The Many Hats of Neoantigen Peptides in Precision Medicine

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Even with standard methods of care, 55% of the 2 million people diagnosed with cancer this year will unfortunately pass. With this devastating statistic in mind, researchers and clinicians worked together to stray from one-size-fits-all treatments and moved to precision medicine. Personalized immuno-oncology utilizes next-generation sequencing technologies (NGS) to determine the genome of a patient’s tumor in order to identify unique biomarkers which can be used to initiate an antitumor immune response.

There are two different types of personalized cancer biomarkers; tumor associated antigens (TAAs) are peptides within cancerous tissue which become overexpressed as a result of an oncogenic mutation, but will also be expressed in wild-type tissue. However, due to their baseline expression, many TAAs fail to induce a strong enough immune response to overcome a cancer cell’s natural immune system defenses. Neoantigens, on the other hand, are mutated peptides, and therefore are only expressed within cancerous tissue. Due to their unique sequences, neoantigens are not recognized by the immune system as “self” and are able to avoid cancer’s anti-immune detection defenses in order to initiate antitumor killing.

The two most common forms of neoantigen- based therapies are personalized cancer vaccines (PCVs) and Allorgeneic/Autologous T-cell therapy (ATCT). PCVs introduce a range of about 20–50 different neoantigen peptides, DNA, RNA or loaded dendritic cells directly into a patient’s lymphatic system in order to induce T-cell mediated tumor killing. During ATCT, patient derived naïve T cells are isolated in order to identify those which when bound to a neoantigen, can initiate a strong immune response when reinfused back into the patient. The first stage of either of these therapeutics is to identify neoantigen candidates through comparative NGS between cancerous and wild-type tissue. These thousands of sequences will be narrowed down through bioinformatics in order to determine which set of neoantigens will be processed by the patient’s dendritic system, presented on their specific human leukocyte antigen (HLA)-typed major histocompatibility complex (MHC), and bind with high affinity and immunogenicity to T-cell receptors (TCRs).

After identifying neoantigen candidates through bioinformatics, most researchers will move on to preclinical in vitro functional screening of 1–1000s of predicted peptides. During PCV development, synthetic long peptides will be produced and incubated with either dendritic cells to analyze antigen presenting cell loading and processing or patient-derived PBMCs to measure T-cell induction through ELISPOT or fluorescently labeled HLA tetramers. For clinicians working on ATCT, preclinical in vitro functional screening includes incubating neoantigen peptide candidates with patient derived T cells in order to identify which neoantigens bind to patient derived TCRs and initiate an immune response. Once bound by neoantigen, each TCR will be analyzed to identify which pairs of TCRs and neoantigens bind with the strongest affinity and immunogenicity through SPR-based affinity measurement and T-cell induction assays as described above. Some researchers will take this process one step further, and optimize the TCR sequences of strong candidates through various affinity maturation technologies prior to expansion and infusion.

Once the top neoantigens are identified for either PCV or ATCT, researchers will move on to drug development and clinical testing. For novel therapeutic platforms, it is important to analyze the safety and efficacy of the specific therapeutic platform in nonhuman primates prior to treatment in a human. In order to accomplish this, researchers will identify neoantigens specific to their nonhuman patient and use them to either generate a vaccine or identify immunogenic TCRs. After treatment with either the PVC or ATCT, patient PBMCs will be isolated and incubated using control “unvaccinated” and testing “vaccinated” peptides. After incubation, the PBMCs will be analyzed for T-cell induction cytokines such as INF-γ and TNF-α through flow cytometry and ELISPOT. The incubated PBMCs will also be co-cultured with patient tumor samples for in vitro cytotoxicity studies in which researchers will identify if the vaccinated peptides were able to prime patient immune cells in order to induce tumor killing upon re-exposure. If the vaccine formulation shows strong efficacy and is well tolerated, the same protocol will be used to begin treating human patients and analyzing their therapeutic efficacy.

Regardless of the therapeutic avenue, one commonality linking all forms of neoantigen treatments is the need for reliable neoantigen peptides as they are the only reagent required for therapeutic discovery, development, and efficacy screening. However, neoantigen peptides are commonly quite difficult to synthesize as they can be highly hydrophobic, vary significantly in length and charge, and have a strong tendency to aggregate. Therefore, neoantigen peptide synthesis can be a significant bottleneck in the timeline of clinicians generating PCVs and ATCTs. Despite these difficulties and its young idealization, the field of neoantigen-based therapeutics has shown more promise for cancer remission and tumor regression than other immuno-oncology therapeutics and even highly used historically efficacious treatments.

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