Essence of PanoPOLY

PanoPOLY is driven by a profound passion for tackling pancreatic cancer, one of the most challenging and currently incurable forms of cancer. Through our participation in the iGEM initiative, we aim to make significant advancements in pancreatic cancer treatment by developing novel and groundbreaking approaches, specifically focusing on overcoming gemcitabine resistance pancreatic cancer. Our ultimate goal is to create new strategies that substantially improve survival rates, offering hope to patients facing this formidable disease. We are committed to improving patient outcomes, striving to make a significant impact on pancreatic cancer treatment. We aspire to bring new hope to patients and families affected by this challenging condition, potentially transforming the landscape of pancreatic cancer care.

Introduction

Although pancreatic cancer is not among the most common malignancies globally, it presents a significant health challenge due to its exceptionally high mortality rate. According to the American Cancer Society, approximately 56,000 cases of pancreatic cancer were diagnosed, with an estimated 45,000 deaths in the United States in 2019 (Mizrahi et al., 2020).

According to the statistics from the World Health Organization (WHO) in 2022, pancreatic cancer ranks as the 14th most common cancer and the 10th leading cause of mortality among all cancers. Notably, its impact is even more pronounced in certain regions, ranking as the 4th leading cause of cancer deaths in the United States and the 3rd in Hong Kong. These figures highlight the disproportionate burden of pancreatic cancer and underscore the critical importance of focused research efforts to address this formidable health challenge.

Heavy cigarette smoking stands out as a primary modifiable risk factor in pancreatic cancer, with a clear positive correlation between smoking intensity and disease incidence (McGuigan et al., 2018b). Other modifiable risk factors, such as obesity and unhealthy dietary habits, also account for the increased risk of pancreatic cancer. Aging and a family history of pancreatic cancer are regarded as non-modifiable risk factors for this disease. Regions with heavy smoking and unhealthy eating habits, such as the US and Denmark, are experiencing higher incidence and mortality rates of pancreatic cancer.

Pancreatic cancer is lethal (high death rate) because it is asymptomatic at an earlier stage and is usually diagnosed at an advanced stage when the tumor is unresectable or has already metastasized (i.e., stage III or IV). For patients diagnosed at an early stage after treatment, the 5-year survival rate ranges from approximately 10% to 20% (Puckett & Garfield, 2022). Pancreatic ductal adenocarcinoma (PDAC) accounts for approximately 90% of pancreatic cancers and is often present at an advanced stage when the cancer is unresectable or has metastasized (Vincent et al., 2011). From this information, we can conclude that the 5-year survival rate is higher for patients diagnosed at earlier stages of pancreatic cancer; however, approximately 90% of patients are diagnosed at an advanced stage, which ultimately decreases the 5-year survival rate of pancreatic cancer. It is predicted to rise from 3rd to 2nd most common cause of cancer death in the US by 2030 (McGuigan et al., 2018).

Current Treatments

The treatment procedure for pancreatic cancer depends on the stage of the tumor at diagnosis, which reflects its size, location, and spread. While surgery can be a curative option, this is generally only true when the cancer is detected at an early stage and has not spread to other parts of the body. Pancreatic cancer can be divided into three types.

Resectable or borderline resectable tumor

For resectable or borderline resectable tumors, surgery is the primary treatment option. Pancreatic duodenectomy (Whipple's procedure) is the standard surgical technique for tumors in pancreatic heads. Similarly, distal pancreatectomy, often combined with splenectomy, is used as the surgical method for the removal of tumors in the body or tail of the pancreas. Currently, there has been more research on laparoscopic pancreaticoduodenectomy, a robot-assisted surgery, and it has been shown that patients have reduced blood loss, a shorter duration of hospital stay, and improved disease-free survival compared to traditional open surgery.

After surgery, patients usually receive chemotherapy or chemotherapy combined with radiotherapy (RT). Adjuvant chemotherapy with gemcitabine or FOLFIRINOX was administered, and a significant increase in the survival rate of patients was observed. The median survival rate of patients receiving adjuvant chemotherapy has increased from 12.8 to 21.6 months (Mizrahi et al., 2020). Systemic chemotherapy and chemoradiotherapy are administered to decrease the chances of cancer relapse and kill the cancerous cells that might be left after surgery.

Locally advanced tumor

Preoperative systemic chemotherapy plays an important role in the treatment of locally advanced tumors. This therapy helps to shrink the size of the tumor and downstage it into resectable tumors. Systemic chemotherapy involves regimens that have been approved for metastatic settings such as gemcitabine plus nab-paclitaxel or FOLFIRINOX. Some patients show a positive response (shrinkage of the tumor) to preoperative systemic chemotherapy and undergo surgical resection.

Tumor that remain unresectable after initial therapy, treatment options include continued systemic chemotherapy or chemoradiotherapy, depending on the patient's condition and tumor characteristics. The role of radiation therapy in this setting remains under investigation.

Metastatic tumor

In this stage, the tumor has already spread to other organs, and surgery is no longer an option. At this stage, the primary treatment regimen is systematic chemotherapy to palliate cancer-related symptoms, aimed at palliating symptoms and prolonging survival. Gemcitabine was given to patients and the median survival was compared between patients treated with these drugs and untreated metastatic pancreatic cancer. It was found that the use of systematic chemotherapy improved the overall median survival from 6.8 to 11.1 months (Mizrahi et al., 2020).

Gemcitabine

Gemcitabine is a pro-drug used in adjuvant and neoadjuvant chemotherapy. This drug requires cellular uptake and serial phosphorylation inside the cell to become pharmacologically active. Gemcitabine exhibits cytotoxic effects by inducing apoptosis.

Gemcitabine induces apoptosis by incorporating itself into an elongating DNA strand during DNA synthesis (usually at the S phase). One of the cellular metabolites of gemcitabine (DFCTB) is a competitive substrate for deoxycytidine triphosphate (dCTP). This allowed the incorporation of dFCTP into elongated DNA strands. Next, a deoxyribonucleotide is added to the progressing chain length, and replication terminates (Mini et al., 2006). In this process of “masked chain termination,” gemcitabine is locked in the DNA and because of the terminal nucleotide, proofreading exonuclease is unable to remove the gemcitabine nucleotide from its penultimate position, ultimately activating the apoptotic pathway in these cells.

Another feature of gemcitabine is its self-potentiation, which means that it can enhance its activity. Dfctp inhibits the enzyme RR, which controls the formation of nucleotide triphosphate and the conversion of CDP to dCDP (Jia & Xie, 2015). This ultimately decreases the concentration of dCTP required for DNA synthesis. When the concentration of dCTP is low, its competitiveness decreases, and dFCTP, that is, gemcitabine, is readily incorporated into the progressing DNA strand.

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Fig 2. Laboratory Environment
Figure 2. Animation showing the action of gemcitabine on gemcitabine-sensitive pancreatic cancer cell.

Gemcitabine Resistance

Conventional chemotherapy with gemcitabine initially yields positive therapeutic effects. Despite its initial effectiveness, prolonged use of gemcitabine can lead to the development of drug resistance, resulting in the formation of gemcitabine-resistant cancer cells. This resistance arises from various mechanisms, including molecular and cellular changes. These changes encompass deactivation of the apoptosis process, increased expression of drug efflux pumps, activation of oncogenic signaling pathways, and alterations in mRNA regulation. One of the mechanisms involves the upregulation of Human Epidermal Growth Factor Receptor 2 (HER2). In gemcitabine-resistant pancreatic cancer cells, there is often an observed increase in HER2 expression and activation. This overexpression of HER2 receptors triggers downstream signaling pathways that promote cell survival, proliferation, and metastasis, effectively circumventing the cytotoxic effects of gemcitabine. The enhanced HER2 signaling can lead to the activation of anti-apoptotic proteins and increased DNA repair mechanisms, further contributing to drug resistance. Understanding this HER2-mediated resistance mechanism has opened new avenues for potential combination therapies, such as coupling gemcitabine with HER2-targeted agents, which may help overcome resistance and improve treatment outcomes in pancreatic cancer patients

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Figure 3. Animation showing no effects seen in gemcitabine-resistant pancreatic cancer cells upon administration of gemcitabine.

Challenges of Current Treatments

The challenge of the current treatment approach lies primarily in the development of drug resistance. As previously mentioned, several molecular signaling pathways contribute to gemcitabine resistance by altering gene expression, thereby complicating the targeting and induction of apoptosis in cancer cells.

Additionally, there are limitations to surgery and radiation therapy, as residual tumor tissue often remains after surgical resection. Such local therapies are less effective due to tumor cell angiogenesis and early-stage metastasis. Furthermore, a unique characteristic of PDAC is the development of a fibrotic barrier around cancer cells composed of fibroblasts, leukocytes, and extracellular matrix (ECM). This fibrotic barrier formation is associated with a desmoplastic reaction triggered by oncogenic signals, notably mutant K-Ras. The desmoplastic stroma formed during this reaction affects the efficacy of drug treatments and has been shown to promote the immunosuppression of T lymphocytes.

Traditional chemotherapy is regarded as a difficult and non-specific approach to differentiate between healthy and cancerous cells, resulting in damage to healthy pancreatic cells. Therefore, there is a significant need to develop targeted therapies for pancreatic cancer.

Overcoming

After conducting multiple research studies and evaluating statistical data, we found that the primary challenges in pancreatic cancer are its limited treatment options and the low efficacy of these treatments. Moreover, there is a concerning trend of decreasing sensitivity to first-line treatments for non-resectable or metastatic pancreatic cancer, primarily due to acquired drug resistance through induced or genetic mechanisms.

In response to these challenges, PanoPOLY aims to address the molecular changes in pancreatic cancer cells that promote tumor proliferation and metastasis, specifically targeting drug-resistant pancreatic cancer cells. We are developing an innovative dual-targeting nanobody called Panobody using a synthetic biological approach. This Panobody comprises two distinct targeting peptides: one arm specifically designed to target the Epidermal Growth Factor Receptor (EGFR), and the other engineered to target Human Epidermal Growth Factor Receptor 2 (HER2). Our team hypothesizes that simultaneously targeting both EGFR and HER2 with our novel nanobody could significantly reduce the growth of pancreatic cancer cells and complement the action of gemcitabine, with the ultimate goal of prolonging the survival of patients with pancreatic cancer.

Solution

Our project adopts an innovative dual-action approach in the development of a novel therapeutic nanobody called Panobody for pancreatic cancer. This strategy aims to simultaneously target cancer cells and enhance their sensitivity to gemcitabine, a standard chemotherapeutic agent. The Panobody is engineered with two distinct targeting moieties, with one of the arms specifically targeting the EGFR and the other targeting HER2. These receptors are often overexpressed in pancreatic cancer and play crucial roles in tumor growth, survival, and drug resistance.

The dual-targeting nanobody called Panobody targets the EGFR and HER2 and inhibits the signaling function of these receptors, dual inhibition is expected to not only slow the proliferation of gemcitabine-resistant cells but also to potentially re-sensitize them to chemotherapy.

To achieve this, we have designed and synthesized a vector carrying the encoding sequence of the Panobody. We are using a plasmid vector. Our designed sequence is inserted into the multiple cloning site of this plasmid and subsequently, transformation is done to introduce the recombinant plasmid into E. coli (BL21). After successful transformation, E. coli with the recombinant plasmid is allowed to grow where they synthesize our protein. This protein which is our dual targeting nanobody is then purified and tested on an in-vitro cell-based model to check its efficacy.

Fig 2. Laboratory Environment
Figure 4. BioBrick design of Panobody targeting HER2 and EGFR.

Figure 5. Predicted 3D structure of Panobody by AlphaFold.

EGFR and HER2

Our genomic bioinformatic analysis of dry lab data revealed significant upregulation of EGFR and HER2 mRNA (p=4.49 10-22 and p=6.44 10-43, respectively) in pancreatic cancer patients, as evidenced by GTEx and TCGA datasets. EGFR and HER2 are cell surface proteins found on cell surfaces that function as tyrosine kinases. These proteins consist of three main parts: an external region that binds to specific molecules, a section that spans the cell membrane, and an internal portion. When particular molecules attach to the external region, it activates the tyrosine kinase function in the membrane-spanning section. This activation results in the addition of phosphate groups to signaling molecules, subsequently stimulating cell growth and division. Our further analysis showed that these two genes not only overexpressed but also independently predicted poor overall survival in patients with pancreatic cancer (p=0.0012 and p=0.026, respectively). Notably, high levels of EGFR and/or HER2 correlate with decreased survival rates in pancreatic cancer (p=0.00012). These findings suggest that EGFR and HER2 represent promising therapeutic targets for improving patient outcomes in pancreatic cancer. To further correlate these two genes and chemoresistance in pancreatic cancer patients, we examined a database of pancreatic cancer cell lines (BxPC-3 and CFPAC-1) that show resistance to gemcitabine treatment. Our findings revealed that EGFR and HER2 are significantly overexpressed in these gemcitabine-resistant pancreatic cancer cells. To determine the clinical relevance of this observation, we conducted an analysis of 107 pancreatic cancer patients who underwent gemcitabine treatment. In line with the cell line data, we observed that EGFR and/or HER2 were more commonly expressed in pancreatic cancer patients who exhibited resistance to gemcitabine treatment compared to those who were sensitive to the treatment (p=0.04). Based on the above pilot data, we hypothesized that targeting EGFR and HER2 simultaneously not only suppresses the pancreatic cancer growth but also sensitize to gemcitabine treatment.

Through our wet lab analysis, we verify that Panobody's concurrent targeting of EGFR and HER2 not only substantially inhibits pancreatic cancer cell growth but also increases their responsiveness to gemcitabine therapy. Overall, this two-pronged strategy shows potential in addressing the absence of targeted treatments and chemotherapy resistance in pancreatic cancer management.

We are committed to sharing the progress, results, and importance of our project with healthcare professionals, researchers, patients, and the general public. We are optimistic that our dual-targeting nanobody will work effectively against gemcitabine-resistant pancreatic cancer and increase the curability and survival of patients.