We present here a flexible vaccine-oriented scheme for conjugating antigen to yeast particles that has undergone multiple refinements with the aim of maximizing cross-presentation. The fusion protein MS74NEY2 represents the latest embodiment, consisting of the following functional elements: MBP, SNAP-tag, CD74 ectodomain, 15-mer CMV-derived surrogate antigen, NY-ESO-1 cancer-testis antigen, glycine/serine linker, ybbR tag, and His6
tag. We have demonstrated cross-presentation of both the surrogate antigen and an NY-ESO-1 HLA-A2 epitope; these antigens can readily be exchanged to suit different immunotherapy applications. The highly hydrophobic NY-ESO-1 protein used in clinical trials is purified from E. coli
but insertion of MBP as the N-terminal domain enabled our fusion proteins to be expressed solubly and also facilitated purification. For other antigens, MBP may be omitted or replaced if desired. A key feature of our conjugation scheme is its site-specific nature, which ensures that antigen can be released in the DC phagosome to translocate to the cytosol for entry into the MHC class I processing pathway. The initial layer of fusion protein is conjugated to BG-derivatized particles through the formation of a stable thioether bond with the SNAP-tag domain. We showed that cross-presentation is highly inefficient when a nonsite-specific chemical conjugation method was used to attach the same protein to yeast. The lack of efficacy is especially noteworthy considering that there were no reactive lysine residues within the CMV peptide and only one in the C-terminal direction. This suggests that merely “tethering” an epitope on both ends with no intervening phagosomal protease sites can impede cross-presentation. To expedite phagosomal antigen release in our scheme, we inserted the extremely CatS-susceptible CD74 ectodomain between the SNAP-tag tether and the antigens, resulting in improved cross-presentation efficiency.
In addition to developing the conjugation scheme with antigen release kinetics in mind, we have also made several improvements to increase antigen dose delivery. The first refinement was to use a yeast strain expressing Aga1p and Aga2p, which provided more free amines on the cell wall for BG and fusion protein conjugation compared with the wild type. Fusing lysine-containing polypeptides to Aga2p did not further increase the antigen density in this first layer, so we devised a novel multilayer conjugation strategy that retains site-specificity. Sfp enzyme is used to attach BG-linked CoA to the ybbR tags of 1 antigen layer, thus permitting the next layer to be added via the SNAP-tag/BG reaction. We experienced no loss in conjugation efficiency in building up to 4 antigen layers (suggesting that further coats can be added almost indefinitely) and demonstrated that cross-presentation levels rose with the number of layers. Finally, breaking up the cell wall into fragments before conjugation further increased the amount of antigen delivered per yeast cell equivalent, probably because the surfaces that were revealed provided additional free amines for BG attachment.
Although the use of yeast hulls instead of whole yeast improves antigen delivery and reduces native yeast protein content, it is at present unclear whether yeast hulls will ultimately be more effective in the setting of immunotherapy. A major reason behind the attractiveness of yeast-based vaccines is their ability to mature DCs and stimulate the secretion of proinflammatory cytokines, obviating the need for additional adjuvants.5
To our surprise, yeast hulls were significantly inferior to whole yeast cells in inducing DC maturation, and more importantly, they elicited little or no IL-12 secretion. In retrospect, this observation is not without precedent as several groups have reported that Zymosan, a preparation of S. cerevisiae
cell wall particles, is a poor inducer of DC IL-12 secretion despite its long-standing reputation as a proinflammatory agent.36-38
IL-12 secretion is likely to be important to vaccine efficacy because it plays a role in enhancing the clonal expansion of primed CD8+
perhaps by increasing resistance to activation-induced cell death.41
The disparity in DC reactions to whole yeast cells and yeast cell wall fragments is puzzling and warrants further investigation. One possibility is that an immunostimulatory component is lacking in yeast hulls, which was either removed with the yeast cytoplasm or washed away from the cell wall fragments. Reevaluation of the wash steps may be helpful in the latter case. In the former case, a possible candidate is double-stranded RNA from viruses that ubiquitously infect yeast cells.42
It has been observed that a combination of Zymosan and poly-I:C (a synthetic double-stranded RNA analog) stimulates far greater levels of IL-12 secretion than either agent alone,38
raising the intriguing possibility that whole yeast cells induce massive IL-12 secretion through a similar synergism. If this is the case, conjugating poly-I:C to yeast hulls may present a simple solution. An alternative explanation stems from the complex structure of the yeast cell wall, with fragmentation resulting in the exposure of different molecular patterns leading to altered signaling cascades in DCs. To elaborate, the outer layer of the S. cerevisiae
cell wall is composed primarily of mannan (hypermannosylated proteins), which in undamaged yeast cells largely masks the inner layer of β1,3-glucan, β1,6-glucan, and chitin.43
Our observation that antigen conjugation (and thus partial disruption of the molecular patterns) had opposite effects on whole yeast cells and yeast hulls could be an indication that DC recognition of the inner cell wall layer partially counteracts the maturation signals derived from ligands in the outer layer.
Vaccination experiments in vivo will be required to discover the effects of yeast particle choice on T-cell memory and protective immunity and to develop an optimal formulation (possibly a mixture of uncoated whole yeast and coated yeast hulls). At the same time, the conjugation scheme presented here is not restricted to yeast and can easily be applied to any particle that is or can be amine-functionalized, including heat-killed bacteria and bacterial ghosts, polymer and inorganic microspheres, ribosomes, and liposomes. Although the ability of antigen-coated particles to induce protective tumor immunity was demonstrated almost 15 years ago,44
particulate antigen delivery vehicles (with the exception of viruslike particles) have received little attention in the field of cancer immunotherapy. Codelivery of adjuvant and antigen in particles holds several advantages over injection of soluble agents, including a high local concentration that does not quickly dissipate, assurance that both adjuvant and a minimum bolus of antigen are delivered to the same cell, and inherent targeting for uptake by phagocytic cells (besides DCs, macrophages45
are also capable of cross-priming). Poor phagosomal escape of antigen represents one barrier to the development of particulate vaccines, and the conjugation strategy described here presents a novel way to break this barrier.