Site-Specific DBCO Modification of DEC205 Antibody for Polymer Conjugation

Abstract

Abstract:The design of multifunctional polymer-based vectors, forming pDNA vaccines, offers great potential in cancer immune therapy. The transfection of dendritic immune cells (DCs) with tumour antigen-encoding pDNA leads to an activation of the immune system to combat tumour cells. In this work, we investigated the chemical attachment of DEC205 antibodies (aDEC205) as DC-targeting structures to polyplexes of P(Lys)-b-P(HPMA). The conjugation of a synthetic block copolymer and a biomacromolecule with various functionalities (aDEC205) requires bioorthogonal techniques to avoid side reactions. Click chemistry and in particular the strain-promoted alkyne-azide cycloaddition (SPAAC) can provide the required bioorthogonality. With regard to a SPAAC of both components, we firstly synthesized two different azide-containing block copolymers, P(Lys)-b-P(HPMA)-N 3 (stat) and P(Lys)-b-P(HPMA)-N 3 (end), for pDNA complexation. In addition, the site-specific incorporation of ring-strained dibenzocyclooctyne (DBCO) moieties to the DEC205 antibody was achieved by an enzymatic strategy using bacterial transglutaminase (BTG). The chemical accessibility of DBCO molecules within aDEC205 as well as the accessibility of azide-functionalities on the polyplex' surface were investigated by various SPAAC experiments and characterized by fluorescence correlation spectroscopy (FCS).

Site-selective modification strategies in antibody–drug conjugates

“…Non-glycosylated approaches to antibody modification are not limited to toxin attachment. Other reports have detailed the attachment of azide polymers, 271 photosensitiser motifs 272 or virus nanoparticles. 273 Recent research offers some reservations concerning the selectivity of mTG for the conserved PWEEQYNST sequence (containing Q295) in the Fc region.…”

HPMA-Based Nanoparticles for Fast, Bioorthogonal iEDDA Ligation

“…Click chemistry can be applied to efficiently labeled nanoparticles with various functional groups for active targeting or with radioactive labels for imaging. − In a rapid labeling approach, this is done ex vivo before administration of the nanomedicine. However, in the case of radiolabeling, this can be disadvantageous, considering that the most commonly utilized radionuclides have a short half-life time like 18 F or 68 Ga with 110 and 68 min, respectively.…”

“…This class of biocompatible reactions is commonly described as bioorthogonal reactions. These reactions can be utilized, for example, for conjugation of biomolecules like antibodies or nanoparticles to radiolabels. ,, In the past decade, many different bioorthogonal reactions have been developed like the Staudinger and traceless Staudinger ligation , and the strain-promoted alkyne–azide cycoaddition (SPAAC, a subclass of “click reactions”). , In addition, the copper-catalyzed alkyne–azide cycloaddition (CuAAC) has been proposed as a bioorthogonal ligation reaction . While these reactions are suited for conjugation in vitro, they have limitations for in vivo applications due to their low reactivity in the case of Staudinger ligations and SPAACs or to the use of cytotoxic copper as a catalyst.…”

Shining Light on Polymeric Drug Nanocarriers with Fluorescence Correlation Spectroscopy

“…Normalized autocorrelation curves measured for freely diffusing AF647 DBCO (blue symbols, Rh = 0.75 nm)and AF647-DBCO conjugated to polyplexes by SPAAC (gray symbols, Rh = 52 nm). Reproduced with permission [85]. Copyright 2018, MDPI.…”

“…b) Normalized autocorrelation curves measured for Oregon Green 488 (yellow symbols, Rh = 0.58 nm), P(Lys)-b-P(HPMA)-N3(stat)OG488 (purple symbols, Rh = 4.3 nm), and conjugate of aDEC205-DBCO and P(Lys)-b-P(HPMA)-N3(stat)OG488(gray symbols, Rh = 10.7 nm). Reproduced with permission [85]. Copyright 2018, MDPI.…”