Naturally Selected CD7 CAR-T Therapy without Genetic editing Exerts Significant Antitumor Efficacy against Relapsed and Refractory Acute Myeloid Leukemia(R/R-AML)

doi:10.21203/rs.3.rs-2110527/v1

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Naturally Selected CD7 CAR-T Therapy without Genetic editing Exerts Significant Antitumor Efficacy against Relapsed and Refractory Acute Myeloid Leukemia(R/R-AML)

https://doi.org/10.21203/rs.3.rs-2110527/v1

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Background

Survival time for patients with relapsed and refractory acute myeloid leukemia (R/R-AML) remains threatened and treatment appears challenging. Chimeric antigen receptor T cell (CAR-T) has been widely used for hematologic malignancies. Current CAR-T therapies for acute myeloid leukemia mostly target myeloid-lineage antigens such as CD123 and CD33, which may have potential hematopoietic toxicity. CD7, as a lineage-specific receptor, is expressed in acute myeloid leukemia cells and T cells, while not expressed in myeloid cells.

Methods

In this report, Immunohistochemistry and flow cytometry were used to analyze CD7 expression in clinical samples from R/R-AML patients and healthy donors. we designed naturally selected CD7 CAR-T to analyze various functions and in vitro anti-leukemic efficacy by flow cytometry, while xenograft models were used to validate in vivo tumor dynamics

Results

we calculated the percentage of CD7 expression in our institution's AML patients with minimal residual disease (MRD) (5/16, 31.25%) and tested CD7 in myeloid and lymphoid lineage cells of R/R-AML patients, concluding that CD7 is expressed in T cells and not in myeloid cells, similar to previous studies. Subsequently, we constructed and designed a naturally selected CD7 CAR-T (CD7 CAR). We did not perform CD7 antigen knockdown on CD7 CAR-T cells, but it naturally eliminated CD7 molecule expression at the end of the culture cycle and then evaluated its ability to target and kill the acute myeloid leukemia cell line MOLM-13 in vitro and in vivo. CD7 CAR-T cells efficiently murder acute myeloid leukemia cells in vitro and significantly inhibit the growth of leukemia cells in xenograft model mice.

Conclusion

Naturally Selected CD7 CAR-T cells were proven to be an effective and safe treatment strategy for relapsed refractory acute myeloid leukemia in preclinical studies.

acute myeloid leukemia

CD7

CAR-T cells

immunotherapy

minimal residual disease (MRD)

Despite standard treatment of chemotherapeutic agents of anthracycline and cytarabine to achieve complete remissions (CR) for patients with acute myeloid leukemia, many patients still relapse within a short period(1, 2).

In recent years, cellular immunotherapy of chimeric antigen receptor T-cell (CAR-T) for relapsed or refractory acute myeloid leukemia(R/R-AML), targeting myeloid-lineage antigens like CD123, CLL-1, and CD33, has shown bright prospects in many pre-clinical pieces of research (3–8). CD123 and CD33 expression in CD34 + hematopoietic stem cells and other normal myeloid cells pose a potential hematopoietic risk as well as a deficiency of evidence for long-term remission, so safety and efficacy need further evaluation (9–11).

Selecting lineage-specific antigens specifically expressed on AML cells and absent from normal myeloid cells may be a promising therapeutic strategy. CD7 is a target that has been used in the therapeutic strategy of peripheral T-cell leukemia with favorable results in preclinical and clinical studies(12–14). Approximately 20–35% of AML has a high expression of CD7, which is frequently associated with a worse prognosis for patients (15–19). Expression of CD7 in NK cells, T cells, and lymphoid progenitors in physiological conditions(20–24). It is involved in the positive regulation of T-cell activity, but its deletion does not interfere with T-cell development and function because the T-cell function of CD7 knockout mice is not impaired(25, 26). Recent clinical trials also have demonstrated that the proliferation of CD7- T cells after CD7 + T cell depletion in patients may compensate for the immune deficiency due to T cell absence (27). These pieces of evidence demonstrate the feasibility of CD7 as a target for CAR-T therapeutic tools in relapsed and refractory acute myeloid leukemia (R/R AML).

Current studies have shown that CD7 gene-edited (CD7KO) and CD7-directed CAR-T has made great progress in the treatment of hematological tumors and increased cost and unknown risks for additional gene knockouts (21, 28–30). In previous studies, we demonstrated in vitro, in vivo, and clinical studies that a naturally selected CD7-targeting CAR-T (CD7 CAR) treatment of T-ALL/LBL can produce promising therapeutic effects(31).

In this experiment, we explored the suitability of CD7 CAR-T cells for the treatment of acute myeloid leukemia. Its various functions have been validated in vitro and xenograft model mice. Its efficient anti-Acute Myeloid Leukemia effect has been demonstrated in the preclinical study phase.

Immunohistochemistry

Bone marrow biopsy samples from the Department of Hematology, Second Hospital of Hebei Medical University. The following immunohistochemical antibodies and reagents are from Leica Microsystems. Bone marrow biopsy samples were processed by Leica Bond MAX Immunostainer. The program was set to peroxide block for 5 min; marker labeling incubation for 15 min; post-primary for 8 min; polymer for 8 min; Mixed DAB Refine for 10 min and finally hematoxylin for 5 min. All the above steps were performed at room temperature. the Olympus microscope (×400 ,×100) was used to observe the samples.

CD7 and AML-MRD Assays

The following experimental flow cytometry antibodies were purchased from BD Bioscience. Bone marrow blood from patients with R/R-AML in the Second Hospital of Hebei Medical University was determined by flow cytometry for CD45-KO, CD117-PE, CD34-APC, CD7-A700 When higher than the isotype control group, the expression was considered positive. Then, Specimens from healthy donors and R/R- AML patients from the Second Hospital of Hebei Medical University were selected and labeled with CD45-V500, CD14-APC, CD16-FITC, CD3-FITC, CD19-APC, CD7-PE, and the proportion of CD7 expression in normal cell populations was detected by flow cytometry (BECKMAN NAVIOS).

CD7 CAR-T Cells Design and Generation

The DNA sequence of the anti-CD7 single-chain variable fragment derived from the CD7-specific mouse monoclonal antibody TH-69 was synthesized and cloned into a CAR composed with a 12AA short hinge-only domain, CD28 transmembrane, 4-1BB, CD3ζ, T2A autocleavage sequences, and endodomain-deleted EGFR (tEGFR)(32–34). CD7 CAR using lentiviral vectors were produced by 293FT cells of transfecting and concentrated by 20000g ultracentrifuging after 2 hours. The Lentivirus loaded with CD7 CARs stored at -80°(35).

Healthy human peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood by Ficoll density centrifugation. CD3 + T cells were obtained from healthy human PBMCs using CD3 microbeads (Miltenyi Biotec) and then activated for 48 hours using CTS CD3/CD28 Dynabeads(GIBCO) in TexMACS (Miltenyi Biotec) supplemented 200IU of IL-2. Two days later, the CD3 + T cells were transduced with the CD7-CAR lentivirus. Three days after transduction, the proportion of CAR-positive T cells was measured utilizing flow cytometry. CD7 CAR-T cells were continuously cultured in TexMACS (Miltenyi Biotec) with 200IU/ml of IL-2 until day 10 post-transduction, and various functional assays in vitro or injections in vivo of CD7 CAR-T cells were performed. Appropriate expansion of initial culture numbers of CD7 CAR-T cells for injection.

Flow Cytometry

The following antibodies are sourced from BioLegend. Anti-humanCD7-APC, CD4-PE-cy7, CD8-PB, CD3-APC, PD-1-PB, CD45RO-Percp, CCR7-PE, ERB-FITC were used to stain cells in DPBS (BI) with 2%FBS (Oricell). The percentage of CD7 CAR was determined by biotinylated Erbitux(34). All flow cytometry data were obtained in a MACSquant with a filter set for eight fluorescence signals and analyzed with FlowJo software (V10.8.1).

Cytotoxicity Assay

Cell line MOLM-13 was purchased from ATCC. The cells were expanded according to ATCC recommendations. A total of 1×105 MOLM-13 cells were co-cultured with CD7 CAR-T cells or non-transduced T cells in RPMI 1640 media (gibco) for 4h according to the efficacy target ratios (E: T) of 0: 1, 0.5:1, 1:1 and 2:1, respectively. At the end of the four-hour co-culture, all cells were stained for PI-PE and annexin-V-APC (BioLegend) and then analyzed by flow cytometry (MACSquant).

Mouse Xenograft Model

six-to 8-week-old nonobese diabetic (NOD)–Prkdcscid-Il2rgem1 (NTG) female mice were purchased from SPF(Beijing) BiotechologyCo., Ltd. and cared for at the Animal Center of the Fourth Hospital of Hebei Medical University. All in vivo studies were done in compliance with the Animal Ethics Committee of the Fourth Hospital of Hebei Medical University. MOLM-13-GFP-luciferase reporter cell line (MOLM-13-GFP-Luc) was constructed for in vivo tumor kinetics. Mice were injected with 5×105 MOLM-13-GFP-Luc cells via tail vein, followed by a single intravenous injection of 1×106 CD7 CAR + T cells was administered, the NTR group was infused with non-transduced T cells, and the vehicle group was infused with DPBS after 5 days. The tumor burden of mice injected intraperitoneally with 150 mg/kg of D-luciferase at the indicated time was monitored using the IVIS imaging system (BERTHOLD LB983 NC100) by recording bioluminescence.

Statistical Analysis

The data are presented as means ± standard deviation (SD). Statistical significance of differences between samples was determined by an unpaired two-tailed Student's t-test and in multiple comparisons by a one-way ANOVA with post-Tukey’s tests. If the variance was not equal, then the unpaired two-tailed Student's t-test with welch's correction was chosen. All P values were calculated using Prism 8 software (GraphPad). The significance of the researches are defined as follows: ns, not significant, p > 0.05; *p < 0.05; **p < 0.01; and ***p < 0.001 and ****p < 0.0001.

CD7 is Expressed in R/R-AML Patients with Minimal Residual Disease (MRD), but not on Normal Cells

We examined CD7 expression in T cells, B cells, monocytes, and neutrophils from normal cell subsets from healthy donors, where CD7 was highly expressed in T cell subsets and low in other myeloid subsets or Expression absent, which is similar from that reported by Diogo Gomes-Silva et al (21). CD7 expression was further determined in normal cell subsets from R/R AML patients and showed similar expression to healthy donors (Fig. 1A-E). A previous study reported that the CD7-positive percentage of acute myeloid leukemia cells detected by flow cytometry was approximately 30–35% (16, 18, 36). We summarized the expression of CD7 in the MRD-positive reports of R/R-AML patients from July 1, 2022, to January 1, 2021, in the Department of Hematology, the Second Hospital of Hebei Medical University, and the results suggested that 5 of the 16 MRD-positive patients (31.25%) had different proportions of CD7 expression, which was also demonstrated by immunohistochemistry (Fig. 1F-G) (Table 1). These results also justify CD7 as a therapeutic target.

Table 1

characteristics of R/R AML
Sample ID	Age	Sex	CD7+	Cytogenetics
1	42	F	Neg	46, XX
2	57	M	Neg	46, XY
3	73	F	79.78%	46, XX
4	67	M	31.38%	46, XY
5	32	F	Neg	46, XX
6	64	F	Neg	46, XX
7	73	M	79.96%	46, XY, del (9) (q22; q34)
8	30	M	Neg	46, XY, t (8;21) (q22; q22)
9	16	M	Neg	45, X, -Y, t (8;21) (q22; q22)
10	71	F	Neg	46, XX
11	34	F	60.75%	46, XX
12	41	M	Neg	47, XY, +8
13	59	M	5.00%	46, XY
14	66	M	Neg	46, XY
15	65	F	Neg	Complex
16	37	M	Neg	Complex

CD7 CAR-T has High CAR and Negative CD7 Expression in vitro Assays

To determine whether normal T cells can redirect, specifically recognize and attack CD7-expressing AML malignant cells upon loading CD7 CAR. We designed a CAR structure including CD7-specific single-chain variable fragments from CD7 hybridoma antibody TH-69, CD28 transmembrane region, 4-1BB, CD3ζ, and T2A autocleavage sequences as well as endodomain-deleted EGFR (tEGFR) as a CAR expression detection marker for CD7 CAR-T (37, 38)(Fig. 2A).We measured the expansion rate of CD7 CAR-T at a variety of time points in vitro and found that CD7 CAR-T had a lower proliferation ratio than NTR group, which was similar to the previous study (12, 21) (Fig. 2B). The viability of CD7 CAR-T cells was lower compared to non-transduced T cells, but the lowest once recorded was higher than 78.3% in post-transduction day7 and 80.7% in day12 (Fig. 2C). We used lentivirus-loaded CD7 CARs to infest normal human T cells and assayed the percentage of CD7 CAR + T cells by flow cytometry on post-transduction days 4 and 12 with the use of tEGFR, a marker carried on the CAR architecture. The results indicated that CD7 CAR + T cells had a high proportion of CD3 + T cells, with the lowest once recorded at 83.6% in post-transduction day4 and 90.8% in day 12, much higher than the control group (Fig. 2D-F). To continuously monitor the CD7 expression of CAR-T cells, we measured the percentage of CD7 by flow cytometry on day 4 and day 12 after transduction, respectively, and the results showed that CD7 CAR-T was mainly dominated by the CD7- T cell subpopulation either day4 or day12 after transduction (Fig. 2G-I). As described in previous studies, this may imply specific binding of CD7 CAR-T to the CD7 + cell population, eliminating the CD7 + T cell subpopulation from the CD7 CAR + T cell population(31).

Expression of CD7 CAR-T Cells Exhaustion Markers and Cell Subpopulation Analysis in vitro

Memory phenotype of T cells is critical for CAR-T anti-tumor efficacy and persistence(39, 40). According to previous researches, TCM cells were defined by CD45RO and CCR7 double positivity (41, 42). We analyzed the CD8 + and CD4 + CD7 CAR-T cell populations separately and concluded that although there were no differences between the CD4 + CAR + T cell subpopulation and the CD8 + CAR + T cell subpopulation, both were dominated by the memory phenotype, either the central (TCM CD45RO + CCR7+) or an effector (TEM CD45RO + CCR7-) memory phenotype (Fig. 3A-D). Finally, detection of programmed cell death protein 1(PD-1) to evaluate CAR-T cell exhaustion and the result shows mean = 0.56% (Fig. 3E).

Evidence of Anti-Tumor Function of CD7 CAR-T in vitro

To verify the anti-leukemic effect of CD7 CAR-T in vitro, we used AML cell line MOLM-13 as target cells and set different efficacy target ratios (E: T = 0:1, 0.5:1, 1:1, 2:1), and found that CD7 CAR-T cells could rapidly eliminate leukemia cells at various dose ratios. As shown in Fig. 4A-D, E: T = 0.5: 1, NTR vs CD7 CAR (mean ± SD = 8.10%±3.76% vs 70.07%±14.69%); E: T = 1:1, NTR vs CD7 CAR (mean ± SD = 9.70%±6.50% vs 89.81%±7.57%); E: T = 2:1, NTR vs CD7 CAR (mean ± SD = 11.60%±8.31% vs 94.12%±2.93%).

CD7 CAR-T Cells Demonstrated Excellent Anti-Leukemic Properties in vivo

Previous in vitro studies have demonstrated the potent anti-tumor ability of CD7 CAR-T cells. To further validate the clearance of CD7 CAR-T cells against acute myeloid leukemia in vivo. We injected 5×105 of MOLM-13-GFP-Luc cell line into the tail vein of each nonobese diabetic (NOD)–Prkdcscid-Il2rgem1 (NTG) mouse to build a xenograft model and injected 1×106 of CD7 CAR-T cells or NTR cells after 5 days, while the vehicle group was injected with an equal dose of DPBS buffer (Fig. 5A, B). No mice died during the 34 days monitoring period, and no evidence of graft-versus-host disease (GVHD) was observed. Using Bioluminescence imaging (BLI), the data indicated that compared to the NTR group, the leukemic cells in the CD7 CAR-T group were suppressed at day 7 after injection, followed by a rapid decrease in tumor burden, which could not be detected in all individual mice of the CD7 CAR-T group at 22 days (Fig. 5B, C).

Acute myeloid leukemia (AML), a heterogeneous disease associated with a wide range of molecular alterations, requires multiple therapeutic strategies in synergy to limit the progression of the disease. Chimeric antigen receptor T-cell (CAR-T) therapy, as an emerging immunotherapeutic tool, is often used as a strategy to reduce the residual chemoresistant tumors in patients with acute myeloid leukemia. i.e. pretreatment of allogeneic hematopoietic stem cell transplantation (allo-HSCT), to minimize relapse rate post-allo-HSCT and avoid the severe side effects (43). And clinical trials of CAR-T targeting CD19 in adults with relapsed or refractory diffuse large B-cell lymphoma have achieved high rates of durable responses, demonstrating CAR-T’s enormous potential to treat hematologic malignancies (44). In the field of acute myeloid leukemia treatment, CAR-T therapy also has some remaining challenges. While CAR-T therapies targeting CD123 and CD33 are already in clinical trials, these candidate targets are also frequently found in hematopoietic stem cells, posing risks associated with potential long-term or permanent myelosuppression (11, 18, 45). In this study, we further explored the availability of naturally selected CD7 CAR-T in the treatment of R/R-AML and demonstrated its great anti-leukemia ability in vitro and xenograft model mice.

CD7, which has been shown to be expressed in approximately 20–35% of AML patients, has been associated with multiple disparate prognoses (15–19). Likewise, we demonstrated this in a subset of minimal residual disease (MRD) from 16 patients with R/R AML. Similar to previous studies, both R/R AML patients and HDs had high expression of CD7 in their normal cell populations such as NK cells, T cells, and in contrast, B cells, monocytes, and neutrophils did not express CD7(21).

First, CD7 positivity does cause diminished proliferation and fratricide for CD7 CAR-T, but similar to our previous report, CD7 CAR-T is dominated by CD7-negative subpopulations after one cycle of in vitro culture, possibly due to antigenic masking/intracellular sequestration by CD7 CAR. secondly, NK cells and T cells, as important immune components in the body, may pose a risk of immunodeficiency to the recipient.

Previous studies have shown that CD7 CAR-T does cause defects in CD7-positive T cells in recipients, but CD7-negative T cell subsets appear to replace their function to some extent, thereby alleviating treatment-related T cell immunodeficiency, and that CD7-positive T cell populations and NK cell populations were restored after bridging allogeneic hematopoietic stem cell transplantation(27, 31). Compared with other myeloid-targeted CAR-T, CD7 CAR-T can avoid damage to normal myeloid cells and reduce hematopoietic toxicity to a certain extent because CD7 is not expressed on other myeloid cells.

Persistence, a key point affecting CAR-T efficacy, has been widely investigated in previous studies, and it has been shown that T cell memory phenotype subpopulation is an important factor in maintaining CAR-T cell persistence(39, 40). In the study, the memory phenotype subpopulation of naturally selected CD7 CAR-T was dominant, which may enable CD7 CAR-T to exert a more durable effect in patients with R/R AML.

In addition, in vitro and xenograft models were tested to determine whether naturally selected CD7 CAR-T would differ from previous studies in its anti-leukemic function, and the results suggested rapid tumor clearance and no abnormal reaction were observed in mice, which provided some evidence of its safety.

In conclusion, patients can benefit from the reduced cost and production cycle of CAR-T as a bridging allogeneic HSCT pretreatment for R/R-AML. Naturally selected CD7 CAR-T, as a CAR-T treatment strategy without additional treatments such as CD7 knockdown, can reduce the cost and additional unknown risks associated with knockdown to some extent. This study demonstrated the feasibility of naturally selected CD7 CAR-T in treating patients with R/R-AML in the preclinical study phase. However, R/R-AML being a complex disease type, the effectiveness of naturally selected CD7 CAR-T therapy needs to be further tested in clinical trials.

R/R AML relapsed and refractory acute myeloid leukemia

CAR-T Chimeric antigen receptor T cell

MRD minimal residual disease

Ethics approval and consent to participate

By the Declaration of Helsinki, the Second Hospital of Hebei Medical University will have reviewed and approved studies involving human participants. Patients/participants provided written informed consent to participate in this study.

Animal trials were reviewed and approved by the Fourth Hospital of Hebei Medical University

Consent for publication

All subjects have written informed consent..

Competing interests

The authors declare that they have no competing interests.

Funding

This study was supported by the Natural Science Foundation of Hebei Province (H2020206403).

Authors' contributions

FW provided funding and designed the experiments and helped revise the manuscript. YuL completed animal testing, clinical sample assays and statistics, and completed the manuscript. SZ and SW assisted with some of the experiments and manuscript writing. yingL and NK completed the CAR construct and completed in vitro experiments, and helped revise the manuscript. All authors contributed to the article and approved the submitted version.

Acknowledgements

We thank members of our laboratory for helpful discussion.

Availability of data and material

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

Dohner H, Estey E, Grimwade D, Amadori S, Appelbaum FR, Buchner T, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129(4):424–47.
Dohner H, Estey EH, Amadori S, Appelbaum FR, Buchner T, Burnett AK, et al. Diagnosis and management of acute myeloid leukemia in adults: recommendations from an international expert panel, on behalf of the European LeukemiaNet. Blood. 2010;115(3):453–74.
Tettamanti S, Marin V, Pizzitola I, Magnani CF, Giordano Attianese GM, Cribioli E, et al. Targeting of acute myeloid leukaemia by cytokine-induced killer cells redirected with a novel CD123-specific chimeric antigen receptor. Br J Haematol. 2013;161(3):389–401.
Pizzitola I, Anjos-Afonso F, Rouault-Pierre K, Lassailly F, Tettamanti S, Spinelli O, et al. Chimeric antigen receptors against CD33/CD123 antigens efficiently target primary acute myeloid leukemia cells in vivo. Leukemia. 2014;28(8):1596–605.
Collinson-Pautz MR, Chang WC, Lu A, Khalil M, Crisostomo JW, Lin PY, et al. Constitutively active MyD88/CD40 costimulation enhances expansion and efficacy of chimeric antigen receptor T cells targeting hematological malignancies. Leukemia. 2019;33(9):2195–207.
Petrov JC, Wada M, Pinz KG, Yan LE, Chen KH, Shuai X, et al. Compound CAR T-cells as a double-pronged approach for treating acute myeloid leukemia. Leukemia. 2018;32(6):1317–26.
Wang J, Chen S, Xiao W, Li W, Wang L, Yang S, et al. CAR-T cells targeting CLL-1 as an approach to treat acute myeloid leukemia. J Hematol Oncol. 2018;11(1):7.
Ataca Atilla P, McKenna MK, Tashiro H, Srinivasan M, Mo F, Watanabe N, et al. Modulating TNFalpha activity allows transgenic IL15-Expressing CLL-1 CAR T cells to safely eliminate acute myeloid leukemia. J Immunother Cancer. 2020;8(2):e001229.
Taussig DC, Pearce DJ, Simpson C, Rohatiner AZ, Lister TA, Kelly G, et al. Hematopoietic stem cells express multiple myeloid markers: implications for the origin and targeted therapy of acute myeloid leukemia. Blood. 2005;106(13):4086–92.
Kenderian SS, Ruella M, Shestova O, Klichinsky M, Aikawa V, Morrissette JJ, et al. CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia. Leukemia. 2015;29(8):1637–47.
Wang QS, Wang Y, Lv HY, Han QW, Fan H, Guo B, et al. Treatment of CD33-directed chimeric antigen receptor-modified T cells in one patient with relapsed and refractory acute myeloid leukemia. Mol Ther. 2015;23(1):184–91.
Gomes-Silva D, Atilla E, Atilla PA, Mo F, Tashiro H, Srinivasan M, et al. CD7 CAR T Cells for the Therapy of Acute Myeloid Leukemia. Mol Ther. 2019;27(1):272–80.
Gomes-Silva D, Srinivasan M, Sharma S, Lee CM, Wagner DL, Davis TH, et al. CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies. Blood. 2017;130(3):285–96.
McMahon CM, Luger SM. Relapsed T, Cell ALL. Current Approaches and New Directions. Curr Hematol Malig Rep. 2019;14(2):83–93.
Zheng J, Wang X, Hu Y, Yang J, Liu J, He Y, et al. A correlation study of immunophenotypic, cytogenetic, and clinical features of 180 AML patients in China. Cytometry B Clin Cytom. 2008;74(1):25–9.
Venditti A, Del Poeta G, Buccisano F, Tamburini A, Cox-Froncillo M, Aronica G, et al. Prognostic relevance of the expression of Tdt and CD7 in 335 cases of acute myeloid leukemia. Leukemia. 1998;12(7):1056–63.
Valet G, Repp R, Link H, Ehninger A, Gramatzki MM, group S-As. Pretherapeutic identification of high-risk acute myeloid leukemia (AML) patients from immunophenotypic, cytogenetic, and clinical parameters. Cytometry B Clin Cytom. 2003;53(1):4–10.
Haubner S, Perna F, Kohnke T, Schmidt C, Berman S, Augsberger C, et al. Coexpression profile of leukemic stem cell markers for combinatorial targeted therapy in AML. Leukemia. 2019;33(1):64–74.
Saxena A, Sheridan DP, Card RT, McPeek AM, Mewdell CC, Skinnider LF. Biologic and clinical significance of CD7 expression in acute myeloid leukemia. Am J Hematol. 1998;58(4):278–84.
Sathe P, Pang SHM, Delconte R, Elwood N, Huntington ND. Identification of Novel Human NK Cell Progenitor Subsets. Int J Mol Sci. 2017;18(12):2716.
Gomes-Silva D, Srinivasan M, Sharma S, Lee CM, Wagner DL, Davis TH, et al. CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies. Mol Ther. 2017;27(1):272–80.
Hauser A, Schrattbauer K, Najdanovic D, Schlossnickel R, Koch A, Hejtman M, et al. Optimized quantification of lymphocyte subsets by use of CD7 and CD33. Cytometry A. 2013;83(3):316–23.
Hao QL, Zhu J, Price MA, Payne KJ, Barsky LW, Crooks GM. Identification of a novel, human multilymphoid progenitor in cord blood. Blood. 2001;97(12):3683–90.
Rabinowich H, Pricop L, Herberman RB, Whiteside TL. Expression and function of CD7 molecule on human natural killer cells. J Immunol. 1994;152(2):517–26.
Bonilla FA, Kokron CM, Swinton P, Geha RS. Targeted gene disruption of murine CD7. Int Immunol. 1997;9(12):1875–83.
Lee DM, Staats HF, Sundy JS, Patel DD, Sempowski GD, Scearce RM, et al. Immunologic characterization of CD7-deficient mice. J Immunol. 1998;160(12):5749–56.
Pan J, Tan Y, Wang G, Deng B, Ling Z, Song W, et al. Donor-Derived CD7 Chimeric Antigen Receptor T Cells for T-Cell Acute Lymphoblastic Leukemia: First-in-Human, Phase I Trial. J Clin Oncol. 2021;39(30):3340–51.
Cooper ML, Choi J, Staser K, Ritchey JK, Devenport JM, Eckardt K, et al. An "off-the-shelf" fratricide-resistant CAR-T for the treatment of T cell hematologic malignancies. Leukemia. 2018;32(9):1970–83.
Png YT, Vinanica N, Kamiya T, Shimasaki N, Coustan-Smith E, Campana D. Blockade of CD7 expression in T cells for effective chimeric antigen receptor targeting of T-cell malignancies. Blood Adv. 2017;1(25):2348–60.
Depil S, Duchateau P, Grupp SA, Mufti G, Poirot L. 'Off-the-shelf' allogeneic CAR T cells: development and challenges. Nat Rev Drug Discov. 2020;19(3):185–99.
Lu P, Liu Y, Yang J, Zhang X, Yang X, Wang H, et al. Naturally selected CD7 CAR-T therapy without genetic manipulations for T-ALL/LBL: first-in-human phase 1 clinical trial. Blood. 2022;140(4):321–34.
Baum W, Steininger H, Bair HJ, Becker W, Hansen-Hagge TE, Kressel M, et al. Therapy with CD7 monoclonal antibody TH-69 is highly effective for xenografted human T-cell ALL. Br J Haematol. 1996;95(2):327–38.
Hudecek M, Sommermeyer D, Kosasih PL, Silva-Benedict A, Liu L, Rader C, et al. The nonsignaling extracellular spacer domain of chimeric antigen receptors is decisive for in vivo antitumor activity. Cancer Immunol Res. 2015;3(2):125–35.
Wang X, Chang WC, Wong CW, Colcher D, Sherman M, Ostberg JR, et al. A transgene-encoded cell surface polypeptide for selection, in vivo tracking, and ablation of engineered cells. Blood. 2011;118(5):1255–63.
Ma F, Ho JY, Du H, Xuan F, Wu X, Wang Q, et al. Evidence of long-lasting anti-CD19 activity of engrafted CD19 chimeric antigen receptor-modified T cells in a phase I study targeting pediatrics with acute lymphoblastic leukemia. Hematol Oncol. 2019;37(5):601–8.
Chang H, Salma F, Yi Q-l, Patterson B, Brien B, Minden MD. Prognostic relevance of immunophenotyping in 379 patients with acute myeloid leukemia. Leuk Res. 2004;28(1):43–8.
Ho JY, Wang L, Liu Y, Ba M, Yang J, Zhang X, et al. Promoter usage regulating the surface density of CAR molecules may modulate the kinetics of CAR-T cells in vivo. Mol Ther Methods Clin Dev. 2021;21:237–46.
Peipp M, Küpers H, Saul D, Schlierf B, Greil J, Zunino SJ, et al. A recombinant CD7-specific single-chain immunotoxin is a potent inducer of apoptosis in acute leukemic T cells. Cancer Res. 2002;62(10):2848–55.
Fraietta JA, Lacey SF, Orlando EJ, Pruteanu-Malinici I, Gohil M, Lundh S, et al. Determinants of response and resistance to CD19 chimeric antigen receptor (CAR) T cell therapy of chronic lymphocytic leukemia. Nat Med. 2018;24(5):563–71.
Louis CU, Savoldo B, Dotti G, Pule M, Yvon E, Myers GD, et al. Antitumor activity and long-term fate of chimeric antigen receptor-positive T cells in patients with neuroblastoma. Blood. 2011;118(23):6050–6.
Kawalekar OU, O'Connor RS, Fraietta JA, Guo L, McGettigan SE, Posey AD Jr, et al. Distinct Signaling of Coreceptors Regulates Specific Metabolism Pathways and Impacts Memory Development in CAR T Cells. Immunity. 2016;44(2):380–90.
Larbi A, Fulop T. From "truly naive" to "exhausted senescent" T cells: when markers predict functionality. Cytometry A. 2014;85(1):25–35.
Daver N, Alotaibi AS, Bucklein V, Subklewe M. T-cell-based immunotherapy of acute myeloid leukemia: current concepts and future developments. Leukemia. 2021;35(7):1843–63.
Schuster SJ, Bishop MR, Tam CS, Waller EK, Borchmann P, McGuirk JP, et al. Tisagenlecleucel in Adult Relapsed or Refractory Diffuse Large B-Cell Lymphoma. N Engl J Med. 2019;380(1):45–56.
Budde L, Song JY, Kim Y, Blanchard S, Wagner J, Stein AS, et al. Remissions of Acute Myeloid Leukemia and Blastic Plasmacytoid Dendritic Cell Neoplasm Following Treatment with CD123-Specific CAR T Cells: A First-in-Human Clinical Trial. Blood. 2017;130(Supplement 1):811-.

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Reviewers agreed at journal
05 Oct, 2022
Reviewers invited by journal
04 Oct, 2022
Editor assigned by journal
01 Oct, 2022
First submitted to journal
28 Sep, 2022

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Naturally Selected CD7 CAR-T Therapy without Genetic editing Exerts Significant Antitumor Efficacy against Relapsed and Refractory Acute Myeloid Leukemia(R/R-AML)

Status:

Version 1

Abstract

Background

Methods

Results

Conclusion

Figures

Background

Materials And Methods

Immunohistochemistry

CD7 and AML-MRD Assays

CD7 CAR-T Cells Design and Generation

Flow Cytometry

Cytotoxicity Assay

Mouse Xenograft Model

Statistical Analysis

Results

Discussion

Abbreviations

Declarations

References

Status:

Version 1