Targeting Trends

Apr-May-Jun 2008Volume 9, Issue 2Targeting TrendsReporting the latest news in Molecular SurgeryInside this issue:Targeting TopicsScientific References 3Targeting TalkCytotoxicity Assays 5Targeting ToolsFeatured Products 7Targeting TeaserWord Quiz 8NewsletterHighlights! ATS Licenses SP-SAP(page 2)! Upcoming Events(page 2)! Teaser Winners(page 2)! mu p75-SAP,WBLyse, CFSE(page 7)Denise Higgins, EditorSelective lesion of basal forebrain cholinergic neurons inmice with the mu p75-saporin immunotoxin:Neuroanatomy and behaviorContributed by Pierre-Henri Moreau, Brigitte Cosquer, Hélène Jeltsch, Jean-Christophe Cassel, Chantal MathisL a b o r a t o i re d’Imagerie et de Neurosciences Cognitives, UMR7191 CNRS, Equipe de Neurobiologie Cognitive etC o m p o rtementale, Université Louis Pasteur, IFR 37 de Neurosciences, GDR 2905 CNRS, 12 rue Goethe, 67000S t r a s b o u rg, FranceThe basal forebrain cholinergic neurons (BFCNs) are dramatically affected in severalneurodegenerative diseases such as Alzheimer’s disease or Rett syndrome. Thecharacterization of the behavioral consequences of selective BFCN lesions is necessary tostudy the implication ofthese neurons in cognitivefunctions. Until recently, thismodel was not available inmice, despite the growinginterest in this species, dueto the creation of a widevariety of geneticallymodified mouse linesmodeling neurodegenerativediseases. The first version ofa new cholinergicimmunotoxin, mu p75-SAP,induced specific lesions ofthe BFCNs associated withdramatic memoryperformance deficits, but italso showed side effects andpoor survival rates (Berger-(continued on page 6)Figure 1. Micrographs of braincoronal sections of mice treated withPBS or mu p75-saporin (SAP). Inlesioned mice, the number of ChATpositiveneurons is dramaticallyreduced in the medial septum (MS)and the diagonal band of Broca(DBB) (a,c) and in the nucleus basalis(NB) (b,d). AChE staining ismassively depleted in the corticalmantle (e,g) and the hippocampus(f,h), but not in the thalamus (Th).Scale bar = 200 µm.

Page 2ATS Licenses SP-SAP to Advanced Pain T h e r a p e u t i c sSubstance P receptor-positiveneuron eliminated with SP-SAPTargeting TrendsAdvanced Targeting Systems is pleased to announce that it has licensed itsSubstance P receptor-targeted chronic pain drug (SP-SAP). Advanced PainTherapeutics, LLC (APT) obtained exclusive, worldwide rights to develop,manufacture, use, and sell SP-SAP for the treatment of severe chronic pain. CatoResearch, a global contract research and development organization, will provide CROservices to the new company.SP-SAP is a single-dose, non-opioid, substance P receptor-targeted treatmentdesigned to specifically bind to and eliminate a subset of neurons that send thechronic pain signal to the brain. Preclinical studies in animal models have shown thatSP-SAP eliminates chronic pain without disrupting other sensory modalities or motorfunction and is well tolerated.“When we partner early, as we have here, we can make a major difference in theoverall development of promising drug candidates such as SP-SAP,” said LyndaSutton, COO of Cato Research and CEO of Advanced Pain Therapeutics. “Theflexible, broad-based relationships among APT, ATS, and Cato Research position us well to execute our innovativedrug development model.”Denise Higgins, Vice President of ATS, affirms that this arrangement allows for a unique synergy between thecompanies. “We are enthusiastic about working with APT and Cato Research to help address the under-served chronicpain population. Our preclinical work with SP-SAP makes us very optimistic about the impact this innovativetreatment can have for those who are suffering.”Chronic disease states and tissue damage can lead to chronic pain. In particular, terminally ill patients oftenexperience severe, chronic pain due to advancement of disease or unwanted side effects of treatment. Although inmany cases, standard treatments, such as opioids, can control pain, there is a significant subset of patients who cannotfind relief through standard care. Use of opioids can also be associated with unwanted, severe side-effects. In thesecases, sedation or cordotomy may be a patient’s only option for pain relief. Hence, severe chronic pain represents aserious, poorly met medical need. SP-SAP offers a novel approach to target a specific set of neurons involved inchronic pain and as such, has the potential to revolutionize the way severe chronic pain is treated.Ta rgeting Teaser Wi n n e r sThe solution to the puzzle was:Across: 1. TARGET5. GROWTH7. EPA8. LEG11. FRACTION13. TOXINDown: 1. TUBE2. RECEPTOR3. ERG4. CAT6. WELL9. GEL10. STUN12. OILCongratulations to the puzzle solversfrom our last newsletter. Each winnerreceives $100 credit towards researchproduct purchases from AdvancedTargeting Systems.WINNERS: Valery Nelson, Panacea Pharmaceuticals, Gaithersburg MD * Barry Marguiles, Towson University,Towson MD * Seto Chice- SUNY HSC at Brooklyn, Brooklyn NYExperimental BiologyApril 5-9, 2008San Diego, CABooth #1135Amer Assoc for Cancer ResearchApril 12-16, 2008San Diego, CABooth #1139

Volume 9, Issue 2Ta rgeting Talk: Cytotoxicity A s s a y sby Dr. Douglas LappiPage 5One of the tests you can use to test yourtargeting agent for internalization is the in vitroCytotoxicity Assay. Protocols to assist in preparingfor, executing and interpreting results are nowposted on our website. From the Home page(www.ATSbio.com) click on Protocols.There are several protocols available.1. Preparing for a Cytotoxicity Assay usingSecondary Conjugates. This protocol will behelpful when using our secondary antibodysaporinconjugates with your primary antibody.These include Anti-M-ZAP (Cat. #IT-30), Goat-ZAP (Cat. #IT-36), Hum-ZAP (Cat. #IT-22),Mab-ZAP (Cat. #IT-04), Rab-ZAP (Cat. #IT-05),and Rat-ZAP (Cat. #IT-26).2. Preparing for a Cytotoxicity Assay usingStreptavidin-ZAP. This protocol will be helpfulwhen using our streptavidin-saporin conjugatewith your biotinylated targeting agent (peptide,ligand, cytokine, growth factor, antibody, etc.).3. Concentration Calculation: Convert molarity tomg/ml and mg/ml to molarity. This protocolwill help in determining the correct amount ofmaterial to use in your assay. There is also alink to an Online Calculator.4. Cytotoxicity Assay for Targeted Toxins in vitro.This protocol includes photos of what yourplates should look like during the assay process.It takes five days to complete this assay. Starton a Monday and develop on Friday. There aremany factors that go into a successfulDay Five. Lighter-colored wells signify dead cells.Your targeted toxin is working.cytotoxicity assay. This protocol should helpyou design and execute appropriately.5. Preparing Cytotoxicity Data. This protocol willgive an example of how to process the datafrom a Cytotoxicity Assay. ATS uses SOFTMaxPro software connected to a plate reader todetermine the A490 value. Then we import thisdata into Prism software (GraphPad) toconduct further data analysis. Here is a figuregenerated with Prism.This graph gives important information abouthow the potency of your targeted toxin. TheED50 is the Median Effective Dose (producesdesired effect in 50% percent of population).The lower this number is, the more potent thetargeted toxin.We hope these protocols will be helpful to youin your research. If there are additionalprotocols or tutorials we can provide, please donot hesitate to ask.Starting a new lab? Waiting for equipment?Let us test your materials for you. ATS is expert atconducting in vitro assays with targeted toxins.Send us your primary antibody, peptide or protein,ligand, or lectin. When the in vitro results confirmthe desired specificity, ATS can prepare acustom saporin conjugate.Email ATS (ats@atsbio.com) or call toll-free (877)889-2288 to discuss your project.All discussions and services can be covered with aconfidentiality agreement.

Page 6Targeting TrendsSelective lesion of BFCN with mu p75-SAP(continued from page 1)Sweeney et al., 2001; Hunter et al., 2004).Therefore, as soon as the improved version of mup75-saporin was released by ATS, we characterizedthe neuroanatomical and behavioral effects of thisnew cholinergic neurotoxin.Mu p75-saporin was administered bilaterally inthe cerebral ventricles of male C57BL/6J at thedose of 0.4 µg/mouse. At this dose, all treated micesurvived and none of them showed abnormal lossof weight or epileptic-like episodes as obtainedwith higher doses or reported with the previousversion of the toxin. The loss of cholineacetyltransferase-immunoreactive neurons wasmore pronounced in the medial septum (-82%, seefigure 1) and the diagonal band of Broca than in thenucleus basalis (-55%). The cholinergic specificityof the lesion was suggested by preservedparvalbumin immunostaining. The hippocampusand several regions of the cortical mantle exhibiteda marked drop in the levels of acetylcholinesterasepositivestaining. This suggests that septohippocampaland basal-cortical projections of theBFCNs underwent massive degeneration. Asopposed to the cholinergic toxin 192 IgG-SAP usedin rats, mouse Purkinje cells remained undamagedby mu p75-saporin as suggested by preserved anticalbindinimmunostaining in the cerebellum(Traissard et al., 2007). The lesion of the BFCNsaffected both locomotor activity as well as learningand memory performances. Nocturnal, and to alesser extent, diurnal locomotor activity wasincreased in lesioned mice. The rate of acquisitionof a water-maze reference memory task wassignificantly slower in mice treated with mu p75-saporin (see figure 2). Acquisition performance wasalso affected in the Barnes maze as suggested by anincrease in the total number of holes and in thelatency to find the target hole. Retentionperformance of lesioned mice was lower than thoseof control mice in both spatial memory tasks,although the effect was significant only in theBarnes-maze probe trial. Motivation, visualcapacities and sensorimotor coordination appearedunaffected in the water-maze visual discriminationtask and the beam-walking test, respectively.The lesion of BFCNs with mu p75-saporininduces behavioral deficits similar to those reportedafter ICV 192 IgG-SAP in rats, but withoutFigure 2. Mu p75-saporin (SAP) treated mice show a slower learning comparedto PBS controls in the water-maze spatial reference memory task. * p < 0.01,significantly different from PBS.Purkinje cell damage. In addition, this new version of themouse immunotoxin has fewer side effects and appears moreefficient than its predecessor. This safer and more powerfultool may be particularly adapted to improve transgenic modelsof AD in which amyloid and/or tangle pathologies areexpressed, but do not result in extensive loss of cholinergicneurons.References1. Berger-Sweeney J, Stearns NA, Murg SL, Floerke-Nashner LR, Lappi DA,Baxter MG. 2001. Selective immunolesions of cholinergic neurons in mice:Effects on neuroanatomy, neurochemistry, and behavior. J Neurosci 20:8164-8173.2. Hunter CL, Quintero EM, Gilstrap L, Bhat NR, Granholm AC. 2004.Minocycline protects basal forebrain cholinergic neurons from mu p75-saporinimmunotoxic lesioning. Eur J Neurosci 12:3305-3316.3. Traissard N, Herbeaux K, Cosquer B, Jeltsch H, Ferry B, Galani R, Pernon A,Majchrzak M, Cassel JC. 2007. Combined damage to entorhinal cortex andcholinergic basal forebrain neurons, two early neurodegenerative featuresaccompanying Alzheimer's disease: effects on locomotor activity and memoryfunctions in rats. Neuropsychopharmacol 4:851-871.Do you have an idea for a new target? Contact us with your suggestion. Ifyour target is chosen for development of a targeted toxin, we will providethe conjugate to you at no charge. Just send an email to Denise Higgins,Vice President of Business Development at: denise@ATSbio.com.

Volume 9, Issue 2Ta rgeting Tools: Featured ProductsPage 7mu p75-SAPIn 2004, ATS re-designed the anti-murine p75-SAP t a rg e t e dtoxin (mu p75-SAP, Cat. #IT-16) and produced a conjugate thatis much more potent in our in vitro cell cytotoxicity assays.P r e v i o u s l y, we used a rat monoclonal antibody. This antibodyhad been outperformed by our rabbit polyclonal (Cat. #AB-N01), in several assays, especially flow cytometry analysis ofmurine p75-expressing cells. This is an important indicator ofbeing able to bind to the cell surface, which is fundamental fora targeted toxin.To create this toxin, we affinity-purified the rabbitpolyclonal (Cat. #AB-N01A P) with the immunogen bound to asolid support, and conjugated the affinity-purified antibody tosaporin. As can be seen in the cytotoxicity assay on the right,the new mu p75-SAP is orders of magnitude more potent thanthe previous conjugate. The new and more active version ofmu p75-SAP has an ED5 0 in the picomolar range compared toan ED5 0 in the nanomolar range for the previous product. Webelieve that the greater potency will translate to smalleramounts used for elimination of p75-positive neurons in themouse brain, and that this will result in a greater index ofe fficacy and lesser non-specific cytotoxicity. (see cover articlefor in vivo r e s u l t s ) .The mu p75-SAP kit includes, in addition to theimmunotoxin, equal aliquots of saporin (Cat. #PR-01), thea ffinity-purified rabbit polyclonal antibody (AB-N01A P), andthe control immunotoxin, Rabbit-IgG-SAP (Cat. #IT- 3 5 ) .Also available are fluorescent conjugates of A B - N 0 1A P:Cy3-labeled Anti-murine NGFr (Cat. #FL-05), and Cy5-labeledAnti-murine NGFr (Cat. #FL-06).Ahh. The joysof Spring!NG3 cells are plated at 1000 cells/well and incubated overnight. Saporin,mu p75-SAP (conjugate of the affinity-purified rabbit polyclonal to mouseNGFr and saporin), and AB-N02-SAP (previous rat monoclonal version ofmu p75-SAP) are added in 10-µl volumes and the plates are incubated 72hours. PMS/MTS developing reagent is added and the plates are incubated1-2 hours, then read at 490 nm.Flow Cytometry To o l sAdvanced Ta rgeting Systems is excited to offer twonew tools for flow cytometry.W B Lyse (Cat. #FL-08) is a gentle erythrocytelysing reagent matched with a leukocyte preservative.This kit can be used to enumerate lymphocyte subsets,detect a large variety of antigens on lymphocytes, and toidentify other leukocyte subsets, including CD34 stemcells, and granulocytes. W B Lyse works on many typesof specimens and is active against all erythrocytes and isavailable in the 100 or 500 test size.The CFSE (carboxyfluorescein-succinimidyl ester)Compensation Kit (Cat. #FL-13) provides a reproducibleand convenient source of CFSE particles for evaluatingand correcting for the spectral overlap between CFSEand other dyes. Fluorescence compensation is the processwherein a portion of the primary dye signal is removedfrom all non-primary dye channels. Compensation isgenerally required whenever more than a single dye(color) is used in flow cytometric analysis. This 1-colorCFSE Compensation Kit is sufficient for 25 tests andcontains negative/unstained microspheres and CFSEmicrospheres. Cells, once labeled with CFSE, can bestimulated in vitro and cell proliferation measured bychanges in the staining intensity of the cells.

Ta rgeting Te c h n o l o g yAdvanced Targeting Systems’ technology -Molecular Neurosurgery - is amodification of one of the most widelyused techniques: surgical lesioning of aregion and observation of the effect.Choose anANTIBODY §specific toyour celltype.The targeting agent is administered to thecells (in vivo or in vitro).Theantibodyseeks outits targetreceptoron the cellsurface.SAPORINis a potentcytotoxin.Safe in thelab. Lethalin the cell.ATS binds SAPORIN with yourANTIBODY to make apowerful targeting agent.§ or anything recognized on thecell surface and internalized.Cells thatdo nothave thereceptorwill not beaffected.The conjugate is internalized andSAPORIN breaks away from the antibody.SAPORINinactivates theribosomes.The result isCELL DEATH.Unscramble these five Jumbles, oneletter to each block, to solve the puzzle.ZENEMYBOATMICELGREENLOLAIRANCHMOODITFENTFEARAnswer:She wanted her lab to be ...WIN$100.00Limit one entry per laboratory.Please correct the addressinformation below andprovide the following:Ta rgeting Te a s e rWhy the scientist made hermedia green.Arrange the circled letters to form theanswer, as suggested by the above clue.1. Solve the puzzle.2. Fax in this entire page withthe correct solution by May 31, 20083. Win $100 credit toward your next purchase.Your Name:Phone:Fax:Email:“Molecular Surgery for Scientists”PRSRT STDUNITED STATESPOSTAGE PAIDSAN DIEGO, CAPERMIT #1235www.ATSbio.com11175-A Flintkote AvenueSan Diego, CA 92121Toll-Free: (877) 889-2288Phone: (858) 642-1988Fax: (858) 642-1989