How can we make an impact on B-cell lymphoma?

Lymphoid cancers include almost 100 variants, and collectively are one of the most common types of cancers and leading causes of cancer death (1). In particular, the incidence rate of the most common subtype, non-Hodgkin B-cell neoplasms, has been increasing in recent years with an estimated 117, 470 new cases in 2016 in the US (1). There are numerous subtypes of this cancer with the most aggressive and common type being diffuse large B-cell lymphoma (DLBCL) comprising 30-40% of all non-Hodgkin lymphomas (1,2). Currently, there are new immunotherapies being developed, including most prominently chimeric antigen receptors (CAR) T cell therapy and anti-CD20 monoclonal antibody rituximab. However, the 5-year survival rate for the most aggressive types is still only 60-70% for DLBCL as of 2018 and around 47-63% for Burkitt lymphoma as of 2016 (3,4).

Our team aims to develop a novel phage-based cancer gene therapy using adeno-associated virus phage (AAVP) vectors. We were motivated by the recent developments in therapeutic targets for B cell lymphoma, and the clinical success of CAR-T cell therapy and antibody-drug conjugates. Despite these advancements, there are still gaps in the field of cancer immunotherapy, making current treatments for B cell lymphomas, such as CAR-T cell therapy, more risky and inaccessible. As a result, we wanted to address problems surrounding limited treatments for B cell and hematological cancers.

Current plan in the lab

The current work in this field involves the transgene delivery of TNF-α into cancerous cells due to the ability of these proteins to mediate apoptotic activity. Based on a study conducted by Staquicini et al., (5) researchers developed an AAVP construct (RGD4C-AAVP-TNFα) to deliver TNF-α into glioblastoma cells. After treatment, researchers observed antitumor activity and induction of cell death in the target glioblastoma cells (5). A similar study was conducted by Lyu et al. (6), where researchers examined the effects of a recombinant construct containing anti–HER-2/neu single-chain antibody fused to TNF-α (scFv23/TNF). Upon treatment of HER2/neu cells with scFv23/TNF, there was an observable increase in TNF-R1 expression and induction of apoptotic activity through cleavage of caspase 8, caspase 3, and poly(ADP-ribose) polymerase (6).

Similarly, several researchers have investigated the ability to deliver GrzB into cancerous cells to induce apoptotic activity. Based on a study conducted by Cheung et al. (7), researchers created a GrzB-Fc-4D5 recombinant fusion construct containing humanized anti-HER2/neu scFv fused to active GrzB via an IgG heavy-chain fragment (7). The researchers observed that GrzB-Fc-4D5 internalized into target SKOV3 cells within 1 hour of treatment, which allowed GrzB to reach the cytoplasmic compartment (7). Upon delivery of the GrzB, there was an observable increase in the caspase-9 activation and inhibition of AKT phosphorylation, characteristic of HER2/neu signaling (7).

As a result, these findings show that transgenic delivery of GrzB or TNF-α into tumor cells via receptor binding has significant potential. However, more work needs to be done to improve delivery efficiency; which we plan to address with the use of an AAVP vector due to the cis form that is present in AAVs (7). Furthermore, there is a lack of research surrounding the use of AAVPs to target hematological malignancies therefore, we intend to leverage the apoptotic mechanisms of GrzB and TNF-α peptides to improve tumor cell killing. As a result, our design builds upon previous work completed involving phage-based vectors by addressing limitations such as improving the effectiveness of gene delivery and increasing binding flexibility by utilizing scFvs to bind to receptors.

Hypotheses

Our project’s focus on using AAVPs as a cancer treatment allows for engineered phages to directly interact with human cancer cells. By engineering an AAVP vector with a transgene cassette and anti-CD22 scfv, the vector will interact with the CD22 receptor to target cancer cells. Our use of AAVPs will improve the effectiveness of gene delivery due to the cis form present in AAVs. Similarly, our use of scFvs to bind to receptors will allow for greater flexibility in binding sites (8). When the AAVP vector is administered intravenously, it will be endocytosed and cause the expression of granzyme B and TNF-α to induce cancer cell killing (9-12). Therefore, our proposed design displays translational potential both in vivo and in vitro, potential for intravenous delivery, increased bioavailability, and the ability to treat various types of cancers, even those that do not present with a tumor (13).

A future implementation may include release into the human or animal body at the clinical level through intravenous administration. Some safety concerns we need to consider include the expression of cytotoxic proteins (perforin and granzyme B) which can cause cell and tissue damage, especially if the targeting molecule does not work. However, our AAVP vectors cannot replicate in mammalian hosts, as human cells are not permissive to the AAVP. This will prevent the environmental spread and human-to-human spread.

Future directions

Looking beyond the scope of the iGEM competition, we see great value in publishing our in vivo and in vitro designs in high-profile journals. We are in the process of consulting with physicians and specialists in B-cell lymphoma to seek feedback on our proposed design and gain a better understanding of the therapeutic context for drug development and evaluation. Similarly, we see great value in speaking with patients and families who are directly impacted by B-cell lymphoma to elicit their perspectives on this novel approach in comparison to current treatments. We anticipate undergoing the drug development process by performing in vivo and in vitro testing, conducting clinical trials with sample patient populations to evaluate safety and efficacy and seeking regulatory approval. Therefore, we intend to utilize the infrastructure provided by the McMaster Innovation Park for the commercial development and clinical translation of our design. Furthermore, this project design is highly transferable to different applications and we aim to continue researching this idea and apply our project to a larger scale to aid cancer patients.

Designing an adeno-associated virus phage with transgene cassette and anti-cd22 scfv, which targets cancer cells by interacting with CD22 receptors. Intravenous delivery of AAVP vector leads to endocytosis of vector and expression of granzyme B and TNF-α to induce cancer cell killing. Figure was created using Biorender adapted from references 8-11.