Ablynx Develops Nano Antibody for Treatment of Rare Clotting Disorder

Last week, Ablynx announced substantial progress in the development of the nano antibody drug caplicizumab to treat acquired thrombotic thrombocytopenic purpura (aTTP), a rare, but life-threatening autoimmune disease. The Belgian biopharmaceutical company has submitted a Marketing Authorization Application (MAA) to the European Medicines Agency (EMA) for approval. If accepted, caplicizumab will not only be the first therapeutic specifically indicated for the treatment of aTTP, but also the first approved nano antibody drug on the market.

aTTP is characterized by the autoimmune impairment of ADAMTS13, an enzyme that normally cleaves multimeric von Willebrand factor (vWF) into its functional form. Without the function of ADAMTS13, multimeric vWF forms aggregates with platelets in the blood. Low free platelet count and excess clotting result in thrombotic complications and a significant risk of organ damage due to the blockages of blood flow to tissues.

The current standard of care for aTTP involves immunosuppression and daily plasma exchange transfusion, in which a patient’s plasma is replaced with donor plasma to remove platelet-vWF aggregates. Caplicizumab is an anti-vWF nano antibody that prevents the formation of aggregates by blocking the interaction of multimeric vWF complexes with platelets.

While dozens of monoclonal antibodies have been approved by the FDA for therapeutic use (with hundreds more undergoing clinical trials), caplicizumab is the first therapeutic nano antibody. Nano antibodies are single-domain antibody fragments that bear full antigen binding capacity like monoclonal antibodies, but have a smaller size and unique structure, giving them features of small-molecule drugs. Nano antibodies are more stable than conventional monoclonal antibodies, allowing for multiple administration routes, and can be humanized to lower toxicity and immunogenicity. Because they are encoded by single genes, nano antibodies are easier and more cost-effective than traditional antibodies to engineer and manufacture.

Currently, caplicizumab is undergoing Phase III clinical trials and a three-year follow-up study has been initiated to determine the long-term safety and efficacy of this drug. Ablynx aims to commercialize caplicizumab in North America and Europe upon the trial’s conclusion and approval of BLA filing in 2018.

With the obvious advantages of nano antibodies over conventional monoclonal antibodies as biological drugs, caplicizumab is likely only the first of many to come.

Allele Researchers Engineer Modified Nanoantibodies to Increase Sensitivity in Biochemical Assays

Researchers at Allele have published new work demonstrating a novel application for nanoantibodies (nAbs) in direct signal amplification. nAbs have distinguishable qualities that set them apart from their traditional IgG counterparts, including significantly smaller size, better stability, and excellent specificity. However, because of their small size, there are no suitable secondary antibodies for traditional assays like immunohistochemistry, immunofluorescence, and other biochemical assays that require an enhanced signal.

The researchers engineered a modified nAb, termed “nAb Plus,” to directly amplify nAb signal detection through the addition of a small scaffolding protein containing numerous reporter binding sites. nAb Plus bypasses the need for secondary antibodies or additional amplification steps, streamlining biochemical assays and decreasing costs of reagents. The authors demonstrate the use of nAb Plus using immunohistochemistry, an assay typically requiring one or more signal amplification steps. However, nAb Plus could also be incorporated in any biochemical assay needing signal enhancement.

Abstract: Revealing the spatial arrangement of molecules within a tissue through immunohistochemistry (IHC) is an invaluable tool in biomedical research and clinical diagnostics. Choosing both the appropriate antibody and amplification system is paramount to the pathologic interpretation of the tissue at hand. The use of single domain VHH nanoantibodies (nAbs) promise more robust and consistent results in IHC, but are rarely used as an alternative to conventional immunoglobulin G (IgG) antibodies. nAbs are originally obtained from llamas and are the smallest antigen-binding fragments available. To determine whether the unique biophysical properties of nAbs give them an advantage in IHC, we first compared a basic fibroblast growth factor nAb to polyclonal IgG antibodies using tissue isolated from pancreatic adenocarcinoma. The nAb was extremely effective in antigen signal detection and allowed for a more streamlined and reproducible protocol. Furthermore, because nAbs are expressed in Escherichia coli from a single gene, they are quite amenable to genetic engineering. As such, we then covalently bound a highly biotinylated amplifier protein to basic fibroblast growth factor and p16 nAbs (termed nAb Plus), resulting in improved IHC sensitivity. The use of a biotinylated nAb Plus not only achieved local, covalent signal amplification, but also eliminated the need for a secondary antibody and subsequent amplification steps. These results highlight nAbs as valuable alternatives to conventional IgG antibodies, decreasing overall processing time and costs of reagents while increasing sensitivity and reproducibility across individual IHC assays.

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Allele Publication Explains cGMP Generation of Induced Pluripotent Stem Cells

The discovery that adult somatic cells can be reprogrammed to pluripotent stem cells has given the biomedical community a powerful platform for personalized medicine. However, the translation of cell therapies from bench to bedside holds a significant challenge. Realizing the clinical potential for stem cells requires their production under current Good Manufacturing Practice (cGMP) regulations enforced by the FDA. A new protocol (http://onlinelibrary.wiley.com/doi/10.1002/cpsc.18/abstract) published by scientists at Allele and detailed in this quarter’s issue of Current Protocols in Stem Cell Biology, reveals key conditions required for converting adult fibroblasts to induced pluripotent stem cells (iPSCs) under cGMP regulations.1

The patent-pending protocol is an update to a previous protocol that describes how to reprogram fibroblasts to iPSCs using mRNA. “The system of using mRNA to reprogram fibroblasts presents itself as a very favorable candidate for generating iPSCs for cell therapy” according to the senior author of the paper and CEO of Allele, Dr. Jiwu Wang, “our company is committed to developing stem cell based therapies using this protocol and through the establishment of our own stem cell GMP facilities here in California”. mRNA transfection is “footprint free”, meaning no insertions or alterations have been made to the genome. Transfection of mRNA is also “cleanup free,” because mRNA transcripts are supplied to the cells in the culture medium only for the time required to induce pluripotency. Furthermore, genomic analyses of iPSCs reprogrammed using mRNA indicate that this method of conversion is unlikely to introduce problematic mutations.2

The new version of the protocol describes reprogramming technology that utilizes all cGMP-certified reagents and vessels, meaning that every material is manufactured under guidelines that allow for ancillary use in manufacturing processes related to cell therapy. All materials described in the protocol – from cell medium and components to the coating for tissue culture plates – were meticulously evaluated at every step of generating and storing iPSCs. For truly cGMP produced cell lines, all processes should take place in certified cleanrooms with qualified equipment and thoroughly trained operators.

Establishing a cGMP process for any product intended for human use is a daunting undertaking. Unlike drugs and small-molecule pharmaceuticals, stem cells are living entities whose production cannot be chemically synthesized. Therefore, special considerations must be made – particularly for making individual cell lines – to help assure the highest safety and quality of downstream stem cell products. Adhering to cGMP regulations infuses high quality into the design and manufacturing process at every step. Through rigorous testing, researchers at Allele have identified critical parameters for generating iPSCs from fibroblasts that are cGMP-compliant, and are optimistic that the methods described in this recent publication will serve as a launch pad for the development of future cell products and therapies.

 

  1. Ni Y, Zhao Y, Warren L, Higginbotham J, Wang J. cGMP Generation of Human Induced Pluripotent Stem Cells with Messenger RNA. Current Protocols in Stem Cell Biology,2016; 39:4A.6.1-4A.6.25.
  2. Bhutani K, Nazor KL, Williams R, et al. Whole-genome mutational burden analysis of three pluripotency induction methods. Nature communications. 2016;7:10536.

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Thursday, November 10th, 2016 cGMP, iPSCs and other stem cells No Comments

The NIH Awards Allele with Grant for the Development of a New Antibody Therapy for Treating Alzheimer’s Disease

SAN DIEGO–(BUSINESS WIRE)–

The National Institute on Aging of the NIH has awarded a grant to Allele Biotechnology and Pharmaceuticals (“Allele”) to develop a new antibody therapy for treating Alzheimer’s disease. Alzheimer’s disease is the most common cause of dementia, but there are currently no treatments to stop or reverse its progression.

Alongside academic collaborators, scientists at Allele have revealed a strong correlation between a previously uncharacterized target gene and Alzheimer’s disease. They discovered that expression of the gene reduces beta-amyloid production and tau phosphorylation, two components of plaque formation in Alzheimer’s disease. Furthermore, high levels of this protein in the brain can counteract loss of synapses and cognitive impairments in mice.

Allele will generate a panel of antibodies that recognize this protein with the goal of employing one of these antibodies as a therapeutic drug candidate. The antibodies’ unique size and shape allow them to pass the blood-brain barrier to reach crucial regions of the brain, and each antibody can be easily modified and engineered to heighten its therapeutic potential. Researchers at Allele hope that an antibody treatment will improve the function of its target protein in the brains of Alzheimer’s patients and ultimately reduce pathogenesis of the disease.

Recombinant antibodies represent one of the most important classes of biological therapeutics: 80% of the best selling drugs on the market are antibodies; immune checkpoint therapies and CAR-T cell therapies rely on antibodies. Continuously seeking unique antibodies against high value targets is a key focus of Allele, along with its induced pluripotent stem cell (iPSC) programs and iPSC-based drug screening projects. With the support of the new NIH grant, Allele will not only move closer to finding antibody drug candidates in fighting one of the most devastating diseases, but also generate long-needed research tools for other scientists to further study Alzheimer’s disease. For example, fusion of these antibodies to fluorescent proteins such as mNeonGreen can be used to image Alzheimer’s disease-related factors in cultured neurons, astrocytes, oligodendrocytes, or “minibrain”-like organoids derived from human iPSCs.

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Allele Receives NIH Award to Fund the Development of Large-Scale Stem Cell Production

SAN DIEGO–(BUSINESS WIRE)–

The NIH’s National Heart, Lung, and Blood Institute has awarded Allele Biotechnology and Pharmaceuticals (“Allele”) a Phase 1 SBIR grant to develop a novel manufacturing system to produce stem cell-derived human tissue and cells for clinical therapy. By increasing the scale of production and reducing the cost of manufacturing, Allele is confident that this system will overcome a considerable roadblock for clinical applications of stem cells, which is to produce a sufficient amount of therapeutic material at a manageable cost.

At the core of translating this potentially game-changing technology into medically-beneficial applications is the use of induced pluripotent stem cells (iPSCs), which hold unprecedented promise of providing any type of immune-matched cells of unlimited quantity. Allele has already developed a patented method of reprogramming somatic cells into iPSCs, secured industrial licensees using this technology, and initiated cGMP procedures for clinical applications.

Further moving iPSCs into commercially viable clinical cell therapies still requires overcoming one major barrier: the prohibitive cost of manufacturing iPSC-derived cells, mostly due to the need of expensive clinical-grade growth factors and cytokines. For example, the estimated cost of the growth factors and cytokines needed to produce a typical transfusion of platelets is $87,252.

Ultimately, Allele’s goal is to create clinical-grade iPSCs and control their differentiation into specific cell types at a scale large enough to satisfy the clinical demand. “We have been diligently working on removing the use of protein factors through our own proprietary protocols to generate many clinically-relevant cell types, including beta cells, mesenchymal stem cells, neural progenitor cells, oligodendrocytes, liver, and heart cells,” said Dr. Jiwu Wang, Allele’s CEO and the Principle Investigator of the new NIH grant. “By developing a recombinant protein-independent, real-time adjusted culture system under this project, we are confident that—as many groundbreaking technologies such as genome sequencing have done—the manufacturing process will mature and the costs will come down to eventually benefit everybody.”

Allele’s plan gained trust from the NIH scientific review panel, which gave it a near-perfect score. With this funding, Allele’s researchers will move even faster towards the goal of bringing iPSC products to clinical applications. Successful efforts will also likely provide a vehicle for genome-editing technologies such as CRISPR to be delivered into patients.

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