Science Pool

Monoclonal antibodies (mAbs) have been a huge success story in the biopharmaceutical industry. They have changed the landscape of biologicals and offered therapies for previously untreatable diseases. Although mAbs have been on the market since the mid 1980s, they have become increasingly sophisticated over the past decades. This also holds true for the technologies enabling the identification, design, preclinical and manufacturing processes of monoclonal antibodies.

While the traditional hybridoma technology is still broadly used for the creation of mAbs, novel technologies such as immune antibody libraries or fully humanoid antibody libraries have emerged in recent years. Moreover, researchers can today leverage a range of powerful approaches to optimize the mAb creation process, e.g., high-throughput screening, state-of-the-art sequencing, AI / machine learning or analytics. This, in turn, speeds up the development cycle, allows for precise selection of the desired mAb properties and ultimately reduces failures of monoclonal antibodies at advanced development stages.

But how to choose from the vast range of technologies for antibody development? This challenge was discussed at Evotec´s recent Innovation Week in a session titled "Translate your idea into a product: AI -driven antibody discovery at Evotec".

The session covered Evotec´s capabilities for supporting all activities across the R&D continuum, i.e.

• Discovery and optimization of novel antibodies for specific disease targets,
• Evaluation of lead antibodies for disease efficacy and safety and
• Process development and manufacturing.

"In many cases, it is not the question of using either one or another technology," says Barbara Bachler-Konetzki, Group Leader In Vitro Pharmacology at Evotec. "Depending on the desired properties of the monoclonal antibody, combining several technologies will do the trick. Therefore, it is important to have access to a comprehensive repertoire of leading-edge, synergistic technologies."

For state-of-the-art monoclonal antibody development, Evotec has established a unique one-stop-shop from target identification to IND. This includes a broad technology platform as well as unparalleled expertise in drug development and even manufacturing, including latest advances in artificial intelligence and machine learning such as generative adversarial networks (GAN) to create synthetic realistic outcomes by machine learning (J.HAL).

The resulting integrated biologics platform is called J.DESIGN and integrates molecular, process and manufacturing design.

The example of Evotec´s internal SARS-CoV-2 campaign shows how its proprietary J.HAL technology can be used to identify antibodies effectively blocking the SARS-CoV-2 infection pathway by binding to SARS spike protein, effectively neutralizing the infectivity across several SARS-CoV-2 strains. The subsequent in silico sequence analysis informs about mAb properties and engineering opportunities to reach the desired properties re immunogenicity, stability etc. Further steps, e.g., sequence and stability optimization, improve manufacturability and yields or pharmacokinetics (PK).

For mAb development, Evotec pursues a translational approach, i.e., leveraging its extensive expertise in various therapeutic areas to facilitate the setup of disease-relevant biological assays. Among others, this allows for PK/PD characterization of biologics early in the R&D process as well as the prediction of downstream in vivo efficacy and demonstration of target engagement. Moreover, Evotec’s pre-clinical department offers the full range of in vitro and in vivo GLP and non-GLP pre-clinical evaluation studies to assess the safety profile of the drug candidate. In addition, Evotec has established several sophisticated manufacturing facilities worldwide to reduce the risk of downstream attrition and delay.

This unparalleled infrastructure and translational expertise put Evotec in a unique position to conduct leading-edge, integrated monoclonal antibody development programs all the way from target identification to manufacturing.

FULL REPLAY

Over recent years, interest in the multi-attribute method (MAM) has grown and the technique has become an important mass spectrometry-based tool for identifying and quantifying the site-specific product attributes and purity information for biotherapeutics such as monoclonal antibodies (mAbs) and fusion molecules. Improving the throughput of sample preparation without introducing chemical modifications and variability will further increase the utility of MAM in drug development.

In this publication, we focus on:

• the development of a fully automated MAM sample preparation protocol incorporating rapid desalting (< 15 min) using miniaturized size-exclusion chromatography columns in pipette tips on an automated liquid handling system which leads to complete rapid digestion of mAbs in approximately 3 hours with excellent reproducibility
• analysis of samples using electrospray ionization mass spectrometry coupled to a U-HPLC system with dual column switching 
• comparison of the new sample preparation method versus manual low artefact sample preparation including analysis of reproducibility, recovery, efficiency and flexibility

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Evotec strives to provide partners and customers with less expensive, faster, and more flexible approaches for discovering, developing and manufacturing biotherapeutics.

With the acquisition of Just Biotherapeutics in 2019, Evotec made a major push into biologics, and has successfully built a fully-integrated platform to drive monoclonal antibody (“mAb”) programmes from concept through to commercialisation. The company generates antibody lead candidates by providing access to in vivo and in vitro sources of antibodies, combined with state-of-the-art technologies to ensure success for a broad range of targets and disease states. In addition, if needed, selected lead candidates can be further optimised using powerful computational platforms such as Evotec’s proprietary Abacus™ in silico tool suite to enhance the productivity, but also ease of manufacturing and formulation stability.

For in vivo immunisation, Evotec is offering the ATX-Gx^TM mouse platform developed by its collaboration partner Alloy Therapeutics. This platform is a suite of immunocompetent transgenic mice for best-in-class in vivo discovery of fully human monoclonal antibodies. These mice are engineered to drive the greatest potential diversity and broadest epitopic coverage of unique human antibodies binding to a specific target of interest.

ATX-Gx^TM mice provide:

• a fully human heavy chain repertoire,
• human kappa and human lambda chain repertoire,
• haplotype diversity, and
• limited immunodominance.

Taken together, these immunocompetent transgenic mice represent the complete functional human antibody repertoire, and their fully functional, robust immune response is equivalent to the response of wild type mice. They are also available on multiple genetic backgrounds such as BALB/c or BL/6.

ATX-Gx^TM has been extensively validated and continuously expanded for more than 12 years and is currently used by over 75 antibody discovery groups in large biopharma and small-to-midsize biotech companies as well as academic research labs.

Following immunisation, Evotec is combining traditional hybridoma technology with automated devices for high throughput clone selection, screening, recombinant expression and purification to shorten and simplify the process of hybridoma establishment. Screening is performed with high-throughput technologies, such as the iQue® Advanced Flow Cytometry Platform. Next-generation sequencing is used to obtain VH/VL information, followed by an in silico analysis using Just – Evotec Biologics Abacus® software tool. Once VH/VL information is available, several hundred mAbs can be produced in parallel in small scale. This high quality material can then be used for further downstream characterisation, such as functional activities that allow the selection of potential lead candidates.

This streamlined workflow to generate high-quality antibody panels for functional assessment adds to Evotec’s comprehensive suite of large molecule discovery tools, disease biology, state-of-the-art cell line development, machine learning design tools, manufacturing, preclinical IND-enabling studies, as well as FIH clinical support, and commercialisation.

Evotec’s AI-driven antibody discovery can be accessed as stand-alone services or through Evotec’s seamlessly integrated antibody drug discovery platform

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The 11^th edition of our Drug Discovery Update (DDup) is dedicated to biotherapeutics discovery and development with a focus on artificial intelligence and machine learning and its opportunity in transforming antibody discovery.

In this edition, it covers:

• an introduction to antibodies including:
  • an overview of monoclonal antibodies as therapeutic agents
  • a history of antibody drug discovery platforms
• how Just - Evotec Biologics are exploiting artificial intelligence and machine learning to generate a novel humanoid antibody library biased towards highly desirable features
• a description of the process from idea to IND and beyond for antibody discovery and development
• facts and figures on the market for therapeutic antibodies
• an overview of function-focused antibody (FFmab) screening
• an interviews with the experts

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Stable expression of recombinant protein therapeutics in CHO cells often relies on random or semi-random genomic integration events which results in a widely heterogenous cell population. This leads to significant effort in clone screening during cell line development in order to identify clones with high expression, growth and product quality. 

In this publication, we focus on:

• the development of two targeted integration systems that express high levels of recombinant protein in CHO cells by: 
  • the installation of rationally designed piggyBac-based chromosomal landing pads
  • the use of site specific recombinases including PhiC31 and CRE 
• demonstration of the functional integration of several donor vectors encoding various therapeutic proteins including two monoclonal antibodies and one Fc-fusion molecule

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Loss of heart myocardium is considered to be an irreversible process which can eventually lead to heart failure. Adult cardiomyocytes divide at a rate of less than 1% per year and no cardiac stem or progenitor cell type contribute significantly to the replacement of lost myocytes. One approach being pursued to replace lost heart muscle and regenerate the heart is the use of stem cell-derived cardiomyocytes.

In this publication, we focus on an in-depth review of the use of human pluripotent stem-cell derived cardiomyocytes in heart regeneration including:

• a background to cardiac regeneration and the approaches used to address this
• preclinical research and achievements in the use of cell therapy and stem cell-derived cardiomyocytes for the replacement of lost heart muscle 
• an overview of existing open questions such as how the technology works, the duration of effect, patient selection, immunological issues and how to reduce risk
• a summary of the clinical trials currently ongoing in this field

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Dendritic Cells (DCs) are very efficient antigen-presenting cells and have long been considered as attractive candidates for cancer immunotherapy. They are obtained from the patient and loaded in vitro with tumour antigens and additional maturation stimuli and subsequently, infused back into the patient. However, after more than 200 clinical trials involving thousands of patients, clinical responses have been disappointing so far.

While the treatment is safe and well-tolerated and often elicits anti-tumour immunity in both patients with advanced stages of disease and those with minimal residual disease following tumour resection, only a minority of patients demonstrates objective response rates. There are several reasons why the results are disappointing, and while scientists have been able to address problems such as active immune suppression and evasion mechanisms of the tumour, some DC therapy-related aspects contributing to the limited clinical efficacy of DC therapy remain to be solved: the choice of the antigen, the method of loading, and, above all, the type of DCs used. Access to the full spectrum of DCs is limited and many subsets known to be very effective simply are not accessible as they can be derived from the patients only in very low quantities.

As a result, sentiment has switched to approaches viewed as more promising, such as checkpoint inhibitors or chimeric antigen receptor (CAR)-T cells.

Exciting advances with iPSCs

However, thanks to the recent advances made with induced pluripotent stem cells (iPSCs) interest in these vaccines has been renewed. iPSCs can be induced to produce dendritic cells and this provides an opportunity for the rational design of DC vaccines displaying additional functionality via genetic engineering technologies. As iPSCs also open up the possibility for the mass production of large numbers of high-quality iPSC-derived DCs, it is now possible to design next generation DC vaccines from engineered DCs.

Moreover, iPSCs also allow for the production of DC subsets that are not accessible as yet for therapeutic development because sufficient quantities could not be obtained. Examples are DCs facilitating anti-viral responses and a certain subset called CD141+ specialised on cross-presentation of antigens. The CD141+ subset, which is found in very low abundance in vivo, is of particular interest for cancer therapy as it induces optimal cytotoxic T lymphocyte (CTL) responses. Thanks to iPSC, these subsets now can be produced under cGMP conditions in bulk quantities.

Off-the-shelf cancer vaccines?

All in all, these advances may provide the opportunity to design off-the-shelf DC products suitable for cancer vaccines.

Evotec therefore has invested in British immune oncology company OXvax Ltd., a spin-out from the University of Oxford focused on the development of an advanced next-generation dendritic cell vaccine platform for the treatment of solid cancers. The company is pioneering the use of iPSCs as a novel source of CD141+ dendritic cells (DC) and is based on intellectual property from the Fairchild laboratory at the Sir William Dunn School of Pathology. OXvax’s technology addresses, among others, the low cross-presentation and the T-cell activation problems of past DC-based cancer treatments. The platform enables the manufacture at scale of an off-the-shelf, highly potent vaccine which addresses the major limitations that have frustrated cancer vaccine development in the past. If the approach is successful in oncology, it can also be expanded to other therapeutic areas, e.g. viral infections.

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Learn more about Human iPSC-Derived Mixed CNS Cells and their advantages including:

• Highly physiological neural networks
• Long term synchronous network activity
• Suitable as seizure liability mode

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