Cancer research is an ever-evolving field. Extensive efforts by countless researchers in the past have opened up diverse options for therapeutic intervention. However, a fundamental issue in drug therapy for cancer is that current chemotherapeutic drugs have poor specificity and indiscriminately affect cancerous and non-cancerous cells. Developing drugs that preferentially target tumour cells represents an open challenge in cancer therapy research that is of significant interest to biomedical researchers, patients, medical practitioners, and pharmaceutical companies. Although we are addressing the broader problem of improving the specificity of chemotherapy, we chose breast cancer as our model due to its importance to our local community.
Improving the specificity of chemotherapy is a longstanding problem relevant to all types of cancers, but we centered our project on breast cancer. On International Women's Day 2021, our team members volunteered for a breast cancer screening camp conducted in Chathancode, Kerala. We realised that even in our small community of Vithura, with a population of 20,000, the number of breast cancer cases was alarming. Soon after, we reached out to many people in Vithura.
Figure 1. Breast cancer screening camp in Chathancode
We spoke with medical professionals who conduct screening and awareness initiatives among the local rural communities. Next, we met with breast cancer survivors to talk about their experiences with chemotherapy. We interacted with the housekeeping staff at our institute to understand their awareness of breast cancer. Each of these interactions provided us with valuable insights into the impact breast cancer has on individuals and communities, but all of them affirmed our decision to focus on breast cancer research.
In 2020, there were 2.3 million new cases of breast cancer and 685,000 deaths globally. Currently, in India, breast cancer has the highest gross incidence and mortality among all cancers. Projections from the WHO Global Cancer Observatory suggest that by 2040, there could be 3.9 million new cases and 1.04 million deaths worldwide. [1]
Figure 2. Breast cancer statistics of India, 2020
Breast cancer is emerging as a major cause of morbidity and mortality in India. Inaccessible health care facilities, lack of awareness, and the stigma associated with breast cancer have led to underdiagnosis and undertreatment. These issues are magnified in our local community of Vithura. In June 2022, we partnered with the Indian Medical Association to conduct a breast cancer screening camp for the housekeeping staff on our campus, who otherwise have limited access to advanced screening facilities. One in seven women screened using the iBreast screening device were suspected of breast cancer, which was much higher than the national average of one in twenty-nine women. In Thiruvananthapuram, breast cancer had the highest incidence among all cancers and accounted for one-fourth of all new cancer cases in 2018-2019. Against this backdrop, we aim to develop an accessible and effective drug delivery system with enhanced specificity.
Socio-economic inequalities compound the issue in India by limiting access to life-saving diagnostics and treatment. Over 40% of households across India resort to distress funding to cover the cost of cancer treatment [2].These issues are magnified in our local community of Vithura. In Thiruvananthapuram, breast cancer had the highest incidence among all cancers and accounted for one-fourth of all new cancer cases in 2018-2019 [3]. As cases continue to rise, the next few years represent a crucial moment to change how we think about breast cancer therapy. As research moves towards optimising drug delivery systems, we believe our project fits with these shifting paradigms in cancer therapeutics and can have a meaningful impact on society.
Cancer cells differentially overexpress specific cell surface markers. Targeted therapies capitalise on this property of cancer cells to preferentially attack cancerous cells. However many non-cancerous cells have basal expression of the cell surface marker and are thereby affected by the drug. We wanted to build a more robust system that delivered and activated by utilising not one, but two targets. The functioning of this system is analogous to an AND gate: the drug is activated only in cells overexpressing both markers, and any cell expressing one or none of the markers remains largely unaffected. This second marker would therefore serve as an additional checkpoint. Breast cancer was chosen to be a model due to its relevance in our local community, but we hope to extend the modular design of Duonco to other types of cancer in the future.
Figure 3. Core idea of project
An ideal target cancer marker for Duonco is one that has shown to be overexpressed in breast cancer, and has an accessible extracellular domain. Among the different molecular subtypes of breast cancer, we chose HER2+ breast cancer due to the availability of such markers. Other subtypes are characterised by nuclear-localised markers (such as estrogen or progesterone) or altogether lack defined markers (as with triple-negative breast cancer), and are not an ideal model system to test Duonco. Among the cell surface markers associated with HER2+ breast cancer, we chose the following:
Human Epidermal Growth Factor Receptor 2 (HER2), also known as ErbB2, is a 185 kDa protein. HER2 plays an essential role in cell differentiation and growth but overexpression of HER2 is associated with malignancy in cells, and occurs in approximately 15-30% of breast cancers [4]. It is strongly associated with increased disease recurrence and a poor prognosis. HER2 is the target of many breast cancer treatments such as trastuzumab [5].
CX3C motif chemokine receptor 1 (CX3CR1), is a transmembrane protein of the G protein-coupled receptor 1. It is responsible for modulating cell activity towards survival, migration and proliferation [6]. CX3CR1 has been shown to be important in driving metastasis in breast cancer. Research on using CX3CR1 as a target is not as abundant as for HER2, but some studies have indicated that it may be a viable target [7].
OMVs (Outer Membrane Vesicles) are non-replicating nanovesicles that are continuously shed by gram-negative bacteria. They have a mean size between 20 to 200 nm. Usually bilayered, they are spherical buds of the outer membrane filled with periplasm [8].
There are several advantages to using OMVs for targeted drug delivery. OMVs can easily be decorated with foreign epitopes and can carry diverse payloads. They protect their contents from the external environment and dilution. In addition, they also have intrinsic immunostimulatory properties, thus synergistically helping the body's innate immune system in the targeting of cancer cells [9].
Figure 4. Structural representation of OMV
Affibodies are small proteins that bind to their target proteins with high affinity, mimicking monoclonal antibodies. They hold many advantages over traditional antibodies: they are smaller (~6 kDa), spontaneously fold into their 3D structures, do not require post-translational modifications, and generally tolerate fusion with other proteins.
For this project, we chose ZHER2:342 (BBa_K4359002) to be expressed on the surface of the OMVs to create Affi-OMVs. ZHER2:342, an affibody protein that is engineered to bind to the extracellular domain of HER2. It binds with remarkable affinity to HER2 (Kd = 22 pM) [10].
Tumor homing peptides (THPs) are oligopeptides, usually consisting of 30 or fewer amino acids that
selectively bind to specific markers on tumour cells. They are discovered via phage display experiments from a
pool of randomly generated peptides. THPs can be used to decorate drug carriers to target them to tumour cells
overexpressing the target market. Their small size, low immunogenicity, high specificity and versatility makes
them ideal for a variety of purposes.
For this project we chose THP Pep-1 (BBa_K4359004) to
be expressed on the surface of OMVs to generate THP-OMVs. THP Pep-1 is a peptide engineered to bind to CX3CR1 [11].
Figure 5. OMVs expressing affinity proteins
The prodrug-enzyme system consists of an inactive prodrug that is activated by its cognate enzyme. This activated form of the drug would then produce a therapeutic effect (cancer cell death). The prodrug-enzyme system enables a more precise regulation of the therapeutic effect by controlling when and where the prodrug and enzyme interact [12].
For this project, we chose cytosine deaminase. Cytosine deaminase converts 5-fluorocytosine (5FC) to 5-fluorouracil (5FU). 5FU traverses into the neighbouring cells. It exerts its anticancer action through inhibition of thymidylate synthase and incorporation of its metabolites RNA and DNA. If injected at the core of the tumour, even a small amount of the activated drug should be enough to cause a significant decrease in the size of the tumour [13].
Proteins such as affibodies and THPs can be expressed on the surface of OMVs by genetically fusing them to a bacterial protein that naturally translocates to the outer membrane, and concomitantly, OMVs. One such bacterial protein is Cytolysin A (ClyA) [14]. A flexible serine-glycine (SG) linker is included to join these domains, as well as an epitope tag for detection. The following composite parts can be generated:
Figure 6. Composite parts
These recombinant proteins are expressed separately in two populations of E.coli, where they translocate to the outer membrane of the bacteria. OMVs bleb out from the surface of the bacteria, generating two types of OMVs: Affi-OMVs expressing the affibody against the HER2 receptor and THP-OMVs expressing THPs against the CX3CR1 receptor. Affi-OMvs are loaded with the prodrug 5FU and THP-OMVs with the enzyme cytosine deaminase respectively.
Figure 7. Affi-OMVs loaded with prodrug and THP-OMVs loaded with enzyme
We hypothesise that when Affi-OMVs and THP-OMVs are administered, and encounter a cancer cell expressing HER2 and CX3CR1, they are simultaneously internalised, and release their contents. The prodrug is converted to its active drug form by the enzyme and exerts its therapeutic effect. In the case of a non-cancerous cell having basal expression of one or both of these receptors, the probability of simultaneous internationalisation is low. Even if one type of OMV is internalised, in the absence of its partner, a negligible effect is expected.
Figure 8. Project work flow