CD24 and its ligand Siglec-10 were described as an innate immune checkpoint in carcinoma. Here, we investigated this axis in B-cell lymphoma by assessing CD24 expression and evaluating pro-phagocytic effects of CD24 antibody treatment in comparison to hallmark immune checkpoint CD47. In mantle cell lymphoma (MCL) and follicular lymphoma patients, high mRNA expression of CD24 correlated with poor overall survival, whereas CD47 expression did not. Conversely, CD24 expression did not correlate with survival in diffuse large B-cell lymphoma (DLBCL), whereas CD47 did. CD24 was also highly expressed on MCL cell lines, where treatment with CD24 antibody clones SN3 or ML5 potently induced phagocytosis, with SN3 yielding >90% removal of MCL cells and triggering phagocytosis of primary patient-derived MCL cells by autologous macrophages. Treatment with CD24 mAb was superior to CD47 mAb in MCL and was comparable in magnitude to the effect observed in carcinoma lines. Reversely, CD24 mAb treatment was less effective than CD47 mAb treatment in DLBCL. Finally, phagocytic activity of clone SN3 appeared at least partly independent of antibody-dependent cellular phagocytosis (ADCP), suggesting CD24/Siglec-10 checkpoint activity, whereas clone ML5 solely induced ADCP. In conclusion, CD24 is an immunotherapeutic target of potential clinical relevance for MCL, but not DLBCL.

Elevated activation of the autophagy pathway is currently thought to be one of the survival mechanisms allowing therapy-resistant cancer cells to escape elimination, including for cytarabine (AraC)-resistant acute myeloid leukemia (AML) patients. Consequently, the use of autophagy inhibitors such as chloroquine (CQ) is being explored for the re-sensitization of AraC-resistant cells. In our study, no difference in the activity of the autophagy pathway was detected when comparing AraC-Res AML cell lines to parental AraC-sensitive AML cell lines. Furthermore, treatment with autophagy inhibitors CQ, 3-Methyladenine (3-MA), and bafilomycin A1 (BafA1) did not re-sensitize AraC-Res AML cell lines to AraC treatment. However, in parental AraC-sensitive AML cells, treatment with AraC did activate autophagy and, correspondingly, combination of AraC with autophagy inhibitors strongly reduced cell viability. Notably, the combination of these drugs also yielded the highest level of cell death in a panel of patient-derived AML samples even though not being additive. Furthermore, there was no difference in the cytotoxic effect of autophagy inhibition during AraC treatment in matched de novo and relapse samples with differential sensitivity to AraC. Thus, inhibition of autophagy may improve AraC efficacy in AML patients, but does not seem warranted for the treatment of AML patients that have relapsed with AraC-resistant disease.

In earlier studies, galectin-9 (Gal-9) was identified as a multifaceted player in both adaptive and innate immunity. Further, Gal-9 had direct cytotoxic and tumor-selective activity towards cancer cell lines of various origins. In the current study, we identified that treatment with Gal-9 triggered pronounced membrane alterations in cancer cells. Specifically, phosphatidyl serine (PS) was rapidly externalized, and the anti-phagocytic regulator, CD47, was downregulated within minutes. In line with this, treatment of mixed neutrophil/tumor cell cultures with Gal-9 triggered trogocytosis and augmented antibody-dependent cellular phagocytosis of cancer cells. Interestingly, this pro-trogocytic effect was also due to the Gal-9-mediated activation of neutrophils with upregulation of adhesion markers and mobilization of gelatinase, secretory, and specific granules. These activation events were accompanied by a decrease in cancer cell adhesion in mixed cultures of leukocytes and cancer cells. Further, prominent cytotoxicity was detected when leukocytes were mixed with pre-adhered cancer cells, which was abrogated when neutrophils were depleted. Taken together, Gal-9 treatment potently activated neutrophil-mediated anticancer immunity, resulting in the elimination of epithelial cancer cells.

Background/Objectives: Point-of-care (PoC) manufactured fresh chimeric antigen receptor (CAR)-T cells are typically formulated in hypothermic preservation formulations (HPFs) and stored under hypothermic conditions (2–8 °C) until administered to the patient. However, in current HPFs the shelf life of fresh CAR-T cells is short (~24–36 h) due to limited CAR-T cell stability, which poses significant time constraints on manufacturing procedures and logistics. The objective of this study was to improve the stability and extend the shelf life of fresh clinical-grade CAR-T cell drug products (DPs). Methods: A novel HPF was developed by supplementing a base HPF with the novel excipient SUL-138, which stabilizes mitochondria during hypothermic storage and subsequent rewarming, alone or in combination with endogenous mitochondrial substrates. This panel of HPFs was first screened for their stability-improving characteristics in the model cell line Jurkat cells. Subsequently, HPFs were assessed for their stability-improving characteristics of clinical-grade CD19 CAR-T cell DPs. Critical quality attributes, including CAR-T cell viability, T-cell differentiation state, exhaustion markers, and functional potency were evaluated in a good manufacturing practice (GMP)-compliant stability study up to 72 h. Results: For Jurkat cells, HPFs supplemented with SUL-138 and a combination of glucose, glutamine, and succinate demonstrated the greatest stability improvement at 2–8 °C, improving cell viability from ~1% to >85% after 72 h. For CAR-T cells, supplementation of HPFs with SUL-138 alone demonstrated the greatest improvement, resulting in a CAR-T cell viability from ~40% to >85% after 72 h of storage at 2–8 °C, while no additional benefits from mitochondrial substrates were observed. The novel HPF did not significantly impact CAR-T cell potency test results, T cell subset distribution, or exhaustion markers compared to control. Conclusions: A novel clinical-grade HPF that significantly improved fresh CAR-T cell stability during hypothermic storage was developed. This novel HPF can aid in the establishment of GMP-compliant and PoC CAR-T cell manufacturing platforms.