BIOCHEMISTRY, BIOMEDICINE & PHARMACEUTICS

In this original research article, Dobnik et al. used the molecular biology technique and the transmission electron microscopy technique to characterize and quantitate the viral vectors used for clinical trials. Accurate Quantification and Characterization of Adeno-Associated Viral Vectors David Dobni, et al 2019. Frontier in Microbiology One of the main challenges in the gene therapy viral vector development is to establish an optimized process for its large scale production. This requires optimization for upstream and downstream processes as well as methods that enable the step-by step analytical characterization of the virus, the results of which inform the iterative refinement of production for yield, purity and potency. The biggest problem here is a plethora of viral vector formulations, many of which interfere with analytical techniques. We took adeno-associated virus (AAV) as an example and showed benefits of combined use of molecular methods and transmission electron mic...

In this review article, Fontana et al. discuss the strategies for reducing immunogenicity for therapeutic proteins using various immunomodulatory regimens. These immunomodulatory strategies may reduce the deleterious effect of the immune response evoked against the therapeutic proteins, and improve the safety and efficacy of therapeutic proteins. The strategies include agents that reduce B-cell proliferation, immune tolerization etc. Approaches to Mitigate the Unwanted Immunogenicity of Therapeutic Proteins during Drug Development Laura I. Salazar-Fontana et al. (AAPS 2017: 19(2):377-385) Abstract All biotherapeutics have the potential to induce an immune response. This immunological response is complex and, in addition to antibody formation, involves T cell activation and innate immune responses that could contribute to adverse effects. Integrated immunogenicity data analysis is crucial to understanding the possible clinical consequences of anti-drug antibody (ADA) responses....

Taking immunogenicity assessment of therapeutic proteins to the next level Büttel IC et al, Biologicals. 2011 Mar;39(2):100-9 Abstract Therapeutic proteins provide innovative and effective therapies for numerous diseases. However, some of these products are associated with unwanted immunogenicity that may lead to clinical consequences such as reduced or loss of efficacy, altered pharmacokinetics (PK), general immune and hypersensitivity reactions, and neutralisation of the natural counterpart (e.g. the physiological hormone). Regulatory guidance on immunogenicity assessment needs to take into consideration a great diversity of products, indications and patient populations as well as constantly advancing manufacturing technologies. Such guidance needs to be sufficiently specific while, at the same time, allowing interactive discussion and adjusted benefit-risk weighing of each product on a case-by-case basis, e.g. for a unique treatment of a life threatening disease acceptable t...

Immunogenicity of biotherapeutics in the context of developing biosimilars and biobetters.  Barbosa MD. Drug Discov Today. 2011 Apr;16(7-8):345-53. Abstract Issues concerning the approval of biosimilars are currently being addressed by the US Food and Drug Administration and the European Medicines Agency. There appears to be a consensus that immunogenicity impacts comparability studies and the interchangeability of biosimilars. In addition, preclinical immunogenicity assessment and mitigation, if validated in clinical studies, might impact patient safety and development costs, and also facilitate the development of 'biobetters' and other protein therapeutics. This review addresses recent advances in the field of biosimilars and focuses on predictive immunology, with an emphasis on preclinical immunogenicity assessments of protein therapeutics other than vaccines and their corresponding clinical outcomes. Click here to read full text

Immunogenicity assessment of biotherapeutic products: An overview of assays and their utility. Wadhwa M, Knezevic I, Kang HN, Thorpe R. Biologicals. 2015 Sep;43(5):298-306.   Biotherapeutic products (BTPs) are the fastest growing medicines in the pharmaceutical market. Despite their clinical success, the immunogenicity of BTPs continues to be a major concern. Assessment of immunogenicity as well as appropriate interpretation of immunogenicity data is therefore, of critical importance for defining safety profile of these products for the purpose of their licensure and use. In the past decade, much progress has been made towards how immunogenicity should be studied. This article reflects the content of the brief presentation on principles of methods used for immunogenicity assessment and their merits and limitations given at the first World Health Organization (WHO) implementation workshop on rDNA derived biotherapeutic products held in the Republic of Korea in May 2014...

Recommendations for the characterization of immunogenicity response to multiple domain biotherapeutics. Gorovits B, Wakshull E, Pillutla R, Xu Y, Manning MS, Goyal J J Immunol Methods. 2014 Jun;408:1-12 Abstract Many biotherapeutics currently in development have complex mechanisms of action and contain more than one domain, each with a specific role or function. Examples include antibody-drug conjugates (ADC), PEGylated, fusion proteins and bi-specific antibodies. As with any biotherapeutic molecule, a multi-domain biotherapeutic (MDB) can elicit immune responses resulting in the production of specific anti-drug antibodies (ADA) when administered to patients. As it is beneficial to align industry standards for evaluating immunogenicity of MDBs, this paper highlights pertinent immunogenicity risk factors and describes steps involved in the design of a testing strategy to detect and characterize binding (non-neutralizing and neutralizing, NAb) ADAs. In a common tier based appro...

A risk-based bioanalytical strategy for the assessment of antibody immune responses against biological drugs.  Shankar G, Pendley C, Stein KE. Nat Biotechnol. 2007 May;25(5):555-61. Bioanalytical assessments of anti-drug antibodies (ADAs) provide an understanding of the immunogenicity of biological drug molecules. The potential to induce ADAs after treatment with biologics is a safety issue that has become an important consideration in the development of biologics and a critical aspect of regulatory filings. US and European regulatory agencies are recommending that sponsors study immunogenicity using a risk-based approach, encouraging sponsors to formulate and implement their own risk management plans and to conduct discussions with the agencies when necessary. It follows from this that the greater the safety risks of ADAs, the more diligently one should clarify the immunogenicity of the product. Here we propose a general strategy to broadly assign immunogenicity risk levels...

Witztum  et al. recently published (New England Journal of Medicine, 2019) results from phase 3 clinical trials on efficacy and safety of antisense oligonucleotide Volanesoren in treatment of familial Chylomicronemia. Familial Chylomicronemia is a genetic disorder caused by mutation of enzyme lipoprotein lipase or associated proteins required for its function. Valonesoren inhibits the synthesis of Apoc-III, thereby decreasing the triglycerides level in patients with hypertriglyceridemia. Based on this clinical study, IONIS pharmaceutical obtained a positive opinion and conditional approval to market the product in the European Union region. ( Committee for Medicinal Products for Human Use- CHMP   Public Assessment Report   ) . The following are the excerpts and the result summary from the study: Witztum et al, 2019, NEJM Journal: New England Journal of Medicine Title: Volanesorsen and Triglyceride Levels in Familial Chylomicronemia Syndrome. Abstract BACKGROUND: F...

Meadows et al. recently published papers (Mol Ther Methods Clin Dev, 2019) that investigated the threshold of antibody titer levels that would limit the transduction efficiency of systematic rAAV9 gene delivery.  The early clinical trials have revealed a potential impact of preexisting antibodies against adeno associated virus in the efficacy of transgene expressions. This study attempts to characterize the correlation of transgene expression (NAGLU)  with preexisting antibody titers against AAV9.  The nonclinical nonhuman primate studies were conducted to evaluate the transduction efficiency after systemic delivery of rAAV9 at varying level of preexisting antibodies and define efficacy threshold if any. The following are the excerpts and the result summary from the study: Journal Title: Molecular Therapy: Methods & Clinical Development Title: Threshold for Pre-existing Antibody Levels Limiting Transduction Efficiency of Systemic rAAV9 Gene Delivery: Releva...

Long et al. & Majowicz et al. recently published papers (Mol Ther Methods Clin Dev, 2019) that reported potential impact of preexisting antibodies against adeno associated virus serotype 5 ( AAV5) in the efficacy of transgene expressions. These nonclinical nonhuman primate studies were conducted to evaluate the efficacy of the gene therapy at varying level of preexisting antibodies. Factor VIII and factor IX activity as measured as a pharmacodynamic marker of efficacy.  The Following are the excerpts and the result summary from the study: Journal: Molecular Therapy Methods & Clinical Development Title:  The Impact of Pre-existing Immunity on the Non-clinical Pharmacodynamics of AAV5-Based Gene Therapy Abstract Adeno-associated virus (AAV)-based vectors are widely used for gene therapy, but the effect of pre-existing antibodies resulting from exposure to wild-type AAV is unclear. In addition, other poorly defined plasma factors could inhibit AAV vector transd...

Bruce et al.  recently published a paper (PLOS Medicine, 2019) that provides a trend on the use of surrogate biomarkers to evaluate clinical benefits. The study primarily reviewed European Public Assessment Reports (EPARs) to identify the use of surrogate biomarkers for the primary endpoint.  The Following are the excerpts and the result summary from the study: Journal: PLOS Medicine Title: The use of validated and nonvalidated surrogate endpoints in two European Medicines Agency expedited approval pathways: A cross-sectional study of products authorized 2011–2018 Abstract BACKGROUND:  In situations of unmet medical need or in the interests of public health, expedited approval pathways, including conditional marketing authorization (CMA) and accelerated assessment (AA), speed up European Medicines Agency (EMA) marketing authorization recommendations for medicinal products. CMAs are based on incomplete benefit-risk assessment data and authorization remai...

GIVLAARI (givosiran) injection, for subcutaneous use GIVLAARI is an aminolevulinate synthase 1-directed small interfering RNA (siRNA), covalently linked to a ligand containing three N-acetylgalactosamine (GalNAc) residues to enable the delivery of the siRNA to hepatocytes. CLINICAL PHARMACOLOGY Mechanism of Action  Givosiran is a double-stranded small interfering RNA that causes degradation of aminolevulinate synthase 1 (ALAS1) mRNA in hepatocytes through RNA interference, reducing the elevated levels of liver ALAS1 mRNA. This leads to reduced circulating levels of neurotoxic intermediates aminolevulinic acid (ALA) and porphobilinogen (PBG), factors associated with attacks and other disease manifestations of AHP.  Pharmacodynamics The pharmacodynamic effects of GIVLAARI were evaluated in chronic high excreters treated with 0.035 to 2.5 mg/kg single dose and AHP patients treated with 2.5 to 5 mg/kg once monthly and 2.5 to 5 mg/kg once Reference ID: 4522564 quarterly dose...

MALS (Multi Angle Stable Light Scattering) 

Liquid Chromatography-Mass Sppectoromtery- It is a powerful analytical technique that combines the resolving power of liquid chromatography with the detection specificity of mass spectrometry. Liquid chromatography (LC) separates the sample components and then introduces them to the mass spectrometer (MS). The MS creates and detects charged ions. The LC/MS data may be used to provide information about the molecular weight, structure, identity, and quantity of specific sample components. Applications of LC-MS Proteomics Metabolomics Protein Identification, Characterization and Quntitation Protein Structural Analysis Triple quadrupole Mass Spectrometry Principle (Watch Video) 6470B Triple quadrupole LC/MS (Agilent) Technical Specification The Agilent 6470B triple quadrupole LC/MS system delivers robust performance and high versatility for commercial, life sciences research, and regulatory applications. It has best-in-class sensitivity, precision, and scan speed.  The 6470B LC/MS/MS t...

FDA Guidance for Biodistribution Studies in Gene Therapy The longer the GT product persists, the greater the duration and degree of risk of delayed adverse events. FDA recommends the clinical biodistribution studies using methods shown to be sensitive and quantitative to detect product sequences. These studies should be designed to determine the distribution of the product in non-target tissues and the persistence of the product in both non-target and target tissues following direct in vivo administration of the product. The studies should employ an animal species that permits vector transduction and/or vector replication and that the animal species be biologically responsive to the specific transgene of interest or to therapeutic components in the product (e.g., for products that may not contain transgenes and only genome editing components). Digital Droplet PCR (ddPCR) Platforms for Viral Biodistribution Studies QX200 AutoDG Droplet Digital PCR System Quantstudio 3D ...

 Zolgensma is the first gene therapy product approved for the treatment of children less than two years of age with Spinal Muscular Atrophy (SMA). Norvastis obtained the approval of the drug May 2019 & has been a blockbuster with a price tag of 2 million dollars. In the recent  FDA inspections, FDA observed that the standards were not meet in accordance to GMP guidance. Those include Failure to follow the standard procedures Failure to report complete data sets for the potency testing of the gene therapy product Failure to thoroughly review any unexplained discrepancy whether or not the batch has been already distributed. Laboratory records do not include completed records of any testing and standardization of laboratory standards. What action FDA is taking? FDA are carefully assessing the issue of the manipulation of the product testing data used in the production process and are conducting a thorough assessment of the information from a recently com...

Identification and engineering of TCR epitopes in Cas9 protein helps reduce T cell response against CRISPR CRISPR is a bacterial-derived gene editing tools that can potentially cure various genetic disorders. CRISPR consist of a Cas9 protein that creates a nick in the specific site of double-stranded DNA that is recognized by guide RNA. During the double strand break repairs, the target site may be engineered to obtain the intended outcomes. The adeno-associated viral vectors are most commonly used for delivery of gene and long term expression of Cas9 proteins. These endogenously synthesized Cas9 proteins are processed and presented via MHC-I in the target cells. Any preexisting reactive T cells recognize to MHC- Cas9 peptide complex and elicit cell-mediated cytotoxicity. This may have a detrimental effect on the tissues that express Cas9 transgene. When T cell encounters the target cells, T cell receptor bind to the MHC class I presented with short antigenic peptides (8-10 amino a...

Gene editing is a genome engineering technology that allows us to make intended changes in the DNA. It includes ablating the target genes, inserting / deleting a segment of DNA to the target site, etc. CRISPR is a bacterial-derived gene-editing technology consisting of cas9 protein and guide RNA. The CRISPR technology is being explored for the curative intervention of genetic diseases including muscular dystrophy. The recent study by Gersbach lab published, in Nature Medicine, evaluated the long-term efficacy of AAV mediated CRISPR delivery in the treatment of Duchene muscular dystrophy.  In this study, the authors reported corroborating data on the risk associated with AAV-CRISPR, a novel approach to evaluate these risks, and interesting results that pave the path for future research. The long-term and persistent expression of Cas9 / gRNA are observed in the mouse model of DMD (mdx null mice). The correction of dmd gene resulted in expression of the full-length dystrophin pro...

AAV5 mediated delivery of micro-RNA for treatment of ALS and FTD with C9orf72 G4C2 expansion Amyotrophic lateral sclerosis (ALS) is a life-threatening neurodegenerative disease characterized by progressive degeneration of upper and lower motor neurons, leading to muscle atrophy and paralysis. A significant number of ALS patients also develop frontal temporal dementia (FTD) caused by progressive degeneration of frontal and temporal lobes. There are no disease-modifying drug and most patients die within 3-5 years after the onset of the disease. The most common genetic cause of ALS and FTD is an expansion of G4C2 repeats (> one hundred repeats) in the first intronic site of the  c9orf72 gene present on chromosome 9.   The two underlying pathogenic mechanisms have been proposed for c9orf72 related neurotoxicity. First, the loss of function of c9orf72 caused by G4C2 expansion disrupts mRNA splicing and decreased protein expression resulting in neurotoxicity. But t...

CRISPR is a gene editing tool that are being explored as a potential gene therapy candidate for gene deletions, corrections etc. The high efficiency and specificity of the CRISPR enable its global use in basic research and drug discovery research. The CRISPR consists of two components a) Cas9, a protein that has a nuclease activity b) Guide RNA that recognizes the target sequence, and enables cleavage of target sequence catalyzed by Cas9. Cas9 is a protein that is originally isolated from bacteria Staphylococcus aureus and Streptococcus pyogenes. Because Cas9 is originated from human pathogenic bacteria, the prior exposure to the protein may result in pre-existing antibodies and T cell response against Cas9. The pre-existing immunogenicity against the Cas9 poses a risk impacting the safety and efficacy of CRISPR mediated gene therapy. Recent studies have reported that the prevalence of pre-existing antibodies against Cas9 proteins. Simhari et al. 2018 reported the prevalence of a...