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Cited by 1CAR-T cell therapy researchImmunotherapy and Immune ResponsesT-cell and B-cell Immunology

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Pre-clinical safety and efficacy evaluation of Helicobacter Pylori neutrophil-activating protein (NAP)-armed CAR-T cells targeting B cell lymphomas

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Ma, Jing, Sarén, Tina, Jin, Chuan, Kim, Hyeong Su, Contreras Pineda, Paola Donaji, de Bernard, Marina, Amini, Rose-Marie, Rondahl, Veronica, Enblad, Gunilla, Yu, Di, Essand, Magnus·2025·Cancer Immunology, Immunotherapy
DOI 10.1007/s00262-025-04112-1PMC PMC12255594

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Abstract

CD19 CAR-T cell therapy shows striking results in treating B cell malignancies. However, approximately two-thirds of the lymphoma patients eventually relapse, with about one-third displaying CD19-negative tumors at relapse. Our previous study showed that CAR-T cells armed with the Helicobacter pylori neutrophil-activating protein (NAP), CAR(NAP)-T cells, can trigger a bystander immune response and eliminate CAR-target-antigen-negative tumor cells. Here, we report the development of CD20-targeted CAR-T cells (CAR20-T cells), with the targeting moiety from rituximab, and the safety and efficacy of NAP-armed CAR-T cells. CAR20-T cells displayed efficient and specific cytotoxic potential against multiple human B cell lymphoma cell lines in vitro. In addition, primary mantle cell lymphoma cells, isolated from a patient who relapsed after rituximab treatment, can also be eliminated by CAR20-T cells. CAR20(NAP)-T cells delayed tumor growth and prolonged survival of mice with lymphoma. No obvious histopathological alteration in major organs were observed in mice treated with CAR(NAP)-T cells. Further, no excessive cytokine release or immune cell activation was observed when human blood from healthy volunteers was exposed to recombinant NAP protein in an ex vivo blood loop assay, suggesting a safe therapeutic profile for NAP. Taken together, these results warrant the clinical investigation of CAR20(NAP)-T cells.

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Fig. 1 Human CAR20-T cells displayed potent cytotoxic capacity towards CD20-expressing lymphoma cells in vitro. A Lentiviral (LV) constructs used for human T cell engineering. LV(CAR20) containing the single chain variable fragment (scFv) derived from rituximab and 4-1BB and CD3ζ intracellular domains together with green fluorescent protein (GFP) for detection of transduced T cells. LV(Mock) only encoding GFP was used as control. B Transduction efficiency of CAR20-T presented as percentage of CAR+ cells out of CD3+ cells. Mock-T cells were used as control. C Mean fluorescent intensity (MFI) of CAR on CAR20-T (CD3+GFP+) and Mock-T (CD3+GFP+) cells. Each dot represents T cells isolated from one healthy donor (n = 6), with bars indicating mean ± SEM. Non-parametric unpaired t-test was used to compare between groups (**: p < 0.01, ****: p < 0.0001). D Representative contour plots of the concordance between GFP and CAR expression in the CD3+ population of CAR20-T and Mock-T cells. E Proportion of CAR20-Ts or Mock-Ts (CD3+GFP+) expressing either 0, 1, 2 or 3 activation/inhibitory receptors (PD-1, TIM-3 and LAG-3) (n = 3). F CD20 expression on five human B cell lymphoma cell lines, DG75, DB, U698, Raji and Daudi. G Relative viability of luciferase-tagged lymphoma target cells after 4 days exposure to CAR20-T cells at the effector to target ratio = 3:1. Each dot represents T cells isolated from one healthy donor (n = 8), with bars indicating mean ± SEM. Non-parametric unpaired t-test was used to compare groups for each cell line (*: p < 0.05, ***: p < 0.001). H Relative viability of scarlet-expressing Daudi cells measured every hour over time by total integrated intensity using Incucyte after exposure to CAR20-T cells or Mock-T cells, prepared from one healthy donor, at the effector to target ratio 5:1. I–L Primary mantle cell lymphoma (MCL) cells, isolated from a patient who had developed resistance towards rituximab (anti-CD20 antibody) treatment, were co-cultured with either CAR20-T cells or Mock-T cells at a 1:1 effector to target cell ratio for 3 days. I Illustration of experimental set up. J Histogram displaying CD20+ tumor cells after co-culture with CAR20-T or Mock-T cells. Each individual histogram represents a co-culture of lymphoma cells and transduced T cells derived from one donor. K Quantification of the percentage of CD20+ tumor cells in panel J. Each dot represents T cells isolated from one healthy donor (n = 3) bars indicating mean ± SEM. Non-parametric unpaired t-test was used to compare groups (**: p < 0.01). L IFN-γ production from T cells as in panel J. (N.D. = not detectable)
Fig. 1 Human CAR20-T cells displayed potent cytotoxic capacity towards CD20-expressing lymphoma cells in vitro. A Lentiviral (LV) constructs used for human T cell engineering. LV(CAR20) containing the single chain variable fragment (scFv) derived from rituximab and 4-1BB and CD3ζ intracellular domains together with green fluorescent protein (GFP) for detection of transduced T cells. LV(Mock) only encoding GFP was used as control. B Transduction efficiency of CAR20-T presented as percentage of CAR+ cells out of CD3+ cells. Mock-T cells were used as control. C Mean fluorescent intensity (MFI) of CAR on CAR20-T (CD3+GFP+) and Mock-T (CD3+GFP+) cells. Each dot represents T cells isolated from one healthy donor (n = 6), with bars indicating mean ± SEM. Non-parametric unpaired t-test was used to compare between groups (**: p < 0.01, ****: p < 0.0001). D Representative contour plots of the concordance between GFP and CAR expression in the CD3+ population of CAR20-T and Mock-T cells. E Proportion of CAR20-Ts or Mock-Ts (CD3+GFP+) expressing either 0, 1, 2 or 3 activation/inhibitory receptors (PD-1, TIM-3 and LAG-3) (n = 3). F CD20 expression on five human B cell lymphoma cell lines, DG75, DB, U698, Raji and Daudi. G Relative viability of luciferase-tagged lymphoma target cells after 4 days exposure to CAR20-T cells at the effector to target ratio = 3:1. Each dot represents T cells isolated from one healthy donor (n = 8), with bars indicating mean ± SEM. Non-parametric unpaired t-test was used to compare groups for each cell line (*: p < 0.05, ***: p < 0.001). H Relative viability of scarlet-expressing Daudi cells measured every hour over time by total integrated intensity using Incucyte after exposure to CAR20-T cells or Mock-T cells, prepared from one healthy donor, at the effector to target ratio 5:1. I–L Primary mantle cell lymphoma (MCL) cells, isolated from a patient who had developed resistance towards rituximab (anti-CD20 antibody) treatment, were co-cultured with either CAR20-T cells or Mock-T cells at a 1:1 effector to target cell ratio for 3 days. I Illustration of experimental set up. J Histogram displaying CD20+ tumor cells after co-culture with CAR20-T or Mock-T cells. Each individual histogram represents a co-culture of lymphoma cells and transduced T cells derived from one donor. K Quantification of the percentage of CD20+ tumor cells in panel J. Each dot represents T cells isolated from one healthy donor (n = 3) bars indicating mean ± SEM. Non-parametric unpaired t-test was used to compare groups (**: p < 0.01). L IFN-γ production from T cells as in panel J. (N.D. = not detectable)CC BY · PubMed Central
Fig. 2 Murine mCAR20-T and mCAR20(NAP)-T cells display cytotoxicity against murine lymphoma engineered with human CD20, A20-hCD20, in vitro and display therapeutic efficacy in vivo. A Schematic illustration of retroviral (RV) constructs used for murine T cell engineering. B Histogram displaying the expression of human (h)CD20 on parental A20 and cells engineered to express human CD20 (A20-hCD20). C Relative target cell viability, D IFN-γ, and E TNF-α secretion in the co-culture where engineered mouse T cells were exposed to luciferase-tagged parental A20 or A20-hCD20 cells at effector to target cell ratios 5:1–20:1 for 4 days. The experiment was performed twice. Data is shown as mean ± SD from one representative co-culture experiment with experimental triplicates. Two-way ANOVA with Tukey’s multiple comparisons test was used to compare between groups. Statistical significance is color-coded between corresponding treatment group and Mock group (**: p < 0.01; ****: p < 0.0001). F–M Mice were implanted subcutaneously with 2 × 105 A20-hCD20 cells and treated three times (3 × 106 T cells/dose) with Mock-T (n = 20), mCAR20-T (n = 20) or mCAR20(NAP)-T (n = 19). F Illustration of treatment schedule. G representative contour plot showing CAR expression level (represented as Thy1.1) in CD3 + cells. All experiments were assessed using a mixed population containing both transduced and untransduced cells. H Weight (mean) of mice after tumor implantation and during treatment. I Tumor size (mean + SEM). J Mouse survival (Kaplan–Meier) curve, and K–M tumor size for individual mice after treatment are presented. Numbers in K–M indicate tumor-free mice. The experiment was performed twice, and data was pooled. Tumor sizes on day 28 (time when the first mouse was sacrificed due to reaching the humane endpoint in Mock-T group) were compared between each treatment group using nonparametric Kruskal–Wallis One-way ANOVA test with Dunn’s correction for multiple comparisons. The survival curves were compared by the Log-rank test (*: p < 0.05, ***: p < 0.001)
Fig. 2 Murine mCAR20-T and mCAR20(NAP)-T cells display cytotoxicity against murine lymphoma engineered with human CD20, A20-hCD20, in vitro and display therapeutic efficacy in vivo. A Schematic illustration of retroviral (RV) constructs used for murine T cell engineering. B Histogram displaying the expression of human (h)CD20 on parental A20 and cells engineered to express human CD20 (A20-hCD20). C Relative target cell viability, D IFN-γ, and E TNF-α secretion in the co-culture where engineered mouse T cells were exposed to luciferase-tagged parental A20 or A20-hCD20 cells at effector to target cell ratios 5:1–20:1 for 4 days. The experiment was performed twice. Data is shown as mean ± SD from one representative co-culture experiment with experimental triplicates. Two-way ANOVA with Tukey’s multiple comparisons test was used to compare between groups. Statistical significance is color-coded between corresponding treatment group and Mock group (**: p < 0.01; ****: p < 0.0001). F–M Mice were implanted subcutaneously with 2 × 105 A20-hCD20 cells and treated three times (3 × 106 T cells/dose) with Mock-T (n = 20), mCAR20-T (n = 20) or mCAR20(NAP)-T (n = 19). F Illustration of treatment schedule. G representative contour plot showing CAR expression level (represented as Thy1.1) in CD3 + cells. All experiments were assessed using a mixed population containing both transduced and untransduced cells. H Weight (mean) of mice after tumor implantation and during treatment. I Tumor size (mean + SEM). J Mouse survival (Kaplan–Meier) curve, and K–M tumor size for individual mice after treatment are presented. Numbers in K–M indicate tumor-free mice. The experiment was performed twice, and data was pooled. Tumor sizes on day 28 (time when the first mouse was sacrificed due to reaching the humane endpoint in Mock-T group) were compared between each treatment group using nonparametric Kruskal–Wallis One-way ANOVA test with Dunn’s correction for multiple comparisons. The survival curves were compared by the Log-rank test (*: p < 0.05, ***: p < 0.001)CC BY · PubMed Central
Fig. 3 Inclusion of NAP in CAR-T cells is unlikely to compromise safety in vivo. A Illustration of experimental set up. B Weight of mice after tumor implantation and treatment in both experiments. Data is shown as mean ± SD (n = 3). C Representative images of Hematoxylin–Eosin (HE) stained heart tissue sections collected from each treatment group, showing mild myxomatous degeneration of cardiac valves. Other examined organs had no lesions (Supplementary Fig. 3A-E). AVV: Atrioventricular valves. Scale bar equals 1 mm in 20 × images and equals 200 µm in zoomed-in images. D Liver enzymes (ASAT, ALAT, ALP) measured at different time points after treatment. Data is shown as mean ± SD (n = 3). ASAT: aspartate aminotransferase; ALAT: alanine aminotransferase; ALP: alkaline phosphatase. Two-way ANOVA with Geisser-Greenhouse correction and Dunnett’s multiple comparison tests were performed to compare matched values between time points (within the same treatment group) and values between groups (at same time point). The statistical labels are colored according to treatment groups (*: p < 0.05; **: p < 0.01). E Systemic cytokine levels measured at different time points after treatment. A panel of 13 cytokines (LEGENDplex) was assessed and the cytokines with detectable level are presented (CXCL9, CXCL10, CCL2). Each dot represents an individual animal with bars indicating mean ± SEM (n = 3). Two-way ANOVA with Geisser-Greenhouse correction and Dunnett’s multiple comparison tests were performed to compare matched values between time points (within the same treatment group) and values between groups (at the same time point). The statistical labels are colored according to treatment groups (*: p < 0.05; **: p < 0.01)
Fig. 3 Inclusion of NAP in CAR-T cells is unlikely to compromise safety in vivo. A Illustration of experimental set up. B Weight of mice after tumor implantation and treatment in both experiments. Data is shown as mean ± SD (n = 3). C Representative images of Hematoxylin–Eosin (HE) stained heart tissue sections collected from each treatment group, showing mild myxomatous degeneration of cardiac valves. Other examined organs had no lesions (Supplementary Fig. 3A-E). AVV: Atrioventricular valves. Scale bar equals 1 mm in 20 × images and equals 200 µm in zoomed-in images. D Liver enzymes (ASAT, ALAT, ALP) measured at different time points after treatment. Data is shown as mean ± SD (n = 3). ASAT: aspartate aminotransferase; ALAT: alanine aminotransferase; ALP: alkaline phosphatase. Two-way ANOVA with Geisser-Greenhouse correction and Dunnett’s multiple comparison tests were performed to compare matched values between time points (within the same treatment group) and values between groups (at same time point). The statistical labels are colored according to treatment groups (*: p < 0.05; **: p < 0.01). E Systemic cytokine levels measured at different time points after treatment. A panel of 13 cytokines (LEGENDplex) was assessed and the cytokines with detectable level are presented (CXCL9, CXCL10, CCL2). Each dot represents an individual animal with bars indicating mean ± SEM (n = 3). Two-way ANOVA with Geisser-Greenhouse correction and Dunnett’s multiple comparison tests were performed to compare matched values between time points (within the same treatment group) and values between groups (at the same time point). The statistical labels are colored according to treatment groups (*: p < 0.05; **: p < 0.01)CC BY · PubMed Central
Fig. 4 Recombinant NAP protein did not induce blood immune cell activation or cytokine release at levels above unmanageable clinical toxicity in the rotating whole human blood loop system. Recombinant NAP protein was evaluated for potential excessive immune activation side-by-side with Alemtuzumab, which has a manageable toxicity profile in the clinic. A Freshly acquired human blood was incubated in the rotating heparin-coated loops with either Phosphate-buffered saline (PBS, negative control), NAP, Alemtuzumab or Lipopolysaccharide (LPS, positive control) for immune activation. B–F Cytokines levels after 4 h incubation (n = 10), B IL-6, C IL-8, D TNFα, E IFN-γ and F IL-2 were analyzed by MSD Discovery® multiplex. The calculated lower limit of quantification (LLOQ) was marked with a dotted line. Each donor was run twice on separate occasions. G C3a and H C5a levels in 15 min plasma samples measured by ELISA. B–H paired Student’s t-test was performed on Log10 transformed values (values below LLOQ were set up to LLOQ) with Holm-Sidak correction for multiple comparisons between PBS and other groups. I Mean fluorescent intensity (MFI) of CD62P on platelets (CD41+CD45−). J–L MFI of CD69 and CD107a on J T cells (CD3+CD56−), K NK cells (CD3−CD56+) and L B cells (CD3−CD19+). M–N MFI of CD11b and CD83 on M granulocytes (CD14−CD66b+) and N monocytes (CD14+CD66B.−). J–N Each dot represents data from a healthy donor (n = 10), with bars indicating mean + SEM. One-way ANOVA was performed with Dunnett’s correction for multiple comparisons between PBS and other groups (* = p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001)
Fig. 4 Recombinant NAP protein did not induce blood immune cell activation or cytokine release at levels above unmanageable clinical toxicity in the rotating whole human blood loop system. Recombinant NAP protein was evaluated for potential excessive immune activation side-by-side with Alemtuzumab, which has a manageable toxicity profile in the clinic. A Freshly acquired human blood was incubated in the rotating heparin-coated loops with either Phosphate-buffered saline (PBS, negative control), NAP, Alemtuzumab or Lipopolysaccharide (LPS, positive control) for immune activation. B–F Cytokines levels after 4 h incubation (n = 10), B IL-6, C IL-8, D TNFα, E IFN-γ and F IL-2 were analyzed by MSD Discovery® multiplex. The calculated lower limit of quantification (LLOQ) was marked with a dotted line. Each donor was run twice on separate occasions. G C3a and H C5a levels in 15 min plasma samples measured by ELISA. B–H paired Student’s t-test was performed on Log10 transformed values (values below LLOQ were set up to LLOQ) with Holm-Sidak correction for multiple comparisons between PBS and other groups. I Mean fluorescent intensity (MFI) of CD62P on platelets (CD41+CD45−). J–L MFI of CD69 and CD107a on J T cells (CD3+CD56−), K NK cells (CD3−CD56+) and L B cells (CD3−CD19+). M–N MFI of CD11b and CD83 on M granulocytes (CD14−CD66b+) and N monocytes (CD14+CD66B.−). J–N Each dot represents data from a healthy donor (n = 10), with bars indicating mean + SEM. One-way ANOVA was performed with Dunnett’s correction for multiple comparisons between PBS and other groups (* = p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001)CC BY · PubMed Central

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Metadata

Authors
Ma, J., Sarén, T., Jin, C., Kim, H. S., Contreras Pineda, P. D., de Bernard, M., Amini, R.-M., Rondahl, V., Enblad, G., Yu, D., & Essand, M.
Affiliations
  • Uppsala University (SE)
  • University of Padua (IT)
  • Swedish University of Agricultural Sciences (SE)
Corresponding author
Yu, Di
ORCID
Venue
Cancer Immunology, Immunotherapy
Publisher
Springer Science and Business Media LLC
Published
2025-07-12
Volume
74
Issue
8
Pages
262-262
ISSN
  • 0340-7004 (ISSN-L)
  • 1432-0851
DOI
10.1007/s00262-025-04112-1
PMC
PMC12255594
OpenAlex
W4412362323
Cited by
1
References
3
Citations by year
2026: 1
FWCI
0.41
Type
journal article
Language
en
Open access
Licence
Locations
  • Cancer Immunology Immunotherapy (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Epsilon Open Archive (Sveriges lantbruksuniversitet biblioteket (Swedish University of Agricultural Sciences)) (acceptedVersion, cc-by)
  • Uppsala University Publications (Uppsala University) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Padua Research Archive (University of Padova) (submittedVersion, cc-by)
Funders
  • Erik, Karin och Gösta Selanders stiftelse (2023)
  • Cancerfonden (22-2229Pj, 19-0184Pj)
  • Barncancerfonden (PR2022-0105, PR2020-0167)
  • VINNOVA (2021-04488)
  • Vetenskapsrådet (2019-01326)
  • Uppsala University
Topics
  • Health Sciences › Medicine › Oncology › CAR-T cell therapy research
  • Life Sciences › Immunology and Microbiology › Immunology › Immunotherapy and Immune Responses
  • Life Sciences › Immunology and Microbiology › Immunology › T-cell and B-cell Immunology
Keywords
chimeric antigen receptor T cells, B-cell lymphoma, mantle cell lymphoma, relapsed lymphoma, rituximab, cytokine release syndrome, tumor growth inhibition
MeSH terms
Rituximab, Receptors, Chimeric Antigen, Animals, Bacterial Proteins, Female, Humans, T-Lymphocytes, Immunotherapy, Adoptive, Lymphoma, B-Cell, Helicobacter pylori, Xenograft Model Antitumor Assays, Cell Line, Tumor, Mice

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CoChat. (2026). Citation check: Pre-clinical safety and efficacy evaluation of Helicobacter Pylori neutrophil-activating protein (NAP)-armed CAR-T cells targeting B cell lymphomas. https://app.cochat.ai/papers/10.1007/s00262-025-04112-1
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Ma, J., Sarén, T., Jin, C., Kim, H. S., Contreras Pineda, P. D., de Bernard, M., Amini, R.-M., Rondahl, V., Enblad, G., Yu, D., & Essand, M. (2025). Pre-clinical safety and efficacy evaluation of Helicobacter Pylori neutrophil-activating protein (NAP)-armed CAR-T cells targeting B cell lymphomas. Cancer Immunology, Immunotherapy. https://doi.org/10.1007/s00262-025-04112-1