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Development of novel therapeutic antibodies that not only kill tumor cells but modulate the adaptive immune response has the potential to produce long term anti-cancer immunity and a durable clinical response. We previously reported the discovery of an anti-complement factor H (CFH) autoantibody in lung cancer patients that were associated with early stage disease and exceptional outcomes. The human monoclonal antibody GT103, produced from a single CFH autoantibody-expressing B cell of a lung cancer patient, recognizes a conformationally distinct epitope on tumor cells, kills tumor cells, and inhibits tumor growth in animal studies. Recent experiments have shown that GT103 restructures the tumor microenvironment and initiates a robust antitumoral adaptive immune response. The current study further elucidates several mechanisms by which GT103 kills tumor cells and drives the immune program. Here we show GT103 has specificity for tumor cells without binding to native soluble CFH or normal tissues. GT103 causes complement C3 split product deposition on tumor cellsin vitroandin vivo,triggers antibody-dependent cellular phagocytosis, and increases translocation of the danger associated molecular pattern molecule calreticulin to the plasma membrane.We also demonstrate that GT103 causes B cell activation and that GT103 antitumor activityin vivois B cell dependent. The complex mechanism of GT103, a tumor specific antibody that kills tumor cells and stimulates an immune response, supports further development of this human-derived antibody as a novel therapeutic option for patients with lung cancer.

BACKGROUND: Biomarkers that predict which patients with early stage NSCLC will develop recurrent disease would be of clinical value. We previously discovered that an autoantibody to a complement regulatory protein, complement factor H (CFH), is associated with early stage, non-recurrent NSCLC, and hypothesized that the anti-CFH antibody inhibits metastasis. OBJECTIVES: The primary objective of this study was to evaluate the anti-CFH antibody as a prognostic marker for recurrence in stage I NSCLC. A secondary objective was to determine if changes in antibody serum level one year after resection were associated with recurrence. METHODS: Anti-CFH antibody was measured in the sera of 157 stage I NSCLC patients designated as a prognostic cohort: 61% whose cancers did not recur, and 39% whose cancers recurred following resection. Impact of anti-CFH antibody positivity on time to recurrence was assessed using a competing risk analysis. Anti-CFH antibody levels were measured before resection and one year after resection in an independent temporal cohort of 47 antibody-positive stage I NSCLC patients: 60% whose cancers did not recur and 40% whose cancers recurred following resection. The non-recurrent and recurrent groups were compared with respect to the one-year percent change in antibody level. RESULTS: In the prognostic cohort, the 60-month cumulative incidence of recurrence was 40% and 22% among antibody negative and positive patients, respectively; this difference was significant (Gray’s test, P= 0.0425). In the temporal cohort, the antibody persisted in the serum at one year post-tumor resection. The change in antibody levels over the one year period was not statistically different between the non-recurrent and recurrent groups (Wilcoxon two-sample test, P= 0.4670). CONCLUSIONS: The anti-CFH autoantibody may be a useful prognostic marker signifying non-recurrence in early stage NSCLC patients. However, change in the level of this antibody in antibody-positive patients one year after resection had no association with recurrence.

Complement-dependent cytotoxicity of A549 lung cancer cells by the alternative pathway.

Characterization of the humoral immune response in selected patients with cancer who uniformly do well may lead to the development of novel therapeutic strategies. We have previously shown an association between patients with early-stage nonmetastatic lung cancer and autoantibodies to complement factor H (CFH). CFH protects normal and tumor cells from destruction by the alternative complement pathway by inactivating C3b, a protein that is essential for formation of a lytic complex on the cell surface. Here, we show that CFH autoantibodies in lung cancer patients recognize a conformationally distinct form of CFH in vitro, are IgG3 subclass, and epitope map to a crucial functional domain of CFH known to interact with C3b. Purified CFH autoantibodies inhibited binding of CFH to A549 lung tumor cells, increased C3b deposition, and caused complement-dependent tumor cell lysis. This work demonstrates that CFH autoantibodies isolated from patients with lung cancer can kill tumor cells in vitro, suggesting that they may perform this function in vivo as well. Development of specific antibodies to the conformationally distinct epitope of CFH may lead to a useful biologic therapy for lung cancer. Cancer Immunol Res; 3(12); 1325–32. ©2015 AACR.

Deposition of C3-related fragments on A549 lung cancer cells.

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Exosomes are a class of extracellular vesicles (EVs) that are mediators of normal intercellular communication, but exosomes are also used by tumor cells to promote oncogenesis and metastasis. Complement factor H (CFH) protects host cells from attack and destruction by the alternative pathway of complement-dependent cytotoxicity (CDC). Here we show that CFH can protect exosomes from complement-mediated lysis and phagocytosis. CFH was found to be associated with EVs from a variety of tumor cell lines as well as EVs isolated from the plasma of patients with metastatic non-small cell lung cancer. Higher levels of CFH-containing EVs correlated with higher metastatic potential of cell lines. GT103, a previously described antibody to CFH that preferentially causes CDC of tumor cells, was used to probe the susceptibility of tumor cell-derived exosomes to destruction. Exosomes were purified from EVs using CD63 beads. Incubation of GT103 with tumor cell-derived exosomes triggered exosome lysis primarily by the classical complement pathway as well as antibody-dependent exosome phagocytosis by macrophages. These results imply that GT103-mediated exosome destruction can be triggered by antibody Fc-C1q interaction (in the case of lysis), and antibody-Fc receptor interactions (in the case of phagocytosis). Thus, this work demonstrates CFH is expressed on tumor cell derived exosomes, can protect them from complement lysis and phagocytosis, and that an anti-CFH antibody can be used to target tumor-derived exosomes for exosome destruction via innate immune mechanisms. These findings suggest that a therapeutic CFH antibody has the potential to inhibit tumor progression and reduce metastasis promoted by exosomes.

Characterization of the humoral immune response in selected patients with cancer who uniformly do well may lead to the development of novel therapeutic strategies. We have previously shown an association between patients with early-stage nonmetastatic lung cancer and autoantibodies to complement factor H (CFH). CFH protects normal and tumor cells from destruction by the alternative complement pathway by inactivating C3b, a protein that is essential for formation of a lytic complex on the cell surface. Here, we show that CFH autoantibodies in lung cancer patients recognize a conformationally distinct form of CFH in vitro, are IgG3 subclass, and epitope map to a crucial functional domain of CFH known to interact with C3b. Purified CFH autoantibodies inhibited binding of CFH to A549 lung tumor cells, increased C3b deposition, and caused complement-dependent tumor cell lysis. This work demonstrates that CFH autoantibodies isolated from patients with lung cancer can kill tumor cells in vitro, suggesting that they may perform this function in vivo as well. Development of specific antibodies to the conformationally distinct epitope of CFH may lead to a useful biologic therapy for lung cancer. Cancer Immunol Res; 3(12); 1325–32. ©2015 AACR.

Rituximab therapy for B cell chronic lymphocytic leukemia (B-CLL) has met with mixed success. Among several factors to which resistance can be attributed is failure to activate complement dependent cytotoxicity (CDC) due to protective complement regulatory proteins, including the soluble regulator complement factor H (CFH). We hypothesized that rituximab killing of non-responsive B-CLL cells could be augmented by a novel human monoclonal antibody against CFH. The B cells from 11 patients with B-CLL were tested ex vivo in CDC assays with combinations of CFH monoclonal antibody, rituximab, and a negative control antibody. CDC of rituximab non-responsive malignant B cells from CLL patients could in some cases be augmented by the CFH monoclonal antibody. Antibody-mediated cytotoxicity of cells was dependent upon functional complement. In one case where B-CLL cells were refractory to CDC by the combination of rituximab plus CFH monoclonal antibody, additionally neutralizing the membrane complement regulatory protein CD59 allowed CDC to occur. Inhibiting CDC regulatory proteins such as CFH holds promise for overcoming resistance to rituximab therapy in B-CLL.

UDP-glucuronosyltransferase A1 (UGT2A1) is expressed in the lung and exhibits activity against polycyclic aromatic hydrocarbons (PAHs), suggesting UGT2A1 involvement in the local metabolism of PAH tobacco carcinogens. The goal of the present study was to investigate the importance of two additional UGT2A enzymes, UGT2A2 and UGT2A3, in tobacco carcinogen metabolism. Real-time polymerase chain reaction suggested that wild-type UGT2A2 had the highest expression in the breast, followed by trachea > larynx > kidney. A novel splice variant of UGT2A2 lacking exon 3 (termed UGT2A2Δexon3) was investigated, with UGT2A2Δexon3 expression determined to be 25–50% that of wild-type UGT2A2 in all tissues examined. UGT2A3 was determined to be well expressed in the liver and colon, followed by pancreas > kidney > lung > tonsil > trachea > larynx. Cell homogenates prepared from human embryonic kidney (HEK)293 cells overexpressing wild-type UGT2A2 (termed UGT2A2_i1) exhibited glucuronidation activity, as observed by reverse-phase ultra-pressure liquid chromatography, against 1-hydroxy-(OH)-pyrene, 1-naphthol, and hydroxylated benzo(a)pyrene metabolites, whereas homogenates prepared from HEK293 cells overexpressing UGT2A3 only showed activity against simple PAHs like 1-OH-pyrene and 1-naphthol. Activity assays showed the UGT2A2Δexon3 protein (termed UGT2A2_i2) exhibited no detectable glucuronidation activity against all substrates examined; however, coexpression studies suggested that UGT2A2_i2 negatively modulates UGT2A2_i1 activity. Both UGT2A2 and UGT2A3 exhibited no detectable activity against complex PAH proximate carcinogens, tobacco-specific nitrosamines, or heterocyclic amines. These data suggest that, although UGT2A1 is the only UGT2A enzyme active against PAH proximate carcinogens (including PAH diols), both UGTs 2A1 and 2A2 play an important role in the local detoxification of procarcinogenic monohydroxylated PAH metabolites.

The UDP-glucuronosyltransferase (UGT) 1A enzymes are involved in the phase II metabolism of many important endogenous and exogenous compounds. The nine UGT1A isoforms exhibit high interindividual differences in expression, but their epigenetic regulation is not well understood. The purpose of the present study was to examine microRNA (miRNA) regulation of hepatic UGT1A enzymes and determine whether or not that regulation impacts enzymatic activity. In silico analysis identified miRNA 491-3p (miR-491-3p) as a potential regulator of the UGT1A gene family via binding to the shared UGT1A 3′-untranslated region common to all UGT1A enzymes. Transfection of miR-491-3p mimic into HuH-7 cells significantly repressed UGT1A1 ( P < 0.001), UGT1A3 ( P < 0.05), and UGT1A6 ( P < 0.05) mRNA levels. For UGT1A1, this repression correlated with significantly reduced metabolism of raloxifene into raloxifene-6-glucuronide (ral-6-gluc; P < 0.01) and raloxifene-4′-glucuronide (ral-4′-gluc; P < 0.01). In HuH-7 cells with repressed miR-491-3p expression, there was a significant increase (∼80%; P < 0.01) in UGT1A1 mRNA and a corresponding increase in glucuronidation of raloxifene into ral-6-gluc (50%; P < 0.05) and ral-4′-gluc (22%; P < 0.01). Knockdown of endogenous miR-491-3p in HepG2 cells did not significantly alter UGT1A1 mRNA levels but did increase the formation of ral-6-gluc (50%; P < 0.05) and ral-4′-gluc (34%; P < 0.001). A significant inverse correlation between miR-491-3p expression and both UGT1A3 ( P < 0.05) and UGT1A6 ( P < 0.01) mRNA levels was observed in a panel of normal human liver specimens, with a significant ( P < 0.05) increase in UGT1A3 and UGT1A6 mRNA levels observed in miR-491-3p nonexpressing versus expressing liver specimens. These results suggest that miR-491-3p is an important factor in regulating the expression of UGT1A enzymes in vivo.

UDP-glucuronosyltransferase (UGT) 2A1 is a respiratory and aerodigestive tract-expressing phase II detoxifying enzyme that metabolizes various xenobiotics including polycyclic aromatic hydrocarbons (PAHs). In the present study, a novel exon 3 deletion splice variant was identified for UGT2A1 (UGT2A1Δexon3). As determined by reverse transcription-polymerase chain reaction (PCR), UGT2A1Δexon3 was shown to be expressed in various tissues including lung, trachea, larynx, tonsil, and colon. The ratio of UGT2A1Δexon3/wild-type UGT2A1 expression was highest in colon (0.79 ± 0.08) and lung (0.42 ± 0.12) as determined by real-time PCR; an antibody specific to UGT2A1 showed splice variant protein (UGT2A1_i2) to wild-type protein (UGT2A1_i1) ratios in the range of 0.5 to 0.9 in these tissues. Using ultra-pressure liquid chromatography, we found that homogenates prepared from UGT2A1_i2-overexpressing human embryonic kidney 293 cells exhibited no glucuronidation activity against PAHs, including benzo[a]pyrene-7,8-dihydrodiol (B[a]P-7,8-diol). An inducible in vitro system was created to determine the effect of UGT2A1_i2 expression on UGT2A1_i1 activity. Increasing UGT2A1_i2 levels resulted in a significant (p0.01) decrease in the UGT2A1_i1 V(max) against 1-hydroxy (OH)-pyrene, 3-OH-benzo[a]pyrene, and B[a]P-7,8-diol; no significant changes in K(M) were observed for any of the three substrates. Coimmunoprecipitation experiments suggested the formation of UGT2A1_i1 and UGT2A1_i2 hetero-oligomers and UGT2A1_i1 homo-oligomers; coexpression of UGT2A1_i1 or UGT2A1_i2 with other UGT1A or UGT2B enzymes caused no change in UGT1A or UGT2B glucuronidation activity. These data suggest that a novel UGT2A1 splice variant regulates UGT2A1-mediated glucuronidation activity via UGT2A1-specific protein-protein interactions, and expression of this variant could play an important role in the detoxification of carcinogens within target tissues for tobacco carcinogenesis.

OBJECTIVE To examine UGT2A1 expression in human tissues, determine its glucuronidation activity against tobacco carcinogens, and assess the potential functional role of UGT2A1 missense single nucleotide polymorphisms on UGT2A1 enzyme activity. METHODS Reverse transcription polymerase chain reaction and real time polymerase chain reaction were used to assess UGT2A1 gene expression in various human tissues. A glucuronidation assay measured by reverse phase ultra-performance liquid chromatography was used to determine UGT2A1 activity. RESULTS UGT2A1 was expressed in aerodigestive tract tissues including trachea, larynx, tonsil, lung, and colon; no expression was observed in breast, whole brain, pancreas, prostate, kidney, liver, or esophagus. UGT2A1 exhibited highest expression in the lung, followed by trachea >tonsil >larynx >colon >olfactory tissue. Cell homogenates prepared from wildtype UGT2A1(75Lys308Gly) overexpressing HEK293 cells showed significant glucuronidation activity against a variety of polycyclic aromatic hydrocarbons including, 1-hydroxy-benzo(a)pyrene, benzo(a)pyrene-7,8-diol, and 5-methylchrysene-1,2-diol. No activity was observed in UGT2A1 overexpressing cell homogenate against substrates that form N-glucuronides, such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), nicotine, or N-OH-2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (N-OH PhIP). A significant (P

UGT2A1 is a phase II enzyme highly expressed in the respiratory and aerodigestive tracts. In the present study, a novel exon 3 deletion splice variant for UGT2A1 (termed ‘UGT2A1Δexon3’) was characterized. As determined by reverse-transcription polymerase chain reaction, UGT2A1Δexon3 was shown to be co-expressed with wild-type UGT2A1 in various tissues including lung, trachea, larynx, tonsil, and colon. The mRNA expression ratio of UGT2A1Δexon3:wild-type UGT2A1 was highest in colon (0.79±0.08) and lung (0.42±0.12) as determined by real-time PCR; splice variant protein (termed ‘UGT2A1_i2’) to wild-type protein (termed ‘UGT2A1_i1’) ratios were in the range of 0.5-0.9 in these tissues as determined by Western blot analysis using an UGT2A1-specific antibody. In contrast to the UGT2A1 wild-type isoform, homogenates prepared from UGT2A1_i2-over-expressing HEK293 cells exhibited no glucuronidation activity against PAHs, including benzo(a)pyrene-7,8-dihydrodiol (B(a)P-7,8-diol). An inducible in vitro system was created to determine the effect of UGT2A1_i2 expression on UGT2A1_i1 activity. Increasing UGT2A1_i2 levels resulted in a significant (p Citation Format: Ryan T. Bushey, Gang Chen, Philip Lazarus. Identification and functional characterization of a novel UGT2A1 splice variant: Potential importance in tobacco-related cancer susceptibility . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2563. doi:10.1158/1538-7445.AM2013-2563

Background: The UDP-Glucuronosyltransferases (UGTs) are phase II metabolic enzymes that prime endogenous and exogenous compounds for excretion from the body. The UGTs are often found to have high inter-individual differences in expression, and this impacts patients’ response to drug therapy. Studies examining the epigenetic regulation of the UGT enzymes have not previously been performed. Recently, it has been shown that the phase I metabolic cytochrome P-450s (CYPs) are negatively regulated by microRNA (miRNA). miRNA are short, endogenous strands of RNA roughly 22-24 nucleotides in length that regulate gene expression. MiRNA specifically bind to the 3’ untranslated region (UTR) of mature messenger RNA (mRNA) and post-transcriptionally repress protein expression. Purpose: The goal of this study is to identify and evaluate miRNA regulation of UGT1A expression and whether this regulation contributes to differences in UGT1A enzymatic activity. Results: miR-491-3p was identified using the miRanda prediction program. Luciferase assays showed a significant reduction in luciferase activity with an attached UGT1A 3’UTR in the presence of miR-491-3p mimic (P Citation Format: Douglas F. Dluzen, Anna Salzberg, Dongxiao Sun, Nathan Jones, Ryan Bushey`, Philip Lazarus. miR-491-3p regulation of the UDP-glucuronosyltransferase (UGT) 1A gene family. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3089. doi:10.1158/1538-7445.AM2013-3089

UDP-glucuronosyltransferases (UGTs) play an important role in the metabolism and excretion of various endogenous and xenobiotic compounds including several carcinogens and chemotherapeutic agents. UGT2A1 is an extra-hepatic member of the UGT2A sub-family that was shown to be expressed in the lung in previous studies. The goal of the present study was to examine UGT2A1 expression in human tissues, assess its glucuronidation activity against exogenous substrates including lung carcinogens, and determine the potential functional role of UGT2A1 polymorphisms on UGT2A1 enzyme activity. Cell homogenates were prepared from HEK293 cells transfected with wild type or variant UGT2A1. Enzyme activity against carcinogens was determined through reverse-phase UPLC. Expression of UGT2A1 was determined by RT-PCR/sequencing, real time PCR (ABI), and western blot using a UGT2A1-specific antibody. In addition to the wild-type UGT2A1*1, two prevalent variants of UGT2A1 were investigated: a variant that causes a lysine to arginine amino acid change at codon 75 (UGT2A1*2), and a variant that causes a glycine to arginine amino acid change at codon 308 (UGT2A1*3). In addition, a newly-identified splice variant isoform lacking exon 3 (allele identified as UGT2A1*1a since it was not yet determined whether this variant was due to a genetic polymorphism or to altered splicing) was also investigated. As determined by RT-PCR and direct sequencing, UGT2A1 was expressed in lung, trachea, larynx, tonsil, mouth, and colon; no expression was observed in breast, whole brain, cerebral cortex or esophageal tissues. Reverse transcription-PCR demonstrated that tissues had variable levels of expression of UGT2A1 and the splice variant encoded by UGT2A1*1a; the UGT2A1*1a encoded variant was expressed in 5 of 6 individual lung specimens and a pooled larynx sample from 10 people. Homogenates of HEK293 cells over-expressing UGT2A1*1 or UGT2A1*2 both exhibited significant activity against 1-naphthol, with a Vmax/KM of 4.0 uL.min−1.ug homogenate protein−1 and 3.5 uL.min−1.ug homogenate protein−1 respectively. No activity was observed for cell homogenates over-expressing the UGT2A1*3 variant or the UGT2A1*1a isoform. A similar pattern of activity was observed for a variety of polycyclic aromatic hydrocarbons (PAHs) including 1-hydroxypyrene, dibenzo(a,l)pyrene-11,12-diol, benzo(a)pyrene-7,8-diol, 5-methylchrysene-1,2-diol, 1-hydroxy (OH)-benzo(a)pyrene, 7-OH-benzo(a)pyrene, and 8-OH-benzo(a)pyrene. No activity was observed against substrates that form N-glucuronides such as NNAL, nicotine or N-OH-PhIP. UGT2A1 is expressed in various target organs for tobacco-induced carcinogenesis and exhibits activity against a variety of PAHs abundant in tobacco smoke. The UGT2A1308Arg variant could play an important role in tobacco-related cancer risk due to a lack of enzyme activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1698.