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Comparative Study
. 2025 Jan;104(1):761-771.
doi: 10.1007/s00277-024-06028-9. Epub 2024 Oct 15.

A comprehensive analysis of the major specificities of anti-human T lymphocyte porcine immunoglobulin (p-ATG)

Haoyong Zou #  1 , Wenqu Yin #  1 , Peng Geng  1 , Li Lin  2 , Xilin Nie  1 , Zui Tao  1 , Gang Chen  3 , Bin Chen  1 , Hao Feng  3 , Kuanhong Xu  1 , Zhi Zhang  4
Affiliations
Comparative Study

A comprehensive analysis of the major specificities of anti-human T lymphocyte porcine immunoglobulin (p-ATG)

Haoyong Zou et al. Ann Hematol. 2025 Jan.

Abstract

Background: Anti-human T lymphocyte porcine immunoglobulin (p-ATG) is a potent immunosuppressive agent derived from porcine sources used in various immunotherapy applications. It is compared with similar products derived from other species, such as rabbit anti-thymocyte globulin (r-ATG) and ATG-Fresenius (ATG-F), which have distinct biological and therapeutic properties. This study aims to elucidate the mechanisms of action and comparative efficacy of p-ATG in relation to r-ATG and ATG-F through a comprehensive in vitro analysis.

Methods: A comparative analysis of p-ATG, r-ATG and ATG-F was performed, focusing on E rosette inhibitory potency, lymphocyte toxic potency, blocking activities of 24 CD molecules, and flow quantitative potency. Flow cytometric analysis was used to quantify these characteristics and assess the potency of the immunoglobulins.

Results: p-ATG exhibited lower E rosette inhibitory and lymphocyte toxic potencies compared to r-ATG but was significantly more potent than ATG-F at equivalent concentrations. At protein concentrations above 12.5 µg/mL, p-ATG showed slightly lower potency than r-ATG and much higher potency than ATG-F in flow cytometry assays. Both p-ATG and r-ATG exhibited similar killing effects on lymphocytes within the peripheral blood mononuclear cells (PBMCs), including CD3 + T cells, with a dose-dependent response. Notably, p-ATG displayed more pronounced blocking activities against CD8, CD99, and TCR α/β compared to r-ATG.

Conclusion: p-ATG offers certain advantages over r-ATG and ATG-F, particularly in its ability to inhibit specific CD molecules and its overall potency in immunosuppressive assays, providing valuable insights for assisting clinical decision-making regarding the selection of ATG types based on patient-specific needs and treatment objectives.

Keywords: ATG-Fresenius (ATG-F); Anti-human T lymphocyte porcine immunoglobulin (p-ATG); Antibody; Horse anti-thymocyte globulin (h-ATG); Organ transplantation; Rabbit anti-thymocyte globulin (r-ATG).

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Conflict of interest statement

Declarations. Ethics approval: The studies involving healthy human serum and PBMCs were generously provided by Tongji Medical College, Huazhong University of Science and Technology. The human samples used in this study were were reviewed and approved by the Medical Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (TJ-IRB2023078). Consent of publication: Not applicable. Competing interests: The authors declare that there are no commercial or financial affiliations that could be construed as a potential conflict of interest in the conduct or reporting of this research.

Figures

Fig. 1
Fig. 1
Comparative analysis of e-rosette inhibition and lymphocyte cytotoxicity tests. (A) E-Rosette Inhibition Test: The inhibition rates for ATG-F, p-ATG, and r-ATG were assessed at concentrations of 6.25 µg/mL and 12.5 µg/mL. At both concentrations, p-ATG demonstrated better inhibition compared to ATG-F, though it was slightly less effective than r-ATG. (B) Lymphocyte Cytotoxicity Test: The cytotoxicity of each product was evaluated at concentrations of 6.25 µg/mL, 12.5 µg/mL, 25 µg/mL, and 50 µg/mL. At concentrations below 12.5 µg/mL, p-ATG exhibited greater cytotoxicity than r-ATG. Above 12.5 µg/mL, p-ATG’s effect was slightly weaker than r-ATG’s but still significantly stronger than ATG-F. Values are presented as mean ± SD
Fig. 2
Fig. 2
Comparison of EC50 for lymphocyte cytotoxicity between different ATG products. The EC50 values for lymphocyte cytotoxicity were determined for three batches (1, 2, 3) of each ATG product to assess batch consistency. (A-C) 4-parameter logistic curves for each of the p-ATG (A), r-ATG (B), and ATG-F (C) products. (D) Comparison of EC50 for lymphocytotoxicity of the three ATG products. No statistically significant differences were observed in the flow cytometry potency among the ATG products from different suppliers. Values are presented as mean ± SD
Fig. 3
Fig. 3
Comparison of pALG and rATG killing ratio on human PBMC and CD3 + T cellsin vitro. (A) The killing ratios of human PBMCs by p-ATG and r-ATG at varying concentrations are shown. The x-axis represents different concentrations, and the y-axis indicates the proportion of PBMC killing. The mean killing rates across six healthy individuals showed no significant differences between r-ATG and p-ATG within the concentration range of 1 to 200 µg. (B) The killing ratios of human CD3 + T cells by p-ATG and r-ATG at various concentrations are presented. The x-axis represents different concentrations, and the y-axis shows the proportion of CD3 + T cell killing. Analysis of nine healthy individuals revealed that r-ATG was significantly more effective than p-ATG at concentrations of 20, 40, and 50 µg, with no significant differences observed at concentrations above 80 µg. Values are presented as mean ± SD
Fig. 4
Fig. 4
The mean concentration of 50% inhibition of each CD antibody on lymphocyte antigens of different ATG products from various suppliers. The x-axis represents different ATG products, and the y-axis indicates the concentration required to achieve 50% inhibition of CD molecules. The 50% inhibition concentration reflects the antibody level targeting specific antigens. Products that did not achieve more than 50% inhibition for CD44, CD99, CD4, and CD50 are shown as 0 on the graph. Testing included three batches of p-ATG, r-ATG, and ATG-F. Values are presented as mean ± SD
Fig. 5
Fig. 5
Correlation analysis of the 50% inhibition concentration between different CD molecules. (A) p-ATG, (B) r-ATG, (C) ATG-F. Pearson correlation analysis results are shown, with correlations significant at P < 0.05 (*) and P < 0.01 (**). Positive correlations are indicated in red, and negative correlations are indicated in blue
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