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CD70-specific CAR T cells have potent activity against acute myeloid leukemia without HSC toxicity.

ABSTRACT: The prognosis of patients with acute myeloid leukemia (AML) remains dismal, highlighting the need for novel innovative treatment strategies. The application of chimeric antigen receptor (CAR) T-cell therapy to patients with AML has been limited, in particular by the lack of a tumor-specific target antigen. CD70 is a promising antigen to target AML, as it is expressed on most leukemic blasts, whereas little or no expression is detectable in normal bone marrow samples. To target CD70 on AML cells, we generated a panel of CD70-CAR T cells that contained a common single-chain variable fragment (scFv) for antigen detection, but differed in size and flexibility of the extracellular spacer and in the transmembrane and the costimulatory domains. These CD70scFv CAR T cells were compared with a CAR construct that contained human CD27, the ligand of CD70 fused to the CD3ζ chain (CD27z). The structural composition of the CAR strongly influenced expression levels, viability, expansion, and cytotoxic capacities of CD70scFv-based CAR T cells, but CD27z-CAR T cells demonstrated superior proliferation and antitumor activity in vitro and in vivo, compared with all CD70scFv-CAR T cells. Although CD70-CAR T cells recognized activated virus-specific T cells (VSTs) that expressed CD70, they did not prevent colony formation by normal hematopoietic stem cells. Thus, CD70-targeted immunotherapy is a promising new treatment strategy for patients with CD70-positive AML that does not affect normal hematopoiesis but will require monitoring of virus-specific T-cell responses.

SUBMITTER: Sauer T

PROVIDER: S-EPMC8323977 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Preclinical evaluation of CD70-specific CAR T cells targeting acute myeloid leukemia.

Project description:BackgroundsChimeric antigen receptor (CAR)-T cell therapy has achieved unprecedented success in treating hematopoietic malignancies. However, this cell therapy is hampered in treating acute myeloid leukemia (AML) due to lack of ideal cell surface targets that only express on AML blasts and leukemia stem cells (LSCs) but not on normal hematopoietic stem cells (HSCs).MethodsWe detected the CD70 expression on the surfaces of AML cell lines, primary AML cells, HSC, and peripheral blood cells and generated a second-generation CD70-specific CAR-T cells using a construct containing a humanized 41D12-based scFv and a 41BB-CD3ζ intracellular signaling domain. Cytotoxicity, cytokine release, and proliferation in antigen stimulation, CD107a assay, and CFSE assays were used to demonstrate the potent anti-leukemia activity in vitro. A Molm-13 xenograft mouse model was established to evaluate the anti-leukemic activity of CD70 CAR-T in vivo. CFU assay was explored to assess the safety of CD70 CAR-T on HSC.ResultsCD70 heterogeneously expressed on AML primary cells, including leukemia blasts, leukemic progenitor, and stem cells, but not expressed on normal HSCs and majority of blood cells. Anti-CD70 CAR-T cells exhibited potent cytotoxicity, cytokines production, and proliferation when incubated with CD70+ AML cell lines. It also displayed robust anti-leukemia activity and prolonged survival in Molm-13 xenograft mouse model. However, such CAR-T cell therapy did not completely eliminate leukemia in vivo.DiscussionOur study reveals that anti-CD70 CAR-T cells are a new potential treatment for AML. However, such CAR-T cell therapy did not completely eliminate leukemia in vivo, suggesting that future studies aiming to generate innovative combinatorial CAR constructs or to increase CD70 expression density on leukemia cell surface to prolong the life-span of CAR-T cells in the circulation will be needed in order to optimize CAR-T cell responses for AML.

| S-EPMC9950117 | biostudies-literature

High-affinity FR?-specific CAR T cells eradicate AML and normal myeloid lineage without HSC toxicity.

Project description:Acute myeloid leukemia (AML) is an aggressive malignancy, and development of new treatments to prolong remissions is warranted. Chimeric antigen receptor (CAR) T-cell therapies appear promising but on-target, off-tumor recognition of antigen in healthy tissues remains a concern. Here we isolated a high-affinity (HA) folate receptor beta (FR?)-specific single-chain variable fragment (2.48?nm KD) for optimization of FR?-redirected CAR T-cell therapy for AML. T cells stably expressing the HA-FR? CAR exhibited greatly enhanced antitumor activity against FR?(+) AML in vitro and in vivo compared with a low-affinity FR? CAR (54.3?nm KD). Using the HA-FR? immunoglobulin G, FR? expression was detectable in myeloid-lineage hematopoietic cells; however, expression in CD34(+) hematopoietic stem cells (HSCs) was nearly undetectable. Accordingly, HA-FR? CAR T cells lysed mature CD14(+) monocytes, while HSC colony formation was unaffected. Because of the potential for elimination of mature myeloid lineage, mRNA CAR electroporation for transient CAR expression was evaluated. mRNA-electroporated HA-FR? CAR T cells retained effective antitumor activity in vitro and in vivo. Together, our results highlight the importance of antibody affinity in target protein detection and CAR development and suggest that transient delivery of potent HA-FR? CAR T cells is highly effective against AML and reduces the risk for long-term myeloid toxicity.

| S-EPMC4889499 | biostudies-literature

STING Pathway Activation Stimulates Potent Immunity against Acute Myeloid Leukemia.

Project description:Type I interferon (IFN), essential for spontaneous T cell priming against solid tumors, is generated through recognition of tumor DNA by STING. Interestingly, we observe that type I IFN is not elicited in animals with disseminated acute myeloid leukemia (AML). Further, survival of leukemia-bearing animals is not diminished in the absence of type I IFN signaling, suggesting that STING may not be triggered by AML. However, the STING agonist, DMXAA, induces expression of IFN-? and other inflammatory cytokines, promotes dendritic cell (DC) maturation, and results in the striking expansion of leukemia-specific T cells. Systemic DMXAA administration significantly extends survival in two AML models. The therapeutic effect of DMXAA is only partially dependent on host type I IFN signaling, suggesting that other cytokines are important. A synthetic cyclic dinucleotide that also activates human STING provided a similar anti-leukemic effect. These data demonstrate that STING is a promising immunotherapeutic target in AML.

| S-EPMC5116809 | biostudies-literature

Memory-like NK cells armed with a neoepitope-specific CAR exhibit potent activity against NPM1 mutated acute myeloid leukemia.

Project description:Acute myeloid leukemia (AML) remains a therapeutic challenge, and a paucity of tumor-specific targets has significantly hampered the development of effective immune-based therapies. Recent paradigm-changing studies have shown that natural killer (NK) cells exhibit innate memory upon brief activation with IL-12 and IL-18, leading to cytokine-induced memory-like (CIML) NK cell differentiation. CIML NK cells have enhanced antitumor activity and have shown promising results in early phase clinical trials in patients with relapsed/refractory AML. Here, we show that arming CIML NK cells with a neoepitope-specific chimeric antigen receptor (CAR) significantly enhances their antitumor responses to nucleophosphmin-1 (NPM1)-mutated AML while avoiding off-target toxicity. CIML NK cells differentiated from peripheral blood NK cells were efficiently transduced to express a TCR-like CAR that specifically recognizes a neoepitope derived from the cytosolic oncogenic NPM1-mutated protein presented by HLA-A2. These CAR CIML NK cells displayed enhanced activity against NPM1-mutated AML cell lines and patient-derived leukemic blast cells. CAR CIML NK cells persisted in vivo and significantly improved AML outcomes in xenograft models. Single-cell RNA sequencing and mass cytometry analyses identified up-regulation of cell proliferation, protein folding, immune responses, and major metabolic pathways in CAR-transduced CIML NK cells, resulting in tumor-specific, CAR-dependent activation and function in response to AML target cells. Thus, efficient arming of CIML NK cells with an NPM1-mutation-specific TCR-like CAR substantially improves their innate antitumor responses against an otherwise intracellular mutant protein. These preclinical findings justify evaluating this approach in clinical trials in HLA-A2+ AML patients with NPM1c mutations.

| S-EPMC9231490 | biostudies-literature

Preclinical development and evaluation of nanobody-based CD70-specific CAR T cells for the treatment of acute myeloid leukemia.

Project description:BackgroundAcute myeloid leukemia (AML) treatment remains challenging. CD70 was reported as a promising AML-specific antigen. Preclinically, CAR T-cell with single-chain-variable fragment (scFv) or truncated CD27 targeting CD70 has been reported to treat AML. However, various disadvantages including spontaneous exhaustion, proteinase-mediated loss of functional receptors, and high immunogenicity, limited its further application to clinical settings. Alternatively, the single-variable domain on heavy chain (VHH), also known as nanobodies, with comparable binding ability and specificity, provides an optional solution.MethodWe generated CD70 knocked-out novel nanobody-based anti-CD70-CAR T-cells (nb70CAR-T) with two different VHHs for antigen detection. Next, we detected the CD70 expression on primary AML blasts by flow cytometry and associated the efficacy of nb70CAR-T with the target antigen density. Finally, epigenetic modulators were investigated to regulate the CD70 expression on AML cells to promote the functionality of nb70CAR-T.ResultsOur nb70CAR-T exhibited expected tumoricidal functionality against CD70-expressed cell lines and primary AML blasts. However, CD70 expression in primary AML blasts was not consistently high and nb70CAR-T potently respond to an estimated 40.4% of AML patients when the CD70 expression level was over a threshold of 1.6 (MFI ratio). Epigenetic modulators, Decitabine and Chidamide can up-regulate CD70 expression on AML cells, enhancing the treatment efficacy of nb70CAR-T.ConclusionCD70 expression in AML blasts was not fully supportive of its role in AML targeted therapy as reported. The combinational use of Chidamide and Decitabine with nb70CAR-T could provide a new potential for the treatment of AML.

| S-EPMC10264288 | biostudies-literature

Demethylating therapy increases cytotoxicity of CD44v6 CAR-T cells against acute myeloid leukemia.

Project description:BackgroundCD44v6 chimeric antigen receptor T (CD44v6 CAR-T) cells demonstrate strong anti-tumor ability and safety in acute myeloid leukemia (AML). However, the expression of CD44v6 on T cells leads to transient fratricide and exhaustion of CD44v6 CAR-T cells, which affect the application of CD44v6 CAR-T. The exhaustion and function of T cells and CD44v6 expression of AML cells are associated with DNA methylation. Hypomethylating agents (HAMs) decitabine (Dec) and azacitidine (Aza) have been widely used to treat AML. Therefore, there may be synergy between CD44v6 CAR-T cells and HAMs in the treatment of AML.MethodsCD44v6 CAR-T cells pretreated with Dec or Aza were co-cultured with CD44v6+ AML cells. Dec or aza pretreated AML cells were co-cultured with CD44v6 CAR-T cells. The cytotoxicity, exhaustion, differentiation and transduction efficiency of CAR-T cells, and CD44v6 expression and apoptosis in AML cells were detected by flow cytometry. The subcutaneous tumor models were used to evaluate the anti-tumor effect of CD44v6 CAR-T cells combined with Dec in vivo. The effects of Dec or Aza on gene expression profile of CD44v6 CAR-T cells were analyzed by RNA-seq.ResultsOur results revealed that Dec and Aza improved the function of CD44v6 CAR-T cells through increasing the absolute output of CAR+ cells and persistence, promoting activation and memory phenotype of CD44v6 CAR-T cells, and Dec had a more pronounced effect. Dec and Aza promoted the apoptosis of AML cells, particularly with DNA methyltransferase 3A (DNMT3A) mutation. Dec and Aza also enhanced the CD44v6 CAR-T response to AML by upregulating CD44v6 expression of AML cells regardless of FMS-like tyrosine kinase 3 (FLT3) or DNMT3A mutations. The combination of Dec or Aza pretreated CD44v6 CAR-T with pretreated AML cells demonstrated the most potent anti-tumor ability against AML.ConclusionDec or Aza in combination with CD44v6 CAR-T cells is a promising combination therapy for AML patients.

| S-EPMC10174291 | biostudies-literature

Demethylating therapy increases anti-CD123 CAR T cell cytotoxicity against acute myeloid leukemia.

Project description:Successful treatment of acute myeloid leukemia (AML) with chimeric antigen receptor (CAR) T cells is hampered by toxicity on normal hematopoietic progenitor cells and low CAR T cell persistence. Here, we develop third-generation anti-CD123 CAR T cells with a humanized CSL362-based ScFv and a CD28-OX40-CD3ζ intracellular signaling domain. This CAR demonstrates anti-AML activity without affecting the healthy hematopoietic system, or causing epithelial tissue damage in a xenograft model. CD123 expression on leukemia cells increases upon 5'-Azacitidine (AZA) treatment. AZA treatment of leukemia-bearing mice causes an increase in CTLA-4negative anti-CD123 CAR T cell numbers following infusion. Functionally, the CTLA-4negative anti-CD123 CAR T cells exhibit superior cytotoxicity against AML cells, accompanied by higher TNFα production and enhanced downstream phosphorylation of key T cell activation molecules. Our findings indicate that AZA increases the immunogenicity of AML cells, enhancing recognition and elimination of malignant cells by highly efficient CTLA-4negative anti-CD123 CAR T cells.

| S-EPMC8575966 | biostudies-literature

GPC2-CAR T cells tuned for low antigen density mediate potent activity against neuroblastoma without toxicity.

Project description:Pediatric cancers often mimic fetal tissues and express proteins normally silenced postnatally that could serve as immune targets. We developed T cells expressing chimeric antigen receptors (CARs) targeting glypican-2 (GPC2), a fetal antigen expressed on neuroblastoma (NB) and several other solid tumors. CARs engineered using standard designs control NBs with transgenic GPC2 overexpression, but not those expressing clinically relevant GPC2 site density (∼5,000 molecules/cell, range 1-6 × 103). Iterative engineering of transmembrane (TM) and co-stimulatory domains plus overexpression of c-Jun lowered the GPC2-CAR antigen density threshold, enabling potent and durable eradication of NBs expressing clinically relevant GPC2 antigen density, without toxicity. These studies highlight the critical interplay between CAR design and antigen density threshold, demonstrate potent efficacy and safety of a lead GPC2-CAR candidate suitable for clinical testing, and credential oncofetal antigens as a promising class of targets for CAR T cell therapy of solid tumors.

| S-EPMC9092726 | biostudies-literature

HSC commitment-associated epigenetic signature is prognostic in acute myeloid leukemia.

Project description:Acute myeloid leukemia (AML) is characterized by disruption of HSC and progenitor cell differentiation. Frequently, AML is associated with mutations in genes encoding epigenetic modifiers. We hypothesized that analysis of alterations in DNA methylation patterns during healthy HSC commitment and differentiation would yield epigenetic signatures that could be used to identify stage-specific prognostic subgroups of AML. We performed a nano HpaII-tiny-fragment-enrichment-by-ligation-mediated-PCR (nanoHELP) assay to compare genome-wide cytosine methylation profiles between highly purified human long-term HSC, short-term HSC, common myeloid progenitors, and megakaryocyte-erythrocyte progenitors. We observed that the most striking epigenetic changes occurred during the commitment of short-term HSC to common myeloid progenitors and these alterations were predominantly characterized by loss of methylation. We developed a metric of the HSC commitment–associated methylation pattern that proved to be highly prognostic of overall survival in 3 independent large AML patient cohorts, regardless of patient treatment and epigenetic mutations. Application of the epigenetic signature metric for AML prognosis was superior to evaluation of commitment-based gene expression signatures. Together, our data define a stem cell commitment–associated methylome that is independently prognostic of poorer overall survival in AML.

| S-EPMC3934187 | biostudies-literature

Tandem CAR-T cells targeting CD70 and B7-H3 exhibit potent preclinical activity against multiple solid tumors.

Project description:Purpose: Given that heterogeneous expression and variants of antigens on solid tumors are responsible for relapse after chimeric antigen receptor (CAR)-T cell therapy, we hypothesized that combinatorial targeting two tumor-associated antigens would lessen this problem and enhance the antitumor activity of T cells. Methods: The co-expression level of CD70 and B7-H3 was analyzed in multiple tumor tissue samples. Further, two putative antigens were identified in The Cancer Genome Atlas and Gene Expression Profiling Interactive Analysis database. Two CD70 targeted CARs with different antigen binding domain, truncated CD27 and CD70 specific single-chain antibody fragment (scFv), were designed to screen a more suitable target-antigen binding moiety. Accordingly, we designed a bivalent tandem CAR (TanCAR) and further assessed the anti-tumor efficacy of TanCAR-T cells in vitro and in vivo. Results: Our results indicated that co-expression of CD70 and B7-H3 was observed on multiple tumor types including kidney, breast, esophageal, liver, colon cancer, glioma as well as melanoma. The CD70 targeted CAR-T cells with binding moiety of CD70 specific scFv exhibit a higher affinity and antitumor effect against CD70+ tumor cells. TanCAR-T cells induced enhanced ability of cytolysis and cytokine release over unispecific CAR-T cells when encountering tumor cells expressing two target-antigens. Further, low doses of TanCAR-T cells could also effectively control the lung cancer and melanoma xenografts and improved overall survival of the treated animals. Conclusion: TanCAR-T cells targeting CD70 and B7-H3 exhibit enhanced antitumor functionality and improve the problem of antigenic heterogeneity and variant in the treatment against solid tumor and melanoma.

| S-EPMC7359081 | biostudies-literature

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