Targeting the mevalonate or Wnt pathways to overcome CAR T-cell resistance in TP53-mutant AML cells.

TP53-mutant acute myeloid leukemia (AML) and myelodysplastic neoplasms (MDS) are characterized by chemotherapy resistance and represent an unmet clinical need. Chimeric antigen receptor (CAR) T-cells might be a promising therapeutic option for TP53-mutant AML/MDS. However, the impact of TP53 deficiency in AML cells on the efficacy of CAR T-cells is unknown. We here show that CAR T-cells engaging TP53-deficient leukemia cells exhibit a prolonged interaction time, upregulate exhaustion markers, and are inefficient to control AML cell outgrowth in vitro and in vivo compared to TP53 wild-type cells. Transcriptional profiling revealed that the mevalonate pathway is upregulated in TP53-deficient AML cells under CAR T-cell attack, while CAR T-cells engaging TP53-deficient AML cells downregulate the Wnt pathway. In vitro rational targeting of either of these pathways rescues AML cell sensitivity to CAR T-cell-mediated killing. We thus demonstrate that TP53 deficiency confers resistance to CAR T-cell therapy and identify the mevalonate pathway as a therapeutic vulnerability of TP53-deficient AML cells engaged by CAR T-cells, and the Wnt pathway as a promising CAR T-cell therapy-enhancing approach for TP53-deficient AML/MDS.

Twisted: Escape of epitope-edited healthy cells from immune attack.

Hematopoietic stem and progenitor cell-derived neoplasia is challenging to target by cell surface-directed immunotherapy due to lack of tumor cell-specific antigen identification. Marone et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20231235) provide a solution by target-epitope resistance editing in healthy hematopoietic stem cells.

Erratum: Anti-CD117 CAR T cells incorporating a safety switch eradicate human acute myeloid leukemia and hematopoietic stem cells.

[This corrects the article DOI: 10.1016/j.omto.2023.07.003.].

Anti-CD117 CAR T cells incorporating a safety switch eradicate human acute myeloid leukemia and hematopoietic stem cells.

Discrimination between hematopoietic stem cells and leukemic stem cells remains a major challenge for acute myeloid leukemia immunotherapy. CAR T cells specific for the CD117 antigen can deplete malignant and healthy hematopoietic stem cells before consolidation with allogeneic hematopoietic stem cell transplantation in absence of cytotoxic conditioning. Here we exploit non-viral technology to achieve early termination of CAR T cell activity to prevent incoming graft rejection. Transient expression of an anti-CD117 CAR by mRNA conferred T cells the ability to eliminate CD117+ targets in vitro and in vivo. As an alternative approach, we used a Sleeping Beauty transposon vector for the generation of CAR T cells incorporating an inducible Caspase 9 safety switch. Stable CAR expression was associated with high proportion of T memory stem cells, low levels of exhaustion markers, and potent cellular cytotoxicity. Anti-CD117 CAR T cells mediated depletion of leukemic cells and healthy hematopoietic stem cells in NSG mice reconstituted with human leukemia or CD34+ cord blood cells, respectively, and could be terminated in vivo. The use of a non-viral technology to control CAR T cell pharmacokinetic properties is attractive for a first-in-human study in patients with acute myeloid leukemia prior to hematopoietic stem cell transplantation.

Impact of Ligand Size and Conjugation Chemistry on the Performance of Universal Chimeric Antigen Receptor T-Cells for Tumor Killing.

All Universal Chimeric Antigen Receptor T-cells (UniCAR T-cells) are T-cells which have been engineered to recognize a haptenated ligand. Due to this feature, UniCAR T-cells have the potential to mediate a potent and selective tumor killing only in the presence of a haptenated tumor ligand, thus avoiding the long-lasting biocidal effects of conventional CAR T-cells. We have used fluorescein-labeled versions of small organic ligands and different antibody formats specific to carbonic anhydrase IX (a tumor-associated antigen) in order to assess whether the killing potential of UniCAR T-cells depended on the molecular features of the haptenated molecule. Both small molecule ligands and larger antibody fragments were potent in mediating tumor cell killing over a broad concentration range. Antibodies could be conveniently used both in IgG format and as smaller diabody fragments. Importantly, the use of site-specific chemical modification strategies for the antibody coupling to fluorescein led to a substantial improvement of tumor cell killing performance, compared to the random modification of primary amino groups on the antibody surface.

Development of a novel fully-human anti-CD123 antibody to target acute myeloid leukemia.

Monoclonal antibodies are being considered as biopharmaceuticals for the in vivo targeting of acute myeloid leukemia. Here we describe the generation and characterization of a fully-human monoclonal antibody specific to CD123, a surface marker which is overexpressed in a variety of hematological disorders, including acute myeloid leukemia. The cloning and expression of the extracellular portion of CD123 as recombinant Fc fusion allowed the selection and affinity maturation of a human antibody, called H9, which specifically recognized the cognate antigen in biochemical assays and on leukemic cells. The H9 antibody and a previously-described anti-CD123 antibody (CSL362) were reformatted into full immunoglobulin human IgG1 formats, including a variant bearing S293D and I332E mutations to enhance antibody-dependent cell-mediated cytotoxicity (ADCC). The two antibodies recognized different epitopes on the surface of the N-terminal domain of CD123, as revealed by crystallography and SPOT analysis. Both H9 and CSL362 in full immunoglobulin format were able to selectively kill leukemic cells in in vitro ADCC assays, performed both with cell lines and with patient-derived AML blasts. Further, the two antibodies, when reformatted as bispecific BiTE™ reagents by fusion with the anti-CD3 scFv(OKT3) antibody fragment, induced selective killing of AML blasts by patient-derived, autologous T-cells in an in vitro setting, but BiTE(CSL362/OKT3) exhibited a 10-fold higher potency compared to BiTE(H9/OKT3). The availability of two classes of CD123-specific biopharmaceuticals, capable of redirecting the cytolytic activity of NK cells and T cells against AML blasts, may enable novel interventional strategies and combination opportunities for the treatment of AML.

At-Home Application of Autologous Platelet Rich Plasma as Treatment for Pressure Sore and Related Anemia.

Pressure sores are a major complication in the bed-ridden older patient. In this report, we present the case of platelet rich plasma (PRP) application for the treatment of a pressure sore in an 88-year-old female affected by transfusion-dependent chronic inflammatory disease anemia associated with the congenital and inherited condition of thalassemic trait carrier. A weekly application schedule was planned athome, given the patient's debilitation and her decreased performance status as well as personal and family difficulties to go as outpatients at our treatment center. After 9 PRP applications, a remarkable sore improvement was achieved so that PRP was discontinued; nevertheless, sore rapidly improved until the full resolution and the complete closing after 4 months from the start of PRP treatment. Noteworthy, transfusion support was interrupted and a significant recovery and a sustained stabilization of hemoglobin (Hb) level at 1 year after ulcer healing were observed. The present case suggests that PRP application, performed athome in our case, is a feasible and effective treatment for pressure sores and related complications.