- Letter to the Editor
- Open Access
CAR T cells targeting CD99 as an approach to eradicate T-cell acute lymphoblastic leukemia without normal blood cells toxicity
Journal of Hematology & Oncology volume 14, Article number: 162 (2021)
CAR T cell therapy has shown dramatic clinical success in relapsed or refractory B-ALL and other hematological malignancies. However, the loss of specific antigens, cell fratricide, T cell aplasia, and normal T cell separation are challenges in treating T cell leukemia/lymphoma with CAR T therapy. CD99 is a promising antigen to target T-ALL and AML as it is strongly expressed on the majority of T-ALL and AML. Here, we isolated a low-affinity CD99 (12E7) antibody, which specifically recognizes leukemia cells over normal blood cells. Moreover, T cells transduced with an anti-CD99-specific CAR that contained the 12E7 scFv expanded with minor fratricide and without normal blood cells toxicity. We observed that our anti-CD99 CAR T cells showed robust cytotoxicity specifically against CD99+ T-ALL cell lines and primary tumor cells in vitro and significantly prolonged cell line-derived xenografts (CDXs) or patient-derived xenografts (PDXs) models survival in vivo. Together, our results demonstrate that anti-CD99 CAR T cells could specifically recognize and efficiently eliminate CD99+ leukemia cells.
To the editor:
T-ALL is an aggressive hematological malignancy accounting for 15% of pediatric and 25% of adult ALL cases [1, 2]. The standard treatment of chemotherapy combined with glucocorticoids has significantly improved survival, but up to 20% of pediatric and 40% of adult T-ALL patients are at risk for relapse [3, 4]. Novel optimal therapeutic strategies need to be developed for T-ALL, particularly for relapsed and refractory T-ALL patients. CARs targeting CD19 have been studied extensively for the treatment of B-ALL [5, 6]. However, CAR T cells share similar antigens with malignant T cells, and translating this approach to T-ALL has been extremely challenging due to fratricide and T cell aplasia .
CD99 has been demonstrated to have stronger expression in newly diagnosed T-ALL and also used as a new tool for the detection of MRD [8, 9]. We also confirmed CD99 up-regulated in transcript and protein levels compared with normal T cells (Additional file 1: Fig S1a, b). Further, we found that the CD99 expression is strong in different subgroups with the meanings of FPKM above 100 (Additional file 1: Fig S1c, d), indicating that CAR T cell therapy based on CD99 is a promising therapeutic strategy for T-ALL rooting out. To avoid T cell fratricide and potential on-target, off-tumor effects, we first identified a lower affinity anti-CD99 mAb (12E7) that specifically recognizes CD99-expressing T-ALL cell lines but not normal blood cells (Fig. 1a). Then, we systematically investigated the sensitivity of 12E7 mAb in normal tissue and observed 12E7 mAb-positive signals only in parts of the thymus, but not in the spleen, liver, kidney or other important organs (Additional file 1: Fig S1e). We also found the 12E7 scFv exhibited a lower binding affinity of 6.97 × 10–8 M, which is lower than the 1021527 (5.76 × 10–9 M) and 3B2/TA8 (1.93 × 10–9 M) (Fig. 1b and Additional file 2: Fig S1f). And there was a strong positive correlation between the 12E7 mAb and 12E7 scFv in the different cell lines according to flow cytometry analysis (Fig. 1c). Together, the results indicate that the 12E7 mAb is an optimal antibody for anti-CD99 CAR T therapy according to its specific target molecule recognition and limited binding to normal cells.
Next, the 12E7 scFv was incorporated into the lentivirus CAR vector to generate anti-CD99 CAR (Fig. 1d). Following activation and transduction of T cells, anti-CD99 CAR T cells were significantly fewer than the anti-CD19 CAR T control (Fig. 1e). Interestingly, the efficacy of transduced CAR+ cells was significantly increased during cell culture, almost 100% in the 12 days (Fig. 1f). And the following results showed that CD99 induced expression after CD3/CD28 beads activation and could be targeted by anti-CD99 CAR T cells (Fig. 1g, h). In contrast, we purified the CAR+ cells after 3 days transduction and found the antigen of CD99 did not express in anti-CD99 CAR T cells and CAR+ cells could not exhibit significant fratricide during the cell culture (Fig. 1i, j). Next, we assessed the antigen specificity and cytotoxic activity of anti-CD99 CAR T cells in NIH 3T3 human CD99 overexpression cell line and MOLT-4 CD99 knockdown cell line and found that the cytotoxicity was strongly correlated with the expression level of CD99 (Additional file 2: Fig. S1g-h). Furthermore, anti-CD99 CAR T cells showed specifically target the T-ALL cell lines and primary cells but with minimal killing of normal blood cells (Fig. 1k–m and Additional file 2: Fig. S1i). In addition, not only the T-ALL, we also found CD99 expressed and induced powerful antitumor activity in AML and a vast majority of solid tumor cells (Additional file 3: Fig. S1j, k), suggesting that CD99 may be a broad spectrum target for CAR T cell therapy.
To assess the effect of anti-CD99 CAR T cells against CD99+ T-ALL cells in vivo, we performed experiments using CDXs and PDXs models created by Jurkat, MOLT-4, and patient samples, respectively (Fig. 2a). Anti-CD99 CAR T cells conferred robust protection against leukemia progression and significantly extended the median survival of the mice in the CDX and PDX models (Fig. 2b–g). Especially in the PDX-1 model, the engraftment of CD99+ primary T-ALL cells gradually increased over time in the PB of the control T cell groups, whereas anti-CD99 CAR T cells significantly delayed leukemia progression (Fig. 2c). Compared with the T cell treatment group, anti-CD99 CAR T cells significantly eliminated infiltrating leukemia cells in the spleen and BM (Fig. 2h, i and Additional file 4: Fig. S2a-e). More importantly, the results showed that CARs persisted at a detectable level in the PB for at least 21 days in the Jurkat CDX model treated with CAR T cells (Fig. 2j) but without a significant change in animal body weight (Fig. 2k).
In summary, we demonstrated that CD99 is an attractive target for the immunotherapy of T-ALL and provided preclinical evidence for the therapeutic and safe use of fratricide-resistant anti-CD99 CAR T cells. Importantly, future clinical trials will need to assess the safety and feasibility of anti-CD99 CAR T cell therapy.
Availability of data and materials
All supporting data are included in the manuscript and supplemental files. Additional data are available upon reasonable request to the corresponding author.
T-cell acute lymphoblastic leukemia
Acute lymphoblastic leukemia
- CAR T:
Chimeric antigen receptor T cell
Minimal residual disease
Single-chain variable fragment
Acute myeloid leukemia
Cell line-derived xenograft
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We thank Peng Zhou, Long Chen and Jian Lin (College of Chemistry and Molecular Engineering, Peking University) for assisting some laboratory experiments. We thank members of our team for critical discussion and suggestions.
This work was supported by the grant from the Wuhan Science and Technology Plan Project (2019030703011533) to Tongcun Zhang and also the grant from Postdoctoral Science Foundation of China (2020M682491), Wuhan Former Funded Science and Technology Program (2020020602012111) and grand science and technology special project carried out by the department of Science and Technology of Hubei Province (2020BCB048) to Haichuan Zhu.
Ethics approval and consent to participate
This study was approved by the Animal Ethics Committee of Wuhan University of Science and Technology of ID WKD-Zhu-1.
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The authors declare that they have no competing interests.
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(a) Relative CD99 expression was calculated as CD99 fragments per kilobase of exon model per million mapped fragments (FPKM) on T-ALL samples (n=264) and normal PBMC samples (n=23). Data from Dvinge H et al. PNAS, 2014 and Liu Y et al. Nature genetic. 2017. (b) The relative CD99 protein level was calculated as the CD99 mean fluorescence intensity (MFI) on T-ALL samples (n=22) and normal T cell samples (n=5) by flow cytometry. (c) Relative CD99 expression was calculated as FPKM on ETP ALL (n=19), nearETP ALL (n=24) and nonETP ALL (n=146) samples. Data from Liu Y et al. Nature genetic. 2017.(d) Relative CD99 expression was calculated as CD99 FPKM on T-ALL subgroup, including LMO2_LYL(n=18), LMO1/2(n=10), HOXA(n=33), TLX3(n=46), TLX1(n=26), NKX2_1(n=14), TAL1(n=87), TAL2(n=8) and unknown(n=22) samples. Data from Data from Liu Y et al. Nature genetic. 2017. (e) Representative immunohistochemistry (IHC) images of human normal paraffin tissue sections with the CD99 (12E7) mAb. IgG as the negative control.
(f) Binding kinetics of anti-CD99 antibodies (1021527 and 3B2/TAB) with CD99 protein. Analysis of the interaction between the antibodies and CD99 protein using BLItz biolayer interferometry. (g) The CD99 expression level in NIH 3T3 human CD99-overexpression cell line, and anti-CD99 CAR T cells specifically lysis efficiency at different effector-to-target ratios (1:1/5:1/25:1). (h) The CD99 expression level in MOLT-4 human CD99 knockdown cell line, and anti-CD99 CAR T cells specifically lysis efficiency at effector-to-target ratios (25:1). (i) The gating strategy of blast cells from T-ALL patients and the CD99 expression level in four patients’ blasts (The detail information showed in the Additional file 5: Table S1).
(j) Upper: Flow cytometry showing CD99 expression in different AML cell lines. Lower: Cytotoxic activity of anti-CD99 CAR T cells against AML cell lines as determined by calcein release assay at different E:T ratios (1:1/5:1/25:1) after 2-3h of co-culture. (k) Upper: Flow cytometry showing CD99 expression in different solid tumour cell lines. Lower: Cytotoxic activity of anti-CD99 CAR T cells against various solid tumour cell lines as determined by calcein release assay at different E:T ratios (1:1/5:1/25:1) after 2-3h of co-culture. ***p ≤ 0.001,**p ≤ 0.01,NS = no significant, Scale bar, 50 μm or 200 μm.
(a) Spleens from T cell and anti-CD99 CAR T cell treatment groups were weighed and photographed from the PDX-1. (b) The proportion of human CD7 positive cells in the spleen of PDX-1 models. (c) Spleens from T cell and anti-CD99 CAR T cell treatment groups were weighed and photographed from the PDX-2. (d) The proportion of human CD7 positive cells in the spleen of PDX-2 models. (e) Histological features of the spleen in the T cell and anti-CD99 CAR T cell treatment groups (Jurkat, MOLT-4, PDX-1 and PDX-2). Scale bar, 50μm or 200μm.
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Shi, J., Zhang, Z., Cen, H. et al. CAR T cells targeting CD99 as an approach to eradicate T-cell acute lymphoblastic leukemia without normal blood cells toxicity. J Hematol Oncol 14, 162 (2021). https://doi.org/10.1186/s13045-021-01178-z
- CAR T
- Antitumor activity