ObjectivesAutologous chimeric antigen receptor (CAR) αβ T‐cell therapies have demonstrated remarkable antitumor efficacy in patients with haematological malignancies; however, not all eligible cancer patients receive clinical benefit. Emerging strategies to improve patient access and clinical responses include using premanufactured products from healthy donors and alternative cytotoxic effectors possessing intrinsic tumoricidal activity as sources of CAR cell therapies. γδ T cells, which combine innate and adaptive mechanisms to recognise and kill malignant cells, are an attractive candidate platform for allogeneic CAR T‐cell therapy. Here, we evaluated the manufacturability and functionality of allogeneic peripheral blood‐derived CAR+ Vδ1 γδ T cells expressing a second‐generation CAR targeting the B‐cell‐restricted CD20 antigen.MethodsDonor‐derived Vδ1 γδ T cells from peripheral blood were ex vivo‐activated, expanded and engineered to express a novel anti‐CD20 CAR. In vitro and in vivo assays were used to evaluate CAR‐dependent and CAR‐independent antitumor activities of CD20 CAR+ Vδ1 γδ T cells against B‐cell tumors.ResultsAnti‐CD20 CAR+ Vδ1 γδ T cells exhibited innate and adaptive antitumor activities, such as in vitro tumor cell killing and proinflammatory cytokine production, in addition to in vivo tumor growth inhibition of B‐cell lymphoma xenografts in immunodeficient mice. Furthermore, CD20 CAR+ Vδ1 γδ T cells did not induce xenogeneic graft‐versus‐host disease in immunodeficient mice.ConclusionThese preclinical data support the clinical evaluation of ADI‐001, an allogeneic CD20 CAR+ Vδ1 γδ T cell, and a phase 1 study has been initiated in patients with B‐cell malignancies (NCT04735471).
Objective-Megakaryocytic cells (Mks) undergo endomitosis and become polyploid. Mk ploidy correlates with platelet production. We previously showed that nicotinamide (NIC) greatly increases Mk ploidy in cultures of human mobilized peripheral blood (mPB) CD34 + cells. This study aims to examine the generality of NIC effects, NIC's impact on Mk ultrastructure, and potential mechanisms for the increased ploidy.Methods-We used electron microscopy to examine Mk ultrastructure and flow cytometry to evaluate NIC effects on Mk differentiation and ploidy in mPB CD34 + cell cultures under diverse megakaryopoietic conditions. Mk ploidy and NAD(H) content were evaluated for NIC and other NAD + precursors. We tested additional inhibitors of the SIRT1 and SIRT2 histone/protein deacetylases and, after treatment with NIC, evaluated changes in the acetylation of SIRT1/2 targets.Results-NIC increased ploidy under diverse culture conditions and did not alter Mk ultrastructure. 6.25 mM NIC increased NAD + levels 5-fold. Quinolinic acid increased NAD + similar to that for 1 mM NIC, but yielded a much smaller ploidy increase. Similar increases in Mk ploidy were obtained using NIC or the SIRT1/2 inhibitor cambinol, while the SIRT2 inhibitor AGK2 moderately increased ploidy. SIRT1/2 inhibition in cells treated with NIC was evidenced by increased acetylation of nucleosomes and p53. Greater p53 acetylation with NIC was associated with increased binding of p53 to its consensus DNA binding sequence. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conflict of Interest DisclosureNo financial interest/relationships with financial interest relating to the topic of this article have been declared. Nicotinamide has diverse functions in cells. NIC inhibits the activity of the silent information regulator 2 (Sir2) family of histone/protein deacetylases (sirtuins or SIRTs) [9][10][11][12]. Sirtuins catalyze a unique NAD + -dependent deacetylation reaction and are important for a wide variety of biological processes including transcriptional silencing, lifespan regulation, and the regulation of apoptosis [13][14][15]. NIC is also a precursor of NAD + via the salvage pathway [16,17]. NAD + regulates a variety of intracellular activities such as the DNA-binding specificity of p53 [18,19]. We have previously established that the p53-mediated Mk apoptotic program is intimately linked with terminal maturation and polyploidization, possibly through the downstream effects of p53 on MDM2 and BCL2 expression [20,21]. NIH Public AccessThe aim of this study was to examine the generality of the NIC effects, NIC's ...
The current standard of care for acute myeloid leukemia (AML) is largely ineffective with very high relapse rates and low survival rates, mostly due to the inability to eliminate a rare population of leukemic stem cells (LSCs) that initiate tumor growth and are resistant to standard chemotherapy. RNA-sequencing analysis on isolated LSCs confirmed C-type lectin domain family 12 member A (CLL1, also known as CLEC12A) to be highly expressed on LSCs but not on normal hematopoietic stem cells (HSCs) or other healthy organ tissues. Expression of CLL1 was consistent across different types of AML. We developed CLT030 (CLL1-ADC), an antibody-drug conjugate (ADC) based on a humanized anti-CLL1 antibody with 2 engineered cysteine residues linked covalently via a cleavable linker to a highly potent DNA-binding payload, thus resulting in a site-specific and homogenous ADC product. The ADC is designed to be stable in the bloodstream and to release its DNA-binding payload only after the ADC binds to CLL1-expressing tumor cells, is internalized, and the linker is cleaved in the lysosomal compartment. CLL1-ADC inhibits in vitro LSC colony formation and demonstrates robust in vivo efficacy in AML cell tumor models and tumor growth inhibition in the AML patient-derived xenograft model. CLL1-ADC demonstrated a reduced effect on differentiation of healthy normal human CD34 cells to various lineages as observed in an in vitro colony formation assay and in an in vivo xenotransplantation model as compared with CD33-ADC. These results demonstrate that CLL1-ADC could be an effective ADC therapeutic for the treatment of AML.
Background-Megakaryopoiesis encompasses hematopoietic stem and progenitor cell (HSPC) commitment to the megakaryocytic cell (Mk) lineage, expansion of Mk progenitors and mature Mks, polyploidization, and platelet release. pH and pO 2 increase from the endosteum to sinuses, and different cytokines are important for various stages of differentiation. We hypothesized that mimicking the changing conditions during Mk differentiation in the bone marrow would facilitate expansion of progenitors that could generate many high-ploidy Mks.
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