Top Selling Monoclonal Antibodies 2014
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Top Selling Monoclonal Antibodies 2014
Top Selling Monoclonal Antibodies 2014
Abstract A review of the best selling monoclonal antibodies in 2014 and 2013 is provided. Humira with sales of over $12.7 billion ($9.3 bn in 2012, $11bn 2013) remains the best selling monoclonal antibody, biologic as well prescription drug brand in 2014 and since 2012. The ranks of the next 4 top selling mabs remained unchanged from the 2012-2013 Table.  Remicade (9.8 Bn), Rituxan (7.6 Bn) , Avastin (7.0 Bn) and Herceptin (6.8 Bn)  were the second, third, fourth and fifth top selling mabs in 2014. The actual sales for 2014 as reported by the companies are provided. The total sales of the top selling blockbuster mabs listed in the Table were $68 billion in 2014. The global sales of all the approved therapeutic monoclonal antibodies were $78 billion in 2014. Besides the top 5 mabs, there was two monoclonal antibody with sales of over $3 billions, three with sales over $ 2 billion and 6 with sales of over $ 1 billion in 2014. In addition 5 recently launched mabs were nearing to reach sales of $ 1 billion this year. Currently 36 monoclonal antibodies are marketed in the US and Europe (January 2015).  FDA approved 6 new monoclonal antibodies in 2014. Alexion Soliris was the most expansive marketed monoclonal antibody with a price tag of $440,000 per year of treatment, a sort of Rolls-Royce of mabs and had sales of $2.2 billion in 2014.
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Best Selling Blockbuster Monoclonal Antibodies 2017

Best Selling Blockbuster Monoclonal Antibodies 2017 | Top Selling Monoclonal Antibodies 2014 | Scoop.it

 

Abstract

 

A review of the best selling monoclonal antibodies in 2017 and previous years is provided. Humira with sales of over $19 billion ($9.3 bn in 2012, $14.5 bn in 2015 and $16 bn in 2016) remains the best selling monoclonal antibody, biologic as well the prescription drug brand in 2017. Humira has remained the top selling global brand since 2012. The ranks of the next 4 top selling mabs changed rankings from the 2012-2016 Table due to competition from biosimilars.  Rituxan (9.2 Bn) , Herceptin (7.4 Bn), Remicade (7.1 Bn) and Avastin (7.1 Bn) and were the second, third, fourth and fifth top selling mabs in 2017. The actual sales for 2017 as reported by the companies are provided. 



The global pharma biotech market for prescription medicinal brands was over $1 trillion dollar in 2017. Biologics accounted for $220 billion and  mabs for $95 billion

 

The total sales of the top selling blockbuster mabs listed in the Table were $95 billion in 2017.  Besides the top 5 mabs with sales over $7 billion, there were two monoclonal antibody with sales of over $4 billions, four with sales over $3 billion, five with sales over $ 2 billion and 3 with sales of over $ 1 billion in 2015. There were 19 blockbuster mabs in 2019. At least 5 new recently launched mabs are likely to cross sales of $ 1 billion in 2018.

 

Currently 73 monoclonal antibodies are marketed in the US and Europe (Dec. 2017).  FDA approved 6 new monoclonal antibodies in 2014, 9 in 2015, 9 in 2016 and 9 in 2017. The FDA has already approved 4 new mabs in 2018 and EMA CHMP has recommended 2 new mabs in the 1Q2018.

 

The FDA has approved only 9 biosimilar so far while EMA/CHMP has cleared 28 biosimilar so far.

 

Alexion Soliris was the most expansive marketed monoclonal antibody with a price tag of $440,000 per year of treatment, a sort of Rolls-Royce of mabs and had sales of $2.6 billion in 2017.

 

The full report is not for public release or for request by email

 

 

 

 

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The full report is not available for public release or for request by email

 

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Structural investigation of human S. aureus-targeting antibodies that bind wall teichoic acid: mAbs: Vol 10, No 7

Structural investigation of human S. aureus-targeting antibodies that bind wall teichoic acid: mAbs: Vol 10, No 7 | Top Selling Monoclonal Antibodies 2014 | Scoop.it
ABSTRACT
Infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are a growing health threat worldwide. Efforts to identify novel antibodies that target S. aureus cell surface antigens are a promising direction in the development of antibiotics that can halt MRSA infection. We biochemically and structurally characterized three patient-derived MRSA-targeting antibodies that bind to wall teichoic acid (WTA), which is a polyanionic surface glycopolymer. In S. aureus, WTA exists in both α- and β-forms, based on the stereochemistry of attachment of a N-acetylglucosamine residue to the repeating phosphoribitol sugar unit. We identified a panel of antibodies cloned from human patients that specifically recognize the α or β form of WTA, and can bind with high affinity to pathogenic wild-type strains of S. aureus bacteria. To investigate how the β-WTA specific antibodies interact with their target epitope, we determined the X-ray crystal structures of the three β-WTA specific antibodies, 4462, 4497, and 6078 (Protein Data Bank IDs 6DWI, 6DWA, and 6DW2, respectively), bound to a synthetic WTA epitope. These structures reveal that all three of these antibodies, while utilizing distinct antibody complementarity-determining region sequences and conformations to interact with β-WTA, fulfill two recognition principles: binding to the β-GlcNAc pyranose core and triangulation of WTA phosphate residues with polar contacts. These studies reveal the molecular basis for targeting a unique S. aureus cell surface epitope and highlight the power of human patient-based antibody discovery techniques for finding novel pathogen-targeting therapeutics.

Keywords: Wall Teichoic Acid, WTA, Staphylococcus aureus, monoclonal antibodies, antibody structure, antibody-carbohydrate interactions
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Probing Conformational Diversity of Fc Domains in Aggregation-Prone Monoclonal Antibodies

Probing Conformational Diversity of Fc Domains in Aggregation-Prone Monoclonal Antibodies | Top Selling Monoclonal Antibodies 2014 | Scoop.it
Abstract
Purpose
Fc domains are an integral component of monoclonal antibodies (mAbs) and Fc-based fusion proteins. Engineering mutations in the Fc domain is a common approach to achieve desired effector function and clinical efficacy of therapeutic mAbs. It remains debatable, however, whether molecular engineering either by changing glycosylation patterns or by amino acid mutation in Fc domain could impact the higher order structure of Fc domain potentially leading to increased aggregation propensities in mAbs.

Methods
Here, we use NMR fingerprinting analysis of Fc domains, generated from selected Pfizer mAbs with similar glycosylation patterns, to address this question. Specifically, we use high resolution 2D [13C-1H] NMR spectra of Fc fragments, which fingerprints methyl sidechain bearing residues, to probe the correlation of higher order structure with the storage stability of mAbs. Thermal calorimetric studies were also performed to assess the stability of mAb fragments.

Results
Unlike NMR fingerprinting, thermal melting temperature as obtained from calorimetric studies for the intact mAbs and fragments (Fc and Fab), did not reveal any correlation with the aggregation propensities of mAbs. Despite >97% sequence homology, NMR data suggests that higher order structure of Fc domains could be dynamic and may result in unique conformation(s) in solution.

Conclusion
The overall glycosylation pattern of these mAbs being similar, these conformation(s) could be linked to the inherent plasticity of the Fc domain, and may act as early transients to the overall aggregation of mAbs.

KEY WORDS
aggregation differential scanning calorimetry Fab domain Fc domain molecular fingerprinting monoclonal antibodies nuclear magnetic resonance spectroscopy storage stability 
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IBCN 2018: PD-L1 Expression According to Five Monoclonal Antibodies in Urothelial Cell Cancer

IBCN 2018: PD-L1 Expression According to Five Monoclonal Antibodies in Urothelial Cell Cancer | Top Selling Monoclonal Antibodies 2014 | Scoop.it
Tissue Microarrays (TMA) containing samples of 139 muscle-invasive UC patients were stained with the anti-PD-L1 antibodies: 22C3, 28-8, SP142, SP263, and E1L3N on the Ventana Benchmark (SP142, SP263) and DAKO platforms (22C3, 28-8, E1L3N). PD-L1 expression was manually scored on tumor cells (TC) and infiltrating immune cells (IC). Next, the PD-L1 status was determined according to corresponding assay specifications used in clinical trials.

PD-L1 expression was higher on TC than IC using antibodies 22C3, 28-8, SP263 and E1L3N, while SP142 demonstrated less PD-L1 expression on TC. PD-L1 status was positive in 20% to 27% of patients. The percentage agreement in PD-L1 status between individual antibody clones: i) varied from 60% to 90%, ii) was lowest for E1L3N and SP142, and iii) was better when based on a higher cutoff value for 22C3 (³10%) and 28-8 (³5%). Fleiss’ Kappa as an index of inter-assay agreement was 0.506 for all antibodies (PD-L1 status identical in 64%), it improved to 0.617 considering 22C3, 28-8, SP-142 and SP263 (PD-L1 status identical in 78%), and was best considering only 22C3, 28-8 and SP263 (Fleiss’ Kappa 0.674, PD-L1 status identical in 84%).

In summary, they found a substantial concordance in PD-L1 status between the most frequently used PD-L1 companion diagnostics. In the majority of cases, the PD-L1 status was similar by each companion diagnostic antibody, indicating that the application of different companion PDL1 antibodies may have limited implications on therapeutic decision making in ICI treatment for UC patients.
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Ultrasensitive Characterization of Charge Heterogeneity of Therapeutic Monoclonal Antibodies Using Strong Cation Exchange Chromatography Coupled to Native Mass Spectrometry - Analytical Chemistry (...

Ultrasensitive Characterization of Charge Heterogeneity of Therapeutic Monoclonal Antibodies Using Strong Cation Exchange Chromatography Coupled to Native Mass Spectrometry - Analytical Chemistry (... | Top Selling Monoclonal Antibodies 2014 | Scoop.it
Ultrasensitive Characterization of Charge Heterogeneity of Therapeutic Monoclonal Antibodies Using Strong Cation Exchange Chromatography Coupled to Native Mass Spectrometry...
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Polyclonal and monoclonal antibodies for the treatment of influenza

Polyclonal and monoclonal antibodies for the treatment of influenza | Top Selling Monoclonal Antibodies 2014 | Scoop.it
Abstract Purpose of review: This review summarizes past and ongoing efforts for using polyclonal and monoclonal antibodies for the treatment of influenza, and is focused on products that have entered clinical trials. Recent findings: At least 3 polyclonal and 8 monoclonal antibody products have been tested in clinical trials for the treatment of influenza. Considered across the two classes of therapeutics, these products appear to be safe and well tolerated. However, the efficacy results have been mixed and inconclusive. To date, no products have consistently shown superiority to currently available antivirals. Summary: No products within these two classes have been licensed, and several products appear to have stopped further clinical development. There are several ongoing studies that are anticipated to be completed or reported in the next 1–2 years which will be critical for understanding the value of polyclonal and monoclonal antibodies in the treatment of influenza.
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Team gets a closer look at how proteins meet on the cell membrane | EurekAlert! Science News

Team gets a closer look at how proteins meet on the cell membrane | EurekAlert! Science News | Top Selling Monoclonal Antibodies 2014 | Scoop.it
At last, the researchers have defined the molecular basis of the cell membrane in integrin activation.
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The Evolution of Antibody Patents - IPWatchdog.com

The Evolution of Antibody Patents - IPWatchdog.com | Top Selling Monoclonal Antibodies 2014 | Scoop.it
As the pharmaceutical industry continues to shift toward biologic-based drugs, including monoclonal antibodies, protecting the underlying technology has been and continues to be a priority for companies.
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Crystal structure of a membrane-bound O -acyltransferase

Crystal structure of a membrane-bound O -acyltransferase | Top Selling Monoclonal Antibodies 2014 | Scoop.it
Crystal structures of DltB, a bacterial membrane-bound O-acyltransferase, are reported both alone and in complex with the d-alanyl donor protein DltC.
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 FDA Approves Hemlibra® (emicizumab-kxwh, Roche) New Prophylactic Treatment For Hemophilia A Without Factor VIII Inhibitors

 FDA Approves  Hemlibra® (emicizumab-kxwh, Roche) New Prophylactic Treatment For Hemophilia A Without Factor VIII Inhibitors | Top Selling Monoclonal Antibodies 2014 | Scoop.it

On October 4, 2018, the FDA approved Hemlibra® (emicizumab-kxwh) for routine prophylaxis to prevent or reduce the frequency of bleeding episodes in adults and children, ages newborn and older, with hemophilia A without factor VIII inhibitors. Hemlibra is now the only medicine approved to treat people of all ages with hemophilia A with and without factor VIII inhibitors.

 

  • FDA approval based on positive results from Phase III studies, including the HAVEN 3 study showing Hemlibra prophylaxis significantly reduced treated bleeds compared to no prophylaxis in people with hemophilia A without factor VIII inhibitors
  • First medicine to significantly reduce treated bleeds compared to prior factor VIII prophylaxis based on a prospective intra-patient comparison
  • Only approved medicine that can be self-administered subcutaneously once weekly, every two weeks or every four weeks for hemophilia A with and without factor VIII inhibitors
  • The efficacy and safety of Hemlibra has been demonstrated in one of the largest pivotal clinical trial programs in hemophilia A
Krishan Maggon 's insight:

Hemlibra was granted Breakthrough Therapy Designation by the FDA for hemophilia A without factor VIII inhibitors. It was also granted Priority Review, a designation given to medicines that the FDA has determined to have the potential to provide significant improvements in the treatment, prevention or diagnosis of a serious disease. Submissions to other regulatory authorities around the world are ongoing.

Hemlibra was approved by the FDA in November 2017 for adults and children with hemophilia A with factor VIII inhibitors. It has been studied in one of the largest pivotal clinical trial programs in people with hemophilia A with and without factor VIII inhibitors, including four pivotal HAVEN studies (HAVEN 1, HAVEN 2, HAVEN 3 and HAVEN 4).

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Regeneron and Sanofi receives first US FDA approval for PD-1 treatment

Regeneron and Sanofi receives first US FDA approval for PD-1 treatment | Top Selling Monoclonal Antibodies 2014 | Scoop.it
The US FDA approved the first drug created by the 2015 partnership between Regeneron and Sanofi to research and develop immuno-oncology therapies....
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A Systematic Screen of FDA-Approved Drugs for Inhibitors of Biological Threat Agents

A Systematic Screen of FDA-Approved Drugs for Inhibitors of Biological Threat Agents | Top Selling Monoclonal Antibodies 2014 | Scoop.it
The rapid development of effective medical countermeasures against potential biological threat agents is vital. Repurposing existing drugs that may have unanticipated activities as potential countermeasures is one way to meet this important goal, since ...
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Bispecific Single-Domain Antibody Fused to Monoclonal Antibody (SMAB): The Natural Form | BioPharm International

Bispecific Single-Domain Antibody Fused to Monoclonal Antibody (SMAB): The Natural Form | BioPharm International | Top Selling Monoclonal Antibodies 2014 | Scoop.it
FDA has approved three bispecific antibodies so far, and 75 bispecifics are in clinical trials. Dr. Li Chen will provide an overview of therapeutic antibodies, discuss the novelty of bispecifics, and present case studies followed by a summary.<br /><br />
Live: Wednesday, Oct.
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High-resolution glycosylation site-engineering method identifies MICA epitope critical for shedding inhibition activity of anti-MICA antibodies: mAbs: Vol 0, No ja

High-resolution glycosylation site-engineering method identifies MICA epitope critical for shedding inhibition activity of anti-MICA antibodies: mAbs: Vol 0, No ja | Top Selling Monoclonal Antibodies 2014 | Scoop.it
ABSTRACT
As an immune evasion strategy, MICA and MICB, the major histocompatibility complex class I homologs, are proteolytically cleaved from the surface of cancer cells leading to impairment of CD8 + T cell- and natural killer cell-mediated immune responses. Antibodies that inhibit MICA/B shedding from tumors have therapeutic potential, but the optimal epitopes are unknown. Therefore, we developed a high-resolution, high-throughput glycosylation-engineered epitope mapping (GEM) method, which utilizes site-specific insertion of N-linked glycans onto the antigen surface to mask local regions. We apply GEM to the discovery of epitopes important for shedding inhibition of MICA/B and validate the epitopes at the residue level by alanine scanning and X-ray crystallography (Protein Data Bank accession numbers 6DDM (1D5 Fab-MICA*008), 6DDR (13A9 Fab-MICA*008), 6DDV (6E1 Fab-MICA*008). Furthermore, we show that potent inhibition of MICA shedding can be achieved by antibodies that bind GEM epitopes adjacent to previously reported cleavage sites, and that these anti-MICA/B antibodies can prevent tumor growth in vivo.

Keywords: Epitope mapping, antibody discovery, binding interface, antibody characterization, FPOP, alanine scan
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Frontiers | Oncolytic Viruses Partner With T-Cell Therapy for Solid Tumor Treatment | Immunology

Frontiers | Oncolytic Viruses Partner With T-Cell Therapy for Solid Tumor Treatment | Immunology | Top Selling Monoclonal Antibodies 2014 | Scoop.it
Adoptive T-cell immunotherapies, including chimeric antigen receptor-modified T-cells (CAR-T cells), have revolutionized cancer treatment, especially for hematologic malignancies. Clinical success of CAR-T cell monotherapy in solid tumors however, has been only modest. Oncolytic viruses provide direct cancer cell lysis, stimulate systemic immune responses, and have the capacity to provide therapeutic transgenes. Oncolytic virotherapy has shown great promise in many preclinical solid tumor models and the first oncolytic virus has been approved by the FDA for the treatment of advanced melanoma. As monotherapies for solid tumors, oncolytic virotherapy provides only moderate anti-tumor effects. However, due to their complementary modes of action, oncolytic virus and T-cell therapies can be combined to overcome the inherent limitations of each agent. This review focuses on the aspects of oncolytic viruses that enable them to synergize with adoptive T-cell immunotherapies to enhance anti-tumor effects for solid tumors.
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Fragmentation of a Monoclonal Antibody by Peroxotungstate

Fragmentation of a Monoclonal Antibody by Peroxotungstate | Top Selling Monoclonal Antibodies 2014 | Scoop.it
Abstract
Purpose
Tungsten and tungsten oxide leachates found in glass pre-filled syringes were identified to initiate protein precipitation and aggregation. Here, we tested the possibility of tungsten and tungsten oxide to induce the chemical degradation of proteins via reaction with hydrogen peroxide, a possible impurity present in protein formulations, to yield peroxotungstate.

Methods
A monoclonal antibody (mAb) was incubated with various concentrations of peroxotungstate and the reaction mixtures analyzed by SDS-PAGE and mass spectrometry.

Results
Exposure of a mAb to 1.07–1070 ppm peroxotungstate (based on tungsten content) at temperatures of 4°C and 22°C (pH 5–7) induced protein fragmentation. The extent of fragmentation increased with higher temperatures, lower pH and higher peroxotungstate concentrations. The mAb fragments were identified to contain different combinations of heavy chains (H) and light chains (L). Analogous mAb fragments were generated when the protein was exposed to H2O2 and orthotungstate at levels as low as 5 ppm. In addition, extracts from tungsten pins used to manufacture glass pre-filled syringes, in combination with H2O2 caused comparable fragmentation of the mAb. Mass spectrometric identification of the fragments suggests fragment generation by oxidative disulfide bond cleavage between the heavy and light chains, confirmed by mass spectrometry data on product formation. The mechanism of oxidative fragmentation was separately confirmed with insulin.

Conclusion
Fragmentation of the mAb by peroxotungstate is proposed to occur through inter-chain disulfide bond oxidation to form thiosulfinate (CyS(═O)SCy) and thiosulfonate [CyS(═O)2SCy], followed by hydrolysis.

Key Words
Fragmentation monoclonal antibody oxidation pre-filled syringes tungsten 
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Biosimilar medicines | European Medicines Agency

Biosimilar medicines | European Medicines Agency | Top Selling Monoclonal Antibodies 2014 | Scoop.it
A biosimilar is a biological medicine highly similar to another already approved biological medicine (the 'reference medicine'). Biosimilars are approved according to the same standards of pharmaceutical quality, safety and efficacy that apply to all biological medicines. The European Medicines Agency (EMA) is responsible for evaluating the majority of applications to market biosimilars in the European Union (EU). Biological medicines offer treatment options for patients with chronic and often disabling conditions such as diabetes, autoimmune disease and cancers. Biological medicines contain active substances from a biological source, such as living cells or organisms (human, animals and microorganisms such as bacteria or yeast) and are often produced by cutting-edge technology. Most biological medicines in current clinical use contain active substances made of proteins. These can differ in size and structural complexity, from simple proteins like insulin or growth hormone to more complex ones such as coagulation factors or monoclonal antibodies. Examples of types of proteins in biological medicines approved in the EU A biosimilar is a biological medicine highly similar to another biological medicine already approved in the EU (called 'reference medicine') in terms of structure, biological activity and efficacy, safety and immunogenicity profile (the intrinsic ability of proteins and other biological medicines to cause an immune response). The EU approved the first biosimilar in 2006. A biosimilar is not regarded as a generic of a biological medicine. This is mostly because the natural variability and more complex manufacturing of biological medicines do not allow an exact replication of the molecular micro-heterogeneity. The EU has pioneered the regulation of biosimilar medicines by establishing a solid framework for their approval and by shaping biosimilar development globally. The evidence acquired over ten years of clinical experience shows that biosimilars approved through EMA can be used as safely and effectively in all their approved indications as other biological medicines. For the list of biosimilar medicines approved via the centralised procedure via EMA, see: Information for patients and healthcare professionals EMA and the European Commission have developed information materials on biosimilar medicines to improve understanding of these medicines in the EU. An animated video for patients explains key facts on biosimilar medicines and how EMA works to ensure that they are as safe and effective as their reference biological medicines. The video is also available in the following European languages: Dutch, English, French, German, Italian, Polish, Portuguese and Spanish. EMA plans to publish further language versions when available. An information guide for patients published by the European Commission explains in a clear, unbiased way what biosimilar medicines are, how they are developed and approved in the EU and what patients can expect in terms of availability and safety. EMA and organisations representing patients contributed to the development of this guide. It is available in 23 official EU languages on the Commission's website.   EMA and the European Commission have published an information guide for healthcare professionals to provide reference information on the science and regulation underpinning the use of biosimilar medicines. EU scientific experts and organisations representing doctors, nurses, pharmacists and patients contributed to the development of this guide. This guide is also available in seven European languages below. EMA plans to publish further language versions when available.
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Latest Cornell dot features a new cancer weapon: antibodies

Latest Cornell dot features a new cancer weapon: antibodies | Top Selling Monoclonal Antibodies 2014 | Scoop.it
Antibody-based imaging of a particularly aggressive form of breast cancer is undergoing clinical trials worldwide, but the path from trial to application is being hampered by a major obstacle: safety.
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Detailed glycosylation analysis of monoclonal antibodies : Alphalyse

Detailed glycosylation analysis of monoclonal antibodies : Alphalyse | Top Selling Monoclonal Antibodies 2014 | Scoop.it
Glycosylation of monoclonal antibodies (mAbs) greatly impacts the activity, efficacy & stability of your mAb.Contact us for glycosylation characterization...
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Monoclonal Antibodies vs. Natural Antibodies | Amgen Science

Monoclonal Antibodies vs. Natural Antibodies | Amgen Science | Top Selling Monoclonal Antibodies 2014 | Scoop.it
Monoclonal antibodies - one of 13 drug formats that Amgen is using to fight disease.
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Xencor’s lead lupus antibody misses phase 2 endpoint | FierceBiotech

Xencor’s lead lupus antibody misses phase 2 endpoint | FierceBiotech | Top Selling Monoclonal Antibodies 2014 | Scoop.it
Topline results from a phase 2 study showed Xencor’s lead CD19-targeting antibody missed its primary endpoint in lupus, but the company described the data as a “positive trend” that still contained a “promising treatment effect” worthy of future study.
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Antibody Structure and Fragments

Antibody Structure and Fragments | Top Selling Monoclonal Antibodies 2014 | Scoop.it
Conventional Antibodies Conventional or full size antibodies are glycoproteins called immunoglobulins that are produced by plasma cells in response to a foreign molecule (immunogen). The primary function of antibodies is to bind specifically to an antigen and elicit an immune response, thereby protecting the host from infection. There are several classes of antibodies, but this summary focuses on the IgG and IgM class antibodies, which are heavily utilized in a multitude of research, diagnostic and therapeutic biomedical applications. Conventional Antibody Structure The basic unit of a complete antibody is a four polypeptide unit containing two heavy chains and two light chains held together via disulfide bonds. The basic shape of an antibody is a Y, and the hinge region at the point of the Y structure has flexibility. Each polypeptide chain has a constant region, which is very similar for all antibodies, and a variable region, which is specific to each particular antibody. The common notation for the light chain variable region is VL and for the light chain constant region is CL (Figure 1 Left panel). The notation is similar for the heavy chain variable (VH) and constant regions (CH). Carbohydrates are normally attached to the CH2 domain of the heavy chains. The fragment crystallizable (Fc) region contains only constant regions from the heavy chains (CH), but the fragment antigen-binding region (Fab) includes both a constant domain and the variable domains of both the heavy and light chains (VH and CL). The fragment variable region Fv region contains only the two variable domains (Figure 1). See mouse antibody for discussion on immunoglobulin isotypes, subclasses, and the number of immunoglobulin domains. A database of all publicly available antibody structures, the Structural antibody database (SAbDab) curated from PDB databank, is available [2]. Conventional Antibody Applications Conventional antibodies have been utilized in research for protein detection via Western blot analyses [3], immunohistochemistry [4] and enzyme-linked immunosorbent assays (ELISA) [5] for decades. Full size antibodies have also been developed for diagnostic applications such as pregnancy tests and detection of bacteria and viruses in blood, such as an ELISA that detects HIV. Additionally, conventional full size antibodies are used commonly in disease therapeutics. For example, Infliximab is one antibody of many available that recognize tumor necrosis factor alpha (TNFα) and it is used in the treatment of Crohn's disease and rheumatoid arthritis [6, 7]. Trastuzumab, or Herceptin, is an antibody used in the treatment of metastatic breast cancer that binds to epidermal growth factor receptor 2 [8]. Additionally, there are several antibody-based therapies given to transplant recipients to prevent allograft rejection, including Muromomab [9]. Advantages of using conventional antibodies include the fact the Fc region engages the body's immune response, and will target bound molecules for destruction. Disadvantages of full size antibodies include their inability to penetrate into certain tissues due to their relatively large size [10]. A disadvantage to using full size antibodies in clinical applications is that the Fc domain will frequently elicit an immunological response, which may be detrimental to the patient's health. The Fc domain often causes significant nonspecific binding, which may impair detection applications. Antibody Fragments Fragments of an antibody can be produced through chemical or genetic mechanisms. Chemical fragmentation utilizes reducing agents to break the disulfide bonds within the hinge region and digestion of the antibody with proteases including pepsin, papain, and ficin. Genetic construction of fragments offers the ability to create a multitude of fragment containing molecules, each with unique binding and functional characteristics. Fab, Fab', (Fab')2, and Fv Chemical and protease digestion of full size antibodies yield antigen binding fragments (Fab), which are generated from the variable regions of IgG and IgM class antibodies (Figure 1, right panel). Fc fragments, composed only of the heavy chain constant regions, are removed from the Fabs. Antigen binding fragments include Fab, Fab', (Fab')2, and Fv. These fragments are able to bind antigen but they each lack the Fc region that contains the heavy chain constant domains 2 and 3. Two individual F(ab) fragments are separated from the Fc region when a full size antibody is digested with papain enzyme. However, a F(ab')2 fragment, which retains a small part of the Fc hinge region, is separated from the rest of the Fc region of an antibody by digestion with pepsin. Although the utilization of biochemical methods to generate antibody fragments produces useful tools for diagnostic and therapeutic applications, it is quite laborious and requires a large quantity of antibody starting material. Monovalent F(ab) fragments have one antigen binding site, whereas divalent F(ab')2 fragments have two antigen binding regions that are linked by disulfide bonds. Reduction of F(ab')2 fragments produces 2 monovalent Fab' fragments, which have a free sulfhydryl group that is useful for conjugation to other molecules. Fv fragments are the smallest fragment made from enzymatic cleavage of IgG and IgM class antibodies. Fv fragments have the antigen-binding site made of the VH and VL regions, but they lack the CH1 and CL regions (Figure 1 right panel). The VH and VL chains are held together in Fv fragments by non-covalent interactions. scFv, diabody, triabody, tetrabody, Bis-scFv, minibody, Fab2, Fab3 Genetic engineering methods allow the production of single chain variable fragments (scFv), which are Fv type fragments that include the VH and VL domains linked by a flexible peptide (Figure 1 right panel) [11]. When the linker is at least 12 residues long, the scFv fragments are primarily monomeric [12]. Manipulation of the orientation of the V-domains and the linker length creates different forms of Fv molecules [12]. Linkers that are 3-11 residues long yield scFv molecules that are unable to fold into a functional Fv domain. These molecules associate with a second scFv molecule, which creates a bivalent diabody [13]. If the linker length is less than three residues, scFv molecules associate into triabodies or tetrabodies. Multivalent scFvs possess greater functional binding affinity to their target antigens than their monovalent counterparts by having binding to two more target antigens, which reduces the off-rate of the antibody fragment [14]. Minibodies are scFv-CH3 fusion proteins that assemble into bivalent dimers. Bis-scFv fragments are bispecific [15]. Miniaturized scFv fragments can be generated that have two different variable domains, allowing these Bis-scFv molecules to concurrently bind to two different epitopes. Genetic methods are also used to create bispecific Fab dimers (Fab2) and trispecific Fab trimers (Fab3). These antibody fragments are able to bind 2 (Fab2) or 3 (Fab3) different antigens at once. Camelid/shark antibodies and nanobodies In addition to conventional antibodies, camelid and shark species contain a subset of peculiar Heavy Chain Antibodies (hcAb) exclusively composed by heavy chain homodimers lacking light chains [16]. The Fab portion of these antibodies is called VHH (variable domain of heavy chain antibodies), being the smallest antigen-binding region naturally found [17]. Nanobodies are VHH-derived recombinant domains able to bind antigens. They are very stable and can be easily produced in huge quantity by using traditional simple systems such as bacteria (regular antibodies with both light and heavy chains are difficult to express in a bacterial system), thus representing a promising tool for research and therapeutic purposes, especially in the areas of super-resolution microscopy, mass spectrometry, and targeted protein degradation [18]. Nanobodies can also be delivered inside living cells through conjugated with peptides [19, 20] or expressed in vivo directly and recognize its targets in vivo, while regular antibodies with both light and heavy chains do not usually work in living cells. For example, anti-GFP nanobody is used to develop an electromagnetic control system for neuronal activity in vivo [21]. Nanobodies against RFP or GFP, when conjugated with far-red dyes Atto, attained 118 fold magnification of fluorescent signals over GFP or RFP, and were used to generate whole-body mouse neuronal connectivity [22]. They have also been used to stablize the active state of proteins in structural studies [23]. Recombinant anti–mouse and anti–rabbit IgG secondary nanobodies have the potential to replace widely used polyclonal secondary antibodies [24]. Cow ultralong CDR3H About 10% of bovine immunoglobulins contain long CDR3H region with multiple cysteine residues [25, 26], and this long-stalk is thought to contribute to the antibody diversity [27]. Antibody Fragment Applications Fragments offer advantages over a full size antibody for some applications. This topic was recently reviewed by Nelson [28]. One advantage of fragments over full size antibodies is that fragments are small enough to infiltrate into some tissues that full size antibodies are unable to penetrate, which aids in both therapeutic and immunohistochemical procedures [10]. Antibody fragments are smaller than conventional antibodies and generally lack glycosylation, allowing their production in prokaryotic expression systems, which provide time and cost savings. However, fragments that lack the Fc domain are degraded in the body much more rapidly than conventional antibodies [29], and are unable to elicit Fc mediated cytotoxic processes unless they are conjugated to an effector moiety [30], which requires further optimization for antibody fragment-based therapeutics. However, the lack of Fc domain is a substantial advantage for primary antibodies used in immunohistochemistry and other detection applications because they have greatly reduced non-specific binding to the Fc receptor. Antibody fragments that are without the Fc region have the advantage of reduced non-specific binding. One scFv that is commonly used in diagnostics is the NC10 antibody against influenza neuraminidase. The MOC-31 antibody against epithelial cell adhesion molecule Ep-CAM is a scFv commonly used in as a cancer therapy. Diabodies, triabodies and tetrabodies have potential uses in applications such as radioimmunotherapy and diagnostic in vivo imaging [31]. Nanobodies can be used for specific purposes in (co)-immunoprecipitation, or coupled to fluorescent proteins tracking in real-time intracellular targets in live cells [32]. Although various antibody fragments offer certain advantages, they are not commonly utilized in experiments. In the 38430 articles surveyed by Labome as of March, 2016, only few articles cited applications of antibody Fab fragments. Roche anti-digoxigenin Fab fragments were used in 1:1000 or 1:2000 to perform in situ hybridization to show that main proximodistal limb subdivision depends on diffusible signals [33], ciliogenesis is regulated by an evolutionarily assembled cis-regulatory module [34], and mimetic wing pattern evolution in butterfly could be regulated by optix gene [35]. They were also used to perform protein folding analysis to study the process of single calmodulin molecule folding through single-molecule force spectroscopy [36]. Roche anti-digoxigenin antibody (catalog number 11093274910) was generated in sheep and produced through digestion with papain. Roche anti-fluorescein alkaline phosphatase Fab fragments were used to perform southern blot to study the function of topoisomerase II [37]. More recent examples of Roche anti-digoxigenin or anti-fluorescein Fab reagents include studies to describe the expression pattern for neuronally expressed connexin 35b [38], to discuss methods for apoptotic cell death research in Drosophila embryo, ovary, and cultured cell lines [39], to test if synaptic GluA2 concentrations regulate protein trafficking or local translation [40], to study the relation between human cortical formation and impaired sonic hedgehog signaling [41], to investigate the mechanism underlying traveling waves of clock gene expression during development [42], to investigate how the interaction between TRF2 and lamin A/C regulates chromosome structure [43], to describe a method to examine the developmental pathways that link specific subtypes of neurons in mice [44], among others [45-55]. Invitrogen Alexa 546-conjugated donkey anti-rabbit IgG Fab fragment was used to perform immunohistochemistry to investigate the role of sFLT-1 in the maintenance of the avascular photoreceptor layer in mouse models [56] and Invitrogen Alexa Fluor 488 (Fab')2 fragment of rabbit anti-goat IgG (H+L) was used in immunohistochemistry to investigate the mechanism of tip link regeneration in auditory hair cells [57]. Fc fragment receptors Fc receptors (FcRs) are molecules expressed primarily on/in innate immune cells, that recognize and bind the Fc domain of antibodies thereby providing them with cell-based systems to elicit an immune response. The diverse functions of FcRs reflect the wide range of protective or modulating roles of antibodies, including mediating the neutralization and clearance of targeted substrates, as well as the programming of adaptive immunity [58]. The biological functions of FcRs are regulated by immunoreceptor tyrosine-based activation motifs (ITAMs) and immunoreceptor tyrosine-based inhibitory motifs (ITIMs), that act as the receptor’s interface with activating and inhibitory signaling pathways, respectively. Thus, signaling by ITAMs can elicit cell activation, phagocytosis and endocytosis, whereas signaling by ITIMs have an inhibitory effect on cell activation [59]. FcRs have been described for all classes of immunoglobulins and some of them are discussed below. IgG receptors This family includes FcγRI, FcγRII, FcγRIII and their isoforms. They are responsible for antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ACDP) [58]. Another receptor that binds IgG is neonatal Fc receptor (FcRn), which is involved in the transfer of passive humoral immunity from a mother to her fetus. FcRn also protects IgG from degradation in vivo, explaining their long half-life in the serum [60]. This phenomenon has led to the development of better therapeutic antibodies by introducing alterations in the Fc region to promote the Fc-FcRn interaction. IgE receptors They include the high affinity FcεRI, which is capable of binding monomeric IgE and the low affinity C-type lectin FcεRII, which interacts preferentially with complex IgE. FcεRI mediates the immediate hypersensitivity response of many allergic reactions by stimulating degranulation and the release of a range of inflammatory mediators on mast cells and basophils [61]. FcεRII exists both in a membrane-bound form that delivers a downregulating signal for IgE synthesis [61], as well as soluble fragments that generate the opposing upregulating effect [62]. Its role in transcytosis of IgE-allergen complexes in human airway and intestinal epitelium is actively being investigated as a potential target for allergic airway inflammation as a result of food allergies [63, 64]. IgA receptors The only member of this group, FcαRI, is expressed only in cells of the myeloid lineage. It plays a role in both pro- and anti-inflammatory responses depending on the state of the IgA bound. While binding of secretory IgA (SIgA) present at mucosal sites has anti-inflammatory effects including prevention of pathogen invasion, binding of serum IgA leads to inflammatory responses. FcαRI also regulates neutrophil viability depending on the inflammatory microenvironment [65]. TRIM21 It can be distinguished from other FcRs as it shows a remarkably broad antibody specificity. It can bind IgG, IgM and IgA [66, 67]. Also, because it is expressed by cells of most histogenic lineages [68]. TRIM2 participate in antibody-mediated interference of viral replication, by targeting cytosolic virus-antibody complexes for proteasomal degradation. Binding of Fc domains to FcRs can have undesired effects in monoclonal antibody-based analytical methods such as immunohistochemistry (IHC), cell cytometry (FACS) and chromatin immunoprecipitation (ChIP). Non-specific binding to FcRs may introduce background noise that can lead to de detection of false positives. Solutions to deal with this problem include the use of (i) isotype controls for gating, (ii) serum to broadly compete for the receptors involved in non‐specific binding, or (iii) purified IgG to specifically block Fc receptors [69]. Innovex Fc Receptor blocker #NB309 can be used to block paraffin or frozen sections during IHC or IC experiments [70] or BD Biosciences #553142 for flow cytometry [70]. Note Dr. Macarena Fritz Kelly from São José dos Campos, SP, Brazil, contributed the section about Fc receptors in September, 2018.
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Humanization of Antibodies using a Statistical Inference Approach

Humanization of Antibodies using a Statistical Inference Approach | Top Selling Monoclonal Antibodies 2014 | Scoop.it

Abstract Antibody humanization is a key step in the preclinical phase of the development of therapeutic antibodies, originally developed and tested in non-human models (most typically, in mouse) [...] Monoclonal antibody humanness 

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New international research collaboration explores blood-based biomarker testing - Cancer Research Institute (CRI)

New international research collaboration explores blood-based biomarker testing - Cancer Research Institute (CRI) | Top Selling Monoclonal Antibodies 2014 | Scoop.it
The study explores a potential non-invasive approach to identify tumor mutations and cancer patients that could potentially respond to immunotherapy.
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Randomized study of the safety and pharmacodynamics of inhaled interleukin-13 monoclonal antibody fragment VR942

This study, considered to be the only example of a dry powder anti-IL-13 fragment
antibody being administered via inhalation, demonstrated that single and repeat doses
were well tolerated over a period of up to 10 days in duration.
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Targeting G protein-coupled receptor signalling by blocking G proteins

Targeting G protein-coupled receptor signalling by blocking G proteins | Top Selling Monoclonal Antibodies 2014 | Scoop.it
G protein-coupled receptors (GPCRs) are the largest class of drug targets, largely owing to their druggability, diversity and physiological efficacy. Many drugs selectively target specific subtypes of GPCRs, but high specificity for individual GPCRs may not be desirable in complex multifactorial disease states in which multiple receptors may be involved. One approach is to target G protein subunits rather than the GPCRs directly. This approach has the potential to achieve broad efficacy by blocking pathways shared by multiple GPCRs. Additionally, because many GPCRs couple to multiple G protein signalling pathways, blocking specific G protein subunits can 'bias' GPCR signals by inhibiting only a subset of these signals. Molecules that target G protein α or βγ-subunits have been developed and show strong efficacy in multiple preclinical disease models and biased inhibition of G protein signalling. In this Review, we discuss the development and characterization of G protein α and βγ-subunit ligands and the preclinical evidence that this exciting new approach has potential for therapeutic efficacy in a number of indications, such as pain, thrombosis, asthma and heart failure.
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