Adam Lowe, Ph.D.

Melan-A is a cancer testis antigen commonly found in melanoma, and has been shown to stimulate the body’s immune response against cancerous cells. We have developed and executed a process utilizing current good manufacturing practices (cGMP) to produce the 6 times-His tagged protein in C41DE3 Escherichia coli for use in Phase I clinical trials. Approximately 11 g of purified Melan-A were produced from a 20 L fed-batch fermentation. Purification was achieved through a three column process… Show more

Melan-A is a cancer testis antigen commonly found in melanoma, and has been shown to stimulate the body’s immune response against cancerous cells. We have developed and executed a process utilizing current good manufacturing practices (cGMP) to produce the 6 times-His tagged protein in C41DE3 Escherichia coli for use in Phase I clinical trials. Approximately 11 g of purified Melan-A were produced from a 20 L fed-batch fermentation. Purification was achieved through a three column process utilizing immobilized metal affinity, anion exchange, and cation exchange chromatography with a buffer system optimized for low-solubility, high LPS binding capacity proteins. The host cell proteins, residual DNA, and endotoxin concentration were well below limits for a prescribed dose with a final purity level of 91%. Show less

NY-ESO-1 is a cancer testis antigen expressed in numerous cancers. Initial tests have shown its efficacy as a cancer vaccine, stimulating the body's own immune response against the invading tumor. To produce enough material for phase I clinical trials, a process using current good manufacturing practices to produce clinical grade material was developed and executed. His-tagged NY-ESO-1 was expressed in C41DE3 Escherichia coli under control of the T-7 promoter. NY-ESO-1 was produced in a 20 L… Show more

NY-ESO-1 is a cancer testis antigen expressed in numerous cancers. Initial tests have shown its efficacy as a cancer vaccine, stimulating the body's own immune response against the invading tumor. To produce enough material for phase I clinical trials, a process using current good manufacturing practices to produce clinical grade material was developed and executed. His-tagged NY-ESO-1 was expressed in C41DE3 Escherichia coli under control of the T-7 promoter. NY-ESO-1 was produced in a 20 L fed-batch fermentation utilizing a pH-stat control scheme. The protein was then purified from inclusion bodies using a three-column process that achieved a yield of over 3.4 g and endotoxin below the detection limit of 0.005 EU/μg protein. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011 Show less

Single nucleotide polymorphisms (SNPs) are one of the key diagnostic markers for genetic disease, cancer progression, and pharmcogenomics. The ligase detection reaction (LDR) is an excellent method to identify SNPs, combining low detection limits and high specificity. We present the first multiplex LDR-surface enhanced Raman spectroscopy (SERS) SNP genotyping scheme. The platform has the advantage in that the diagnostic peaks of Raman are more distinct than fluorescence, and in theory, a… Show more

Single nucleotide polymorphisms (SNPs) are one of the key diagnostic markers for genetic disease, cancer progression, and pharmcogenomics. The ligase detection reaction (LDR) is an excellent method to identify SNPs, combining low detection limits and high specificity. We present the first multiplex LDR-surface enhanced Raman spectroscopy (SERS) SNP genotyping scheme. The platform has the advantage in that the diagnostic peaks of Raman are more distinct than fluorescence, and in theory, a clinically significant number of markers can be multiplexed in a single sample using different SERS reporters. Here we report LDR-SERS multiplex SNP genotyping of K-Ras oncogene alleles at 10 pM detection levels, optimization of DNA labeling as well as Raman conditions, and the linear correlation of diagnostic peak intensity to SNP target concentration in heterozygous samples. Genomic DNA from typed cells lines was obtained and scored for the K-Ras genotype. These advances are significant as we have further developed our new SNP genotyping platform and have demonstrated the ability to correlate genotype ratios directly to diagnostic Raman peak signal intensity. Show less

Recombinant immunotherapeutics are important biologics for the treatment and prevention of various diseases. Immunotherapy can be divided into two categories, passive and active. For passive immunotherapy, the successes of antibody and cytokine therapeutics represent a promising future and opportunities for improvements. Efforts, such as cell engineering, antibody engineering, human-like glycosylation in yeast, and Fab fragment development, have led the way to improve antibody efficacy while… Show more

Recombinant immunotherapeutics are important biologics for the treatment and prevention of various diseases. Immunotherapy can be divided into two categories, passive and active. For passive immunotherapy, the successes of antibody and cytokine therapeutics represent a promising future and opportunities for improvements. Efforts, such as cell engineering, antibody engineering, human-like glycosylation in yeast, and Fab fragment development, have led the way to improve antibody efficacy while decreasing its high manufacturing costs. Both new cytokines and currently used cytokines have demonstrated therapeutic effects for different indications. As for active immunotherapy, recently approved HPV vaccines have encouraged the development of preventative vaccines for other infectious diseases. Immunogenic antigens of pathogenic bacteria can now be identified by genomic means (reverse vaccinology). Due to the recent outbreaks of pandemic H1N1 influenza virus, recombinant influenza vaccines using virus-like particles and other antigens have also been engineered in several different recombinant systems. However, limitations are found in existing immunotherapeutics for cancer treatment, and recent development of therapeutic cancer vaccines such as MAGE-A3 and NY-ESO-1 may provide alternative therapeutic strategy. Show less

Genomics provides a comprehensive view of the complete genetic makeup of an organism. Individual sequence variations, as manifested by single nucleotide polymorphisms (SNPs), can provide insight into the basis for a large number of phenotypes and diseases including cancer. The ability rapidly screen for SNPs will have a profound impact on a number of applications, most notably personalized medicine. Here we demonstrate a new approach to SNP detection through the application of surface-enhanced… Show more

Genomics provides a comprehensive view of the complete genetic makeup of an organism. Individual sequence variations, as manifested by single nucleotide polymorphisms (SNPs), can provide insight into the basis for a large number of phenotypes and diseases including cancer. The ability rapidly screen for SNPs will have a profound impact on a number of applications, most notably personalized medicine. Here we demonstrate a new approach to SNP detection through the application of surface-enhanced Raman scattering (SERS) to the ligase detection reaction (LDR). The reaction uses two LDR primers, one of which contains a Raman enhancer and the other a reporter dye. In LDR, one of the primers is designed to interrogate the SNP. When the SNP being interrogated matches the discriminating primer sequence, the primers are ligated and the enhancer and dye are brought into close proximity enabling the dye's Raman signature to be detected. By detecting the Raman signature of the dye rather than its fluorescence emission, our technique avoids the problem of spectral overlap which limits number of reactions which can be carried out in parallel by existing systems. We demonstrate the LDR-SERS reaction for the detection of point mutations in the human K-ras oncogene. The reaction is implemented in an electrokinetically active microfluidic device that enables physical concentration of the reaction products for enhanced detection sensitivity and quantization. We report a limit of detection of 20 pM of target DNA with the anticipated specificity engendered by the LDR platform. Show less

(-)-Deoxypseudophrynaminol 1 possesses 43-fold greater antibacterial potency than the racemate toward Staphylococcus aureus, indicating that the (-)-enantiomer is the biologically active isomer in this assay. Comparison of the percent growth inhibition by derivatives of 1 indicates that prenylation of N8 and replacement of N1-methyl by methyl carbamate are detrimental to antibacterial potency. (-)-1 is a promising lead structure for the development of the novel hexahydropyrrolo[2,3-b]indole… Show more

(-)-Deoxypseudophrynaminol 1 possesses 43-fold greater antibacterial potency than the racemate toward Staphylococcus aureus, indicating that the (-)-enantiomer is the biologically active isomer in this assay. Comparison of the percent growth inhibition by derivatives of 1 indicates that prenylation of N8 and replacement of N1-methyl by methyl carbamate are detrimental to antibacterial potency. (-)-1 is a promising lead structure for the development of the novel hexahydropyrrolo[2,3-b]indole class of antibacterial agents. Show less

This paper presents a brief review of metal-citrate coordination chemistry and its relevance to secondary metal-citrate transporters, a recently discovered family of proteins, designated as CitMHS. The potential role of CitMHS in gram positive immunology, for example in treatment of MRSA, is also discussed.

This book chapter presents practical information and protocols for removing endotoxin from biopharmaceutical preparations.