ALBERT

Description: Background: CD33, the target for the anti-AML immunoconjugate, gemtuzumab ozogamicin (GO; Mylotarg™), contains two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). We have previously shown that these motifs control uptake of antibody-bound CD33 and GO-induced cytotoxicity. In this study, we determined which phosphorylation state favors uptake of antibody-bound CD33, identified proteins that bind to CD33 in an ITIM-dependent manner, and assessed their importance for CD33 internalization by siRNA-based gene silencing. Methods: Internalization of anti-CD33 antibodies was measured by flow cytometry in the presence or absence of the tyrosine phosphatase inhibitor, pervanadate, in human CD33+ AML cell lines (ML-1, HL-60, NB4, U937, TF-1) and CD33− Jurkat T cells infected with wild-type and mutant CD33. Pull-down experiments were performed with glutathione S-transferase (GST) proteins fused to phosphorylated cytoplasmic tails of CD33, using human myeloid cell lysates. Co-immunoprecipitations were performed with myeloid cell lines expressing HA-tagged wild-type CD33. Lentivirus-based siRNA constructs were generated for gene silencing, and expressed in human CD33+ AML cell lines. Results: Pervanadate significantly increased uptake of anti-CD33 antibodies in human AML cell lines; this effect was dependent upon the integrity of the ITIMs and was prevented by co-treatment with the Src tyrosine kinase inhibitor PP2, suggesting that Src family kinase-dependent phosphorylation of the ITIMs critically controls uptake of antibody-bound CD33, possibly by altering which proteins binds to CD33 or by facilitating binding of adaptor-proteins required for endocytosis. We identified several proteins, including the tyrosine phophatases, SHP-1 and SHP-2, and the non-receptor tyrosine kinase, Syk, which bound to phosphorylated wild-type and mutant CD33 in a manner that paralleled the endocytic properties of the corresponding CD33 protein. Since these three proteins have been implicated in endocytic processes of other cell surface proteins, we assessed their role in uptake of antibody-bound CD33 by siRNA-mediated gene silencing. Simultaneous depletion of SHP-1 and SHP-2, but not SHP-1 or SHP-2 alone, significantly increased internalization of antibody-bound CD33 in the two AML cell lines with the highest cell surface expression of CD33, whereas no effect was seen in two other cell lines with lower CD33 expression levels. In contrast, depletion of Syk, whose expression has previously been correlated to the inhibitory effect of anti-CD33 antibodies on AML cell growth, failed to affect antibody internalization in the cell lines assessed. Conclusion: These studies indicate that the phosphorylation status of the ITIMs controls uptake of antibody-bound CD33. In line with this model, SHP-1 and SHP-2, which have been shown to dephosphorylate CD33 in vitro, can affect this endocytic process. Thus, our data imply manipulation of the phosphorylation state of CD33, e.g. by activating Src family kinases or interfering with phosphatases as a novel means to increase uptake of anti-CD33 antibody-based therapeutics such as GO. Finally, the variable effect of SHP-1 and SHP-2 depletion suggests that there are significant cell-type specific differences in the response to anti-CD33 antibody ligation, for example differences in tyrosine phosphorylation levels and/or activation/recruitment or redundancies of tyrosine phosphatases.

Description: Background: The anti-acute myeloid leukemia (AML) immunoconjugate, gemtuzumab ozogamicin (GO; Mylotarg™), contains a humanized anti-CD33 antibody (hP67.6) to facilitate uptake of the toxic calicheamicin-γ1 derivative in CD33-positive AML cells. This putative mechanism implies a critical role for the intracellular accumulation of the toxic moiety for GO-induced cytotoxicity. Indeed, drug efflux by P-glycoprotein (Pgp) mediates resistance to GO and correlates with clinical outcome after GO monotherapy. Furthermore, recent in vitro data obtained in human myeloid cell lines have unequivocally demonstrated a quantitative relationship between CD33 expression and GO-induced cytotoxicity. In light of these findings, we have now re-examined the significance of CD33 expression levels on AML blasts and relationship with Pgp activity for clinical outcome of patients treated with GO monotherapy. Methods: Pre-treatment bone marrow samples from patients enrolled in multicenter phase II protocols evaluating the safety and efficacy of GO monotherapy (generally 2 doses of 9 mg/m2 14 days apart) were used for analysis. Relative CD33 expression was quantified by flowcytometry immunophenotyping using the hP67.6 antibody, and linear fluorescence values used for calculations. Pgp function was cytofluorometrically determined by efflux of the fluorescent dye, DiOC2. Results are presented as mean values and 95% confidence intervals. Unpaired t-tests, Pearson correlations, and logistic regression models were used for statistical analysis. Results: Patients achieving a complete remission (CR) or CR with incomplete recovery of platelet counts (CRp) had statistically significantly higher mean CD33 expression levels (71.20 [57.20–85.19], n=69) compared to non-responders (54.44 [48.38–60.51], n=203; p=0.01). There was an inverse relationship between CD33 expression and Pgp efflux (r=−0.23) and this contributed to responders having a statistically significantly lower mean Pgp efflux (1.40 [1.28–1.52], n=57) compared to non-responders (1.83 [1.72–1.95], n=173; p

Description: Hard X-ray imaging of the Galactic plane by the INTEGRAL satellite is uncovering large numbers of 20-100 keV "IGR" sources. We present results from Chandra, INTEGRAL, optical, and IR observations of four IGR sources: three sources in the Norma region of the Galaxy(1GR J16195-4945,IGR J16207-5129, and IGR J16167-4957) and one that is closer to the Galactic center (IGR 5171 95-4100). In all four cases, one relatively bright Chandra source is seen in the INTEGRAL error circle, and these are likely to be the soft X-ray counterparts of the IGR sources. They have hard 0.3-10 keV spectra with power-law photon indices of Gamma = 0.5-1.1. While many previously studied IGR sources show high column densities (NH approx. 10(exp 23)-10(exp 24)/sq cm), only IGR J16195-4945 has a column density that could be as high as 10(exp 23)/sq cm. Using optical and IR sky survey catalogs and our own photometry, we have obtained identifications for all four sources. The J-band magnitudes are in the range 14.9-10.4, and we have used the optical/IR spectral energy distributions (SEDs) to constrain the nature of the sources. Blackbody components with temperature lower limits of 〉9400 K for IGR J16195-4945 and 〉18,000 K for IGR J16207-5129 indicate that these are very likely high-mass X-ray binaries (HMXBs). However, for IGR 516167-4957 and IGR J17195-4100, low extinction and the SEDs indicate later spectral types for the putative companions, suggesting that these are not HMXBs.