Abstract

This year, our team focuses on engineering B-cells as living and evolving drugs. We propose engineered, antibody-producing B-cells as prophylactic treatment for neurodegenerative diseases.

The Problem

Neurodegenerative diseases pose a significant threat to our increasingly ageing society. The global prevalence of all-cause dementia is projected to increase from 55 million people in 2023 to a staggering 113 million in 2050.1 As one of the most prevalent neurodegenerative disorders, Alzheimer’s disease is closely linked to the overall epidemiology of dementia, accounting for the majority of cases.2

The impact of Alzheimer’s disease goes beyond its prevalence and reaches into various aspects of society. It is currently the seventh leading cause of death worldwide. Its associated economic burden is also significant, with global costs estimated to reach around $1.3 trillion in 2023.3

In light of the escalating challenges posed by neurodegenerative diseases, particularly Alzheimer’s, there is a pressing need for comprehensive scientific projects aimed at tackling this silent pandemic.

Project Inspiration

However, effectively targeting the root cause of this devastating disease remains a formidable challenge. At the heart of Alzheimer’s disease is its defining hallmark: the presence of extracellular β-amyloid (Aβ) plaques and intracellular neurofibrillary tangles containing tau proteins.4

In recent years, considerable effort has been devoted to exploring various therapeutic approaches, including the development of monoclonal antibody-based therapies directed against β-amyloid plaques. Notably, two such therapies have received FDA approval, marking significant progress in the field.5 Despite these developments, several shortcomings limit the efficacy and practicality of these treatments. Because symptoms often do not manifest until years after the physiological onset of the disease, treatment usually follows a late diagnosis and therefore has limited clinical efficacy. In addition, while these monoclonal antibody treatments provide temporary relief, they do not confer lasting ‘immunity’ against Alzheimer’s disease.6

The limitations of existing therapies have been the driving force behind our research efforts. By addressing the shortcomings of current treatments, we aim to develop an innovative therapeutic approach.

Our Solution

To address the major challenge in biomedicine posed by neurodegenerative diseases our team is developing a novel cell therapy approach based on highly innovative B-cell engineering.

B-cell engineering – living and evolving drugs

B-cells are vital components of the adaptive immune system, producing antibodies in response to antigens. The use of B-cell engineering has been recently implemented against HIV in mouse models and has proven to be a potent and scalable method.7,8 Our project therefore aims to expand on a novel cell therapy approach based on engineered B-cells as viable antibody-producing drugs against neurodegenerative diseases.

Once implemented, the therapy will involve extracting B-cells from a patient’s body and introducing specific antibody sequences into the B-cells’ genome. These engineered cells would then be reinfused into the patient. Upon contact with their target antigen, the B-cells will produce the inserted antibodies. The engineered B-cells then outcompete the body’s response and eventually differentiate into antibody-producing plasma cells and persist as long-lasting memory B-cells.9

Advantages over conventional antibody therapies

B-cell engineering as promising therapeutic and prophylactic approach against various targets

B cell engineering is very flexible and could be applied for virtually any target and can differentiate into long-lived plasma cells, protecting the host for decades, sometimes even lifelong 8,10. To demonstrate the flexibility, we decided look at a viral target, a bacterial target and a cancer target.

Alzheimer’s diseases

In the context of Alzheimer’s diseases in particular, the usefulness of antibody therapies is debated, as they need to be applied in the early stages of the disease. We therefore propose to engineer B-cells as viable, long-living, antibody-producing cells that can be used as a “vaccine” against Alzheimer’s disease.8 Anti-amyloid antibody sequences could be incorporated into a patient’s B-cells, enabling the body to secrete antibodies in response to antigen encounters. Antibodies are produced as soon as Aβ plaques begin to form. The engineered B-cells could be used as a prophylactic measure without the need for prior diagnosis, allowing a therapeutic response even in the early, non-symptomatic stages of the disease.

Hepatitis B virus

Hepatitis B virus (HBV) attacks the liver and is according to the WHO responsible for more than 800.000 deaths a year mainly from liver cirrhosis and heptatocellular carcinoma 11. The infection can be either acute or chronic. Approximately 300 Mio. individuals are affected from such a chronic infection, putting these people at a high risk of deceasing due to cirrhosis and heptatocellular carcinoma. Even though a very successful vaccine exists, chronic infection are still not curable 12. The hepatitis B surface antigen (HBsAG) has been identified to be a promising target for a monoclonal antibody (mAB) therapy. These mABs bind to the HBsAG and inhibit entry, allowing for eventual clearance of the infection 13. However large amounts of antibodies are needed since HBV deploys decoy particles coated in HBsAG as a part of its immune escape strategy, requiring large amounts of the mAB to successfully clear the infection 14. This could be tackled by using engineered B cells which continuously produce the antibody, abolishing the need for constant administration of the therapeutic antibody.

α-Hemolysin of Staphylococcus aureus

Our bacterial model of choice is Staphylococcus aureus, especially it’s toxin α-hemolysin. This pathogenic and commensal bacterium lives on our skin and in our gastrointestinal tract, but can cause disease in some cases 15,16. If infected with a normal S. aureus strain, this doesn’t pose a great treat to our health since it can be nicely counteracted using methicillin 17. However, the emergence of MRSA (methicillin-resistant S. aureus) posed a great challenge when it comes to treatment of these infections, often leading to severe outcomes such as pneumonia or even death 18. Thus, novel therapeutics are needed and α-hemolysin proved to be a promising candidate, due to it being highly conserved between S. aureus strains 19. Not only is this toxin highly preserved, but also an important virulence factor for the bacterium allowing it to cause symptoms described above 20,21. Monoclonal Antibodies against exactly this protein proved to be a very promising approach to target these germs 19,22,23. Taken together this allows us to use α-hemolysin as a good bacterial model system.

CAIX-expressing cancers

Monoclonal Antibodies are already in use for treatment of specific cancers by targeting neoantigens or overexpressed surface proteins such as HER2 in HER2-positive breast cancers 24,25. Similar to HER2 Carbonic anhydrase IX (CAIX) is a transmembrane protein especially present under hypoxic conditions as it can be the case in cancer. Thus these cancer cells show high levels of CAIX expression 26, which is unusual since CAIX is mainly found in the digestive tract 27. Therefor CAIX could be a potential candidate for monoclonal antibody therapy for example in clear cell renal cell carcinoma (ccRCC), which is why we selected it as a model target for cancer in our project.

Platform approach – Blood-brain barrier transport and improved safety of B-cell therapy

A major challenge in the treatment of neurodegenerative diseases with antibodies is the delivery across the blood-brain barrier. Therefore, we aim to introduce antibodies into B-cells that specifically target Aβ plaques and contain binding sites for transferrin receptor-mediated transcytosis into the brain.28

Other bench-to-bedside optimizations include the controlled induction of class switching or the fine-tuning of the antibody concentration secreted by the B-cell using genetic switches. Thereby, antibody treatment could be customised to the patient.29

To ensure the safety of B-cell therapy, we also propose to integrate a medically inducible kill switch. We aim to adapt an inducible system based on caspase-9 overexpression that has already been established for CAR T-cell therapy.30 This ensures that therapeutic B-cells can be eliminated if adverse effects occur.

Experimental Execution

For our proof of concept, we aim to generate antibodies specifically designed to target Aβ plaques and optimise their ability to traverse the blood-brain barrier via transferrin receptor-mediated transcytosis. We are thus producing various antibody constructs using an Expi293 cell model and evaluating their binding affinity towards the target molecule and transcytosis efficacy.

Antibody constructs exhibiting the highest affinity will be selected for integration into the antibody locus of RAMOS B-cells using CRISPR/Cas technology, ensuring stable integration in the genome. Utilising available assay systems, we will initially focus on antibodies directed against alpha-hemolysin, an exotoxin produced by S. aureus. The most effective antibody constructs will then be adapted to target the Aβ plaques and introduced into B-cells following the strategy depicted in Figure 3.

From bench to bedside

B cell engineering can be realized in a similar manner as therapeutic T cell engineering such as CAR T cells. First blood is taken from the patient and peripheral blood mononuclear cells (PBMCs) are isolated and B cells are purified e.g., via FACS or MACS 31. These cells then need to be expanded and activated to allow gene transfer 32. Once the B cells are successfully modified, they can be infused back into the patient. To deliver the gene, various methods are in place such as Lentiviruses or transposons, however rAAV based vectors have been proven to be a good system in gene therapy and vaccine development 33. The major upside of rAAVs is that they usually don’t integrate their genome, however if the genome is modified in a way that it carries homology arm, genome integration can be achieved site-specifically, which is not the case for Lentiviruses which integrate randomly 7,8. In future it is even thinkable that patients won’t need to undergo this procedure since rAAV-vaccines grow in popularity. One could simply vaccinate individuals with AAVs showing tropism for B cells and carrying genetic information for the production of antibodies against desired targets 34. This additionally has the advantage that one could produce vaccines in large amounts, abolishing the need for personalized therapy which is expensive and laborious 35. B cell engineering could also provide a great opportunity, when looking at secondary immunodeficiencies that could arise with increasing age. By applying such treatments one could compensate for the loss of BCR repertoire diversity which in turn counteracts secondary immunodeficiencies 36,37.

B cell engineering is challenging

B cell engineering sadly isn’t as straight forward as it might seem. Multiple challenges are encountered in the process of generating such cells. A major issue is the danger of mispairing of the endogenous BCR light & heavy chain with the additionally introduced ones. This results in poor expression of the desired BCR and ultimately the poor expression of the antibody 7,38-39. Luckily there are ways to cope with this issue, an idea that immediately comes to mind is knocking-out the endogenous chains and replacing them with orthotopically with the BCR of interest e.g., via CRISPR-mediated HR. That way one the artificial BCR is being expressed and the problem of mispairing is solved 40. Alternatively one can introduce a linker between the engineered light and heavy chain, connecting them covalently, thus prohibiting mispairing with the endogenous proteins 39. Another hurdle is the safety aspect. What if the engineered cell happens to target self-peptides? What if these cells happen to turn dangerous for the patient? There have to be measures in place to prevent this or at the very least to interrupt any of these events. One way of doing this is the introduction of a kill-switch, which relies on Caspase-9 to selectively induce apoptosis in the engineered cells 41-42. Such a switch consists of an extracellular domain drug-binding domain and an intracellular Caspase 9 domain. Upon administration of the drug, this artificial receptor dimerizes, Caspase 9 gets active leading to Caspase 3 activation and apoptosis 39. Alternatively, one could think of an activation switch rather than a kill switch to increase safety of these cells. The activation switch could be a chimeric receptor composed of the intracellular & transmembrane domain of the BCR, however its extracellular domain has been exchanged to an scFv- or drug-binding-domain. If the specific drug is now delivered to these cells the make use of the classical BCR-signaling pathway, simulating engagement of actual BCR, leading to antibody secretion so to say on demand independently of encounter of its target 41,42.

Footnotes

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  7. Nahmad, A. D. et al. Engineered B cells expressing an anti-HIV antibody enable memory retention, isotype switching and clonal expansion. Nat. Commun. 11, 5851 (2020). ↩ ↩2 ↩3

  8. Nahmad, A. D. et al. In vivo engineered B cells secrete high titers of broadly neutralizing anti-HIV antibodies in mice. Nat. Biotechnol. 40, 1241–1249 (2022). ↩ ↩2 ↩3 ↩4

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  28. Rofo, F. et al. A Brain-Targeting Bispecific-Multivalent Antibody Clears Soluble Amyloid-Beta Aggregates in Alzheimer’s Disease Mice. Neurotherapeutics 19, 1588–1602 (2022). ↩

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