Multiple myeloma (MM) is a disease with known immune dysregulation. Natural killer (NK) cells have shown preclinical activity in MM. We conducted a first‐in‐human study of umbilical cord blood‐derived (CB) NK cells for MM patients undergoing high dose chemotherapy and autologous haematopoietic stem cell transplantation (auto‐HCT). Patients received lenalidomide (10 mg) on days −8 to −2, melphalan 200 mg/m² on day −7, CB‐NK cells on day −5 and auto‐HCT on day 0. Twelve patients were enrolled, three on each of four CB‐NK cell dose levels: 5 × 10⁶, 1 × 10⁷, 5 × 10⁷ and 1 × 10⁸ CB‐NK cells/kg. Ten patients had either high‐risk chromosomal changes or a history of relapsed/progressed disease. There were no infusional toxicities and no graft‐versus‐host disease. One patient failed to engraft due to poor autologous graft quality and was rescued with a back‐up autologous graft. Overall, 10 patients achieved at least a very good partial response as their best response, including eight with near complete response or better. With a median follow‐up of 21 months, four patients have progressed or relapsed, two of whom have died. CB‐NK cells were detected in vivo in six patients, with an activated phenotype (NKG2D⁺/NKp30⁺). These data warrant further development of this novel cellular therapy.

Chimeric antigen receptor (CAR) engineering of natural killer (NK) cells is promising, with early-phase clinical studies showing encouraging responses. However, the transcriptional signatures that control the fate of CAR-NK cells after infusion and factors that influence tumor control remain poorly understood. We performed single-cell RNA sequencing and mass cytometry to study the heterogeneity of CAR-NK cells and their in vivo evolution after adoptive transfer, from the phase of tumor control to relapse. Using a preclinical model of noncurative lymphoma and samples from a responder and a nonresponder patient treated with CAR19/IL-15 NK cells, we observed the emergence of NK cell clusters with distinct patterns of activation, function, and metabolic signature associated with different phases of in vivo evolution and tumor control. Interaction with the highly metabolically active tumor resulted in loss of metabolic fitness in NK cells that could be partly overcome by incorporation of IL-15 in the CAR construct.

Integration of single-cell multiomics profiles generated by different single-cell technologies from the same biological sample is still challenging. Previous approaches based on shared features have only provided approximate solutions. Here, we present a novel mathematical solution named bi-order canonical correlation analysis (bi-CCA), which extends the widely used CCA approach to iteratively align the rows and the columns between data matrices. Bi-CCA is generally applicable to combinations of any two single-cell modalities. Validations using co-assayed ground truth data and application to a CAR-NK study and a fetal muscle atlas demonstrate its capability in generating accurate multimodal co-embeddings and discovering cellular identity.

Key Points: Cytomegalovirus reactivation and interleukin 15 are major contributors to NK cell repertoire diversity and maturation after CBT. An immature NK cell subset characterized by low diversity index and poor effector function was highly predictive of relapse after CBT.

SARS-CoV-2 vaccines pose as the most effective approach for mitigating the COVID-19 pandemic. High-degree efficacy of SARS-CoV-2 vaccines in clinical trials indicates that vaccination invariably induces an adaptive immune response. However, the emergence of breakthrough infections in vaccinated individuals suggests that the breadth and magnitude of vaccine-induced adaptive immune response may vary. We assessed vaccine-induced SARS-CoV-2 T cell response in 21 vaccinated individuals and found that SARS-CoV-2–specific T cells, which were mainly CD4 ⁺ T cells, were invariably detected in all individuals but the response was varied. We then investigated differentiation states and cytokine profiles to identify immune features associated with superior recall function and longevity. We identified SARS-CoV-2–specific CD4 ⁺ T cells were polyfunctional and produced high levels of IL-2, which could be associated with superior longevity. Based on the breadth and magnitude of vaccine-induced SARS-CoV-2 response, we identified 2 distinct response groups: individuals with high abundance versus low abundance of SARS-CoV-2–specific T cells. The fractions of TNF-α– and IL-2–producing SARS-CoV-2 T cells were the main determinants distinguishing high versus low responders. Last, we identified that the majority of vaccine-induced SARS-CoV-2 T cells were reactive against non-mutated regions of mutant S-protein, suggesting that vaccine-induced SARS-CoV-2 T cells could provide continued protection against emerging variants of concern.

Purpose: Acute myeloid leukemia (AML) is characterized by frequent mutations in FMS-like tyrosine kinase 3 (FLT3), overexpression of murine double minute 2 (MDM2), and TP53 wild-type (WT). Monotherapies targeting FLT3 frequently result in the development of resistant disease. In this study, we investigated the antileukemic efficacy of co-targeting FLT3 and MDM2 with quizartinib and milademetan (Q/M) in FLT3 internal tandem duplication (FLT3-ITD) AML cell lines, xenograft and patient-derived xenograft (PDX) models, and a phase I clinical trial. Experimental Design: Preclinical studies used human and murine cell lines carrying FLT3-ITD and/or tyrosine kinase domain mutations, TP53 WT/knockdown, leukemia cell xenograft models, and a PDX model. Assays were conducted using milademetan (DS-3032b) and murine-specific MDM2 inhibitor (DS-5272). An open-label, phase I, dose-escalation clinical trial (ClinicalTrials.gov NCT03552029) was conducted. Results: Dual inhibition of FLT3-ITD and MDM2 synergistically induced apoptosis in FLT3-ITD/TP53 WT AML and venetoclax-resistant cell lines, reduced tumor burden, and improved survival in xenograft and PDX models of FLT3-ITD AML. Phase I clinical data indicated favorable safety and tolerability for the Q/M combination treatment. Complete responses with incomplete hematologic recovery were achieved in 40% of patients with relapsed/refractory AML. Unsupervised single-cell proteomic analysis showed that Q/M treatment decreased the expression of prosurvival proteins (p-ERK, p-AKT, and Mcl-1) and activated protein signaling downstream of p53 including p53 upregulated modulator of apoptosis. YTHDF2 was increased after therapy in resistant cells. The Q/M combination demonstrated higher activity in CD34+ versus CD34− leukemia blasts. Conclusions: Preclinical and mechanistic rationale and preliminary clinical data support the future development of MDM2/FLT3-targeting strategies for FLT3-mutant AML.