Single-Walled Carbon Nanotubes Deliver Peptide Antigen into Dendritic Cells and Enhance IgG Responses to Tumor-Associated Antigens

Active specific immunotherapies (vaccines) take advantage of the ability of the immune system to recognize and react with foreign antigens. In the case of cancer vaccines, the antigens typically are not foreign, but overexpressed self-antigens on the surface or inside the tumor cell, some of which are then presented on the cell surface in the context of HLA molecules for recognition by T-cells.1,2 The generation of an immune response generally requires peptide presentation by dendritic cells (DCs), which are the most effective antigen presenting cells (APCs).3,4 Tumor self-antigens are generally poorly immunogenic and the host bearing the tumor either does not recognize the antigen or is tolerant to it.5,6 While a number of strategies have been used to enhance the immunogenicity of tumor peptide antigens and break the self-tolerance, soluble peptides are poor immunogens, and effective immunization strategies require delivery formulations that include carrier molecules or adjuvants. Great advances have been made within the past decade in the discovery of new adjuvants suitable for clinical use, including cytokines,(7) saponins, CpG motifs,(8) and heat shock proteins(9) that can stimulate the innate immune system and induce strong adaptive immune responses. However, their biological activities are normally short-lived, requiring several doses to be administered. Materials made from synthetic and natural sources such as dendrimers or keyhole limpet hemocyanin (KLH), have been proposed as potential carrier molecules.10,11 The potent immunogenicity and lack of flexibility of these carrier molecules can lead to the problem of carrier-induced suppression. The host immune system becomes overwhelmed with the maintenance of a polyclonal response to the carrier molecule, thereby limiting the ability to generate a focused response to the antigen of choice, which is detrimental to the efficacy of the vaccine.(12) Thus, there is a need for new carriers, especially those that efficiently deliver the antigens into professional APCs, such as DCs. A promising approach to improve the immunogenicity of proteins and peptides for cancer vaccines is the use of particulate vaccines.(13) Conjugation of antigenic epitopes to particulate scaffolds has shown that the particles improve immune responses and that the response is most improved when the particles are on the nanoscale.(14) This discovery has introduced the concept of nanovaccines, where changes in the nanoscale architecture of particulate vaccine materials can direct the characteristics of the immune response.(15) An important characteristic of nanovaccines is their enhancement of delivery to APCs. Antigen delivery through nanoparticles also alters cellular trafficking and can serve as an intracellular depot of antigen, both of which improve the immune response to the delivered antigen. However, most studies of nanoparticulate vaccines have used the strongly immunogenic, egg protein, OVA as the model antigen in initial investigations; peptides derived from this foreign protein are already known to be highly immunogenic. As a nanomaterial of unique physical and chemical properties, carbon nanotubes have attracted considerable interest in biomedical applications,(16) including their use as nanovaccine scaffolds. Because of their propensity to internalize into a wide variety of cell types and through several mechanisms,17−19 they appear to be particularly suited to deliver antigenic epitopes to APCs. In addition, carbon nanotubes have a unique geometry (very high aspect ratio) that may inherently alter the immunogenicity of appended antigens. Single-walled carbon nanotubes (SWNTs) also have a large available surface area for chemical modification, with every atom inherently at the nanotube surface. Furthermore, well-functionalized, dispersed carbon nanotubes do not appear to have inherent toxicity(20) (in contrast to unmodified carbon nanotubes(21)), and can bear large numbers of peptide ligands. There is also evidence that carbon nanotubes can produce immune responses when covalently linked to highly immunogenic peptide sequences.(22) However, there are no reports on the use of carbon nanotubes (CNTs) to deliver antigen to primary human DCs, nor the demonstration of improvement of immunogenicity of therapeutically relevant antigens administered with common adjuvants. Furthermore, there is continuing debate about the inherent immunogenicity of SWNTs and their ability to augment immune responses.(23) In the current study, we aimed to demonstrate that carbon nanotube–peptide constructs could improve the immunogenicity of a weakly immunogenic, clinically relevant cancer-associated peptide. Wilm’s tumor antigen (WT1) is widely used in human trials as a cancer vaccine.24−26 A 19 amino acid peptide named WT1Pep427 derived from WT1 protein was chosen for study because the peptide has high binding affinity across multiple human HLA-DR haplotypes and induces strong CD4 T cell responses in humans, but is a poor binder to both I-Ed and I-Ad class II molecules of BALB/c mice. We used spectrally quantifiable chemical approaches to covalently append large numbers of peptide ligands onto solubilized SWNT scaffolds. The SWNTs were covalently modified on their sidewalls with hydrophilic ethylene glycol using 1,3-dipolar cycloaddition of azomethine ylides. We then investigated the kinetics and cellular pharmacology of the nanotube interaction with APCs in vitro; finally we asked whether these nanotube–peptide conjugates could improve immune responses in vivo in mice. We also demonstrated that the nanotube scaffold itself is noncytotoxic to human dendritic cells and is nonimmunogenic.