In the lab: preclinical development of the DRibbles vaccine

Evidence supporting the utility of the DRibbles concept for priming T cell responses was first demonstrated in a series of in vitro experiments using a modified OVA-expressing HEK 293 T tumor cell model [2]. The OVA gene was engineered to produce “short-lived” OVA proteins that would become poly-ubiquinated and degraded by proteasomes under physiologic conditions [2, 3]. Whole cells were treated with bortezomib (Velcade®, Takeda, Osaka, Japan) and ammonium chloride (NH₄Cl), which block proteasome activity and lysosomal digestion of autophagosomes, respectively. Then, the treated cells were mechanically disrupted and fractionated by centrifugation to harvest an autophagosome-enriched product (Fig. 1a). This product was termed “DRibbles,” an acronym for “DRiPs and SLiPs-containing blebs.” The short-lived OVA proteins were found to be enriched in this DRibbles autophagosome product, compared to non-treated cells or non-disrupted bortezomib/NH₄Cl-treated cells. Furthermore, DRibbles vaccine was superior in priming OVA-specific T cells compared to non-treated or non-disrupted cells. These data suggested that DRibbles could be an effective vaccine against endogenous tumor-associated short-lived proteins.

Next, the DRibbles vaccine was evaluated for in vivo efficacy. DRibbles can either be produced based on an autologous concept (i.e. making the vaccine from a patient’s own tumor) or an allogeneic concept (i.e. making an “off-the-shelf” vaccine from one or more tumors to be administered to many patients). To model the autologous concept, DRibbles vaccine was generated from a 3LL Lewis lung cancer cell line and was shown to delay tumor growth and improve survival in that cancer model [4]. Next, to model the allogeneic concept, DRibbles vaccine was generated from multiple implantable methylcholantherene (MCA)-induced sarcoma cell lines. The long-standing paradigm was that whole-cell MCA vaccine would be effective only against homologous tumors [5]. However, vaccination with DRibbles derived from unrelated MCA-induced sarcomas was also effective in slowing tumor growth of other, independently-derived MCA sarcomas [3]. T cells isolated from these mice released interferon gamma against both homologous and independently-derived tumors, suggesting they had been cross-primed to a broader array of antigens present across a variety of sarcomas. This phenomenon was called ‘cross-protection,’ and provided evidence that an allogeneic DRibbles vaccine might serve as an “off-the shelf” vaccine in the clinic.

Further work was conducted to characterize components of the DRibbles vaccine. It was confirmed in various cell lines that DRibbles contain long-lived proteins (i.e. proteins not destined for rapid poly-ubiquitination and degradation), and are enriched for short-lived proteins, short-protein fragments, and poly-ubiquitinated proteins [4]. In addition to these potential antigens, the murine DRibbles product contained various damage-associated molecular pattern (DAMP) signals including heat shock proteins, high-mobility group box 1 protein (HMGB1) and calreticulin, suggesting that DRibbles could potentially mediate both adaptive and innate immunity. Finally, the DRibbles autophagosome surfaces were found to contain CLEC9A ligands, which have been shown to bind CLEC9A receptor [6] and facilitate antigen uptake by a subset of dendritic cells that play an important role in cross-presentation [7] (Fig. 1b). In summary, DRibbles was found to be composed of microvesicles that efficiently deliver a variety of antigens to pAPCs in ways that traditional liposomal and cellular vaccines do not.

Bench to bedside: translational data regarding the DRibbles vaccine

The results of this characterization, combined with the promise of ‘cross-protection’, led to the development of various human DRibbles autophagosome vaccine formulations for the treatment of human subjects. The first allogeneic human DRibbles vaccine, named DPV-001, was derived from autophagosome products of two human cancer cell lines: UbiLT3 and UbiLT6. UbiLT3 was derived from a non-small cell lung carcinoma (NSCLC) of mixed histology, whereas UBiLT6 was derived from an NSCLC adenocarcinoma. Liquid chromatography tandem mass spectrometry and western blotting techniques have been used to quantitatively catalogue over 2400 of the most common protein constituents in DPV-001. Of these most common proteins, there are over 25 published cancer-associated antigens, including at least 12 proteins that are among the NCI’s list of prioritized cancer antigens [8] such as TP53, survivin, EphA2, cyclin B1, XAGE1, Her2/neu, RhoC, Mesothelin, Legumain, PDGFRb, FOSL1 and KRAS [9].

Whole exome sequencing was used to show that many of the UBiLT3/6 genes are mutated or polymorphic versus the reference human genome (hg19). Thus, the DPV-001 DRibbles vaccine likely contains protein variants that are foreign to vaccinated patients. The UbiLT3/6 sequences were compared to 520 unique lung adenocarcinoma sequences from The Cancer Genome Atlas (TCGA) [10]. In addition to containing commonly observed oncogene mutations (for example KRAS G12C, found in 6.8 % of adenocarcinomas in TCGA, http://www.cbioportal.org/index.do, accessed February 6, 2016), the UbiLT3/6 cell lines also shared polymorphisms with the identified non-synonymous mutations from each lung adenocarcinoma in the TCGA. This suggests that DRibbles may serve as an off-the-shelf vaccine against “private antigens” found in individual patients. Furthermore, non-exact foreign protein variants (for example other KRAS G12 codon point mutations) may function as altered-peptide ligands which stimulate immune responses that spread to a patient’s own tumor-specific neo-epitopes [11, 12].

In the clinic: development of human DRibbles vaccines

In humans, the DRibbles vaccine was first evaluated as an autologous vaccine manufactured with tumor cells isolated from pleural effusions of patients with NSCLC. In this phase I clinical trial, autologous DRibbles vaccine was found to be safe when combined with docetaxel plus GM-CSF [13]. Autologous DRibbles vaccines, while providing a potential opportunity to vaccinate against patient-specific neo-epitopes, have proven difficult to manufacture consistently. Furthermore, a recent study suggested that in melanoma patients, CD8+ T cells may more frequently recognize non-mutated antigens such as NY-ESO-1 and GP100, rather than neoepitopes [14]. Subsequent trials have focused on allogeneic DRibbles products, which contain numerous non-mutated self-antigens. Studies are underway to evaluate the role of allogeneic DRibbles in malignancies such as prostate adenocarcinoma and NSCLC (Table 1). These studies are also assessing the DRibbles vaccine in conjunction with low-dose cyclophosphamide and various adjuvants such as topical imiquimod or GM-CSF.

In addition to DRibbles, a multitude of clinical trials are underway evaluating safety and anti-tumor efficacy of other modulators of autophagy such as hydroxychloroquine and alpha-tocopheryloxyacetic acid (alpha-TEA) [15–17]. Besides DRibbles, there are reports of alternative potentially effective cell-derived vesicle vaccines. For example, melanoma patients have been treated with an autologous product of dendritic cell-derived exosomes pulsed with tumor antigen peptides [18]. More recently, another group has shown that tumor-derived exosomes could be a more effective method for priming anti-tumor immune responses compared to tumor lysate alone [19].

One major barrier for all cancer vaccine clinical trials is the difficulty in demonstrating efficacy in early stage disease, especially in tumor types with low recurrence rates or prolonged latency periods. Therefore, scientifically grounded immune monitoring strategies must be used to inform early development of vaccines, allowing for smaller trials designed to facilitate optimization of the vaccine and perhaps identify indirect evidence of efficacy. Because DRibbles vaccines are multi-valent, and because relevant antigenic targets may vary across patients, next-generation high-throughput technologies such as seromic protein arrays are being used to evaluate patient-specific immune responses and discover relevant antigens. The phase II non-small cell lung cancer DRibbles adjuvant trial serves as an example of how a seromics-based approach might be used to monitor immune response. This trial was designed to detect enhancement in antigen-specific adaptive immunity using serum protein arrays that measure antibody reactivity against a panel of over 8,000 normal human protein isoforms. The rationale is that the most robust immune responses might be integrated with concomitant CD4 T-helper, CD8 cytotoxic, and humoral immune responses [20], and therefore antibody reactivity may serve to identify antigen-specific immune responses associated with therapy. Using the protein array, several of the 9 treated DRibbles patients were found to exhibit robust (i.e. >10-fold increase from baseline) antibody responses to multiple antigens following vaccination [10].

Ethics approval and consent to participate

Not applicable.