Notes+from+various+documents+11-18-12

overview identify tumor specific not just upregulated cancer antigens highthroughput manner (pooled tumor cDNA library lysates, phage, random peptide arrays)

Cancer vaccines -vaccines can beat infectious diseases as smallpox, which was eradicated, and polio, which is near eradication -how tumor is like a pathogen (causes harm, some unique mutations, and frameshifts) -how tumor can be targeted (common mutations and frameshifts) -common tumor frameshifts -prophylactic cancer vaccines work in animal models --vaccination with a tumor associated antigen was able to repress mammary tumors in rats (Vaccination with soluble lowmolecular weight tumor-associated proteins suppresses chemically-induced mammary tumorigenesis in rats,”) --whole cell tumor lysate (Immunotherapeutic potential of whole tumour cells,”) --Self tumor mixtures are being tested in clinical trials (Genetic fusion of chemokines to a self tumor antigen induces protective, T-cell dependent antitumor immunity,”) -some of these methods are impractical for humans

-good to find specific antigens (other antigens might cause auto-immunity or prevent patient from overcoming autoimmunity) -breast cancer associated antigens have been identified such as MUC1, HER2/neu, and MAGE3 (Cancer vaccines: between the idea and the reality) -tumor associated antigens also occur in normal cells -tumor specific antigens (mutations, frameshifts, chromosomal rearrangements, aberrant protein modifications) -a lymphoma tumor specific antigen (Tumor-specific antigen of murine T-lymphoma defined with monoclonal antibody.)

Serological analysis notes 1-27-13

Protein microarray -example of binding sera to a human protein microarray (Protein microarrays for highly parallel detection and quantitation of specific proteins and antibodies in complex solutions) -random peptides can act as mimotopes (even if the original epitope contains post translational modifications such as glycosylations.) -example of our use of the random peptide array (Bacterial glycoprofiling by using random sequence peptide microarrays) -ability of array to distinguish diseases (good for diagnoses) --treatment often most effective when started early -cell lysate arrays will contain unannotated proteins and mutated proteins which may not be on databases and would not be on protein microarrays -methods for recognizing antibody antigen interactions (ELISAs, phage display, ribosome display, beads, and microarrays). They all have different pros and cons in different areas. Billings et al., 1983. . (Usuda et al., 1999 . . L-F. and M., 2004-01-01 . . (Schimmele and Pluckthun, 2005. . Lam et al., 1996. . (Hueber et al. -survey target binding with in vitro proteins or peptides (Hueber et al.; Lam et al., 1996). --decoding what is bound can be difficult. decoding what is bound through peptide-sequencing, chemical-tracking, or other reading methods. The synthesis, binding, and decoding steps tend to be laborious, time consuming, and often lack reproducibility (Gao et al., 2004). --antigens or epitopes may not be known due to mutant, unknown -with a random peptide library one library can be used for all screens -antibody panned against phages with random peptide fusions can reveal mimotopes Folgori et al., 1994) -microarrays with random peptides or peptoids can find ligands Lim et al., 2009 Probing of a SPOT synthesis array carrying 5520 random 15-mers with three different monoclonal antibodies followed by substitutional analysis was able to identify mimotopes of the known wild type epitopes (Reineke et al., 2002). -our labs technology: 10,000 random peptides printed onto a functionalized glass slied -patterns of binding against the 10k peptides can be used for classifying diseases and diagnoses (Stafford et al., 2012)

SMC1 -SMC first found necessary for nuclear division (Larionov et al., 1985; Strunnikov et al., 1993). -SMC1 needed for nuclear division (SMC1: an essential yeast gene encoding a putative head-rod-tail protein is required for nuclear division and defines a new ubiquitous protein family,”) -many functions of SMC1 involved in the cell division of human cells where it serves many functions such as stabilizing the sister chromatids during their replication and separation, helping to repair DNA, helping to activate the S phase check point, and regulating gene transcription by interaction with CTCF which is a transcriptional insulator (SMC1 is a downstream effector in the ATM/NBS1 branch of the human S-phase checkpoint) -SMC1 may be involved in tumors (Shen's work) -mutation may lead to the progression of cancer (Yazdi et al., 2002) -we have correlated increased levels of a particular aberrant transcript encoding a frame-shifted SMC1A (SMC1Afs) with tumor cells relative to normal cells (Luihen Shen, manuscript in prep). -This exon 4 reading frame shift produces a truncated protein ending with 17 unique amino acids.

Phage Antibody Libraries -Phage allow amplification in an abbreviated form expressed by a bacteriophage (Making antibodies by phage display) -structure of antibodies and phage -just variable regions are included (not constant regions) -immunoglobulin-specific primers can amplify RNA from B cells to make library (Optimization of primer sequences for mouse scFv repertoire display library construction) -disadvantages: work involved, and binding may not be exactly the same in every case. -original used against library of peptides to bind to a given antibodiy (Scott and Smith, 1990) -libraries are "panned" against a particular molecule of interest - Phages are washed across an isolated, immobilized target; bound recombinant phage are collected and amplified in bacteria for additional rounds of panning (Coomber, 2002). - some disadvantages of phage include viability of bacteria, whether scFv is displayed well on surface, etc.