CD44v6-targeted CAR T-cells specifically eliminate CD44 isoform 6 expressing head/neck squamous cell carcinoma cells

Haist, C; Schulte, Elena; Bartels, Nina; Bister, A; Poschinski, Zoe; Ibach, Tabea C.; Geipel, Katja; Wiek, Constanze; Wagenmann, Martin; Monzel, Cornelia; Scheckenbach, Kathrin; Hanenberg, Helmut

doi:10.1016/j.oraloncology.2021.105259

Cited by 27 publications

(37 citation statements)

References 60 publications

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“…Negative controls in these assays were non-transduced T-cells as well as CD19 CAR T-cells. We initially confirmed that our flow cytometry-based (26) and our 96-well plate cytotoxicity assay (29) provided similar results (Supplementary Figure S2A). As the plate cytotoxicity assay facilitated a much higher throughput of samples, all consecutive cytotoxicity assays were analyzed with this methodology.…”

Section: Primary Human T-cells Expressing Egfr and Cd44v6 Cars Effectively Kill Uccsupporting

confidence: 74%

“…To generate CAR T-cells, we employed previously established CARs against EGFR, CD44v6, and CD19 ( 29 ) (Haist et al submitted). The standard lentiviral vectors ( 26 ) expressed second-generation CARs containing the CD8 leader peptide, the single-chain variable fragments (scFvs) of a EGFR-, CD44v6-, or CD19-specific monoclonal antibody, an extracellular hinge region using 99 amino acids from human CD34 ( 29 ), the CD28 transmembrane and co-stimulatory domains as well as the CD3ζ signaling domain ( Figure 2A ) . Three days after transduction, CAR-expressing T-cells were purified.…”

Section: Resultsmentioning

confidence: 99%

“…As the search for ideal target antigens to achieve specific antitumor activity still is a major challenge for solid cancers, we evaluated well-established surface molecules, EGFR and CD44v6, as possible target antigens for CAR T-cell therapy of UC. Both antigens are overexpressed on solid cancers ( 17 , 19 ), and we already had constructed second-generation CARs in lentiviral vectors that efficiently killed human head and neck squamous cell carcinomas ( 29 ) (Haist et al submitted). Using a set of UCC we demonstrated that EGFR was robustly expressed on all malignant UCC and that the expression levels were higher compared to non-malignant uroepithelial HBLAK cells.…”

Section: Discussionmentioning

confidence: 99%

“…After prestimulation on immobilized CD3/CD28 monoclonal antibodies, T-cells were transduced with lentiviral particles on the recombinant fibronectin fragment Retronectin ® as described ( 26 , 32 ). Subsequently, T-cells were cultured in medium containing 100 IU/ml IL-2 for 72 h. To obtain >98% pure CAR T-cells, transduced T-cells were incubated with magnetic microbeads coupled to the CD34 QBEND10 antibody (Miltenyi Biotec) ( Supplementary Table S1 ), which recognizes a 99 amino acid sequence that we have included as novel hinge domain in our CARs ( 29 ) (Bister et al, in press; patent EP3293199). Subsequently, CD34 microbead-stained T-cells were purified on MACS MS columns according to the manufacturer’s instructions (Miltenyi Biotec).…”

Section: Methodsmentioning

confidence: 99%

“…The CD19 and CD44v6 CAR constructs were described previously (26)(27)(28)(29). The EGFR CAR lentiviral vector contains optimized sequences for the heavy-chain and light-chain variable region segments derived from the monoclonal antibody of Cetuximab (30).…”

Section: Generation Of Lentiviral Constructs and T-cell Transduction Selection And Functional Assaysmentioning

confidence: 99%

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Epigenetic Priming of Bladder Cancer Cells With Decitabine Increases Cytotoxicity of Human EGFR and CD44v6 CAR Engineered T-Cells

et al. 2021

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BackgroundTreatment of B-cell malignancies with CD19-directed chimeric antigen receptor (CAR) T-cells marked a new era in immunotherapy, which yet has to be successfully adopted to solid cancers. Epigenetic inhibitors of DNA methyltransferases (DNMTi) and histone deacetylases (HDACi) can induce broad changes in gene expression of malignant cells, thus making these inhibitors interesting combination partners for immunotherapeutic approaches.MethodsUrothelial carcinoma cell lines (UCC) and benign uroepithelial HBLAK cells pretreated with the DNMTi decitabine or the HDACi romidepsin were co-incubated with CAR T-cells directed against EGFR or CD44v6, and subsequent cytotoxicity assays were performed. Effects on T-cell cytotoxicity and surface antigen expression on UCC were determined by flow cytometry. We also performed next-generation mRNA sequencing of inhibitor-treated UCC and siRNA-mediated knockdown of potential regulators of CAR T-cell killing.ResultsExposure to decitabine but not romidepsin enhanced CAR T-cell cytotoxicity towards all UCC lines, but not towards the benign HBLAK cells. Increased killing could neither be attributed to enhanced target antigen expression (EGFR and CD44v6) nor fully explained by changes in the T-cell ligands PD-L1, PD-L2, ICAM-1, or CD95. Instead, gene expression analysis suggested that regulators of cell survival and apoptosis were differentially induced by the treatment. Decitabine altered the balance between survival and apoptosis factors towards an apoptosis-sensitive state associated with increased CAR T-cell killing, while romidepsin, at least partially, tilted this balance in the opposite direction. Knockdown experiments with siRNA in UCC confirmed BID and BCL2L1/BCLX as two key factors for the altered susceptibility of the UCC.ConclusionOur data suggest that the combination of decitabine with CAR T-cell therapy is an attractive novel therapeutic approach to enhance tumor-specific killing of bladder cancer. Since BID and BCL2L1 are essential determinants for the susceptibility of a wide variety of malignant cells, their targeting might be additionally suitable for combination with immunotherapies, e.g., CAR T-cells or checkpoint inhibitors in other malignancies.

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Section: Primary Human T-cells Expressing Egfr and Cd44v6 Cars Effectively Kill Uccsupporting

confidence: 74%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Generation Of Lentiviral Constructs and T-cell Transduction Selection And Functional Assaysmentioning

confidence: 99%

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Epigenetic Priming of Bladder Cancer Cells With Decitabine Increases Cytotoxicity of Human EGFR and CD44v6 CAR Engineered T-Cells

et al. 2021

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CD44‐targeted chimeric antigen receptor‐T eradicates acute myeloid leukaemic stem cells

Shahoumi

2022

Clinical and Translational Dis

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Acute myeloid leukaemia (AML) is the most common, aggressive hematologic malignancy of adults in the United States. Globally, leukaemia is estimated to be the 11th most frequent cancer-related mortality, accounting for 311 594 deaths. 1 More than 20 000 new cases of AML and approximately 11 000 AML-related deaths are estimated to occur in the United States. 2 Although different therapeutic strategies and novel drug combinations have led to higher response rates and improved survival rates in AML patients, relapse remains a major clinical challenge. A considerable body of evidence suggests that disease initiation and progression, therapeutic resistance and relapse of AML are mainly driven by leukaemia stem cells, which are characterized by their unlimited capacity for self-renewal, tumour initiation and differentiation. [3][4][5] In addition, AML patients with DNMT3A and FLT3 mutations have significantly higher recurrence rates and worse survival, and these mutations appear to confer a poor prognosis. 6,7 Despite significant progress in the development of newer FLT3 inhibitors with greater potency and specificity, drug toxicity and the emergence of resistance pose significant challenges. 8 Thus, there is a critical need for therapeutic strategies that decrease the risk of relapse and improve the survival of AML patients. As an alternative to these inhibitors, more efficient and less harmful immunotherapy-based approaches such as adoptive transferring T cell therapy are in development for the treatment of AML. Chimeric antigen receptor (CAR)-T cells areThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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CD44v6 chimeric antigen receptor T cell specificity towards AML with FLT3 or DNMT3A mutations

Tang

Huang

Tang

et al. 2022

Clinical & Translational Med

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Background Chimeric antigen receptor T‐cell (CAR‐T) therapy for acute myeloid leukaemia (AML) has thus far been elusive, in part due to target restriction and phenotypic heterogeneity of AML cells. Mutations of the FMS‐like tyrosine kinase 3 (FLT3) and DNA methyltransferase 3A (DNMT3A) genes are common driver mutations that present with a poor prognosis in AML patients. We found that AML patients with FLT3 or DNMT3A mutations had higher expression of CD44 isoform 6 (CD44v6) compared to normal specimens. Therefore, we intended to demonstrate CD44v6 could be a specific option for AML with FLT3 or DNMT3A mutations. Methods Internal tandem duplication (ITD) mutations of FLT3 (FLT3/ITD) knock‐in clone and DNMT3A‐R882H mutant clones of SKM‐1 cells were generated using CRISPR/Cas9 and lentiviral transfection, respectively. CD44v6 CAR‐T cells were constructed by transfecting T cells with lentivirus containing CD44v6 CAR. CD44v6 expression in AML cell lines, AML patients and healthy donors was evaluated by flow cytometry. DNA methylation assays were used to analyse the mechanisms of FLT3 and DNMT3A mutations affecting CD44v6 expression. Results Aberrant overexpression of CD44v6 was observed in AML cell lines with FLT3 or DNMT3A mutations compared to the wild‐type SKM‐1 or K562 cells. AML patients with FLT3 or DNMT3A mutations had higher expression of CD44v6 compared to normal specimens. Then we constructed CD44v6 CAR‐T cells and found that CD44v6 CAR‐T specifically lysed CD44v6+ cells, accompanied by cytokines release. No significant killing effect was observed from CD44v6‐ AML cells and normal cells after co‐culture with CD44v6 CAR‐T. These results were also observed in vivo. Furthermore, we found that FLT3 or DNMT3A mutations induced CD44v6 overexpression by downregulating the CpG methylation of CD44 promoter. Conclusions Collectively, CD44v6 is a promising target of CAR‐T for AML patients with FLT3 or DNMT3A mutations.

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CD44v6-targeted CAR T-cells specifically eliminate CD44 isoform 6 expressing head/neck squamous cell carcinoma cells

Cited by 27 publications

References 60 publications

Epigenetic Priming of Bladder Cancer Cells With Decitabine Increases Cytotoxicity of Human EGFR and CD44v6 CAR Engineered T-Cells

Epigenetic Priming of Bladder Cancer Cells With Decitabine Increases Cytotoxicity of Human EGFR and CD44v6 CAR Engineered T-Cells

CD44‐targeted chimeric antigen receptor‐T eradicates acute myeloid leukaemic stem cells

CD44v6 chimeric antigen receptor T cell specificity towards AML with FLT3 or DNMT3A mutations

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