The motivation for this project stems from the urgent need to tackle the problem of acquired drug resistance to EGFR-targeted therapy in non-small cell lung carcinoma (NSCLC) patients, which is an obstacle to achieve efficient cancer treatment leading to poor patients’ survival in the world, including Hong Kong. By understanding the acquired drug resistance mechanisms of NSCLC and exploring novel therapeutic approaches, we aim to develop a more accessible and effective treatment for the affected population.

NSCLC is one of the most common and deadliest types of cancer. In 2020, approximately 2.2 million new cases of lung cancer were diagnosed globally [1]. Regions with high rates of smoking and exposure to environmental pollutants are more affected by this disease, including East Asia, North America, and Europe, which have the highest incidence of NSCLC.

As a city in East Asia, lung cancer was the leading cause of cancer-related deaths in Hong Kong in 2021, with 4,037 fatalities, accounting for 26.7% of total cancer deaths [2]. NSCLC is the most common type of lung cancer. In 2020, it comprises 81.3% of all cancer cases in Hong Kong.

A significant proportion of NSCLC patients, 44.8% in Hong Kong [3], is reported to harbor epidermal growth factor receptor (EGFR) gene mutation.

NSCLC is a type of cancer that is often diagnosed in late stage, presenting significant challenges to treatment. Stage IV NSCLC, also known as metastatic NSCLC, is the last stage of the disease, characterized by the spread of cancer from the lungs to other parts of the body, such as the liver, bones, or brain. Unfortunately, the five-year survival rate for patients with Stage IV NSCLC is only around 9%, emphasizing the need for more effective treatment options for these patients.

The primary goal of treatment for stage IV NSCLC is to prolong the patient's life, relieve symptoms, and improve their quality of life. The treatment approach depends on several factors, including the patient's overall health, the extent of the cancer, and the presence of specific genetic mutations.

Early-stage of NSCLC (Stage I and II): If the cancer is diagnosed at early stage, surgery is often chosen to be the first-line treatment. A portion or the entire lung of the patient may be removed depending on the size and location of the tumor. In some cases, chemotherapy might be recommended following surgical treatment in efforts to decrease the probability of tumor recurrence.

Locally advanced NSCLC (Stage III): If the cancer has spread to nearby lymph nodes or tissues, a combination of chemotherapy and radiation therapy is often adopted. Such combinatory therapy may be followed by surgical treatment in some cases.

Advanced NSCLC (Stage IV): If the cancer has spread to other parts of the body, most patients with NSCLC at Stage IV are treated with systemic medicines such as chemotherapy, targeted therapy, or immunotherapy.

Chemotherapy is a treatment option for Stage IV non-small cell lung cancer when there are no identifiable genetic mutations or when cancer progresses following targeted therapy. Chemotherapy is regarded as a conventional and affordable cancer treatment modality that eliminates fast-growing cells through the inhibition of macromolecular synthesis or neoplastic cell function [5].

However, chemotherapy works on all types of active cells due to its lack of specificity. Healthy cells that have high rates of growth and division are always harmed, such as hair follicle cells, skin cells, and cells lining the digestive tract. As a result, various common side effects include nausea and vomiting, loss of appetite, diarrhea, hair loss, mouth sores, skin dryness, and fatigue.

Another main concern regarding chemotherapy for NSCLC is its limited effectiveness. According to a study conducted in 2009, NSCLC patients with EGFR mutation showed a higher response rate of 71.2% with gefitinib treatment, when compared to 47.3% with carboplatin-paclitaxel chemotherapy, and their progression-free survival was extended [6].

Currently, the presence of targeted therapy, involving tyrosine kinase inhibitors (TKIs), remains the standard first-line treatment modality for patients diagnosed with metastatic, non-squamous NSCLC with EGFR mutation.

Several generations of EGFR TKIs have been adopted throughout the years for NSCLC treatment. Both gefitinib and erlotinib are known as first-generation EGFR TKIs, which inhibits EGFR through competitive binding action with ATP binding site of the TK receptor. The first generation of TKIs have demonstrated an increase of progression-free survival (PFS) when compared to platinum doublet chemotherapy.

Meanwhile, second-generation EGFR TKIs, such as Afatinib and Dacomitinib, operates through irreversible binding towards EGFR. Accordingly, clinical trials results have exhibited that second-generation TKIs were more effective in terms of improving patient overall survival rate when compared to the first-generation drugs. In particular, the median overall survival of patients treated with dacomitinib was found to be 34.1 months, which is longer than 26.8 months that gefitinib has to offer [7].

Despite the considerable improvement in overall survival achieved by Dacomitinib, it has a relative higher toxicity compared with gefitinib. Moreover, about 50% of patients with NSCLC have developed the EGFR T790M mutation [8]. Aside from known toxicity, Dacomitinib is also unable to efficiently combat the developed EGFR mutation. Fortunately, third generation TKI, including Osimertinib, might be useful in combating the acquired EGFR T790M mutation. Osimertinib is currently applied as the first-line treatment to Stage IV NSCLC patients, where the mode of action is through competitive inhibition of mutated EGFR activity. As a result, the growth of the tumor might be slowed down or halted.

Figure 6 EGFR-TKI Treatment in Lung Cancer.

Although Osimertinib has shown promising results in clinical trials as a first-line targeted therapy for EGFR T790M mutation-positive NSCLC, drug resistance inevitably develops among patients.

Osimertinib resistance leads to the emergence of acquired new EGFR mutations, C797S gene mutation, or the activation of bypass signalling pathways, such as MET amplification, which limits the effectiveness of Osimertinib. Hence, it highlights the demand for ongoing research to overcome this issue.

To overcome the Osimertinib resistance in NSCLC, a new treatment strategy using bispecific antibodies to co-target EGFR mutations and MET amplification has been proposed and has shown promising outcomes in clinical trials [9]. However, there are only eight clinically approved bispecific antibodies currently available for targeted therapy. Obviously, the limited available choices reflect there is a constant and immediate demand of novel and potential bispecific antibody candidates for clinical trials.

Moreover, the therapy cost of bispecific antibodies ranges from 30k to 170k USD per treatment, which is barely affordable for most patients. This high therapy cost is neither covered by the public health system nor charities in well-developed cities such as Hong Kong. We believe this extremely high therapy cost reduces the chance for the underprivileged patients to receive proper and timely treatment. Hence, there is an urged need to reduce the inequality by developing potent but low-cost bispecific antibodies.

After conducting thorough interviews with medical professionals and understanding the challenges faced by NSCLC patients, we have identified the development of acquired drug resistance as a major obstacle to treatment success. Despite advances in targeted therapies and immunotherapies, many patients continue to suffer from low survival rates due to the eventual emergence of drug resistance.

Our primary objective is to develop a bispecific antibody that, when used in combination with Osimertinib, enhances its therapeutic effect and overcomes drug resistance. By targeting both EGFR and c-MET, two cell surface tyrosine kinase receptors preferentially overexpressed in Osimertinib-resistant NSCLC cells, we aim to extend the median survival rate of NSCLC patients with Osimertinib resistance.

In a recent clinical trial involving more than 550 patients diagnosed with advanced NSCLC, Osimertinib treatment yielded a median overall survival rate of 38.6 months [10].

Our goal is to significantly expand this median survival rate by incorporating our bispecific antibody into the treatment regimen.

To achieve this, we have outlined the following future goals:

• Conduct preclinical trials to test the safety and efficacy of our bispecific antibody in combination with Osimertinib.
• Investigate the potential of our newly-developed drug as a breakthrough treatment option for NSCLC patients.
• Carry out clinical trials to validate the success of our combination therapy in improving survival rates for patients with advanced NSCLC.
• Obtain FDA approval for our drug, paving the way for widespread utilization and improving the lives of NSCLC patients around the world.

To address such issues related to acquired drug resistance development and affordability, it is of great importance to develop an effective therapeutic alternative with the goal of increasing the survival of these patients. Based on previous studies, we found that EGFR and tyrosine-protein kinase Met (c-MET) were preferentially upregulated in drug-resistant cell lines. Targeting these two proteins at the same time can be achieved by conventional bispecific antibodies which are currently being tested in clinical trials. However, the production cost of the conventional bispecific antibody is high and the procedures are tedious. For example, conventional bispecific antibody production relies on the co-culture of two different cell types that expresses half of the light-heavy chain of the antibody, and the subsequent dimerization of the half antibody secreted by the two cell types. This process is complicated and time-consuming. Therefore, we would like to employ a synthetic biology strategy to construct an innovative bispecific antibody against c-MET and EGFR.

Our team will work on developing an innovative approach to construct a bispecific antibody to combat Osimertinib resistance in non-small cell lung carcinoma (NSCLC). Bispecific antibodies (BsAb) are a large family of molecules designed to recognize two different antigens. This property has been exploited to target two types of membrane receptors of within a cell.

Traditional bispecific antibodies are formed by the combination of two different light-heavy chain antibody moieties. In our project, we propose a novel bispecific antibody design that consists of two major components, a synthetic cyclic peptide and a native antibody.

The cyclic peptide is presumably conjugated to the antibody to form a secondary antigen binding site (bispecific). Since linear peptides are prone to proteolytic degradation and the conjugation efficiency varies depending on the circularization of target peptide, we employ a synthetic biology approach to design and build an intein splicing BioBrick that is capable of one-step circularization and purification of the target peptide for subsequent antibody conjugation.

Figure 8First model: plasmid design for cyclic peptide with his tag or GST tag for purification.

Figure 9 Second model: plasmid design for cyclic peptide with two flanking inteins and Chitin binding domain (CBD) for purification.

Figure 10 Second model: Improved design with superior intein sequence and Chitin binding domain (CBD) for purification.

Figure 11 Rational BioBrick design of the novel bispecific antibody.

In addition to designing and building synthetic BioBricks to express a functional cyclic peptide for effective antibody conjugation, we will also characterize this novel bispecific antibody and examine its effects in a cell-based model. Our project will harness the power of synthetic biology to develop a novel therapeutic bispecific antibody against resistant NSCLC, with the ultimate goal of overcoming drug resistance. This approach may overcome Osimertinib resistance in NSCLC through a relatively low-cost production method, significantly improving treatment outcomes and accessibility for patients.

We are dedicated to sharing our progress and achievements with stakeholders, patients, and the global community. Through transparent and engaging communication, we aim to foster awareness of our innovative treatment approach and its potential to transform the lives of NSCLC patients. By collaborating with healthcare professionals, researchers, and organizations, we strive to usher in a new era of hope and healing for those affected by NSCLC.

Together, we believe that our combinatory therapeutic approach and biological synthesis of antibodies will not only overcome acquired drug resistance but also improve treatment outcomes and accessibility for millions of NSCLC patients worldwide. As we embark on this ambitious journey, we remain steadfast in our pursuit of a brighter, healthier future for all.