Engineered T cells expressing chimeric antigen receptors (CARs) on their cell surface can redirect antigen specificity. This ability makes CARs one of the most promising cancer therapeutic agents. CAR-T cells for treating patients with B cell hematological malignancies have shown impressive results. Clinical manifestation has yielded several trials, so far five CAR-T cell therapies have received US Food and Drug Administration (FDA) approval. However, emerging clinical data and recent findings have identified some immune-related toxicities due to CAR-T cell therapy. Given the outcome and utilization of the same proof of concept, further investigation in other hematological malignancies, such as leukemias, is warranted. This review discusses the previous findings from the pre-clinical and human experience with CAR-T cell therapy. Additionally, we describe recent developments of novel targets for adoptive immunotherapy. Here we present some of the early findings from the pre-clinical studies of CAR-T cell modification through advances in genetic engineering, gene editing, cellular programming, and formats of synthetic biology, along with the ongoing efforts to restore the function of exhausted CAR-T cells through epigenetic remodeling. We aim to shed light on the new targets focusing on acute myeloid leukemia (AML).

Increasing knowledge of cancer immunology has led to the design of therapies using immune cells directly or manipulating their activity, collectively termed immunotherapy. In the field of immuno-oncology, research on adaptive immune T cells has led to the development of CAR-T cells. Innate immune cells such as NK cells can also eliminate oncogenically transformed cells and regulate cells of the immune system. Considering NK cells as a live drug, numerous methods for the isolation and activation of NK cells have been shown to be clinically and therapeutically relevant. In such processes, various cytokines and antibodies present a source of stimulation of NK cells and enhance the efficacy of such treatments. The ex vivo expansion and activation of NK cells, along with genetic modification with CAR, enhance their antitumor activity. Recent preclinical studies have shown an antitumor effect through extracellular vesicles (EVs) derived from NK cells. Work with autologous NK cells has provided insights for clinical applications. In this review, we outline the recent advances of NK-cell-based immunotherapies, summarizing CAR-NK cells, BiKEs, and TriKEs as treatment options against cancer. This review also discusses the challenges of NK cell immunotherapy.

Purpose: Acute myeloid leukemia (AML) is a highly aggressive form of leukemia that results in poor survival outcomes. Currently, diagnosis and prognosis are based on invasive single-point bone marrow biopsies (iliac crest). There is currently no AML-specific non-invasive imaging method to assess disease in the whole body, including in extramedullary organs, representing an unmet clinical need. About 85-90% of human myeloid leukemia cells express CD33 (an accepted biomarker for AML) cell surface receptors, highlighting CD33 as an ideal candidate for AML immunoPET. Experimental Design: In this study, we evaluated if ⁶⁴ Cu-DOTA-anti-CD33 murine monoclonal antibody (mAb) can be used for ImmunoPET-CT imaging of AML in preclinical model. For translational purpose, a humanized anti-CD33 antibody was produced, and after confirming its anti-CD33 PET-CT imaging ability we further explored its therapeutic potential. Results: ⁶⁴ Cu-DOTA-anti-CD33 based PET-CT imaging detected CD33 ⁺ AML in mice with high sensitivity (95.65%) and specificity (100%). In the entire skeletal system, CD33 ⁺ PET activity was significantly high in the bones of AML bearing mice over non-leukemic control mice; femur (p<0.00001), tibia (p=0.0001), humerus (p=0.0014), and lumber spine (p<0.00001). Interestingly, the imaging showed CD33 ⁺ PET activity prevelant in epiphysis/diaphysis of the bones, indicating spatial heterogeneity. Anti-CD33 therapy using newly developed humanized anti-CD33 mAb as an ADC (p=0.02) and ²²⁵ Ac-anti-CD33-RIT (p<0.00001), significantly reduced disease burden over that of respective controls. Conclusion: We have successfully developed a novel anti-CD33 immunoPET-CT based non-invasive imaging tool for diagnosis of AML and its spatial distribution, which indicates a preferential skeletal niche. Translational Relevance: Acute myeloid leukemia (AML) exhibits extensive heterogeneity in therapeutic response, even in patients sharing similar characteristics, indicating the inadequacy of currently used biopsy-based prognostic tools. There is a critical need for non-invasive quantitative imaging modality specific to AML, which is able to assess this disease in the whole body, including in extramedullary organs and to longitudinally monitor treatment response. CD33, an accepted AML biomarker is expressed on more than 85-90% of blast cells and Mylotarg (anti-CD33-ADC) is FDA approved immunotherapy for AML. In this preclinical study using anti-human CD33 mAb ( ⁶⁴ Cu-DOTA-anti-CD33), a CD33 PET-CT imaging modality was developed that detected AML with high sensitivity and specificity. The approach also indicated spatial heterogeneity in leukemic disease, dependent on disease burden, which warrants caution in analyzing results from single point bone marrow biopsies. Therefore, this imaging modality can be used to non-invasively detect AML in humans and also monitor treatment response, an unmet clinical need.

Vascular permeability, tissue transfer rate (K _trans ), fractional extracellular tissue space (ν _ec ) and blood perfusion are crucial parameters to assess bone marrow vasculature (BMV) function. However, quantitative measurements of these parameters in a mouse model are difficult because of limited resolution of standard macroscopic imaging modalities. Using intravital multiphoton microscopy (MPM), live imaging of dextran transfer from BMV to calvarium tissue of mice bearing acute lymphoblastic leukemia (ALL) was performed to obtain BMV parameters. Mice bearing ALL had increased BMV permeability, altered K _trans , increased ν _ec , decreased blood perfusion, and increased BMV permeability resulting in reduced drug uptake. Targeted 2 Gy radiation therapy (RT) to mice bearing ALL increased local BMV perfusion and ALL chemotherapy uptake (P<0.0001 and P=0.0036, respectively), suggesting RT prior to chemotherapy treatment may increase treatment efficacy. Developed MPM techniques allow for a quantitative assessment of BMV functional parameters not previously performed with microscopic or macroscopic imaging.