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

Die Autoren bedanken sich bei Andres Salazar (Oncovir, Inc.) für das Geschenk des Poly-ICLC danken.

1. Isolation und Kryokonservierung von PBMCs 4 <ol><li> Aseptisch Spike einer der Access-Ports in der Leukapherese Tasche mit einem Plasma-Transfer-Set. Mit einem 60-ml-Spritze, übertragen Sie die Leukapherese von Patienten in eine sterile 500 ml-Flasche erhältlich. </li><li> Stellen Sie die Lautstärke des Leukapherese auf sein ursprüngliches Volumen mit Raumtemperatur RPMI 2x. Gründlich mischen. </li><li> Vorsichtig mischen die Flasche Ficoll-Paque PLUS. In 12 ml Ficoll-Paque PLUS in sterile 50 ml kon...

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B., 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=Toll-like+receptor+agonists:+are+they+good+adjuvants?.">Toll-like receptor agonists: are they good adjuvants?.</a> Cancer J. 16, 382-391 (2010).</li><li>Kedl, R. M., Kappler, J. W., Marrack, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epitope+dominance,+competition+and+T+cell+affinity+maturation.">Epitope dominance, competition and T cell affinity maturation.</a> Curr. Opin. 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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. 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Phase-I-und-II-Studien von Monozyten-abgeleiteten DCs haben gezeigt, dass sie Immunreaktionen induzieren in Patienten jedoch klinische Erfolg wurde 1 beschränkt. Dies kann zum Teil auf den Mangel an Konsens darüber, wie die optimalen DCs für die Tumor-Immuntherapie Verwendung erzeugen. Obwohl es zahlreiche Möglichkeiten, um klinische Studien zu DCs zu erzeugen, unterscheiden sich diese Verfahren im Hinblick auf die Verwendung von Zytokinen, die Monozyten, Stimuli, um die Reifung zu induzieren, und die Met...

<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

Obwohl klinische Studien festgestellt haben, dass Antigen-beladenen DC Impfstoffe sicher und viel versprechende Therapie für Tumore 1 sind, bleibt ihre klinische Wirksamkeit festgelegt werden. Das nachfolgend beschriebene Verfahren, hergestellt gemäß Good Manufacturing-Verfahren (GMP)-Richtlinien, ist eine Optimierung der häufigste ex vivo Herstellungsverfahren zur Erzeugung einer großen Anzahl von DCs für klinische Studien 2. Unsere Methode nutzt die synthetischen TLR-Agonisten Polyinosin 3-Polycytidylsäure-Poly-L-Lysin Carboxymethylcellulose (Poly-ICLC), um die DCs zu stimulieren. Unsere früheren Studie festgestellt, dass Poly-ICLC die stärkste einzelne Reifung Stimulus für den menschlichen DCs ist, wie durch eine Hochregulation von CD83 und CD86, Induktion von Interleukin-12 (IL-12), Tumor-Nekrose-Faktor (TNF), Interferon-gamma-induzierten beurteilt Protein 10 (IP-10), interleukmin 1 (IL-1) und Typ-I-Interferonen (IFN) und minimal Interleukin 10 (IL-10) Produktion. </p&gt; DCs sind aus gefrorenem peripheren mononukleären Blutzellen (PBMCs) von Leukapherese erhalten differenziert. PBMCs durch Ficollgradientenzentrifugation isoliert und in Aliquots eingefroren. Am Tag 1 werden PBMCs aufgetaut und überzogen auf Gewebekulturflaschen für Monozyten, die auf der Kunststoffoberfläche nach 1-2 h Inkubation bei 37 ° C in der Gewebekulturinkubator haften wählen. Nach der Inkubation werden die Lymphozyten durch Waschen entfernt und die anhaftenden Monozyten werden für 5 Tage in Gegenwart von Interleukin-4 (IL-4) und Granulozyten-Makrophagen-Kolonie-stimulierender Faktor (GM-CSF) zu unreifen DC differenzieren kultiviert. Am Tag 6 werden unreife DCs mit Keyhole-Limpet-Hämocyanin (KLH)-Protein, das als eine Kontrolle für die Qualität der Impfstoff dient und die Immunogenität der Impfstoff 3 steigern gepulst. Die DCs stimuliert werden, um zu reifen, beladen mit Peptid-Antigene, und über Nacht inkubiert. Am 7. Tag wurden die Zellen gewaschen und in 1 ml eingefroren Aliquots, welche4 - 20 x 10 6 Zellen mit einer kontrollierten Rate Gefrierschrank. Lot Release-Tests für die Chargen von DCs durchgeführt und müssen Mindestanforderungen erfüllen, bevor sie in die Patienten injiziert werden.

Zwischen 10 - 20% Ausgangsmaterial PBMCs differenzieren in DCs am Ende der Kulturperiode. Reife DCs sind CD11c +, CD14-, CD83 +, CD40 + und CCR7 + (Abbildung 1). Sie exprimieren hohe Mengen von MHC-Klasse-I und-II-Molekülen und den kostimulatorischen Moleküle CD80 und CD86. Poly-ICLC auch unteren Ebenen der PDL-1 induziert zu anderen TLR-Agonisten 14 verglichen. Darüber hinaus führen diese Poly-IC-gereiften DCs sezernieren große Mengen an IL-12 (Abbildung 2 und 15,1...

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

Das am häufigsten verwendete Verfahren zum Erzeugen einer großen Anzahl von autologen dendritischen Zellen (DCs) zur Verwendung bei Tumor-Immuntherapie beschrieben. Das Verfahren nutzt IL-4 und GM-CSF zu DCs aus Monozyten differenzieren. Die unreifen DCs sind, um zu reifen und dann mit Antigenen beladen stimuliert, bevor sie zurück in den Patienten injiziert werden.

Herstellung von Tumor-Antigen-beladenen reifen dendritischen Zellen für die Immuntherapie

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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