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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.

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.

Vascular permeability, tissue transfer rate (Ktrans), fractional extracellular tissue space (νec) 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 Ktrans, increased νec, 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

Mixed chimerism is a promising approach to inducing allograft and xenograft tolerance. Mixed allogeneic and xenogeneic chimerism in mouse models induced specific tolerance and global hyporesponsiveness, respectively, of host mouse natural killer (NK) cells. In this study, we investigated whether pig/human mixed chimerism could tolerize human NK cells in a humanized mouse model. Our results showed no impact of induced human NK cell reconstitution on porcine chimerism. NK cells from most pig/human mixed chimeric mice showed either specifically decreased cytotoxicity to pig cells or global hyporesponsiveness in an in vitro cytotoxicity assay. Mixed xenogeneic chimerism did not hamper the maturation of human NK cells but was associated with an alteration in NK cell subset distribution and interferon gamma (IFN-gamma) production in the bone marrow. In summary, we demonstrate that mixed xenogeneic chimerism induces human NK cell hyporesponsiveness to pig cells. Our results support the use of this approach to inducing xenogeneic tolerance in the clinical setting. However, additional approaches are required to improve the efficacy of tolerance induction while ensuring adequate NK cell functions.

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.

CD19 targeted 2nd generation chimeric antigen receptor T (CAR T) cells have been successful against relapsed and/or refractory B cell malignancies. The pending FDA-approval of 2 separate CD19 targeted CAR T products highlight the need to understand the biology behind this novel therapy. CAR design includes a single-chain variable fragment, which encodes antigen-binding, fused to a transmembrane domain, co-stimulatory domain, and CD3ζ activation domain. The two CARs likely to be approved as standard of care include a 41BB or CD28 co-stimulatory domain. CD28 is a critical co-stimulatory receptor required for full T cell activation and persistence, while 4-1BB is a member of the tumor necrosis factor receptor family and also a critical T cell co-stimulatory factor. Early evaluation of the co-stimulatory domains role in CAR design confirmed that they are required to enhance T cell function, but lacked insight regarding their mechanism for this enhancement. Furthermore, clinical outcomes suggest that the co-stimulatory domains in CARs support different T cell functions in patients. For example, while overall outcomes are similar between 41BB (19BBz) and CD28-containing CARs (1928z), 19BBz CAR T cells can persist for years in patients, but functional 1928z CAR T cells rarely persist longer than a month. Recent studies are providing insight to these differences and have demonstrated that 4-1BB-containing CARs reduce T cell exhaustion, enhance persistence, and increase central memory differentiation and mitochondrial biogenesis, while CD28-containing CARs support robust T cell activation and exhaustion, and are associated with effector-like differentiation. However, these studies have been performed mostly in vitro or in immune deficient mice, which limits their ability to model complex immune biology. Therefore, we evaluated murine CD19-targeting CARs with a 4-1BB (m19BBz) or CD28- (m1928z) co-stimulatory domain in relevant animal models of immunity. We directly compared m19BBz and m1928z CAR T cell immune phenotype, cytotoxicity, cytokine production, gene expression, intracellular signaling, and in vivo persistence, expansion, and B cell acute lymphoblastic leukemia (B-ALL) eradication. In vitro assays revealed that m1928z CAR T cells had enhanced cytotoxicity and cytokine production compared to m19BBz CAR T cells. Also, evaluation of m1928z and m19BBz CAR T cells displayed similar immune phenotypes, but markedly different gene expression with m1928z CAR T cells upregulating genes related to effector function and exhaustion, while m19BBz CAR upregulated genes critical for NFkB regulation, T cell quiescence and memory. In vivo, both m1928z and m19BBz CAR T cells supported equivalent protection against B-ALL. Similar to patients, in our mouse models there are functional differences between the mouse CD19-targeted CAR T cells. At 1 week post-infusion m19BBz CAR T cells are present in the blood of mice at significantly greater levels than m1928z CAR T cells. Furthermore, m19BBz CAR T cells enhance proliferation and/or anti-apoptosis protein expression to enhance B cell killing, which is evidenced by our observation that irradiation significantly weakens the in vivo efficacy of m19BBz but not m1928z CAR T cells. Our results suggest that B cell killing by m1928z CAR T cells is not impacted by irradiation because of their efficacious cytotoxicity of B cells. In contrast, m19BBz CAR T cells have enhanced viability and anti-apoptosis protein expression, which allows them to compensate for reduced effector function. We investigated potential mechanisms for the enhanced viability and anti-apoptosis of m19BBz CAR T cells and determined that NFkB signaling is upregulated much greater by m19BBz than m1928z. We have observed this difference in both a reporter cell line and primary mouse T cells. We are now dissecting what cellular components mediate increased NFkB signaling by the m19BBz CAR. Our animal models recapitulate equivalent anti-leukemia efficacy of CD19-targeted CAR T cells regardless of co-stimulatory domain, but underscore that anti-leukemia killing is mediated by different methods depending on the co-stimulatory domain. Our work sheds light on how 4-1BB mechanistically regulates and impacts CAR T function and has implications for future CAR design and evaluation. Disclosures No relevant conflicts of interest to declare.