Description
LACMA: Lactate-responsive Module and Application
Project Description
1. The Problem
Tumor cells rely heavily on anaerobic glycolysis to produce lactate through the "Warburg effect" [1]. This leads to a significant increase in lactate concentration in the tumour microenvironment (TME) — up to 20-40 mM, compared to 1.5-3 mM in normal tissues — as well as a decrease in local pH to 6.0-6.5.
In the tumor microenvironment (TME), lactate and its induced acidic environment primarily regulate immune cells in two ways: They inhibit effector immune cells—high lactate concentrations impair CD8+ T cell cytotoxicity and induce exhaustion (while low concentrations promote their mitochondrial oxidation), suppress NK cell activity and trigger apoptosis, and weaken dendritic cell (DC) antigen presentation by downregulating key molecules like MHC-II. They also enhance immunosuppressive cells—regulatory T cells (Tregs) take up lactate via MCT1 to boost their suppressive function, myeloid-derived suppressor cells (MDSCs) produce ROS/NO via HIF-1α, and tumor-associated macrophages (TAMs) polarize to the M2 immunosuppressive phenotype. Additionally, lactylation modulates immune cell gene expression, further reinforcing the immunosuppressive TME[2].
It has been demonstrated that lactate is not completely toxic to the organism: within physiological concentrations, lactate can regulate key physiological processes such as cellular metabolism, signal transduction, and epigenetics by mediating protein lactylation modifications, and is one of the important molecules for maintaining normal physiological functions of the organism. This suggests that when intervening against lactate accumulation in the tumour microenvironment (TME), it is necessary to avoid the complete elimination of physiological concentrations of lactate and the normal physiological pathways it mediates, and instead focus on the strategy of "precisely regulating the lactate level in the TME" — i.e., reducing the local excess of lactate in the tumour to relieve immunosuppression, while preserving the physiological lactate concentration and related physiological functions, to avoid the imbalance of physiological homeostasis caused by excessive removal[3].
In summary, the accumulation of lactic acid in the tumor microenvironment (TME) creates a highly inhibitory microenvironment that significantly impairs the survival and function of effector immune cells, representing one of the barriers to the current suboptimal efficacy of tumor immunotherapy.
2. Our Solution
To address the problem of TME lactate-suppressed immunity, we propose a synthetic biology-based strategy for intracellular lactate degradation: a designed LACMA module (Lactate-responsive Module and Application) is integrated into therapeutic immune cells (e.g., CAR-T) to enable their specific initiation and degradation of lactate in acidic tumour environments The LACMA module drives the expression and secretion of secretory lactate oxidase (sLOx, secrete lactate oxidase) under acidic low pH conditions, catalysing the oxidation of local lactate to pyruvate and hydrogen peroxide, thereby reducing lactate concentration within the TME.
Studies have shown that delivery of sLOx to the tumour site effectively depletes lactate and releases immunostimulatory hydrogen peroxide, significantly alleviating lactate-mediated immunosuppression and enhancing the efficacy of immune checkpoint inhibitors[4, 5].
Similarly, we expect that after LACMA intervention, immune cells (e.g. CAR-T) will have higher survival and functional activity in the TME, be able to kill tumour cells more efficiently and significantly improve the overall immunotherapeutic efficacy.
Module expression and activation:
Integration of the LACMA gene sequence into the genome of immune cells, which relies on acidic signals to initiate module activation after its migration into the tumour.
Lactate degradation mechanism:
LACMA drives secreted LOx to continuously produce and degrade lactate in the extracellular space of tumours. The hydrogen peroxide produced both enhances local immunogenicity and assists in weakening inhibitory signals under quantitative control.
TME remodelling:
by depleting lactate, the LACMA module will neutralise the acidic microenvironment, inhibit inhibitory pathways and remodel the anti-tumour-friendly microenvironment.
Taken together, the LACMA module is expected to significantly enhance the efficacy of immunotherapies, such as chimeric antigen receptor T cells, by degrading lactate in the TME in a targeted, secretory manner, directly counteracting the lactate-mediated immunosuppressive effects.
3. LACMA
LACMA consists of three functionally synergistic core modules. The component architecture and overall mechanism of each module are as follows:
I. Component Construction of Each Module
a. Front-end Lactate Sensor Module:
Using two fusion proteins as core functional units, we first cleaved the lactate sensor (Lactate-induced Dimerizer) and TEV enzyme into inactive N-terminal fragments (TEV-N) and C-terminal fragments (TEV-C), respectively; Following optimization screening, the lactate sensor N-terminus is fused to TEV-N, and the C-terminus to TEV-C, yielding two fusion proteins: TEV-N Lac-sensor-C and TEV-C Lac-sensor-N.
b. Intermediate Signal Transduction Module:
We engineered the GV-ERT2 fusion protein as the intermediate signal transduction module. "GV" represents the Gal4-VP64 transcription activator containing a DNA-binding domain and a transcription activation domain, while 'ERT2' denotes the endoplasmic reticulum retention signal domain containing the TEV cleavage site (TEVS). These two components were genetically fused in series to form the "GV-ERT2" structure.
c. Downstream functional response module:
We concatenated the secretory lactate oxidase (sLOx) with a promoter driven by the Gal4 protein-specific binding sequence (5×UAS) to construct the "5×UAS-sLOx" expression cassette, which was stably integrated into the host cell's nuclear genome.
II. Overall Mechanism of Action
Under normal lactate concentrations, the two fusion proteins in the front-end lactate sensing module (TEV-N Lac-sensor-C + TEV-C Lac-sensor-N) remain spatially separated due to the lactate sensor lacking substrate binding. TEV-N and TEV-C cannot assemble, rendering the TEV enzyme inactive. At this stage, the GV-2ER fusion protein in the intermediate signaling module remains anchored in the cytoplasm due to interactions between ERT2 and endoplasmic reticulum-associated heat shock proteins (HSPs), preventing nuclear entry. Consequently, the downstream functional response module—the "5×UAS-sLOx" expression cassette—remains silenced.
When lactate concentration increases in the environment (e.g., tumor microenvironment), lactate molecules bind specifically to the lactate sensor, triggering a conformational change in the sensor. This drives the two fusion proteins closer spatially, allowing TEV-N and TEV-C to reassemble into a complete and active TEV enzyme. The active TEV enzyme specifically recognizes and cleaves the TEVS site on GV-2ER, causing dissociation between the GV transcription activator and the ERT2 domain. The released GV transcription factor exposes its nuclear localization signal, enters the nucleus via the nuclear pore complex, and binds specifically to the 5×UAS sequence of the downstream "5×UAS-sLOx" expression cassette via its Gal4 domain. Concurrently, the VP64 domain recruits the transcription initiation complex, initiating transcription and translation of sLOx. The resulting sLOx protein is secreted into the extracellular space, where it catalyzes the degradation of lactate within the tumor microenvironment.
References
- Walenta, S. and W.F. Mueller-Klieser, Lactate: mirror and motor of tumor malignancy. Seminars in Radiation Oncology, 2004. 14(3): p. 267-274.
- Gu, X.Y., et al., Impact of lactate on immune cell function in the tumor microenvironment: mechanisms and therapeutic perspectives. Front Immunol, 2025. 16: p. 1563303.
- Li, X., et al., Lactate metabolism in human health and disease. Signal Transduct Target Ther, 2022. 7(1): p. 305.
- Cao, Z., et al., Lactate oxidase nanocapsules boost T cell immunity and efficacy of cancer immunotherapy. Sci Transl Med, 2023. 15(717): p. eadd2712.
- Choi, H., et al., Lactate oxidase/catalase-displaying nanoparticles efficiently consume lactate in the tumor microenvironment to effectively suppress tumor growth. J Nanobiotechnology, 2023. 21(1): p. 5.