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Type 1 interferons (IFN) have been shown to be efficacious against many viruses. However, a significant drawback is the short duration of exposure. We have improved the PK of IFN ALFACON1 (CIFN) by glycoengineering. ALS-624 was generated by introducing four glycosylation sites, which demonstrated a significantly increased Molecular Weight of 30–45kD compared to 19kD for CIFN. ALS-624 was purified and fractionated to yield 4-Gly, 3-Gly and 2-Gly fractions by size exclusion chromatography. In a rat PK study, four groups were subcutaneously dosed with the three glycosylated ALS-624 fractions plus a non-glycosylated CIFN control. The high MW fraction of 4-Gly exhibited a 6-fold increase of AUC and over an 11-fold increase in T1/2 over CIFN suggesting the potential for QW dosing. In a Yellow Fever Virus study, hamsters were injected intraperitoneally with virus. All compounds were administered i.p. at a dose of 1.25×106 IU/kg/dose, given QW (4Gly and Peg-IFN-α-2a), BIW (3Gly), or QD (IFN alfacon-1 and placebo) beginning at −4h and ending 14days post infection. Animals were observed for mortality for 21days. QW dosing of 4-Gly achieved similar efficacy to QD CIFN and QW Peg-IFN-α-2a in overall survival rate and reduction of ALT level. Further refinement resulted in development of ALS-683 which has a MW in the 60–90kD range and maintains undiminished biological potency. In mouse PK/PD study, both ALS-683 mouse homolog and unglycosylated mouse IFNα1 were dosed at 1000IU/g. Animals (N =3) were sacrificed at pre-dose and various time points post-dose. Mouse plasma and liver were analyzed for PK and PD markers. ALS-683 homolog has ∼37-fold improvement in T1/2 and ∼33-fold improvement in AUC when compared with unglycosylated mouse IFNα1. Significantly improved PD responses were also observed. ALS-683 can be further developed for possible once a month dosing. [ABSTRACT FROM AUTHOR]

A functional cure of chronic hepatitis B requires eliminating the hepatitis B virus (HBV)-encoded surface antigen (HBsAg), which can suppress immune responses. STOPS are phosphorothioated single-stranded oligonucleotides containing novel chemistries that significantly reduce HBsAgs produced by HBV-infected liver cells. The STOPS molecule ALG-10000 functions inside cells to reduce the levels of multiple HBV-encoded molecules. However, it does not bind HBV molecules. An affinity resin coupled with ALG-10000 was found to bind several proteins from liver cells harboring replicating HBV. Silencing RNAs targeting host factors SRSF1, HNRNPA2B1, GRP78 (HspA5), RPLP1, and RPLP2 reduced HBsAg levels and other HBV molecules that are concomitantly reduced by STOPS. Host proteins RPLP1/RPLP2 and GRP78 function in the translation of membrane proteins, protein folding, and degradation. ALG-10000 and the knockdowns of RPLP1/2 and GRP78 decreased the levels of HBsAg and increased their ubiquitination and proteasome degradation. GRP78, RPLP1, and RPLP2 affected HBsAg production only when HBsAg was expressed with HBV regulatory sequences, suggesting that HBV has evolved to engage with these STOPS-interacting molecules. The STOPS inhibition of HBsAg levels in HBV-infected cells occurs by sequestering cellular proteins needed for proper expression and folding of HBsAg., Competing Interests: All authors declare no competing interests., (© 2021 The Author(s).)

Aim: To conduct a retrospective study in 400 chronic hepatitis B patients in order to identify hepatitis B viral factors associated with complications of liver disease or development of hepatocellular carcinoma., Methods: The mean follow-up time was 83.6+/-39.6 mo. Alpha-fetoprotein test and abdominal ultrasound were used for cancer surveillance. Hepatitis B basal core promoter mutants, precore mutants, genotypes, hepatitis B viral DNA (HBV DNA) level and hepatitis B e antigen (HBeAg) were measured. Univariate analysis and logistic regression were used to assess odds ratios for viral factors related to liver deaths and hepatocellular carcinoma development., Results: During follow-up, 38 patients had liver deaths not related to hepatocellular carcinoma. On multivariate analysis, older age [odds ratio: 95.74 (12.13-891.31); P<0.0001], male sex [odds ratio: 7.61 (2.20-47.95); P=0.006], and higher log10 HBV DNA [odds ratio: 4.69 (1.16-20.43); P<0.0001] were independently predictive for these liver related deaths. Also, 31 patients developed hepatocellular carcinoma. Multivariate analysis showed that older age [odds ratio: 26.51 (2.36-381.47); P=0.007], presence of precore mutants [odds ratio: 4.23 (1.53-19.58); P=0.02] and presence of basal core promoter mutants [odds ratio: 2.93 (1.24-7.57); P=0.02] were independent predictors for progression to hepatocellular carcinoma., Conclusion: Our results show that high levels of baseline serum HBV DNA are associated with non-hepatocellular carcinoma-related deaths of liver failure, while genetic mutations in the basal core promoter and precore regions are predictive for development of hepatocellular carcinoma.

Background Patients with chronic hepatitis B virus (HBV) infection are at risk for death from complications of liver disease and development of hepatocellular carcinoma (HCC). To identify the time course and risk factors associated with these events, we conducted a prospective study in chronic hepatitis B patients referred to our clinic. Methods From January 1989 to March 1998, 400 hepatitis B surface antigen (HBsAg)-positive patients were classified into three categories: inactive carriers (N=110), chronic hepatitis (N=151), and cirrhosis (N=139). These patients were observed at 3- to 6-month intervals with liver tests, alpha-fetoprotein (AFP) levels, and ultrasound examinations. The study endpoints were death from liver disease complications and development of HCC. Results The patients were followed for a mean time (± SD) of 83.6 ± 39.6 months. During this period, no liver-related deaths or HCC were noted in inactive carriers. However, 38 of 139 (27.3%) patients with cirrhosis died from non-HCC-related liver complications. Multivariate analysis demonstrated that male sex (odds ratio [OR] 5.9; 95% confidence interval [CI], 2.0-22.6; P =.003), decreased initial serum albumin (OR 69.1; 95% CI, 11.5-486.4; P =.0009), low platelet count (OR 8.8; 95% CI, 0.96-92.9; P =.05), and presence of cirrhosis (OR 14.2; 95% CI, 3.4-111.8; P =.0009) were independently associated with increased mortality from chronic hepatitis B. During the same time period, nine of 151 (6.0%) chronic hepatitis patients and 22 of 139 (15.8%) patients with cirrhosis developed HCC. By multivariate analysis, progression to HCC was associated with advanced age (OR 19.7; 95% CI, 1.9-231.9; P =.01) and presence of cirrhosis (OR 3.6; 95% CI, 1.6-8.9; P =.003). Patients positive for hepatitis B early antigen (HBeAg) and HBeAg antibodies experienced liver-related deaths and developed HCC at similar rates. Conclusions This prospective study from the United States confirms previous observations of the high risk of mortality and development of HCC in patients infected with HBV. To decrease the risk of these complications, antiviral therapy should be initiated early in the course of the disease. In addition, surveillance for HCC must be performed at least every 6 months in patients with chronic hepatitis and cirrhosis.

Breast cancer accounts for 30-40% of all deaths from cancers in females. In an effort to identify tumor associated antigens that may be useful for immunotherapy, we utilized serological analysis of recombinant cDNA expression libraries (SEREX) technique to identify breast cancer-associated antigens. SEREX screening of cDNA expression libraries derived from 3 breast cancer patients identified a total of 88 positive clones (bcg-1 to bcg-88), including 27 hitherto unknown sequences. The cDNA sequences and mRNA expression patterns were characterized. Seroreactivity of the SEREX clones were determined in sera from 75 breast cancer patients, 75 colon cancer patients, and 25 healthy donors. Expression analysis on a cDNA panel from 17 different normal tissues by reverse transcription-PCR (RT-PCR) revealed tissue restricted mRNA expression of 2 of the 27 unknown antigens. Bcg-72 is expressed only in breast, prostate and thymus, while bcg-84 is expressed at moderate levels in testis, spleen and breast. The other 25 unknown antigens were expressed in most other tissues. Serologic assay revealed that 7 out of the 88 clones showed reactivity to at least one serum from either 75 breast or 75 colon cancer patients. These clones did not react with sera from a panel of 25 healthy adult individuals. Our results demonstrate the utility of the SEREX approach for the identification of potential tumor associated antigens in human breast cancer.