NY-ESO-1 and LAGE-1 are cancer testis antigens with an ideal profile for tumor immunotherapy, combining up-regulation in many cancer types with highly restricted expression in normal tissues and sharing a common HLA-A*0201 epitope, 157–165. Here, we present data to describe the specificity and anti-tumor activity of a bifunctional ImmTAC, comprising a soluble, high-affinity T-cell receptor (TCR) specific for NY-ESO-1157–165 fused to an anti-CD3 scFv. This reagent, ImmTAC-NYE, is shown to kill HLA-A2, antigen-positive tumor cell lines, and freshly isolated HLA-A2- and LAGE-1-positive NSCLC cells. Employing time-domain optical imaging, we demonstrate in vivo targeting of fluorescently labelled high-affinity NYESO-specific TCRs to HLA-A2-, NY-ESO-1157–165-positive tumors in xenografted mice. In vivo ImmTAC-NYE efficacy was tested in a tumor model in which human lymphocytes were stably co-engrafted into NSG mice harboring tumor xenografts; efficacy was observed in both tumor prevention and established tumor models using a GFP fluorescence readout. Quantitative RT-PCR was used to analyze the expression of both NY-ESO-1 and LAGE-1 antigens in 15 normal tissues, 5 cancer cell lines, 10 NSCLC, and 10 ovarian cancer samples. Overall, LAGE-1 RNA was expressed at a greater frequency and at higher levels than NY-ESO-1 in the tumor samples. These data support the clinical utility of ImmTAC-NYE as an immunotherapeutic agent for a variety of cancers.Electronic supplementary materialThe online version of this article (doi:10.1007/s00262-012-1384-4) contains supplementary material, which is available to authorized users.

Elevated tumor interstitial fluid pressure (TIFP) is a characteristic of most solid tumors. Clinically, TIFP may hamper the uptake of chemotherapeutic drugs into the tumor tissue reducing their therapeutic efficacy. In this study, a means of modulating TIFP to increase the flux of macromolecules into tumor tissue is presented, which is based on the rationale that elevated plasma colloid osmotic pressure (COP) pulls water from tumor interstitium lowering the TIFP. Concentrated human serum albumin (20% HSA), used as an agent to enhance COP, reduced the TIFP time-dependently from 8 to 2 mm Hg in human tumor xenograft models bearing A431 epidermoid vulva carcinomas. To evaluate whether this reduction facilitates the uptake of macromolecules, the intratumoral distribution of fluorescently conjugated dextrans (2.5 mg/ml) and cetuximab (2.0 mg/ml) was probed using novel time domain nearinfrared fluorescence imaging. This method permitted discrimination and semiquantification of tumor-accumulated conjugate from background and unspecific probe fluorescence. The coadministration of 20% HSA together with either dextrans or cetuximab was found to lower the TIFP significantly and increase the concentration of the substances within the tumor tissue in comparison to control tumors. Furthermore, combined administration of 20% HSA plus cetuximab reduced the tumor growth significantly in comparison to standard cetuximab treatment. These data demonstrate that increased COP lowers the TIFP within hours and increases the uptake of therapeutic macromolecules into the tumor interstitium leading to reduced tumor growth. This model represents a novel approach to facilitate the delivery of therapeutics into tumor tissue, particularly monoclonal antibodies.

There is a lack of available methods to noninvasively quantify lymphatic function in small experimental animals, a necessity for studies on lymphatic system pathophysiology. We present a new method to quantify lymph flow in mice and rats, based on optically monitoring the depot clearance of near-infrared fluorescently labeled albumin and subsequent calculation of removal rate constants (k). BSA was conjugated with Alexa680 NHS ester and remained stable in protein-rich solutions without free dye dissociation. To assess lymph flow, mice or rats were imaged every 30 or 60 min during a 3- to 6-h period following an intradermal injection of 0.5 or 1 μl Alexa680-albumin. Mice were awake between measurements, whereas rats were anesthetized throughout the experiment. The k, a parameter defined as equivalent to lymph flow, was calculated from the slopes of the resultant log-linear washout curves and averaged -0.40 ± 0.03 and -0.30 ± 0.02%/min for control C57BL/6 and C3H mice, respectively. Local administration of the vasoconstrictor endothelin-1 in mice led to a significant reduction in k, whereas overhydration in rats increased k, reflecting the coupling between capillary filtration and lymph flow. Furthermore, k was 50% of wild type in lymphedema Chy mice where dermal lymphatics are absent. We conclude that lymph flow can be determined as its rate constant k by optical imaging of depot clearance of submicroliter amounts of Alexa680-albumin. The method offers a minimally invasive, reproducible, and simple alternative to assess lymphatic function in mice and rats.

Key Points• Multiplexing antibodies against common human epitopes all labeled with the same fluorophore facilitates optical imaging of heterogenous AML.• Multiplexing fluorescently labeled monoclonal antibodies permits optical imaging of primary patient xenograft pathology/therapy response.Antibodies play a fundamental role in diagnostic immunophenotyping of leukemias and in cell-targeting therapy. However, this versatility is not reflected in imaging diagnostics. In the present study, we labeled anti-human mAbs monochromatically against selected human myeloid markers expressed on acute myeloid leukemia (AML) cells, all with the same near-infrared fluorochrome. In a novel "multiplexing" strategy, we then combined these mAbs to overcome the limiting target-to-background ratio to image multiple xenografts of AML. Time-domain imaging was used to discriminate autofluorescence from the distinct fluorophore-conjugated antibodies. Imaging with multiplexed mAbs demonstrated superior imaging of AML to green fluorescent protein or bioluminescence and permitted evaluation of therapeutic efficacy with the standard combination of anthracycline and cytarabine in primary patient xenografts. Multiplexing mAbs against CD11b and CD11c provided surrogate imaging biomarkers of differentiation therapy in an acute promyelocytic leukemia model treated with all-trans retinoic acid combined with the histone-deacetylase inhibitor valproic acid. We present herein an optimizedapplication of multiplexed immunolabeling in vivo for optical imaging of AML cellxenografts that provides reproducible, highly accurate disease staging and monitoring of therapeutic effects. (Blood. 2013;121(7):e34-e42) IntroductionDespite modern advances in therapeutics and improvement in the diagnosis of acute myeloid leukemia (AML) subtypes, the majority of patients die from their disease. 1,2 Thus, in the absence of definitive in vitro models of human AML and failure of significant numbers of new drugs late in clinical trials, 3,4 it is essential that murine AML models are further developed to exploit more specific, targeted therapeutics. Although preclinical development of therapeutics has previously exploited genetic and syngeneic models of AML, 5,6 subcutaneous xenografting of human cell lines in immunodeficient mice has been a popular screening tool. 7 However, the ease of measuring therapeutic efficacy with simple caliper measurements in subcutaneous tumors is offset by the recognition that these models do not typify clinical disease. 8,9 True evaluation of therapeutic potential in this disease necessitates preclinical screening in multiple, systemic, or orthotopic xenograft models of AML, increasingly with primary patient cells that reflect the heterogeneity of the disease. 10 To facilitate the use of humanized orthotopic AML models in therapeutic settings, investigators have increasingly turned to noninvasive preclinical imaging techniques to define efficacy. 11 Optical imaging of reporter gene expression 12,13 is the preferred modality owing to low cost an...