Lipid-based nanosystems: the next generation of cancer immune therapy.

Immunotherapy has become an important part of the oncotherapy arsenal. Its applicability in various cancer types is impressive, as well as its use of endogenous mechanisms to achieve desired ends. However, off-target or on-target-off-tumor toxicity, limited activity, lack of control in combination treatments and, especially for solid tumors, low local accumulation, have collectively limited clinical use thereof. These limitations are partially alleviated by delivery systems. Lipid-based nanoparticles (NPs) have emerged as revolutionary carriers due to favorable physicochemical characteristics, with specific applications and strengths particularly useful in immunotherapeutic agent delivery. The aim of this review is to highlight the challenges faced by immunotherapy and how lipid-based NPs have been, and may be further utilized to address such challenges. We discuss recent fundamental and clinical applications of NPs in a range of areas and provide a detailed discussion of the main obstacles in immune checkpoint inhibition therapies, adoptive cellular therapies, and cytokine therapies. We highlight how lipid-based nanosystems could address these through either delivery, direct modulation of the immune system, or targeting of the immunosuppressive tumor microenvironment. We explore advanced and emerging liposomal and lipid nanoparticle (LNP) systems for nucleic acid delivery, intrinsic and extrinsic stimulus-responsive formulations, and biomimetic lipid-based nanosystems in immunotherapy. Finally, we discuss the key challenges relating to the clinical use of lipid-based NP immunotherapies, suggesting future research directions for the near term to realize the potential of these innovative lipid-based nanosystems, as they become the crucial steppingstone towards the necessary enhancement of the efficacy of immunotherapy.

The interaction mechanism of plasma iron transport protein transferrin with nanoparticles.

In the biological systems, exposure to nanoparticles (NPs) can cause complicated interactions with proteins, the formation of protein corona and structural changes to proteins. These changes depend not only on NP physicochemical properties, but also on the intrinsic stability of protein molecules. Although, the formation of protein corona on the surface of NPs and the underlying mechanisms have been fully explored in various studies, no comprehensive review has discussed the direct biochemical and biophysical interactions between NPs and blood proteins, particularly transferrin. In this review, we first discussed the interaction of NPs with proteins to comprehend the effects of physicochemical properties of NPs on protein structure. We then overviewed the transferrin structure and its direct interaction with NPs to explore transferrin stability and its iron ion (Fe3+) release behavior. Afterwards, we surveyed the various biological functions of transferrin, such as Fe3+ binding, receptor binding, antibacterial activity, growth, differentiation, and coagulation, followed by the application of transferrin-modified NPs in the development of drug delivery systems for cancer therapy. We believe that this study can provide useful insight into the design and development of bioconjugates containing NP-transferrin for potential biomedical applications.

T Cells Expressing a TCR-Like Antibody Selected Against the Heteroclitic Variant of a Shared MAGE-A Epitope Do Not Recognise the Cognate Epitope.

Antibodies-recognising peptides bound to the major histocompatibility complex (pMHC) represent potentially valuable and promising targets for chimeric antigen receptor (CAR) T cells to treat patients with cancer. Here, a human phage-Fab library has been selected using HLA-A2 complexed with a heteroclitic peptide variant from an epitope shared among multiple melanoma-associated antigens (MAGEs). DNA restriction analyses and phage ELISAs confirmed selection of unique antibody clones that specifically bind to HLA-A2 complexes or HLA-A2-positive target cells loaded with native or heteroclitic peptide. Antibodies selected against heteroclitic peptide, in contrast to native peptide, demonstrated significantly lower to even negligible binding towards native peptide or tumour cells that naturally expressed peptides. The binding to native peptide was not rescued by phage panning with antigen-positive tumour cells. Importantly, when antibodies directed against heteroclitic peptides were engineered into CARs and expressed by T cells, binding to native peptides and tumour cells was minimal to absent. In short, TCR-like antibodies, when isolated from a human Fab phage library using heteroclitic peptide, fail to recognise its native peptide. We therefore argue that peptide modifications to improve antibody selections should be performed with caution as resulting antibodies, either used directly or as CARs, may lose activity towards endogenously presented tumour epitopes.

Liposomes targeted to MHC-restricted antigen improve drug delivery and antimelanoma response.

PURPOSE: Melanoma is the most aggressive form of skin cancer. Chemotherapy at a late stage fails due to low accumulation in tumors, indicating the need for targeted therapy. MATERIALS AND METHODS: To increase drug uptake by tumor cells, we have targeted doxorubicin-containing liposomes using a T-cell receptor (TCR)-like antibody (scFv G8 and Hyb3) directed against melanoma antigen A1 (MAGE-A1) presented by human leukocyte antigen A1 (M1/A1). With the use of flow cytometry and confocal microscopy, we have tested our formulation in vitro. In vivo pharmacokinetics was done in tumor-free nu/nu mice, while biodistribution and efficacy study was done in nu/nu mice xenograft. RESULTS: We demonstrated two to five times higher binding and internalization of these immunoliposomes by M1+/A1+ melanoma cells in vitro in comparison with nontargeted liposomes. Cytotoxicity assay showed significant tumor cell kill at 10 µM doxorubicin (DXR) for targeted vs nontargeted liposomes. In vivo pharmacokinetics of nontargeted and targeted liposomes were similar, while accumulation of targeted liposomes was 2- to 2.5-fold and 6.6-fold enhanced when compared with nontargeted liposomes and free drug, respectively. Notably, we showed a superior antitumor activity of MAGE-A1-targeted DXR liposomes toward M1+/A1+ expressing tumors in mice compared with the treatment of M1-/A1+ tumors. Our results indicate that targeted liposomes showed better cytotoxicity in vitro and pharmacokinetics in vivo. CONCLUSION: Liposomes decorated with TCR-mimicking scFv antibodies effectively and selectively target antigen-positive melanoma. We showed that DXR-loaded liposomes coupled to anti-M1/-A1 scFv inflict a significant antitumor response. Targeting tumor cells specifically promotes internalization of drug-containing nanoparticles and may improve drug delivery and ultimately antitumor efficacy. Our data argue that targeting MAGE in A1 context, by nanosized carriers decorated with TCR-like antibodies mimicking scFv, can be used as a theragnostic platform for drug delivery, immunotherapy, and potentially imaging, and diagnosis of melanoma.

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

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.