Pinpoint targeting for high affinity IL-2 receptor using biocompatible MPC polymer conjugated with interleukin-2 contained PTX

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Purpose This study was intended to classify the characteristics of a novel immunosuppressive agent was produced, which was using watersoluble and biocompatible 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer conjugated with interleukin-2 (IL2). Materials and Methods poly (MPC-co-BMA-co-NPMA) (PMBN) was constructed from about a 40 mol % MPC unit, a 50 mol % n-butyl methacrylate (BMA) unit, and a 10 mol % p-nitrophenylcarbonyloxyethyl methacrylate (NPMA) unit. The PMBN-IL-2 conjugate was purified by dialysis cassette, which removed free IL-2 and p-nitrophenol. Moreover, PMBN-IL-2 conjugate and a given amount of paclitaxel (PTX) was mixed, for which ethanol was removed by evaporation to obtain the drugs incorporated in IL-2-conjugated PMBN Presupposing that the concentration of PMBN and PMBN-IL-2 conjugate were fixed, Each reagent (PMBN-IL2 conjugate containing PTX, PMBN containing PTX, and conventional PTX) added to each well, and cells (MJ cells, OKM2T, and MOLT3) were incubated. MJ and OKM-2T, cutaneous T cell lymphoma and ATL respectively, overexpress IL-2Rα. MOLT-3, a T cell lymphoblastic leukemia, is HTLV-1 negative and has undetectable high affinity IL-2Rα. Cells viability was measured by Cell Counting Kit. Mononuclear cells were isolated from HLA-incompatible blood samples from healthy donors. Stimulators and responders were plated. The same reagent such as above was added in a single dose. The inhibition of the mixed lymphocyte reaction (MLR) was assessed by measurement of cell ploriferation. Results The PMBN-IL-2 conjugate containing PTx inhibited the proliferations of MJ and OKM-2T overexpressing IL-2R at lower concentration than the PMBN containing PTX and the conventional PTX respectively. The IC50 of the PMBN-IL-2 conjugate containing PTX, PMBN containing PTX and conventional PTX in MJ cells was respectively 4.0 nM, 50.1 nM and 56.2 nM, and the IC50 of the same reagents in OKM-2T was respectively 5.0 nM, 77.6 nM, and 91.2 nM, Whereas No inhibition was detectable in MOLT-3 by these reagents containing PTX at the almost same concentration as conventional. The PMBN-IL2 conjugate containing PTx inhibited the proliferations of human alloreactive cells at lower concentration than the PMBN containing PTX and the conventional PTX respectively. The IC50 of the PMBN-IL-2 conjugate containing PTX, PMBN containing PTX and conventional PTX in human MLR inhibition was respectively 2.5 nM, 19.9 nM and 10.0 nM. Conclusion This novel agent may be useful to selectively eliminate activated lymphocytes hyperproducing high affinity IL-2 receptors, as in allograft rejection, graft-versus-host disease, autoimmune disorders, etc. As an entirely human “immunotoxin analogue” it may alleviate the dose limiting toxicity and immunogenicity of conventional immunotoxins.