TILT Biotherapeutics (TILT) is a Finland-based private biotechnology spin-out from the University of Finland focusing on the development of cancer immunotherapies utilizing oncolytic viruses encoded with a variety of cytokines to treat various cancers. The company was founded in 2013 by Akseli Hemminki, a cancer clinician, and aims to use an oncolytic virus to enhance T-cell therapy, including tumour-infiltrating lymphocytes (TILs), chimeric antigen receptor T cells (CAR-Ts), and checkpoint inhibiting antibody therapy of solid tumours (1).

The therapeutic benefit of various oncolytic viruses has been widely published in the literature, and researchers have improved the efficacy of first-generation products using genetic engineering (2). TILT coordinated a European Union (EU)-funded project, UNLEASHAD, which has developed an adenovirus-based oncolytic product, TILT-123 (igrelimogene litadenorepvec), that specifically targets and lyses cancer cells. It also releases TNF-α and interleukin-2 (IL-2) cytokines into the microenvironment, providing an alert and attracting immune cells to the tumour. This strategy ensures anti-tumour immunity and may be suitable for tumours that are refractory to other types of immunotherapies (3).

TILT-123 is currently in Phase I clinical trials as monotherapy and in combination with TIL therapy in Europe in metastatic melanoma. Preclinical results have been published, and the virus backbone has been demonstrated to be safe and well-tolerated in humans. Trials for other solid tumour indications are planned in combination with TILT-123 and a checkpoint modulator or a CAR-T product (2), and the company has other patent-protected products in the pipeline, including oncolytic viruses coding for bispecific antibodies TILT-321 and TILT-322 (3).

In May 2023, the company announced that the first patient had been dosed in a Phase I solid tumour trial, which will assess TILT-123 in combination with avelumab in squamous cell carcinoma of the head and neck (SCCHN) and melanoma in patients with advanced solid tumours refractory to, or progressing after, anti-programmed cell death ligand 1 (anti-PD-L1) (4). The study is part of TILT’s agreement with Merck KGaA, Darmstadt, Germany, and Pfizer, and the European trial is underway at the Docrates Cancer Center (Helsinki, Finland). The company expects to open its US site in the third quarter of 2023 (5). In November 2022, TILT opened an office in Boston to support its US clinical trial programme.

According to CEO Aseli Hemminki, “The oncolytic virus is designed with T cells in mind, and this design is based on patient observations, not just laboratory data. TILT-123 is armed with the most potent cytokines in the context of adaptive anti-tumour responses; it is genetically modified for improved access to cancer cells and for improved safety. Moreover, adenovirus is the most immunogenic virus type and is possible to upscale in manufacturing for industrial production. Importantly, TILT’s virus backbone has already been demonstrated safe in humans and can access distant tumours by vascular route. Therefore, in addition to intratumoural administration, the product can be administered by systemic route” (1).

TILT has partnered with Germany-based Merck KGaA, Switzerland-based MSD International GmbH, and US-based Pfizer (Merck-Pfizer Alliance). TILT is collaborating with the Cancer Gene Therapy Group at the University of Helsinki and with the University of Pennsylvania, where they are exploring the use of TILT‑123 in combination with CAR-Ts in solid tumours (6).

Since its conception, TILT has attracted more than US$42 million (€39 million) in private investment. On 7 Feb. 2023, it raised €22 million (US$23.8 million) from a financing round led by Finland’s Lifeline Ventures and €5.9 million (US$6 million) equity from the European Innovation Council (EIC) Fund as well as a €2.1 million (US$2.3 million) grant from EIC’s Accelerator programme. In addition, the company has received investments from Stephen Industries Inc Oy and ACME Investments SPF Sàrl. These funds will be used to advance the development of TILT-123 to Phase II clinical trials (7). This follows on from the first funding round of €10 million (US$10.9 million) in June 2022.

There is significant interest in TIL‑based therapeutics, and several companies both in Europe and the United States are exploring this route. In the US, TIL-based therapies are under development from: CTRL Therapeutics (8), Iovance Biotherapeutics (9), Obsidian Therapeutics (10), and Turnstone Biologics (11). Recently, Concerta Bioscience acquired Jounce Therapeutic to gain access to its first-in-class immunotherapy, which is designed to selectively deplete immunosuppressive tumour-infiltrating T regulatory cells in the tumour microenvironment (12).

Over the past few years, R&D efforts in the field of oncolytic viruses have received support from private investors and pharmaceutical companies. TILT’s leading programme, TILT-123, is progressing steadily through Phase I clinical development, and the company has raised sufficient funds to progress into Phase II trials. However, competition within this field is increasing as Concerta Biosciences, CTRL Therapeutics, and Obsidian Therapeutics have pre‑clinical candidates, and Turnstone Biologics has two Phase I trials underway with TILs. Meanwhile, Iovance Biotherapeutics has several Phase II trials nearing completion that are evaluating TILs as monotherapy and in combination with PD-1 or IL-2 analogues. Therefore, investors will be keen to see updates on TILT’s Phase II trial (NCT04217473), which is due for completion in February 2025 (13), and will be closely monitoring other competitor products as they progress through clinical development.

. 2017 13 (5), 970–971.
2. TILT Biotherapeutics. Next-generation Armed Oncolytic Immunotherapies. 

. May 2022.
3. EC. New Immunotherapy Tools to Fight Cancer. UNLEASHAD Project. Results in brief. H2020. CORDIS. 1 July 2018.
4. TILT Biotherapeutics. TILT Biotherapeutics Announces First Patient Dosed in Phase I Solid Tumours. Press Release, 15 May 2023.
5. TILT Biotherapeutics. TILT Biotherapeutics Announces Boston Office Opening as it Expands its Clinical Trials in the US. Press Release, 11 Sep. 2022.
6. TILT Biotherapeutics. Pipeline. 

 (accessed 17 Aug. 2023).
7. TILT Biotherapeutics. TILT Biotherapeutics Announces Final Close of €22 Million Financing. Press Release, 7 Feb. 2023.
8. cTRL Therapeutics. Accessible Cell Therapy (accessed 17 Aug. 2023).
9. Iovance Biotherapeutics, Investigating TIL Therapy & PBL Therapy for Cancer (accessed 17 Aug. 2023).
10. Obsidian Therapeutics. Our Lead Clinical Program (accessed 17 Aug. 2023).
11. Turnstone Biologics. Leading the Fight Against Solid Tumours (accessed 17 Aug. 2023).
12. Jounce Therapeutics. Jounce Therapeutics Enters Into an Agreement to Be Acquired by Concentra Biosciences for $1.85 in Cash per Share Plus Contingent Value Rights. Press Release, 27 Mar. 2023.
13. A Phase 1, Open-Label, Dose-Escalation Clinical Trial of Tumour Necrosis Factor Alpha and Interleukin 2 Coding Oncolytic Adenovirus TILT‑123 in Melanoma Patients Receiving Adoptive Cell Therapy With Tumour Infiltrating Lymphocytes. 

classic.clinicaltrials.gov/ct2/show/NCT04217473

Cheryl Barton is director of Pharmavision, info@pharmavision.co.uk.

Articles

Features

New routes to cancer treatment can be found with the help of tumor-infiltrating lymphocyte (TIL) therapies.

Building a New Arsenal of Therapeutics from Oncolytic Viruses

Monoclonal antibodies (mAbs) are an important class of therapeutic proteins used for treating various diseases such as cancers, autoimmune diseases, and genetic disorders (1,2). Therapeutic mAb products are known to have different variants, such as conformational isomers, charge variants, glycan variants, and size variants (3). Size variants, such as aggregates and fragments, are formed when samples containing mAbs undergo stresses, such as those encountered during the various stages of the product life cycle, including cell culture, purification, storage, formulation, transportation, administration, and handling. The formation of aggregates as the result of various stress factors can lead to the reduction of process yield and product shelf life. The administration of samples containing mAb aggregates to patients may also result in adverse immune responses such as anaphylaxis and neutralization of endogenous proteins. Hence, monitoring, analysis, and control of mAb stability is a critical challenge, and researchers and manufacturers continue to show interest in it as well (3–5).

Monoclonal antibody aggregation is influenced by a variety of factors, such as buffer composition, ionic strength, protein concentration, mechanical stress, temperature, interfacial surfaces, and chemical contaminants. Aggregates could vary in size, structure, and morphology (2). They can be soluble oligomers (less than 100 nm size) or particles of sub-visible (0.1–100 µm) and visible (more than 100 µm) size range (6). Therapeutic protein aggregates of different size ranges are analyzed using an array of techniques such as size exclusion chromatography (SEC), dynamic light scattering (DLS), asymmetrical flow field-flow fractionation, electrophoresis, analytical ultracentrifugation, light obscuration, nanoparticle tracking analysis, microscopes, and flow-based imaging techniques (7,8). Microscopes and flow-based imaging techniques are widely used and play a crucial role in aggregate analysis. The application of different microscopes to analyze aggregates of mAb has also been mentioned in the 

Optical, electron, and atomic force microscopy are some of the commonly used microscopes to visualize aggregates of therapeutic proteins (8). Electron microscopes, such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM) can be used in the visualization of specimens as small as 1 nm to a few microns. TEM and SEM can further be used to determine the size, shape, and morphology of aggregates. Optical microscopes can be easily used to visualize particles having sizes of more than 1 µm. Brightfield and fluorescence microscopes are commonly available optical microscopes. In brightfield imaging, aggregates can be visualized as dark spots against a bright background, whereas in a fluorescence microscope, aggregates can be visualized by labeling them with fluorescent dyes. However, the use of microscopes to analyze therapeutic protein aggregates has a general disadvantage in that only a small fraction of the sample can be analyzed at a time, and, hence, the output might not be a true representation of the entire sample. Further, manual identification of subtle changes in the size, shape, and distribution of protein aggregates from a large number of microscopic images would be cumbersome, and would also be susceptible to human error, variability, and biases (2,8–11).

For this reason, image analysis algorithms are being increasingly used to characterize aggregates. These algorithms are able to analyze a large number of microscopy images via automatic quantitation for various attributes such as size, count, shape, and morphology. Image analysis algorithms offer outputs with high accuracy and reproducibility that would be otherwise challenging to achieve manually, thereby facilitating high throughput screening of protein aggregates (9–11). These algorithms can be used either as a complementary tool to already established techniques used for therapeutic protein aggregate characterization, or to address research applications where other existing techniques cannot be used (9). The advantages of using image analysis algorithms with microscopic techniques are summarized in 

A fluorescence microscope is a type of optical microscope that enables selective determination of specimens due to tagging with a fluorescent dye. The microscope works on the principle of fluorescence where the analyte is irradiated using light from an excitation source followed by the emission of light having a higher wavelength, which is selectively detected (9). Apart from tagging the sample with fluorescent dye, this technique does not require any other complex sample preparation steps (8,11). A simple widefield fluorescence microscope is cheap and easy to maintain and use. Wider illumination areas combined with lesser operational complications are also its distinctive features. Moreover, images in a fluorescence microscope can be acquired with reduced exposure times. However, as explained in the previous section, the use of fluorescence microscopes, in general, has a disadvantage in that only a small fraction of the sample can be analyzed at a time (9–11). The fluorescence microscope has an additional drawback wherein its images are corrupted by noise and blurred particles from out of the focal plane. The noise results in images with very low signal-to-noise ratio (9).

A novel semi-automated image analysis algorithm that removed different noises in the fluorescence microscope image and enabled the analysis of therapeutic mAb aggregates has been proposed recently. In this approach, fluorescent microscope images of aggregates captured using a widefield fluorescence microscope were used (9). The images were captured at x4 magnification and had a resolution of 1536 x 2048 and a scale-to-pixel ratio of 133:50 (m/pixels). In the algorithm (illustrated in 

), the first step was importing the microscope images containing fluorescent dye-tagged mAb aggregates into MATLAB (MathWorks) as a 3D matrix. The two dimensions of the matrix corresponded to the number of pixels along the length and breadth of the image. The third dimension of the matrix was designated to correspond to red, green, and blue channels. The contribution of the green channel was taken forward for processing the images. Median filtering was then applied, followed by the application of a total variation algorithm and fast Fourier transform. The image obtained after denoising was used to normalize the background. After denoising, either weighted median or modified 2D Otsu thresholding was done. Thresholding was followed by standard binary morphological processing steps such as closing, filling, and cleaning, which were used to label individual active regions in the image for subsequent analysis. The scale-to-pixel ratio of the image was used to normalize the data from pixel to metric format. The details of aggregate particles obtained from the images in the form of circularity, diameter, perimeter, and area were arranged in ascending order and sorted into various groups for plotting size distribution graphs. A flowchart showing different steps used in fluorescence microscope image processing is depicted (

), and a representative fluorescence microscope image, binary image obtained after its processing, and the size distribution of aggregates obtained from a set of images are also shown (

). A detailed description of all the steps used in fluorescence image processing has already been published (9). 

Brightfield microscopy is a widely used imaging technique (8,12). It is the simplest of all microscopy techniques, which makes it widely popular. In this technique, the sample is illuminated with white light, which ultimately results in showing off specimens as dark images in the sample. Brightfield microscopy can be used to visualize and analyze sub-visible and visible aggregates. Generally, brightfield images of protein aggregates appear as dark spots depending on the refractive index of the protein aggregates and the surrounding medium (12). However, brightfield microscopy may not be suitable for detecting aggregates with low refractive index or with low contrast between the aggregate and the background. Monoclonal antibody aggregates are usually translucent in nature, and hence their effective visualization in brightfield images requires filtering the sample containing the aggregates onto a membrane followed by staining with a dye. The translucent nature, random irregular shapes of the aggregates, low contrast between background and aggregate, and inconsistent stray light can make aggregate analysis tedious (8,12–14). Hence, as per the authors’ knowledge, there have been severely limited efforts to automatically quantify information from brightfield images of therapeutic mAb aggregates that have not been stained with any dye.

A brightfield image analysis algorithm that can analyze mAb aggregates of more than 1-µm size was recently published using images of aggregates captured by an imaging system (Cytell Cell Imaging System, GE Healthcare) (10). The acquired images at x4 magnification had a resolution of 1920 x 1440 and a scale-to-pixel ratio of 11:10 (m/pixels). The images were called in for image analysis on MATLAB, and the processing algorithm involved several steps, starting from converting the brightfield image to grayscale image. This conversion step was followed by determining the number of pixels corresponding to each color code from 0 to 255, where 0 represents black and 255 represents white. The pixel-color distribution was plotted and was used to segregate the aggregates, background, and hemocytometer lines using thresholding. The aggregates are dark colored and cover the left portion of the peak while the background and particle boundary were included in the peak and on the right side of it (

). Following thresholding, morphological operations were performed to improve the images. The particles in the output binary images were then labeled, and the area corresponding to each label was determined. The scale-to-pixel ratio was used to normalize the data from pixel to metric format. From the extracted data, aggregate size distribution was obtained. A flowchart showing overall steps used in the brightfield image processing is depicted in 

, and a detailed description of all the steps used for image processing has been published elsewhere (10). 

An electron microscope is used to obtain high-resolution images of biological specimens utilizing electrons used as the source of illumination. Transmission electron microscopy (TEM) is a type of electron microscope used to view thin specimens through which electrons can pass to form the image. TEM is used to analyze the interior of cells, structure of proteins, viral structures, and nanoparticles. Electron microscopes can provide visual information about mAb aggregates in the submicron size range. However, the application of output micrographs is usually limited to visual assessment of aggregates, and, thus, there have been limited efforts to quantify the information from electron micrographs. An image analysis algorithm for automated detection and numerical size distribution of therapeutic mAb aggregates was therefore developed. The acquired micrographs were subjected to de-noising, low-pass filtering, background normalization, conversion to binary images, and automatic determination of heterogeneous aggregate sizes and shapes to obtain aggregate distribution. A detailed description of the TEM image processing has been published elsewhere (15).

Image analysis algorithms have been applied to different projects to analyze mAb aggregates. Three case studies are presented in this article. In the first case study, application of the brightfield image analysis algorithm to characterize mAb aggregates obtained by subjecting the mAb samples to rapid interfacial agitation through bubbling stress is shown. Application of the fluorescence microscope image processing algorithm to analyze mAb aggregates in serum and phosphate solubilizing buffer (PBS) is demonstrated in the second case study. Third case study depicts the aggregation of therapeutic mAb in serum having the combined presence of ferrous ion (Fe2+) and hydrogen peroxide (H2O2).

mAb aggregation by air/liquid interfacial agitation stress through air bubbling

The exposure of mAb-containing samples to various interfacial surfaces is known to impact the product’s stability. Interfacial surfaces encountered by mAbs during various stages of manufacturing, transportation, and handling occur in the form of solid/liquid, liquid/liquid, and air/liquid surfaces. Air/liquid interface is commonly observed in the form of air bubbles and headspaces. The combination of agitation and interfacial stress is highly detrimental to the stability of mAbs and could occur during various operations including sparging, mixing, pumping, shaking, filling, transportation, and handling (16). In this study, samples containing 1 mg/mL of mAb in PBS at 37 °C were subjected to rapid air/liquid interfaces and agitation stress using a peristaltic pump (

) followed by analysis using an array of techniques, including brightfield microscope imaging at x4 magnification and image processing to analyze aggregate size in the sub-visible and visible ranges. All the images of samples that were subjected to stress for different time points were analyzed. Brightfield images of samples subjected to stress resulted in dark-colored aggregates. The output images obtained after image analysis showed white particles against a black background. In the case of samples containing mAb subjected to stress for 60 minutes, image processing resulted in aggregate sizes up to 20 µm. Aggregates having sizes of up to 60 µm were obtained from samples containing mAb subjected to stress for more than 60 minutes. A representative image of a sample containing mAb that was stressed for 120 minutes, the output binary image obtained after image processing, and the size distribution of aggregates obtained from several images are shown in 

. The average diameter of aggregates obtained by the algorithm at different time points ranged from 5–9 m. 

Comparing aggregate size distribution in serum vs. in PBS

The aggregate size distribution in the sample containing mAb after administration into the human body could vary and requires deeper investigation (17,18). Buffers such as PBS and 

 models such as serum, plasma, cerebrospinal fluid, and synovial fluid can be used to mimic physiologically relevant conditions to determine the stability of therapeutic proteins (19). Serum is a complex biological matrix that contains many components, including proteins, lipids, and small molecules, and it interferes with the analysis of therapeutic mAb when using techniques such as size exclusion chromatography (SEC) and dynamic light scattering (DLS) (19,20). Hence, tagging the therapeutic protein with a suitable fluorescent dye and analyzing it using a fluorescence-based technique are required to analyze mAb aggregates in serum (19). This case study explored the comparison of aggregate size distribution of mAb in PBS and in serum using the fluorescence microscope image processing algorithm (

). The mAb was tagged by fluorescein isothiocyanate (FITC), a fluorescent dye. Among various samples containing mAb subjected to different stresses, the samples subjected to stirring and interfacial agitation stress resulted in green-colored FITC-tagged aggregates at all the analysis time points in both PBS and serum. Further, serum samples containing mAb aggregates obtained by stirring and interfacial agitation resulted in a higher percentage of particles having more than 10 µm size compared to PBS samples at all the analysis time points. A representative image of serum and PBS containing FITC-tagged mAb subjected to stirring stress that was captured at 48 hours and the size distribution of aggregates obtained by compiling several images captured at this time point are shown in 

. Overall, the case study showed that the aggregate size distribution of mAb in different fluids, such as PBS and serum, that are used to show physiologically relevant conditions would be different. 

Aggregation of therapeutic IgG in the combined presence of Fe2+ and H2O2

Chemicals such as H2O2 and Fe2+ are known to cause degradation of proteins. H2O2 in the presence of Fe2+ leads to Fenton reaction, resulting in the formation of hydroxyl radicals (•OH) that are known to cause oxidative damage to biomolecules such as DNA, proteins, and cells (21). The degradation of biomolecules in the combined presence of H2O2 and Fe2+ is known to be higher than the degradation in the presence of only H2O2 or Fe2+ (22). This case study explored the aggregation of mAbs in the combined presence of H2O2 and Fe2+ in saline, artificially prepared extracellular fluid, macromolecule-free serum, and serum (23). In the case of human serum, the aggregation of FITC-tagged mAb in the presence of both H2O2 and Fe2+ were explored using the fluorescence microscope analysis algorithm (

). It was observed in fluorescence microscope that serum samples containing mAb having the presence of both 0.8 mM Fe2+ and 0.375% H2O2 resulted in the presence of green-colored aggregates (

), whereas serum samples containing mAb that did not have both H2O2 and Fe2+ did not result in aggregates that were in the analysis range of fluorescence microscope image analysis. The processing of fluorescence microscope images of serum containing H2O2 and Fe2+ showed that aggregates having sizes up to 60 µm were observed, with average aggregate size being 12.5 µm. 

Apart from the application of established analytical techniques, mAb aggregates can also be analyzed using image analysis algorithms coupled to microscopy images. The application of algorithms to process microscopic images can be utilized either as a complementary tool to already established techniques, or to address research applications where existing tools cannot be applied.

1. Bansal, R.; Dash, R.; Rathore, A. S. Impact of mAb Aggregation on Its Biological Activity: Rituximab as a Case Study. 

 (9), 2684–2698. DOI: 10.1016/j.xphs.2020.05.015
2. Sreenivasan, S.; Jiskoot, W.; Rathore, A. S. Rapid Aggregation of Therapeutic Monoclonal Antibodies by Bubbling Induced Air/Liquid Interfacial and Agitation Stress at Different Conditions. 

, 97–109 . DOI: 10.1016/j.ejpb.2021.08.010
3. Sreenivasan, S.; Kumar, D.; Malani, H.; Rathore, A. S. Does Interaction of Monoclonal Antibody Charge Variants with VEGF-A and ELISA Reagents Affect its Quantification? 

, 113513. DOI: 10.1016/j.ab.2019.113513.
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6. Kannan, A.; Shieh, I. C.; Hristov, P.; Fuller, G. G. In-Use Interfacial Stability of Monoclonal Antibody Formulations Diluted in Saline I.V. Bags. 

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7. Bansal, R.; Gupta, S.; Rathore, A.S. Analytical Platform for Monitoring Aggregation of Monoclonal Antibody Therapeutics. 

, 152. DOI: 10.1007/s11095-019-2690-8
8. Zölls, S.; Tantipolphan, R.; Wiggenhorn, M. Particles in Therapeutic Protein Formulations, Part 1: Overview of Analytical Methods. 

 (3), 914–935. DOI: 10.1002/jps.23001
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, 11375. DOI: 10.1038/s41598-021-90623-7
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, 1747–1763. DOI: 10.1007/s11095-021-03108-7
11. Demeule, B.; Gurny, R.; Arvinte, T. Detection and Characterization of Protein Aggregates by Fluorescence Microscopy. 

 (1–2), 37–45. DOI: 10.1016/j.ijpharm.2006.08.024
12. Selinummi, J.; Ruusuvuori, P.; Podolsky, I.; et al. Bright Field Microscopy as an Alternative to Whole Cell Fluorescence in Automated Analysis of Macrophage Images. 

 (10), e7497. DOI: 10.1371/journal.pone.0007497
13. Buggenthin, F.; Marr, C.; Schwarzfischer, M.; et al. An Automatic Method for Robust and Fast Cell Detection in Bright Field Images from High-Throughput Microscopy. 

, 297. DOI: 10.1186/1471-2105-14-297
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15. Kumar, M.; Pant, A.; Bansal, R.; et al. Electron Microscopy-Based Semi-Automated Characterization of Aggregation in Monoclonal Antibody Products. 

, 1458–1465. DOI: 10.1016/j.csbj.2020.06.009
16. Li, J.; Krause, M. E.; Chen, X.; et al. Interfacial Stress in the Development of Biologics: Fundamental Understanding, Current Practice, and Future Perspective. 

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17. Schuster, J.; Koulov, A.; Mahler, H. C.; et al. 

Anurag S. Rathore, asrathore@biotechcmz.com, is a professor in the Department of Chemical Engineering at the Indian Institute of Technology Delhi and a member of 

®’s Editorial Advisory Board, and Shravan Sreenivasan is a graduate student working under the supervision of Prof. Rathore.

Therapeutic protein aggregates can be characterized using image analysis algorithms coupled with microscopy techniques.

Utilizing Image Analysis Algorithm for Therapeutic mAb Aggregate Analysis

On Oct. 2, 2023, The Nobel Prize in Physiology or Medicine was awarded jointly to Katalin Karikó and Drew Weissman for their work on nucleoside base modifications that allowed the development of effective messenger RNA (mRNA) vaccines against COVID-19. The duo’s groundbreaking discoveries were crucial in the current international understanding of how mRNA interacts with the immune system; their work played a crucial role in the rapid rate of vaccine development during the global pandemic.

Karikó and Weissman began their collaboration during the 1990s at the University of Pennsylvania. Karikó, then an assistant professor, was committed to developing mRNA as a therapeutic, despite difficulties in receiving funding. Enthusiasm for mRNA research was limited at the time; though the introduction of 

transcription in the 1980s spurred molecular biology development, 

 transcribed mRNA was unstable, requiring lipid systems to encapsulate the mRNA. The treatment also heightened inflammatory reactions. But Weissman, a new colleague of Karikó, proved to be the perfect partner for her research. As an immunologist, he worked primarily with dendritic cells, which are vital to immune surveillance and activating vaccine-induced immune responses. Together they began to focus on the interaction of different RNA types with the immune system.

Karikó and Weissman’s breakthrough came when they began studying why dendritic cells recognized 

transcribed mRNA as a foreign substance, while mRNA from mammalian cells did not have the same response. They knew that the bases of RNA from mammalian cells are often chemically modified, while in 

 transcribed mRNA is not. Karikó and Weissman produced variants of mRNA with different chemically altered bases and found that when those mRNA were delivered to dendritic cells, the inflammatory reactions were almost completely abolished. This discovery fundamentally altered the understanding of how cells recognized and respond to mRNA.

In 2005, Karikó and Weissman published the results of their research, 15 years before the COVID-19 pandemic began. In later studies published in 2008 and 2010, they proved that mRNA with base modifications shows a significant improvement in protein production compared to unmodified mRNA. Thanks to their findings, the two major roadblocks in mRNA development had been eliminated, and several companies began working to develop the method. And after the COVID-19 outbreak, two base-modified mRNA vaccines were able to be developed at record speed. The Nobel prize honors the work of Karikó and Weissman, and the critical role it played in combatting one of the greatest health crises of the modern age.

News

The prize was awarded jointly to Katalin Karikó and Drew Weissman for their groundbreaking discovery regarding modification of the bases in mRNA. 

2023 Nobel Prize in Medicine Awarded for Development of mRNA Vaccines Against COVID-19

Ginkgo Bioworks announced a collaboration on Sept. 27, 2023 with Pfizer under which Pfizer will use Ginkgo's proprietary RNA technology to advance the discovery and development of novel RNA molecules across priority research areas.

Pfizer will pay Ginkgo an upfront payment. Ginkgo is eligible to receive research fees and development and commercial milestone payments. All told, the company may receive up to an aggregate total of $331 million across three programs. In addition, Ginkgo is entitled to potential royalties on sales further downstream.

Ginkgo's RNA technology combines high-throughput screening that evaluates the behavior of RNA constructs with a multi-parameter design framework. The technology allows the company to identify novel natural and synthetic elements that are optimal for a particular application. Ginkgo will apply its technology to achieve efficient production, circularization, improved stability, and enhanced translation of each RNA construct.

"RNA therapeutics are proving to be an important platform to advance the world of scientific innovation, and with progress in synthetic biology we have the potential to create new RNA treatments that may benefit patients worldwide," said Will Somers, head, Biomedicine Design, Pfizer, in a company press release. "Access to Ginkgo's proprietary platform will help enable Pfizer to search for novel and exciting RNA constructs with improved stability and expression that could lead to more effective treatments."

"Billions of patients around the world have already benefited from advances in RNA-based technologies. We're thrilled to be able to help enable the discovery and development of novel drugs using this powerful modality by applying our platform technologies, which are designed to program RNA for maximum therapeutic effect," said Jason Kelly, CEO and co-founder, Ginkgo Bioworks, in the release. "Ginkgo's approach enables and accelerates discovery across different disease areas and modalities for our partners, who bring deep expertise in drug development."

Pfizer has entered into a collaboration with Ginkgo Bioworks to discover novel RNA molecules across priority research areas.

Pfizer and Ginkgo Bioworks to Collaborate on Multi-Target RNA Discovery 

It is common knowledge that messenger RNA (mRNA) advances saved lives and limited the extent of the COVID-19 pandemic. Far less recognized, even in the science and technology community, is the decade-long nurture and support governmental organizations provided the mRNA ecosystem and the remarkable range of therapeutic advances that mRNA (and mRNA coupled to other platforms) could solve for, going forward.

On Aug. 23, 2023, the latter was made explicit when, as part of the Biden administration moonshot initiative, the Advanced Research Projects Agency for Health (ARPA-H) announced the launch of “Curing the Uncurable via RNA-Encoded Immunogene Tuning” (CUREIT) project. This effort, led by a team at Emory University in Atlanta, Ga., with up to $24 million in new funding, has the goal of developing “… generalizable mRNA platforms that can be harnessed to train the immune system to more effectively fight cancer, … other applications could extend to autoimmune disorders, transplants, and infectious diseases” (1).

As mRNA and RNA approaches surge in investment dollars and drug candidates moving into clinical trials, the Alliance for mRNA Medicines (AMM), initiated its “DC Day” meetings with a discussion with Peter Marks, MD, PhD, director, Center for Biologics Evaluation and Research, FDA. Top of mind for Marks was the possibility that public and political resistance to advanced therapies could be an obstacle to therapeutic efficacy, saying “We don't want the benefits of mRNA technology wasted due to poor uptake. We need to help people understand how well-established this technology is, and how it is not altering the human genome.”

On the side of production expedience, economics, and workforce efficiencies, Marks avers that, “mRNA vaccines have already shown tremendous capabilities which demonstrate a strong value proposition,” compared to more traditional approaches.

In relation to why there was the need for a new advocacy group, such as AMM, Marks says that while recognizing “When a group such as [AMM] is formed, it floats all boats,” he particularly emphasizes, “it’s very helpful to have a single unified voice to streamline engagement of expert working groups, build consensus, and relay authoritative guidance, fundamental facts (and constraints), to both the general public and also the pertinent governmental agencies. I look forward to regular working discussions with AMM—and other participants—in this field.”

Excellent and exhaustive communication was a hallmark for vaccine strategy during the past pandemic. Taking hard-won lessons forward, groups such as AMM will doubtless play a central role in coordination and consolidation for future efforts. Government agencies such as FDA, the Biomedical Advanced Research and Development Authority, the Office of Science and Technology Policy, and the US Army Medical Research Institute of Infectious Diseases, deserve to take a bow for having helped establish the necessary pre-conditions to allow pharmaceutical sciences to move smoothly between discovery, development, and deployment stages. As for the next generation of life saving medicines, AMM and partners should provide that clear voice to this shared vision for innovative mRNA and RNA therapies.

1. The White House, As Part of President Biden’s Unity Agenda, Biden Cancer Moonshot Announces Launch of ARPA-H’s CUREIT Project Led by Emory University to Develop New Tools to Strengthen the Immune System and Save Lives. Press Release, Aug. 23, 2023.

Chris Spivey is the editorial director of 

Government provides a spoonful of sugar, and genuine leadership, for good medicines.

FDA Gets Its Arms Around Advanced Therapy Medicinal Products Early

On Sept. 28, 2023, Sartorius and UK-based startup SPARTA Biodiscovery (SPARTA) announced that they have entered into a partnership for SPARTA’s analysis platform. The platform helps speed the development, manufacture, and quality control of nanoparticles (small polymer or lipid capsules that act as carriers to deliver active agents to target cells) for biopharmaceutical drugs, including gene therapies, vaccines, and cancer drugs.

Under the agreement, Sartorius will invest up to £3.5 million (US$4.3 million) in a minority shareholding in SPARTA through Sartorius Ventures, its corporate investment unit. SPARTA, which was spun off from Imperial College London, developed its core technology, the single particle automated Raman trapping analysis, and developed a prototype for the platform at the college. The technology was established in the group of co-founder Professor Molly Stevens Fellow of the Royal Society, Fellow of the Royal Academy of Engineering. Single particle automated Raman trapping analysis uses spectroscopy to quickly and automatically analyze populations of individual nanoparticles at high throughput. Its mode of action helps accelerate nanoparticle development and optimize quality control during production, according to a company press release.

“The SPARTA platform will help customers optimize the design of nanoparticles at an early stage, even before the drug candidates move on to the clinic. We have teamed up to bring additional product commercialization and application expertise to the table to facilitate a swift transitioning from prototype to launch,” said Oscar-Werner Reif, head of corporate research and chief technology officer at Sartorius, in the press release.

“Often the challenge in bringing new therapies to life is not the development of new active agents, but safe and robust delivery of such agents into the body, e.g., through nanoparticles. We see an unmet need in R&D, manufacturing, and quality control in analyzing the composition of these nanoformulations. Together with Sartorius, we will be able to bring this technology to customers faster,” said Jelle Penders, CEO and co-founder of SPARTA Biodiscovery, in the release.

Under the partnership, Sartorius will leverage SPARTA Biodiscovery’s technology to accelerate development of nanoparticles used for drug delivery.

Sartorius and SPARTA Biodiscovery Partner on Novel Nanoparticle Analysis Platform 

On Sept. 27, 2023, Gritstone bio, a clinical-stage biotechnology company, announced it has been awarded a contract by the Biomedical Advanced Research and Development Authority (BARDA) for the randomized Phase IIb study of its self-amplifying mRNA (samRNA) vaccine. The samRNA vaccine contains Spike antigen (similar to first-generation COVID-19 vaccines), as well as other viral targets to defend against COVID-19. The contract, valued at up to $433 million, is part of ‘Project NextGen’, a US Department of Health and Human Services (HHS) initiative that aims to push forward new and innovative vaccines and therapeutics providing larger and more long-lasting defenses for COVID-19.

As agreed upon in the contract, Gritstone bio is set to run a 10,000-participant, randomized Phase IIb double-blinded study that will compare its next-generation COVID-19 vaccine candidate against an approved COVID-19 vaccine. The study will test the immunogenicity, safety, and efficacy of the candidate, and it will be run in the United States alongside the COVID-19 Prevention Network (CoVPN). The CoVPN, which is supported by the National Institute of Allergy and Infectious Diseases (NIAID), is a network of clinical trial sites located at Fred Hutchinson Cancer Center with experience conducting large-scale COVID-19 vaccine trials.

This new vaccine candidate is a part of Gritstone bio’s CORAL program. The CORAL program uses self-amplifying mRNA and novel immunogens containing Spike antigen and additional viral targets to enhance B and T cell immunity. As of the publication of this article, the program has conducted three Phase I trials across a variety of patient populations and settings, including vaccine-naïve healthy and HIV+ subjects in South Africa, and healthy volunteers after primary series of approved COVID-19 vaccines. The results of these studies have showed high neutralizing antibody levels for at least six months and wide T cell responses. The CORAL program is supported by BARDA, NIAID, the Coalition for Epidemic Preparedness Innovations, and the Bill & Melinda Gates Foundation.

samRNA is a quickly emerging platform technology that can be used to develop multiple vaccines by changing the sequence of the antigen encoded in the vector RNA and delivered in a lipid nanoparticle. samRNA vaccines, much like traditional mRNA vaccines, use the translation system of the host to change mRNA to protein target antigens, encouraging immunity. But unlike mRNA, samRNA makes multiple copies of the antigen RNA once in the cell, which can lead to longer and amplified antigen expression.

The contract is part of US Department of Health and Human Services’ ‘Project NextGen’ initiative, aimed at pushing forward new and stronger COVID-19 vaccines and therapies.

BARDA Awards Gritstone bio $433 Million Contract for Phase IIb Study of Next-Gen COVID-19 Vaccine

PeptiDream, a public Kanagawa, Japan-based biopharmaceutical company, has entered into a new multi-target collaboration and license agreement with Genentech, a member of the Roche Group, in which the companies will work to discover and develop novel macrocyclic peptide-radioisotope (peptide-RI) drug conjugates.

Under the agreement, PeptiDream will utilize its proprietary Peptide Discovery Platform System (PDPS) technology to discover, optimize, and develop macrocyclic peptide candidates. The candidates will be used as peptide-RI drug conjugates against targets of interest to Genentech. PeptiDream will lead early preclinical development, and Genentech will take over further development and commercialization of any peptide-RI drug conjugate products that result from the collaboration. However, PeptiDream will retain the right to develop and commercialize such peptide-RI drug conjugate products in Japan, according to a Sept. 20, 2023 company press release.

According to the transaction terms, PeptiDream will receive an upfront payment of $40 million from Genentech. PeptiDream will be eligible for potentially up to $1 billion in payments based on certain development, regulatory, and commercial milestones. PeptiDream is also eligible to receive tiered royalties on net sales (ex-Japan) of products arising from the collaboration.

This new collaboration and license agreement follows a long-term partnership between the companies that started out as a multi-target collaboration and license agreement in December 2015, after which Genentech licensed PeptiDream’s PDPS technology in 2016. The companies initially expanded that original collaboration in 2018.

“We are extremely excited to announce this new collaboration with Genentech for the discovery and development of peptide-RI drug conjugates. Combining this expertise with Genentech’s powerful clinical development and commercialization capabilities, we hope to bring innovative first-in-class peptide radiopharmaceuticals to patients worldwide,” said Patrick C. Reid, president and CEO, PeptiDream, in the press release.

Under a new pact, Genentech and PeptiDream will collaborate to discover and develop novel peptide-radioisotope drug conjugates.

Genentech Forms Collaboration with PeptiDream for Peptide-Radioisotope Drug Conjugates

BenevolentAI, a company that is a part of the development of advanced AI to accelerate biopharma discovery, announced on Sept. 20, 2023 that it has signed a strategic collaboration with Merck.

This collaboration will help Merck leverage BenevolentAI’s AI platform capabilities and access a team of interdisciplinary drug discovery scientists to identify and develop innovative compounds through Hit Identification, or Hit ID, to the pre-clinical stage. Further, BenevolentAI will leverage its suite of AI chemistry design tools in combination with its fully equipped wet lab facility in Cambridge, UK, to deliver small molecule drug development candidates into the Merck pipeline, according to the press release.

The terms of the agreement outline that BenevolentAI will be eligible for payments of up to $594 million, which consists of a low double-digit million dollar upfront payment on signing and then potentially discovery, development, and commercial milestones, according to the release. Further, tiered royalties will also be payable on net sales of any commercialized products.

“Our strategic collaboration with Merck exemplifies BenevolentAI’s unique capability to leverage AI to accelerate drug discovery. Our proprietary technology and extensively validated approach of combining AI, molecular biology, medicinal chemistry, and in vivo pharmacology supports the discovery of innovative drug candidates in fewer cycles,” said Joanna Shields, chief executive officer of BenevolentAI, in a press release. “We look forward to working with the Merck team and are excited at the opportunities in our partnership.”

BenevolentAI will leverage its suite of AI chemistry design tools in combination with its fully equipped wet lab facility in Cambridge, UK, to deliver small molecule drug development candidates into the Merck pipeline, according to the press release.

BenevolentAI Signs Collaboration with Merck

CN Bio, a organ-on-a-chip (OOC) company specializing in the design and manufacture of single-and multi-organ microphysiological systems (MPS), has partnered with LifeNet Health LifeSciences (LifeNet Health), a non-profit provider of all-human research solutions, to supply validated primary human cells. The cells will be used with CN Bio’s PhysioMimix OOC range of MPS.

Under the agreement, LifeNet Health’s MPS-validated, hepatic cells will be made available either within CN Bio’s non-alcoholic steatohepatitis (NASH) “in-a-box” kit or directly from LifeNet Health. The companies expect that this partnership will provide a streamlined solution for the recreation of CN Bio’s advanced 

 liver models and that the combination will maximize assay success.

According to a Sept. 6, 2023 company press release, CN Bio will use a stringent process to pre-qualify select hepatocyte, stellate, and Kupffer cells that are proven to thrive in extended 3D MPS culture. These cells will have to show that they maintain function and phenotype for a minimum of two weeks. It is known that lot validation in standard static 2D or 3D cell cultures does not guarantee the performance of primary cells in perfused MPS cultures. CN Bio carried out an analysis over a five-year period where it demonstrated that greater than 60% of hepatocyte cell lots across various suppliers failed to meet acceptable performance criteria for MPS assays. Thus, pre-validating cell lots for use in MPS assays is necessary, and it ensures that robust and reliable human-relevant data can be generated, according to the company press release. However, the validation process itself requires that additional resource, time, and budget be factored in.

“LifeNet Health and CN Bio share a commitment to helping researchers bring safer, more effective therapies to patients sooner, with no unnecessary animal testing,” said Tomasz Kostrzewski, chief scientific officer, CN Bio, in the press release. “In our studies, LifeNet Health’s cells have consistently shown high success rates, and we are confident this will lead to more reliable, reproducible results. Our partnership represents an important milestone for existing customers, alongside those looking to adopt OOC technology into their own labs. Validation of cells is a costly and laborious process, but one that is imperative for assay success. Harnessing our combined technical expertise and customer support, we can offer researchers a more complete solution to easily recreate our industry-leading liver models, removing the risk of failed experiments due to untested cells and the burden of in-house quality control testing—so that they can focus their efforts on new discoveries.”

“This partnership represents a joint investment to set a new standard for cell quality across the industry, enabling customers across a broad range of market segments to realize the potential of OOC, without the need for costly in-house validation,” said Ed LeCluyse, chief scientist, LifeNet Health LifeSciences, in the release. “Our cells are fully characterized for quality and performance, to ensure they can perform within advanced platforms, to more effectively mimic 

CN Bio and LifeNet Health LifeSciences have teamed up to provide direct access to a portfolio of highly characterized hepatic cells for the creation of advanced liver-on-a-chip assays.

BioPharm International's BP Elements, October 2023