An atlas of bloodstream-accessible bone marrow proteins for site-directed therapy of acute myeloid leukemia.

The concept of arming antibodies with bioactive payloads for a site-specific therapy of cancer has gained considerable interest in recent years. However, a successful antibody-based targeting approach critically relies on the availability of a tumor-associated target that is not only preferentially expressed in the tumor tissue but is also easily accessible for antibody therapeutics coming from the bloodstream. Here, we perfused the vasculature of healthy and acute myeloid leukemia (AML)-bearing rats with a reactive ester derivative of biotin and subsequently quantified the biotinylated proteins to identify AML-associated bone marrow (BM) antigens accessible from the bloodstream. In total, >1400 proteins were identified. Overall, 181 proteins were >100-fold overexpressed in AML as compared with normal BM. Eleven of the most differentially expressed proteins were further validated by immunohistochemistry and confocal microscopic analyses, including novel antigens highly expressed in AML cells (for example, adaptor-related protein complex 3 ß2) and in the leukemia-modified extracellular matrix (ECM) (for example, collagen-VI-α-1). The presented atlas of targetable AML-associated BM proteins provides a valuable basis for the development of monoclonal antibodies that could be used as carriers for a site-specific pharmacodelivery of cytotoxic drugs, cytokines or radionuclides to the BM in AML.

Noninvasive Imaging of Acute Renal Allograft Rejection by Ultrasound Detection of Microbubbles Targeted to T-lymphocytes in Rats.

PURPOSE: We propose CD3-antibody-mediated contrast-enhanced ultrasonography using human T-lymphocytes for image-based diagnosis of acute allograft rejection (AR) established in a rat renal transplantation model. MATERIALS AND METHODS: 15 minutes after tail vein injection of 30 × 10(6) human T-lymphocytes, contrast media/microbubbles conjugated with an anti-human CD3 antibody was applied to uni-nephrectomized 10-week-old allogeneically transplanted male rats (Lewis-Brown Norway (LBN) to Lewis, aTX) and ultrasound was performed to investigate the transplanted kidney as well as the native kidney. In vivo results were confirmed via immunohistochemical stainings of CD3 after post mortem dissection. Syngeneically transplanted rats (LBN to LBN, sTX), rats with ischemia/reperfusion injury (IRI, 45 min. warm ischemia), and rats subjected to acute cyclosporin A toxicity (CSA) (cyclosporine 50 mg/kg BW for 2 days i. p.) served as controls. RESULTS: Accumulation of human T-lymphocytes was clearly detected by antibody-mediated sonography und was significantly increased in allografts undergoing AR (5.41 ±â€Š1.32 A. U.) when compared to native control kidneys (0.70 ±â€Š0.08 A. U.). CD3 signal intensity was low in native kidneys, sTX (0.99 ±â€Š0.30 A. U.), CSA (0.10 ±â€Š0.02 A. U.) and kidneys with IRI (0.46 ±â€Š0.29 A. U.). Quantification of the ultrasound signal correlated significantly with the T-cell numbers obtained by immunohistochemical analysis (R2 = 0.57). CONCLUSION: Contrast-enhanced sonography using CD3-antibodies is an option for quick and highly specific assessment of AR in a rat model of renal transplantation.

Renal Contrast-Enhanced Sonography Findings in a Model of Acute Cellular Allograft Rejection.

Noninvasive methods to diagnose and differentiate acute cellular rejection from acute tubular necrosis or acute calcineurin inhibitor toxicity are still missing. Because T lymphocytes play a decisive role in early states of rejection, we investigated the suitability and feasibility of antibody-mediated contrast-enhanced ultrasound by using microbubbles targeted to CD3(+) , CD4(+) , or CD8(+) T cells in different models of renal disease. In an established rat renal transplantation model, CD3-mediated ultrasound allows the detection of acute rejection as early as on postoperative day 2. Ultrasound signal intensities increased with the severity of inflammation. Further, an early response to therapy could be monitored by using contrast-enhanced sonography. Notably, acute tubular necrosis occurring after ischemia-reperfusion injury as well as acute calcineurin inhibitor toxicity could easily be differentiated. Finally, the quantified ultrasound signal correlated significantly with the number of infiltrating T cells obtained by histology and with CD3 mRNA levels, as well as with chemokine CXCL9, CXCL11, and CCL19 mRNA but not with KIM-1 mRNA expression, thereby representing the severity of graft inflammation but not the degree of kidney injury. In summary, we demonstrate that antibody-mediated contrast-enhanced ultrasound targeting T lymphocytes could be a promising tool for an easy and reproducible assessment of acute rejection after renal transplantation.