Mid- and Low- Rectal Cancer

Cancer has emerged as a critical global public health issue, profoundly impacting physical health, economic stability, and social development across the globe. As the world's population ages, projections indicate that the burden of cancer is set to increase by approximately 50% by 2040 compared to 2020, with nearly 30 million new cases. This alarming trend is particularly pronounced in countries undergoing rapid social and economic transformation, such as China, where lifestyle contributes to rising incidence rates.1The increase in cancer cases poses significant economic pressures on families and society, necessitating urgent attention and action.

Figure 1: Colorectum cancer ranked 3rd in the new incidence of cancer in 2022 in China.

In China, the incidence of colorectal cancer ranks third among malignant tumors, with over 60% of these cases being rectal cancer. The most common subtype of rectal cancer is mid- and low- rectal cancer. In China, the number of patients diagnosed with mid- and low- rectal cancer each year is about 440,000.2 Due to the specific characteristics of lifestyle factors, the incidence of mid-to-low rectal cancer is gradually increasing, which presents a significant public health challenge for Chinese society. Furthermore, that will impose a substantial burden on patients.

To eradicate mid- and low- rectal cancer, patients often need extensive surgical interventions, including the removal of the anus and the creation of a permanent colostomy on the abdomen. This means that they have to rely on colostomy bags for excretion for the rest of their lives. This will have a significant impact on patients both psychologically and physiologically. Patients often experience anxiety and depression as a result. Additionally, the stoma is easy to be infected and irritated, and it may also lead to gastrointestinal disorders. The care and adaptation required for the stoma can also cause inconveniences into the patients' daily lives.

We aim to treat more patients and alleviate the significant burden that cancer imposes on the nation, society, and families, while also enabling more patients to retain anal function and maintain a high quality of life. This is why our project places particular emphasis on mid- and low- rectal cancer.

Shortcomings of Current Treatments

Currently, the primary therapy for mid- and low-rectal cancer involves surgical intervention, often supplemented by adjuvant radiotherapy, adjuvant chemotherapy, and adjuvant immunotherapy.

However, after discussions with doctors, we identified several critical issues associated with these existing treatment:3

• Traditional surgical treatments can have a relatively high recurrence rate.
• Adjuvant radiotherapy can increase the risk of complications such as anastomotic leakage after surgery, which can cause great inconvenience to patients.
• Adjuvant chemotherapy is known to cause substantial side effects, including nausea, fatigue, and immunosuppression, which can adversely affect patients' quality of life during treatment.
• Adjuvant immunotherapy is prone to causing related adverse reactions.

Given these challenges, there is a pressing need for the development of innovative cancer therapies that help protect patients` lives and well-being. Consequently, we have recently focused our attention on the emerging field of bacterial therapy, which holds promise for more targeted and effective treatment options.

Bacterial Therapy

Bacterial therapy is an innovative and attractive strategy for cancer treatment. Due to the hypoxic conditions, low pH, necrosis, abundant metabolic byproducts, and immunosuppressive state within the tumor microenvironment, bacteria possess intrinsic characteristics that allow for targeted localization in this environment, thereby making bacterial therapy possible.4

As for the effect, bacteria can induce direct tumor cell killing through mechanisms such as oncolysis, immune activation, or production of anti-tumor agents.5 As reported before, bacteria are modified to produce therapeutic molecules, such as cytokines, toxins, or prodrug converting enzymes, enabling highly localized drug delivery and minimizing systemic toxicity.56

Here, we chose Escherichia coli Nissle 1917 (EcN) as the bacterial chassis, a widely recognized probiotic with a well-established human safety record.7Several progress had been made in engineering EcN to treat cancer, ranging from achieving drug delivery, tumor surface hallmark recognition, and promoting immune cell infiltration.689

Although currently only Bacillus Calmette-Guérin (BCG) has received FDA approval for the treatment of bladder cancer, several bacterial therapies based on strains such as Salmonella and Escherichia coli are in clinical trials for various cancers, including lipomas, colorectal cancer, liver cancer, melanoma, and pancreatic cancer. Several of these clinical trials have reached phase III, indicating that bacterial therapies show promising potential for the future.10

Based on these studies, we firmly believed bacterial therapy`s efficacy in cancer treatment.

Type VIII Secretion System

As mentioned aboved, several approaches through bacterial engineering have been proposed to enhance the body's immune system to achieve anti-tumor effects. Among these, bacterial secretion systems have garnered our particular interest. During bacterial evolution, highly specialized nanomachines have been developed to secrete virulence factors into the environment, classified into the Type I to IX Secretion Systems. 11 Each type exhibits varying structural complexities and functions, playing crucial roles in bacterial growth, environmental adaptation, and pathogenicity.

However, for our engineering, we sought a secretion system that fulfills the following criteria:

• it must be amenable to genetic engineering and manipulation;
• it should have a relatively simple and well-characterized structure, ideally with available crystal structures in the Protein Data Bank (PDB);
• it must not be critical for bacterial survival, such that genetic modifications would not compromise bacterial viability.

Based on these considerations, we focused on the Type VIII Secretion System, also known as the Curli system.

First described in 1989 by the Staffan Normark group, curli fibers were identified as a novel surface organelle distinct from flagella and fimbriae.12 Structural studies revealed that Curli is composed of amyloid fibers located on the bacterial cell wall (Figure 2).13

Figure 2: CsgA form the curli on the bacterial surface.

In E. coli, the Curli system is encoded by seven Curli-specific genes (Csg) in two operons: CsgBAC and CsgDEFG. Among these, CsgA is the primary structural component that forms the Curli fiber. Remarkably, the crystal structure of CsgA demonstrates a β-sheet-rich amyloid configuration, consistent with its secretion through CsgG, followed by self-assembly on the bacterial surface. 14

Due to this surface display feature, several studies have explored engineering CsgA for the display of proteins, peptides, or other molecules of interest to confer functional properties.15-17 Among them, we were particularly impressed by the work from Neel S. Joshi's group, in which they incorporated the nonstandard amino acid p-azido-L-phenylalanine (pAzF) into CsgA for labeling with dibenzocyclooctyl (DBCO)-Cy5, thereby enabling in vivo microbial tracking.18 Given the feasibility of in vivo tracking, we wonder whether it would be possible to achieve in situ prodrug activation by leveraging the EcN colonization preference for TME, and thus killing tumor cells effectively.

Prodrug Activation Through IEDDA Reaction

As mentioned above, we used the prodrug strategy, which has been reported to not only reduce the “on-target but off-tumor” toxicity after systematic delivery, but also help prolong the drug circulating time so thus enrich the concentration of drug in tumor site rather than other organs.19 As certain conditions must be meet to achieve transforming the prodrug from an inactive form to an active form, considering the classical characteristic of tumor microenvironment, researchers have developed many tumor-associated response pattern, such as acid environmental response, specific tumor-associated enzyme response and so on.20-22 In deed, these strategies have made great progress and laid solid foundation for further study; but it should be noted that most conditions mentioned above are tumor-associated but not tumor-specific, which means that the risk of “on-target but off-tumor” also existed and this problem was more obvious and tricky in clinical as the result of tumor heterogeneity. Furthermore, the activation rate under these conditions was usually low, which hindered the treatment efficiency and potency.

So how should we solve these problems? Interestingly, we noticed that researchers used to dig out those existed features in the TME, but ignore the fact that we could develop new chemical reaction with high efficiency and specificity as the “key” to activate prodrug. It's worth to be mentioned that the inverse electron demand Diels–Alder (IEDDA) reaction fits that need perfectly.

The “click-to-release” reaction between trans-cyclooctene (TCO) and tetrazine is a prime example of IEDDA cycloaddition (Figure 3). In this process, the strained alkene of TCO reacts with the electron-deficient tetrazine ring in a highly efficient and selective manner. The IEDDA reaction proceeds through a concerted mechanism, where TCO acts as the dienophile and tetrazine as the diene. Upon cycloaddition, the reaction releases significant ring strain from the TCO, driving the reaction forward at an exceptionally fast rate, often with second-order rate constants about 10 ⁶M^-1s^-1.23 This reaction is notable for its bioorthogonality, as it occurs with high specificity under physiological conditions without interfering with biological molecules, so thus widely used in biological modification, drug delivery and so on.24-25

Based on this strategy, Raffaella Rossin et al. proposed to conjugate TCO with monomethyl auristatin E (MMAE), forming the pro-Antibody Drug Conjugate(ADC), which showed prolonged circulation and tumor-enriched; and after adding tetrazine variants, the prodrug was transformed into the active form in high efficiency and exerted tumor-killing effects.26 For our engineering, we expected to introduce TCO into doxorubicin (DOX) to form the TCO-DOX “caged” complex; and once the complex met the tetrazine, the active form of DOX could be restored and exerted its anti-tumor effect.

Figure 3: Overview of click-to-release reaction.

Genetic Codon Expansion

Following above, in Raffaella Rossin's work, the tetrazine variants were added after the pro-ADC finishing pre-targeting, in another word, drug release in situ depended on the antibody moiety rather than tetrazine. However, considering the reaction between TCO and tetrazine was bioorthogonal, we thought that there might be much simpler approach, as long as we could achieve directly introducing the tetrazine group into CsgA, which we thought was the most important part in our project.

Genetic codon expansion technology is a genetic engineering approach that allows for the incorporation of unnatural amino acids (UAAs) into proteins by expanding the genetic code beyond the 20 standard amino acids. This is achieved through the introduction of orthogonal tRNA and aminoacyl-tRNA synthetase (aaRS) pairs that recognize a specific, non-standard codon (typically the amber condon TAG) and charge the corresponding tRNA with the desired UAA. The bioorthogonal nature of the tRNA/aaRS pair ensures that it does not interfere with the host's endogenous translational machinery (Figure 4).27

Following previous work done by Ryan A. Mehl's group, we chose to apply the evolved Methanococcus jannaschii (Mj) tyrosyl tRNA synthetase (RS)/tRNACUA pair to incorporate Tet v2.0 into CsgA on EcN surface.28

Figure 4 | Overview of genetic codon expansion.