Although the presence of an unmutated IgV(H) gene is strongly associated with the expression of ZAP-70, ZAP-70 is a stronger predictor of the need for treatment in B-cell CLL.

No abstract

IntroductionThe B cells of patients with chronic lymphocytic leukemia (CLL) can be segregated into one of at least 2 major subsets based upon the mutational status of the expressed immunoglobulin (Ig) heavychain variable region genes (IgV H ). 1 Patients with CLL cells that express unmutated IgV H tend to have a relatively aggressive clinical course when compared with patients who have CLL cells that express IgV H with somatic mutations. 2,3 Although these 2 types share a common gene expression profile, isolated CLL B cells of these 2 subgroups can be distinguished from each other by the differential expression of a relatively small subset of genes. 4 One of these genes encodes the zeta-associated protein of 70 kDa (ZAP-70), a receptor-associated protein tyrosine kinase (PTK) expressed by T lymphocytes and natural killer cells (NK cells) but not by normal B cells or most cases of CLL with mutated IgV H .The functional significance of ZAP-70 gene expression in this subset of CLL B cells is unknown. Irrespective of the expression of ZAP-70, CLL cells generally express similar levels of p72 Syk , a related PTK. 5,6 B cells use p72 Syk for signal transduction via the B-cell receptor (BCR) complex. 7 Following BCR ligation, p72 Syk is recruited to the phosphorylated immunoreceptor tyrosine-based activation motifs (ITAM) of the activated BCR complex, where it subsequently becomes phosphorylated and activated. 8 In normal B cells, activated p72 Syk phosphorylates several proteins, including BLNK (for B-cell linker protein, also known as SLP-65, BASH, or BCA). 9-11 BLNK serves as a docking site for a number of signaling molecules, including Btk, Vav, and phospholipase C-gamma (PLC␥). Phosphorylation and activation of PLC␥ lead to hydrolysis of the polyphosphoinositides and subsequent production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), which serve to increase intracellular calcium (Ca 2ϩ ) and activate protein kinase C (PKC) and Ras, respectively.Recent studies indicate that CLL cells that express ZAP-70 mRNA have levels of ZAP-70 protein that are similar to those of normal blood T cells. 6,12,13 Furthermore, treatment of ZAP-70 ϩ CLL cells with anti-induced significantly greater tyrosine phosphorylation of cytosolic proteins, including p72 Syk , than CLL cells that lacked ZAP-70. 6 Moreover, following treatment with anti-, ZAP-70 underwent tyrosine phosphorylation and became associated with surface and CD79b, arguing that this PTK might enhance BCR receptor signaling in CLL B cells. However, the contributions of ZAP-70 to the activation of downstream adaptor proteins, such as BLNK or PLC␥, or to changes in intracellular Ca 2ϩ in response to Ig receptor signaling in CLL have not been resolved.Resolution of the role that ZAP-70 plays in Ig receptor signaling in CLL is important, as this protein tyrosine kinase is not an obvious candidate to enhance BCR signaling, given that CLL cells generally also express p72 Syk . 6 Indeed, p72 Syk has an approximately 100-fold greater intrinsic PTK activity than...

IntroductionZAP-70 is a 70-kDa T-cell antigen receptor (TCR) z-chainassociated cytoplasmic protein tyrosine kinase (PTK) that initially was identified in T lymphocytes. 1,2 Following ligation of the TCR, tyrosine-containing immunoreceptor tyrosine-based activation motifs (ITAMs) within the cytoplasmic tails of the CD3 molecules and the TCR zeta chain (CD247) are phosphorylated by the Src kinase, Lck. 3 ZAP-70 is recruited to the phosphorylated ITAMs and becomes activated via tyrosine phosphorylation. The activated ZAP-70 in turn can induce activation of downstream signaling pathways, such as the phospholipase Cg/Ca 2ϩ (PLC-␥) signaling pathway and the Ras/mitogen-activated protein kinase (MAPK) pathway. 4 B cells generally lack ZAP-70, but instead use a related PTK, called p72 Syk , to mediate signaling via the B-cell receptor (BCR) complex. 5 Similar to p72 Syk is recruited to the phosphorylated ITAMs of the accessory molecules of the BCR complex, namely CD79a and CD79b, whereupon p72 Syk becomes activated. 6,7 As such, ZAP-70 and p72 Syk play similar roles in antigen-receptor signaling pathways.Previous studies demonstrated that chronic lymphocytic leukemia (CLL) B cells that express unmutated immunoglobulin heavychain variable region genes (IGHV) generally express ZAP-70, in contrast to normal B cells or most patients with CLL that use mutated IGHV, 8,9 allowing ZAP-70 to be used as a surrogate marker for expression of unmutated IGHV. 10 Patients with CLL cells that use unmutated IGHV and/or express ZAP-70 have a relatively short median time from diagnosis to initial treatment than patients with leukemia cells that use mutated IGHV. Recent studies have found that leukemia-cell expression of ZAP-70 actually might be a stronger risk factor for aggressive disease than use of unmutated IGHV genes by CLL cells. 11,12 Conceivably, ZAP-70 contributes to the relatively aggressive clinical behavior of CLL cells that express unmutated IGHV genes.Indeed, the repertoire of Ig expressed in CLL is highly restricted, suggesting that leukemia cells are selected based upon their capacity to interact with some unknown antigen(s). 13,14 More recent studies have found that expression of ZAP-70 in CLL is associated with enhanced Ig receptor signaling. 8,15,16 This is despite the fact that most CLL cells also express p72 Syk at levels similar to that of normal B cells, which do not require ZAP-70 for efficient BCR signaling. Moreover, the introduction of ZAP-70 into CLL cells that previously lacked this tyrosine kinase could enhance their capacity to undergo phosphorylation of P72 Syk , B-cell linker protein (BLNK), and PLC-␥, and to experience intracellular calcium flux in response to surface IgM ligation. 17 These studies demonstrated that ZAP-70 could enhance BCR signaling in CLL B cells. However, they did not establish whether this effect was dependent upon the kinase activity of ZAP-70. This would be important to resolve prior to the development of specific inhibitors of the ZAP-70 kinase for the treatment of patients with ...

High sensitivity imaging tools could provide a more holistic view of target antigen expression to improve the identification of patients who might benefit from cancer immunotherapy. We developed for immunoPET a novel recombinant human IgG1 (termed C4) that potently binds an extracellular epitope on human and mouse PD-L1 and radiolabeled the antibody with zirconium-89. Small animal PET/CT studies showed that 89Zr-C4 detected antigen levels on a patient derived xenograft (PDX) established from a non-small-cell lung cancer (NSCLC) patient before an 8-month response to anti-PD-1 and anti-CTLA4 therapy. Importantly, the concentration of antigen is beneath the detection limit of previously developed anti-PD-L1 radiotracers, including radiolabeled atezolizumab. We also show that 89Zr-C4 can specifically detect antigen in human NSCLC and prostate cancer models endogenously expressing a broad range of PD-L1. 89Zr-C4 detects mouse PD-L1 expression changes in immunocompetent mice, suggesting that endogenous PD-1/2 will not confound human imaging. Lastly, we found that 89Zr-C4 could detect acute changes in tumor expression of PD-L1 due to standard of care chemotherapies. In summary, we present evidence that low levels of PD-L1 in clinically relevant cancer models can be imaged with immunoPET using a novel recombinant human antibody.

The ultrasmall nanoparticle AGuIX is a versatile platform that tolerates a range of chemical diversity for theranostic applications. Our previous work showed that AGuIX clears rapidly from normal tissues, while durably accumulating within the tumor microenvironment. On this basis, AGuIX was used to detect tumor tissue with Gd(3+) enhanced MRI and can sensitize tumors to radiation therapy. As we begin the translation of AGuIX, we appreciated that coupling AGuIX to a long-lived radioisotope would help to more completely measure the magnitude and duration of its retention within the tumor microenvironment. Therefore, we developed (89)Zr-DFO-AGuIX. AGuIX was coupled to DFO and then to (89)Zr in ∼99% radiochemical yield. Stability studies showed that (89)Zr-DFO-AGuIX did not dissociate after 72 h. In animals bearing U87MG xenografts, it was detectable at levels above background for 72 h. Lastly, (89)Zr-DFO-AGuIX did not accumulate in inflammatory abscesses in vivo, highlighting its specificity for well vascularized tumors.

Purpose mTOR regulates many normal physiological processes and when hyperactive, can drive numerous cancers and human diseases. However, it is very challenging to detect and quantify mTOR signaling non-invasively in clinically relevant animal models of disease or man. We hypothesized that a nuclear imaging tool measuring intracellular mTOR activity could address this unmet need. Experimental Design Although the biochemical activity of mTOR is not directly amenable to nuclear imaging probe development, we show that the transferrin receptor can be used to indirectly measure intracellular changes in mTOR activity. Results After verifying that the uptake of radiolabeled transferrin (the soluble ligand of the transferrin receptor) is stimulated by active mTORC1 in vitro, we showed that 89Zr-labeled transferrin can measure mTORC1 signaling dynamics in normal and cancerous mouse tissues with positron emission tomography (PET). Lastly, we show that 89Zr-Tf can detect the upregulation of mTORC1 by tumor cells to escape the antitumor effects of a standard of care antiandrogen, which is to our knowledge the first example of applying PET to interrogate the biology of treatment resistant cancer. Conclusions In summary, we have developed the first quantitative assay to provide a comprehensive measurement of mTOR signaling dynamics in vivo, in specific normal tissues and during tumor development in genetically engineered animal models using a nuclear imaging tool that is readily translatable to man.

The glucocorticoid receptor (GR) is an emerging drug target for several common and deadly solid tumors like breast and prostate cancer, and clinical trials studying the antitumor effects of GR antagonists are beginning. Since GR expression can be variable in tumor cells, and virtually all normal mammalian tissues express some GR, we hypothesized that an imaging tool capable of detecting GR positive tumors and/or measuring GR occupancy by drug in tumor and normal tissues could improve the precision application of anti-GR therapies in the clinic. To this end, we developed a fluorine-18 labeled corticosteroid termed GR02 that potently binds the endogenous ligand binding pocket on full length GR. Binding of 18F-GR02 was suppressed in many normal tissues by co-treatment with mifepristone, a GR antagonist in human use, and was elevated in many normal tissues among mice lacking circulating corticosteroids due to adrenalectomy. 18F-GR02 also accumulated in GR positive subcutaneous and subrenal capsule prostate cancer models, and uptake in tumors was competed by mifepristone. Combined with a straightforward and high yielding radiosynthesis, these data establish the foundation for near-term clinical translation of 18F-GR02.