longitudinal plasma DNA samples.  

Results  We set the criteria for a positive call when the following conditions were satisfied: (1) T790M mutation frequency > 0.098% (3 standard deviations above the background signal); (2) at least two positive droplets in duplicate ddPCR reactions. Among the 62 patients with advanced NSCLC exhibiting resistance to TKI treatment, 15 had one or more serial plasma samples that tested positive for T790M. T790M mutation was detected in the plasma as early as 205 days (median 95 days) before disease progression, determined by imaging analysis. Plasma T790M concentrations also correlated with intervention after disease progression.  

Conclusions We developed a ddPCR assay to quantify the T790M mutation in plasma. Quantification of longitudinal plasma T790M mutation may allow noninvasive assessment of drug resistance and guide follow-up treatment in TKI-treated patients with NSCLC.

Trial Registration Clinical Trials.gov identifier: NCT02804100. 

cfDNA was extracted from 2–6 ml of plasma using an Apostle MiniMax High Efficiency cfDNA Isolation Kit (standard edition) (Apostle, Silicon Valley, CA, USA) according to the manufacturer’s instructions.