Fremstilling af tumorantigen-loaded dendritiske celler til immunterapi
Summary
Den mest almindeligt anvendte metode til at generere mange autologe dendritiske celler (DC'er) til anvendelse i tumor immunterapi er beskrevet. Fremgangsmåden anvender IL-4 og GM-CSF at differentiere udviklingslandene fra monocytter. De umodne DC'er stimuleres til modne og derefter fyldt med antigener, før de injiceres tilbage i patienten.
Abstract
Mens kliniske undersøgelser har fastslået, at antigen-loaded DC vacciner er sikre og lovende behandling for tumorer 1, deres kliniske effekt er endnu ikke fastslået. Den nedenfor beskrevne metode, aflægges i overensstemmelse med Good Manufacturing Process (GMP) retningslinjer, er en optimering af de mest almindelige ex vivo forberedelse metode til at generere et stort antal udviklingslandene for kliniske forsøg 2.
Vores metode udnytter den syntetiske TLR 3 agonist Polyinosinic-Polycytidylic Syre-poly-L-lysin Carboxymethylcellulose (Poly-ICLC) at stimulere udviklingslandene. Vores tidligere undersøgelse fastslået, at poly-ICLC er den mest potente individuel modning stimulans for humane DC'er som vurderet ved en opregulering af CD83 og CD86, induktion af interleukin-12 (IL-12), tumornekrosefaktor (TNF), interferon gamma-inducerede protein 10 (IP-10), interleukmin 1 (IL-1), og type I-interferoner (IFN) og minimal interleukin 10 (IL-10) produktion. </p>
DC'er er differentieret fra frosne perifere mononukleære blodceller (PBMC'er) opnået ved leukaferese. PBMC'er isoleres ved Ficoll-gradient centrifugering og frosset i portioner. På dag 1, er PBMC'er optøet og udpladet på vævsdyrkningskolber at selektere for monocytter, der klæber til plastoverfladen efter 1-2 timers inkubation ved 37 ° C i vævskultur inkubator. Efter inkubering lymfocytter vaskes og vedhængende monocytter dyrkes i 5 dage i nærværelse af interleukin-4 (IL-4) og granulocyt makrofag-koloni-stimulerende faktor (GM-CSF) til at differentiere til umodne DC'er. På dag 6, er umodne DC'er pulses med keyhole limpet hæmocyanin (KLH)-protein, der tjener som en kontrol for kvaliteten af vaccinen og kan øge immunogeniciteten af vaccinen 3.. DCS stimuleres til at modnes, lastet med peptidantigener, og inkuberes natten over. På dag 7, vaskes cellerne, og frosset i portioner på 1 ml indeholder4 – 20 x 10 6 celler med en kontrolleret-sats fryser. Lot release test for partierne af udviklingslandenes udføres, og skal opfylde mindstekrav specifikationer, inden de bliver sprøjtet ind patienterne.
Protocol
Representative Results
Discussion
Fase I og II kliniske forsøg med monocytafledte DC'er har vist, at de inducerer immunrespons hos patienter, men klinisk succes har været begrænset 1.. Dette kan til dels skyldes den manglende konsensus om, hvordan at generere de optimale DC'er til tumor immunterapeutisk brug. Selvom der er mange måder at generere klinisk-grade DC'er, disse metoder variere med hensyn til anvendelsen af cytokiner anvendes til at differentiere monocytter, stimuli, der bruges til at inducere modning og fremg…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Forfatterne vil gerne takke Andres Salazar (Oncovir, Inc.) for gaven af Poly-ICLC.
Materials
| Reagent/Supplies/Equipment | Manufacturer | Catalog No. | |
| RPMI-1640 medium with L-glutamine | BioWhittaker | 12-702F | |
| 1M HEPES buffered saline | BioWhittaker | 17-737E | |
| Phosphate buffered saline (PBS) | BioWhittaker | 17-516F | |
| Human albumin, 25% solution USP | Aventis Behring | ||
| Ficoll-Hypaque PREMIUM | GE Healthcare | 17-5442-03 | |
| Human AB serum | Valley Biomedical | HP1022 | |
| Sterile saline USP | Hospira | ||
| CryoMACS DMSO | Miltenyi Biotec | 170-076-303 | |
| Leukine GM-CSF, 0.5 mg/ml | Berlex | A02266 | |
| MACS GMP IL-4 | Miltenyi Biotec | 170-076-101 | |
| Hiltonol, Poly-ICLC, 2 mg/ml | Oncovir | NA | |
| VACMUNE KLH | Biosyn | ||
| 225 sq cm EasyFlasks | Nalgene Nunc | 159934 | |
| Falcon 6-well tissue culture plates | Becton Dickinson | 353046 | |
| 1.8 ml CryoTube vials | Nalgene Nunc | 377267 | |
| Controlled Rate Freezer | Thermo | CryoMed |
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