In recent years, kynureninase (KYNase), as a key enzyme in the tryptophan metabolic pathway, has gradually attracted extensive attention in the field of cancer immunotherapy. Its therapeutic advantage mainly lies in its targeted clearance of the immunosuppressive metabolite - kynurenine (Kyn). We utilized synthetic biology methods and engineered bacteria to express KY Nase, regulating the metabolism of the KP pathway within tumor cells, consuming its downstream metabolite Kyn. This inhibits tumor immune escape, enhances the host's immune system response, and achieves tumor cell death. Through reasonable design, we can achieve unique combined functions for the treatment of cancer. Here, we utilized Escherichia coli BL21 (DE3) as the chassis cell and the hypoxic and high-lactic-acid regulatory ALPaGA subsystem as promoter, to express KYNase in the tumor microenvironment. We also made semi-rational modifications to KYNase, hoping to obtain an enzyme with better catalytic effect. We designed and built a PhiX174E lysis protein module to release KYNase extracellularly at the right time. For biosafety, we designed a suicide switch to prevent engineered strains from being released into the environment. Our project is expected to promote the treatment of colorectal cancer and provide a new idea and method for cancer treatment.

One of our team members visited a cancer ward during a summer project, following a doctor on rounds, organizing medical cases, and attending discussions. It was his first time facing the true nature of cancer: the pain, death, and despair.

A colorectal cancer patient under 30, with liver metastases, was in critical condition and could barely walk. Immune checkpoint inhibitors failed. Usually, T cells release signals instructing cancer cells to die, but here, the cancer cells ignored them as their pathways are blocked.

Why do some tumors resist immunotherapy? Could we reactivate immunity at its source? We need a solution.

With that in mind, we focused on kynurenine (KYN), a molecule secreted by tumors via the IDO pathway that suppresses T cells and silences the immune system. KYNase, an enzyme from Pseudomonas fluorescens that degrades KYN, appeared in our sight.

KYNase is rare, however. Its expression system is unstable, and its side effects are unclear. In order to ensure safety, we used BL21 (DE3) as the chassis bacteria, linking KYNase release to the ALPaGA lactate-responsive promoter, restricting activity to the tumor microenvironment. A multilayered suicide system was built to prevent bacterial leakage.

Tumor treatment is a crucial area in the medical field. With the advancement of technology, treatments have evolved from conventional surgery, radiotherapy, and chemotherapy to novel strategies, such as targeted therapy and immunotherapy, significantly improving patients’ survival rate and quality of life. Surgery is the first choice for early-stage solid tumors, with robotic-assisted surgery enhancing precision and safety. Radiotherapy kills tumor cells through high-energy rays, and intensity-modulated radiotherapy (IMRT) as well as proton therapy can reduce the damage to normal tissues. Chemotherapy uses cytotoxic drugs to inhibit tumor growth, but because of its side effects, it is gradually used in combination with targeted drugs to improve efficacy. Small molecule inhibitors (e.g., EGFR, ALK inhibitors) and monoclonal antibodies (e.g., anti-HER2 trastuzumab) have achieved remarkable therapeutic effects in tumors such as lung and breast cancer. Protein-based biopharmaceuticals, such as kynureninase, have been developed for more effective and specific therapies with fewer side effects than conventional approaches, and are being actively investigated in preclinical and clinical settings.

Kynureninase (KYNase) is a PLP-dependent enzyme that is a member of the transaminases or alpha family, subgroup IV, the largest family of PLP-dependent enzymes, with a monomeric molecular weight of approximately 45 kDa. Characteristic of the transaminase family, the monomeric form of KYNase contains a large structural domain and a small structural domain, with the active site located in the cleft between the structural domains at the interface of the subunits, containing residues from the residues from both subunits. KYNase exhibits good catalytic activity towards KYN and can effectively eliminate KYN from TME.

Kynurenine has been largely implicated in a variety of pathologies, such as metabolic, neurological, cardiac, and renal diseases. This enzyme can use KYN or 3-HK as substrates to produce o-aminobenzoic acid (AA) and 3-HANA, respectively [1]. Specifically, 3-HANA has been described as a modulator of the immune response, as 3-HANA induces apoptosis in monocytes and macrophages under inflammatory conditions, is enhanced by ferrous or manganese ions [2] , and triggers the death of activated T-cells by depleting intracellular GSH [3]. In addition, 3-HANA inhibits dendritic cell maturation and suppresses T cell stimulation [4]. A recent report showed that KYNase expression correlated with poor overall survival in lung adenocarcinoma, promoting tumor immunosuppression by inducing tumor-infiltrating Treg, accompanied by a consistent increase in PD1 and PD-L1 protein levels [5].

Chae et al. developed nanoparticles containing kynurenine ammonia lyase for effector T cell proliferation in TME [6]. Zeng et al. developed a near-infrared (NIR)-responsive semiconducting polymer nanoenzymes integrating three functional modules: a NIR-absorbing semiconducting polymer nanoparticle core, recombinant kynurenine ammonia lyase, and a PEG-modified 1O2-cleavablelinker. This design significantly enhanced the anti-tumor immune response through the synergistic effect of photodynamic therapy and enzymatic immunometabolic modulation [7]. Based on these studies, a "kynurenine starvation therapy" strategy was proposed to target TME using a combination of IDO1 inhibitors and kynurenine enzymes. The two-pronged therapeutic paradigm not only reduces Trp depletion by inhibiting IDO1, but also attenuates the over-accumulation of KYN by the KYNase catalysis on the Trp-KYN axis, and leads to significant changes in TME by blocking the KYN-AhR signaling pathway [8].

Using kynureninase as the basis for the therapy, we are working to develop a synthetic biology tumor therapy strategy: the KINETiC (Kynurinine-depleting Immuno-reversal Network in E.coli for Therapy in Colorectal Cancer).

In addition to malignant cells, tumors are composed of a variety of normal cells. These include blood vessels, fibroblasts, and immune cells of the myeloid or lymphoid lineages. The pathway that attracts tumors is usually associated with the secretion of soluble signaling molecules (e.g., chemokines or cytokines) secreted by cancer cells, immune cells, fibroblasts, and mesenchymal stem cells. This secretion permits crosstalk to occur within the TME. Soluble factors not only attract immune cells but also reprogram their functions [9]. Upon recognition of tumor antigens, myeloid cells, especially dendritic cells (DCs) and macrophages, produce inflammatory cytokines such as type 1 IFN, IL-12, IL-15, IL-18, and IL-21, which are involved in the tumor-killing response of natural killer (NK) cells [10, 11]. CCR2 inflammatory monocytes are recruited by ccR2 into tumors [12], and macrophages through SDF-1/CXCL12 expression is attracted by cancer-associated luteinizing cells (CAF), and indeed, SDF-1 amplifies macrophage polarization into tumor-associated macrophages (TAM) (producing high levels of IL-10). In addition, the chemokines CCL5, CCL20 and CCL22 recruit Treg and activate their inhibitory effects by producing IL-10 and TGF-β1 [13]. Tumor growth can also radically alter myelopoiesis in the bone marrow (BM), leading to the production of myeloid-derived suppressor cells (MDSC).The cytokines GM-CSF, G-CSF, and IL-6 produce MDSC from precursors, apparently reprogramming the BM and altering the composition of circulating myeloid cells [14, 15]. MDSC can also differentiate into TAM, which is capable of directly inhibiting CD8 cells through nitric oxide synthase-2 (NOS-2) and arginase (ARG-1) secretion [16]. TME is characterized not only by animmunosuppressive environment but also persistent inflammation. This inflammatory state attracts neutrophils in the TME in an IL-8-dependent manner. Once in the TME, TAN maintains inflammation by releasing nitric oxide (NO) and reactive oxygen species (ROS). TAN also induces T-cell apoptosis releasing TNF-α, inhibits the proliferation of ARG-1-secreting T-cells (by regulating PD-1/PD-L1 signaling), and participates in Treg recruitment, which further induces a state of immunosuppression through the production of CCL17 [17].

Tryptophan metabolism in the tumor microenvironment is one of the key mechanisms regulating immune response and tumor progression, which is mainly mediated through the indoleamine 2,3-dioxygenase (IDO1/IDO2) and tryptophan 2,3-dioxygenase 2 (TDO2) pathways to form an immune-suppressive microenvironment. IDO1/IDO2 catalyzes the degradation of tryptophan along the kynurenine (KYN) pathway, which depletes the environmental tryptophan while generating the product kynurenine [18-22].

The details of this pathway were first elaborated by Alexander Braunstein in the late 1940s [15]. O2 cleavage of the pyrrole ring is followed by N-formyl-L-kynurenine catalyzed by blood proteins, TDO2 in the liver, or IDO1/IDO2 in other tissues. After hydrolysis of the formamide, L-kynurenine is subsequently hydroxylated by kynurenine monooxygenase, yielding 3-hydroxy-L-kynurenine 3-hydroxy-L-kynurenine, which undergoes hydrolytic cleavage of the Cβ--Cγ bond catalyzed by pyridoxal phosphate (PLP)-dependent kynurenine ammonia-lyase [EC3.7.1.3] to yield 3-hydroxy-octopine benzoate and L-Ala. Another PLP-dependent enzyme, kynurenine aminotransferase (KAT), transaminates kynurenine or 3-hydroxykynurenine to give kynurenine or xanthate, respectively. 3-Hydroxy-phthalamidobenzoate is converted by heme-iron-free 3-hydroxy-phthalamidobenzoate dioxygenase to 2-amino-3-carboxy-phthalamidobenzoate semialdehyde, which is spontaneously cyclized to quinolinic acid ester [23], or enzymatically decarboxylated to 2-amino-phthalamidobenzoate semialdehyde. Quinolinic acid phosphoribosyltransferase is ultimately converted to NAD(P)+, but it is also a neurotoxin due to its agonist activity on the NMDA receptor, whereas 2-amino-muconate semialdehyde is subsequently fully catabolized and metabolized to CO2 and NH3. Thus, the kynurenine pathway is an ab initio source of NAD(P)+ from tryptophan, whereas nicotinic acid is converted to NAD(P)+ by the remedial pathway. The major organ for tryptophan metabolism in the kynurenine pathway is the liver. Recently, it has been found that kynurenine pathway enzymes are elevated in response to inflammatory processes, including Huntington's chorea [24], Alzheimer's disease [25], multiple sclerosis [26], ALS [27], and HIV-associated dementia [28]. The kynurenine pathway has been shown to be induced by γ-interferon [29] and Alzheimer's peptide Aβ (1-42) [4]. Kynurenine is produced from kynurenine by KAT, which is also neuroactive as an antagonist of NMDA receptors and α7 nicotinic acetylcholine receptors. Thus, kynurenine pathway enzymes have recently emerged as potential new drug targets for these diseases.

Depletion of tryptophan inhibits tryptophan-dependent T-cell activation, leading to T-cell arrest in T1 phase, whereas accumulation of kynurenine leads to activation of the aryl hydrocarbon receptor (AhR), which promotes regulatory T cell (Treg) differentiation and myeloid-derived suppressor cell (MDSC) recruitment, enhancing immunosuppression. Moreover, kynurenine also promotes the release of angiogenic factors through AhR signaling, contributing to tumor metastasis.

The relative significance of the tryptophan starvation response versus KYN accumulation in TME as a driver of immunosuppression has been debated for nearly two decades. Although the idea that local tryptophan starvation is the primary cause of impaired T-cell function in tumors remains prevalent, quantitative arguments and recent experimental evidence have begun to challenge this hypothesis.

Colorectal cancer is a type of malignant tumor that occurs in the colon (large intestine) or rectum. As one of the common malignant tumors worldwide, it can cause severe damage to the body and even lead to death. The risk of developing this disease increases with age, and most cases are diagnosed in people over 50 years old. There are often no significant clinical manifestations in the early stage. Its common clinical symptoms include: changes in bowel habits (such as diarrhea, constipation, or thinning of stools); blood in the stool (rectal bleeding), which can be bright red or dark tarry; persistent abdominal cramps, pain, or bloating; unexplained sudden weight loss without active weight loss efforts; frequent fatigue and lack of energy even after adequate rest; and iron deficiency anemia caused by chronic bleeding (manifested as fatigue, weakness, and pallor).

Tumor proliferation can lead to intestinal lumen stenosis and obstruction, causing intractable abdominal pain, bloating, and changes in bowel habits. Ulcerative lesions are prone to inducing gastrointestinal bleeding, which can result in iron deficiency anemia in the long term, characterized by decreased hemoglobin levels and reduced oxygen-carrying capacity, thereby triggering hypoxic damage to multiple organs. Advanced tumors often metastasize to key organs such as the liver, lungs, and bones. For example, liver metastasis can lead to liver failure, lung metastasis causes respiratory insufficiency, and bone metastasis is accompanied by severe pain and pathological fractures, significantly shortening the patient's survival period. Clinical data show that the 5-year survival rate of patients with distant metastasis is only 14% (based on statistics from the SEER database from 2016 to 2022). Meanwhile, tumor metabolites and immunosuppressive factors can induce cachexia, manifested as progressive weight loss, muscle atrophy, and impaired immune function, increasing the patient's susceptibility to infections and further exacerbating the deterioration of the condition. In addition, the comprehensive treatment cost of colorectal cancer is high, with an annual medical expenditure ranging from 100,000 to 300,000 yuan, which significantly increases the family's economic pressure and harms the patient's mental health. The incidence of anxiety and depression after diagnosis is as high as 42%, and the sense of hopelessness in advanced patients can induce post-traumatic stress disorder, imposing additional psychological pressure on the family support system.

We modularly disassembled the KINETiC therapy and constructed it through multiple DBTL cycles. Specifically, we determined the induction concentration of the lactate-inducible promoter ALPaGA, successfully achieved the expression of kynureninase (KYNase), verified its activity, and performed enzyme modification on it to improve its activity. Meanwhile, we designed the PhiX174E lysis module for better release of therapeutic proteins, and finally designed a suicide switch module. Firstly, we designed the KYNase expression gene circuit based on synthetic biology theory. To achieve better protein expression, we first used BL21 (DE3) as the chassis cell and combined the J23119 strong constitutive promoter with the T7 terminator to efficiently express antimicrobial peptides. After cell disruption, we can obtain antimicrobial peptides and prepare them into injectable biological agents. Through communication with Professor Jingning Zhu, an expert in molecular biology, we learned that the strain which can induce the expression of KYNase by characteristic substances in the tumor microenvironment has the advantage of "single administration for long-term prevention or treatment" in terms of therapy. Therefore, we decided to upgrade the strain. We learned that the tumor microenvironment has the characteristics of hypoxia and high lactate. Thus, we used the ALPaGA inducible promoter, which can be induced by a high-lactate environment, as the regulatory element to synthesize and secrete KYNase.

The degradation rate of kynurenine is closely related to the catalytic activity of KYNase. Therefore, improving the catalytic activity of KYNase is of great significance for tumor treatment through kynurenine degradation. Semi-rational protein design is a protein engineering method that combines computational simulation and experimental verification, standing between fully rational design and directed evolution. It utilizes bioinformatics, structural biology, and molecular simulation technologies to purposefully modify key sites of proteins, supplemented by experimental screening and optimization to improve protein stability, activity, specificity, or develop new functions. Semi-rational protein design first requires analyzing the structure and function of the target protein, then using computer-aided design, and finally constructing mutants for experimental verification and optimization. To enable the intracellularly produced KYNase to be released at an appropriate time, we constructed a lysis module based on the PhiX174E lytic protein. In the latest DBTL cycle, we have accepted the suggestions from the NMPA (National Medical Products Administration) and experts from traditional Chinese medicine hospitals, and plan to improve the suicide switch module to ensure its biological safety. In the future, we will integrate these modules into a complete engineered bacterium to realize the full KINETiC therapeutic strategy. The complete KINETiC therapy will integrate the modules described above, combining the BL21 (DE3) chassis cell, the ALPaGA lactate-induced KYNase expression module, the lysis module, and the suicide switch module, and utilize these modules to implement a synthetic biology therapy targeted at the tumor microenvironment.