Biochemical and Immunological Properties of Cytokines Conjugated to Dendritic Polymers

Abstract

Here we describe a post-translational modification of SC-63032, a variant of the species restricted, multi-lineage hematopoeitic factor human interleukin-3 (hIL-3). We have made two new dendritic polymer (polyamidoamine or PAMAM dendrimers, generation 5)-SC-63032 bioconjugates. Using two distinct chemistries (one of which is novel to this work), we achieved site-specific conjugation with respect to the amino acid in the proteins ligated to the dendrimers. In both bioconjugates, conjugated cytokine maintains its ability to bind the hIL-3 alpha receptor subunit, but is significantly (about 10-fold) less potent in inducing hIL-3 dependent in vitro cell proliferation than is the free cytokine. In vivo data indicates that conjugation decreases the immunogenicity of the conjugated cytokine modestly. In the absence of pharmacokinetic or biodistribution effects associated with the bioconjugates that increase their potency in vivo (which can only be tested in a higher primate, due to the species restriction of hIL-3 and its derivatives), these immune mitigation effects may be too small to be therapeutically significant. Though unmodified PAMAM dendrimers fail to elicit an antibody response in mice, protein conjugation to dendrimers haptenizes them, and a dendrimer-specific antibody response is produced. In toto, the principal limitation of the dendrimer-cytokine bioconjugates herein is in their reduced receptor affinity and potency in vitro. Were the in vivo potency of the bioconjugates to parallel the in vitro potency of the conjugates reported here, it is likely that particular dendrimer bioconjugates could not justify their higher costs of goods relative to the parent SC-63032 molecule, though retention of SC-63032 biological activities in conjugates suggests that other cytokine-dendrimer bioconjugates may be bioactive. This is good news to the nanotechnology community, in as much as PAMAM dendrimers are among the monodisperse polymeric nanomaterials available, and these results show that they can be used successfully in conjugates to bioactive proteins.

Phase Detection of the Two-Port FPW Sensor for Biosensing

“…chemical warfare agents, and biological species [1]- [4]. They are used mostly to detect the adsorption or attachment of protein molecules [5], cells [6], bacteria [7], or the immunological properties of cytokines conjugated to organisms on the plate surface of two-port IDTs [8].…”

Tailored Control and Optimisation of the Number of Phosphonic Acid Termini on Phosphorus‐Containing Dendrimers for the Ex‐Vivo Activation of Human Monocytes

“…This collection is based on a unique set of dendritic structural parameters-cyclo(triphosphazene) core, benzylhydrazone branches and phosphonic acid surface-and was designed to study the influence of phosphonate (phosphonic acid) surface loading towards the activation of human monocytes ex vivo. Starting from the versatile hexachloro-cyclo(triphosphazene) N 3 P 3 Cl 6 , six first-generation dendrimers were obtained, bearing one to six full branches, that lead to 4,8,12,16,20 and 24 phosphonate termini, respectively. The surface loading was also explored at the limit of dense packing by means of a first-generation dendrimer having a cyclo(tetraphosphazene) core and bearing 32 termini, and with a first-generation dendrimer based on a AB 2 /CD 5 growing pattern and bearing 60 termini.…”

“…The in vitro immunostimulating properties of this conjugate were reported to be lower than those of free hIL-3. [20] However, we have recently shown that phosphorus-containing dendrimers [21] decorated with suitable amino-bismethylene phosphonic acid derivatives on the surface were able to both promote the amplification of fully functional NK cells [22] and to provide the activation of monocytes from healthy human peripheral blood mononuclear cells (PBMC) in culture, [23] whereas the corresponding surface monomers were unable to provide such activities. Monocytes are a pivotal subpopulation of the innate immune system.…”

Dendrimers in Cancer Treatment and Diagnosis

“…In the past, natural proteins have commonly been used as carriers to generate antibodies to small molecules; now an alternative strategy using dendrimers has been demonstrated. In particular, unmodified PAMAM dendrimers that fail to elicit an antibody response on their own become haptenized upon protein conjugation and generate a dendrimer-dependent antigenic response [86,87]. A specific example of this technique is provided by the dendrimeric presentation of antigenic HIV peptides, which proved superior to other multimeric presentation strategies, such as conjugation to dextran [88].…”