L A C M A

Background

Cancer has become the second leading cause of death worldwide. According to WHO statistics, more than 20 million new cancer cases were diagnosed in 2022, with nearly 10 million deaths.
Beyond the loss of life, cancer imposes an enormous socioeconomic burden. The global financial loss caused by cancer exceeds USD 1.2 trillion annually, including direct medical expenditures, caregiving costs, and indirect economic impacts from productivity loss.
Confronted with this dual health and financial crisis, the development of innovative therapeutic strategies is urgently needed.

Background

Traditional cancer treatments, including surgery, radiotherapy, and chemotherapy, have numerous limitations. Cancer immunotherapy has ushered in a new era of oncological treatment by activating or enhancing the patient’s immune system to recognize and eliminate cancer cells.
A notable example is the chimeric antigen receptor T (CAR-T) cell therapy, which has demonstrated a profound impact across various cancers, achieving breakthroughs particularly in hematologic malignancies.
However, the efficacy of immunotherapy is still limited by tumor-induced immunosuppression.

Background

During the inception of our project, we learned that Professor Long Liang from our institute was conducting a research project related to cancer.
We engaged in an in-depth discussion with him, during which he emphasized that tumor immunosuppression is predominantly linked to cellular metabolism.
We then performed an extensive literature review and discussion, identifying a key mechanism of tumor-induced immunosuppression: excessive lactate accumulation in the tumor microenvironment (TME).

Background

Driven by the Warburg effect, cancer cells rely on high-rate glycolysis, producing substantial quantities of lactate.
Consequently, lactate concentrations within the TME rise sharply, ranging from 10–30 mM in the serum of cancer patients and reaching up to 50 mM within tumor tissues, which is markedly higher than in normal tissues.
The lactate-rich conditions directly inhibit T-cell metabolism and effector function, severely reducing cytotoxic activity.
Therefore, although CAR-T technology provides strong tumor-targeting capacity, its therapeutic efficacy remains significantly hindered by the high lactate levels within the TME.

Design

To address this issue, we designed a lactate-sensing and lactate-degrading auxiliary module, aiming to provide an effective tool for advancing cancer immunotherapy.
When integrated into immune cells (e.g., CAR-T cells), this module enables secretion of lactate oxidase (LOx), which degrades extracellular lactate and restores its concentration to physiological levels.

Design

Through protein engineering, we constructed a split, two-component system consisting of a lactate sensor and an inactivated TEV protease.
When lactate levels become abnormally elevated, lactate induces reconstitution of the split components, thereby restoring TEV protease activity.
Activated TEV specifically cleaves the GV-2ER fusion protein localized in the endoplasmic reticulum, releasing the transcription factor GV (Gal4-VP64).
Free GV translocates into the nucleus and activate downstream expression of secreted lactate oxidase (sLOx).
Extracellular LOx then catalyzes lactate degradation, mitigating the immunosuppressive microenvironment and enhancing the anti-tumor activity of immune cells.

Lab Work

We have selected the lactate-sensing protein LIdR (Lactate-induced Dimerizer) as the primary component of our lactate sensor.
Additionally, we have chosen the engineered secreted lactate oxidase (sLOx), which incorporates an N-terminal secretion signal peptide, as the lactate-degrading enzyme.
Human embryonic kidney 293T cells have been identified as the target host cells for this system.
A variety of transfer plasmids, including Stev-lac-1, Stev-lac-2, Stev-lac-3, Stev-lac-4, Stev-lac-5, Stev-lac-6, Stev-lac-7, Stev-lac-8, pcDNA3.1(+), GV-2ER, pGL4.35, renilla, UAS-sLOx, PASS-sLOx, and TEV, have been meticulously designed and constructed to establish the overall system. Lab

Model

Building on the experimental design, our modeling group adopted a modular framework comprising four units: lactate sensing (S), protease assembly (P), gene regulation (G), and metabolic output (M).
Using ordinary differential equations and enzyme kinetics, we simulated the genetic circuit, performed sensitivity analysis, and constructed a Markov model.
Structural docking and parameter estimation predicted dynamic responses of eight fusion protein configurations, which were evaluated by sensitivity, sLOx yield, leakage, and response speed to identify the optimal design.
This framework provided predictive guidance for experiments, and inspired the development of software to assess fusion protease stability and catalytic efficiency. Model

HP Work

Our Human Practices (HP) team prioritized science communication and public engagement, continuously refining project design based on community feedback.
In education and outreach, we disseminated knowledge across multiple platforms and employed diverse approaches including board games, online lectures, teaching in rural areas, myth-busting manuals, interactive exhibitions, and public science booths to promote synthetic biology and project awareness, garnering broad recognition.
At the broader human practices level, we integrated our project with society through public surveys, expert interviews, field visits to pharmaceutical companies and participation in biosafety forums, thereby ensuring comprehensive alignment between our research and societal needs. HP