Eric J. Lepin

Phosphorylated S6 Ribosomal Protein: A Novel Biomarker of Antibody‐Mediated Rejection in Heart Allografts

We tested the hypothesis that phosphorylation of S6 ribosomal protein (S6RP), a downstream target of the PI3K/Akt/mTOR pathway, is a biomarker of antibody‐mediated rejection (AMR) in heart allografts. Primary cultures of human aortic and microvascular endothelial cells (EC) were treated with anti‐HLA class I and class II antibodies (Ab) and cell lysates were studied for phosphorylation of S6 ribosmal protein at Serine235/236 (p‐S6RP). Treatment of cultured EC with anti‐class I and class II Ab stimulated S6RP phosphorylation. Immunohistochemical techniques were used to detect the level of p‐S6RP in endomyocardial biopsies (n = 131) from 46 heart transplant recipients and the results were correlated with histopathological diagnosis of rejection, C4d staining, production of posttransplant anti‐HLA Ab and clinical outcome. Increased phosphorylation of S6RP in endomyocardial biopsies was significantly associated with the diagnosis of AMR (p < 0.0001). No significant association between acute cellular rejection (ACR) and p‐S6RP was observed. C4d staining was positively associated with both AMR and p‐S6RP. Posttransplant anti‐HLA class II Ab production was also significantly associated with a positive p‐S6RP status in cardiac biopsies. These results indicate that p‐S6RP is a useful biomarker for the diagnosis of AMR.

Humanized radioiodinated minibody for imaging of prostate stem cell antigen-expressing tumors.

Purpose: Prostate stem cell antigen (PSCA) is a cell surface glycoprotein that is overexpressed in prostate cancer, including hormone refractory disease. Previous preclinical studies showed the intact anti-PSCA antibodies, 1G8 and hu1G8, localized specifically to PSCA-expressing xenografts. Optimal micro positron emission tomography (microPET) imaging using hu1G8, however, required a delay of 168 hours postinjection. In this study, the 2B3 minibody (an 80-kDa engineered antibody fragment) has been produced for rapid targeting and imaging. Experimental Design: A gene encoding a PSCA-specific minibody, VL-linker-VH-hinge-huIgG1 CH3, was assembled. The minibody was expressed by secretion from mammalian cells and purified by cation exchange chromatography. Relative affinity and specificity were determined by competition ELISA and flow cytometry. Serial microPET imaging using a 124I-labeled minibody was conducted at 4 and 21 hours in mice bearing LAPC-9 AD, LAPC-9 AI, PC-3, and LNCaP-PSCA human prostate cancer xenografts. Tumor and tissue biodistribution was determined, and region of interest analysis of the images was conducted. Results: Yields of 20 mg/L purified 2B3 minibody were obtained that showed specific binding to LNCaP-PSCA cells. Purified 2B3 minibody showed specific binding to LNCaP-PSCA cells with an apparent affinity of 46 nmol/L. Radioiodinated 2B3 minibody showed rapid nontarget tissue and blood clearance kinetics (t1/2β = 11.2 hours). MicroPET scanning using the 124I-2B3 minibody showed both androgen-dependent and -independent tumors as early as 4 hours and excellent high contrast images at 21 hours postinjection. Conclusions: Imaging PSCA-positive prostate cancer is feasible using an intermediate size antibody fragment at 21 hours.

Cys-diabody Quantum Dot Conjugates (ImmunoQdots) for Cancer Marker Detection

The present work demonstrates the use of small bivalent engineered antibody fragments, cys-diabodies, for biological modification of nanoscale particles such as quantum dots (Qdots) for detection of target antigens. Novel bioconjugated quantum dots known as immunoQdots (iQdots) were developed by thiol-specific oriented coupling of tumor specific cys-diabodies, at a position away from the antigen binding site to amino PEG CdSe/ZnS Qdots. Initially, amino PEG Qdot 655 were coupled with reduced anti-HER2 cys-diabody by amine-sulfhydryl-reactive linker [N-ε-maleimidocaproyloxy] succinimide ester (EMCS) to produce anti-HER2 iQdot 655. Spectral characterization of the conjugate revealed that the spectrum was symmetrical and essentially identical to unconjugated Qdot. Specific receptor binding activity of anti-HER2 iQdot 655 was confirmed by flow cytometry on HER2 positive and negative cells. Immunofluorescence results showed homogeneous surface labeling of the cell membrane with Qdot 655 conjugate. In addition, cys-diabodies specific for HER2, as well as prostate stem cell antigen (PSCA), were conjugated successfully with amino PEG Qdot 800. All of these iQdots retain the photoluminescence properties of the unconjugated Qdot 800 as well as the antigen binding specificity of the cys-diabody as demonstrated by flow cytometry. Simultaneous detection of two tumor antigens on LNCaP/PSCA prostate cancer cells (which express PSCA and HER2) in culture was possible using two iQdots, anti-HER2 iQdot 655 and anti-PSCA iQdot 800. Thus, these iQdots are potentially useful as optical probes for sensitive, multiplexed detection of surface markers on tumor cells. The present thiol-specific conjugation method demonstrates a general approach for site-specific oriented coupling of cys-diabodies to a wide variety of nanoparticles without disturbing the antigen binding site and maintaining small size compared to intact antibody.

Microfluidic-based 18F-labeling of biomolecules for immuno-positron emission tomography.

Methods for tagging biomolecules with fluorine 18 as immuno–positron emission tomography (immunoPET) tracers require tedious optimization of radiolabeling conditions and can consume large amounts of scarce biomolecules. We describe an improved method using a digital microfluidic droplet generation (DMDG) chip, which provides computer-controlled metering and mixing of 18F tag, biomolecule, and buffer in defined ratios, allowing rapid scouting of reaction conditions in nanoliter volumes. The identified optimized conditions were then translated to bench-scale 18F labeling of a cancer-specific engineered antibody fragments, enabling microPET imaging of tumors in xenografted mice at 0.5 to 4 hours postinjection.

Diabodies Targeting Epithelial Membrane Protein 2 Reduce Tumorigenicity of Human Endometrial Cancer Cell Lines

Purpose: Endometrial cancer is the most common gynecologic malignancy. One promising biomarker is epithelial membrane protein 2 (EMP2), and its expression is an independent prognostic indicator for tumors with poor clinical outcome expression. The present study assesses the suitability of EMP2 as a therapeutic target. Experimental Design: Human monovalent anti-EMP2 antibody fragments were isolated from a human phage display library and engineered as bivalent antibody fragments (diabodies) with specificity and avidity to both EMP2 peptides and native cell-surface EMP2 protein. Diabodies were assessed using cell death and apoptosis assays. In addition, the efficacy of EMP2 diabodies on endometrial cancer tumors was determined using mouse xenograft models. Results: Treatment of human endometrial adenocarcinoma cell lines with anti-EMP2 diabodies induced significant cell death and caspase-3 cleavage in vitro. These responses correlated with cellular EMP2 expression and were augmented by progesterone, which physiologically induces EMP2 expression. In vivo, treatment of subcutaneous human xenografts of HEC-1A cell lines with anti-EMP2 diabodies suppressed tumor growth and induced cell death in the xenograft. Conclusions: These findings suggest that EMP2 may be a potential pharmacologic target for human endometrial cancer.

Fluorescent Image–Guided Surgery with an Anti-Prostate Stem Cell Antigen (PSCA) Diabody Enables Targeted Resection of Mouse Prostate Cancer Xenografts in Real Time

Purpose: The inability to visualize cancer during prostatectomy contributes to positive margins, cancer recurrence, and surgical side effects. A molecularly targeted fluorescent probe offers the potential for real-time intraoperative imaging. The goal of this study was to develop a probe for image-guided prostate cancer surgery. Experimental Design: An antibody fragment (cys-diabody, cDb) against prostate stem cell antigen (PSCA) was conjugated to a far-red fluorophore, Cy5. The integrity and binding of the probe to PSCA was confirmed by gel electrophoresis, size exclusion, and flow cytometry, respectively. Subcutaneous models of PSCA-expressing xenografts were used to assess the biodistribution and in vivo kinetics, whereas an invasive intramuscular model was utilized to explore the performance of Cy5-cDb–mediated fluorescence guidance in representative surgical scenarios. Finally, a prospective, randomized study comparing surgical resection with and without fluorescent guidance was performed to determine whether this probe could reduce the incidence of positive margins. Results: Cy5-cDb demonstrated excellent purity, stability, and specific binding to PSCA. In vivo imaging showed maximal signal-to-background ratios at 6 hours. In mice carrying PSCA+ and negative (−) dual xenografts, the mean fluorescence ratio of PSCA+/− tumors was 4.4:1. In surgical resection experiments, residual tumors <1 mm that were missed on white light surgery were identified and resected using fluorescence guidance, which reduced the incidence of positive surgical margins (0/8) compared with white light surgery alone (7/7). Conclusions: Fluorescently labeled cDb enables real-time in vivo imaging of prostate cancer xenografts in mice, and facilitates more complete tumor removal than conventional white light surgery alone. Clin Cancer Res; 22(6); 1403–12. ©2015 AACR. See related commentary by van Leeuwen and van der Poel, p. 1304

Complement-independent mechanisms of antigraft antibodies in transplant arteriosclerosis and accommodation

Purpose of reviewPatients exhibiting a humoral immune response to the allograft demonstrate a lower graft survival and an increased risk of the development of transplant arteriosclerosis. Unfortunately, current immunosuppressive regimens are not effective in controlling the humoral response to the allograft. This article focuses on the recent progress made toward understanding the role of antigraft antibodies in chronic rejection and graft accommodation. Recent findingsRecent studies suggest that antigraft antibodies play an important role in controlling endothelial and smooth muscle cell homeostasis, in the absence of complement, by binding to ligands on the surface of the cell and transducing intracellular signals. Antigraft antibodies exhibit two primary effector functions: stimulation of cell proliferation and upregulation of cell survival genes. The intracellular events initiated by antibody ligation appear to be influenced by the cell type, specificity, and concentration of the antibody and the degree of molecular aggregation. SummaryCharacterizing the response of endothelial and smooth muscle cells to antigraft antibodies and elucidating the signaling pathways have the potential to identify novel immunotherapies to promote cell survival while preventing transplant arteriosclerosis.

Positron Emission Tomographic Imaging of Iodine 124 Anti-Prostate Stem Cell Antigen-Engineered Antibody Fragments in LAPC-9 Tumor-Bearing Severe Combined Immunodeficiency Mice

The humanized antibody (hu1G8) has been shown to localize to prostate stem cell antigen (PSCA) and image PSCA-positive xenografts. We previously constructed hu1G8 anti-PSCA antibody fragments and tested them for tumor targeting and the ability to image prostate cancer at early and late time points postinjection by positron emission tomography (PET). We now then compare the PET imaging and the radioactivity accumulation properties in prostate cancer tumors and nontarget tissues to determine the superior 124I-labeled hu1G8 antibody format. 124I-labeled diabody, minibody, scFv-Fc, scFv-Fc double mutant (DM), and parental IgG were administered into severe combined immunodeficiency (SCID) mice bearing LAPC-9 xenografts and followed by whole-body PET imaging of mice at preselected time points. Regions of interest were manually drawn around tumor and nontarget tissues and evaluated for radioactivity accumulation. The 124I-hu1G8 IgG has its best time point for tumor high-contrast imaging at 168 hours postinjection. The 124I-hu1G8 minibody at 44 hours postinjection results in superior tumor high-contrast imaging compared to the other antibody formats. The 124I-hu1G8 minibody at 44 hours postinjection also has comparable percent tumor radioactivity compared to 124I-hu1G8 IgG at 168 hours postinjection. The 124I-hu1G8 minibody is the best engineered hu1G8 antibody format for imaging prostate cancer.

Abstract 4283: Evaluation of PSCA-specific minibody for in vivo imaging

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Introduction: Molecular targeted therapy of advanced prostate cancer requires the ability to classify tumors at the molecular level without invasive tissue sampling. Noninvasive molecular imaging can yield information on tumor localization, phenotype, and response to therapy. Previously we have generated humanized antibody to prostate stem cell antigen (PSCA), a cell surface marker highly elevated in prostate cancers, and used the antibody to detect tumors by PET imaging. To improve affinity, minibody variants were obtained by yeast display, and one clone, A11, demonstrated high imaging contrast in PSCA positive xenograft tumors. However, xenograft tumors are limited as model of natural disease progression, and cannot be used to assess background uptake of antibody in normal tissues, given that the humanized antibody does not recognize the murine (m)PSCA. This study aims to evaluate the biodistribution of A11 minibody in a mouse model expressing human (h)PSCA. Such model is important for assessing minibody performance prior to further clinical development. Methods: hPSCA knock-in (KI) mouse was generated by replacing exon 1 of mPSCA with hPSCA cDNA thereby driving hPSCA expression under mPSCA promoter. hPSCA-KI line was evaluated for hPSCA expression in major organs by RT-PCR and immunohistochemistry. For imaging study, groups of 8 weeks old hPSCA-KI and wild-type mice were injected with radiolabeled (I-124) minibody A11 and imaged by microPET at 4 and 20 hrs post injection. Mice were then sacrificed and major organs collected, weighed, and counted to determine biodistribution of A11. Results: By immunohistochemistry, hPSCA is expressed in prostate, bladder and stomach of heterozygous hPSCA-KI mice as expected. Other organs showed much lower or no expression of hPSCA, as confirmed by RT-PCR. MicroPET imaging showed specific uptake of I-124 labeled A11 in the prostate, bladder and stomach of hPSCA-KI mice. A11 biodistribution (% injected dose/gram of tissue) was significantly higher in hPSCA-KI mice compared to wild-type mice: prostate (0.83 ± 0.13 vs. 0.49 ± 0.04), bladder (3.21 ± 0.42 vs. 1.66 ± 0.25) and stomach (2.00 ± 0.22 vs. 0.79 ± 0.05). There was no uptake difference between groups for other major organs including pancreas, lung, kidney, spleen and liver. Conclusions: The minibody uptake in hPSCA-KI mice was specific to normal site of expression (prostate, bladder and stomach). While the % uptake was significantly higher than wild-type controls, the actual levels were modest and should not necessarily interfere with tumor imaging. This part of the study remains to be investigated. PSCA has been shown to be elevated in the PTEN conditional knockout prostate cancer mouse model. Thus, hPSCA-KI;PTEN-knockout mice are being generated and will be used to evaluate the PSCA-specific minibody as a tumor imaging tool. PSCA minibody imaging may be useful as a surrogate to diagnose and monitor response to targeted therapy of this type of prostate cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4283. doi:10.1158/1538-7445.AM2011-4283