MicroSPECT/CT Imaging of Human Leukemia Engraftment In NOD-Scid Mice Using [111In]-Labeled 7G3 Anti-CD123 Antibodies

Abstract 968

Radioimmunotherapy (RIT) of acute myelogenous leukemia (AML) is a promising strategy that could improve the long-term outcome of patients. A limitation of current therapies is their inability to eradicate the leukemic stem cell (LSC), which is responsible for causing and propagating the disease and the failure to achieve long-term survival. Monoclonal antibody (mAb) 7G3 recognizes CD123 (IL-3R α) in the absence of CD131 (IL-3R β) a LSC characteristic but not of normal hematopoietic stem cells. Our laboratory is developing ¹¹¹In-labeled 7G3 modified with nuclear localization sequence (NLS) peptides for Auger electron RIT of AML aimed at the LSC. These electrons have only nanometer-micrometer range but are lethal when released inside the cell nucleus. Our objective was to examine the ability of ¹¹¹In-NLS-7G3 to localize at sites of AML engraftment in NOD-scid mice taking advantage of the concurrent gamma emissions of ¹¹¹In for microSPECT/CT imaging. These properties were examined in NOD-scid mice engrafted with CD123⁺/CD131^- AML cell lines which avoids the challenges of engrafting primary AML specimens including their variability in engraftment efficiency and heterogeneity in CD123 expression. Alternatively, mice bearing s.c. CD123-transfected Raji (Raji-CD123) tumors were studied. 7G3 was reacted with sulfo-SMCC to introduce maleimide groups for conjugation to NLS peptides (CGYGPKKKRKVGG) then directly derivatized with DTPA for complexing ¹¹¹In. The pharmacokinetics (PK) of elimination from the blood of ¹¹¹In-NLS-7G3 intact IgG2a and F(ab’)₂ fragments were studied in NOD-scid and Balb/c mice. The effect of pre-administration of a 10-fold excess of unlabeled BM4 IgG2a or IgG1 irrelevant mAbs on the PK was assessed. Imaging and biodistribution studies were performed at 48 to 72 h p.i. of ¹¹¹In-NLS-7G3 IgG2a and F(ab’)₂ in AML-engrafted mice. Sites of engraftment visualized on the images were analyzed for AML by flow cytometry. PK studies revealed an unusually rapid elimination of ¹¹¹In-NLS-7G3 IgG2a but not F(ab’)₂ in NOD-scid compared to Balb/c mice with blood radioactivity levels at 18 h 4-fold lower (3.9 ± 0.9 vs. 17.0 ± 4.4% i.d./ml, respectively). This rapid elimination prevented uptake into Raji-CD123 xenografts and did not allow their visualization by microSPECT/CT. The elimination of ¹¹¹In-NLS-7G3 F(ab’)₂ was not significantly different in NOD-scid or Balb/c mice (0.60 ± 0.17 vs. 0.87 ± 0.06 %i.d./ml, respectively at 72 h p.i.). Pre-administration of unlabeled BM4 improved the retention of ¹¹¹In-NLS-7G3 in the blood by 3.2-fold and allowed for high uptake (14.7 ± 2.8 % i.d./g) and imaging of tumors. Xenografts also had high uptake (7.2 ± 3.8 % i.d./g) and imaged with ¹¹¹In-NLS-7G3 F(ab’)₂. AML-3, -4 and -5 cells engrafted into bone marrow (BM) and NOD-scid mice assessed by the proportion of CD45⁺ cells (12%, 0.5% and 63%, respectively). CD45⁺ cells in the spleen for AML-5 engraftment was 83%. Uptake of ¹¹¹In-NLS-7G3 F(ab’)₂ in the spleen and BM of AML-3 engrafted mice were 5-fold and 1.5-fold higher than ¹¹¹In-NLS-BM4 control F(ab’)₂. The spleen was visualized by microSPECT/CT in engrafted mice receiving ¹¹¹In-NLS-7G3 F(ab’)₂ but not with ¹¹¹In-NLS-BM4 F(ab’)₂. In AML-3 engrafted mice imaged with ¹¹¹In-NLS-7G3 IgG2a, multiple intense foci of radioactivity were noted in the spine, pelvis and femur, representing leukemia in the marrow. Extramedullary (EM) disease was also imaged in the brain and lymph nodes. Uptake in the femur was 2-fold higher in engrafted mice compared to non-engrafted mice. In AML-5 engrafted mice, outgrowths of AML at the femoral heads was imaged with ¹¹¹In-NLS-7G3 IgG2a following pre-administration of an excess of unlabeled BM4. No visualization of these AML growths was noted for mice receiving ¹¹¹In-NLS-BM4. We conclude that microSPECT/CT allowed sensitive detection of the specific uptake of ¹¹¹In-NLS-7G3 and its F(ab’)₂ fragments in AML engrafted mice. PK and Raji-CD123 xenograft microSPECT/CT studies identified a tumor delivery barrier. A robust AML engraftment model was established with cells displaying the CD123⁺/CD131^- phenotype. These results are promising for preclinical evaluation of ¹¹¹In-NLS-7G3 for Auger electron RIT of AML and suggest that imaging is a useful tool to assess engraftment in NOD-scid mice as well as for evaluating response to ¹¹¹In-NLS-7G3. Preliminary results of studies to examine ¹¹¹In-NLS-7G3 for RIT of AML will also be presented.