Science Pool

Full poster title: Development of recombinase-based targeted integration systems for production of exogenous proteins using transposon-mediated landing pads

Summary of the poster:

• We demonstrated proof of principle of targeted integration systems in our CHO host cell line with consistent genome integration into expected landing pad sites
• Test cases using three antibody or antibody-fusion therapeutic molecules showed similar levels of productivity
• We also show preliminary data from ongoing work to build upon these targeted integration systems, which includes isolating a single-copy landing pad cell line and developing a CHO display platform

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About the Webinar

• At Evotec we discover, develop and manufacture novel medicines for hard-to-treat diseases through efficient partnerships with academia, biotechs and pharma
• For antibody-based modalities, Evotec has developed an integrated discovery platform ranging from target validation, antibody discovery and optimization, in vitro/in vivo pharmacology, PK, and toxicology to select the best clinical candidates
• We apply both humanized, mouse-derived hybridoma and phage display (from immune and synthetic antibody libraries) coupled to automated HTS antibody screening and AI/ML-based optimization, antibody hits are selected to best fit their intended modality, (e.g. mAbs, ADCs, bispecifics)
• A particular emphasis is placed on integrating good manufacturing properties for the selected antibody sequence

About the Speaker

Thierry Wurch SVP, Integrated Biologics Discovery | Evotec Thierry joined Evotec in May 2022 as SVP Integrated Biologics Discovery. He has more than 22 years of expertise in the antibody R&D space primarily in Oncology and immune-oncology. Thierry held positions of growing strategic importance from head of an antibody discovery and engineering department (Pierre Fabre, 2003-2011), head of immunotherapy discovery research activities (Servier, 2011-2017) then moving to BD-oriented activities and heading external innovation for Oncology (Servier, 2017-2020 and Ipsen 2020-2022). Thierry is also Chairman of the antibody sub-committee at NC-IUPHAR, member of the Editorial board of mAbs Journal and Distinguished Advisor of The Antibody Society.

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Antibodies generated in the lab are important as potential treatments for a broad spectrum of diseases, in particular infectious diseases caused by viruses. They can be obtained either by animal-derived B cells or from antibody library display platforms. Evotec’s strategy for the optimal path to obtain lead candidates is offering access to both sources of antibodies for discovery, coupled with the exploitation of state-of- the-art technologies to ensure success for a broad range of targets and disease states. In addition, selected lead candidates can be further optimized using powerful computational platforms to enhance productivity, manufacturability, and formulation stability. This is the end-to-end J.Design biologics platform, which is fueled by the front-end discovery platform, J.HAL™ (Just Humanoid Antibody Library) and associated data-driven, company-wide machine learning methodology.

By using artificial intelligence (AI) and machine learning (ML), J.HAL can generate novel, humanoid antibody sequences that both represent natural repertoires and are biased towards desirable features. To enable properties such as broad target and epitope engagement, focused efficacy, and suitable developability, Just-Evotec Biologics has devised an Antibody-GAN (Generative Adversarial Network), a new synthetic approach to designing a novel class of antibody therapeutics, which is termed humanoid antibodies.

At the conferences International Conference on Antiviral Research (ICAR) 2021 and Antibody Engineering & Therapeutics Europe 2022, researchers from Evotec and Just-Evotec Biologics introduced results obtained by using GAN to generate novel sequences, which mimic natural human response and provide the necessary diversity and developability features.

Competing Neural Networks

GAN is based on competing, deep layer neural networks that learn and produce the features of the mature human antibody repertoire, including sequence characteristics and structure properties, allowing for the encoding of key properties of interest into diverse libraries for a feature-biased discovery platform. It works to:

• capture the complexity of the entire variable region of the standard human antibody sequence space,
• provide a basis for generating novel antibodies that span a larger sequence diversity than standard in silico generative approaches, and
• incorporate transfer learning, a critical feature for antibody discovery to bias the physical properties of the generated antibodies towards broader efficacy traits such as CDR lengths and surface properties, improved developability (e.g., improved thermal and pH stability), and diverse chemical and biophysical properties.

The GAN network is trained by using hundreds of thousands of human antibody sequences to recognize legitimate human v-genes. The generator network generates random sequences to fool the discriminator while continually receiving feedback from the discriminator on sequence validity. Over time, the two networks get progressively better at their tasks. After full training, the Antibody-GAN generator is eventually able to produce fully human, novel antibody sequences for the germline for which the GAN was trained.

Antibodies targeting SARS-CoV-2

To demonstrate the usefulness of this platform, the researchers used their newly constructed, 1 billion theoretical diversity phage Fab library with the intent to discover antibodies to the SARS-CoV-2 spike protein. Candidates that specifically bound SARS-CoV-2 spike protein and did not bind an irrelevant antigen were further characterized for dose-dependent binding using AlphaLISA technology. In the primary “yes/no” binding screen a total of 73 unique antibody sequences specific for SARS-CoV-2 spike protein were identified. The researchers then performed binding assays using unpurified transfection supernatants and later reproduced the results with purified material. The candidate antibody supernatants that specifically bound SARS-CoV-2 spike protein were subsequently tested for their ability to block binding of this protein to human ACE-2 receptor. The team identified multiple antibodies that effectively blocked spike human ACE2 receptor interaction, demonstrating the feasibility to screen unpurified transfection supernatants for functional activity. After further rounds of panning, the top candidates expressed at flask scale were purified and tested for SARS-CoV-2 neutralization ability across multiple strains. The researchers identified multiple candidates with neutralizing activity against several strains of SARS-CoV-2. Nine of these antibodies exhibited blocking activity of the spike protein to the ACE2 receptor in an in vitro functional assay. Of note, all antibody data shown here were from native library candidates without any affinity maturation.

The presentation demonstrates that applying machine learning algorithms in antibody discovery “promotes efficient learning from the least expensive and most abundant data encoded in the DNA of antibodies, to validation of this learning through less abundant, more expensive, but most relevant data from GMP manufacturing at full commercial scale,” stated James N. Thomas, retired Executive Vice President, Global Head of Biotherapeutics and President U.S. Operations at Just - Evotec Biologics. “This is a systems approach to platform definition and continuous improvement, and it is unique in the industry, made possible by a number of factors that will be difficult for others to replicate."

To learn more about Evotec's capabilities read our related poster.

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Key Takeaways:

• We have developed an AI-generated antibody library platform, which we call J.HAL^®,  utilizing a Generative Adversarial Network (GAN) that generates novel sequences which mimic natural human response, as well biasing toward diversity and developability features.
• The resulting Humanoid Antibody Library was successfully screened to obtain a panel of novel, diverse and pharmacologically active human antibodies against SARS-CoV-2.

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Key Takeaways:

• Non-infectious, purified RVLPs can be used in place of model viruses to predict process performance for viral clearance.
• Initial screenings show that clearance also trends similarly between bench screenings and higher throughput plate screenings.
• Plate-based screening of RVLPs in-process can examine up to 24 different run conditions simultaneously and uses less viral surrogate compared to bench scale runs, allowing for greater evaluation and confidence going into formal viral clearance studies.

Improving virus clearing studies in recombinant protein production

Chinese hamster ovary (CHO) cells are the most frequently used mammalian host cells for the industrial manufacturing of recombinant protein therapeutics. They can produce recombinant proteins on the scale of up to 10 gram per liter of culture. However, they are also known to contain type‐C endogenous retrovirus (ERV) sequences in their genome and to release retroviral‐like particles. Although evidence for their infectivity is missing, this has raised safety concerns, and regulatory agencies require demonstration that the purification process removes or inactivates viruses.

Viral clearance validation is assessed through “spiking studies”, whereby model mammalian viruses are introduced into process material which then undergoes the purification technique to be tested. Viral quantity before and after processing is determined through infectivity or qPCR assay. As these studies use live viruses, they require specialized Biological Safety Level laboratories (BSL) and experienced personnel and can create a substantial bottleneck because typically only 3rd party facilities are qualified to perform these studies.

As an alternative, Just - Evotec Biologics is in the early stages of establishing a high-throughput process using commercially available purified retrovirus-like particles from Cygnus Technologies LLC. These particles are non-infectious and mimic the physicochemical properties of live infectious viruses. By using these particles as spiking agents, the retroviral clearance capability of downstream unit operations can be studied, assessed, and quantified by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Usually, this is performed at bench scale using chromatography columns.

In a poster presented at this year’s ACS spring conference entitled High throughput optimization of chromatography steps for viral clearance using retrovirus-like particles (RVLPs), researchers from Just-Evotec Biologics detailed the high-throughput workflow for the analysis of RVLP content for rapid analysis of in-process samples.

The research team compared common bench scale chromatography runs with a plate-based screen using resin-loaded filter plates and a liquid handling robot. While at bench scale, only a single set of run conditions can be tested at a time, the plate-based screening can examine up to 24 different run conditions simultaneously. It also uses less RVLP stock solution. The researchers expect that plate-based screening of RVLPs will not only save time and costs, but also allows for better evaluation and confidence before formal viral clearance studies.

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iPSC and age-related diseases: Case study AMD
Age-related macular degeneration (AMD) is a leading cause of vision loss in people over the age of 50, accounting for 90% of blindness in this population. There are dry and wet forms of the disease and to date, there are limited treatment options for the wet form and none for dry AMD. The exact cause of the disease is unknown, but it is suspected that it results from a combination of hereditary and environmental factors, with smoking and diet being implicated. Globally, more than 190 million people are affected by AMD and this figure is expected to increase to more than 285 million by the year 2040. The estimated global cost of the disease currently stands at $343bn, with $255bn in direct healthcare costs.

Significant research effort has been targeted towards identification of the genes involved in permanent vision loss through photoreceptor and/or retinal pigment epithelium (RPE) cell dysfunction or cell death. So far, mutations in over 250 genes are known to be involved. Despite a good understanding of the genotype-phenotype relationship in AMD, this has not translated into predictive in vitro and in vivo models to understand disease mechanisms.

Partnership on iPSC-derived RPE cells
To drive the search for treatments for this devastating disease, it is vital to identify and develop new models which enable elucidation of retinal disease mechanisms and reliably predict the efficacy of therapeutic compounds. To address these challenges, Evotec has teamed up with the Centre for Regenerative Therapies (CRTD) in Dresden, Germany. The CRTD has a longstanding interest in degenerative processes and has contributed significantly to our understanding of retinal diseases in recent years. The joint TargetRD project is based on Evotec’s know-how in induced pluripotent stem cell (iPSC) technology, generating iPSC-derived RPE cells from AMD patients.

RPE cells are essential for visual function and a key component for light detection by photoreceptor neurons. They also are crucial for maintaining the blood-retina-barrier, for the transport of diverse biomolecules, ions, and fluids in and out of the retinal tissue, and recycling of the visual chromophore retinal molecule.

Even more important is their ability for phagocytosis: A single RPE cell is in contact with about 30 photoreceptor cells and responsible for the phagocytosis and removal of the distal portions of the photoreceptor outer segments that are phagocytozed in the course of a day. This is an important process with up to 10% eliminated daily, meaning the entire population of photoreceptor outer segments is turned over every 2 weeks. Maintaining, repairing, or replacing RPE cells therefore is crucial for the management of AMD, but also for other retinal degenerative diseases.

So far, research has been hampered by limited access to RPE cells. There are immortalized retinal cell lines available, but they lack the typical cell morphology or function of their in vivo counterparts, e.g. pigmentation, polarization, and expression of certain proteins. Likewise, artificial organoids, so-called 3D-cups, are not ideal since they are difficult to grow and differentiate and not suited for high-throughput profiling of compounds.

This situation is now improving as Evotec and CRTD succeeded in developing a protocol to efficiently and robustly produce high quality human iPSC-derived RPE cells at industrial scale from patient cells. This means that partners can not only investigate disease pathology directly in this highly relevant retinal cell type but for the first time also study disease phenotypes and mechanisms within the context of a patient’s genome. Moreover, the TargetRD platform enables the study of individual, overlapping functional and morphological changes from iPSC-RPE cells derived from patients with different genetic backgrounds, providing an opportunity to unravel complex AMD disease phenotypes.

Promising first results
Already, the partners were able to show the utility of the TargetRD platform by analyzing iPSC-RPE cells carrying a patient mutation resulting a lysosomal storage disorder. Amongst other symptoms, patients with this type of mutation develop severe retinal degeneration, leading to complete blindness early in life. Using the various phenotypic and functional assays established, it was possible to demonstrate impaired trans-epithelial resistance in patient cells. This indicates that in this case, patient RPE cells are unable to form the tight monolayer required for normal RPE cell function. Furthermore, patient RPE cells have, as expected, impaired lysosomal activity and are unable to phagocytose photo- receptor outer segments (POS) at the same rate as control RPE cells. Since all assays are able to support high-throughput screening, the partners can use the TargetRD platform to identify phenotypic and functional disease phenotypes from patient cells, enabling novel drug discovery approaches.

Furthermore, the project combines Evotec’s drug discovery expertise and the academic excellence of the CRTD to achieve significant progress towards developing therapies much needed by the patients.

Both partners believe this is a very promising approach that enables successful drug discovery programs for retinal degenerations coupled to a high likelihood of successful translation into the clinic.

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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