BIOCHEMISTRY, BIOMEDICINE & PHARMACEUTICS

Multiple Choice Questions on Major discoveries in Microbiology  1) The Nobel prize winner physician and microbiologist Robert Koch was the first scientist to identify the causative agent for the infectious disease called anthrax in humans. Which other microorganisms were first reported (isolated) and identified by him?  Select all the correct answers: a) Mycobacterium tuberculosis b) Bacillus anthracis   c) Bacillus cereus d) Vibrio cholerae   e) Escherichia Coli 2) The discovery of the antibiotic penicillin in treating infection caused by Staphylococcus spp  is considered a revolutionary contribution in the field of medicine.  Who was the first scientist to discover the antibiotic penicillin ? a) Edward Jenner b) Alexander Fleming c) Paul Ehlrich d) Selman Waksman e) Louis Pasteur 3) Which of the following scientist had a major contribution in the field of science who developed a compound microscope and coined the term ‘cell’? a) Robert Koch b) An...

          Bispecific antibodies consist of two small-chain fragments variable (scFv) of the antibody that has binding affinities to different target antigens. T cell engagers are a class of bispecific antibodies that selectively recruit and activate T cytotoxic cells in the tumor tissue and mediate cytotoxicity. In T cell engagers, one scFv domain is designed to bind to CD3 molecules in the T cell, and the second scFv domain binds to a tumor-associated antigen expressed in the tumor tissue. Certain tumor-associated antigens are known to express in specific cancer types. These tumor-associated antigens have been targeted for the treatment of these specific cancers.  These tumor antigens include CD19, BCMA, PSMA, HER2, TROP2, etc.            Bispecific antibodies, similar to any protein therapeutics, can evoke the generation of anti-drug antibodies. Therefore, detection and monitoring of anti-drug antibodies is an i...

Duchenne muscular dystrophy (DMD) is a rare, X-linked, fatal, degenerative neuromuscular disease caused by dystrophin gene (DMD) mutations. The Estimated incidence of DMD worldwide is 1 in 5000 live male births.  The DMD gene encodes for the cytoskeletal protein dystrophin. Dystrophin is a 427-kDa cytoskeletal protein required for sarcolemmal stability. The deficiency of the protein leads to susceptibility to repeated cycles of necrosis and regeneration as well as diminished regenerative muscle capacity, resulting in muscle fibrosis. DMD is a progressive disorder with the loss of ambulation between age 9 and 14 years, followed by respiratory complications and cardiac function decline, and ultimately death. Gene Therapy for DMD The dystrophin is one of the large proteins, and the packaging of cDNA that encodes the full protein into the AAV vector (size limit- 4,500 to 5000 bp) is not possible. Evidence suggests that the truncated dystrophin are partially functio...

Lumasiran Lumasiran, an investigational, subcutaneously administered RNAi therapeutic targeting hydroxyacid oxidase 1 (HAO1) – the gene encoding glycolate oxidase (GO) – in development for the treatment of adults and children with primary hyperoxaluria type 1 (PH1). Primary hyperoxaluria type 1 (PH1) PH1 is an ultra-rare orphan disease characterized by excessive oxalate production, which can lead to end-stage renal disease (ESRD) and other systemic complications. PH1 affects approximately 3.5 to 4 individuals per million in Europe and the United States. Heterogeneity in disease manifestation often contributes to delays in diagnosis, with a median time to diagnosis of approximately six years. PH1 leads to progressive kidney damage, and patients with advanced kidney disease require intensive dialysis to help filter waste products from their blood until they are able and eligible to receive a dual or sequential liver/kidney transplant, an invasive procedure associated with a high risk of ...

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...

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...

 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...

Vestronidase alfa, an enzyme replacement therapy, is an approved drug for the treatment of MPS VII. The approval of this drug has set a precedent for future drug development for the regulators and drug developers. First, the pivotal clinical trial was conducted to evaluate the totality of the clinical data for determining the efficacy of the treatment, therefore, no primary clinical endpoint was established. Second, the drug obtains approval positive outcome on the secondary endpoint; changes in the urinary glycosaminoglycan, from placebo control. The glycosaminoglycan is also the pharmacodynamic biomarker that reflected the efficacy of the drug. Third, the novel multi-domain clinical responder index was designed to evaluate the treatment outcomes and included six minutes walk test (6MWT), forced vital capacity (FVC), shoulder range motion, visual acuity, BOT-2 fine, and gross motor activity. While MRDI evaluation per subject suggested the stabilization of clinical condition if no...

What are Hemophilia disorders ? Hemophilia is the group of bleeding disorders that are caused by a deficiency of proteins (factors) of the blood clotting cascade. Hemophilia A is caused by the deficiency of factor VIII protein and Hemophilia B is caused by a downstream protein factor IX. The deficiency of the factors VIII and IX affect the clotting cascade, resulting in higher and spontaneous bleeding episodes. The severe bleeding disorder leads to spontaneous bleeding in joints and soft tissue resulting in arthropathy; and increased risk of intravascular and intracranial hemorrhage. Why the development of new treatment paradigm is necessary? The standard treatment for hemophilia (A and B type) is the intravenous infusion of an exogenous clotting protein more than once a week to prevent bleeding. In addition, the development of inhibitors decreases the efficacy of the exogenous replacement factor and the severe hemophilia patients do not respond to these therapies.  There...

Inherited Biallelic RPE65 Mutation-Associated Retinal Dystrophy Inherited retinal dystrophies are a group of rare blinding conditions that are associated with progressive visual dysfunction. This rare genetic disorder is classified based on the phenotype, and the mutation in any one of more than 220 different genes are causal for this rare blinding condition. Retinitis pigmentosa is the most common subgroup of inherited retinal dystrophy characterized by reduced ability to perceive light and progressive loss of visual field. RP is genetically heterogeneous, and mutations in any one of over one hundred genes can cause the phenotype. RPE65 gene encodes all-trans-retinyl ester isomerase, an enzyme crucial to the retinoid cycle Autosomal recessive biallelic mutations in this gene lead to the inability to regenerate 11-cis-retinal, via 11-cis-retinol, in the retinal pigment epithelial cells RPE cells. This impairs the ability of RPE cells to respond to light, which disrupts the vis...