Biomedical subjects

Luigi Grasso

Publications and source records attributed to Luigi Grasso.

4 recordsLinked to original sources

PubMed2006

Human antibodies for immunotherapy development generated via a human B cell hybridoma technology.

Current strategies for the production of therapeutic mAbs include the use of mammalian cell systems to recombinantly produce Abs derived from mice bearing human Ig transgenes, humanization of rodent Abs, or phage libraries. Generation of hybridomas secreting human mAbs has been previously reported; however, this approach has not been fully exploited for immunotherapy development. We previously reported the use of transient regulation of cellular DNA mismatch repair processes to enhance traits (e.g., affinity and titers) of mAb-producing cell lines, including hybridomas. We reasoned that this process, named morphogenics, could be used to improve suboptimal hybridoma cells generated by means of ex vivo immunization and immortalization of antigen-specific human B cells for therapeutic Ab development. Here we present a platform process that combines hybridoma and morphogenics technologies for the generation of fully human mAbs specific for disease-associated human antigens. We were able to generate hybridoma lines secreting mAbs with high binding specificity and biological activity. One mAb with strong neutralizing activity against human granulocyte-macrophage colony-stimulating factor was identified that is now considered for preclinical development for autoimmune disease indications. Moreover, these hybridoma cells have proven suitable for genetic optimization using the morphogenics process and have shown potential for large-scale manufacturing.

Amino Acid Sequence↗

PubMed2005

Rapid generation of plant traits via regulation of DNA mismatch repair.

The reversible inhibition of DNA repair is a novel approach to maximize genetic diversity within a plant's genome in order to generate offspring exhibiting important de novo output traits. This process is based on the inhibition of the evolutionarily conserved mismatch repair (MMR) system. In this process, a human dominant negative MMR gene allele is introduced into the germline of a target plant, yielding progeny that can be screened to identify variants with commercially important agronomic output traits. Using this novel strategy, we generated MMR-deficient Arabidopsis thaliana plants that showed genome-wide instability of nucleotide repeats associated with chromosomal microsatellites, in addition to base substitution mutations. Functional screenings of the MMR-deficient Arabidopsis offspring identified variants expressing selectable traits (ethylene insensitivity and salt tolerance), as well as plants exhibiting altered morphologic traits (albinos and dwarfs). We determined by segregation analyses of variant plants that the de novo phenotypes were due to both recessive and dominant genetic mutations. Mutations caused by MMR deficiency showed a different spectrum compared with those derived using ethylmethane sulphonate (EMS) mutagenesis. Our finding demonstrates the feasibility of using reversible MMR deficiency via transient expression of a single human gene product to enhance genetic diversity in plants.

Journal Article↗

PubMed2005

Morphogenics as a tool for target discovery and drug development.

Mutations in DNA mismatch repair (MMR) genes lead to genetically hypermutable cells. Germline mutations in MMR genes in man have been linked to the genetic predisposition to hereditary nonpolyposis colon cancer and a number of other inherited and sporadic malignancies. The ability to modulate the MMR process (referred to as morphogenics) in model systems offers a powerful tool for generating functional diversity in cells and multicellular organisms via the perpetual genomewide accumulation of randomized point and slippage mutation(s). Morphogenics is a platform process that employs a dominant negative MMR gene to create genetic diversity within defined cellular systems and results in a wide range of phenotypes, thus enabling the development and improvement of pharmaceutical products and the discovery of new pharmaceutical targets. Libraries of morphogenics-derived siblings are generated through random mutagenesis from naturally occurring DNA polymerase-induced mutations that occur during DNA replication. Morphogenic cells are screened in high-throughput assays to identify subclones with desired phenotypes for pathway discovery and/or product development. Morphogenics has been successfully applied to a wide range of hosts, including mammalian cells, transgenic mice, plants, yeast, and bacteria. Manipulation of these systems via morphogenics has led to the discovery of novel disease-associated phenotypes in targeted model systems. Moreover, morphogenics has been successfully applied to antibody-producing cell lines to yield subclones producing antibodies with enhanced binding affinities for therapeutic use, as well as to derive subclones with enhanced titers that are suitable for scaleable manufacturing. The selective manipulation of the MMR process via morphogenics is a platform technology that offers many advantages for the discovery of druggable targets, as well as for the development of novel pharmaceutical products.

Base Pair Mismatch↗

PubMed2004

Gene expression analysis of tumor spheroids reveals a role for suppressed DNA mismatch repair in multicellular resistance to alkylating agents.

Drug resistance is a major obstacle in the successful treatment of cancer. Thus, elucidation of the mechanisms responsible is a critical first step in trying to prevent or delay such manifestations of resistance. In this regard, three-dimensional multicellular tumor cell spheroids are intrinsically more resistant to virtually all anticancer cytotoxic drugs than conventional monolayer cultures. We have employed the EMT-6 subline PC5T, which forms highly compact spheroids, and differential display to identify candidate genes whose expression differs between monolayer and spheroids. Approximately 5,000 bands were analyzed, revealing 26 to be differentially expressed. Analysis of EMT-6 tumor variants selected in vivo for acquired resistance to alkylating agents identified eight genes whose expression correlated with drug resistance in tumor spheroids. Four genes (encoding Nop56, the NADH SDAP subunit, and two novel sequences) were found to be down-regulated in EMT-6 spheroids and four (encoding 2-oxoglutarate carrier protein, JTV-1, and two novel sequences) were up-regulated. Analysis of the DNA mismatch repair-associated PMS2 gene, which overlaps at the genomic level with the JTV-1 gene, revealed PMS2 mRNA to be down-regulated in tumor spheroids, which was confirmed at the protein level. Analysis of PMS2(-/-) mouse embryo fibroblasts confirmed a role for PMS2 in sensitivity to cisplatin, and DNA mismatch repair activity was found to be reduced in EMT-6 spheroids compared to monolayers. Dominant negative PMS2 transfection caused increased resistance to cisplatin in EMT-6 and CHO cells. Our results implicate reduced DNA mismatch repair as a determinant factor of reversible multicellular resistance of tumor cells to alkylating agents.

Alkylating Agents↗