Intracellular Routing in Breast Cancer Cells of Streptavidin-Conjugated Trastuzumab Fab Fragments Linked to Biotinylated Doxorubicin-Functionalized Metal Chelating Polymers

Abstract

We describe the synthesis of a heterotelechelic metal-chelating polymer (Bi-MCP-Dox), a polyacrylamide with a number average degree of polymerization DPn = 50 (PDI = 1.2), with biotin (Bi) and doxorubicin (Dox) as functional chain ends and diethylenetriaminepentaacetic acid (DTPA) pendant groups as the binding sites for metal ions. We compared its behavior in cell-uptake experiments with a similar polymer (Bi-MCP) without Dox. These MCPs were complexed with trastuzumab Fab (tmFab) fragments covalently linked to streptavidin (SAv) to form tmFab-SAv-Bi-MCP-Dox and tmFab-SAv-Bi-MCP via the strong affinity between Bi and SAv. tmFab targets human epidermal growth factor receptor-2 (HER2), which is overexpressed on certain human breast cancer cells. Surface plasmon resonance (SPR) experiments with the extracellular domain (ECD) of HER2 showed that incorporation of the MCPs in these complexes had no significant effect on the association or dissociation rate with the HER2 ECD and the dissociation constants. The tmFab-complexed MCPs were subsequently labeled with (111)In (an Auger electron emitting radionuclide). Auger electrons can cause lethal DNA double strand breaks (DSBs) but only if they are emitted intracellularly and especially, in close proximity to the nucleus. To evaluate the cellular and nuclear uptake of tmFab-SAv-Bi-MCP-Dox, we incubated HER2+ SK-BR-3 human breast cancer cells with the complexes saturated with stable In(3+) and visualized their distribution by confocal fluorescence microscopy, monitoring the fluorescence of Dox. In parallel, we carried out cell fractionation studies on tmFab-SAv-Bi-MCP-Dox and on tmFab-SAv-Bi-MCP labeled with (111)In. Both radiolabeled complexes showed cell internalization and nuclear localization. We conclude that metal-chelating polymers with this composition appear to encourage internalization, nuclear uptake, and chromatin (DNA) binding of trastuzumab fragments modified with streptavidin in human breast cancer cells expressing HER2. Further study is needed to understand the impact of polymer charge on cellular uptake and distribution to intracellular compartments.

Telechelic polymers from reversible-deactivation radical polymerization for biomedical applications

“…3). 27 Dox was further linked at the o-chain-end via thiol-maleimide chemistry. The polymer backbone also contained pendant metal-chelating moieties (MCP) functionalized with diethylenetriaminepentaacetic acid (DTPA) to chelate 111 In, an Auger electron emitting radionuclide to cause lethal DNA double strand breaks.…”

In Situ Quenching of Trialkylphosphine Reducing Agents Using Water-Soluble PEG-Azides Improves Maleimide Conjugation to Proteins

“…Early reports on the use of TCEP in protein conjugation strategies suggested that this phosphine was compatible with maleimide and did not need to be removed before the addition of the Michael acceptor. 2 , 11 − 13 A number of recent reports, however, have confirmed that TCEP and THPP do indeed react with maleimides to reduce conjugation yields significantly. 8 , 14 − 17 Importantly, it has also been demonstrated that ylenes formed between maleimide and TCEP, such as 3 , are remarkably stable under physiological conditions and can remain incorporated in the products of some protein conjugations ( Figure 1 b).…”

Characterization of Reactions between Water-Soluble Trialkylphosphines and Thiol Alkylating Reagents: Implications for Protein-Conjugation Reactions

“…15 The two most commonly used water soluble alkylphosphines are tris-(2-carboxyethyl)phosphine (TCEP) 1 and tris- In addition to the favourable physico-chemical properties of TCEP and THPP, these reagents are also considered generally to provide methodological advantages over thiol based reducing agents, with reports in the literature that TCEP is unreactive towards maleimide and numerous examples of maleimide-based conjugations being performed in the presence of TCEP. [18][19][20][21][22][23][24] Contrary to this however, a number of groups have reported decreased yields of conjugation when using maleimide in the presence of TCEP, suggesting possible reaction between the two reagents. [25][26][27][28] Recently, Sánchez et al have reported the observation of a phosphonium-ion adduct between TCEP and maleimide on the basis of mass spectrometry, though this adduct was not fully characterised.…”