The development of IL-2 conjugated liposomes for therapeutic purposes

Abstract

A unique immunoliposome has been developed as a drug delivery vehicle for immunotherapy. Human recombinant interleukin-2 (IL-2) has been chemically coupled to the external surface of small unilamellar vesicles (SUVs) containing methotrexate as a candidate immunosuppresive agent in order to specifically direct the drug-bearing liposome to activated T-cells expressing the high affinity IL-2 receptor. This drug delivery system is designed to deliver an immunosuppressive agent to those cells that actively participate in disorders such as graft rejection without delivering an effective but potentially toxic drug to all cells of the immune system as well as other healthy tissues. IL-2 was chemically modified with succinimidyl 4-[p-maleidophenyl butyrate](SMPB) while the receptor binding domain on IL-2 was protected by monoclonal anti-IL-2 bound to Protein A-Silica Gel. The antibody recognizes the receptor binding domain of the IL-2 molecule. The IL-2 was derivatized with S-succinimidyl-S-thioacetate (SATA) in order to add an acetyl thioester group to the lipid and create the complex. The derivatized lipid (SATA-PE) was then part of the liposome formulation containing DSPC:cholesterol: SATA-PE at a mole ratio of 1.5:1.0:0.26. SMPB-IL-2 was covalently coupled to the external surface of the SUV after deacetylation of the thioester moiety at pH 7.4 in PBS. Liposomes prepared by sonication or extrusion had an average diameter of 46-50 nm. SUV-IL-2 bound to the high affinity IL-2 receptor as measured by competitive binding assays and Scatchard analysis using 111InCl2-loaded liposomes The preparation exhibited a binding constant of 30 pM, consistent with values for free IL-2 cited in the literature. SUV IL-2 could be used as the sole source of IL-2 for the murine CTLL-2 T-cell line or for human mitogen-activated PBLs. The presence of IL-2 coupled to the surface was absolutely required for delivery of the drug to the cell. When methotrexate was encapsulated within the internal aqueous space, receptor-mediated endocytosis led to the inhibition of proliferation due to delivery of MTX to the cytoplasm of the cell. More than 90% of the methotrexate was retained within the liposome during storage over a 24-h period at 4 degrees C. This immunoliposome represents a new class of cell specific immunoliposomes whose entry into the cell is controlled by a cell surface receptor.

Modeling the receptor pharmacology, pharmacokinetics, and pharmacodynamics of NKTR-214, a kinetically-controlled interleukin-2 (IL2) receptor agonist for cancer immunotherapy

“…The effect is fundamentally different than a slow release of free-IL2 from a formulated matrix [30, 31] or from free-IL2 linked to an Fc to improve t ½ [32, 33]. Therefore, we developed specialized bioanalytical assays to characterize relevant groups of conjugated-IL2 species derived from NKTR-214.…”

“…For example, IL2 has previously been formulated to allow slow release from a polymer matrix [30, 31, 36]. -IL2 release from such a matrix does not create an array of conjugated-IL2 species such as that produced from NKTR-214 in vivo .…”

Nukleinsäure‐basierte molekulare Werkzeuge

“…[283] Tatsächlich wurden Folsäure-modifizierte DNA-Nanoröhren erfolgreich in Zellen eingebracht. [284] Ebenso wie funktionalisierte Liposomen zur gewebespezifischen Freisetzung von Wirkstoffen Verwendung finden, [285] lassen sich auch molekulare Nukleinsäuremotive als Navigationsmodule für eine gewebespezifische Abgabe einer molekularen Ladung in vivo einsetzen. Zellen, die zu unterschiedlichen Gewebetypen gehören, exprimieren unterschiedliche Zelloberflächenrezeptoren.…”

Cytotoxicity of doxorubicin‐loaded Con A‐liposomes

“…No significant increase in the size of vesicles was observed for Con Aconjugated liposomes prepared by sonication, which have a diameter size of 300 nm, while the unconjugated liposomes formed from DPPE-MBS had a diameter size of 200 nm [Francis and Jones, 1990;Francis et al, 1992;Jones et al, 1993]. In contrast, proteoliposomes prepared with antibody fragments or other small molecules such as interleukin-2 presented a small size (46-60 nm) in comparison with lectin-conjugated liposomes [Koningsberg et al, 1998]. …”