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.

Keywords:

• cancer
• immunotherapy
• nanoparticles
• cancer testis antigens
• HLA-A1 targeting

Supplementary materials

Table S1 Stability of immunoliposomes over 2 weeks

Figure S1 pMHC expression on tumor cell lines.

Notes: We determined the expression of MAGE A1 and HLA-A1 individually on the melanoma cell lines in use by PCR. Expression of HLA-A1 is prevalent in all cell lines shown in upper gel row, whereas the lower row shows MAGE A1 peptide expression.

Abbreviations: pMHC, peptide:MHC; MHC, major histocompatibility complex; MAGE A1, Melanoma AntiGEn A1; HLA-A1, human leukocyte antigen A1; PCR, polymerase chain reaction.

Figure S2 Off-target binding of liposomes-scFv Hyb3 on APD cells by flow cytometry.

Notes: (A) This panel shows liposomes incubated with APD cells pulsed with peptide gated against APD cells without peptide incubated with liposomes at 4°C for 2 hours. Solid black line in each histogram indicates APD cells without peptide incubated with respective sample. Blue filled histogram represents APD cells pulsed with peptide incubated with nontargeted liposomes, green filled histogram represents APD cells pulsed with peptide incubated with liposomes-scFv Hyb3, red filled histogram represents APD cells pulsed with peptide incubated with liposomes-scFv G8. (B) This panel shows the same samples gated against unstained APD cells. Solid black line in each histogram indicates unstained APD cells without peptide incubation. Blue histogram represents APD cells pulsed with peptide incubated with nontargeted liposomes, green histogram represents APD cells pulsed with peptide incubated with liposomes-scFv Hyb3, red histogram represents APD cells pulsed with peptide incubated with liposomes-scFv G8.

Abbreviation: scFv, single-chain variable fragment.

Figure S3 Stability of immunoliposomes over 2 week by flow cytometry.

Notes: (A) First experiment done with a batch of liposomes, 1 week postpreparation, showing a binding of up to 60% with tumor cells at 37°C for 2 hours. (B) Same batch of liposomes tested on same cells 1 week later, 2 weeks postpreparation, showing decreased binding to cells.

Acknowledgments

The authors thank Dr Ann Seynhaeve at Experimental Surgery Laboratory for her help and advice. The study was financially supported by MRACE grants (Erasmus MC, Rotterdam, the Netherlands).

Disclosure

The authors report no conflicts of interest in this work.