Targeting melanoma with immunoliposomes coupled to anti-MAGE A1 TCR-like single-chain antibody

Abstract

Therapy of melanoma using T-cells with genetically introduced T-cell receptors (TCRs) directed against a tumor-selective cancer testis antigen (CTA) NY-ESO1 demonstrated clear antitumor responses in patients without side effects. Here, we exploited the concept of TCR-mediated targeting through introduction of single-chain variable fragment (scFv) antibodies that mimic TCRs in binding major histocompatibility complex-restricted CTA. We produced scFv antibodies directed against Melanoma AntiGEn A1 (MAGE A1) presented by human leukocyte antigen A1 (HLA-A1), in short M1/A1, and coupled these TCR-like antibodies to liposomes to achieve specific melanoma targeting. Two anti-M1/A1 antibodies with different ligand-binding affinities were derived from a phage-display library and reformatted into scFvs with an added cysteine at their carboxyl termini. Protein production conditions, ie, bacterial strain, temperature, time, and compartments, were optimized, and following production, scFv proteins were purified by immobilized metal ion affinity chromatography. Batches of pure scFvs were validated for specific binding to M1/A1-positive B-cells by flow cytometry. Coupling of scFvs to liposomes was conducted by employing different conditions, and an optimized procedure was achieved. In vitro experiments with immunoliposomes demonstrated binding of M1/A1-positive B-cells as well as M1/A1-positive melanoma cells and internalization by these cells using flow cytometry and confocal microscopy. Notably, the scFv with nonenhanced affinity of M1/A1, but not the one with enhanced affinity, was exclusively bound to and internalized by melanoma tumor cells expressing M1/A1. Taken together, antigen-mediated targeting of tumor cells as well as promoting internalization of nanoparticles by these tumor cells is mediated by TCR-like scFv and can contribute to melanoma-specific targeting.

<p>Liposomes targeted to MHC-restricted antigen improve drug delivery and antimelanoma response</p>

“…Preparation, purification, and characterization of scFv proteins scFv G8 and Hyb3 were produced by our research group and collaborators as described previously. 24 Both scFv-DNAs were cloned into the pABC4 vector (adding a HIS-tag and cysteine) and put into BL21 bacteria. Bacteria were grown at 37°C until an optical density of 0.6-0.8 at 600 nm was reached.…”

“…Both scFvs were characterized and evaluated for in vitro binding to APD cells before coupling to liposomes as has been shown previously. 24 Preparation and characterization of liposomes A neutral liposome composition, typically HSPC, Ch, DSPE-PEG 2000 , and DSPE-PEG 2000 maleimide, was used in a molar ratio of 55:40:4:1, respectively. 30,31 Liposomes were prepared by a film hydration method as previously described.…”

“…22 Additional library engineering and selections yielded antibodies with different M1/A1 affinities, 23 which enabled studies into the relevance of antibody affinity for the binding and antitumor efficacy of multivalent nanoparticles. We investigated scFv G8 and Hyb3 because they are specific to pMHC complexes and have previously 24 shown promising results in vitro.…”

Construction and preliminary identification of a prokaryotic expression single-chain antibody fragments library against Streptococcus pneumoniae from antibody-producing cells in human tonsil

“…The single-chain antibody is a kind of genetic engineering antibody and has many advantages like lower molecular weight, stronger tissue penetration, and faster body clearance [7,8]. It has the potential value for clinical diagnosis and treatment [9][10][11][12][13].…”

Análise do papel de genes <i>bir</i> no processo adaptativo ao hospedeiro e desenvolvimento de proteolipossomos para potencial uso vacinal com foco em reinvasão sanguinea de <i>Plasmodium</i>.

“…(NIZARD et al, 1998), tendo a vantagem de evitar conjugações químicas covalentes (FRIEDMAN et al, 2013;TABATA et al, 2013). Imunolipossomo (como são chamados lipossomos direcionados por anticorpos) tem grande papel na busca de alvos específicos na entrega de drogas tanto em estratégias refinadas contra tumores (HOSOKAWA et al, 2003;SAEED et al, 2016) quanto contra o próprio gênero Plasmodium (URBÁN et al, 2011(URBÁN et al, , 2015. Por carregarem um grande números de moléculas em seu interior, vesículas direcionáveis podem atingir alvos de difíceis acesso, incluindo tecidos cerebrais protegidos por barreira hemato-encefálica (MARUYAMA et al, 1990;PARDRIDGE, 2007).…”