Name: preparation of tumor antigen-loaded mature dendritic cells for immunotherapy
Uploaded: 2013-08-01T12:00:00
Description: Watch this Scientific Journal Video about Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy at JoVE.com

Forfatterne ønsker å takke Andres Salazar (Oncovir, Inc.) for gaven av Poly-ICLC.

En. Isolasjon og Nedfrysing av PBMCs 4 <ol><li> Aseptisk pigg en av adkomstveiene havner i leukaferese bag ved hjelp av en plasma overføring sett. Ved hjelp av en 60 ml sprøyte, overfører leukaferese oppnådd fra pasienter i en steril 500 ml flaske. </li><li> Juster volumet på leukaferese til 2x sin opprinnelige volumet ved hjelp romtemperatur RPMI. Bland godt. </li><li> Bland flaske Ficoll-Paque PLUS. Legg 12 ml Ficoll-Paque PLUS i sterile 50 ml konisk tube. </li><li> Forsiktig lag 30 ml av det fortynn...

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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clinical+grade+cocktail+of+cytokines+and+PGE2+results+in+uniform+maturation+of+human+monocyte-derived+dendritic+cells:+implications+for+immunotherapy.">A clinical grade cocktail of cytokines and PGE2 results in uniform maturation of human monocyte-derived dendritic cells: implications for immunotherapy.</a> Vaccine. 20, A8-A22 (2002).</li><li>Bhardwaj, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+immune+system+to+treat+cancer.">Harnessing the immune system to treat cancer.</a> J. Clin. Invest. 117, 1130-1136 (2007).</li><li>Gnjatic, S., Sawhney, N. 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Immunol. 163, 57-61 (1999).</li><li>Rouas, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(I:C)+used+for+human+dendritic+cell+maturation+preserves+their+ability+to+secondarily+secrete+bioactive+IL-12.">Poly(I:C) used for human dendritic cell maturation preserves their ability to secondarily secrete bioactive IL-12.</a> Int. Immunol. 16, 767-773 (2004).</li><li>Jongmans, W., Tiemessen, D. M., van Vlodrop, I. J., Mulders, P. F., Oosterwijk, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th1-polarizing+capacity+of+clinical-grade+dendritic+cells+is+triggered+by+Ribomunyl+but+is+compromised+by+PGE2:+the+importance+of+maturation+cocktails.">Th1-polarizing capacity of clinical-grade dendritic cells is triggered by Ribomunyl but is compromised by PGE2: the importance of maturation cocktails.</a> J. Immunother. 28, 480-487 (2005).</li><li>Krause, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostaglandin+E2+is+a+key+factor+for+monocyte-derived+dendritic+cell+maturation:+enhanced+T+cell+stimulatory+capacity+despite+IDO.">Prostaglandin E2 is a key factor for monocyte-derived dendritic cell maturation: enhanced T cell stimulatory capacity despite IDO.</a> J. Leukoc. Biol. 82, 1106-1114 (2007).</li><li>Morelli, A. E., Thomson, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cells+under+the+spell+of+prostaglandins.">Dendritic cells under the spell of prostaglandins.</a> Trends Immunol. 24, 108-111 (2003).</li><li>Adams, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+cell+(DC)+based+therapy+for+cervical+cancer:+use+of+DC+pulsed+with+tumour+lysate+and+matured+with+a+novel+synthetic+clinically+non-toxic+double+stranded+RNA+analogue+poly+[I]:poly+[C(12)U]+(Ampligen+R).">Dendritic cell (DC) based therapy for cervical cancer: use of DC pulsed with tumour lysate and matured with a novel synthetic clinically non-toxic double stranded RNA analogue poly [I]:poly [C(12)U] (Ampligen R).</a> Vaccine. 21 (12), 787-790 (2003).</li><li>Colombo, M. P., Trinchieri, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-12+in+anti-tumor+immunity+and+immunotherapy.">Interleukin-12 in anti-tumor immunity and immunotherapy.</a> Cytokine Growth Factor Rev. 13, 155-168 (2002).</li><li>Mayordomo, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow-derived+dendritic+cells+pulsed+with+synthetic+tumour+peptides+elicit+protective+and+therapeutic+antitumour+immunity.">Bone marrow-derived dendritic cells pulsed with synthetic tumour peptides elicit protective and therapeutic antitumour immunity.</a> Nat Med. 1, 1297-1302 (1995).</li><li>Dhodapkar, M. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+generation+of+broad+T-cell+immunity+in+humans+after+a+single+injection+of+mature+dendritic+cells.">Rapid generation of broad T-cell immunity in humans after a single injection of mature dendritic cells.</a> J. Clin. Invest. 104, 173-180 (1999).</li><li>Schuler-Thurner, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+induction+of+tumor-specific+type+1+T+helper+cells+in+metastatic+melanoma+patients+by+vaccination+with+mature,+cryopreserved,+peptide-loaded+monocyte-derived+dendritic+cells.">Rapid induction of tumor-specific type 1 T helper cells in metastatic melanoma patients by vaccination with mature, cryopreserved, peptide-loaded monocyte-derived dendritic cells.</a> J. Exp. Med. 195, 1279-1288 (2002).</li></ol>

Fase I og II kliniske studier av blodmonocyttavledede DCs har vist at de indusere immunresponser hos pasienter imidlertid klinisk suksess har vært begrenset en. Dette kan delvis skyldes mangel på enighet om hvordan å generere de optimale DCs for svulst immunterapeutisk bruk. Selv om det er mange måter å generere klinisk grad DCS disse metodene varierer når det gjelder bruken av cytokiner som brukes til å differensiere monocytter er stimuleringen brukes til å indusere modningen og fremgangsmåter for a...

<table border="1">
<tbody>
 <tr>
 <td fo:width="2in">Reagent/Supplies/Equipment</td>
 <td>Manufacturer</td>
 <td>Catalog No.</td>
 <td></td>
 </tr>
 <tr>
 <td>RPMI-1640 medium with L-glutamine</td>
 <td>BioWhittaker</td>
 <td>12-702F</td>
 <td></td>
 </tr>
 <tr>
 <td>1M HEPES buffered saline</td>
 <td>BioWhittaker</td>
 <td>17-737E</td>
 <td></td>
 </tr>
 <tr>
 <td>Phosphate buffered saline (PBS)</td>
 <td>BioWhittaker</td>
 <td>17-516F</td>
 <td></td>
 </tr>
 <tr>
 <td>Human albumin, 25% solution USP</td>
 <td>Aventis Behring</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Ficoll-Hypaque PREMIUM</td>
 <td>GE Healthcare</td>
 <td>17-5442-03</td>
 <td></td>
 </tr>
 <tr>
 <td>Human AB serum</td>
 <td>Valley Biomedical</td>
 <td>HP1022</td>
 <td></td>
 </tr>
 <tr>
 <td>Sterile saline USP</td>
 <td>Hospira</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>CryoMACS DMSO </td>
 <td>Miltenyi Biotec</td>
 <td>170-076-303</td>
 <td></td>
 </tr>
 <tr>
 <td>Leukine GM-CSF, 0.5 mg/ml</td>
 <td>Berlex</td>
 <td>A02266</td>
 <td></td>
 </tr>
 <tr>
 <td>MACS GMP IL-4</td>
 <td>Miltenyi Biotec</td>
 <td>170-076-101</td>
 <td></td>
 </tr>
 <tr>
 <td>Hiltonol, Poly-ICLC, 2 mg/ml</td>
 <td>Oncovir</td>
 <td>NA</td>
 <td></td>
 </tr>
 <tr>
 <td>VACMUNE KLH</td>
 <td>Biosyn</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>225 sq cm EasyFlasks</td>
 <td>Nalgene Nunc</td>
 <td>159934</td>
 <td></td>
 </tr>
 <tr>
 <td>Falcon 6-well tissue culture plates</td>
 <td>Becton Dickinson</td>
 <td>353046</td>
 <td></td>
 </tr>
 <tr>
 <td>1.8 ml CryoTube vials</td>
 <td>Nalgene Nunc</td>
 <td>377267</td>
 <td></td>
 </tr>
 <tr>
 <td>Controlled Rate Freezer</td>
 <td>Thermo</td>
 <td>CryoMed</td>
 <td></td>
 </tr>
 </tbody>
</table>

preparation of tumor antigen-loaded mature dendritic cells for immunotherapy

Mens kliniske studier har fastslått at antigen-lastet DC vaksiner er trygge og lovende behandling for svulster 1, forblir deres kliniske effekten skal etableres. Metoden er beskrevet nedenfor, utarbeidet i samsvar med Good Manufacturing Process (GMP) retningslinjer, er en optimalisering av de vanligste ex vivo forberedelse metode for å generere et stort antall utviklingsland for kliniske studier to. Vår metode benytter syntetisk TLR 3 agonist Polyinosinic-Polycytidylic Acid-poly-L-lysin Carboxymethylcellulose (Poly-ICLC) for å stimulere DCs. Våre tidligere undersøkelser fastslår at Poly-ICLC er den mest potente individuell modning stimulans for humane DC som vurdert av en oppregulering av CD83 og CD86, induksjon av interleukin-12 (IL-12), tumornekrosefaktor (TNF), interferon gamma-indusert protein 10 (IP-10), interleukmin 1 (IL-1) og type I-interferoner (IFN), og den minimale interleukin 10 (IL-10)-produksjon. </p&gt; DCs er differensiert fra frosne perifere mononukleære celler (PBMCs) fremstilt ved leukaferese. PBMC blir isolert ved Ficoll-gradientsentrifugering, og frosset i alikvoter. På dag 1 blir PBMC tint og platet ut på vevskulturflasker å selektere for monocytter som festes til plastoverflaten etter 1-2 timers inkubasjon ved 37 ° C i vevskultur-inkubator. Etter inkubasjon blir lymfocyttene vasket av, og de adherente monocytter dyrkes i 5 dager i nærvær av interleukin-4 (IL-4) og granulocytt-makrofag-kolonistimulerende faktor (GM-CSF) for å differensiere til umodne DCS. På dag seks, er umodne DCs pulserende med Nøkkelhullet limpet hemocyanin (KLH) protein som fungerer som en kontroll for kvaliteten på vaksinen, og kan øke effekten av vaksinen tre. DCS blir stimulert til å modne, lastet med peptid antigener, og inkubert over natten. På dag 7 ble cellene vasket, og frosset i en ml alikvoter inneholdende4-20 x 10 6 celler ved hjelp av en kontrollert-rate fryser. Lot utgivelsen testing for de grupper av utviklingsland er utført og må oppfylle minimum spesifikasjoner før de blir injisert i pasienter.

Mellom 10-20% av start PBMC differensierer til DC ved slutten av kulturen periode. Modne DCs er CD11c +, CD14-, CD83 +, CD40 +, og CCR7 + (Figur 1). De uttrykker høye nivåer av MHC klasse I og II molekyler og de kostimulerende molekyler CD80 og CD86. Poly-ICLC også indusert lavere nivåer av PDL-1 i forhold til andre TLR-agonister 14. I tillegg viser disse poly-IC-modnet DCS utskiller store mengder av IL-12 (figur 2 og 15,16) og indusere proliferasjon av allogen...

Watch this Scientific Journal Video about Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy at JoVE.com

Preparation of Tumor Antigen-loaded Mature Dendritic Cells for Immunotherapy

Den mest benyttede metode for generering av et stort antall autologe dendrittiske celler (DC) for anvendelse i tumor immunoterapi er beskrevet. Metoden bruker IL-4 og GM-CSF for å skille DCs fra monocytter. Umodne DC blir stimulert til å modne og deretter lastet med antigener før de blir injisert tilbake i pasienten.

Utarbeidelse av Tumor Antigen-lastet Moden dendrittiske celler for Immunterapi

While clinical studies have established that antigen-loaded DC vaccines are safe and promising therapy for tumors 1, their clinical efficacy remains to be ...

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