Adenovirus (Ad) vectors are used to develop gene therapy products and vaccines. Decades of research stand behind these vectors which have gone through several iterations and improvements to make them what they are now. As mentioned in a previous article, the E1 proteins which control the adenoviral replication cycle were removed in very early iterations of the adenovirus vectors. This prevented the virus from replicating in humans, thus only allowing the virus to deliver its genetic payload. In addition, the E3 region, which is not essential for viral propagation, was removed. The space freed up in the wildtype virus genome by the removal of E1 and E3 allowed for the insertion of up to 6.5 kb of foreign DNA.

This was the first generation of Ad vectors. In the second generation more improvements were made when E2a, E2b and E4 were removed. The removal freed up yet more space in the genome for up to 10.5 kb of transgene DNA to be inserted into the chromosome. This was particularly relevant for researchers desiring to put multiple antigens into one vector. For instance, in pursuit of AIDS vaccines it was shown that a combination of HIV virus-derived envelope together with Gag and Pol provided broad immune responses. The deletion of more genes encoding for E proteins from the viral backbone also improved the safety of Ad vectors by making it less likely that spontaneous recombination events during the vector propagations would lead to replication-competent viral particles. The deletion of multiple E-encoding genes from the viral genome also led to a significant reduction in viral gene expression in host cells, lowering a cytotoxic T-lymphocyte response against the vector itself. This made second generation Ad vectors much less likely to be cleared by the immune system.

Third generation Ad vectors are void of all viral sequences except for the inverted terminal repeats, which are the signals for the DNA to be effectively packaged into the adenoviral capsid proteins. These high-capacity adenovirus vectors can package up to 36 kb of foreign genetic material. To produce these vectors, adenoviral helper viruses are required to produce the proteins needed for replication and packaging of the Ad vector genome.

Recombinant Ad vectors have been around for a long time and the active research around them has led to great leaps forward in their usage and in the fields of gene therapy and vaccine production. Currently, it is possible to make conditionally replicating Ad vectors, which only replicate inside tumor cells. When this is combined with the effective cell targeting provided by transgenic knob proteins on the capsid surface, Ad vectors become a truly powerful tool for cancer therapies as well.

Main advantages for Adenovirus vectors

Ad vectors have advanced further than any other vector system owing to continued active research from academia and industry together. This research has made Ad vectors more effective while also making them safe. Modern Ad vectors have four key advantages in gene therapy and vaccine development.

1. High transduction efficiency in dividing and quiescent cells

This is one of the reasons that adenoviruses were first considered as vectors for gene therapy and vaccines. Ad vectors can deliver genetic cargo to cells very efficiently, so that therapeutic levels can be achieved with fewer viral particles. This is extremely important for in vivo systemic applications where high concentrations of Ad vector are typically more challenging.

2. Epichromosomal persistence in cells

This feature of Ad vector has led to some very specific and highly sought-after applications. Persistence of the vector is important to allow the genetic payload to be delivered, transcribed, and expressed as therapeutic proteins. Without the ability to persist, these therapies would not be present long enough to be effective. However, a common mode of persistence for viruses is chromosomal integration. This is often undesirable or unnecessary in gene therapy and carries many safety concerns specifically for DNA vectors. Because Ad vectors do not integrate into the chromosome of the host cells, there is no risk that they will permanently alter the host genetic make-up.

3. Broad tissue tropism

A wide variety of wild-type adenoviruses have been modified to create Ad vectors, providing a range of different tissue tropisms. More specific tropism has been developed in many therapeutic cases thanks to the genetic manipulation of the knob protein, which is the protruding end of the fiber capsid that is primarily responsible for host cell attachment. This protein is the key to unlock the entry into specific cells through receptor-specific binding. By modifying the knob protein, it is possible to make Ad vectors that can be precisely targeted to specific tissues.

4. Scalable production

Gene therapy products that cannot be scaled to commercial manufacture cannot be used to treat real-world diseases. As we saw during the rollout of the COVID-19 vaccines, Ad vector-based products can be rapidly scaled to meet market demand. To meet demand for the COVID-19 vaccine, each 5-day production cycle readily delivered over 15 million doses of vaccine to battle SARS-CoV2 virus.

Together, the described benefits of Ad vectors provide a broad and versatile platform to achieve sought-after clinical breakthroughs in the treatment and prevention of debilitating and life-threatening diseases

Clinical applications

Clinical applications of Ad vectors can be broadly split into two categories: vaccines and gene therapies.

Vaccines

Basic research in pursuit of improved Ad vectors revealed that the high prevalence of wild-type adenoviruses in the human population has led to widespread pre-existing immunity, particularly to some human serotypes like Adenovirus serotype 5. Developments in the field have turned this potential drawback into an important feature of Ad vector-based vaccines.

With respect to vaccine development, the main purpose of a DNA-based vector is to deliver epitopes from other viruses to host cells and to ensure production of such epitopes in order to raise an broad and effective immune response against the virus. Over the iterations of development, Ad vectors have become better at delivering increasingly large nucleic acid payloads. In addition, the immunogenicity of some Ad vectors has been tweaked and harnessed to boost the production of pro-inflammatory cytokines that can enhance the humoral and cellular immune responses.

A successful application of this approach is the Ad vector-based Ebola vaccine developed by Janssen. This vaccine, which takes advantage of Janssen’s rare Ad26 serotype, induces specific antibody and T-cell responses against Ebola virus. Clinical results demonstrated that the vaccine elicits a powerful humoral immune response in humans that persists for more than a year. Rapid and durable T-cell responses using Ad26 were also seen in adenovector-based COVID-19 vaccines, which induced strong humoral and cellular immune responses in 100% of clinical trial participants after two doses.

Ad vectors have also been widely used for the production of cancer vaccines. Current vaccine research is focused on prostate cancer, human papilloma virus, colorectal, and pancreatic cancers. In addition to cancer prevention through vaccines, Ad vectors are also used for anticancer therapies.

Oncolytic virotherapy

Anticancer therapies using adenovirus vectors fall broadly into three main categories:

1. Delivery of suicide genes

Many tumor cell types proliferate rapidly and have a dysfunctional p53 tumor suppressor pathway. Ad vectors can be engineered to induce p53 expression inside tumors, triggering cell death. Another successful application has been using Ad vectors to deliver genes that convert a pro-drug into an active cytotoxic agent. For example, the enzyme purine nucleoside phosphorylase converts the pro-drug fludarabine monophosphate into fluoroadenine, which kills proliferating cells. Trials using this enzyme have already been conducted using Ad vectors. The ability of Ad vectors to remain localized is extremely valuable when delivering cytotoxic therapies that can harm heathy cells if the treatment is not constrained to the target cell populations.

2. Delivery of immune-regulatory genes

Ad vectors can also be loaded with genes that stimulate an antitumor immune response. Antitumor interferon-β and interferon-α-2b have both been safely delivered to the lungs of patients via intrapleural injection.

3. Chimeric and tropism-modified oncolytic Ad vectors

A problem that often arises during cancer therapies is poor recognition of tumors by immune cells and Ad vectors. In this case, Ad vector knob proteins can be modified to bind more strongly to receptors on the surface of the tumor. One example of this is in an Ad vector used to treat ovarian cancer where the entire fiber knob domain of Ad5 was replaced with that of Ad3. The result was targeted ablation of ovarian cancer cells displaying elevated levels of Ad3 receptors.

Adenoviral vectors have come a long way since their initial use several decades ago. Research has shown that they can be used extremely effectively for a number of gene therapy and vaccine applications.

Adenoviruses have been shown to be very effective vehicles for delivering genetic material to cells and are therefore in demand to develop gene therapies and vaccine products. The road from early-stage R&D to final production of an adenoviral vector product is well trodden, and there is a lot of detailed information out there to help you on your journey.

That said, taking an adenoviral product to the clinic always requires some process development prior to bringing the vector into GMP. This is based on the many examples in practice where the transgene had detrimental effects on vector yield or purity once inserted into the vector. For instance, the foreign gene may encode for a protein that interferes with HEK293 cell propagation, resulting in low yield of vector particles per cell. Likewise, the protein produced may bind to the Adenoviral vector capsid proteins, which are generally carry a net negative charge that can substantially affect the purification strategy.  A good way of mitigating risk is to seek expertise at the relevant stages of development to fill informational gaps and reduce the possibility of unexpected problems while moving from bench to clinic.

Working with a center of excellence contract development and manufacturing organization (CDMO) is an attractive option for many who embark on the adenoviral production journey. Other than a general vendor type CDMO, A center of excellence CDMO has in-depth experience and can help guide the process by providing practical solutions to challenges that may pop up.   If you are developing a therapy from the very beginning, a “center of excellence CDMO” can often complement your expertise and help you navigate the complexities of the scale-up process.

In this article, we highlight how a “center of excellence CDMO” can help you to smoothly navigate the transitions from R&D to scale-up and clinical manufacturing. The information here will provide you with a stable foundation when you start a conversation with a CDMO, allowing you to identify important traits that will give you a strong relationship going forward.

Common challenges that a CDMO can overcome

Concentration challenges

One of the most important considerations that need to be made early in the product development trajectory is the concentration of the vector needed to be an effective therapy in the clinic (part of Target Product Profile setting).

For adenovector based vaccines, a dose of 10¹⁰-10¹¹ virus particles per milliliter typically suffices for systemic administration i.e. intramuscular injection. For oncolytic therapies, either higher or lower doses could be required, depending on the route of administration and the target tissue. For instance, treatment of a wound-bed post cancer surgery may require doses of up to 10¹³ virus particles per milliliter to effectively deliver a high dose in a confined location with minimal spill-over to surrounding tissue.

At such high vector concentrations, aggregation of virus particles may occur, which reduces therapeutic efficacy. The high concentrations needed for many oncolytic and gene therapies exacerbate this problem. Aggregation can manifest itself in quite spectacular fashion, as concentrated adenovirus preparations become cloudy before “snow” appears on the bottom of the vial. Understanding how adenovirus particles behave and good forward planning are essential to reduce the chances of aggregation.

On the other end of the spectrum, there is also a risk that during the scale-up process the concentration step will not be sufficiently effective, leading to viral titers that are lower than expected. This is a critical issue that can arise due to the significant alterations that are often required during scale-up of concentration steps, such as switching from ultracentrifugation to chromatography.

Since the stakes and capital investment are always higher the further down the scale-up path you are, it pays to address any issues relating to concentration as early as possible.

Release of intracellular DNA

Eukaryotic cells have to be lysed in order to release the adenoviruses, and in the process the release of intracellular DNA is inevitable. The more viral particles needed, the greater the number of cells that must be lysed, and therefore the more host cell DNA is released. Unfortunately, DNA often forms an intricate and sticky web that easily traps adenovirus particles, causing them to aggregate. As discussed in the previous paragraph, adenoviruses are already prone to aggregation, so adding DNA into the mix only increases the risk. The DNA webs also are notorious for clogging filters, which further hampers the purification process. These problems can be circumvented in the process development stages by adding a DNA reduction step such as the introduction of benzonase. With careful quality control, interventions like this can mitigate the negative effects of extracellular DNA.

Process yield & scalability

Perhaps the biggest consideration when manufacturing Ad vectors is the process yield at large scales. In the R&D phase, simply ensuring there is “enough” virus to perform preclinical experiments is acceptable. However, production at larger scales inherently leads to larger losses. During the R&D phase it is therefore essential to prioritize yield. This can be done by reducing the number of empty capsids that are present in your viral preparation and ensuring that your process is ready for the switch to large scale production. This often requires changing from ultracentrifugation, which is a common way to concentrate adenovirus in the research lab but not appropriate for scaling. With technologies used for scale up such as chromatography, it is often difficult to achieve the same yield as ultracentrifugation which is why process development is so important.

How does a CDMO help?

Understanding the different applications for adenoviral vectors is a good way of highlighting how a good relationship with a center of excellence CDMO should work. The center of excellence will have industry experts who can save you a lot of time and expense in developing robust and scalable manufacturing processes for your vector. For example, at Batavia, our in-house experts have extensive experience in developing adenoviral vector-based therapies be it vaccines or oncolytic products. These experts are deployed to relevant projects to help guide the strategy and ensure the highest likelihood of manufacturing success.

Clear communication is a must for a good center of excellence CDMO. Clearly articulating the challenges and opportunities of a scale-up project is the only way to make effective progress. While good communication may seem less relevant than the technical capabilities of a prospective CDMO, it is nevertheless important and very easy to assess early on your interactions. Pay close attention to how your CDMO communicates. Make sure that misunderstandings are quickly resolved, and that communication is clear and timely. The way a CDMO communicates and structures their meeting and interactions is a reflection of how they operate, so if you are not satisfied, it’s best to move on.

A good CDMO should bring balance and clarity to the scale-up project. When you partner with a center of excellence CDMO, you should feel confident that you are in good hands. You should also feel that you acutely understand the process going forward, including the potential risks.

At some point when moving from R&D to scale-up and production, you will meet a whole range of different regulations that have to be thoroughly studied and strictly adhered to, if your adenovirus vector is to make it successfully into the clinic. A good center of excellence CDMO has experience with the relevant and most up-to-date regulations and requirements, and will help you navigate the complex landscape of rules and paperwork. Always ask about track record.

Choosing a CDMO

If you decide that working with a center of excellence CDMO is the right path for you, the next question is who should you choose? Fortunately, the CDMO field is competitive, and you do have a choice. It’s important that you exercise this choice and do your due diligence by thoroughly researching and interviewing your CDMO candidates. Pay close attention to the way they communicate with you and how they discuss your project and bring up challenges. Most importantly pay close attention to the quotation and make sure to read the small footnotes to understand what is included in price and what is excluded and needs to be paid additionally.

It’s never too early to begin discussions with a partner to help you scale your adenoviral vector product. Even informal conversations early on can give you insights and help you make decisions that will have a positive impact on the future of your project. To start with, why not book a call with one of our experts. We’re looking forward to hearing about your project and you can be sure that we’ll give you clear advice and an all-included costing overview for your project.