Cynthia A. Challener is a contributing editor to Pharmaceutical Technology.

Cynthia A. Challener

There is no doubt that chimeric antigen receptor (CAR) T-cell therapies have had a tremendous positive impact on patients that have received them; however, autologous cell therapies produced in centralized manufacturing facilities have inherent limitations that inhibit widespread availability and access. Allogeneic, or off-the-shelf versions, are one potential solution, but developers of these therapies must overcome different challenges, such as graft-versus-host disease, the potential for introduction of chromosomal abnormalities, ensuring successful gene editing of all donor cells, and problems with T-cell exhaustion (1).

Another option is to produce autologous therapies at the point of care (POC) (2). Companies such as Miltenyi Biotech, Lonza, SQZ Biotech, Orgenesis, and Ori Biotech have developed or are commercializing closed, automated cell- and gene-therapy production platforms with small footprints that can be installed at hospitals and institutions for good manufacturing practice (GMP) production of CAR T-cell therapies onsite. There are various university hospitals around the world exploring the potential of these systems. Orgenesis and aCGT Vector are establishing networks of POC sites leveraging their own production technologies.

A third approach being explored by several companies eliminates the need for gene editing of immune cells outside the body altogether. These solutions involve in vivo generation of CAR cells. This approach also has its challenges, but offers the potential to combine some of the benefits of both autologous and allogeneic cell therapies.

Autologous cell therapies today require collection of immune cells from the patient that are then shipped to a central manufacturing facility. Most patients are extremely ill and have endured multiple previous therapies, leading to variability in the starting material and the potential for manufacturing failures. Production slots are also limited, often creating delays that such ill patients cannot generally afford. Facilities that offer cell therapy are limited as well, and as a result, patients must travel to them, adding to the burden placed on them and their families. Furthermore, the cost of gene-modified cell therapies is high.

An IQVIA survey (3), notes Gregory Frost, chairman and CEO of EXUMA Biotech, revealed that the patient’s health status, cost to patient, and geographic distance for the patient to travel were the top three reasons why community oncologists do not refer patients for CAR-T therapy.

Allogenic cell therapies offer compelling benefits relative to autologous CAR T-cell treatments for certain indications. As off-the-shelf products, they are available immediately, eliminating the logistics complexities that add time and cost to autologous therapies. They also do not suffer from the variability associated with patient-derived cells.

Avoiding the natural immune response to foreign CAR-T cells has, however, proven much more challenging than past industry hurdles such as the advancement from mouse antibodies to human antibodies, according to Frost. “Although many different strategies are being explored, the only clinically validated solutions to this challenge to date has been sustained immunosuppressive treatment regimens to eliminate the body’s T and NK cell function, thereby increasing the risk of opportunistic infections without transplant-ward-level care,” he says. Generally as a result, the time that must be spent in the specialized care setting is often extended and introduces additional burdens for patients and their families that do not live near one of these specialized facilities.

 CAR therapies have the potential to offer the benefits of both autologous and allogeneic therapies. “The most apparent advantage that 

 CAR therapy could provide over autologous or allogeneic cellular therapy is to capture the durability benefits of autologous cell therapy with the reduced cost and complexity of an off-the-shelf product,” contends Frost.

If other safety parameters can be maintained clinically, Frost explains a CAR therapy that delivers a cost-of-goods-per-patient-treated on par with biologics may change the efficacy bar from 30–50% durable complete responses to a 30+% improvement in progression-free survival or overall survival, a particularly relevant goal for indications in solid tumors. “Moreover,” he adds, “lymphodepletion may not be necessary for 

 therapies, thereby potentially expanding the setting of care beyond tertiary care centers with intensive-care units and extending CAR therapy into indications not previously addressed (e.g., autoimmune disease) due to an unfavorable benefit-to-risk ratio.” Furthermore, as a true off-the-shelf option

 CAR therapy products are unburdened by the complex logistics for cryopreservation of a cellular drug product.

 CAR therapy “may address the significant concerns associated with autologous and allogenic gene-modified cell therapies and thus function to expand patient access to CAR therapy by making it safer, potentially more efficacious, less financially toxic, and available in settings more convenient to patients and their families,” Frost comments.

It is also worth noting that in addition to the reduced cost and complexity of therapy and the potential to expand to a wider array of care settings

 CAR/T-cell receptor (TCR) therapies reduce the complexity of cell processing validation, according to Frost. “While the product is the process,” he observes, “the 

 approach does not require the associated costs and time to develop an optimized and validated manufacturing cell process, which can introduce new challenges in comparability assessment with subsequent process changes.”

Frost also points out that enhancements and minor modifications to the transduction program encoded in the vector can easily be made, allowing for iterative evaluation preclinically and potentially clinically. “The versatility of the technology is anticipated to extend the technology broadly into academia and small and large biotechnology companies, which could result in the accelerated discovery and development of transformative therapies at substantially reduced costs,” he concludes.

 gene-modified cell therapies face their own set of technical challenges. A key issue, according to Frost, is the inability to perform preparative lymphodepletion of the patient prior to the infusion of the engineered cells. This procedure removes other immune cells that would otherwise compete with the CAR cells for growth factors and other nutrients, reducing their chances to survive, expand, and exhibit antitumor function. “Lymphodepletion is not a viable option for 

 CAR therapy since it would kill off the very cells targeted for in vivo engineering. As such, any 

 approach will need a means for ensuring robust proliferation and survival in an environment that is rife with competition for the same nutrients,” Frost says.

It is also essential that the vehicle delivering the genetic material for reengineering of the target immune cells is highly selective for the right cells. “Whether the vehicle is a viral vector or lipid nanoparticle (LNP), it must target delivery to the specific cell type to be re-engineered (e.g., T cell, natural killer cell) in order to prevent any unintended gene modification,” comments Frost.

Viral vectors and lipid nanoparticles represent two different approaches being explored for in vivo engineering. Nonviral approaches typically involve LNPs encapsulating nucleic acids, while viral approaches are generally based on the same retroviral vectors used in current 

Non-viral approaches, notes Frost, have the possible advantage of a more defined regulatory pathway based on the recent approvals of the COVID-19 vaccines. Non-integrating approaches may, however, not provide a sustained clinical response given that CAR transcripts become diluted (and eventually lost) as the cells divide, he observes.

Such therapies would likely require persistent retreatment in the absence of a clinical response, especially for the treatment of solid tumors, which require a prolonged T-cell response. “Integrating vectors, such as lentiviral (LV) vectors, may offer the benefit of stably
integrating into the host-cell genome to allow for a sustained response with a single dose,” says Frost. As such, he anticipates further investigations into the tropism of viral approaches to reduce the risks of adverse events associated with non-target cell integration.

The level of investment in in vivo gene-modified cell therapies suggests that this new approach truly does have real potential. Several startups have been established in the past few years with strong financial backing.

 CAR/TCR therapy platform, GCAR, comprises a lentivector with a CD3- targeting/activation element on its surface to target T cells and provide them with their initial proliferation stimulus. The genetic payload within the LV encodes for either a CAR or TCR, as well as EXUMA’s intracellular “DRIVER” signaling domain. Once integrated and expressed by T cells, DRIVER signaling and initial CD3 activation causes robust in vivo proliferation with persistence and antitumor activity in a lymphoreplete environment occurring from continued antigen engagement, according to Frost.

“Our proprietary LV design elements address the challenges faced by 

 CAR therapy, as the DRIVER allows for proliferation and persistence in the midst of competing cells, and the CD3-targeting moiety directs the lentivector to T cells,” Frost states. In preclinical animal models, EXUMA has confirmed enhanced tropism to T cells, circulating CAR-positive cells, and a potent, dose-dependent elimination of target cells upon 

Umoja Biopharma has developed its VivoVec surface-engineered LV vector-based off-the-shelf viral-vector particles for the generation of CAR-T cells 

 (4). The particles include rapamycin-activated cytokine receptors (RACRs) designed to selectively provide costimulatory molecules to CAR-T cells in the presence of rapamycin. Preclinical study results indicate the particle design demonstrates enhanced T-cell binding and activation both in vitro and 

, as well as generation of large numbers of CAR-T cells, thus enabling enhanced antitumor activity at lower doses.

Vector BioPharma’s Shielded, Retargeted Adenovirus (SHREAD) platform uses engineered adenoviral vectors combined with exogenous, high-avidity adapter proteins (in the form of virus-like particles) to achieve delivery to defined biomarkers on specific cells or tissues of DNA encoding various genes and regulators, allowing local production of potent drugs with reduced risk of systemic toxicities (5,6). The company was established in August 2022 to commercialize technology developed in the laboratory of Andreas Plückthun, PhD at the University of Zürich (7).

Ensoma, launched in February 2021, is also leveraging adenoviral vectors (8). Ensoma’s Engenious vectors are devoid of any viral genome, thus minimizing the chance of an immune response and freeing up to 35 kilobases of DNA packaging capacity to deliver a diverse range of genome modification technologies. The vectors can be used in vivo to engineer hematopoietic stem cells as well as erythroid, lymphoid (e.g., T cells, B cells), and myeloid (e.g., macrophages, microglia) cell types with precision. The company has entered into a strategic collaboration with Takeda Pharmaceutical Company (9).

Capstan Therapeutics, which was co-founded by Carl June, one of the original innovators in the autologous CAR T-cell-therapy space, and launched in September 2022 (10), is using mRNA technology to generate CAR-T cells 

. In one example, they injected CD-5 targeted mRNA-LNPs to reduce fibrosis and restore cardiac function in a mouse model of heart disease (11).

Interius BioTherapeutics is commercializing technology developed by Saar Gill at the University of Pennsylvania (12). The company is initially focused on treating hematologic malignancies using an in vivo CAR treatment, but hopes to expand to applications beyond immuno-oncology that address diseases not amenable to current gene-therapy modalities. It has provided minimal information about the specific technology involved.

gene-modified cell therapies at the preclinical development stage. SG295 is an 

 CAR T with CD8-targeted fusogen delivery of a CD19-targeted CAR for treatment of patients with B cell malignancies and for which the company expects to file an investigational new drug application (IND) in 2023 (13). SG418 is a hematopoietic stem cell-targeted fusosome with the ability to deliver gene-editing material 

 to repair genetic abnormalities such as those that cause sickle cell disease and beta-thalassemia. Sana hopes to demonstrate preclinical proof of concept in 2023.

Gene therapy provides a potential regulatory roadmap

gene-modified cell therapies constitute a novel drug class, they are related to direct gene therapies, several of which have received regulatory approval in the United States, European Union, and other countries around the world. These gene-therapy approvals, according to Frost, provide a helpful roadmap for regulatory evaluation of 

 CAR therapies. “While each product and platform has its own unique considerations that must be addressed for a specific indication, the FDA has also published numerous guidance documents regarding gene therapy that are helpful to sponsors developing 

As is the case with recent regulatory reviews of retroviral gene vectors in cells other than T lymphocytes, Frost expects that regulators will expect developers of 

 therapies based on retroviral vectors to demonstrate that those vectors are indeed transducing the target cell type (e.g., T cells) and not other cell types, such as hematopoietic stem cells or gametes. In this context, he notes that significant consideration must be given to the tropism of the delivery vehicle, whether viral or non-viral.

Clinical candidates anticipated in the next few years

Given the growing number of companies developing 

 CAR therapies using many different approaches, Frost anticipates candidates entering clinical trials within the next several years. The data gathered during those trials will serve to better refine the science. “With the advances that will be achieved and the growing body of evidence that will be generated as a result of these studies, it can be expected that the overall industry will become more comfortable with 

 gene therapy, ultimately facilitating the progression of other products to the clinic and eventually the market. The widespread use and breadth of enhancements to monoclonal antibodies, which were first discovered in 1975 and first approved in 1986, is suggestive of the potential for in vivo CAR therapy. With today’s technology and know-how, the innovation cycle for this technology is expected to be dramatically accelerated,” he concludes.

Preparing for the Allogeneic Cell Therapy Tipping Point

Alvaro, D. and Challener, C.A. Bedside Cell Therapies: Point-of-Care Manufacturing Is Slowly Becoming a Reality. Pharma’s Almanac, Nov. 15, 2022.

Saez-Ibañez, A. R.; Upadhaya, S.; Partridge, T.; Shah, M.; Correa, D.; and Campbell, J.Landscape of Cancer Cell Therapies: Trends and Real-world Data. Nature Reviews Drug Discovery, 2022. 21, 631-632, Supplementary Fig. 9,. doi: https://doi.org/10.1038/d41573-022-00095-1.

Umoja Biopharma. Umoja Biopharma Presents New Preclinical Data on its Integrated In Vivo CAR T and Engineered Induced Pluripotent Stem Cell Platform Technologies at the Society for Immunotherapy of Cancer (SITC) 37th Annual Meeting. Press Release, Nov. 10, 2022.

Freitag, P. C. et al. Modular Adapters Utilizing Binders of Different Molecular Types Expand Cell-Targeting Options for Adenovirus Gene Delivery. Bioconjug Chem. 2022 Sep 21;33(9):1595-1601. doi: 10.1021/acs.bioconjchem.2c00346.

Smith, P. C.et al. The SHREAD Gene Therapy Platform for Paracrine Delivery Improves Tumor Localization and Intratumoral Effects of a Clinical Antibody. Proc Natl Acad Sci U S A. 2021 May 25;118(21):e2017925118. doi: 10.1073/pnas.2017925118.

Vector BioPharma AG. Versant Ventures Launches Vector BioPharma AG. Press Release. August 10, 2022.

Ensoma. Ensoma Launches to Pioneer Next-Generation 

 Approach to Deliver First “Off-the-shelf” Genomic Medicines. Press Release, Feb. 11, 2021.

Ensoma. Ensoma Announces Strategic Collaboration with Takeda to Accelerate Next-Generation 

 Gene Therapies. Press Release, February 2021.

Capstan Therapeutics. Capstan Therapeutics Launches with $165 Million to Deliver on the Clinical Promise of Precise 

 Cell Engineering. Press Release, Sept. 14, 2022.

Rurik, J.G. et al., CAR T Cells Produced 

 to Treat Cardiac Injury. Science. 2022. 375 (6576), pp. 91-96, Jan. 6, 2022. DOI: 10.1126/science.abm0594

Interius BioTherapeutics. Interius BioTherapeutics Raises $76M Series A Financing to Advance Breakthrough Cell and Gene Therapy Platform. Press Release, May 18, 2021.

Sana Biotechnology. Sana Biotechnology Confirms Key Program Timelines and Announces Portfolio Prioritization. Press Release, Nov. 29, 2022.

Cynthia A. Challener, PhD has been a freelance technical writer for over 20 years, leveraging her education from Stanford University (BS) and University of Chicago (PhD) and 10+ years of industry experience. She currently focuses on pharma/biopharma topics, writing technical articles, white papers, blogs, and other content for a variety of clients in addition to contributing regularly to 

Articles

Key challenges posed to autologous and allogeneic treatments could be resolved by in-vivo CAR-T gene therapies.


Advances in In Vivo CAR T-cell Therapies

Drug delivery to the human eye is a rapidly growing area in the pharmaceutical industry, but it is also one of the most challenging. The inherent structure and physiology of the eye make it difficult for drugs to reach the site of action and persist over time. Despite these constraints, ocular therapies continue to garner significant interest due to growing patient need, according to Ashley Rezak, Global Market Manager for Topical Drug Delivery at Lubrizol Life Science Health (LLS Health). Drug product developers are actively seeking options for overcoming these barriers and effectively delivering ophthalmic drugs.

Ocular injectable products in particular are experiencing strong growth with the development of gene and other advanced therapies. Even so, topicals (mostly liquids including solutions and suspensions, but also semi-solids) remain the most prominent dosage form for ophthalmic commercialized products and those in development, says Dr. Robert Lee, President of LLS Health’s CDMO Division.

Topicals are appealing due to their ease of administration, non-invasive nature, and ability to be administered at the site of action, all of which make them attractive to treat eye diseases such as conjunctivitis, glaucoma, and dry eye, among many others. The big challenge is delivery, according to Gregg J. Berdy, MD, voluntary associate professor of clinical ophthalmology in the Department of Ophthalmology at Washington University School of Medicine and a physician with Ophthalmology Associates of St. Louis, Missouri.

Formulations must be stable and homogeneous and designed such that when a drop leaves the medication container and hits the surface of the eye, it has the appropriate concentration of API and can reach the intended target within the eye—the surface, the anterior segment tissues (cornea, aqueous humor, iris, ciliary body, and lens), or the posterior segment tissues (vitreous humor, retina, and choroid)—providing the correct dosage.

Patient centricity an important driver in topical development

One of the challenges with topical ophthalmic drugs is ensuring patient compliance. Topical drugs are easy for patients to apply themselves, but there is no way to ensure that patients do so at the proper frequency, according to Berdy.

“Studies have shown that patients don’t generally take medicines more than twice per day, so it can be difficult to get them to apply ophthalmic drugs as often as needed in some cases. In addition, refill rates for many basic drugs are well below 50%. There is no way to guarantee medication adherence, which requires that patients buy/order, pickup, and then take their prescriptions. Whether the reason may be price, pain, or forgetfulness, compliance is a big challenge with self-administered treatments,” Berdy explains.

For this reason, Berdy notes that physicians often prefer intraocular injectable drugs that can be administered in the office. With these medications, proper dosing is assured. In many cases, he adds that the injectable treatment schedule may be every three months to one year, and patients don’t need to worry about refilling prescriptions on a monthly basis for a prolonged period. Furthermore, there is no worry of accidental harm to the ocular surface while patients apply topical solutions.

Given the benefits and concerns associated with topicals, it is not surprising that much research has focused on developing topical ophthalmic drug products that are easier to use, more comfortable, and specifically reduce the frequency of administration. Such efforts include developing sustained/extended release and increasing the bioavailability of the active drug substances in formulations.

Natural barriers create delivery challenges

Frequent dosing requirements for topical ophthalmic drugs can be attributed to the fact that typically only approximately 5% of the of the administered drug substance in a standard eye drop is absorbed at
the site of action, according to Rezak. There are several reasons why this issue exists. Primarily, she observes, it is due to the tricky path a drug must take to effectively penetrate the target tissue.

In addition, the average eye drop is 40–50 μL, whereas the tear film capacity of the eye is only 7 μL, and the eye can only hold about 20–25 μL, according to Berdy. This situation results in a large waste of product and presents a significant delivery limitation, Rezak notes. Furthermore, the little liquid that does remain is subjected to innate solution drainage and blinking, making it difficult to keep a topical drug product in place long enough to be absorbed.

Given that there are significant protective barriers and mechanisms on the ocular surface and achieving relevant drug concentrations in various parts of the eye is so challenging, it is crucial to understand the pharmacokinetics of any topical ophthalmic drug (1). Part of this knowledge must include the ability to correlate data generated in animal models—often rabbits because of anatomical and physiological similarities—to humans (2).

As a result, some suggest that successful topical ophthalmic drugs for the treatment of diseases of the anterior and posterior segments of the eye must be highly potent and administered only to the eye, avoiding delivery of the topical dose to systemic circulation (1).

“Without incorporating differentiated ingredients or technologies that help overcome these challenges, frequent administration of the drug product is required to maintain an effective drug level,” says Rezak. “In addition to decreased patient compliance due to the added inconvenience of more frequent administrations, toxicity and overdosing can become an issue, as can the increased cost if dealing with an expensive API,” she continues.

Early products focus on outer eye conditions

Most topical ophthalmic drugs are formulated as liquids delivered as drops to the eye. These drugs generally treat issues involving the eye surface or anterior segment tissues, including both acute problems (conjunctivitis, keratitis, iritis) and chronic conditions (dry eye syndrome, glaucoma).

Eye drops have also been formulated to treat surface abrasions and infections and to prevent swelling of the retina following surgery, according to Berdy. The most common use of eye drops today, he observes, is for the treatment of glaucoma. These treatments take many forms, but all must reach the cornea and penetrate into the anterior chamber to be effective.

The first topical treatment for presbyopia (age-related hardening of the lens) was approved in 2021 (3), and several other candidates are in development. These drugs function by modulating the size of
the pupil or changing the thickness of the optical lens. There are also several new treatments for dry eye disease currently advancing through the clinic (4).

Bioadhesion one way to widen topical applications

Despite the challenges to treating diseases at the back of the eye with topical drugs, there are several candidates in development for the treatment of ocular posterior segment diseases including age-related macular degeneration, diabetic retinopathy, diabetic macular edema, retinal vein occlusion, and optic neuropathy glaucoma disease (1).

“The back of the eye is regarded as one of the most difficult areas to effectively treat. Compared to the anterior portion, the posterior segment generally cannot be accessed via a topical route due to the natural impermeability of the eye exterior and the distance a drug would have to travel to reach the site of action, which is usually the retina. However, the industry is making great strides in the area of exploring topicals to treat both anterior and posterior diseases of the eye,” Lee contends.

The best way to enhance drug delivery of ocular topicals, Lee continues, is by prolonging a drug’s residence time on the surface of the eye. “For eye drops, the key is thus muco- or bioadhesion,” he states.

Bioadhesion is commonly defined as the adherence between two surfaces, at least one of which is biological. In the case of topical ophthalmic delivery, the surface of the liquid drug product and the front of the eye are the relevant interfaces, Lee explains. Bioadhesion can be introduced in a formulation via a variety of ingredients and delivery systems.

There are some drug product developers working on advanced delivery systems to instill bioadhesive properties, such as those utilizing solid-lipid nanoparticles, micelles, and liposomes, according to Lee. “Some of these approaches are very promising, and it is exciting to see so much innovation in the ophthalmic space,” he says.

Both small-molecule drugs and biologics can feasibly be delivered using these systems, Lee adds. He does note, however, that the specific active ingredient must be evaluated in the given system to assess suitability, and biologics are often much more challenging to deliver topically due to their large molecule size.

With respect to successful bioadhesive solutions, Rezak stresses that one of the most effective and well-established methods to impart muco- or bioadhesion to a formulation is through strategic excipient selection. “There are many polymers a formulator could select for establishing bioadhesion in a formulation, such as carbomers, carboxymethylcellulose, polycarbophil, and sodium alginate, to name a few,” she comments.

LLS Health provides muco- and bio-adhesive excipients designed to increase the effectiveness of ophthalmic topical therapies. They have unique functionalities that impact the mucoadhesive product profile and come in multiple grades so that profiles can be fine-tuned for the individual needs of different products, according to Rezak. “LLS Health has conducted studies (5) that show formulations containing its Carbopol polymers and Noveon polycarbophil excipients display significantly longer product retention at the target site than those containing alternative polymers,” she says.

In addition to the use of excipients to improve bioadhesion, other innovative approaches to improving the performance of topical ophthalmic drugs are being investigated. Two primary approaches include decreasing the particle size to allow greater penetration through the mucous barrier in the eye and the use of hydrophilic coatings to increase API solubility and bioavailability, particularly for hydrophobic drug substances, according to Berdy.

Some specific examples include nanospheres, nanocapsules, nanomicelles, nanovesicles, dendrimers, in situ gels, and microneedles (2). In situ gels are applied as solutions but once in the eye form a gel in the target tissue in response to a specific stimulus (pH, temperature, or solvent change, ultraviolet irradiation, the presence of specific ions or molecules, etc.) The application of physical or electrical forces (iontophoresis and sonophoresis) to enhance API penetration are also under investigation. Extracellular vesicles, biological particles including exosomes, ectosomes, microvesicles, microparticles, oncosomes, and apoptotic bodies, are also being explored as delivery vehicles in topical ophthalmic drug formulations.

“Every major company in the ophthalmic drug sector is developing topical formulations aimed at a wide range of diseases from those more traditionally treated with drops to disorders of the posterior of the eye. They are exploring both novel compounds and new delivery technologies that will enable both greater efficacy and improved ease of use and comfort for patients,” Berdy concludes.

del Amo, E. M. Topical Ophthalmic Administration: Can a Drug Instilled onto the Ocular Surface Exert an Effect at the Back of the Eye? 

8 Sept. 2022. https://doi.org/10.3389/fddev.2022.954771.

Lixiang Wang, L; Zhou, M. B.; and Zhang, H.The Emerging Role of Topical Ocular Drugs to Target the Posterior Eye. 

 Sep; 10(3): 465–494. doi: 10.1007/s40123-021-00365-y

Novack, G.D. Eyes on New Product Development: Long-Acting Ocular Drug Delivery Technologies Addressing Unmet Needs in Ophthalmology. 

Journal of Ocular Pharmacology and Therapeutics

. 38 (6). https://doi.org/10.1089/jop.2022.29093.gdn

Lubrizol Life Science. Mucoadhesive Polymers in Pharmaceutical Formulations. Technical Brief. 2021.

 
Vol. 47, No. 2 
February 2023 
Pages: 18–19

When referring to this article, please cite it as Challener, C.A. Aiming for Improved Efficacy and Patient Compliance for Topical Ophthalmics. 

Topical eye treatments can be beneficial for patients with a growing array of eye diseases, but only if taken appropriately.

Aiming for Improved Efficacy and Patient Compliance for Topical Ophthalmics


Vol. 35, No. 2
February 2023
Pages: 11–14

When referring to this article, please cite it as Challener, C.A. Technology for 

In vivo CAR-T gene therapies could resolve challenges faced by autologous and allogeneic treatments.

Technology for In Vivo CAR T-cell Therapy Advances

The COVID-19 pandemic remains a global threat due to continued transmission of the SARS-CoV-2 virus despite the availability of several intramuscular vaccines. Vaccines administered intramuscularly via injection can elicit strong systemic responses and thus prevent the occurrence of severe disease. These vaccines don’t generally, however, lead to the development of mucosal immunity, which neutralizes the pathogen at the point of entry into the body. Mucosal immunity reduces viral replication at the level of the mucosa, which results in lower carriage by the vaccinated individual, hence reducing transmission.

Effective vaccines delivered via inhalation can lead to both strong mucosal and systemic immunity. The first such product against COVID-19 (Convidecia Air)—a collaborative effort between pharma company CanSino Biologics (CanSinoBIO, Tianjin, China) and respiratory device developer Aerogen (Galway, Ireland)—received regulatory approval in China in early September 2022 (1).

However, developing these vaccines can be challenging. Active substance formulation and delivery-device development must be pursued simultaneously. Once delivered, the vaccination agent must penetrate protective mucosal barriers and survive long enough to stimulate the desired immune response.

Mucosal membranes line the respiratory, digestive, and urogenital tracts and form an interconnected network of mucosal compartments (2–4). Vaccination that leads to mucosal immunity at one of these mucosal sites thus leads to immunity at distant mucosal sites. Mucosal immune responses involve activation of antigen-presenting cells (APCs) and the production of many different types of immune cells, antibodies, and inflammatory cytokines/chemokines.

Targeting mucosal layers in the upper and lower respiratory tract and lungs is an attractive approach to vaccination because most airborne pathogens enter the body through this route and infect these tissues (5). “Generation of mucosal immunity results in activation of many parts of the immune system, not just antibodies. In addition, replication of viruses is prevented in the respiratory system, enabling interruption of the transmission cycle,” explains J. Robert Coleman, CEO of Codagenix.

Numerous advantages to inhalation vaccines

Vaccines delivered via inhalation enable targeting of the respiratory tract mucosa and generation of both humoral and cell-mediated immunity, according to Pierre A. Morgon, executive vice president, Portfolio Strategy and Supranational Affairs and managing director, CanSinoBIO Europe. It is also possible to achieve good drug absorption, fast onset of action, and high bioavailability with inhaled vaccines (6).

In addition, they eliminate the need for needle-based administration, reducing the risk of needle-stick injuries and allowing avoidance of invasive injections, fear of which may keep some people from getting vaccinated (7).

In fact, inhaled vaccines delivered as liquids using nebulizers can potentially be administered with lower requirement for extensive healthcare infrastructure or as many trained healthcare personnel within an immunization center (2). Delivery by inhalation should, therefore, make large-scale mass vaccination easier to conduct, according to Ronan MacLoughlin, associate director of R&D, science and emerging technologies at Aerogen.

Importantly, less vaccine is needed for delivery via inhalation—typically less than 0.2 mL vs. 0.5 mL for injection, says Morgon. “Lower doses means more doses can be produced per batch, and mass vaccination can thus be achieved more cost-efficiently,” he says.

Furthermore, administration via inhalation can be theoretically achieved through the mouth or nose. The preferred route is, says MacLoughlin, entirely dependent on the target receptors for the vaccine. “If nasally expressed, then nasal should be the preferred route. Inhalation into the lung via the nose would allow for both nasal and lung deposition. Oral inhalation allows for a larger dose to be delivered to the lung, given that avoiding the nasal passages will increase the available fraction of aerosol,” he comments.Aerogen’s proprietary aerosol-generating technology converts the liquid vaccine into fine aerosol droplets that are suitable for delivery of the vaccine deep within the lung.

Some are of the opinion that vaccination through the mouth may result in enhanced induction of mucosal immunity. Researchers at McMaster University, for instance, have shown inhalation of a tuberculosis vaccine to be more effective than delivery via nasal sprays, because the vaccine penetrates much deeper into the airway (8).

Because inhaled vaccines provide local immunity to the respiratory tract, they are seen to be ideal solutions for interrupting the spread of viruses with high transmission rates and the potential to lead to global pandemics (9).

The specific types of vaccines that can be formulated for inhalation delivery is not fully understood yet, according to Jean-Denis Shu, SVP of Medical Affairs with CanSinoBIO. “The critical factors are determined by the target receptor, the vaccine’s stability in the lung, and whether sufficient dose can be given to illicit the immune response,” he observes. CanSinoBIO’s inhaled COVID-19 vaccine, now approved in China, is based on an adenovirus-vectored vaccine (10).

“Inhalation delivery offers the potential advantage of bypassing anti-vector immunity for viral vector vaccines for which people may have already developed some immunity through, for instance, exposure to cold viruses,” Morgon adds. The formulation of CanSinoBIO’s inhaled vaccine (Convidecia Air) is the exact same as that of its intramuscular vaccine (Convidecia) and there is no need for an adjuvant to boost immunogenicity.

Codagenix’s inhaled COVID-19 vaccine candidate is a live, attenuated virus that, according to Coleman, safely and stably brings not only the SARS-CoV-2 spike protein, but many other proteins of the virus to the immune system, allowing for broad protection.

Beyond these two approaches, there are inhalation vaccines under development based on attenuated influenza virus, parainfluenza virus (PIV) 5, lentiviruses, Newcastle disease virus (NDV), and vesicular stomatitis virus (VSV); bacterium vectors, nucleic acids (messenger RNA, DNA), and protein subunits (3). Unlike the CanSinoBIO vaccine, the latter require formulation with adjuvants to ensure sufficient immunogenicity (4,11). A range of adjuvants have been explored, such as polyethyleneimine, N-[1-(2,3-Dioleoyloxy) propyl]-N,N,N-trimethylammonium chloride, chitosan, toxoids, cytokines, saponins, lipids, and toll-like receptor agonists.

The need for effective delivery technologies remains the main challenge for realizing successful mucosal vaccines, including those administered via inhalation (12). Not only are strong adjuvants required for many types of vaccines, it can be difficult to maintain the integrity of the vaccines. The vaccination agent must resist degradation in the harsh mucosal environment in order to have time to reach the immune inductive tissue and elicit the desired immune response while also ensuring that oral tolerance does not develop (11). Overall, therefore, appropriate formulation of inhalation vaccines is crucial for their success (13).

Inhalation vaccines can be formulated as liquids or powders. Dry powders have the potential for long-term storage without cold-chain requirements, while liquid formulations are generally simpler to develop and implement, notes MacLoughlin. Dry powder formulations can be generated by various methods, such as spray drying and thin-film freezing (4,6). It is crucial to control the particle size and particle size distribution to achieve the desired release profiles and distribution in the lung.

The aqueous nature of liquid formulations, meanwhile, allows for rapid interaction with the airway epithelium. “Such interactions may be subdued for dry powders due to local conditions in the airway surface lining fluid,” Morgon observes. He also points out that the need for cold-chain management is not an automatic disadvantage for liquid vaccines, because storage and handling capabilities at 3 to 8 °C are generally available at most locations.

Certain attributes are essential for successful inhalation delivery

The physicochemical attributes of vaccine actives delivered intranasally must allow the vaccine to mimic the wild type natural virus that comes in through the airway, according to Coleman. “Such behavior is essential to induce very broad immunity and stop onward transmission,” he contends. He adds that Codagenix chose to use an attenuated live virus rather than a viral vector or protein subunit for this reason. “Using an attenuated virus allows for presentation of multiple viral proteins and avoids any underlying immunity issues, affording increased vaccine uptake,” states Coleman.

Development of optimized vaccine/device combination essential

A consideration equally important to the design of the vaccine active and formulation is the development of an optimized vaccine/device combination, according to Shu. “Picking any delivery device off-the-shelf and applying that increases the risk that the immunization is not effective. A solid understanding of how the vaccine and device interact are critical,” Shu avers.

Morgon agrees. “Understanding formulation, device performance, delivered dose, distribution of dose within the airways, etc. all impact the
likelihood of success.”

“Co-development, such as that conducted by Aerogen and CanSinoBio, has enabled an effective immunization approach that uses a fraction of the injected dose by volume or mg of drug substance,” MacLoughlin concludes.

Devices use for the delivery of inhalation vaccines should, ideally, be patient agnostic. “Taking the variability out of the patient input in the process is a key requirement in order to ensure consistent, reproducible, and reliable dose across a population,” MacLoughlin observes. To this end, Aerogen has focused on development of devices that require the patient to make only a single inhalation for their entire vaccine dose. “This way, there is no requirement for coordination of breath with aerosol generation, which is a key failing of pressurized metered-dose inhalers, and every patient has the same dose available to them for inhalation,” he notes.

Development of mucosal vaccines, including inhalation vaccines, faces an additional hurdle that vaccines administered by other routes generally do not. “The historic body of work in vaccinology has focused on the production of antibodies in the bloodstream. Successful vaccines are determined to be so because they result in high levels of antibodies,” Coleman says.

Some mucosal vaccines, however, may generate lower levels of antibodies in the blood. That does not mean they are less effective, but regulators may not be aware of the differences and thus still compare the data for mucosal vaccines to those for injectable versions, Coleman adds. In the case of CanSinoBIO’s Convidecia Air, the humoral and cellular responses are higher with the inhaled booster than with the injectable one, Morgon comments.

One program designed to address this concern has been established by the World Health Organization (WHO). Its Solidarity Trial Vaccines program supports the development of second-generation COVID-19 vaccines with greater efficacy, greater protection against variants of concern, longer duration of protection, and improved storage and/or simplified delivery with needle-free administration (14). In the Phase III trial of Codagenix’s CoviLiv vaccine candidate, both antibody generation and actual efficacy (determined by comparing vaccinated and placebo groups) are being evaluated, so the actual efficacy rate will be measured. CanSinoBIO has also agreed to the request from WHO to include Convidecia Air in the Solidarity trial.

Several other inhaled COVID-19 vaccines in development

The same group of researchers at McMaster University that compared the delivery of a tuberculosis vaccine via nasal spray and inhalation also have an inhaled, adenoviral-vectored, aerosol COVID-19 candidate in a Phase I clinical trial (8,15).

Scientists at MIT have shown that peptide vaccines with albumin-binding lipid tails when formulated with the adjuvant CpG and administered via inhalation generate long-lasting immune responses (5). A group at University of North Carolina-Chapel Hill, meanwhile, used lung-derived exosomes, or nanoscale vesicles secreted from lung spheroid cells (LSC-Exo), to effectively deliver nucleotide and protein-based vaccines to the lungs via an inhaler (8,16).

In addition to the above inhalation vaccines under development for COVID-19, several groups are investigating the potential of intranasal vaccines intended to induce mucosal immunity. Some of these have been found wanting, most recently the intranasal version of the AstraZeneca viral-vector vaccine (17). These disappointing results, says Morgon, may drive a heightened focus toward the inhalation route.

At the same time, the approval of the CanSinoBIO inhaled COVID-19 vaccine provides evidence that inhalation delivery can be successful at generating mucosal immunity. “We now have significant data on the safety and efficacy of an inhaled vaccine. These data should give others confidence to proceed to trial with what is known to be a broad pipeline of vaccines that may have utility if inhaled,” Morgon says. “Indeed,” he adds, “significant activity has begun/restarted in the area, given that this new route of administration has been shown to be feasible, and on a global mass immunization scale.” Furthermore, if Convidecia Air is convincingly shown to materially reduce viral carriage in vaccinated individuals and therefore curb transmission, CanSinoBIO believes it will turn out to be a blockbuster product.

“The future of inhaled vaccines has just received a significant boost of more useful rapid methods, although further development is required to improve accuracy and repeatability,” he highlights. “Of the different ‘PAT [Process Analytical Technologies] methods’, ATP bioluminescence technology offers a higher sensitivity compared with colorimetric methods.”

In terms of management of cleaning validation processes, software developments are piquing Sandle’s interest, including some that are employing artificial intelligence. “Such packages can help to sort equipment into types where a matrix approach is preferred, as well as enabling scheduling and for setting re-validation targets,” he states.

At its very essence, cleaning validation will require greater consideration, simply as a result of the evolving therapeutic landscape, Kolbert asserts. “The increased use of biological therapeutic agents in pharmaceutical manufacturing will require increased complexity of analytical techniques as activity of the residue is as important as chemical presence,” he summarizes.

Pharmaceutical Cleaning Validation Market: Industry Analysis and Forecast (2021–2027) by Product Type, Validation Test, and Region. Report

CFR Title 21, 210 Part 133.4 (Government Printing Office, Washington, DC).

Validation of Cleaning Processes (7/93), Inspection Guide. 


Vol. 47, No. 1
January 2023
Page: 26–29, 39

When referring to this article, please cite it as C. Challener. Inhalation Vaccine Development. 

Inhaled vaccines must resist degradation and penetrate the mucosal lining in the airways and lungs. 

Inhalation Vaccine Development

Oligonucleotide drug substances generally are unstable at room temperature, and some are also degraded in the presence of certain enzymes. That is why, in part, they are formulated using special delivery solutions such as lipid nanoparticles (LNPs). (These delivery technologies also facilitate entry of nucleotide APIs into target cells.) Degradation can occur via loss of terminal nucleotides or certain functional groups, oxidation, and other mechanisms. Oligonucleotide–oligonucleotide interactions that can occur at higher concentrations are an added concern. These instability issues must be managed throughout the entire manufacturing process.

The challenges are particularly great for messenger RNA (mRNA), which can be degraded by enzymes found commonly in the environment, temperature, and due to the overall shear sensitivity of the long-chained molecule.

Variety of oligonucleotide drug substances

The landscape of nucleotide-based therapeutics is based on more than half a century of research and development in these modalities, according to Julian Mochayedi, strategic marketing manager for mRNA solutions with MilliporeSigma, the Life Science business of Merck KGaA, Darmstadt, Germany. This knowledge translates to the clinic and beyond, with several approved therapeutic products on the market.

The different types of nucleotide-based therapeutics can be broadly classified by the type of nucleic acid, Mochayedi continues, such as those with DNA or RNA as a basis. Alternatively, they can be differentiated by their functional design, which determines the mode of action. “One can cite DNA-based adeno-associated virus (AAV) vectors for gene delivery, antisense oligonucleotides (ASOs), small-interfering RNAs (siRNAs) for gene expression disruption, and most recently, mRNA to express a protein of interest such as for immunization purposes, as the successful mRNA-based COVID-19 vaccines have demonstrated,” he explains.

mRNA instability particularly challenging

DNA-based oligonucleotide drug substances, such as AAV vectors and plasmid DNA, share the relatively stable characteristics of DNA, Mochayedi notes. The stability of shorter chemically synthesized RNAs such as ASOs and siRNA can be improved through chemical modifications during synthesis. Particularly long biologically synthesized mRNA molecules, however, are inherently unstable, and thus require additional management during manufacturing.

All nucleic acid-based platforms, however, must be formulated using delivery technologies that protect the therapeutic from degradation. Unique solutions such as LNPs help ensure stability in the patient setting, API stability and, ultimately, the efficacy of the therapeutic approach.

There are several reasons why mRNA is inherently unstable. The physiochemical properties of single-stranded mRNA molecules that lead to their instability include their very large size, high negative charge, sensitivity to rapid degradation by omnipresent enzymes such as endonucleases, shear sensitivity, and high viscosity through the formation of secondary structures in solution, according to Mochayedi.

Manufacturing strategies for managing mRNA instability

One of the main culprits causing mRNA degradation is the enzyme RNase. Avoiding it is difficult, however, because this enzyme is ubiquitous. “It is very difficult for mRNA to move freely in the environment,” Christian Cobaugh, CEO and founder of Vernal Biosciences, comments. “Extreme precautions must therefore be taken to avoid introducing this enzyme into manufacturing spaces or enclosures,” he stresses. This issue can often, he notes, be a barrier to entry into the mRNA market for laboratories not experienced in mRNA production.

Use of qualified processes, including sterile single-use technology, should be applied to mitigate contamination and carryover of endonucleases, Mochayedi says. Liberal use of RNase degrading chemicals for cleaning equipment surfaces, operator gloves, and so on is also essential, Cobaugh adds.

The instability of mRNA at room temperature creates further challenges, as the time available for holding generated mRNA sequences at room temperature is fairly restricted, according to Cobaugh. The drug substance must be moved seamlessly to downstream purification and formulation or quickly frozen.

“The choice of adopting a seamless manufacturing approach or having unit operations separated by controlled freezing and subsequent thawing steps must be considered early on, as the decision will impact the design and operation of the mRNA manufacturing facility, and once implemented, that design will essentially be locked in,” Cobaugh contends.

“Ideally, it is best to limit the number of freezing and thaw steps, as they can impact the quality of the mRNA drug substance and formulated mRNA-LNP products,” Cobaugh adds. Furthermore, gentle techniques must be employed. Continuous movement of the mRNA drug substance to purification and then LNP formulation avoids all but the final freezing step. “It is possible, though, with the right engineering solutions and validation procedures to surmount the challenges presented by either approach to mRNA manufacturing,” he concludes.

In addition, stringent optimization of reaction conditions and the process parameters for key unit operations, such as the synthesis step (in vitro transcription) and filtration (via tangential-flow filtration), can overcome viscosity challenges and ensure low shear during processing to maintain the stability of the mRNA at each stage of the manufacturing process, Mochayedi remarks. “Bespoke process development and optimization are necessary for each unique mRNA sequence to address and overcome the highlighted challenges,” he concludes.

While the concept of leveraging mRNA for the prevention or treatment of various diseases is straightforward, the safe and effective delivery of these fragile nucleic acids into cells where their message can be translated into proteins has proved to be a huge challenge, Mochayedi observes. “For the COVID-19 mRNA vaccines, the solution to this problem came through the development of LNPs. These particles, typically composed of four different lipids (ionizable lipids whose positive charges bind to the negatively charged backbone of mRNA, pegylated lipids that help stabilize the particle, and phospholipids and cholesterol molecules that contribute to the particle’s structure), provide a protective bubble for the delicate mRNA molecules, enabling their safe and efficient delivery into cells,” he explains.

The structure of the nucleotide API has a strong impact on the selection of the excipients for the formulation. The manufacturing process must, in addition, be highly controlled. “The buffer solution containing the mRNA drug substance used to produce the LNPs is of low pH (typically aqueous sodium citrate) that places additional stress on the nucleotide’s stability, leading to even more rapid breakdown than occurs at neutral or slightly basic pH,” Cobaugh says.

A low pH of approximately 4.5 is needed to ionize the mRNA so that it forms nanoprecipitates with the lipids. The use of stabilizing process aids is, Cobaugh adds, generally avoided because they would interfere with the ionization of the mRNA and could also potentially end up in the product. Most molecules that would stabilize mRNA against pH-mediated degradation would, furthermore, likely be hazardous chemicals.

The key to successful LNP production, therefore, is minimization of the time required for the LNP manufacturing process. That, Cobaugh emphasizes, requires extensive understanding of the process from a quality-by-design basis combined with thorough process validation. “It is absolutely essential to have a deep awareness of what levers can be pulled from both process and engineering perspectives to minimize the time for all aspects of the formulation process,” he states.

With respect to fill/finish of mRNA therapeutics and vaccines, the strategy is generally no different than that of any other biological API, according to Mochayedi. The goal, agrees Cobaugh, is to fill the product—whether drug substance in sterile single-use plastic bags and containers or formulated drug product into sterile glass vials—and then freeze them as rapidly as possible but in a controlled manner. In this case, the challenges are largely engineering and equipment-related.

For storage and shipment of mRNA, the requirement to maintain the cold chain to ensure its stability and integrity all the way to the patient is of utmost importance, Mochayedi says. “Central storage is usually performed at very low temperatures (-80 °C). Similarly, formulations containing nucleotides require shipment at low temperatures, which is typically achieved using dry-ice packaging,” he notes. In some cases, mRNA drug products may require the addition of a cryoprotectant to protect the particles. The adjustment of excipients to protect the nanoparticles may also enable some formulations to be shipped at approximately 4 °C.

The production of mRNA-LNP formulations that are stable at warmer temperatures is a key focus of developers of candidate therapeutics and vaccines. One approach noted by Cobaugh involves the development of higher-quality DNA templates that are free of breaks (commonly referred to as nicks) that result in the shorter, more unstable mRNA sequences. Mochayedi points to new types of modified nucleotides and new capping technologies to physically altered forms of mRNA, such as circular RNA. “These new approaches may overcome some of the stability issues and enable a broader application of mRNA as a technology platform,” he believes.

Lyophilization of mRNA products as an alternative to freezing is the other main area of investigation. “Theoretically, there is no reason why freeze-dried mRNA products cannot be stored at room temperature as long as water is kept out of the storage containers,” Cobaugh says. He notes that there has been preliminary success in this area, and he is hopeful that lyophilization will be applicable for mRNA products in the not-too-distant future.

Mochayedi agrees, noting that significant investigations into formulation optimizations to overcome the ultra-low temperature requirements of mRNA drug products during shipment and storage are underway across the industry, with lyophilization of formulated mRNA-LNPs as an extremely promising alternative for liquid-based products. “The complete drying of formulations afforded by freeze-drying would unlock the possibility of much more easily providing mRNA therapeutics in countries with higher temperatures,” he remarks. 


Vol. 46, No. 12
December 2022
Pages: 22–23, 27

When referring to this article, please cite it as C. A. Challener, “Managing mRNA Instability During Formulation, Manufacturing, and Shipment,” 

Unique solutions are required to protect inherently unstable messenger RNA.

Managing mRNA Instability During Formulation, Manufacturing, and Shipment

Poor solubility issues are often associated with small-molecule APIs formulated for oral administration. Solubility is also important for parenteral products as well. Injectable products need to be free of particles (foreign substances and active ingredients) and often are high-concentration formulations, and both can be difficult to achieve when drug substances have poor water solubility. Special excipients can help overcome these solubility problems. Top candidates include polymeric micelles and lipid-based formulations.

The online version of this article has been updated from the print version to include additional content.

Poor solubility is a general issue in drug development

Solubility is a challenge that most newly discovered drug candidates face, irrespective of the intended route of administration, according to Philip Schäfer, head of process and formulation materials at MilliporeSigma, the Life Science business of Merck KGaA. In fact, approximately 40% of APIs on the market and around 90% of those in development are poorly water soluble (1).

“Today’s approach of target-oriented high throughput screening aimed at identifying more lipophilic compounds combined with the trend towards larger and more complex molecules both contribute to the development of APIs with reduced solubility,” says Schäfer. More specifically, during drug development, explains Shreya Shah, associate director of formulation services at LLS Health, many molecular targets, such as drug/enzyme binding sites, are hydrophobic. “As like attracts like, the main structure of candidate APIs is also often hydrophobic, making them poorly soluble in aqueous media,” she observes.

Molecular weight, chemical structure, and chemical-group lipophilicity are key factors affecting the solubility of small-molecule APIs, adds Philipp Heller, project manager innovation at Evonik Health Care. He notes that some peptide-based drugs can also suffer from poor solubility depending on their amino acid composition, while proteins have solubility challenges as well.

In the case of peptides and proteins, poor solubility generally occurs when the tertiary structure of these molecules includes large hydrophobic portions on the surface and thus exposed to aqueous media, according to Vincenza Pironti, senior staff scientist, research and development, pharma services at Thermo Fisher Scientific. “Often this aspect can be circumvented by conducting pH or ionic strength studies to find the right window where the protein structure contains exposed charged portions,” she comments.

Solubility is important not just for orally administered drugs; it is also essential for parenteral formulations because these product solutions must be particle-free, Schäfer notes. That must be achieved even for high-concentration products—a growing formulation trend, which further increases the need for high solubility.

“There are several strategies used to improve the solubility of hydrophobic compounds and each has its limitations. Safety is always a key concern in the formulation of novel therapeutics, but it is even more crucial for parenteral formulations that bypass the gastrointestinal tract and are delivered directly to receptor sites via the bloodstream. Therefore, not all strategies are practical, especially when formulators are trying to achieve high drug loading, for example, to reduce the frequency of administration,” comments Shah.

High-concentration injectable formulations present unique challenges.