The development and characterisation of porphyrin isothiocyanate–monoclonal antibody conjugates for photoimmunotherapy

Abstract

A promising approach to increase the specificity of photosensitisers used in photodynamic therapy has been through conjugation to monoclonal antibodies (MAb) directed against tumour-associated antigens. Many of the conjugations performed to date have relied on the activated ester method, which can lead to impure conjugate preparations and antibody crosslinking. Here, we report the development of photosensitiser -MAb conjugates utilising two porphyrin isothiocyanates. The presence of a single reactive isothiocyanate allowed facile conjugation to MAb FSP 77 and 17.1A directed against internalising antigens, and MAb 35A7 that binds to a non-internalising antigen. The photosensitiser -MAb conjugates substituted with 1 -3 mol of photosensitiser were characterised in vitro. No appreciable loss of immunoreactivity was observed and binding specificity was comparable to that of the unconjugated MAb. Substitution with photosensitiser had a minimal effect on antibody biodistribution in vivo for the majority of the conjugates, although a decreased serum half-life was observed using a cationic photosensitiser at the higher loading ratios. Tumour-to-normal tissue ratios as high as 33.5 were observed using MAb 35A7 conjugates. The internalising conjugate showed a higher level of phototoxicity as compared with the non-internalising reagent, using a cell line engineered to express both target antigens. These data demonstrate the applicability of the isothiocyanate group for the development of high-quality conjugates, and the use of internalising MAb to significantly increase the photodynamic efficiency of conjugates during photoimmunotherapy.

Enhancing Tumor Drug Delivery by Laser-Activated Vascular Barrier Disruption

“…Similar results were also reported in studies comparing aluminium tetrasulfophthalocyanine (AlPcS 4 ) immunoconjugates with a noninternalizing MAb 35A7 recognizing carcinoembryonic antigen (CEA) and an internalizing HER2 MAb FSP 77 and 17.1A. 96,97 This hypothesis was further tested with two hydrophilic photosensitizers. 98,99 The rationale for using hydrophilic photosensitizers is obvious, because this type of photosensitizer generally has low photocytotoxicity due to low cell membrane affinity, and they are good candidates for photoimmunoconjugation because of good water solubility.…”

Targeted photodynamic therapy with multiply‐loaded recombinant antibody fragments

“…21 Antibody fragments such as scFvs (single-chain Fv fragments) have been shown to be superior to whole antibodies in many aspects of tumor targeting such as speed of penetration and tumor:normal tissue specificity, 22,23 making them ideal vehicles for targeted PDT if sufficient numbers of PS can be attached to these small 30 kDa proteins. Photocoagulation of ocular vasculature 24 and tumor destruction 25 have been demonstrated by Neri and coworkers using the antibody fragment L19; however, this and other research 17 illustrates the challenge in that only 1-2 PS molecules could be attached directly to L19.…”

“…14 Photosensitizers conjugated to whole antibodies can target a range of cancers 12 or pathogens 15 ; however, experiences so far has shown that many technical problems exist in coupling high numbers of PS to whole antibodies. These include impaired antibody binding, reduced solubility [16][17][18][19] and unfavorable pharmacokinetics due to the resulting high molecular weight of such photoimmunoconjugates (PICs). Some researchers have tried using carrier moieties such as branched carbohydrate or polylysine chains, which are in turn linked to the antibody.…”

Cancer, Photodynamic Therapy and Porphyrin-Type Derivatives

“…21) (Pereira et al 2014a). In fact, porphyrins and their derivatives conjugated with human and bovine serum albumin (HSA and BSA, respectively) have been successfully prepared and have demonstrated remarkable cell type specificity towards tumoral macrophage-like cells when compared to cancer cells (Hamblin and Newman 1994, Sutton et al 2002, Hudson et al 2005. This fact, was justified by the high accumulation of albumin in tumor tissues due to the EPR effect (enhanced permeability and retention of macromolecules in tumor tissues) (Kratz 2008, Maeda et al 2000.…”

“…It takes into consideration the improvement mainly related with PS transport and specificity for tumors (Josefsen andBoyle 2008, Senge 2012). New or already known PSs are conjugated, for example, with antibodies (Kudarha and Sawant 2017, Hudson et al 2005, Pereira et al 2014 or nanoparticles (Yang et al 2017, Ma et al 2017). Targeting strategies have already shown that the PS affinity for tumors increases in significant ways.…”