Using medium analysis to aid in the identification of CAR-T cell metabolic mechanisms.

I. Abstract:

With the advancement of CAR-T cell therapy, testing different CAR-T cells and correlating their metabolic characteristics with the phenotypes of CAR-T cells that perform well and meet expectations will optimize the CAR construct and manufacturing processes.

Vasoactive intestinal peptide (VIP) is an emerging signaling pathway for examining T-cell function.

VIP is expressed in both T cells and tumor cells and is highly upregulated during TCR activation. During the expansion of primary human T cells, the VIP/VIPR axis enhances the differentiation status of less-differentiated T cell phenotypes, as well as their antitumor cytotoxicity and in vivo persistence.

By engineering modifications, CAR-T cells can secrete a novel, potent VIPR antagonist peptide. These cells can overcome the immunosuppressive effects of tumor microenvironments rich in VIP (Figure 1). Blocking the VIP/VIPR axis in T cells can enhance their tumor-killing capacity mediated by T cells.

In this part of the project, we are investigating the characterization of enhanced CAR-T cell manufacturing processes through amino acid profiling of culture media—correlating metabolic characteristics with the desired CAR-T cell population that exhibits low differentiation and non-exhausted phenotypes.

II. Experimental Methods

To investigate whether VIPR antagonists affect metabolic pathways, we performed analysis of the reaction medium using the Rapid Culture Medium Amino Acid and Vitamin Analyzer Rebel (908 Devices) to identify differences in amino acid utilization between CAR and CAR/VIPRa T cells. The culture medium analysis provided data on the consumption and accumulation of compounds in the extracellular environment of the cells.

Analysis of fresh culture media and reaction solutions revealed differences in the consumption of amino acids such as alanine, glutamate, and serine. This prompted us to further investigate the differences in metabolic pathway utilization and metabolite alterations within these CAR-T cells. We will employ LC/MS metabolomics and cellular bioenergetics to elucidate the differences in metabolic states between CAR and CAR/VIPRa T cells.

The complete study includes the following descriptive steps:

In vivo CAR-T cell function and efficacy, as well as lifespan

Fluorescently labeled tumor imaging

Microscopic visual assessment of cells (digital form, shape, and expression of biomarkers)

Assessing T-cell peptide secretion

RNA sequencing
Hippocampal mitochondrial stress testing assesses metabolic respiratory status and stress responses.
Antigen stimulation

Trial activation (e.g., antigen-stimulated cytokine and activation assays)

Cytotoxicity

Proliferation

Flow cytometer – Number and proportion of cells with specific markers
Rebel—Amino Acid Analysis of Fresh Culture Media and Reaction Liquids
Protein Binding Assay—Potency and Activity Determination, in Relation to Antigens, Antibodies, and Substrates, etc.

3. Rebel Rapid Medium Amino Acid and Vitamin Analyzer: Provides parameter guidance to help you fine-tune your biological processes at any time.

Rapid separation of amino acids, vitamins, and biogenic amines
Minimum required sample: as low as 10 µL
Simple sample preparation: Dilute after centrifugation or filtration.
Integrated Automatic Analyzer: Automated Sample Injection, Separation, Detection, Analysis, and Report Generation
Analysis runtime: ~10 minutes per sample
Kit consumables: 200 analyses

IV. Results

Figure 3. A: The initial concentrations of amino acids in the cell culture medium were analyzed using fresh culture medium (RPMI + 10% fetal bovine serum). Notably, arginine was present at a high concentration, whereas alanine was absent.

Figure 3.B and 3.C: Analysis of reaction solutions from different donors (3.B—Sample 29 and Sample 3; 3.C—Sample 51) reveals remarkably similar levels of AA concentration at the end of the culture period. Notably, there is a significant increase in the production/secretion of alanine and a marked consumption of arginine in the reaction solutions, as well as differences between the stressed-cell and control-cell samples.

Figure 3.D Further analysis of the amino acids comprising the VIP receptor antagonist peptide structure revealed differences in the levels of amino acids such as alanine, glutamate, and serine at the end of the culture period.

Figure 4. A employs LCMS analysis to study intracellular metabolism, determining the utilization of different metabolic pathways (glycolysis, TCA cycle, PPP, AMP/ATP) by quantifying the abundance of intracellular metabolites.

Figure 4. The mitochondrial stress assay measures glycolysis and oxidative phosphorylation utilization in antigen-stimulated and control cells through functional assays.

V. Summary

We found that when cells are stimulated by antigens, CAR/VIPRa T cells exhibit a significant increase in energy metabolism. In contrast, CAR-T cells become less energetic and show increased glycolysis—a well-known hallmark of T-cell exhaustion. Moreover, metabolic assessments conducted using multiple approaches yielded results consistent with these observations, such as measurements of oxygen consumption and levels of certain amino acids in the reaction medium. Finally, evaluations of CAR-T cell activity, potency, and cytotoxicity revealed correlations with antitumor efficacy. In summary, this study demonstrates that distinct metabolic signatures underpin the enhanced tumor-targeting ability of CAR/VIPRa T cells upon antigen stimulation.

Keywords:

Optimizing Key Process Parameters for Scale-Up Production of Insulin-Synthesizing β Cells Derived from iPSCs

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