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. 2024 Jan 15;15(1):519.
doi: 10.1038/s41467-024-44786-2.

Generation and optimization of off-the-shelf immunotherapeutics targeting TCR-Vβ2+ T cell malignancy

Jingjing Ren #  1 , Xiaofeng Liao #  2 , Julia M Lewis  3 , Jungsoo Chang  3 , Rihao Qu  4 , Kacie R Carlson  3 , Francine Foss  5 , Michael Girardi  6
Affiliations

Generation and optimization of off-the-shelf immunotherapeutics targeting TCR-Vβ2+ T cell malignancy

Jingjing Ren et al. Nat Commun. .

Abstract

Current treatments for T cell malignancies encounter issues of disease relapse and off-target toxicity. Using T cell receptor (TCR)Vβ2 as a model, here we demonstrate the rapid generation of an off-the-shelf allogeneic chimeric antigen receptor (CAR)-T platform targeting the clone-specific TCR Vβ chain for malignant T cell killing while limiting normal cell destruction. Healthy donor T cells undergo CRISPR-induced TRAC, B2M and CIITA knockout to eliminate T cell-dependent graft-versus-host and host-versus-graft reactivity. Second generation 4-1BB/CD3zeta CAR containing high affinity humanized anti-Vβ scFv is expressed efficiently on donor T cells via both lentivirus and adeno-associated virus transduction with limited detectable pre-existing immunoreactivity. Our optimized CAR-T cells demonstrate specific and persistent killing of Vβ2+ Jurkat cells and Vβ2+ patient derived malignant T cells, in vitro and in vivo, without affecting normal T cells. In parallel, we generate humanized anti-Vβ2 antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) by Fc-engineering for NK cell ADCC therapy.

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Conflict of interest statement

J.R., X.L., J.M.L. and M.G are inventors on patent applications filed by Yale University. Application # 63/484,916. The remaining authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Generation of mCAR-T cells targeting the TCR-Vβ2 chain.
a TRBV usage frequency in CTCL cells from 72 patients seen at the Yale Photopheresis Unit from 2016 to 2022 as determined by anti-Vβ antibody staining (Beckman Coulter IOTest Beta Mark) and flow cytometry. b Distribution of TRBV frequency in total CD4 T cells from a Vβ2+ Sézary Syndrome (SS) patient determined by anti-Vβ antibody set staining and flow cytometry (top) or by paired single cell mRNA/TCR sequencing (bottom,). c Live CTCL cell counts from three Vβ2+ and one Vβ13.2 + CTCL patients after overnight in vitro culture with allogeneic mCAR-Vβ2 T cells with (right) or without (left) knockout (KO) of the endogenous T cell receptor alpha constant (TRAC) region at different effector-to-target (E:T) ratios, as determined by flow cytometry. d, e Live fraction of Vβ2+ CTCL cells or normal Vβ2+ cells (red), Vβ2- normal T cells (blue), and non-T cells (black) from PBMC of two Vβ2+ CTCL patients (d) and healthy control (HC)1 (e) at different E:T ratios after overnight co-culture with allogeneic triple KO (TRAC/B2M/CIITA) mCAR-Vβ2 T cells, as determined by flow cytometry. f Live fraction of each detectable Vβ subtype+ cells from total T cells of HC2 at different E:T ratios after overnight co-culture with allogeneic triple KO mCAR-Vβ2 T cells, as determined by flow cytometry. c–f N = 3 replicates of each E:T condition. Data are presented as mean values +/− SEM. *p < 0.05, **p < 0.01 and ****p < 0.0001 by two-way ANOVA. All replicates are independent samples. Source data and exact p-values are provided as a Source Data file.
Fig. 2
Fig. 2. Efficient and specific elimination of Vβ2+ malignant T cells in vivo by allogeneic CAR-Vβ2 T cells.
a Schematic of the NSG mouse model used to assess in vivo Vβ2+ T lymphoma killing by mCAR-Vβ2 T cells. Illustrations were created with BioRender.com and Adobe Illustrator. b–d Analysis of NSG bone marrow (BM) 10 days post inoculation of Jurkat-TRBV20-1 cells and subsequent treatment with triple KO CD8 mCAR-Vβ2 T cells vs no-treatment control (NC). b Representative flow cytometry quantified in (c, d). c Vβ2+ Jurkat-TRBV20-1 cell count, and (d) mCAR-Vβ2 T cell count with (red) or without (black) mCAR-Vβ2 treatment. e–l Analysis of NSG mice carrying CD4+ T cells from a Vβ2+ PTCL patient 1 week post treatment with triple KO CD8 mCAR-Vβ2 T cells vs no-treatment control (NC). e Representative NSG spleen cell flow cytometry quantified in (f–h) showing Vβ2+ PTCL cells, Vβ2-negative normal T cells and CD8 mCAR-Vβ2. f Vβ2+ PTCL cell count, (g) Vβ2-negative normal CD4+ T cell count, and (h) mCAR-Vβ2 T cell count in spleen with (red) or without (black) mCAR-Vβ2 treatment. i Representative NSG BM flow cytometry quantified in (j–l) showing Vβ2+ PTCL cells, Vβ2-negative normal T cells and CD8 mCAR-Vβ2. j Vβ2+ CTCL cell count (k) Vβ2- normal CD4+ T cell count, and (l) mCAR-Vβ2 T cell count in BM with (red) or without (black) mCAR-Vβ2 treatment. c–l N = 3 mice per group. Data are presented as mean values +/− SEM. *p < 0.05 and **p < 0.01 by two-sided t-test. Source data and exact p-values are provided as a Source Data file.
Fig. 3
Fig. 3. Humanization of CAR-Vβ2 T cells with preserved efficacy.
a, b Representative flow cytometry of (a) BM and (b) spleen, showing CD3+ Vβ2+ CTCL cells, CD3+ Vβ2-negative normal T cells and CD3-negative hCAR-Vβ2 T cells 3 days after in vivo hCAR-Vβ2 treatment, nonspecific CAR-CD19 treatment, or no-treatment control (NC). c Vβ2+ CTCL cells (d) Vβ2-negative normal T cells (e) CD8 hCAR-T and (f) CD4 hCAR-T cells in NSG BM 3 days after in vivo allogeneic triple-KO hCAR-Vβ2 (red) or CAR-CD19 (blue) pan-T cell treatment generated from a healthy donor compared to no-treatment control (NC, black). g Vβ2+ CTCL cell counts (h) Vβ2-negative normal T cell counts (i) CD8 CAR-T cell counts and (j) CD4 CAR-T cell count in spleen of the same mice. c–j N = 3 mice per group. Data are presented as mean values +/− SEM. *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA. Source data and exact p-values are provided as a Source Data file.
Fig. 4
Fig. 4. CRISPR-AAV system for CAR-Vβ2 T cell generation and treatment of Vβ2+ PTCL PDX.
a Adeno-associated virus (AAV) chimeric antigen-receptor (CAR) template structure and the strategy to integrate into the TRAC region using Cas9-TRAC-sgRNA. b Representative flow cytometry showing CD3- purity, CD4 and CD8 population percentages and CAR expression of AAV-dependent allogeneic hCAR-Vβ2 T cells compared to lentiviral-dependent allogeneic CAR-CD19 T cells. c Live PTCL/CTCL cell counts from two Vβ2+ patients and (d) live Jurkat-TRBV20-1 (Vβ2+, left) or Jurkat-TRBV6-2 (Vβ13.2+, right) cell counts after overnight in vitro culture with allogeneic lenti-CAR-CD19 T cells (black) or AAV-hCAR-Vβ2 T cells (red) at different E:T ratios, determined by flow cytometry. e–k Total CD4 T cells isolated from a Vβ2+ PTCL patient were adoptively transferred into groups of NSG mice that were then treated with allogeneic triple-KO AAV-hCAR-Vβ2 (red) or lenti-CAR-CD19 (blue) generated from healthy donor pan T cells, compared to no-treatment control (NC, black). Three days post-treatment (e) Vβ2+ CTCL cells (f) Vβ2- normal T cells (g) CD69+ % in CD8 CAR-T cells, and (h) CD69+ % in CD4 CAR-T cells in spleen were quantified by flow cytometry, as were (i) Vβ2+ CTCL cells (j) CD69+ % in CD8 CAR-T cells, and (k) CD69+ % in CD4 CAR-T cells in BM. l–o Long-term bioluminescence monitoring (IVIS) of Jurkat-TRBV20-1-lucifer cell-bearing NSG mice, at (l–n) ventral or (m–o) dorsal position, without treatment (NC, black) or following treatment with CAR-CD19 T cells (blue), lenti-hCAR-Vβ2 T cells (purple) or AAV-hCAR-Vβ2 T cells (red). p, Body weight and (q) survival of the same mice. c, d n = 3 replicates in each group. ***p < 0.001 and ****p < 0.0001 by two-way ANOVA. e-k, n = 3 mice in each group. *p < 0.05, **p < 0.01 and ***p < 0.001 by one-way ANOVA. n–q n = 5 mice in each group. n, o *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 by two-way ANOVA. q *p < 0.05 and **p < 0.01 by survival analysis (Kaplan-Meier). c–k–n–q Data are presented as mean values +/− SEM. All replicates are independent samples. Source data and exact p-values are provided as a Source Data file.
Fig. 5
Fig. 5. Cytokine optimization for hCAR-Vβ2 T cell expansion in vitro.
a Schematic of the timeline of cytokine optimization and functional analysis. b Live total T cell counts during in vitro expansion of hCAR-Vβ2 T cells from three different donors, determined by trypan blue cell counting. c Jurkat-TRBV20-1 cell killing % and (d) live CD3- hCAR-Vβ2 T cell counts in acute repeated Jurkat-TRBV20-1 killing assay, determined by trypan blue cell counting and flow cytometry. e CD45RA+ CD45RO+ % of CD8 + CD3- hCAR-Vβ2 T cells 4 days post repeated Jurkat-TRBV20-1 cell killing assay, determined by flow cytometry. b–e N = 3 replicates in each group. Data are presented as mean values +/− SEM. f relative Jurkat-TRBV20-1 killing by chronically stimulated hCAR-Vβ2 T cells from two healthy donors generated in seven cytokine conditions, with each killing round normalized to the first round. g summary of optimized cytokine combinations for allogeneic hCAR-Vβ2 T cell expansion in vitro. Illustrations in Fig. 5a, g were created using Adobe Illustrator. All replicates are independent samples. Source data are provided as a Source Data file.
Fig. 6
Fig. 6. Humanized anti-Vβ2 therapeutic antibody with enhanced ADCC.
a An anti-Vβ2 antibody competition assay, in which 1.25μg/ml mouse anti-Vβ2-FITC antibody was mixed with varying concentrations of humanized anti-Vβ2-PE antibody and used to stain CTCL cells from a Vβ2+ patient, with Vβ2 MFI determined by flow cytometry. b ADCC assay luminescence signal after a 6 h co-culture of patient derived Vβ2+ target CTCL cells and Jurkat-NFAT-luciferase effector cells mixed with either 100 ng/ml mouse anti-Vβ2 antibody (black) or humanized anti-Vβ2 antibody (red). PBMC from Vβ2+ CTCL patient 1 (c–e) or from Vβ2+ CTCL patient 2 (f–h) were cultured overnight with NK effector cells from a healthy donor at different E:T ratios without antibody addition (black) or with addition of mouse anti-Vβ2 antibody (blue) or humanized anti-Vβ2 antibody (red), then live CTCL cells (c–f) Vβ2- normal T cells (d–g) and non-T cell PBMC (e–h) counts were determined by flow cytometry. c–h N = 3 replicates in each group. Data are presented as mean values +/− SEM. ***p < 0.001 and ****p < 0.0001 by two-way ANOVA. All replicates are independent samples. Source data and exact p-values are provided as a Source Data file.
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References

    1. Safarzadeh Kozani P, Safarzadeh Kozani P, Rahbarizadeh F. CAR-T cell therapy in T-cell malignancies: is success a low-hanging fruit? Stem Cell Res. Ther. 2021;12:527. doi: 10.1186/s13287-021-02595-0. - DOI - PMC - PubMed
    1. Neelapu SS, et al. Axicabtagene Ciloleucel CAR T-cell therapy in refractory large B-cell Lymphoma. N. Engl. J. Med. 2017;377:2531–2544. doi: 10.1056/NEJMoa1707447. - DOI - PMC - PubMed
    1. Schuster SJ, et al. Chimeric antigen receptor T cells in refractory B-cell lymphomas. N. Engl. J. Med. 2017;377:2545–2554. doi: 10.1056/NEJMoa1708566. - DOI - PMC - PubMed
    1. Ellebrecht CT, et al. Reengineering chimeric antigen receptor T cells for targeted therapy of autoimmune disease. Science. 2016;353:179–184. doi: 10.1126/science.aaf6756. - DOI - PMC - PubMed
    1. Mackensen A, et al. Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus. Nat. Med. 2022;28:2124–2132. doi: 10.1038/s41591-022-02017-5. - DOI - PubMed

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