Nucleic acid-based therapeutics have been stepping up into the spotlight following the deployment of messenger RNA (mRNA) COVID-19 vaccines in 2021 and the rise in clinical development programs for gene therapies. These areas of development may just be the surface of the types of therapeutic applications where nucleic acid-based therapeutics can come into play. Tackling the development challenges of these therapeutics can potentially benefit more expansive areas of unmet medical need, particularly in difficult-to-treat patient populations.

Current immune therapies in development may often fail or not work for everyone. The result of these failures, in part, is what may be driving the R&D engine for alternative modalities, including nucleic acid-based therapeutics.

“We do not yet fully understand why current immune therapies are highly efficacious in some patients while other patients fail to respond, but research scientists are exploring several hypotheses. With cancer immunotherapies, it is important to remember that tumors are highly heterogeneous, in respect to the expression of tumor antigens and their effects on the immune system that enable tumor cells to escape immune surveillance,” explains John Lewis, CEO of Entos Pharmaceuticals.

Lewis points out that some cancers may not respond to current immuno-oncology (IO) drugs because these cancers do not express the target protein, or because they suppress immune surveillance and inhibit T-cell responses. Mutational burden, which is the number of mutations present in a cancer cell, may also play a role in response to IO therapies, especially checkpoint inhibitors, he adds.

“Checkpoint pathways evolved to regulate the T-cell response to cancer cells. Some tumors suppress T-cell response, so checkpoint inhibitors can overcome this suppression. While this allows normal T-cell anti-tumor activity to develop, this activity appears more effective in cells with a higher mutational burden,” says Lewis.

Another reason for failure is that current immune therapies often have safety issues or lack efficacy, or a combination of both, points out Gerrit Dispersyn, CEO, Phio Pharmaceuticals. Regardless of background, he explains, there is a need for more refined therapies to reprogram immune cells and tumor cells. Therefore, there is a need to target the internal cell machinery that can be achieved with oligonucleotide (i.e., nucleic acid)-based therapeutics.

Currently available commercial IO products mainly fall into two categories: immune checkpoint antibodies and chimeric antigen receptor T cell (CAR-T) therapy, Dispersyn notes. He explains that immune checkpoint antibodies “remove the brakes” of all T cells, including the auto-reactive ones, which leads to dose-limiting toxicity. Meanwhile, CAR-T therapies have limited access to solid tumors. Thus, the use of oligonucleotides has the potential to improve efficacy against solid tumors.

“Of the different platform technologies, it is becoming more and more clear that the shortcomings of the currently used platforms—whether it’s antibodies, cell-based therapeutics, or gene therapy—are being realized. Therefore, new approaches to fine tuning or helping overcome these challenges is partially explaining the turn to RNA-based therapeutics,” says Dispersyn.

“Triggering immune responses to fight disease is incredibly challenging, and we do not fully understand the complexities behind it,” adds Matthew Scholz, CEO of Oisin Biotechnologies, a US-based late preclinical stage company specializing in therapeutics for age-related diseases, and OncoSenX, a US-based late-stage pre-clinical cancer company. Scholz notes that there is significant variability in the population as well as heterogeneity within tumors. A therapeutic that targets a surface protein or requires functional T-cell infiltration can therefore be rendered ineffective in many patients. Targeting disease at the cellular level using RNA and DNA to “code” for a desired response is a more specific and nuanced approach, he asserts.

“With nucleic acid-based technologies, only the code for proteins is delivered. With DNA as a payload, tissue-specific expression of the cargo can be achieved through rational promoter selection as well,” says Scholz. “Think of it as a software approach to mobilizing the immune system: nucleic acids provide the program, and the immune system interprets the program and responds accordingly. The caveat of this approach is the nucleic acids must be delivered to the right cells in the body for the program to run effectively, in this case T cells for immune modulation and tumor cells themselves to eliminate tumors.”

For oncology indications, some immunotherapies are powerful at activating immune responses to attack and destroy tumor cells, specifies Jake Becraft, PhD, co-founder and CEO of Strand Therapeutics, an emerging biopharmaceutical company. However, one of the major shortcomings of these therapeutic agents is unexpected, off-target effects that can cause serious adverse reactions and life-threatening toxicities, he adds. The major advantage of nucleic acid-based therapeutics lies in the fact that they can be used to accurately target a tumor or tissue, then have a specific therapeutic protein, biologic, or immune engager expressed only at the site of interest. “This localization dramatically decreases the risks of off-target effects and improves safety and efficacy,” Becraft states.

While nucleic acid-based therapies may not overcome all their challenges, they enable more precise cellular targeting and extended activity than small-molecule or protein-based therapies, adds Lewis. According to Lewis, preclinical studies demonstrate that therapeutic gene expression can be targeted to specific tissue types with tissue-specific promoters, which can potentially improve efficacy while reducing side effects that result from off-target activity. Furthermore, cancer cells have high turnover rates, and DNA-based therapies provide a longer duration of activity than protein and small-molecule therapies, Lewis adds.

Much of the focus today is on RNA and RNA-related technology for developing “next-generation” biotherapeutics. The benefits of RNA technology, according to Dispersyn, is that it does not require modification of the genome. For instance, Phio is using an RNA interference (RNAi) approach to developing therapeutics because RNAi is transient and not permanent. In comparison, gene-editing approaches may result in permanent issues or toxicity.

“In cases where permanent modification is not required, we have the option to use RNA-based therapeutics. This is because DNA is not directly linked to proteins. RNA is a required intermediary step between genetic material and the protein. It is the proteins that make a disease present or absent. Since every translation of the genome goes to RNA, theoretically, almost everything can be solved using RNA therapies. This may be a more helpful solution than editing DNA, which is permanent and the long-term effects of which are largely unknown at this time,” Dispersyn says.

With much of the focus on RNA-based approaches for developing nucleic acid-based therapeutics, where does DNA fit in?

Scholz states that, while DNA is harder to deliver than RNA, DNA does offer many advantages over RNA, such as durability of expression, reduced manufacturing costs, increased stability, and contextual control of expression. “DNA-based vaccines are in clinical trials now, and Oisin Biotechnologies has a pipeline of candidates targeting indications including chronic kidney disease, metabolic diseases, and sarcopenia, to name a few,” says Scholz. “As these technologies are currently being evaluated in human clinical trials, we expect it will not be long before pharmaceutical companies broadly adopt this technology to deliver DNA payload.”

Scholz explains that the key to delivering DNA is to have a non-toxic delivery system that can easily get into cells without causing damage. “These technologies are in humans now, and we expect it won’t be long before more pharmaceutical companies adapt this technology to deliver DNA payloads,” he states. DNA payloads permit an additional layer of selective expression, thus paving the way for encoded therapeutics that not only target and kill tumor cells while sparing healthy cells, but also deliver key immune modulators to healthy cells, including immune cells, which can enable multi-pronged therapeutic approaches, he adds.

Becraft also notes that there are a number of DNA-based approaches currently in development, including T-cell therapies, genome editing and writing, and clustered regularly interspaced short palindromic repeats (CRISPR). Becraft adds that viral-based gene therapies that use adeno-associated virus (AAV) for diseases such as muscular dystrophy use a DNA-based payload. “While DNA and RNA-based therapeutics have similarities, the main difference is that DNA needs to be delivered further into the nucleus. This presents a lot of challenges to overcome due to the strength and defense of the nuclear membrane,” says Becraft.

DNA offers a variety of benefits compared with RNA, remarks Lewis. Benefits from using DNA include more durable expression, the ability to design a single DNA therapy that expresses multiple therapeutic molecules, and less complex supply chain management. “To date, a challenge in realizing the full potential of DNA-based therapies has been developing safe and highly effective delivery systems,” says Lewis.

Lipid-based systems, for example, are associated with liver toxicity while viral delivery systems trigger immune responses (which can result in serious adverse events) that limit repeat dosing, Lewis explains. He further adds that, while the DNA-based gene therapies approved to date have shown promising long-term data, it remains to be seen if a single-dose provides life-long therapy or if repeat dosing will be needed following longer periods of time.

“Given the enormous potential of DNA-based therapies, there are many academic and industry efforts to develop new delivery strategies, either through innovation of novel delivery systems or improved approaches to mitigating the safety and efficacy limitations of current approaches,” says Lewis.

Larry Pitcher, senior director and general manager, microbial manufacturing services, Thermo Fisher Scientific also emphasizes the fact that plasmid DNA (pDNA)-based vaccines have been around since the dawn of recombinant DNA technology. He also notes that pDNA is increasingly being used as a raw material or therapeutic agent in gene therapies and certain vaccines, and that its use is driven by the growth and transformational benefits of cell and gene therapies as well as the expanded application of mRNA.

“Its [pDNA’s] advantages, including weak immunogenicity, increased safety, and ease of manufacture, have dramatically increased demand for materials and manufacturing capacity globally. Investments in state-of-the-art manufacturing ensure [that clients] have reliable access to the high-quality materials and capabilities that have become vital for the production of these new lifesaving medicines and the patients in need,” says Pitcher.

Among the key challenges for developers of nucleic acid-based therapeutics will be the need to scale up to meet current demand and to evolve as market needs change, highlights Serena Fries Smith, director of Market Development & Strategy, mRNA Vaccines and Therapeutics, Thermo Fisher Scientific. Smith explains that, to meet current demand and evolve with the market, developers need a reliable supply of high-quality, good manufacturing practice (GMP)-grade raw materials.

Without reliable materials supply, developers can encounter manufacturing disruptions, including deviations, that can result in costly delays.

“Another challenge is the need for in-region manufacturing capacity and distribution to improve global access, including for people in lower income regions. To help address barriers such as lack of a cold supply chain, we are seeing smaller facilities popping up globally,” Smith says. “Stability and shelf-life are also challenges, but researchers have learned to alter chemical features of the mRNA transcript to improve the stability. In addition, improvements in delivery can protect mRNA against degradation.”

Historical and current challenges to nucleic acid-based therapy development include the production process itself, in terms of scalability, standardized quality metrics, and delivery, emphasizes Jordana Henderson, associate director of Research and Development at TriLink Biotechnologies (part of Maravai LifeSciences), a contract development and manufacturing organization specializing in the synthesis of nucleic acids, nucleoside triphosphates, and mRNA capping analogs. Henderson notes, however, that mRNA production scale-up, specifically, is quickly being overcome through optimized 

 transcription reactions followed by simplified purification processes. Optimal on-target delivery and standardization of drug substance analytical procedures, however, are still a work in progress, she adds.

“To make a functional message, it is critical that the synthetic mRNA contains all the structural elements of a mature post-transcriptionally modified mRNA found in eukaryotes. This includes a 5’ cap structure (Cap 1 or 2), 5’ untranslated region (UTR), coding sequence, 3’ UTR, and poly-adenosine tail,” Henderson explains. She also specifies that generating the appropriate 5’ Cap 1 structure 

 once required additional enzymes and purification steps in the manufacturing process. Now, however, using co-transcriptional cap analogues (e.g., CleanCap, TriLink) can initiate the formation of a Cap 1 structure in a one-pot reaction via the RNA polymerase that is used to transcribe the message of interest, she further elucidates.

 transcription] reaction has enabled streamlined and scaleable purification processes for quick manufacturing of high-quality mRNA products. Ongoing and future work on the mRNA platform is focused on developing safe modifications to the nucleic acid that increase the molecules stability and translatability,” says Henderson.

Henderson also points out that several groups, including academic and industry alike, are actively working on lipid nanoparticle delivery system improvements, while regulatory leaders, including the US Pharmacopeial Convention and FDA, are looking to standardize mRNA drug substance specifications. “Altogether, these efforts will contribute to much needed advances in medical treatment options,” Henderson states.

Becraft points to the specificity of therapeutic protein expression as another major challenge in developing nucleic-acid agents. By using synthetic biology, however, the industry can address this roadblock and improve safety and efficacy. “Synthetic biology coupled with mRNA guarantees that the molecule will be encoded with the correct genetic information. We can also engineer the mRNA to be context-dependent so the protein will only be expressed inside of certain cell types. This logic-based genetic programming allows us to have precise control of the location, timing, and level of protein expression,” Becraft states.

Lewis also hones in on the limitations in tolerability and immunogenicity, as well as scalability, of current delivery methods, as being among the major challenges to nucleic acid-based therapy development. “Lipid-based systems are associated with liver toxicities and viral-based systems lead to immune responses that can both limit initial transduction efficiency and prevent redosing. Entos was established to address these limitations and broadly enable the next generation of nucleic medicines,” he states.

“We are in the process of publishing preclinical data demonstrating the significant potential of our Fusogenix proteolipid vehicle (PLV) platform in diverse indications. These data show that this novel delivery system leads to broad distribution across tissue types and organ systems with minimal toxicity. Gene expression can be targeted using tissue-specific promoters, and delivery of DNA with the Fusogenix PLV platform [e.g., Entos’ DNA vaccine candidate] leads to sustained gene expression out to one-year post-dosing,” Lewis says.

The data from Entos’ as-yet-unpublished preclinical studies also demonstrate that the Fusogenix PLV platform can effectively deliver RNA as well. “Importantly, these studies also show that the low levels of immunogenicity and high levels of activity are maintained after extended periods of repeat dosing,” says Lewis. “Additional studies demonstrate the feasibility of using the Fusogenix platform to enable DNA vaccines against SARS-CoV-2.”

Entos’ DNA vaccine candidate includes the full-length viral spike protein, which may provide enhanced and broad immune responses compared with protein vaccines that use spike protein fragments, Lewis adds. DNA vaccines can also encode genetic adjuvants that can enhance neutralizing anti-SARS-CoV-2 antibody responses in mice and non-human primates. “Data demonstrates that our Fusogenix PLV DNA vaccine candidate protected against SARS-CoV-2 infection in an animal model. We are currently conducting a Phase II trial of our SARS-CoV-2 DNA vaccine candidate,” Lewis states.

Historically, the delivery of oligonucleotides into the cell has been an issue for RNA therapeutics, says Dispersyn. Companies have taken different approaches to address this issue, such as the use of lipid nanoparticles as the delivery vehicle leading to liver accumulation because of its role in first-pass metabolism. “As a consequence the current RNAi approaches most companies utilize are liver specific, even though RNAi can work equally well in non-liver targets if you can deliver the RNA molecules there. Phio’s solution to this non-liver delivery challenge was to develop a RNAi platform with structural and chemical modifications that allowes for spontaneous uptake to cells without the need for a delivery vehicle,” Dispersyn explains.

Oisin’s PLV delivery technology, meanwhile, has enabled the company to develop systemic DNA therapeutics. “Once the first generation of nucleic acid therapeutics have been developed for obvious disease targets, second-generation therapeutics will need to focus on delivering optimized DNA/RNA ‘software’ sequences for more complex diseases,” says Scholz. “Next-generation sequencing and analysis for new unique targets, along with software-based synthetic promoter design, will help us explore different programming techniques for specific disease burdens in the future.”

Of the nucleic acid-based approaches to biotherapeutic development, RNAi has been especially pegged for its potential role in IO therapies. For instance, RNAi can be introduced into T cells. Fundamentally, T cells are like any other cell in that they can be programmed by nucleic acid therapies, points out Scholz. Often in cancer, T cells are overwhelmed by tumor cells, and because of a built-in fail-safe mechanism, T cells can shut down when overwhelmed, that is, they can go into T cell exhaustion. T cell exhaustion can be counteracted with the help of RNAi, however. “In essence, with careful selection of the nucleic acid payload, delivery of RNAi to T cells could be used to combat T cell exhaustion by breaking the inhibitory circuit from the inside, releasing T cells to seek out and fight both the primary tumor and metastases alike,” Scholz emphasizes.

Scholz cautions, however, that RNAi technology has been limited in its ability to target and kill tumor cells directly simply because RNAi technology cannot distinguish a healthy cell from a tumor cell. “Our preclinical studies clearly demonstrate that a plasmid DNA-based suicide gene approach can distinguish tumor cells and effectively eliminate them. Using RNA and DNA in tumors has enormous potential. If you can deliver a precise payload to these tumors that target only that tumor’s microenvironment you could, in theory, come up with many different ways to attack cancer at the cellular level. This, in essence, could get to the root cause of cancer. Being able to target specific cancer traits systemically may also help to tackle metastatic cancers,” Scholz explains.

Conventional RNAi technology has limitations that prevent effective application in immuno-oncology, including challenges with delivery and stability, however, notes Dispersyn. Phio’s platform allows RNAi to be used for increasing the tumor cell killing activity of immune cells, as well as for increasing the vulnerability of tumor cells toward such cell killing. For example, Phio’s focus in adoptive cell therapy includes using its technology to reduce the therapeutic immune cell’s expression of immunosuppressive proteins, which can allow these immune cells to overcome tumor resistance mechanisms and improve their ability to destroy tumor cells.

“Similarly, we can reprogram cells used in adoptive cell therapy, such as CAR T-cells, to increase their actvity towards solid tumors. After expanding these immune cells 

, and enhancing them with our RNAi compounds, the cells are returned to the patient for treatment,” Dispersyn says.

“RNAi could improve the efficacy and safety of current T-cell directed IO therapies by inhibiting the expression of cancer-related proteins rather than inhibiting active proteins contributing to disease pathology,” adds Lewis. Moreover, tissue-specific promoters may provide more precise control of this inhibitory activity to target cells and reduce off-target side effects. “As an example, some checkpoint inhibitors block CTLA-4 [checkpoints T-lymphocyte-associated protein 4] activity on T cells to overcome T-cell suppression and stimulate a T-cell response against the tumor. RNAi designed to inhibit CTLA-4 expression can achieve the same objective but be more effective because it prevents the CTLA-4 signaling pathway from turning on rather than inhibiting signaling once the pathway is active,” Lewis explains.

The value of mRNA therapeutics in immuno-oncology, meanwhile, lays in mRNA’s ability to express therapeutic proteins directly from inside the tumor, initiating the immune response that destroys cancer cells, adds Becraft. This approach enables the encoding of any specific protein sequence that can then be delivered to the tumor itself. In this case, the tumor begins to directly secrete its own anti-tumor IO therapy, and it is this localized expression that ensures the immune system can recognize and attack the tumor cells. What’s more, this immune response can commence without the off-target effects that plague current immunotherapies that must circulate in the blood stream before reaching the tumor microenvironment, notes Becraft.

“This same philosophy can also be adapted to CAR T-cell therapy, where synthetic protein receptors are expressed and embedded on the surface of a patient’s T cells that then can recognize and kill off certain types of cancer cells. However, to accomplish this, current CAR-T therapies use a production method where the cells are extracted from the patient’s body, genetically engineered, then transplanted back into the patient. This approach is difficult to scale and control due to infrastructure and reproducibility challenges,” Becraft states. “At Strand, we believe we can bypass this issue using our self-amplifying mRNA therapeutics to deliver the instructions directly into the patient so that the T cells can make these protein receptors and become armed with the tools to recognize and kill the cancer cells.”

Though the use of nucleic acids in biotherapeutic development has long been studied in research, the practical use of such therapeutics is still in its infancy at the industry level. However, progress toward commercialization is expected to move forward in light of growing interest, investment, and success thus far of early entrants, such as mRNA vaccines.

Feliza Mirasol is the science editor for 

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Further development of nucleic acid-based therapeutics has been accelerated by the recent success of mRNA vaccines.

Shining a Spotlight on Nucleic Acid-Based Therapeutics

As cell and gene therapies continue to grow as novel treatments for unmet medical needs, biopharmaceutical companies will need to assess their options for meeting production demand. Two categories of cell therapies represent different production processes: autologous products use the patient’s own cells, and allogeneic products are manufactured using tissue from a healthy donor.

spoke with Joe Codamo, PhD, head of Global Cell Therapy Business, Catalent, and Jose Cruz, director of Vector Manufacturing at Genezen, about the challenges of scaling up the production of cell therapies and ensuring capacity is met.

 What are the specific needs for scaling up commercial production of cell therapies?

 Autologous and allogeneic therapies are at various stages of their product lifecycles, with some shared and some unique manufacturing needs. The starting material for autologous products can be highly variable given the disease state of the patient. The ‘vein-to-vein’ process lead time is critical, so the manufacturing, product testing, and release timelines should be compatible. For allogeneic therapies, the scale-up and generation of a reproducible cell expansion process is key, as well as robust formulation and high-throughput fill/finish capacity. Shared needs include implementing a secure, robust supply chain that can support the necessary scale, throughput, and reproducibility needed once a product has been commercialized. The associated cost of goods must be appropriate, especially as some may become second- or even first-line treatments.

 There are several challenges that all developers can expect to face when scaling up cell therapies to commercial production. A key need is to manage variability seen in development data that can be magnified upon scale-up. A structured quality-by-design (QbD) development approach fit to transition into commercial manufacturing upon regulatory approval will also need to be implemented. With increasing IND [investigational new drug] applications for novel and innovative cell therapies, it is likely that existing processes will not easily fit to new projects. It is therefore essential that critical core technologies are developed in a way to allow quick tailoring to these specialized processes.

 Are there specific concerns regarding capacity for cell therapies?

The upsurge of IND applications in the cell therapy space has led to an increasingly crowded pipeline. Cell manufacturing technologies are typically very specialized, as opposed to ‘one-size-fits-all,’ resulting in further capacity challenges due to the requirement for highly customized processes.

It is essential that pharmaceutical companies can quickly and thoroughly characterize and release vector technologies. This will enable generation of qualified data that can sustain a candidate through clinical trials, as well as supporting the transition into commercial manufacturing. However, increasingly crowded pipelines can impede these processes.

With cell therapies using autologous (patient) cells specifically, such as CAR [chimeric antigen receptor] therapies, we see a need to ensure sufficient physical capacity to manage the different cells for facility processing. This is due to the requirement for isolation and separate cleanrooms when working with patients’ cells for clinical trials.

This work is predominantly manual, but there are self-contained systems available; there are moves towards automating transduction and expansion of patient’s cells in the future.

 As the number of cell therapies in the clinical pipeline grows and more reach later-phase studies, demand for late-stage and commercial-scale manufacturing is growing correspondingly. This demand will only increase as market acceptance expands and cell therapies are potentially introduced at earlier stages of a patient’s treatment. The CDMO [contract development and manufacturing organization] industry has taken steps to help address the growing demand, as evidenced by the significant number of acquisition and expansion announcements in manufacturing capacity made over the past few years. Getting new facilities online, staffing with appropriately experienced scientific and technical personnel, and having a robust supply chain to meet the markets’ needs are concerns for all advanced therapies, including cell-based ones.

What are the steps for ensuring a contract partner has the capacity to manufacture cell therapies to commercial scale?

When evaluating a potential manufacturing partner, it is important to understand what its scale-up strategy is. For example, has it designed or optimized its infrastructure to deliver larger-scale and/or higher throughput, and is it working with its own supply chain to help assure high-quality production and consistent material quality? CDMOs should have a clear technical transfer and ramp-up strategy to go from clinical to commercial manufacturing. This [strategy] includes a process validation strategy and the supportive studies to demonstrate process and analytical robustness. Finally, the CDMO should have a global strategy to prepare for, and support, commercial launch, including topics spanning communication with regulatory authorities to a robust strategy for business continuity (staffing, material contingency, facility operations, etc.).

 One of the biggest concerns highlighted by clients is the need to find a CDMO offering both the roadmap to commercially manufacture cell therapies as well as the technical skill set to solve the inevitable challenges along the way. Selecting a partner with future commercial capacity while demonstrating cross-industry experience in the clinic, regulatory, and manufacturing sciences is key to successful commercialization, but they can be difficult to find.

Once a suitable partner is identified, early engagement between developers and the technical team is needed to develop a ‘best-launch’ process with an overarching aim to create a ‘best-in-class’ scope of work.

What are the benefits of outsourcing the production of cell therapies?

 Outsourcing cell therapy production has many benefits, particularly for smaller and younger companies with limited financial capital aiming to minimize the fixed costs that can be associated with building physical infrastructure.

Working with a dedicated CDMO partner also provides access to cross-functional expertise from process development, manufacturing sciences, operations, engineering, quality assurance, and regulatory. The seasoned expertise offered by CDMOs can act to bridge the path between discovery and clinical, as well as development and commercial manufacturing.

Working with CDMOs that have standardized development processes
built on QbD principles while offering tailored-fit technologies to manage inherent variability of cell processes will also help to increase speed to market.

An outsourced manufacturing partner can offer scalability, quality, and the regulatory experience gained from producing a variety of products and modalities. A CDMO provides opportunities for supply-chain streamlining and the leveraging of strong, global supply arrangements, as well as the necessary manufacturing capacity to help an early-stage cell therapy company ready itself for production within fast-paced timelines. CDMOs that can support end-to-end services including starting materials (plasmid DNA, viral vectors) and clinical logistics can simplify the supply chain. Outsourcing allows companies to avoid investing in CGMP [current good manufacturing practice] facilities, which may not be cost effective, and pose a financial risk when product demand and its clinical or market success
is unknown.

What are the challenges of outsourcing cell therapy production?

 These can include the management of timelines and communication with manufacturing partners, especially if multiple partners are involved in the process. Gene-enabled cell therapies require viral vector manufacturing as well as the production of the final product, and manufacturing of the cell therapy cannot begin until the viral vector is available. The cell therapy company may need to manage the communication between the CDMOs and their timelines, or if using a single manufacturing partner with integrated services, ensure that the partner provides one main point of contact and streamlines the integration between the supply chain stages.

 As well as the challenge of identifying a CDMO offering both expertise and capacity, it is also necessary to ensure the availability of this capacity in the required timeframe and availability of analytical technologies.

These analytical methods include master cell bank (MCB) and vector product release testing, RCL assays, HEK293T host cell protein (HCP) assays, and others that are essential in determining quality at different stages of the process.

Developers looking for a CDMO partner should also consider whether they are looking for a short or long-term investment. If they envisage the project scaling up from discovery to clinical supply and then to commercial manufacturing, they must determine whether they wish to eventually bring specific steps in-house. If aiming to bring the product to market with support from a CDMO, they should determine whether their partners have these capabilities early in the project’s lifetime to ease transitions and avoid delays.

 What is the ratio of in-house vs. outsourced production of cell therapies? Do you see the industry increasing the amount of outsourcing for these therapies?

For small-scale needs, such as in the early-phase studies, we estimate that more in-house production is currently conducted. However, once either larger batch sizes are needed, or a wider distribution of manufacturing is necessary, the ratio of in-house to outsourced manufacturing changes. Outsourcing partners with established clinical supply capabilities as well as manufacturing expertise are better suited to manage larger scale or larger numbers of batches and the vein-to-vein logistics. For those with in-house capabilities, there is the potential for secondary and back-up supplies to be provided from outsourcing partners as therapies receive worldwide approval.

Currently, the ratio of in-house to outsourced viral vector manufacturing is around 40:60 with lentiviral vector and adeno-associated viral vector manufacturing constituting 66% of total projects (1). Outsourcing is expected to further increase to 68% in the next five years.

Cell therapies using allogeneic (donor) cells, as opposed to autologous cells, could drive greater outsourcing. As managing or handling autologous cells is a critical step, companies are often keen to keep this process in-house. Unlike autologous cells, allogeneic cells have the potential to be produced in one large batch before being packaged into individual doses. Although less personalized, these therapies require processes that are much more manageable at large scale as compared with autologous cell handling and may be more suited to outsourcing.

1. E. Harris, “A Summary of Cell & Gene Therapies Market Outlook Report,” 

Outsourcing partners are ready to fill the market need for greater cell therapies capacities.

Scaling Up Cell Therapies Capacity

908 Devices, a US-based company specializing in chemical and biomolecular analytical devices, has formed a collaboration with CPI to optimize mammalian cell-culture media and feeding strategies. The aim of the project is to improve growth, protein titer, and protein quality attributes.

Under the agreement, CPI researchers will employ 908 Devices’ REBEL desktop analytical device for at-line cell-culture media analysis, which will help to identify and subsequently control key process parameters in bioprocessing workflows, the companies stated in an April 21, 2022 press release. Currently, 908 Devices and CPI scientists are implementing at-line media analysis for a Chinese hamster ovary cell line expressing a monoclonal antibody in small-volume cell culture bioreactor systems through to process scale-up. The scientists are monitoring nutrient levels critical to process stability and product quality and are ultimately controlling outcomes through the design of media formulations and the implementation of optimized feeding strategies, according to the release.

Time course data from the REBEL device provides information and insights into the cell metabolism, in addition to typical cell-culture parameters, such as cell count, viability, and basic metabolite data. These data sets are being mined with the goal of understanding specific amino acid depletion. Amino acid depletion could correlate with low titer and growth as well as potential protein quality attributes.

“The measurements from the REBEL device have enabled us to gain an improved understanding of the cell culture environment within a bioreactor,” said Bethany Kerr, team leader, Upstream Development at CPI, in the press release. “It has also enabled us to assess the impact of additional control over key process parameters. Data generated from the REBEL can be used to develop predictive models for feed control.”

Editors Note: A previous version of this story errenously stated that the companies partnered to develop cell culture media. This has been updated to clarify that the companies are partnering to optimize cell culture media.

News

908 Devices and CPI have formed a collaboration to optimize cell culture media to allow for improved process control.

908 Devices and CPI Partner to Optimize Cell Culture Media for Improved Process Control

Lonza and Integral Molecular recently announced a strategic collaboration wherein they will work together to assess the risks of off-target binding of biologic drug candidates. The collaboration aims to provide a better method of assessing immunogenicity, immunotoxicity, and cross-reactivity potential early in a biologic’s development cycle.

According to a press release from April 25, 2022, the companies feel that their respective expertise complement one another in this endeavor. Lonza’s early development services focuses on cell-based assays, which evaluate the activity of biologic therapies upon binding. Integral Molecular, on the other hand, detects off-target binding liabilities via its Membrane Proteome Array platform.

“Early immunogenicity de-risking significantly increases chances of the future success of therapies as they progress through the development pathway,” said Yvette Stallwood, head of early development services, Lonza, in the press release. “Lonza’s 14 years of expertise in 

 immunogenicity and immunotoxicity assessments complements Integral Molecular’s valuable insights into off-target binding using [its] Membrane Proteome Array.”

“We are pleased to offer complementary expertise to Lonza’s cell-based assay experience for preclinical safety profiling of biologics, thereby offering drug developers a more complete evaluation methodology to select the optimal candidate to move forward to IND [investigational new drug] filing.” said Benjamin Doranz, CEO and co-founder, Integral Molecular, in the press release.

Lonza and Integral Molecular will collaborate to better assess the risks of off-target binding of biologic drug candidates.

Lonza and Integral Molecular Enter Biotherapeutics De-Risking Collaboration

MilliporeSigma, the US and Canada life science business of Merck KGaA, announced that its ZooMAb recombinant antibodies earned an accountability, consistency, and transparency (ACT) label from My Green Lab, a non-profit focused on promoting sustainability in science. ZooMAb is the first-ever antibody to receive the ACT label designation, according to MilliporeSigma in an April 14, 2022 press release.

According to the press release, the antibodies received the lowest environmental impact factor (EIF) scores in the chemicals and reagents category. MilliporeSigma attributes multiple characteristics of the antibodies to this score:

As recombinant antibodies, they can be shipped in room temperature packaging. Traditional monoclonal antibodies (mAbs) must be shipped in specially insulated, ice-cooled containers, which contribute to high packaging material consumption and transport emissions.

The antibodies weigh 2.5 grams, which allows for more opportunities to eventually ship the product in much smaller packaging. MilliporeSigma estimates that this would prevent the emission of around 175 metric tons of CO2 per year by 2025.

While the antibodies are based on animal cells, like traditional mAbs, the recombinant technology they employ provides a greater, more consistent supply. It also reduces the use of animals in research.

In addition to these factors, the manufacturing facility where the antibodies are produced has implemented energy, water and waste reduction measures, produces renewable energy from a wind farm, and has an environmental management system program that is International Organization for Standardization 14001 certified. The antibodies also do not contain any Red List chemicals, carcinogenic, mutagenic or toxic to reproduction substances, polybutylene terephthalate, or GreenScreen Benchmark-1 chemicals, further contributing favorably to its low EIF score.

“Helping our customers and suppliers to reduce their impact on the environment is a key part of our sustainability strategy,” said Jeffrey Whitford, head of sustainability and social business innovation, MilliporeSigma, in the press release. “By showing how products rate in different categories, such as manufacturing; energy and water use; packaging; and end-of-life, the ACT label gives scientists another data point to help increase their sustainability. We are proud to be part of this program and are thrilled to achieve one of the top ranking EIF scores for our ZooMAb antibodies.”

MilliporeSigma’s ZooMAb antibodies have been recognized for their low environmental impact by My Green Lab.

MilliporeSigma’s ZooMAb Antibodies Earn ACT Label from My Green Lab

Standard BioTools, previously known as Fluidigm, announced the launch of the Hyperion+ Imaging System, a high-plex spatial imaging tool, in April 2022. Relative to the original Hyperion Imaging System, the Hyperion+ provides lower limits of detection, improved sample capacity, and quicker time to results.

According to an April 11 press release from the company, the system can process over 100 samples per week, approximately twice the speed of their current model. It also has a 1.6x lower limit of detection, enabling detection of dim markers, which can be important when analyzing the lower-expressing markers often used in translational studies.

The device allows for scanning of eight to over 40 targets in a single run and can easily be scaled to more than 100 targets. Additionally, by using an efficient, simple stain, image, and analyze workflow, it reduces expenses up to 10x relative to immunohistochemistry, which uses complicated cyclic immunofluorescence or digital profiling approaches, according to the release.

“Two critical challenges in realizing the full transformative potential of this remarkable technology are reducing the time to answer key biological questions and detecting important biomarkers that are expressed at low levels,” said Michael Egholm, CEO and president, Standard BioTools, in the press release. “The Hyperion+ Imaging System is designed to solve these challenges with faster time to results and a lower limit of detection than the current Hyperion Imaging System. These capabilities are key to quickly uncovering important spatial relationships with high-plex spatial imaging of 40-plus markers simultaneously at subcellular resolution.”

“My research group has been able to rapidly conduct exquisitely detailed phenotyping studies using the speed enhancements now available in the Hyperion+ Imaging System, which give us unprecedented insight into how cells within the tumor microenvironment function and interact with each other and the extracellular matrix,” said Bernd Bodenmiller, professor at the Department of Quantitative Biomedicine, University of Zurich, and at the Institute of Molecular Health Sciences, ETH Zurich, in the press release. “These insights are the foundation for innovating the next generation of cancer diagnostics and therapeutics and improving outcomes for patients with cancer.”

New Technology

Standard BioTools’ new Hyperion+ Imaging System can process more samples and has lower limits of detection than their current product.

Standard BioTools Launches Hyperion+ Imaging System

In April 2022, Bio-Rad Laboratories introduced three new StarBright Dyes: StarBright UltraViolet 510, StarBright UltraViolet 665, and StarBright UltraViolet 795. These dyes are designed for use with a 355-nanometer ultraviolet (UV) laser in flow cytometry applications. StarBright UltraViolet 510, 665, and 795 dyes were designed to offer enhanced brightness with narrow excitation and emission profiles, allowing for precise resolution.

The StarBright line of dyes offers researchers unique fluorescent nanoparticles conjugated to highly validated flow antibodies, which are compatible with most experimental protocols and flow cytometers, including Bio-Rad’s ZE5 Cell Analyzer and S3e Cell Sorter, according to the company’s April 19, 2022 press release. The dyes are resistant to photobleaching and are highly stable with no loss of signal in fixation. Furthermore, the dyes minimize lot-to-lot variation, which helps to ensure consistent staining and reliable results.

“We are proud to add three new dyes to Bio-Rad’s StarBright UltraViolet portfolio, following its launch last year, and are now better positioned to support researchers with their flow cytometry workflows by providing a range of high-performance dyes excitable by the 355 nanometer UV laser to existing alternatives,” said Mike Blundell, PhD, product manager, flow cytometry, Life Science Group, Bio-Rad, in the press release. “The unrivaled brightness of the dyes enables enhanced resolution of low-density antigens and rare populations while maintaining consistent and reproducible staining without the need for special buffers.”

Bio-Rad Laboratories introduces three new fluorescent dyes under the StarBright product range to support multicolor flow cytometry panels.

Bio-Rad’s Three New Fluorescent Dyes are Intended to Enhance Multiplex Flow Cytometry

Otsuka Pharmaceutical announced on April 15, 2022 that it has entered an exclusive, worldwide license agreement with Osaka University to use R8H283, a novel antibody that recognizes the CD98 heavy chain identified by Osaka University, in its pharmaceutical and medical products.

An Osaka University research group led by Professor Naoki Hosen (Department of Hematology and Oncology, Osaka University Graduate School of Medicine) identified R8H283 as an anti-CD98 heavy chain antibody. CD98 heavy chain is a protein expressed in a wide range of tissues, but R8H283 specifically binds to multiple myeloma. R8H283 was shown to have significant anti-tumor effects in an animal model study, Osaka University announced in February 2022.

Under the terms of the agreement, Otsuka will make an upfront payment, development and sales-milestone payments, and sales royalties to Osaka University. Otsuka will utilize R8H283 exclusively to perform non-clinical research, clinical development, manufacturing, and commercialization.

"R8H283, a unique multiple myeloma-specific antibody, has the potential to be applied not only to antibody drugs but also to various other medical products, including CAR-T cells,” said Professor Naoki Hosen in a press release. “In addition, this antibody could be applicable to other types of cancer. We believe that this agreement is very important in bringing these products to patients as soon as possible. We will continue our efforts to conduct research that will be useful in the treatment of patients suffering from cancer."

Otsuka Pharmaceutical and Osaka University have entered an exclusive license agreement on a new anti-tumor antibody.

Otsuka Pharmaceutical and Osaka University Enter Exclusive License Agreement 

Thermo Fisher Scientific has introduced its new GMP-manufactured Gibco CTA TrueCut Cas9 protein to support genome editing for research and manufacturing applications. The proteins are manufactured with 

) standards in mind, including traceability documentation, aseptic manufacturing, and safety testing.

High-quality ancillary materials and documentation are critical as researchers using genome editing tools move from basic research to clinical settings, including Chimeric antigen receptor (CAR) T-cell therapy research. The Gibco CTS TrueCut Cas9 protein provides greater than 90% efficacy in human primary T-cells, making it a promising solution for CAR T-cell therapy research, according to the company. Along with the company’s recently launched Gibco CTS Xenon Electroporation System, the GMP-manufactured Cas9 protein can aid Thermo Fisher in supporting researchers across the autologous T-cell workflow.

"As genome editing quality and regulatory requirements continue to evolve, we are focused on providing solutions to meet scientists' needs as they transition from the bench to the clinic," said Brandon Pence, vice president and general manager of cell biology at Thermo Fisher Scientific, in a press release. "By adding the Gibco CTS TrueCut Cas9 Protein to our portfolio of genome editing solutions, we can now support scientists in their journey to develop personalized CAR T-cell therapeutics."

Thermo Fisher Scientific has launched its first GMP-manufactured Cas9 protein for clinical genome editing research.

Thermo Fisher Scientific Launches New GMP-manufactured Cas9 Protein

VectorBuilder, a global gene delivery solutions company, announced on April 11, 2022 the construction of a new $500 million R&D and manufacturing center in Guangzhou, China. The ‘Gene Delivery Research and Manufacturing Campus’ will aid in the growth of the company, expanding its R&D capabilities and production capacity for various gene delivery vectors.

The campus will include a state-of-the-art contract development and manufacturing organization facility designed for current good manufacturing practice manufacturing of plasmids, messenger RNA (mRNA), AAV, lentivirus, cell lines, and other types of viral and non-viral vectors. The campus will also house a research institution dedicated to developing new gene delivery techniques, vector-based vaccines, virus-based cancer therapeutics, and training for scientists and engineers joining the gene delivery field.

Construction of the campus will be split into two phases over the next four years with an expected cost of $500 million and 100,000m2 of floor space. The project is part of a global expansion of the company, which is also planning new manufacturing sites in the United States, Europe, and Japan.

“Modern biology is largely built on gene delivery technologies, but until recently, such technologies are mostly limited to research use,” said Dr. Bruce Lahn, chief scientist at VectorBuilder, in a press release. “With the recent advancement of genetic medicine, gene vectors are now rapidly moving into clinical use, including CAR-T, gene therapy, mRNA vaccines, and oncolytic viruses. Some experts predict that in 10 to 20 years, vector-based drugs will become the third pillar of medicine, after small-molecule drugs and protein-based biologics. We are therefore expanding our R&D capabilities, as well as our manufacturing capacity, to continue leading the way in the development of innovative gene delivery technologies that will make research more efficient, and genetic medicine more effective and affordable.”

VectorBuilder will expand with the construction of new $500 million Gene Delivery Research and Manufacturing Campus in Guangzhou, China.

VectorBuilder to Expand with $500 Million Gene Delivery Research and Manufacturing Campus

SphingoTec, a German diagnostics company, announced on April 11, 2022 the implementation of two critical care biomarkers to guide the application of a potential new drug candidate for the treatment of COVID-19 patients. The trial will be led by University Hospital Hamburg-Eppendorf (UKE).

The trial will evaluate one of the first personalized approaches to treat moderate to severe COVID-19 with the drug candidate Adrecizumab (HAM8101; INN: enibarcimab) (1). The trial will use bioactive adrenomedullin (bio-ADM) as a biomarker to help with the early identification of endothelial dysfunction, a condition that has been shown to play a central role in the pathophysiology of COVID-19. Adrecizumab can then target high levels of bio-ADM in patients who have gone into septic shock. The use of this drug candidate has already shown favorable outcomes in a case series of eight severe COVID-19 patients.

The second biomarker in the trial will be dipeptidyl peptidase 3 (DPP3); but in this case, the second biomarker will be used to identify and exclude patients who exhibit high levels of DPP3. This is because DPP3 indicates that there is another underlying disease mechanism existing within the patient that can lead to cardiac depression. In that case, that patient would require a different therapeutic option than the Adrecizumab used in the trial. Both bio-ADM and DPP3 levels will be measured using SphingoTec’s rapid point of care platform Nexus IB10.

“Not all COVID-19 patients have the same disease course, and the pathology is very different,” said PD Dr. med. Mahir Karakas, part of the Department of Intensive Care Medicine at UKE. “Therefore, we have to adapt the treatment to each individual patient. We will now use in a double blind randomized multicenter trial the biomarker bio-ADM and DPP3 which proved to be useful for patient enrichment in a Phase II trial for treatment of septic shock.”

“The innovative biomarkers generate better insights into the pathophysiology of critically ill patients,” added Dr. Andreas Bergmann, founder of SphingoTec and co-founder of Andrenomed. “This is translated into clear decisions that allow a better treatment guidance and ultimately reduce mortality. This study represents a leap from “one drug fits all” to a personalized approach in treating COVID-19.”

1. Adrenomed, “Research & Development,” 

Biomarker-guided trial led by University Hospital Hamburg-Eppendorf (UKE) opens new avenues for personalized medicine for treatment of COVID-19.

BioPharm International's BP Elements, May 2022