Antibody targeting of E3 ubiquitin ligases for receptor degradation.

Most current therapies that target plasma membrane receptors function by antagonizing ligand binding or enzymatic activities. However, typical mammalian proteins comprise multiple domains that execute discrete but coordinated activities. Thus, inhibition of one domain often incompletely suppresses the function of a protein. Indeed, targeted protein degradation technologies, including proteolysis-targeting chimeras1 (PROTACs), have highlighted clinically important advantages of target degradation over inhibition2. However, the generation of heterobifunctional compounds binding to two targets with high affinity is complex, particularly when oral bioavailability is required3. Here we describe the development of proteolysis-targeting antibodies (PROTABs) that tether cell-surface E3 ubiquitin ligases to transmembrane proteins, resulting in target degradation both in vitro and in vivo. Focusing on zinc- and ring finger 3 (ZNRF3), a Wnt-responsive ligase, we show that this approach can enable colorectal cancer-specific degradation. Notably, by examining a matrix of additional cell-surface E3 ubiquitin ligases and transmembrane receptors, we demonstrate that this technology is amendable for 'on-demand' degradation. Furthermore, we offer insights on the ground rules governing target degradation by engineering optimized antibody formats. In summary, this work describes a strategy for the rapid development of potent, bioavailable and tissue-selective degraders of cell-surface proteins.

Role of chemical structures and the 1331T>C bile salt export pump polymorphism in idiosyncratic drug-induced liver injury.

BACKGROUND & AIMS: Several pharmaceutical compounds have been shown to exert inhibitory effects on the bile salt export pump (BSEP) encoded by the ABCB11 gene. We analysed the combined effect on drug-induced liver injury (DILI) development of the ABCB11 1331T>C polymorphism and the presence of specific chemical moieties, with known BSEP inhibiting properties, in the causative drug. METHODS: Genotyping using a TaqMan 5' allelic discrimination assay was performed in 188 Spanish DILI patients, 219 healthy controls and 91 sex-, age- and drug-matched controls. A chemical structure analysis was performed for each individual causative drug. RESULTS: The CC genotype was significantly associated with hepatocellular damage [odds ratio (OR) = 2.1, P = 0.001], particularly in NSAID DILI cases (OR = 3.4, P = 0.007). In addition, the CC genotype was found to be significantly linked to DILI development from drugs causing <50% BSEP inhibition (OR = 1.8, Pc = 0.011). Of the BSEP inhibitory chemical moieties, 59% of the causative drugs contained a carbocyclic system with at least one aromatic ring, corresponding to 61% of the total cases. The C allele was significantly more frequent in DILI cases containing this chemical moiety, which appear to be conditioned on the ABCB11 1331T>C polymorphism in the absence of other BSEP inhibitory structures. CONCLUSION: Patients carrying the C allele in the ABCB11 1331T>C polymorphism are at increased risk of developing hepatocellular type of DILI, when taking drugs containing a carbocyclic system with aromatic rings.

Bacillus Calmette-Guerin immunotherapy of bladder cancer induces selection of human leukocyte antigen class I-deficient tumor cells.

Bacillus Calmette-Guerin (BCG) immunotherapy is a standard treatment for high-risk non-muscle-infiltrating bladder cancer patients. Although the outcomes are good, cancer relapse is observed in around 40% of patients. We present the comparative analysis of human leukocyte antigen (HLA) class I expression in recurrent bladder tumors in patients treated with mitomycin or BCG. HLA class I expression was analyzed by RT-Q-PCR and immunohistochemical techniques. Loss of heterozygosity (LOH) was determined by microsatellite amplification of markers in chromosome 6 and 15. More profound alterations in HLA class I expression were found in post-BCG recurrent tumors than in pre-BCG lesions, whereas mitomycin treatment did not change the HLA class I expression pattern. Post-BCG recurrent tumors also showed a higher incidence of structural defects underlying altered HLA class I expression. We hypothesize that the immunotherapy-activated immune system recognizes and eliminates tumor cells with reversible ("soft") HLA class I changes but not transformed cells with additional, irreversible ("hard") alterations. To our knowledge, this is the first clinical evidence of immunotherapy-induced immunoselection of HLA class I loss tumor variants in bladder cancer, although the study involved a small number of patients.

Leukocyte infiltrate in gastrointestinal adenocarcinomas is strongly associated with tumor microsatellite instability but not with tumor immunogenicity.

PURPOSE: To analyze the correlation of genomic instability with leukocyte infiltrate in gastrointestinal carcinomas (GIACs) and with tumor immunogenicity, e.g., HLA class I cell surface expression defects and galectin-3 and PDL-1 expression. EXPERIMENTAL DESIGN: Lymphocyte and macrophage infiltrations were immunohistochemically studied in HLA class I negative GIACs with sporadic high-level microsatellite instability (MSI-H) or microsatellite stability (MSS). RESULTS: Tumors with MSI-H were associated with the following: dense infiltration (CD45, P < 0.001); cytotoxic CD8-positive lymphocytes (P < 0.001); and a complete absence of HLA class I cell surface expression, due to inactivating ß2-microglobulin (ß2-m) mutation in 50% of cases. In contrast, HLA class I negative tumors with MSS were significantly associated with fewer CD8-positive lymphocytes. There was no association between microsatellite instability and other molecular features of the tumor cells, including expression of galectin-3. Finally, macrophage infiltrate in the tumors was not correlated with microsatellite instability or HLA class I cell surface expression (CD64, P = 0.63; CD163, P = 0.51). CONCLUSIONS: Microsatellite instability appears to be the most important factor determining the composition, density, and localization of leukocyte infiltrate, which is independent of other molecular features such expression of HLA class I cells, galectin-3, or programmed death ligand-1. Accordingly, the strong intratumoral CD8+ T infiltration of MSI-H tumors may be produced by elevated levels of specific inflammatory chemokines in the tumor microenvironment.

Importance of host genetic factors HLA and IL28B as predictors of response to pegylated interferon and ribavirin.

OBJECTIVES: Viral factors are considered the best predictors of response to treatment for chronic hepatitis C (CHC), but genetic factors are known to have an important role in this respect. This paper investigates the relationships among the host genetic factors HLA and IL28B, viral factors, and the outcome of combination therapy. METHODS: A multicenter retrospective cohort of 428 previously untreated CHC patients was treated with pegylated interferon/ribavirin (pegIFN/RBV) for 48 weeks. In all, 378 (88%) of these patients were genotype 1 or 4, and 50 (12%) were genotype 2 or 3. RESULTS: Multivariate logistic regression showed the rs12979860 CC genotype (adjusted odds ratio (aOR)=4.3, 95% confidence interval (95% CI): 2.6-7), the HLA-DQB1*0301 allele (aOR=2.08, 95% CI: 1.2-3.5) and age, viral genotype, and viral load levels to be significantly associated with sustained virological response (SVR). When the variable rs12979860 was eliminated, the area under the receiver operating characteristic (ROC) curve (AUC) decreased significantly (0.76 vs. 0.69; P=0.03). AUC values derived from viral factors were lower than those corresponding to host genetic factors (0.67 vs. 0.72, respectively; P=0.04). The HLA-DQB1*0301 and A*0201 alleles were associated with rs12979860 CC genotype and SVR (P<0.0001). CONCLUSIONS: The HLA-DQB1*0301 allele and IL28B genotype are factors that are associated independently with SVR. There is a synergism between the HLA-DQB1*0301 and HLA-A*0201 alleles with polymorphism rs12979860 CC, which increases the SVR rate. IL28B genotype is the best predictor of SVR.

Polymorphisms of CD16A and CD32 Fcγ receptors and circulating immune complexes in Ménière's disease: a case-control study.

BACKGROUND: Autoimmune diseases with elevated circulating autoantibodies drive tissue damage and the onset of disease. The Fcγ receptors bind IgG subtypes modulating the clearance of circulating immune complexes (CIC). The inner ear damage in Ménière's disease (MD) could be mediated by an immune response driven by CIC. We examined single-nucleotide polymorphism (SNPs) in the CD16A and CD32 genes in patients with MD which may determine a Fcγ receptor with lower binding to CIC. METHODS: The functional CD16A (FcγRIIIa*559A > C, rs396991) and CD32A (FcγRIIa*519A > G, rs1801274) SNPs were analyzed using PCR-based TaqMan Genotyping Assay in two cohorts of 156 mediterranean and 112 Galicia patients in a case-control study. Data were analyzed by χ2 with Fisher's exact test and Cochran-Armitage trend test (CATT). CIC were measured by ELISA for C1q-binding CIC. RESULTS: Elevated CIC were found in 7% of patients with MD during the intercrisis period. No differences were found in the allelic frequency for rs396991 or rs1801274 in controls subjects when they were compared with patients with MD from the same geographic area. However, the frequency of AA and AC genotypes of CD16A (rs396991) differed among mediterranean and Galicia controls (Fisher's test, corrected p = 6.9 × 10-4 for AA; corrected p = 0.02 for AC). Although genotype AC of the CD16A receptor was significantly more frequent in mediterranean controls than in patients, [Fisher's test corrected p = 0.02; OR = 0.63 (0.44-0.91)], a genetic additive effect for the allele C was not observed (CATT, p = 0.23). Moreover, no differences were found in genotype frequencies for rs396991 between patients with MD and controls from Galicia (CATT, p = 0.14). The allelic frequency of CD32 (rs1801274) was not different between patients and controls either in mediterranean (p = 0.51) or Galicia population (p = 0.11). CONCLUSIONS: Elevated CIC are not found in most of patients with MD. Functional polymorphisms of CD16A and CD32 genes are not associated with onset of MD.

A polymorphism in the interleukin-10 promoter affects the course of disease in patients with clear-cell renal carcinoma.

In the tumor microenvironment, interleukin (IL)-10 production has a pleiotropic ability to positively and negatively influence the function of innate and adaptive immunity against cancer. This study investigated whether IL-10 genetic polymorphisms that influence gene expression levels play a role in the risk and clinical course of clear-cell renal cell carcinoma (RCC). We analyzed the allelic and haplotype frequency formed by alleles at -1082(G/A), -819(C/T), and -592(C/A) of the IL-10 gene in RCC (n = 126) and healthy individuals (n = 176). The frequency of IL-10 polymorphic variants was similar between patients and controls. However, -1082 G/A IL-10 genotype showed a significant association with three prognostic indicators: advanced disease stage (p = 0.002), higher tumor size (p = 0.001), and presence of adenopathy (p = 0.006). Our results can be explained by the contradictory antitumor or pro-tumorigenic relationship between this molecule and cancer. Genotypes associated with high or low levels of IL-10 gene expression (GG or AA-1082 IL-10) were both associated with a more favorable course of the disease. We propose the hypothesis that the -1082 GA medium expression genotype confers a tumor-promoting phenotype, likely resulting from the immunosuppressive effects of anti-tumor Th-1 responses in conjunction with the insufficient inhibition of tumor angiogenesis at this intermediate level of IL-10 expression.

[Polymorphisms in inflammatory response genes in metastatic renal cancer]. / Polimorfimos en genes de respuesta inflamatoria en cáncer renal metastásico.

Inflammation has been implicated as an etiological factor in different human cancers. Allelic variations in the genes implicated in inflammation are candidates as genetic determinants or markers of renal carcinoma risk. The present stud investigates whether polymorphisms of the genes that give rise to increases in the levels of proinflammatory cytokines and chemokines are associated with an increased risk of renal carcinoma. To this effect, a number of case-control studies were designed to assess the correlation between renal carcinoma and polymorphisms IL10-1082 A/G (rs 1800896), IL10-592 A/C (rs 1800872), IL10-819 C/T (rs 1800871), IL10-1082 A/G, IL4-590 C/T (rs 2243250), TNF-A-308 A/G (rs 1800629), RANTES-403 G/A (rs 2107538), IL1-A-889 C/T (rs 1800587), MCP-1 2518 G/A (rs 1024611), CTLA-4/+49 A/G (rs 231775) and CTLA-4 CT60 A/G (rs 3087243) in 127 renal carcinoma patients and in 176 healthy subjects. The results obtained in relation to cytokine polymorphism IL-10-1082 A/G indicate that AG heterozygosity status is the principal risk factor in relation to locally advanced or metastatic tumor stage and renal carcinoma. In the case of the molecule CTLA4, the results obtained in renal cancer reveal an association between the polymorphisms of the CTLA-4 gene and an increased risk of developing renal cell carcinoma. A high genotypic frequency of polymorphisms CTLA4/CT60-AA and CTLA4/A49G-AA is observed in patients with renal cell carcinoma versus the controls. An association has been established between polymorphism CTLA4/CT60 and tumor grade in patients with renal cell carcinoma. Logistic regression analysis has confirmed these data, demonstrating a high frequency of the AA genotype in patients with high-grade tumors. The results obtained support the hypothesis that different genetic factors implicated in the regulation of adaptive immune responses, stromal cell composition and local cytokine production levels may be crucial elements in the modification of the clinicopathological parameters of renal carcinoma.

Genetic polymorphisms of RANTES, IL1-A, MCP-1 and TNF-A genes in patients with prostate cancer.

BACKGROUND: Inflammation has been implicated as an etiological factor in several human cancers, including prostate cancer. Allelic variants of the genes involved in inflammatory pathways are logical candidates as genetic determinants of prostate cancer risk. The purpose of this study was to investigate whether single nucleotide polymorphisms of genes that lead to increased levels of pro-inflammatory cytokines and chemokines are associated with an increased prostate cancer risk. METHODS: A case-control study design was used to test the association between prostate cancer risk and the polymorphisms TNF-A-308 A/G (rs 1800629), RANTES-403 G/A (rs 2107538), IL1-A-889 C/T (rs 1800587) and MCP-1 2518 G/A (rs 1024611) in 296 patients diagnosed with prostate cancer and in 311 healthy controls from the same area. RESULTS: Diagnosis of prostate cancer was significantly associated with TNF-A GA + AA genotype (OR, 1.61; 95% CI, 1.09-2.64) and RANTES GA + AA genotype (OR, 1.44; 95% CI, 1.09-2.38). A alleles in TNF-A and RANTES influenced prostate cancer susceptibility and acted independently of each other in these subjects. No epistatic effect was found for the combination of different polymorphisms studied. Finally, no overall association was found between prostate cancer risk and IL1-A or MCP-1 polymorphisms. CONCLUSION: Our results and previously published findings on genes associated with innate immunity support the hypothesis that polymorphisms in proinflammatory genes may be important in prostate cancer development.

The role of Toll-like receptor polymorphisms in acute pancreatitis occurrence and severity.

OBJECTIVES: Toll-like receptors (TLRs) are damage-associated molecular patterns receptors, which are essential in the activation of the inflammasome cascade, required for the initiation of inflammation. We hypothesized that changes in the function of these receptors caused by genetic polymorphisms in their encoding genes could determine acute pancreatitis (AP) incidence or severity. METHODS: Two hundred sixty-nine patients and 269 controls were included. Acute pancreatitis diagnosis criteria were abdominal pain, increased serum amylase levels, and positive findings on abdominal imaging. The patients were observed until discharge. Blood samples were obtained, determining the following TLRs: TLR1 rs5743611, TLR2 rs5743704, TLR3 rs3775291, TLR3 rs5743305, TLR4 rs4986790, TLR4 rs4986791, TLR5 rs5744174, TLR6 rs5743795, TLR7 rs2302267, TLR9 rs352140, and TLR10 rs4129009. RESULTS: No TLR polymorphism was related to AP incidence. Regarding severity, CC genotype patients in TLR3 rs3775291 had an increased risk for severe pancreatitis (CC odds ratio [OR], 2.426; P = 0.015). In addition, TLR6 rs5743795 GG genotype patients had a lower risk for severe AP (GG OR, 0.909; P < 0.05). Intensive care unit admission was related to TLR5 rs5744174 homozygote TT carriers (TT OR, 3.367; P = 0.036). CONCLUSIONS: Our article points to genetic polymorphisms in TLR3 and TLR6 as having a plausible role in the occurrence of severe AP.

Selected ABCB1, ABCB4 and ABCC2 polymorphisms do not enhance the risk of drug-induced hepatotoxicity in a Spanish cohort.

BACKGROUND AND AIMS: Flawed ABC transporter functions may contribute to increased risk of drug-induced liver injury (DILI). We aimed to analyse the influence of genetic variations in ABC transporters on the risk of DILI development and clinical presentations in a large Spanish DILI cohort. METHODS: A total of ten polymorphisms in ABCB1 (1236T>C, 2677G>T,A, 3435T>C), ABCB4 (1954A>G) and ABCC2 (-1774G>del, -1549A>G, -24C>T, 1249G>A, 3972C>T and 4544G>A) were genotyped using Taqman 5' allelic discrimination assays or sequencing in 141 Spanish DILI patients and 161 controls. The influence of specific genotypes, alleles and haplotypes on the risk of DILI development and clinical presentations was analysed. RESULTS: None of the individual polymorphisms or haplotypes was found to be associated with DILI development. Carriers homozygous for the ABCC2 -1774del allele were however only found in DILI patients. Hence, this genotype could potentially be associated with increased risk, though its low frequency in our Spanish cohort prevented a final conclusion. Furthermore, carriers homozygous for the ABCC2 -1774G/-1549A/-24T/1249G/3972T/4544G haplotype were found to have a higher propensity for total bilirubin elevations when developing DILI. CONCLUSIONS: Our findings do not support a role for the analysed polymorphisms in the ABCB1, ABCB4 and ABCC2 transporter genes in DILI development in Spanish patients. The ABCC2 -1774deldel genotype was however restricted to DILI cases and could potentially contribute to enhanced DILI susceptibility.

VEGF polymorphisms are not associated with an increased risk of developing renal cell carcinoma in Spanish population.

PURPOSE: Vascular endothelial growth factor (VEGF) plays a central role in promoting angiogenesis and is over-expressed in renal cell cancer (RCC). Published data on the association between polymorphisms of vascular endothelial growth factor (e.g., -2578C/A [rs699947], -460T/C [rs833061], +405C/G [rs2010963], and +936C/T [rs3025039]) and the risk of renal cell carcinoma are ambiguous and controversial. The aim of this investigation was to investigate this relationship in a series of Caucasian Spanish patients. MATERIALS AND METHODS: A case-control study was performed with 216 cases and 280 controls, genotyping subjects for VEGF polymorphisms using the predesigned TaqMan single nucleotide polymorphism (SNP) genotyping assay (Applied Biosystems, Foster City, CA, USA). The combined effect of the four gene polymorphisms on overall survival was studied by haplotype analysis. RESULTS: The overall results suggest that polymorphisms or haplotypes in the VEGF gene do not modify the risk of RCC. We were unable to replicate the association of the -460T/C (rs833061) polymorphism with renal cancer risk. Data were also gathered on clinical-pathological results, tumor size, clinical stage, histological grade, and survival. CONCLUSIONS: According to our analysis of their contribution to prognostic factors, VEGF polymorphisms do not appear to exert a significant influence on RCC progression or prognosis. This finding might be explained by the tumor biology and pathogenesis of clear cell RCC. Additional studies with larger sample sizes are needed in different ethnic groups to further assess this association.

Association between C13ORF31, NOD2, RIPK2 and TLR10 polymorphisms and urothelial bladder cancer.

Several evidences have been published linking polymorphism in genes involved in chronic or recurrent inflammation with increased tumor risk and progression. Nevertheless the influence of innate immune receptors in urothelial cancer risk and characteristics has not been sufficient explored. We studied the possible association of polymorphisms in genes encoding NOD2, RIPK2, TLR10 and C13ORF31 with the risk, clinical/pathological characteristics and outcomes of urothelial cancer. We have found association between RIPK2 (rs42490) and cancer risk (AA vs AT&TT, p=0042). In addition, we found statistical differences in TLR10 (rs4129009) gen between low and high tumor infiltration stage (p=0.033). NOD2 (rs9302752) and RIPK2 (rs42490) were found to be associated with development of lymph node metastasis (p=0.011 and p=0.015). Importantly we detect association of TLR10 (Log Rank=0.035) and RIPK2 (Log Rank=0040) with overall survival. Multivariate Cox analysis revealed that both SNPs were survival prognosis factor independent of tumor stage and grade. Our results indicate that innate immunity receptors play a role in modulating urothelial cancer risk and progression.

Association of a functional polymorphism of PTPN22 encoding a lymphoid protein phosphatase in bilateral Meniere's disease.

OBJECTIVES/HYPOTHESIS: Bilateral Meniere's disease (BMD) is a severe disease that usually results in bilateral severe or profound sensorineural hearing loss and chronic disequilibrium with loss of vestibular function. We examined single nucleotide polymorphisms (SNPs) in the PTPN22 and CTLA4 genes in Caucasian patients with BMD to assess the possible association between these polymorphism and the predisposition and clinical expression of this disease. STUDY DESIGN: A case control study. METHODS: The functional protein tyrosine phosphatase type 22 (PTPN22) SNP (rs2476601, 1858C/T) and CTLA4 SNP (rs231775, 49A/G) were analyzed in 52 patients with BMD and 348 healthy controls by a TaqMan 5' allelic discrimination assay. Data were analyzed by a chi(2) test with Fisher exact test. RESULTS: No association was found between the +49A/G CTLA4 genotype and BMD patients. However, the heterozygote PTPN22 1858C/T genotype was present at a significantly higher frequency in BMD patients than in controls (odds ratio = 2.25, 95% confidence interval: 1.09-4.62; P = .04). CONCLUSIONS: These results suggest that the PTPN22 1858C/T genotype may confer differential susceptibility to BMD in the Spanish population and support an autoimmune etiology for BMD.

Impact of interleukin-18 polymorphisms-607 and -137 on clinical characteristics of renal cell carcinoma patients.

Current evidence suggests that chronic inflammation is associated with tumor development and progression. Interleukin-18 (IL-18) plays a central role in inflammation and the immune response, contributing to the pathogenesis and pathophysiology of infectious and inflammatory diseases. The objective of this study was to determine whether the presence of IL-18 polymorphisms -137 G/C (rs187238) and -607 A/C (rs1946518) was associated with size, grade, TNM stage, and survival in patients with renal cell carcinoma (RCC). The study cohort included 158 patients with RCC. Control group consisted of 506 samples from Spanish population. The studied IL-18 gene polymorphisms did not influence susceptibility to RCC in the analyzed group of patients (IL-18-607, p = 0.318; IL-18-137 p = 0.740) but may contribute to disease onset and aggressiveness. IL-18-607 CC genotype was significantly associated with higher tumor size (p = 0.001), grade (p = 0.030), T (p = 0.001), M (p = 0.012), and stage (p = 0.002). IL-18-103 GG genotype was correlated with higher tumor size (p = 0.036), grade (p = 0.017), T (p = 0.026), and stage (p = 0.011). The Cox proportional hazard model showed that nuclear grade and stage grouping were independent prognostic factors but IL-18 polymorphism was not. Polymorphism variants in the IL-18 gene (IL-18-607 and IL-18-137) may be associated with a worse prognosis for RCC. High levels of IL-18 production may play a major role in the growth, invasion and metastasis of renal cancer.