Foundational Ideas

Abstract: Designing a novel targeted B-cell lymphoma therapeutic gene delivery platform using AAVP vectors

onCAP is a novel, phage-based cancer gene therapy using adeno-associated virus phage (AAVP) vectors to treat B-cell lymphoma. We constructed our phage combining two modified fd-tet plasmids, fMCS and f3-55nm, as well as an expression cassette engineered from the pAAV plasmid to include granzyme B and TNF-α. This plasmid encodes for a phage displaying anti-CD22 short chain variable fragment on its viral coat, which will lead to the release of the plasmid into mammalian cells. Upon plasmid release in human cancer cells, the proteins granzyme B and TNF-α will be expressed to induce cell death. We hypothesize that an AAVP therapeutic will show high specificity for cancerous B cells with limited off-target effects, because our target marker is primarily expressed on B cells and AAVPs are generally well-tolerated by the immune system. Our goal is to create a proof-of-concept demonstrating the potential of phage therapy in targeting aggressive B-cell cancers.

Rationale

Given the current limitation of B-cell lymphoma treatments and the promise of phage therapies, we hypothesize that adeno-associated virus phages (AAVPs) can provide a new specific, effective, and safe avenue of treatment.

Inspiration

Our idea started from the protocol for designing AAVPs by Hajitou et al. (Hajitou, A., Rangel, R., Trepel, M. et al. Design and construction of targeted AAVP vectors for mammalian cell transduction. Nat Protoc 2, 523–531 (2007). https://doi.org/10.1038/nprot.2007.51), from which we based our initial design and construction of our plasmid. We were fortunate to get into contact with Dr. George P. Smith, who developed the phage display method. He provided us with more current plasmids for AAVPs and helped us modify the protocol to display our targeting anti-CD22 scFv. Building from this foundation, onCAP is a novel application of AAVPs to the field of B-cell lymphoma therapeutic research.

Background

Cancer is the leading cause of death (1), however, current treatments leave the patients to suffer major side effects. Coupled with the lack of information pertaining to drug effects, alternative treatments and appropriate follow up care (2), the side effects of treatment are an unfortunate but necessary aspect of the harsh reality in cancer treatment, one that often severely limit day-to-day ability and quality of life for the patients (2). As such, our team is developing a novel cancer gene therapy using AAVP vectors, adeno-associated virus phage vectors (AAVP) to target B cell lymphoma.

Although molecular and immuno-based therapies reduce the effects that cancer treatment has on patients’ livelihoods, they possess issues of their own. Most commonly, recognition, penetration and general delivery of the drugs to the tumor sites is often subject to a multitude of forces from the body’s immune system and the tumor sites themselves (3). This proves as a barrier for these drugs to be effective on their own, requiring a multitude of treatments and their accompanying side effects to be employed on the patient.

Why are we targeting B-cell Lymphoma?

B cell lymphoma is the cancer of immune cells known as B lymphocytes or B cells. It has been estimated that 80,470 people in the United States will be diagnosed with non-hodgkin’s lymphoma (NHL) in 2022 (4). It is also known that B cell lymphomas make up around 85% of all NHLs (5), with diffuse large B cell lymphoma (DLBCL) being the most common, representing 25% - 30% of all NHLs (6). As such, our therapeutic targets B cell lymphomas with an emphasis on DLBCL. Similar to other cancers, if left untreated B cell lymphoma is fatal.

At a cellular level, this type of lymphoma compromises the normal function of these cells. As a result, B cell lymphoma patients have lowered immunity and higher susceptibility for infection. In physical manifestation, patients commonly experience fevers, night sweats, and swelling of the lymph nodes (7). Tumors are often found in the lymph nodes, and have the ability to metastasize anywhere in the body.

Under regular expression, B cells produce antibodies that can target antigens found on foreign pathogens. One specific method is by tagging these foreign antigens, allowing other immune cells to recognize and phagocytose the intruder. Each pathogen have multiple specific antigens, requiring antibodies with diverse binding for a functioning immune system (8). V(D)J recombination of the V (variable), D (diverse), and J (joining) gene segments in developing B cells is the process that rearranges antibody coding regions of the DNA, resulting in a unique binding site on the antibody protein. This process involves many breaks and ligations of the DNA sequence to produce a random combination of the V, D, and J gene segments for conversion into RNA. This unique sequence is later to be translated into the antibody protein that will be displayed on the surface of the immature B cell. The random selection of these V, D, and J segments is facilitated by the recombination activating genes (RAGs) (9).

In the case of B cell lymphoma, genetic mutations result in improperly functioning B cells. Due to the many breaks and ligations involved in V(D)J recombination, B cells are very susceptible to errors. Abnormal activity of RAGs could possibly result in inactivation of tumor suppressor genes, which compromises the cell’s ability to halt/slow down proliferation. Alternatively, malfunction of RAGs could also lead to activation of oncogenes or the creation of chimeric oncogenes; genes that cause highly accelerated and uncontrolled cell proliferation. Malfunction of RAGs has been shown to be a key contributor to the development of many human and mice lymphomas (10).

Current therapies & limitations to B cell lymphoma

Current therapies for B cell lymphoma follow those of other cancers including but not limited to chemotherapy, radiation, immunotherapy and an assortment of drugs and treatment cycles (11).

CAR-T cell therapy

A newer and rising immunotherapy called CAR-T cell treatment has gained popularity as it involves genetically modifying the patient's own highly specialized immune cells, T cells, to recognize and attack the cancer in their bodies (12). However, with 40-50% of patients reaching lasting immunity, clinical data overlooks patients who relapse (13). The main areas of concern in CAR-T cell treatment are tumor intrinsic factors, host-related factors and its inadequacy (13).

Tumour intrinsic factors refers to the changes of epitope expression, commonly found on the surface of the cells. As antibody binding to these epitopes is crucial for CAR-T treatment success, changes in this expression affect the multiple mechanisms and ultimate failure of the treatment. Similarly, the microenvironments that are crucial for CAR-T cells as they proliferate. It is possible for the increase of these cells in the microenvironment to cause inhibitory signals around them, resulting in suppression of CAR-T cells (13). Host-related factors refers to the patient's baseline characteristics, such that there is impact to the patient’s capacity to respond to the treatment.

CAR-T cell therapy is commonly followed by chemotherapy, because CAR-T and other immunotherapies are not adequate enough to treat B cell lymphoma. When chemotherapy is combined with CAR-T cell therapy, T cells with in the patient’s body may be deleted or destroyed affecting the CAR-T treatment efficacy. The general inadequacies of this treatment include the CAR-T manufacturing of the cells, changes in tumour environment, previous treatments, effects of the neighboring cells can all result in CAR-T cell exhaustion. This is when the CAR-T cells are unable to maintain previous levels of proliferation or their counterparts. Other inadequacies include the translational abilities and profile of the cells seeing as how many patients express higher levels of immunity related genes (13). The majority of the clinical research done on CAR-T cell therapy is done on successful patients, where unsuccessful patients must revert back to more common but much more harsh treatments.

Common Treatments

Meanwhile, there are major undesirable and harsh side effects to common cancer therapies for B cell lymphoma. One immune chemotherapy routine that is commonly used for B cell lymphoma is CHOP (14) and acts by (12) killing fast dividing cells such as cancerous B cells through various drugs. While this treatment has been utilized since the early 1900s (15), and can have 60-70% progression-free complete remission (16). The side effects render the treatment as a life-altering method. The patient's quality of life decreases with the progression of chemotherapy due to hair loss, nausea, mouth sores, weaker immune systems and more (15). Alternatively radiation therapy, more commonly used for treatment in early stage lymphoma, places patients at risk of experiencing similar major side effects in addition to its own set of side effects (14).

Immunotherapy, where the patient's immune system is boosted with naturally or using manmade agents to aid in phagocytosis often needs to be reevaluated on the specific of the agents and their surface profiles as the treatment process takes place (12). Predictions on success, delivery or reaction from the system are difficult to estimate due to unique immune profiles. Additional agents and/or reaction catalysts may be recruited to counter suppressive cells from the tumour site, adding on to the major side effects the patient experiences alongside increasing the risk of infection (11). While other therapies for B cell lymphoma are in use, all come with similar and unique side effects that affect the livelihood of the patient, and affect the efficiency and success of the treatments such that pursuing specific treatments are met with hesitation (11).

As treatment research progresses, coupled with the rise of CAR-T cell treatment, molecular-based treatments are being developed that have shown to have have higher specificity for cancer targets and reduced side effects on healthy cells (3,17). However, these therapies are not currently efficient enough to be the sole treatment due to difficulty of drug delivery and recognition of tumor sites (12).

AAVP in onCAP: why we chose it

Bringing attention to onCAP, we use adeno-associated virus phage vectors (AAVP) as our novel cancer gene therapeutic delivery platform. AAVPs contain single-stranded DNA with up to 5 kb pairs of space for the insertion of therapeutic genes (18). These are eukaryotic viruses created by combining adenoviruses and bacteriophages. Normally bacteriophages are only able to infect bacteria, as such AAVPs typically co-infect with adenoviruses to permit entry of the virus into mammalian hosts. However, genetic and chemical modifications allow them to infect human cells and transduce therapeutic DNA cassettes for gene therapy in human cells (19, 20, 21).

Compared to previous vectors, AAVPs are advantageous due to controlled expression as a result of replication inability. Their capsids initiate weaker immune responses than other therapeutic phages, and their simple structure allows for greater efficiency during infection (18, 22).

Like any phage vector, AAVPs meet the requirements of infection through target cell entry through receptor binding, lysosomal breakdown evasion in the intracellular environment, and the transport of genetic information to the host cell nucleus (23, 24). Current limitations of AAVP use include their susceptibility to proteasomal degradation (25). As such, the AAVP may have to be combined with proteasome-inhibitors for effective anticancer treatment.

In brief, this vector should allow for target recognition and delivery while preventing grand immune reactions. Additionally, we should observe infection into the cancer cells and the release of desired genetic information. We hope to use this evector to release an expression cassette that should induce apoptosis of the cancer cell.

Results of previous literature are able to further support the use of AAVPs. Specifically, one article (26) looked at the transduction efficacy of AAVPs to transduce cells in a mice model of human glioblastoma. Their vectors carried either cytotoxic tumor necrosis factor (TNF) transgenes or a theranostic approach consisting of Herpes simplex virus thymidine kinase (HSVtk) followed by ganciclovir (GCV) transgenes. Efficacy of both vectors were comparable to preclinical efficacy.

Therefore, we hypothesize that our AAVP vector will demonstrate high specificity and immunotolerance (27) to deliver therapeutic genes to induce the apoptosis of cancerous cells. We hope our AAVP vector-based therapeutic delivery platform will decrease side effects and efficiency issues of current B cell lymphoma therapies to increase patients’ quality of life while undergoing treatment.