Neoantigens are peptides expressed on cancer cells that are generated from mutated genes, and can potentially be identified as foreign by the body’s immune system, which then triggers an attack against the cancer cell. It is these neoantigens that are thought to be responsible for the success of the new generation of checkpoint inhibitor immunotherapies against some forms of cancer.
Scientists at Arizona State University’s Biodesign Institute now report on the identification of more than 200,000 cancer neoantigens, which could feasibly lead to the development of broad-spectrum cancer vaccines, as well as tumor type-specific treatments or patient-personalized vaccines. “In a cancer cell, it turns out that all levels of information transfer from DNA to RNA to protein become more error-prone,” said research lead Stephen Albert Johnston, PhD, center director and professor, Biodesign Center for Innovations in Medicine. “We proposed that these mistakes made in cancer cells may also be the source to make a cancer vaccine.”
Johnston and his team at the Biodesign Institute have spent more than a decade working towards the goal of developing a universal vaccine that can prevent cancer. They report on their latest studies in Scientific Reports, in a paper titled, “RNA Transcription and Splicing Errors as a Source of Cancer Frameshift Neoantigens for Vaccines.”
The success of checkpoint inhibitor therapy against cancer is largely attributed to activation of the patient’s immune response to tumor neoantigens that result from DNA mutations in the cancer cells, the authors explained. However, while checkpoint inhibitor immunotherapies are “revolutionizing” how we treat cancer,” about 50–80% of patients with even the most responsive tumor types won’t respond well to treatment. “A surprising finding in the analysis of these patients was that one of the best correlates of response has been the total number of neoantigens in the tumor,” the team stated. The realization that these DNA mutations have such immunological importance has accelerated research efforts to develop personal cancer vaccines. It’s a promising approach, but in reality, “ … a major problem is that the majority of tumors will not have enough neoantigen-generating mutations to sustain development of a personalized vaccine.”
With this in mind, the researchers set out to look for an alternative source of neoantigens that could possibly broaden the scope of neoantigen-based cancer vaccines. They were particularly interested in how disrupted RNA processes can lead to the production of frameshift (FS) mutated peptides, and the exposure of these peptides to the immune system. As they pointed out, “In the process of becoming a tumor, not only does the DNA mutation rate increase with faster cell divisions, but also there is a disruption of basic cellular functions, including RNA transcription, splicing, and the quality control system on peptides.”
They reasoned that frameshift variants produced by errors in RNA processing might be a source of cancer neoantigens, and they also assumed that there is a general increase in error rates in cancer cells. For the most part these errors can be managed and cleaned up by the cell’s own quality control machinery. However, as cancer progresses, these mutated peptides can build up and swamp the cell’s ability to deal with them, so aberrant proteins are then exposed and recognized by the immune system.
“These overwhelm the quality control systems of a cell, producing mistakes in RNA and proteins that are released from the cancer cell, and the immune system can respond to,” said Johnston.
To quickly identify frameshift and splicing mutations, Johnston’s research team designed an array to detect all possible predicted frameshift peptides that any tumor cell could potentially produce. They custom-build this frameshift array, which ended up containing almost 400,000 frameshift peptides, and screened these against the blood samples of cancer patients (and healthy samples as a control) to look for antibodies against the peptides.
“We analyzed the specific IgG reactivities to these FSPs in 64 noncancer control samples and a total of 85 cancers from five different late-stage cancer types with 17 samples each (LC: lung cancer, BC: breast cancer, GBM: glioblastoma, GC: gastric cancer, PC: pancreatic cancer) and 12 stage I pancreatic cancer samples,” the authors noted.
This approach is less complex than extracting, purifying, and then sequencing tumor DNA, which is the typical starting point for the development of personal cancer vaccines. “Personal cancer vaccines are complicated and expensive,” said Johnston. “Also, only about 40% of tumors have enough mutations in the DNA to make a vaccine from. We discovered that even ‘cold tumors’ at the DNA level make lots of mistakes at the RNA level. And the mistakes we focus on are frameshift peptides which are much more immunogenic than the point mutations used in personal cancer vaccines. Most importantly, we can make off-the shelf vaccines for therapeutic or even preventative vaccines which will be much less expensive.”
The results of their screens indicated that all five cancer types, with the exception of glioblastoma, had significantly more peptides reacting with antibodies in the cancer patients than controls. There were also three basic patterns seen among patients with each cancer type. First, the vast majority of the frameshift peptides (69–80%) were personal, or unique to that individual. Second, about 16–19% of the positive peptides were shared between two samples within the same cancer type, and thirdly, 1.5–6.9% were shared between three or more samples (with gastric cancer having the highest, at 6.9%). Strikingly, one of the hardest to treat cancers, glioblastoma, had the greatest potential for personalized vaccines. Of the 17 glioblastoma patient samples studied, each patient had 5,800 frameshift peptides, and of these, 4,500 were unique to that patient.
The team wanted to see how the frameshift mutations compared between early- and late-stage cancers. A comparison of the 20,000 peptides that they identified in late-stage and stage 1 pancreatic cancer showed little overlap, implying that a vaccine for early-stage cancer would have to be different to that for late-stage cancer.
Interestingly, studies in mouse models found that the newly discovered antigens were protective against both breast cancer and melanoma. Johnston’s group has pioneered genetic immunization using gene gun technology, which they used for their experiments to shoot gold nanoparticles containing the most promising vaccines. In a typical experiment, six-week-old mice received one genetic immunization in the pinna of the ear. After four weeks, they were challenged with cancer-causing cells, and then twice received booster shots, two days apart. The results showed that the prototype vaccines could all significantly delay or even prevent tumor growth or progression. Most importantly, in the mouse vaccine challenges, they found that pooling multiple frameshift peptides resulted in a more effective vaccine, with additive effects further delaying tumor growth.
The team has used their work to date as the basis for a large preclinical trial, in dogs, funded by the Open Philanthropy Project, which is evaluating a vaccine candidate designed to be a broadly protective, prophylactic pan-cancer vaccine. Johnston has also established a spinout company, Calviri, to continue cancer vaccine development.
From their screening results and analyses of the different cancer samples, and the mouse cancer vaccine challenges, the Johnston group now has a top 100 peptide list for each of the five human cancers. “We strongly believe that the data presented, as well as more to be submitted, support bringing FS antigen cancer vaccines to clinical trial,” the authors wrote. “We have recently initiated a large dog clinical trial of a pan-cancer prophylactic vaccine and will soon submit protocols for both dog and human therapeutic trials of cancer-type specific vaccines.”
Johnston acknowledged that even optimistically it would be five to 10 years before human use. However, he concluded, “This is probably the only approach to a broadly preventative cancer vaccine, so we feel we have to try it. The implications of success would be quite large—for dogs and people.”