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Total body irradiation (TBI) is a commonly used conditioning regimen for hematopoietic stem cell transplant (HCT), but dose heterogeneity and long-term organ toxicity pose significant challenges. Total marrow irradiation (TMI), an evolving radiation conditioning regimen for HCT can overcome the limitations of TBI by delivering the prescribed dose targeted to the bone marrow (BM) while sparing organs at risk. Recently, our group demonstrated that TMI up to 20 Gy in relapsed/refractory AML patients was feasible and efficacious, significantly improving 2-year overall survival compared to the standard treatment. Whether such dose escalation is feasible in elderly patients, and how the organ toxicity profile changes when switching to TMI in patients of all ages are critical questions that need to be addressed. We used our recently developed 3D image-guided preclinical TMI model and evaluated the radiation damage and its repair in key dose-limiting organs in young (~8 weeks) and old (~90 weeks) mice undergoing congenic bone marrow transplant (BMT). Engraftment was similar in both TMI and TBI-treated young and old mice. Dose escalation using TMI (12 to 16 Gy in two fractions) was well tolerated in mice of both age groups (90% survival ~12 Weeks post-BMT). In contrast, TBI at the higher dose of 16 Gy was particularly lethal in younger mice (0% survival ~2 weeks post-BMT) while old mice showed much more tolerance (75% survival ~13 weeks post-BMT) suggesting higher radio-resistance in aged organs. Histopathology confirmed worse acute and chronic organ damage in mice treated with TBI than TMI. As the damage was alleviated, the repair processes were augmented in the TMI-treated mice over TBI as measured by average villus height and a reduced ratio of relative mRNA levels of amphiregulin/epidermal growth factor (areg/egf). These findings suggest that organ sparing using TMI does not limit donor engraftment but significantly reduces normal tissue damage and preserves repair capacity with the potential for dose escalation in elderly patients.

Sickle cell disease (SCD) is a serious global health problem, and currently, the only curative option is hematopoietic stem cell transplant (HCT). However, myeloablative total body irradiation (TBI)-based HCT is associated with high mortality/morbidity in SCD patients. Therefore, reduced-intensity (2–4 Gy) total body radiation (TBI) is currently used as a conditioning regimen resulting in mixed chimerism with the rescue of the SCD disease characteristic features. However, donor chimerism gradually reduces in a few years, resulting in a relapse of the SCD features, and organ toxicities remained the primary concern for long-term survivors. Targeted marrow irradiation (TMI) is a novel technique developed to deliver radiation to the desired target while sparing vital organs and is successfully used for HCT in refractory/relapsed patients with leukemia. However, it is unknown if TMI will be an effective treatment for a hematological disorder like SCD without adverse effects seen on TBI. Therefore, we examined preclinical feasibility to determine the tolerated dose escalation, its impact on donor engraftment, and reduction in organ damage using our recently developed TMI in the humanized homozygous Berkley SCD mouse model (SS). We show that dose-escalated TMI (8:2) (8 Gy to the bone marrow and 2 Gy to the rest of the body) is tolerated with reduced organ pathology compared with TBI (4:4)-treated mice. Furthermore, with increased SCD control (AA) mice (25 million) donor BM cells, TMI (8:2)-treated mice show successful long-term engraftment while engraftment failed in TBI (2:2)-treated mice. We further evaluated the benefit of dose-escalated TMI and donor cell engraftment in alleviating SCD features. The donor engraftment in SCD mice completely rescues SCD disease features including recovery in RBCs, hematocrit, platelets, and reduced reticulocytes. Moreover, two-photon microscopy imaging of skull BM of transplanted SCD mice shows reduced vessel density and leakiness compared to untreated control SCD mice, indicating vascular recovery post-BMT.

Purpose Although vascular alterations in solid tumor malignancies are known to decrease therapeutic delivery, the effects of leukemia-induced bone marrow vasculature (BMV) alterations on therapeutic delivery are not well known. Additionally, functional quantitative measurements of the leukemic BMV during chemotherapy and radiation therapy are limited, largely due to a lack of high-resolution imaging techniques available preclinically. This study develops a murine model using compartmental modeling for quantitative multiphoton microscopy (QMPM) to characterize the malignant BMV before and during treatment. Methods and Materials Using QMPM, live time-lapsed images of dextran leakage from the local BMV to the surrounding bone marrow of mice bearing acute lymphoblastic leukemia (ALL) were taken and fit to a 2-compartment model to measure the transfer rate (Ktrans), fractional extracellular extravascular space (νec), and vascular permeability parameters, as well as functional single-vessel characteristics. In response to leukemia-induced BMV alterations, the effects of 2 to 4 Gy low-dose radiation therapy (LDRT) on the BMV, drug delivery, and mouse survival were assessed post-treatment to determine whether neoadjuvant LDRT before chemotherapy improves treatment outcome. Results Mice bearing ALL had significantly altered Ktrans, increased νec, and increased permeability compared with healthy mice. Angiogenesis, decreased single-vessel perfusion, and decreased vessel diameter were observed. BMV alterations resulted in disease-dependent reductions in cellular uptake of Hoechst dye. LDRT to mice bearing ALL dilated BMV, increased single-vessel perfusion, and increased daunorubicin uptake by ALL cells. Consequently, LDRT administered to mice before receiving nilotinib significantly increased survival compared with mice receiving LDRT after nilotinib, demonstrating the importance of LDRT conditioning before therapeutic administration. Conclusion The developed QMPM enables single-platform assessments of the pharmacokinetics of fluorescent agents and characterization of the BMV. Initial results suggest BMV alterations after neoadjuvant LDRT may contribute to enhanced drug delivery and increased treatment efficacy for ALL. The developed QMPM enables observations of the BMV for use in ALL treatment optimization.

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).

Relapse of leukemic cells that do not express the antigen targeted by chimeric antigen receptor (CAR) is still a risk. As is the potential for targeting hematopoietic stem cells (HSCs) that share the same antigen expression, off-tumor on-target toxicity. Further, CAR T cells that bind different epitopes of the same antigen can have different tumor-killing efficacies. Therefore, we screen murine single chain variable fragment (scFv) based for indirect affinity to identify a CAR that targets Acute myeloid leukemia (AML), while minimizing toxicities. Also, recent advances in CAR design have demonstrated that the requirement of two separate tumor antigens to be ligated by CARs can increase the specificity for tumor targets. So designing a CAR that only activates a T cell when it binds two separate AML antigens will allow T cells to enhance safety. Therefore, we set out to develop a affinity based multi-antigen CAR T cell therapy that targets well described antigens for AML, including CD33 and CD123. Mice were immunized with these antigens, spleens collected, and fused with myeloma cell lines. The antibodies of fused hybridomas were screened for binding and activation against antigens by high throughput flow cytometry. After screening, we derived multiple de novo CD33, and CD123 scFvs by sequencing. We incorporated CD33 and CD123 scFvs into standard mono-specific CARs utilizing a 41BB co-stimulatory domain to validate antigen-specificity. Gene transfer assessment of CAR T cells demonstrated about 50-80% transduction efficiency for CD33 and CD123 scFvs. There were no differences in CD4 and CD8 proportions in these CAR T cells. We next examined the CARs for their cytotoxic ability using a Real-Time Cell Analysis (RTCA) system. For the CD33 CARs, 2 (6A11-1 and 27A3-1) out of 5, and for the CD123 CAR, 2 (15A12-11 and 15 A12-12) out of 8, scFv sequences transduced into T cells were highly efficacious at killing target cells and generated significant amounts of cytokines such as IFN-g, TNF- a, and IL-6. CAR T cells with these same scFv sequences were able to proliferate better in response to targeted antigen. To find the best possible combination of CD33 and CD123 scFvs we double transduced T cells with four selected CD33 or CD123 scFvs each with only one co-stimulation domain either CD3z or 4-1BB in "AND" gate fashion. Clear differences in cytotoxic ability and cytokine production were observed. We selected 12 combination of CD33/123 CARs bi-specific CARs to evaluate in vitro efficacy, polyfunctionality, and safety. Finally, to find the best combination of CARs that would be less toxic to HSCs, we performed a Colony Forming Unit (CFU) assay with CD34+ bone marrow stem cells and found 5 bi-specific pairs that were less toxic to HSCs . Based on the CFU assay and PSI index, we were able to select the combination of CD33/123 scFvs that would target AML but minimize the killing of HSCs.

Purpose: Acute myeloid leukemia (AML) is a highly aggressive form of leukemia, which 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 noninvasive imaging method to detect disease, including in extramedullary organs, representing an unmet clinical need. About 85% to 90% of human myeloid leukemia cells express CD33 cell surface receptors, highlighting CD33 as an ideal candidate for AML immunoPET. Experimental Design: We evaluated whether [64Cu]Cu-DOTA-anti-CD33 murine mAb can be used for immunoPET imaging of AML in a preclinical model. MicroCT was adjusted to detect spatial/anatomical details of PET activity. For translational purposes, a humanized anti-CD33 antibody was produced; we confirmed its ability to detect disease and its distribution. We reconfirmed/validated CD33 antibody-specific targeting with an antibody–drug conjugate (ADC) and radioimmunotherapy (RIT). Results: [64Cu]Cu-DOTA-anti-CD33–based PET-CT imaging detected CD33+ AML in mice with high sensitivity (95.65%) and specificity (100%). The CD33+ PET activity was significantly higher in specific skeletal niches [femur (P < 0.00001), tibia (P = 0.0001), humerus (P = 0.0014), and lumber spine (P < 0.00001)] in AML-bearing mice (over nonleukemic control mice). Interestingly, the hybrid PET-CT imaging showed high disease activity in the epiphysis/metaphysis of the femur, indicating regional spatial heterogeneity. Anti-CD33 therapy using newly developed humanized anti-CD33 mAb as an ADC (P = 0.02) and [225Ac]Ac-anti-CD33-RIT (P < 0.00001) significantly reduced disease burden over that of respective controls. Conclusions: We have successfully developed a novel anti-CD33 immunoPET-CT–based noninvasive modality for AML and its spatial distribution, indicating a preferential skeletal niche.

Introduction Acute myeloid leukemia (AML) is a highly aggressive form of leukemia with poor long-term survival. Our clinical development of a targeted radiation treatment for relapsed/refractory AML disease led to an impressive two-year overall survival (OS) rate of 41% (Stein et al., 2019) in contrast to Methods We first developed a TMI treatment methodology using a Precision X-RAD small animal irradiator. A whole body CT image was acquired, and three-dimensional (3D) dose calculations were performed using a Monte Carlo dose engine-based SmART-Plan treatment system (van Hoof et al, 2013; Downes et al., 2009; Faddegon et al., 2009). Before treatment, mice were further imaged to verify target anatomy prior to delivering precise radiation doses to the entire skeletal system and spleen while sparing vital organs (lungs, liver, gut). We used a Cbfb-MYH11/Mpl (CMM)-induced mice leukemia model. At 5-10% CMM-GFP cells in peripheral blood (~20-30% in bone marrow; high disease burden), the mice were treated with three doses of CSI (2.5mg/kg) on alternate days with or without 4 Gy radiation (TMI or total body irradiation [TBI]). Cy3 labeled CSI uptake studies were carried out 48h post RT by flow cytometry, and immune cell trafficking and activation studies were conducted by harvesting bone marrow and spleen cells on day 8 post RT. Results Dose volume histogram (DVH) and radiation dose painting show that the TMI treatment plan significantly reduced doses to critical organs (lungs, liver, gut) while maintianing the same dose to the skeleton and spleen, unlike TBI (same dose to entire body) (Figure 1A-D). In TMI-treated mice, the CSI-Cy3 uptake was significantly higher in CMM cells than in the CMM cells of mice treated with CSI alone, indicating improved delivery post RT (Figure 1E, F). The trafficking of T helper cells (CD4+) and cytotoxic T lymphocytes (CD8+; CTLs) as well as effector (CD62L-CD44+) and effector memory T cells (CD62L+CD44+) is significantly augmented in the bone marrow of TMI+CSI treated mice compared to levels in mice treated with TBI+CSI or CSI alone (Figure 1G, H). An increased total number of IFNγ secreting CTLs, and a higher CD8:Treg (CD4+CD25+FOXP3+) ratio indicate enhanced anti-tumerogenic activity in TMI+CSI treated mice over that of mice treated with TBI+CSI or CSI alone (Figure 1I, J). Similarly, myeloid cell trafficking and activation was augmented in TMI-treated bone marrow (data not shown). The benefit of augmented immune modulation in TMI+CSI combinatorial therapy is reflected in a significantly increased survival (~39 median survival days) over untreated mice and those treated with CSI or TMI alone ( ~8-9 days median survival) (Figure 1K). Conclusion This is the first report of a novel radio-immunotherapy using a systemic targeted precise RT (TMI) in combination with STAT3 down-regulation (CSI) in AML. As hypothesized, low-dose targeted RT in combination with blocking of STAT3 signaling improved immune cell trafficking and activation, thereby enhancing the efficacy of this combinatorial therapy in high-disease burden. Further, newly developed low-dose TMI shows enhanced immune modulation over conventional TBI, suggesting the benefits of localized targeted RT in hematological malignancies. Disclosures Hui: Janssen Research & Development, LLC: Consultancy, Honoraria.