Evaluation of the DLL3-targeting antibody-drug conjugate rovalpituzumab tesirine in preclinical models of neuroblastoma.

Neuroblastomas have neuroendocrine features and often show similar gene expression patterns to small cell lung cancer including high expression of delta-like ligand 3 (DLL3). Here we determine the efficacy of rovalpituzumab tesirine (Rova-T), an antibody drug conjugated (ADC) with a pyrrolobenzodiazepine (PBD) dimer toxin targeting DLL3, in preclinical models of human neuroblastoma. We evaluated DLL3 expression in RNA sequencing data sets and performed immunohistochemistry (IHC) on neuroblastoma patient derived xenograft (PDX), human neuroblastoma primary tumor and normal childhood tissue microarrays (TMAs). We then evaluated the activity of Rova-T against 11 neuroblastoma PDX models using varying doses and schedules and compared anti-tumor activity to expression levels. DLL3 mRNA was differentially overexpressed in neuroblastoma at comparable levels to small cell lung cancer, as well as Wilms and rhabdoid tumors. DLL3 protein was robustly expressed across the neuroblastoma PDX array, but membranous staining was variable. The human neuroblastoma array, however, showed staining in only 44% of cases, whereas no significant staining was observed in the normal childhood tissue array. Rova-T showed a clear dose response effect across the 11 models tested, with a single dose inducing a complete or partial response in 3/11 and stable disease in another 3/11 models. No overt signs of toxicity were observed, and there was no treatment-related mortality. Strong membranous staining was necessary, but not sufficient, for anti-tumor activity. Rova-T has activity in a subset of neuroblastoma preclinical models, but heterogeneous expression in these models and the near absence of expression seen in human tumors suggests that any DLL3-targeting clinical trial should be only performed with a robust companion diagnostic to evaluate DLL3 expression for patient selection.

ABBV-011, A Novel, Calicheamicin-Based Antibody-Drug Conjugate, Targets SEZ6 to Eradicate Small Cell Lung Cancer Tumors.

In the past year, four antibody-drug conjugates (ADC) were approved, nearly doubling the marketed ADCs in oncology. Among other attributes, successful ADCs optimize targeting antibody, conjugation chemistry, and payload mechanism of action. Here, we describe the development of ABBV-011, a novel SEZ6-targeted, calicheamicin-based ADC for the treatment of small cell lung cancer (SCLC). We engineered a calicheamicin conjugate that lacks the acid-labile hydrazine linker that leads to systemic release of a toxic catabolite. We then screened a patient-derived xenograft library to identify SCLC as a tumor type with enhanced sensitivity to calicheamicin ADCs. Using RNA sequencing (RNA-seq) data from primary and xenograft SCLC samples, we identified seizure-related homolog 6 (SEZ6) as a surface-expressed SCLC target with broad expression in SCLC and minimal normal tissue expression by both RNA-seq and IHC. We developed an antibody targeting SEZ6 that is rapidly internalized upon receptor binding and, when conjugated to the calicheamicin linker drug, drives potent tumor regression in vitro and in vivo. These preclinical data suggest that ABBV-011 may provide a novel treatment for patients with SCLC and a rationale for ongoing phase I studies (NCT03639194).

Delta-Like Protein 3 Expression and Targeting in Merkel Cell Carcinoma.

PURPOSE: Delta-like protein 3 (DLL3) is being developed as a predictive biomarker for DLL3-targeting antibody-drug conjugate and other therapies. Given the neuroendocrine features of Merkel cell carcinoma (MCC), we sought to evaluate DLL3 expression and its role in MCC. EXPERIMENTAL DESIGN: Formalin-fixed and paraffin-embedded MCC cases were consecutively selected. Immunohistochemistry was performed for DLL3 (SC16.65 antibody) and polyomavirus large T-antigen (sc-136172 antibody). Slides were read out for percentage of positive tumor cells. Cox proportional hazards model was applied to assess the association between DLL3 expression and overall survival (OS). A patient with a DLL3-expressing MCC was treated with rovalpituzumab tesirine (Rova-T) in the "other tumor" cohort of NCT02709889 and assessed for response. RESULTS: The median H-score of DLL3 expression of 65 patients included was 60 (interquartile range, 30-100). Fifty-eight cases (89%) had ≥1% tumor cells positive for DLL3 expression with any intensity, of which the median DLL3 expression was 50% (interquartile range, 25%-70%). Thirty-four cases (52%) had ≥50% tumor cells positive for DLL3 expression with any intensity. Higher H-score of DLL3 expression was associated with higher polyomavirus nuclear expression (p = .003) when it was dichotomized to negative versus positive. H-score of DLL3 expression did not predict OS of patients with MCC (p = .4) after being adjusted for common clinicopathological factors. A patient treated with Rova-T for refractory metastatic MCC achieved partial response. CONCLUSIONS: DLL3 overexpression is very common in MCC by immunohistochemistry. The response to treatment suggests that DLL3 expression may have predictive relevance for DLL3-targeting therapies in MCC. IMPLICATIONS FOR PRACTICE: Delta-like protein 3 (DLL3) is being developed as a predictive biomarker to identify patients for treatment with DLL3-targeting agents. Merkel cell carcinoma (MCC) is an aggressive neuroendocrine carcinoma of the skin. It was found that DLL3 overexpression is very common in MCC by immunohistochemistry and significantly associated with Merkel cell polyomavirus expression. Despite the lack of prognostic significance in this cohort, DLL3 expression may have predictive relevance for DLL3-targeting therapies in MCC. The high levels of DLL3 expression in a subset of MCC may potentially be used to select patients to receive DLL3-targeting therapies.

Delta-like protein 3 expression and therapeutic targeting in neuroendocrine prostate cancer.

Histologic transformation to small cell neuroendocrine prostate cancer occurs in a subset of patients with advanced prostate cancer as a mechanism of treatment resistance. Rovalpituzumab tesirine (SC16LD6.5) is an antibody-drug conjugate that targets delta-like protein 3 (DLL3) and was initially developed for small cell lung cancer. We found that DLL3 is expressed in most of the castration-resistant neuroendocrine prostate cancer (CRPC-NE) (36 of 47, 76.6%) and in a subset of castration-resistant prostate adenocarcinomas (7 of 56, 12.5%). It shows minimal to no expression in localized prostate cancer (1 of 194) and benign prostate (0 of 103). DLL3 expression correlates with neuroendocrine marker expression, RB1 loss, and aggressive clinical features. DLL3 in circulating tumor cells was concordant with matched metastatic biopsy (87%). Treatment of DLL3-expressing prostate cancer xenografts with a single dose of SC16LD6.5 resulted in complete and durable responses, whereas DLL3-negative models were insensitive. We highlight a patient with neuroendocrine prostate cancer with a meaningful clinical and radiologic response to SC16LD6.5 when treated on a phase 1 trial. Overall, our findings indicate that DLL3 is preferentially expressed in CRPC-NE and provide rationale for targeting DLL3 in patients with DLL3-positive metastatic prostate cancer.

Cell Surface Notch Ligand DLL3 is a Therapeutic Target in Isocitrate Dehydrogenase-mutant Glioma.

PURPOSE: Isocitrate dehydrogenase (IDH)-mutant glioma is a distinct glioma molecular subtype for which no effective molecularly directed therapy exists. Low-grade gliomas, which are 80%-90% IDH-mutant, have high RNA levels of the cell surface Notch ligand DLL3. We sought to determine DLL3 expression by IHC in glioma molecular subtypes and the potential efficacy of an anti-DLL3 antibody-drug conjugate (ADC), rovalpituzumab tesirine (Rova-T), in IDH-mutant glioma. EXPERIMENTAL DESIGN: We evaluated DLL3 expression by RNA using TCGA data and by IHC in a discovery set of 63 gliomas and 20 nontumor brain tissues and a validation set of 62 known IDH wild-type and mutant gliomas using a monoclonal anti-DLL3 antibody. Genotype was determined using a DNA methylation array classifier or by sequencing. The effect of Rova-T on patient-derived endogenous IDH-mutant glioma tumorspheres was determined by cell viability assay. RESULTS: Compared to IDH wild-type glioblastoma, IDH-mutant gliomas have significantly higher DLL3 RNA (P < 1 × 10-15) and protein by IHC (P = 0.0014 and P < 4.3 × 10-6 in the discovery and validation set, respectively). DLL3 immunostaining was intense and homogeneous in IDH-mutant gliomas, retained in all recurrent tumors, and detected in only 1 of 20 nontumor brains. Patient-derived IDH-mutant glioma tumorspheres overexpressed DLL3 and were potently sensitive to Rova-T in an antigen-dependent manner. CONCLUSIONS: DLL3 is selectively and homogeneously expressed in IDH-mutant gliomas and can be targeted with Rova-T in patient-derived IDH-mutant glioma tumorspheres. Our findings are potentially immediately translatable and have implications for therapeutic strategies that exploit cell surface tumor-associated antigens.

Transcriptomic and Protein Analysis of Small-cell Bladder Cancer (SCBC) Identifies Prognostic Biomarkers and DLL3 as a Relevant Therapeutic Target.

PURPOSE: Transcriptomic profiling can shed light on the biology of small-cell bladder cancer (SCBC), nominating biomarkers, and novel therapeutic targets. EXPERIMENTAL DESIGN: Sixty-three patients with SCBC had small-cell histology confirmed and quantified by a genitourinary pathologist. Gene expression profiling was performed for 39 primary tumor samples, 1 metastatic sample, and 6 adjacent normal urothelium samples (46 total) from the same cohort. Protein levels of differentially expressed therapeutic targets, DLL3 and PDL1, and also CD56 and ASCL1, were confirmed by IHC. A SCBC PDX model was utilized to assess in vivo efficacy of DLL3-targeting antibody-drug conjugate (ADC). RESULTS: Unsupervised hierarchical clustering of 46 samples produced 4 clusters that correlated with clinical phenotypes. Patients whose tumors had the most "normal-like" pattern of gene expression had longer overall survival (OS) compared with the other 3 clusters while patients with the most "metastasis-like" pattern had the shortest OS (P = 0.047). Expression of DLL3, PDL1, ASCL1, and CD56 was confirmed by IHC in 68%, 30%, 52%, and 81% of tissue samples, respectively. In a multivariate analysis, DLL3 protein expression on >10% and CD56 expression on >30% of tumor cells were both prognostic of shorter OS (P = 0.03 each). A DLL3-targeting ADC showed durable antitumor efficacy in a SCBC PDX model. CONCLUSIONS: Gene expression patterns in SCBC are associated with distinct clinical phenotypes ranging from more indolent to aggressive disease. Overexpression of DLL3 mRNA and protein is common in SCBC and correlates with shorter OS. A DLL3-targeted ADC demonstrated in vivo efficacy superior to chemotherapy in a PDX model of SCBC.

Prevalence of Delta-like protein 3 expression in patients with small cell lung cancer.

OBJECTIVES: Rovalpituzumab tesirine (Rova-T), an antibody-drug conjugate directed against Delta-like protein 3 (DLL3), is under development for patients with small cell lung cancer (SCLC) positive for this protein. However, the prevalence of DLL3 expression and its association with patient ethnicity and other characteristics have remained unclear. MATERIAIS AND METHODS: Tumor samples from 63 patients with SCLC were subjected to immunohistochemical staining for DLL3. The relation of patient characteristics including sex, age, disease stage, and smoking history to DLL3 expression status was analyzed. RESULTS AND CONLUSIONS: Fifty-two patients (83%) were positive for DLL3 expression, with 20 patients (32%) being positive in at least 50% of cancer cells (DLL3-high). DLL3 expression was not associated with any of the patient characteristics examined. In addition, overall survival did not differ between DLL3-high and DLL3-low patients. Our results reveal that DLL3 is expressed in tumor specimens from most patients with SCLC, and they should inform the undertaking of clinical trials of Rova-T including an ongoing phase I study (NCT03086239) in Japan as well as global phase III trials (NCT03061812 and NCT03033511).

Noninvasive Interrogation of DLL3 Expression in Metastatic Small Cell Lung Cancer.

The Notch ligand DLL3 has emerged as a novel therapeutic target expressed in small cell lung cancer (SCLC) and high-grade neuroendocrine carcinomas. Rovalpituzumab teserine (Rova-T; SC16LD6.5) is a first-in-class DLL3-targeted antibody-drug conjugate with encouraging initial safety and efficacy profiles in SCLC in the clinic. Here we demonstrate that tumor expression of DLL3, although orders of magnitude lower in surface protein expression than typical oncology targets of immunoPET, can serve as an imaging biomarker for SCLC. We developed 89Zr-labeled SC16 antibody as a companion diagnostic agent to facilitate selection of patients for treatment with Rova-T based on a noninvasive interrogation of the in vivo status of DLL3 expression using PET imaging. Despite low cell-surface abundance of DLL3, immunoPET imaging with 89Zr-labeled SC16 antibody enabled delineation of subcutaneous and orthotopic SCLC tumor xenografts as well as distant organ metastases with high sensitivity. Uptake of the radiotracer in tumors was concordant with levels of DLL3 expression and, most notably, DLL3 immunoPET yielded rank-order correlation for response to SC16LD6.5 therapy in SCLC patient-derived xenograft models. Cancer Res; 77(14); 3931-41. ©2017 AACR.

Tissue-type plasminogen activator suppresses activated stellate cells through low-density lipoprotein receptor-related protein 1.

Hepatic stellate cell (HSC) activation and trans-differentiation into myofibroblast (MFB)-like cells is key for fibrogenesis after liver injury and a potential therapeutic target. Recent studies demonstrated that low-density lipoprotein receptor-related protein 1 (LRP1)-dependent signaling by tissue-type plasminogen activator (t-PA) is a pro-fibrotic regulator of the MFB phenotype in kidney. This study investigated whether LRP1 signaling by t-PA is also relevant to HSC activation following injury. Primary and immortalized rat HSCs were treated with t-PA and assayed by western blot, MTT, and TUNEL. In vitro results were then verified using an in vivo, acute carbon tetrachloride (CCl4) injury model that examined the phenotype and recovery kinetics of MFBs from wild-type animals vs mice with a global (t-PA) or HSC-targeted (LRP1) deletion. In vitro, in contrast to kidney MFBs, exogenous, proteolytically inactive t-PA suppressed, rather than induced, activation markers in HSCs following phosphorylation of LRP1. This process was mediated by LRP1 as inhibition of t-PA binding to LRP1 blocked the effects of t-PA. In vivo, following acute injury, phosphorylation of LRP1 on activated HSCs occurred immediately prior to their disappearance. Mice lacking t-PA or LRP1 retained higher densities of activated HSCs for a longer time period compared with control mice after injury cessation. Hence, t-PA, an FDA-approved drug, contributes to the suppression of activated HSCs following injury repair via signaling through LRP1. This renders t-PA a potential target for exploitation in treating patients with fibrosis.

IRF-1 promotes liver transplant ischemia/reperfusion injury via hepatocyte IL-15/IL-15Rα production.

Ischemia and reperfusion (I/R) injury following liver transplantation (LTx) is an important problem that significantly impacts clinical outcomes. IFN regulatory factor-1 (IRF-1) is a nuclear transcription factor that plays a critical role in liver injury. Our objective was to determine the immunomodulatory role of IRF-1 during I/R injury following allogeneic LTx. IRF-1 was induced in liver grafts immediately after reperfusion in both human and mouse LTx. IRF-1 contributed significantly to I/R injury because IRF-1-knockout (KO) grafts displayed much less damage as assessed by serum alanine aminotransferase and histology. In vitro, IRF-1 regulated both constitutive and induced expression of IL-15, as well as IL-15Rα mRNA expression in murine hepatocytes and liver dendritic cells. Specific knockdown of IRF-1 in human primary hepatocytes gave similar results. In addition, we identified hepatocytes as the major producer of soluble IL-15/IL-15Rα complexes in the liver. IRF-1-KO livers had significantly reduced NK, NKT, and CD8(+) T cell numbers, whereas rIL-15/IL-15Rα restored these immune cells, augmented cytotoxic effector molecules, promoted systemic inflammatory responses, and exacerbated liver injury in IRF-1-KO graft recipients. These results indicate that IRF-1 promotes LTx I/R injury via hepatocyte IL-15/IL-15Rα production and suggest that targeting IRF-1 and IL-15/IL-15Rα may be effective in reducing I/R injury associated with LTx.

Breast tumor kinase/protein tyrosine kinase 6 (Brk/PTK6) activity in normal and neoplastic biliary epithelia.

BACKGROUND & AIMS: Breast tumor kinase (BRK) augments proliferation and promotes cell survival in breast cancers via interactions with SH2 and SH3 ligand-containing proteins, such as receptor tyrosine kinases (RTK; e.g. EGFR, ErbB2/neu). Since RTK contribute to cholangiocarcinoma (CC) evolution we probed BRK protein expression and function in normal and CC livers. METHODS: Immunohistochemical staining of normal livers and CC (n=93) in a tissue microarray and three CC and an immortalized human cholangiocyte cell lines (real-time PCR, Western blotting, siRNA) were used to study the functional relationships between BRK, EGFR, ErbB2, SAM68, and SPRR2a. RESULTS: BRK protein was expressed in normal human intrahepatic bile ducts; all CC cell lines and a majority of CC showed strong BRK protein expression. Multiplex immunostaining/tissue cytometry and immunoprecipitation studies showed: 1) BRK co-localized with EGFR and ErbB2/neu; 2) BRK(high)/EGFR(high)-co-expressing CC cells had significantly higher Ki67 labeling and; 3) stronger BRK protein expression was seen in perihilar and distal CC than intrahepatic CC and directly correlated with CC differentiation. In cell lines, BRK expression augmented proliferation in response to exogenous EGF, whereas BRK siRNA significantly reduced growth. The SH3 ligand-containing, SPRR2A activated pTyr342 BRK, which in turn, phosphorylated SAM68, causing nuclear localization and increased cell proliferation similar to observations in breast cancers. CONCLUSION: BRK expression in a majority of CC can interact with RTK, augmenting growth and interfering with proliferation inhibitors (SAM68). Therapeutically targeting BRK function (in addition to RTK) should be of benefit for CC treatment.

CD39 deficiency in murine liver allografts promotes inflammatory injury and immune-mediated rejection.

Adenosine triphosphate (ATP), an essential metabolic energy source, is released following cell apoptosis or necrosis. It acts as a damage-associated molecule pattern to stimulate innate immune cells. The ectonucleotidase CD39 regulates immune activation by hydrolysis of extracellular ATP. We have shown previously that CD39 expression by donor livers helps protect syngeneic grafts with extended (24h) cold preservation time from ischemia reperfusion injury. Given its immune regulatory properties, we hypothesized that CD39 expression in donor livers might modulate transplant tolerance that occurs following mouse allogeneic liver transplantation (LTx). Livers from C57BL/6 (B6) wild-type (WT) or CD39 KO mice were transplanted into normal C3H recipients with minimal (approximately 1h) cold ischemia. Serum alanine aminotransferase levels at day 4 post-LTx were significantly higher in animals given CD39KO compared with WT livers. Moreover, IFN-γ production by liver-infiltrating CD8(+) T cells at day 4 was significantly higher in CD39KO than in WT grafts. Furthermore, splenic T cells from CD39KO liver recipients exhibited greater proliferative responses to donor alloantigens than those from mice given WT grafts. By contrast, there was a concomitant significant reduction in the frequency of regulatory T cells (Treg) in CD39KO than in WT livers. Whereas WT liver allografts survived >100 days, no CD39KO grafts survived beyond 40 days (median survival time [MST]: WT: >100 days vs CD39KO: 8 days; p<0.01). In addition, soluble CD39 administration significantly prolonged CD39KO liver allograft survival (MST: 27.5 days). These novel data suggest that CD39 expression in liver allografts modulates tissue injury, inflammation, anti-donor effector T cell responses and Treg infiltration and can suppress transplant rejection.

Acute liver allograft antibody-mediated rejection: an inter-institutional study of significant histopathological features.

Acute antibody-mediated rejection (AMR) occurs in a small minority of sensitized liver transplant recipients. Although histopathological characteristics have been described, specific features that could be used (1) to make a generalizable scoring system and (2) to trigger a more in-depth analysis are needed to screen for this rare but important finding. Toward this goal, we created training and validation cohorts of putative acute AMR and control cases from 3 high-volume liver transplant programs; these cases were evaluated blindly by 4 independent transplant pathologists. Evaluations of hematoxylin and eosin (H&E) sections were performed alone without knowledge of either serum donor-specific human leukocyte antigen alloantibody (DSA) results or complement component 4d (C4d) stains. Routine histopathological features that strongly correlated with severe acute AMR included portal eosinophilia, portal vein endothelial cell hypertrophy, eosinophilic central venulitis, central venulitis severity, and cholestasis. Acute AMR inversely correlated with lymphocytic venulitis and lymphocytic portal inflammation. These and other characteristics were incorporated into models created from the training cohort alone. The final acute antibody-mediated rejection score (aAMR score)--the sum of portal vein endothelial cell hypertrophy, portal eosinophilia, and eosinophilic venulitis divided by the sum of lymphocytic portal inflammation and lymphocytic venulitis--exhibited a strong correlation with severe acute AMR in the training cohort [odds ratio (OR) = 2.86, P < 0.001] and the validation cohort (OR = 2.49, P < 0.001). SPSS tree classification was used to select 2 cutoffs: one that optimized specificity at a score > 1.75 (sensitivity = 34%, specificity = 86%) and another that optimized sensitivity at a score > 1.0 (sensitivity = 81%, specificity = 71%). In conclusion, the routine histopathological features of the aAMR score can be used to screen patients for acute AMR via routine H&E staining of indication liver transplant biopsy samples; however, a definitive diagnosis requires substantiation by DSA testing, diffuse C4d staining, and the exclusion of other insults.

Optimal lung inflation techniques in a rat lung transplantation model: a revisit.

BACKGROUND: Most of the experimental work assessing optimal lung inflation during lung graft preservation was performed in the late 1990s. Since that time, lung preservation before transplantation has been more standardized, and the optimal lung inflation techniques used during lung preservation in the current clinical setting remain undefined. Nonetheless, lung inflation during storage may play a pivotal role in optimal lung preservation. MATERIALS AND METHODS: Lewis rat lungs were perfused with and stored in cold, low-potassium dextran solution (Perfadex, Vitrolife, Göteborg, Sweden) for 6 hours at different levels of lung inflation (25, 50, 75, or 100% of vital capacity [VC]). Orthotopic left lung transplantation using cuff techniques was performed in syngeneic Lewis rats. Posttransplant allograft function, expression of proinflammatory mediators, and expression of lung surfactants were evaluated. RESULTS: Lungs inflated to 75 or 100% VC showed a significantly better oxygenation in blood gas analysis than lungs inflated to 25 or 50% VC. The levels of mRNAs for tumor necrosis factor-α, pro-interleukin-1ß, intracellular adhesion molecule 1 were attenuated in lungs inflated to 75 or 100% VC as compared with deflated lungs, suggesting reduced ischemia/reperfusion injury. In addition, transmission electron microscopy demonstrated better preserved lung surfactants in the alveolar space in the lungs inflated to 75 or 100% VC. CONCLUSIONS: Inflating lungs to 75 or 100% VC during preservation may be beneficial and result in better posttransplant allograft function through attenuated reperfusion injury and better preserved lung surfactants.

Low TCR signal strength induces combined expansion of Th2 and regulatory T cell populations that protect mice from the development of type 1 diabetes.

AIMS/HYPOTHESIS: Weak stimulation of CD4(+) T cells induces expansion of CD4(+) forkhead box P3(+) regulatory T cells (Tregs) and can also promote T helper (Th) 2 responses, which have demonstrable beneficial effects on autoimmune diabetes. This study explored the feasibility of combined Treg/Th2 expansion for immunotherapy of type 1 diabetes in NOD mice. METHODS: We compared Treg and Th responses to dendritic cells (DC) presenting scaled antigen doses to islet-specific NOD CD4(+) T cells. Flow cytometric and Luminex analyses were performed to determine the phenotype and cytokine profile of expanded T cells. The ability of expanded T cells to prevent type 1 diabetes was tested in an adoptive transfer model. RESULTS: In vitro studies revealed a hierarchical, selective expansion of Treg and T effector (Teff) populations at different antigen doses. Thus, a single low dose produced a mixture of Tregs Th2 and type 1 regulatory (Tr1) cells, which prevented diabetes in NOD-SCID mice and increased the ratio of Treg/Teff cells infiltrating the pancreatic islets. Subcutaneous injection of DC, previously shown to prevent diabetes in NOD mice, induced expansion of the same mixture of Tregs Tr1 and Th2 cells. Low-dose expansion of Treg required MHC-T cell receptor interaction and was partly dependent on T cell derived TGF-ß and IL-2. Autocrine IFN-γ was required for the promotion of diabetogenic Th1 cells at high antigen doses. CONCLUSIONS/INTERPRETATION: Weak stimulation of CD4(+) T cells with DC and low-dose antigen expands a combination of antigen-specific Tregs Th2 and Tr1 cells that prevent autoimmunity, without the need to target or purify specific Treg populations.

Increased soluble CD154 (CD40 ligand) levels in xenograft recipients correlate with the development of de novo anti-pig IgG antibodies.

BACKGROUND: De novo anti-pig antibodies are associated with acute humoral xenograft rejection. We explored the relative efficacy of CD40/CD154-pathway blockade versus CD28/B7-pathway blockade in the prevention of de novo anti-pig IgG antibodies in xenograft recipients. METHODS: After α1,3-galactosyltransferase gene-knockout pig artery patch xenotransplantation, recipient baboons received no immunosuppression (IS; n=3), or anti-CD154mAb-based (n=5) or CTLA4-Ig-based (n=5) IS. CD4 T-cell and CD20 B-cell numbers in blood were determined. Serum anti-pig IgG antibodies and serum soluble (s)CD154 levels were measured. In lymph nodes, germinal center formation was examined and numbers of proliferating cells were evaluated by Ki-67 staining. RESULTS: After transplantation, with no IS, CD4 T-cell and CD20 B-cell numbers were increased, but were reduced by IS.In lymph nodes, with no IS, there was enhanced germinal center formation, which was significantly reduced by anti-CD154mAb-based (P<0.01) or CTLA4-Ig-based (P<0.01) IS. With no IS, there was strong expression of Ki-67-positive cells in lymph nodes, indicating extensive cellular proliferation. Ki-67-positive cells were significantly reduced by anti-CD154mAb-based (P<0.05) but not by CTLA4-Ig-based IS. High mean levels of sCD154 were detected with no IS (3324 pg/mL), in comparison to naive control baboons (214 pg/mL). With anti-CD154mAb-based IS, sCD154 was reduced to less than 1 pg/mL and with CTLA4-Ig-based IS to 65 pg/mL. There was significant positive correlation between sCD154 and anti-pig IgG levels (P<0.01). CONCLUSIONS: In xenograft recipients, anti-CD154mAb may reduce class-switching of anti-pig antibodies by binding both T-cell surface CD154 and circulating sCD154, thus preventing subsequent stimulation of B cells and activation of lymphoid follicles in secondary lymphoid tissues.

Distribution of non-gal antigens in pig cornea: relevance to corneal xenotransplantation.

PURPOSE: The aim of this study was to investigate the distribution of antigens other than galactose-α-1,3-galactose (Gal) (non-Gal) recognized by human and rhesus monkey serum antibodies in the α-1,3-galactosyltransferase gene-knockout (GTKO) pig cornea. METHODS: The distribution of non-Gal, specifically N-glycolylneuraminic acid (NeuGc), in the corneas from wild-type (WT) and GTKO pigs was identified. Corneal sections from WT and GTKO pigs were incubated with human or rhesus monkey serum to determine immunoglobulin (Ig)M and IgG binding to corneal tissue by means of fluorescent microscopy. RESULTS: Strong expression of NeuGc was found in all layers of both WT and GTKO pig corneas. In both humans and monkeys, antibody binding (IgG > IgM) to GTKO was found to be weaker than that to entire WT pig corneas, but in both, most antibody binding, especially IgG, was to the epithelium. There was weak diffuse antibody binding, especially of IgG, to the corneal stroma, suggesting binding to antigens expressed on collagen. There was no or minimal binding of IgM/IgG to the corneal endothelium. CONCLUSIONS: Although the cornea is avascular, antibodies in primate serum can bind to pig antigens, especially on epithelial cells and stromal collagen. Although the binding to entire GTKO corneas was weaker than that to WT corneas, deletion of the expression of NeuGc and expression of human complement-regulatory proteins in the pig cornea will be important if prolonged clinical corneal xenograft survival is to be achieved.

Small proline rich protein 2a in benign and malignant liver disease.

UNLABELLED: STAT3-driven expression of small proline rich protein 2a (SPRR2a), which acts as an src homology 3 (SH3) domain ligand, induces biliary epithelial cell (BEC) epithelial-mesenchymal transition (EMT), which, in turn, promotes wound healing. SPRR2a also quenches free radicals and protects against oxidative stress and DNA damage in nonneoplastic BEC. Sprr2a-induced EMT also increases local invasiveness of cholangiocarcinomas (CC), but prevents metastases. Understanding SPRR2a regulation of EMT has potential for therapeutic targeting in both benign and malignant liver disease. Molecular mechanisms responsible for SPRR2a-induced EMT were characterized, in vitro, and then evidence for utilization of these pathways was sought in human intrahepatic CC, in vivo, using multiplex labeling and software-assisted morphometric analysis. SPRR2a complexes with ZEB1 and CtBP on the microRNA (miR)-200c/141 promoter resulting in synergic suppression of miR-200c/141 transcription, which is required for maintenance of the BEC epithelial phenotype. SPRR2a induction promotes dephosphorylation and nuclear translocation of the SH3-domain containing protein GRB2 and an SH3-domain ligand in ZEB1 is required for SPRR2a-induced synergic suppression of miR-200c/141. Multiplex protein labeling of CC and morphometric analyses showed: 1) up-regulation of ZEB-1, and 2) down-regulation of CK19 in intrahepatic CC compared to nonneoplastic BEC, consistent with previous CC proteomic studies showing EMT during cholangiocarcinogenesis. CONCLUSION: SPRR2a modulates ZEB-1 signaling by way of miR-200c/141-associated EMT through SH3-domain networks and contributes to benign and malignant BEC wound-healing responses.

SPRR2A expression in cholangiocarcinoma increases local tumor invasiveness but prevents metastasis.

Cholangiocarcinoma morbidity and mortality is attributable to local invasiveness and regional lymph node and distant organ metastasis. Cholangiocarcinoma progression follows a series of sequential events that resemble wound healing reactions: local invasion resembles the epithelial migration phase involving epithelial-mesenchymal transition (EMT); colonization at distant sites resembles epithelial restitution seen during the reverse process, mesenchymal-epithelial transition (MET). In this study we compare the in vivo local and metastatic growth potential of cholangiocarcinoma cell lines with respect to expression of a novel pSTAT3-dependent, biliary epithelial cell wound healing protein, small proline-rich protein 2A (SPRR2A). SPRR2A has been associated with local aggressiveness, but decreased metastatic capabilities in other cancers. Stable SPRR2A transfection into two cholangiocarcinoma cell lines (SG231 and HuCCT-1), previously shown by us to induce permanent EMT, resulted in local aggressiveness but an inability to form metastases. In contrast, SPRR2A-negative epithelial control cells showed relatively poor local aggressiveness, but readily formed metastatic tumors. Post-intrasplenic injection cell tracking showed that: (a) mesenchymal (SPRR2A+) cells were not trapped in the liver, but were rapidly cleared through mesenteric lymph nodes and did not form metastases; whereas (b) epithelial (SPRR2A-) controls were primarily entrapped within MUC-1-associated liver "micro-infarcts" that later evolved into metastatic colonies. SPRR2A-associated tumor behavior was mimicked by MUC1 shRNA, which induced EMT and, like SPRR2A+ cells, showed reduced metastatic capabilities. Cholangiocarcinoma local invasion involves EMT processes, whereas MET and MUC1 expression promote metastasis. A better understanding of disease progression should help target treatment for this deadly neoplasm.

Tissue biopsy monitoring of operational tolerance in liver allograft recipients.

PURPOSE OF REVIEW: Highly selected, long-surviving, liver allograft recipients with normal/near normal liver injury tests can be weaned from immunosuppression. Baseline biopsies document changes before weaning and can help stratify risk of rejection or dysfunction after weaning; biopsies after weaning are used to study mechanisms of operational tolerance and to monitor for subclinical events. RECENT FINDINGS: Clinicopathological features associated with successful weaning include a lack of sensitization [negative donor-specific antibodies (DSA) and lack of tissue C4d deposits]; 'inexperienced' recipient immune system with limited potential for cross-reactivity (less immunological memory; infant recipients); noninflamed allograft in those with nonviral, nonimmunological original diseases; upregulation of liver genes associated with iron metabolism; allograft colonization with 'immunosuppressive' cells (Treg and γδ-1>γδ-2); and longer time on immunosuppression, which might signal slow clonal deletion or silencing. The differential diagnosis of histopathological findings detected before and after weaning includes emerging infections, typical and atypical cellular rejection, indolent antibody-mediated rejection, 'autoimmunity', and other causes of progressive fibrosis. SUMMARY: Operationally tolerant liver allograft recipients can be successfully managed with very low, and sometimes no immunosuppression, but challenges exist. Newer approaches to tissue pathology and tissue, serum, and cross-platform analytics are needed to predict successful weaning and to monitor for subclinical events.