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9 Review 2017/03/03 Neoepitopes as immunotherapy targets: key challenges and opportunities

Immunotherapy Over the last half century, it has become well established that can elicit a Cory A Brennick‡,1, Mariam M host immune response that can target them with high specificity. Only within the last George‡,1, William L Corwin1, decade, with the advances in high-throughput sequencing and bioinformatics Pramod K Srivastava1 & *,1 approaches, are we now on the forefront of harnessing the host’s to Hakimeh Ebrahimi-Nik 1Department of Immunology, & Carole & treat cancer. Recently, some strides have been taken toward understanding effective Ray Neag Comprehensive Cancer Center, tumor-specific MHC I restricted or neoepitopes. However, many fundamental University of Connecticut, School of questions still remain to be addressed before this therapy can live up to its full clinical Medicine, Farmington, CT 06030–1601, potential. In this review, we discuss the major hurdles that lie ahead and the work USA being done to address them. *Author for correspondence: Tel: +1 860 679 6020 Fax: +1 860 679 8839 First draft submitted: 16 December 2016; Accepted for publication: 21 February 2017; [email protected] Published online: 17 March 2017 ‡Authors contributed equally

Keywords: binding affinity • cancer Immunotherapy • checkpoint blockade • neoepitope •

It is now appreciated that cancer is a pro- a framework to understand not only tumor foundly patient-specific disease, where no development but also treatment resistance two tumors are alike. The foundation of and metastasis. In response to these insights, this insight stemmed from the early murine considerable efforts have begun to develop work of Gross [1], Prehn and Main [2], and and adopt ‘precision medicine’ or cancer Klein et al. [3] that clearly demonstrated therapies that aim to treat in a patient-spe- that the tumor is not only cific manner. To this end, a number of cancer patient specific but inherently specific to immunotherapies have been developed that 4 the individual tumor itself. Continued can- look to exploit the immune system’s exquisite cer research in areas, such as, immunology, ability to selectively target cancer cells [7,8]. genomics, , metabolomics, and While considerable gains have been made so on have further enhanced our appreciation with regard to treatment outcomes for many 2017 of the vast complexity of this disease. patients, there still remains a fundamen- Efforts at with ‘tumor anti- tal deficiency in our knowledge of the pre- gens’ have had limited success in cancer cise immunological underpinnings behind immunotherapy. This is largely because the this tumor specificity observed over a half a main focus has been on shared ‘tumor anti- c­entury ago. gens’, which are actually self-. It is Since the time it was initially proposed only within the last decade that it has become over two decades ago [9], it has now become apparent that common tumor antigens are well established that somatic or passenger not suitable targets for vaccination [4,5]. within the tumor give rise to new Further, the concept of immunoediting [6] epitopes or neoepitopes. Further, evidence or the selective pressure of the immune sys- supports that neoepitopes are recognized tem to drive tumor evolution, has helped as by the as ‘mutated part of

10.2217/imt-2016-0146 © 2017 Pramod K Srivastava & Hakimeh Ebrahimi-Nik Immunotherapy (2017) 9(4), 361–371 ISSN 1750-743X 361 Review Brennick, George, Corwin, Srivastava & Ebrahimi-Nik

self’ and serve as the means by which immune sys- Although the binding affinity of a to tems can differentiate cancer from normal cells [10] . MHC I is one of the most studied criteria in the search Thus, neoepitopes may make strong candidates for for neoepitopes, many other factors discussed here play personalized cancer immunotherapy . With an important role in whether a neoepitope is finally the recent advancements in genomic sequencing and presented on the tumor cells and whether it is taken bioinformatic approaches, the feasibility of personal- up by APCs and cross-presented in order to activate ized therapy in the clinical setting is now on the fore- neoepitope-specific CD8+ T cells. Thus far, there are front. However, there are still major obstacles that need no reliable methods to accurately verify that in silico to be addressed before this therapy is introduced as a identified neoepitopes can actually elicit tumor regres- common practice. To this date, we do not have any sion or an immune response. As mentioned earlier, the means to predict which one of the putative neoepit- released by the proteasome are transported to opes will elicit protective immune response against the ER from the cytosol by TAP. TAPPred is an online tumor growth in the host [11] . Issues ranging from the tool to predict binding affinity of peptides toward the accurate identification all the somatic mutations in TAP [13] . However, these tools for proteasomal cleav- a given tumor sample to the prediction of the ‘best’ age or TAP binding and transport are limited and not neoepitopes and translation of that information into accurate, and most neoepitope prediction algorithms the most effective treatment illustrate the hurdles that do not take into consideration this aspect [14] . Peptide remain. In this review, we will discuss these challenges trimming by ERAP is important for the loading of the (Figure 1) and the work being done to address them in correct-sized peptide onto the peptide-binding cleft of an effort to truly understand what makes a good neo- MHC I. Thus far, no prediction algorithm accurately and how best to translate this knowledge into incorporates the function of ERAP into predicting effective treatment modalities. Class I restricted neoepitopes. It has been found, in various solid and hemato- From ubiquitination to recognition logical tumors, that some of the mutations occur in Cancer immunotherapy includes the use of many that are involved in presentation. If this immune cell types to increase the tumor-killing capac- phenomenon occurs, then predicting neoepitopes for ities of T cells, B cells, natural killer (NK) cells and such tumors and using them for immunotherapy may dendritic cells (DCs). Cytotoxic CD8+ T cells (CTLs) not result in an immune response or tumor regression are the most potent cell type which when activated, are because the presentation of neoepitopes on the cell able to recognize tumor-specific antigens and distin- surface is abrogated. The aberrations in the antigen- guish between normal and cancerous cells. To achieve presentation machinery range from defects in protea- this tumor-specific killing, cancer vaccines should be somal subunit patterns, TAP, ERAP, other chaperone able to target MHC class I restricted epitopes which and MHC I. They occur individually or in then activate CD8+ T cells to specifically kill cancerous combination, and the frequency and nature of the cells. This involves a number of steps which are sum- defects vary according to the type of tumor. Defects marized as follows. Degradation of ubiquitinated pro- in surface expression of MHC I molecules have been teins by the proteasome in the cytosol, chaperoning of demonstrated in a large variety of human tumors [15] . peptides by HSP90 in the cytosol, active transport into As with the above mentioned defects in the antigen- endoplasmic reticulum (ER) by the transporter associ- presentation machinery, particularly loss of MHC I ated with antigen processing (TAP) and trimming to surface expression, the use of neoepitopes that, in silico, appropriate length by ER aminopeptidases associated are predicted to be recognized by the immune system, with antigen processing (ERAP), loading onto the based on MHC I structure, may not necessarily be pre- peptide-binding cleft of the MHC I molecules with the sented by the tumor cells. In such cases, immunother- help of chaperone proteins, such as, tapasin and trans- apy with these neoepitopes will not elicit an immune port to the cell surface which is then recognized by the response or tumor regression. The addition of mass T-cell receptor (TCR) on CD8+ T cells. Neoepitopes spectrometry (MS) may play a key role in prediction are those peptides that arise from somatic mutations pipelines to account for the antigens that are actually and recognized as different from self and presented by presented on the tumor surface. It must be mentioned antigen-presenting cells (APCs), such as, DCs and the that this method has not yet reached the level of being tumor cells itself [9]. Here, cross-presentation plays an a high-throughput approach. However, improved important role as the APC is able to translocate exog- sensitivity of MS in identifying tumor-derived neo- enous antigens from the phagosome into the cytosol epitopes in patient’s tumors validated by neoepitope- for proteolytic cleavage into MHC I epitopes by the specific antitumor immune response has been recently proteasome [12] . reported [16] .

362 Immunotherapy (2017) 9(4) future science group Neoepitopes as cancer immunotherapy targets Review

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Tumor microenvironment: • M2 MDS C Effective neoepitope dosage without tolerizatio n Finding the best neoepitope delivery system Vaccine delivery: • • Inhibition Stimulation Figure 1. The current challenges of neoepitope-based of challenges therapies. current The 1. Figure immunotherapies. Each step in this figure illustrates keythe challenges that need to be addressed for improved clinical translation. See the text for in-depthCTL: Cytotoxic description. T lymphocyte; DC: Dendritic cell; NKC: Natural killer cell; M2: M2 macrophage; MDSC: Myeloid-derived suppressor cell; TCR: T-cell receptor.

future science group www.futuremedicine.com 363 Review Brennick, George, Corwin, Srivastava & Ebrahimi-Nik

Another major aspect to eliciting a sufficient T-cell the international Immunogenetics information system response against a neoepitope is its ability to bind its (IMGT)/HLA database [26], and the less common cognate TCR. It has been clearly demonstrated the HLA’s peptide-binding kinetics being poorly studied, TCR affinity and avidity are crucial to the T-cell accurately predicting a peptide’s ability to bind to its response amounted [17–19] . A great effort has been made cognate MHC I has been very difficult in itself. For to develop programs that can predict the ability of an the more commonly studied MHC I molecules, stan- MHC I with its peptide to bind to a known TCR. Thus dard criteria have been established to predict the likely far the programs available are CTLPan and NetCTL- immunogenic peptides that can be applied to all HLAs Pan, but with a limited known TCR peptide–MHC I as more are characterized. interactions known, these programs are limited in their The thresholds used today to determine which accuracy [20]. With new technologies emerging for the peptides are most likely to elicit a CTL response were identification of TCRs to their cognate antigen, these established over two decades ago, and set the standard

programs may become of great use to predict putative that an IC50 ≤500 nM, preferably 50 nM or less, is con- neoepitopes. sidered a strong to intermediate binder and determines The development of prediction algorithms or pro- an epitope’s capacity to elicit a CTL response [27]. This grams that incorporate not only the binding affinity of criterion has been highly validated in viral and model the neoepitope on MHC I but also the other steps of antigen systems where there is no ‘self’ counterpart. antigen processing, presentation and TCR recognition Since neoepitopes arise from altered self-peptides, remains an important step toward improving the accu- neoepitope-specific T cells potentially face thymic racy of predicting successful neoepitopes. deletion and tolerization. This issue was addressed by Duan et al. who created a differential agretopicity Are we being misguided by binding affinity? index score, which compares the binding affinity of the In an effort to better predict putative neoepitopes from mutated peptide with the reference peptide [28]. This the large set of somatic mutations called for a given score indirectly accounts for how different the peptide tumor, the criterion of binding affinity of a neoepit- is from ‘self’. In that study, they identified three neoepi- ope to its cognate MHC I is applied. The most readily topes that were able to effectively inhibit tumor growth

used technique for measuring binding affinityin vitro in two different BALB/c tumor models, yet had IC50 is by measuring the concentration of a given peptide scores significantly above the 500 nM threshold. By needed to compete with a standardized peptide already current standards, these neoepitopes would have been bound to a given MHC I allele [21] . This results in an overlooked and not considered to be immunogenic

IC50 score for a given peptide and its cognate MHC based on their binding affinity alone. I. It is important to note that this technique does not On the contrary, studies by Gubin et al. [29] and account for the highly complex peptide loading path- Yadav et al. [30] have each identified neoepitopes that fit way in vivo [22], which could aid in the loading of less the strong affinity criterion that were able to effectively stable peptides [23]. It has been demonstrated that the target the tumor. It is worth noting that work done by peptide-loading complex, most importantly tapasin, Assarsson et al. [31] in a viral model system established plays a crucial role in the loading of endogenous pep- that about 7.5% of the predicted high-affinity bind- tide, where in vitro studies would consider that peptide ers are actually recognized by the immune system, yet unable to bind MHC I [24]. With current methodolo- in the tumor model system, the frequency of validated gies unable to recapitulate the peptide-loading complex high-affinity binders to those predicted to be immu- in vitro, measuring whether or not a peptide will bind nogenic is significantly lower. This observation is sup-

to MHC I is limited to the measurement of the IC50 for ported by the work of van Rooij et al. that reported a given peptide. Furthermore, these in vitro measured that less than 1% of the high-affinity binders predicted

IC50, from known viral and model antigens, are used from a melanoma patient were able to be recognized by to train current binding affinity prediction algorithms the patient’s T cells [32]. used today. Surprisingly, it was recently found that proteasome- There are currently more than a dozen various pre- generated spliced epitopes account for a fourth of the diction algorithms for predicting the binding affinity HLA class I immunopeptidome in terms of abun- of peptides, with NetMHC [25] being the most widely dance [33]. This accounts for a larger repertoire of used and validated algorithm for neoepitope prediction proteasomal spliced peptides than originally thought pipelines. Like most prediction algorithms, NetMHC and current prediction methods do not take these has been trained with an artificial neural network, peptides into consideration, as criteria for what pep-

and outputs a predicted IC50. With over 11,000 vari- tides get spliced have not been established. Moreover, ants at the human class I loci identified, according to post-translationally modified proteome substrates are

364 Immunotherapy (2017) 9(4) future science group Neoepitopes as cancer immunotherapy targets Review of importance and currently overlooked. It has been It is also important that the adjuvant delivers optimum clearly demonstrated that post-translational modifi- amount of the antigen by controlling antigen persis- cations, such as, phosphorylation, methylation and tence and concentration [38]. There should be a bal- glycosylation, can be recognized by the immune sys- ance between antigen persistence and delivery. Long- tem as foreign compared with their unmodified coun- term antigen persistence at the site of injection may terpart [34]. With the advancement of MS, it is now have adverse results in tumor rejection and cause CD8 possible to identify these noncanonical antigens and T-cell dysfunction and deletion [39]. Adjuvants func- develop programs that consider these modifications. tion in different ways including: releasing the anti- The dependence on using binding affinity as a major gen gradually [40,41], stimulating pattern recognition predictor of putative neoepitopes, while useful in some receptors on APCs [42,43], protection of antigens from cases, may be limiting our ability to accurately iden- rapid degradation and therefore extending the antigen tify all neoepitopes. The criteria of what makes a good ­presentation time [44,45]. neoepitope have yet to be established, but with the Different types of adjuvants have been tested in can- identification of more neoepitopes and consideration cer therapy vaccines, such as, mineral adjuvants and of weak-affinity and noncanonical neoepitopes, we can cytokines [46], RNA-based adjuvants [47], liposomes [48], start to identify and set the criteria that can help pre- tensoactive agents [49] and bacterial products [50]. In dict neoepitopes more accurately. Only then will we some studies, different types of cells were pulsed with further understand what makes a good neoepitope. neoepitopes for immunization. These cells including B cells [51], macrophages [52], splenocytes [28] and DCs [53] How should we immunize against serve as both delivery system and adjuvant. neoepitopes? DCs as professional APCs play a major role in ini- Since neoantigens are small pieces of peptides harbor- tiating immune response [54]. The first cancer APCs/ ing tumor mutations, immunization with them usually DC-based vaccine (sipuleucel-T) received the US FDA needs strong immunostimulatory agents to produce approval [55]. However, sipuleucel-T failed to achieve efficient immune response. Peptides as vaccines may commercial success in part due to a perception of lim- not be able to stimulate the immune system powerfully ited clinical activity [56]. Since DCs are able to effi- enough on their own. Therefore, it is usually required ciently uptake, process and present the antigen and ini- to use an adjuvant in combination in order to elicit an tiate the immune response, they may be considered as effective immune response [35]. natural adjuvants [57,58]. Although there are many stud- In order to activate naive cytotoxic T cells and ies using DCs as delivery system for different cancer have a robust immune response, usually stimulation immunotherapies that are showing promising results of T helper (Th) cells is needed. Synthetic peptides on CTL response [59–61], most of them failed to induce designed to stimulate cell-mediated immunity often good tumor rejection [62–64]. One of the challenges in do not have a proper Th-epitope and therefore they using DCs as delivery system in neoepitope cancer are not efficient by themselves. Even with the proper therapy is providing enough functional DCs for each T cytotoxic and Th-cell epitopes, an adjuvant is still patient that may not be achieved by isolating patient required in order to have an effective peptide-based blood DCs [65]. The number of blood DCs in cancer vaccine. Based on our unpublished data in BALB/c patients often is reduced [66] and they may be func- mice, using a potent immunostimulatory adjuvant and tionally impaired due to tumor microenvironment delivery system for neoepitope immunization seems (TME) systemic effect [67]. There are other ways to crucial to have an effective tumor rejection response. get syngeneic DCs including in vitro differentiation + The type of induced CD8 T cells against the tumor of blood monocytes [68], hematopoietic stem cells [69] (whether these cells have cytotoxic function or they are or bone marrow derived stem cells [70], in which each dysfunctional), may have an important role in patient have their own hurdles. Different variables using response to cancer immunotherapy and clinical out- DCs play a major role in the final antitumor immu- come [36]. nity caused by DC neoepitope based cancer vaccine, Recent studies show that immune checkpoint block- such as, DC maturation status [61,71], DC type, num- ade (CB) of CTLA4 or PD1, such as, ipilimumab (for ber of the injected DCs and injection location [72]. It CTLA4) or nivolumab (for PD1) can be effective in is clear that having the appropriate tumor neoepit- cancer therapy and enhance immune response [37]. ope is one of the most important elements in cancer In order to get a maximized T-cell response in cancer immunotherapy by DCs. However, determining the immunotherapy, besides proper neoepitope, appropri- exact DC subtype that works the best as adjuvant for ate adjuvant that can induce production of cytokines immunotherapy may be one of the future directions in and costimulatory molecules from APCs is required. DC‑based vaccines.­

future science group www.futuremedicine.com 365 Review Brennick, George, Corwin, Srivastava & Ebrahimi-Nik

Combinatorial therapies landscape plays an important role for whether or not a It is now beyond doubt that the immune system, and patient will have a positive outcome to CTLA4 block- more specifically, the T-cell compartment has the ability ade. It was observed that patients with a high muta- to control and eliminate tumor lesions. Neoepitopes sit at tional and putative neoepitope burden correlated with the forefront of cancer immunotherapy, in that they are a positive response to CTLA4 blockade [83]. the prospective means by which the immune system can Determining biomarkers that can identify which distinguish normal self from mutated self and selectively subset of patients respond to treatment has become eliminate cancerous cells [73,74]. Many clinical trials for critical for both improving treatment outcomes and vaccination with neoepitopes are still in their infancy, but understanding their actions mechanistically. To this they will help us further understand the feasibility of this end, studies examining anti-CLTA4 antibody therapy modality. Further, many immuno­therapies, traditional have demonstrated that its efficacy may be a result of cancer treatment modalities (e.g., chemo­therapy and depleting Treg from the TME and not simply taking radiation) and disease progression all potentially affect the ‘brakes’ off of effector T cells [84–86]. The lack of responses to neoepitopes either directly or indirectly. a universally effective treatment method owes in part However, even armed with the knowledge of neoepitopes to the complexity of the tumor and its microenviron- and improved techniques and tools for identifying and ment. This complexity has been demonstrated not only targeting them in vivo, no ‘silver bullet’ treatment has in the significant disparities between different tumor emerged for treating all cancers. types, but also in the vast heterogeneity within a single As discussed in the preceding sections, several patient’s lesion(s) [87]. Numerous factors, such as, differ- advances have been made in the last few years with ences in tumor cell clonality, immune cell infiltration, regard to the identification, importance and utility of angiogenic factors, mutational burden, etc. contribute to neoepitopes. However, there remain significant hurdles treatment response and u­ltimately determine treatment in leveraging this information into effective treatment outcomes. modalities. A particular challenge is with the treatment It is becoming increasingly evident that while individ- of solid tumors. While other related immunotherapies, ual immunotherapeutic regimes have exciting potential, such as, chimeric antigen receptor T cells, have shown it will likely take a combinatorial, and more importantly, considerable improvements in outcomes for many liq- patient-specific approach to achieve optimal treatment uid or blood tumors, solid tumors have been more dif- outcomes on a patient-by-patient basis [88]. To this end, ficult to target with this approach [75] given that most trials examining the combination of checkpoint inhibi- solid tumors lack the well-characterized surface markers tion therapies using antibodies targeting CTLA4 and found on blood cell subtypes. Further, factors within PD1/PDL1 have shown synergistic improvements for the TME can work in concert to create an immunosup- some patients [89]. Yet, considerable side effects of such pressive milieu that can limit the local effectiveness of a combinatorial treatments remain a challenge. The devel- strong, systemic antitumor immune response [76]. TME opment of other therapies that target additional coin- elements, such as, suppressor cell types (Treg, myeloid- hibitory or costimulatory molecules is also underway. derived suppressor cells, etc.), suppressive cytokines, Other T-cell surface molecules, such as, the coinhibi- extracellular matrix and vasculature remodeling, T-cell tory markers LAG-3 and TIM-3 and the costimulatory exclusion, and others are all means by which cancer cells receptors OX40, GITR and 4–1BB are currently under can alter their environment and co-opt the host immune evaluation individually and in combination with other system for their protection. checkpoint-based therapies for their utility as anticancer Specific therapies, such as, CB, have been developed agents [90]. Given the specific TME milieu for each indi- to target immunosuppressive components. The idea vidual tumor, precise checkpoint targeting may improve behind these treatments is that by targeting the coin- outcomes for certain subsets of patients. hibitory molecules CTLA4 and PD1 found on T cells, Even as other immunotherapies become more the inhibitory signaling or ‘brakes’ can be removed ‘patient specific’, neoepitopes endure as the most tar- from these cells thus generating a stronger antitumor geted approach to cancer treatment. The identification response [77,78]. These therapies have generated exciting of patient-specific neoantigens allows for a directed and durable responses for many different types of can- immune response to tumor-specific epitopes. There cer [37,79–82]. However, these observed partial responses are many different approaches examining how best and the more striking complete responses, in particu- to elicit a strong, specific and long-lasting neoepitope lar, remain limited to a subset of patients. Further, the immune response. One set of methodologies revolves percentage of responding patients can differ signifi- around a vaccine-based approach with CB as a com- cantly between tumor types. It has been observed from binatorial regimen. As previously described in this completed trials for CB therapy that the neoepitope review, a host of different vaccination strategies from

366 Immunotherapy (2017) 9(4) future science group Neoepitopes as cancer immunotherapy targets Review synthesized peptides to whole cell or cell lysate prod- approaches have moved from the laboratory to the ucts in a mixture with adjuvant and in combination clinical setting, outcomes are still immature and with CB can be used as cancer-targeted vaccines. Other uncertain. Neoepitope-focused strategies are likely ex vivo based methods, such as, adoptive cell transfer dependent on the context of the tumor and the TME. or chimeric antigen receptor T cell aim to selectively As more information continues to clarify our under- expand or engineer T cells with specificity to neoepi- standing of the inter-related mechanisms, such as, topes and reinfuse these cell products back into the TME-associated suppression, T-cell exclusion, T-cell patient. The overarching goal of neoepitope thera- exhaustion, immune checkpoint regulation, tumor pies is to effectively ‘educate’ the immune system to heterogeneity, etc., no single approach may be suffi- the best tumor-specific targets while eliciting a strong cient to treat the vast majority of cancers. Moving for- T-cell-based response that can hopefully overwhelm a ward, optimal therapeutic advances will likely include tumor’s defense mechanisms and elicit complete tumor multiple immune-directed treatments that are tailored rejection [11] . not only to the patient’s specific neoepitope repertoire but also to the unique suppressive landscape of the Conclusion individual TME. It is evident that all the components of a neoepitope- based therapy, from their in silico identification to opti- Future perspective mal immune system activation, are critical to r­ealizing Accurate identification of neoepitopes and their sub- their therapeutic potential. While n­eoepitope‑t­argeted sequent use in cancer therapy is still in its nascent

Executive summary From ubiquitination to recognition • Thus far, there are no reliable methods to accurately verify that in silico identified neoepitopes can actually elicit tumor regression or a tumor-specific immune response. • Programs, such as, NetMHC and TAPPred account only for peptide-binding affinity to MHC and transporter associated with antigen processing, respectively. There is urgency for a program that encompasses all aspects of antigen processing and presentation to improve the accuracy of predicting successful neoepitopes. • Whether a cognate T-cell receptor is present against a particular neoepitope is a question yet to be answered. A way to address this may be to develop algorithms that can accurately predict if a cognate T-cell receptor is available for a particular neoepitope. Are we being misguided by binding affinity? • A binding affinity threshold of less than 500 nM may be overlooking many putative neoepitopes. • Although the binding affinity of a peptide to MHC I is one of the most studied criteria in the search for neoepitopes, many other factors play an important role in the presentation of neoepitopes and specific activation of immune response. • Putative tumor antigens need to not only consider neoepitopes but also should consider proteasome-spliced and post-translationally modified peptides. How should we immunize against neoepitopes? • Immunization with neoepitopes usually needs strong immunostimulatory adjuvants to produce a robust immune response. • Adjuvants function in different ways which make a balance between antigen delivery and persistence. • Different types of adjuvants have been tested in cancer immunotherapy vaccines. • Although research on the use of dendritic cells as natural adjuvants has been done for decades, most of the studies failed to induce good tumor rejection despite having a promising cytotoxic T-lymphocyte response. Combinatorial therapies • The recent successes of immunotherapies (e.g., immune checkpoint blockade, adoptive cell transfer, chimeric antigen receptor T cells, etc.) have revitalized the field of oncoimmunology and its focus on personalized medicine. • Neoepitopes function as the unique tumor-specific antigens by which the adaptive immune system can selectively target cancer cells. As such, neoepitopes are central to all forms of immunotherapy. • While emerging immunotherapies have demonstrated significant improvements over conventional therapeutic modalities, complete responses remain suboptimal and limited to a subset of patients. • Future strategies will need to be both targeted and combinatorial in order to achieve optimal outcomes for more patients. A combination of methodologies that are targeted to a patient’s specific neoepitope repertoire and tumor microenvironment will increase our chances of overcoming the confounding factors that have made cancer so recalcitrant to treatment.

future science group www.futuremedicine.com 367 Review Brennick, George, Corwin, Srivastava & Ebrahimi-Nik

stages. Currently, the neoepitope prediction tools uti- in antigen spreading and improved immunotherapy. lized following high-throughput sequencing do not Therefore, finding the ideal, patient-specific combi- accurately identify all of the expressed neoepitopes. nation of neoepitope vaccines, chemotherapy, radia- With recent advances in MS, faster and more precise tion and immune CB will be critical to the future of identification of all expressed neoepitopes may be pos- cancer therapy. sible in the near future. This would add another layer of accuracy to exome and RNA sequencing in the Financial & competing interests disclosure search for clinically relevant, patient-specific neoepi- The authors have no relevant affiliations or financial in- topes. Further analysis of these peptides in conjunc- volvement with any organization or entity with a financial tion with in vivo animal models will help to establish interest in or financial conflict with the subject matter or the criteria for tumor rejecting neoepitopes which in m­aterials discussed in the manuscript. This includes employ- turn will assist in educating in silico algorithms to ment, consultancies, honoraria, stock ownership or options, better predict effective neoepitopes. That being said, expert t­estimony, grants or patents received or pending, or direct neoepitope-targeting therapies alone may not ­royalties. be sufficient to eradicate established tumors, hence No writing assistance was utilized in the production of this combination therapy might be an ideal approach manuscript. to treat cancer. CB that targets immunosuppressive components may elicit a stronger immune response Open access and with targeted neoepitope therapies may enhance This work is licensed under the Attribution-NonCommercial- tumor rejection as well. Chemotherapy and radia- NoDerivatives 4.0 Unported License. To view a copy of this li- tion could cause tumor cell death, which may result cense, visit http://creativecommons.org/licenses/by-nc-nd/4.0

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