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Neoantigens encoded in the cancer genome
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ScienceDirect Neoantigens encoded in the cancer genome Ton N Schumacher1 and Nir Hacohen2,3 Somatic mutations in the genome represent one of the major drivers of malignancy. However, non-synonymous mutations are also a source of mutated peptides that are presented by HLA molecules to induce protective CD4 and CD8 T cell responses. Consistent with this notion, the mutation burden of a tumor is correlated with local immunity as well as outcome of therapy and patient survival. Furthermore, neoantigen-specific T cells appear sufficient to control tumors prophylactically and therapeutically. While the role of neoantigens as a determinant of the foreignness of human cancers is now well established, major questions, including the relative importance of clonal vs subclonal neoantigens, and CD4 vs CD8 T cells, remain unanswered. We expect continued animal studies to address some of the open issues and ongoing clinical trials to establish the utility of therapeutic strategies to enhance neoantigenspecific T cell responses in human cancer. Addresses 1 Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands 2 Cancer Center and Center for Cancer Immunology, Massachusetts General Hospital, Boston, MA, USA 3 Broad Institute of Harvard and MIT, Cambridge, MA, USA Corresponding authors: Schumacher, Ton N ([email protected]) and Hacohen, Nir ([email protected])
Current Opinion in Immunology 2016, 41:98–103 This review comes from a themed issue on Special section: Cancer immunology: Genomics & biomarkers Edited by Ton N Schumacher and Nir Hacohen For a complete overview see the Issue and the Editorial Available online 9th August 2016 http://dx.doi.org/10.1016/j.coi.2016.07.005 0952-7915/# 2016 Elsevier Ltd. All rights reserved.
Introduction Antibodies against molecules such as PD-1 and CTLA-4 that block the inhibitory signals that T cells receive are revolutionizing cancer treatment, with clinical activity being observed across a range of tumor types [1]. It is evident however, that blockade of such inhibitory signals can only be of value if tumors do express antigens that are seen as foreign by T cells, and that can thereby give the ‘Signal 1’ that is essential for T cell activation. Put differently, the ‘foreignness’ of human cancers, as determined by the antigens they express, is one of the essential components of effective cancer immunotherapy [2]. As detailed further below, a large body of recent data suggests Current Opinion in Immunology 2016, 41:98–103
that the neoantigens that arise as a consequence of tumorspecific DNA alterations are an important driver of the currently used cancer immunotherapies. This realization has spurred a major push in both academia and pharma to develop technologies to monitor T cell responses against this class of antigens, and to enhance such T cell responses. In this review, we have chosen not to provide a comprehensive overview of the history of the field — a number of recent reviews have covered this area extremely well. Rather, we will focus on a handful of recent findings and open questions that we feel will in part determine the future development of the field. Before getting to this, we would like to outline our view on a few hotly debated topics in this research area. First, a number of processes such as aberrant RNA splicing or post-translational protein modifications have also been suggested to lead to the formation of epitopes that should be considered neo-antigens [3]. While some of these alterations may indeed not be present in healthy tissues (and T cell recognition of such antigens may contribute to tumor control, see next point below), currently available technologies do not allow us to accurately determine which of these antigens meet the criterion of being truly ‘‘new’’ relative to other tissues in the same individual. Thus, in the current review, we reserve the term ‘neoantigen’ to denote antigens that result from somatic mutations (not only point mutations, but all DNA alterations that lead to the production of novel protein sequence) encoded in the cancer genome or that are derived from foreign material, such as viral antigens. Second, the available data on cancer neoantigens do not exclude a role of T cell recognition of non-mutated self antigens in tumor control. As described below, there is now very substantial data arguing in favor of T cell recognition of neoantigens in the effects of clinically active immunotherapies. These data however do not address whether such neoantigen recognition is paralleled by clinically meaningful recognition of non-mutant self antigens. Rather, the full tumor specificity of neoantigens makes them attractive targets for therapeutic interventions regardless of whether tumor-associated self antigens play an additional role in the final tumor regression process.
Evidence that neoantigen load predicts intratumoral immunity, patient outcome, and response to immunotherapy If neoantigens are critical targets for protective immunity against tumors, a simple prediction is that the abundance of tumor neoantigens (the ‘neoantigen load’) would correlate with first, tumor-associated immune responses; second, overall patient survival irrespective of treatment www.sciencedirect.com
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strategy; and third, regression of tumors in response to immunotherapy. A steadily increasing number of studies have analyzed patient cohorts to test these three hypotheses. In these studies, both mutational burden and the number of neo-antigens predicted to arise from this mutational burden are generally analyzed. We note that these two factors are highly correlated, and there is presently little evidence to suggest that predicted neoantigen counts have superior value (something that could for instance be supported by comparing the predictive power of patient-matched versus randomly selected HLA alleles in neoantigen predictions). A series of studies has demonstrated a relationship between mutational burden/predicted neoantigen load and both the intratumoral immune infiltrate and patient survival. A long-term study of immunity in 619 CRC patients [4] in the absence of immunotherapies found that higher predicted neoantigen load was associated with lymphocytic infiltration of tumors and with disease-specific survival, even for microsatellite-stable tumors. Tumors with a high predicted neoantigen load were enriched in CD45RO+ T cells and harbored a high frequency of HLA mutations, suggestive of Darwinian selection by immune pressure. Consistent with these results, a high neoantigen load in endometrial cancer [5] was also associated with MSI status and elevated CD3+ and CD8+ T cell infiltrates. Similarly, predicted neoantigen load in ovarian cancer was associated with mutations in DNA repair pathways, increased expression of T cell cytotoxicity genes, increased CD3+ and CD8+ T cell infiltrates and improved overall survival. Specifically, tumors with BRCA1/2 mutations exhibited higher CD8+ T cell infiltrates, suggesting that this subclass of tumors may be more sensitive to checkpoint blockade therapy [6]. A study of renal clear cell cancer [7] showed that a combined score of neoantigen load and HLA expression was a predictor of overall survival in patients undergoing surgery and standard therapy. In contrast, a cytolytic T cell score (consisting of the GZMA and PRF1 genes) was not correlated or negatively correlated with outcome, perhaps reflecting immunosuppression. The importance of neoantigens was further assessed in a genetic analysis that showed that that the number of neoantigens predicted to bind autologous HLA Class I proteins in colorectal cancer and kidney clear cell cancer was significantly lower than expected by chance based on the synonymous mutation rate [9], providing the first genetic evidence for immune editing at the level of neoantigen loss. Interestingly, this study and a second study [8] also found that tumors with at least 150–200 non-synonymous (NS) somatic mutations are routinely associated with an elevated cytolytic T cell score [9], suggesting that with this level of DNA damage, foreignness of tumors due to the formation of neoantigens may be highly common. Based on the fact that generation of www.sciencedirect.com
neo-antigens will be a probabilistic process, lower numbers of NS mutations can still be associated with neoantigen-driven immune responses but at increasingly lower rates [10]. A series of studies in the past years have also addressed the relationship between mutational burden or predicted neoantigen load and outcome upon immunotherapeutic intervention. Early studies supported the hypothesis that the extent of DNA damage is correlated with response to therapy in melanoma patients treated with anti-CTLA4 [11], and NSCLC patients treated with anti-PD1 [12]. A more recent study also found predictive power for neoantigen load in anti-CTLA4-treated melanoma patients [13], albeit more weakly. Of note, this study did not observe an association with the class of tetrapeptide motifs in presumed neo-antigens that had in earlier work been proposed to be enriched in clinical responders to anti-CTLA4 [11]. This discrepancy may simply be explained by the lack of a proper validation cohort within the first report (see the correction to [11]). Related to this, the variability in peptide binding motifs of different HLA alleles also makes the occurrence of such motifs across HLA-disparate patients unlikely [14]. Most dramatically, comparison of the effects of PD-1 blockade in patients with mismatch repair proficient and deficient cancers has shown a strong association between clinical response and mismatch repair deficiency [15]. An analysis of advanced urothelial carcinoma patients treated with anti-PDL1 [16] found that higher predicted neoantigen load was predictive of response to therapy regardless of immune infiltrate. Finally, a study in melanoma patients treated with anti-PD1 [17] showed improved overall survival in patients with high-mutation load tumors, especially when combined with short-term response scores, which were independent of mutation load. The aggregation of these studies, some of which were re-analyzed in [18] (see below), thus demonstrates a robust positive relationship between predicted neoantigen load, immunity and overall survival. We should stay open to the possibility that the extent of DNA damage may also influence immune recognition through factors that are unrelated to neoantigen formation, such as detection of cellular stress by innate immune cells. In addition, it is unclear why neoantigen load is not in all cases correlated with T cell infiltration or with shortterm clinical responses [16,17]. One possibility is technical: either the relevant biopsy timepoints were not sampled in these studies, or the prediction of neoantigen targets is insufficiently accurate. As a second possibility, other antigens may also drive the strength of T cell infiltrates. Finally, the specific oncogenic pathways activated within a tumor can influence T cell priming and attraction capacity, thereby creating a barrier against immune control of intrinsically foreign tumors [19,20]. Current Opinion in Immunology 2016, 41:98–103
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Conceivably, such factors may be a significant determinant of short-term clinical response, with mutational burden more prominently determining the subsequent likelihood of outgrowth of antigen-loss variants (see also below). Future studies will need to identify the relative role of the different determinants of cancer–immune interaction (such as T cell priming capacity, local cytokine production, tumor-derived innate immune activation) that together with tumor foreignness determine the extent of T cell control [2].
The effect of genetic heterogeneity In the above studies, the relationship between genomic changes and both immunological properties and clinical outcome was analyzed without taking into account the fact that significant genetic heterogeneity is present within individual human tumors. Intratumoral heterogeneity (ITH) can be expected to significantly influence the outcome of immunotherapies, and recent work from the Swanton group is providing first insights into this [18]. In this work, survival of lung adenocarcinomas was shown to be better predicted by mutational burden when ITH was taken into account. Likewise, reanalysis of some of the earlier studies that examined the relationship between response to checkpoint blocking antibodies and mutational burden showed an increased predictive value when ITH was also assessed. Finally, T cell responses against (a small number of) clonal neoantigens were observed in this study, whereas no responses against non-clonal neoantigens were identified. If a predominant T cell reactivity against clonal neoantigens is confirmed in larger studies, it will be important to understand the mechanistic basis for this bias. Taking into account that protein production levels vary over orders of magnitude, presence of an antigen in either a subset of tumor cells or in all tumor cells seems unlikely to influence antigen load in a decisive way. Conceivably, T cell priming may be most efficient at an early phase during the oncogenic process, at which point clonal mutations will be more prevalent. In addition, an early T cell response against a clonal neoantigen may potentially suppress T cell responses against subsequently developing subclonal mutations through immunodominance. Importantly, even if T cell responses initially are primarily formed against clonal neoantigens, such mutations may become subclonal or lost subsequently. Prior work in mouse models has provided evidence that T cellrecognized neoantigens can be lost from a tumor cell population when exposed to T cell pressure [21]. Furthermore, recent work analyzing the fate of T cell recognized neoantigens in two melanoma patients has also provided evidence for significant dynamics within the human neoantigen landscape. Specifically, of the 6 T cell-recognized neoantigens that were followed longitudinally in these patients, 4 changed over time, either through altered RNA expression or through loss of the Current Opinion in Immunology 2016, 41:98–103
mutant allele [22]. The dynamics of the CD8+ T cellrecognized neoantigen landscape as seen in these analyses indicates that therapeutic targeting of multiple neoantigens will likely be essential to avoid escape from CD8+ T cell pressure. In summary, these recent data suggest that genetic heterogeneity will be of substantial importance in determining outcome upon immunotherapy. More data will be required to understand whether intratumoral heterogeneity will primarily influence patient outcome through its effect on T cell-recognized neoantigens [18], [22], or mostly will lead to therapy resistance by other mechanisms. By analogy with acquired resistance upon targeted therapies, we speculate that the escape pathways that will be identified in the coming years will be diverse.
The role of CD4s and CD8s Analysis of naturally occurring neoantigen specific T cell responses in patients with melanoma, and GI tract cancers has provided evidence for both CD8+ and CD4+ T cell recognition of neoantigens [23–25]. Within these studies, the number of T cell responses against both class I and class II restricted mutant epitopes appears roughly similar. A surprising observation has therefore been that neoantigen vaccine-induced T cell responses in a mouse model were heavily biased toward class II restricted epitopes, also when specifically aiming to induce CD8+ T cell responses [26]. This pronounced CD4+ T cell reactivity may not necessarily be detrimental, as the importance of CD4+ T cells in control of even HLA class II-negative tumors has been established long ago [27]. In addition, the clinical activity of neoantigen-specific CD4+ T cells was recently demonstrated by Rosenberg and colleagues [28]. Nevertheless, it will be important to understand the mechanistic explanation for this apparent discrepancy. As a first possible explanation, natural neoantigen-specific immune responses may only cover a small part of the HLA class II-presented neoantigen repertoire, and by vaccination this limitation is overcome. As a second, and less favorable, explanation, the uptake and processing of antigen by APCs within the tumor microenvironment may be fundamentally different from uptake and processing of vaccine antigens by APCs at a vaccination site, significantly restricting or altering the repertoire of class II-associated peptides, and many vaccine-elicited Class II-restricted T cell responses may thereby be functionally irrelevant. Clearly, only in the former model, the induction of broad CD4+ T cell responses by neoantigen vaccines should be seen as an unexpected asset. A second major surprise in the analysis of naturally occurring neoantigen specific T cell responses in patients has been that only a tiny fraction of predicted neoantigens leads to detectable T cell reactivity. Only part of this low ‘hit rate’ appears to be explained by suboptimal precision www.sciencedirect.com
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of the epitope prediction pipelines, suggesting that there may be tumor-presented neoantigens that do not lead to detectable natural immune responses. To address this question experimentally, Immune responses against series of predicted neoantigens were recently induced in T cell populations from healthy individuals that were matched for the relevant HLA allele. These experiments demonstrated the induction of CD8+ T cell reactivity against many additional neoantigens, including neoantigens that were endogenously presented by tumor cells [29], thereby providing evidence for the existence of a neglected neoantigen repertoire on human tumors that may be exploited therapeutically. It will be important to understand why some MHC-associated neoantigens present on tumor cells do not induce detectable T cell reactivity in patients. As a first explanation, in many human cancers T cell priming may be inefficient, for instance because of lack of sufficient inflammatory signals. In addition, immunodominance may prevent the broadening of T cell reactivity beyond a handful of epitopes. In both these scenarios, efforts to increase the breadth of neoantigen specific T cell responses by vaccination would appear an attractive strategy to circumvent the limitations of the endogenous T cell response. As alternative explanations, lack of detectable T cell reactivity against such neglected neoantigens could also reflect holes within the TCR repertoire of that patient, or could reflect prior tolerization of the relevant neoantigen reactive T cells. Should either of these models prove correct, only the supply of TCRs obtained from an exogenous source could be utilized to achieve reactivity against this class of neoantigens.
Therapeutic approaches to enhance neoantigen-specific T cell responses What kind of neoantigen-based therapies can be envisioned? In the majority of proposed pipelines, a set of target antigens is identified by sequencing tumor and normal tissue, and predicting which mutated epitopes, including unique and shared neoantigens, would be processed and bind the HLA alleles of the patient. Alternatively, as technology advances, mass spectrometry may be used to directly identify HLA-presented neoantigens in individual tumors (see review by Bassani-Sternberg in this issue). The resulting set of predicted or identified tumor-specific neoepitopes can then be used to create a personalized therapy using different strategies. In the first and simplest strategy, a personalized vaccine that combines the selected neoantigens with immunological adjuvant is manufactured and delivered to patients to induce CD4+ and CD8+ T cell responses. These antigens may be delivered in DNA, RNA or peptide form using either synthetic or viral formulations, or may be loaded on antigen-presenting cells. Several proof of principle studies in mice have demonstrated prophylactic and therapeutic protection against tumor growth of such vaccines, and synergy with T cell checkpoint blockade [30–33]. www.sciencedirect.com
Recently, a human trial that utilized DCs loaded with mutated peptides demonstrated induction of neoantigenspecific T cells in 3 patients, with selective recognition of mutated over native peptides and a diverse T cell receptor repertoire per antigen [34]. Of note, recognition of autologous tumor cells by such vaccine-induced T cell responses remains to be demonstrated, and recent work underscores the view that many predicted neoantigens that are immunogenic may not be relevant for tumor control [35]. We expect future trials to further optimize this class of personalized vaccines through selection of better adjuvants, antigen delivery systems, antigenpresenting cells and combination with checkpoint blockade and other therapies (e.g. [36]). In the second approach, T cells are removed from a patient, stimulated with neoantigens ex vivo and infused back into the patient. A clinical trial using adoptive T cell therapy recently demonstrated effective control of a tumor in a patient with metastatic cancer by infusion of neoantigen-specific CD4 T cells [28]. Furthermore, analysis of intratumoral T cell populations from additional patients with either melanoma or GI tract cancers has shown the presence of both CD4+ and CD8+ T cells against neoantigens in most patients [37,25,38,39]. In a third approach, neoantigenspecific TCRs may be identified from either patients [37,25] or healthy individuals [29], cloned and expressed in T cells, and these T cells could then be expanded and transferred into patients. Several challenges need to be addressed to make neoantigen-targeting therapies more practical. The first is the development of rapid and cost-effective GMP-compliant manufacturing processes for these personalized products. A classical vaccine formulation ‘antigen + adjuvant’ is likely to be the most economical, but its ability to mount sufficiently strong T cell responses remains to be established, and conceivably combination with checkpoint blockade therapy will prove critical. Delivery of ex vivo expanded or genetically engineered neoantigen specific T cells can likely be used to create very profound T cell responses, however, logistic hurdles are even more significant, and generation of a sufficiently diverse T cell response may be difficult. The second challenge is to predict the most immunogenic neoantigens per patient. While this problem has been addressed by numerous groups for several decades [40], more comprehensive analyses of endogenously presented peptides (see review by Bassani-Sternberg in this issue) and their immunogenicity [41,42,43,29] will without doubt lead to more reliable predictions.
Conclusion A role for tumor neoantigens in anti-tumor immunity is supported by at least four types of evidence: a sometimes high frequency of neoantigen-specific T cells in patients undergoing checkpoint blockade therapy and TIL therapy; a correlation between mutation load, T cell infiltration Current Opinion in Immunology 2016, 41:98–103
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and survival in several tumor types; the ability of neoantigen vaccines to induce tumor regression in mouse models; and the tumor control achieved upon infusion of neoantigen-specific T cells in a case report. Many questions remain open, including the importance of clonal vs. subclonal mutations; dominant vs. subdominant responses; highly vs. lowly expressed neoantigens and the potential accompanying role of tumor-associated self antigens. At present, many clinical trials are underway (see [44] for description of ongoing trials) and planned, and their success will in large part depend on our ability to address these questions.
Acknowledgements This work was supported by the Dutch Cancer Society Queen Wilhelmina Award NKI 2013-6122 and Dutch Cancer Society grant NKI 2012-5463 (to TNS), and the Blavatnik Family Foundation (to NH). We would like to thank E. Fritsch (Neon Therapeutics) for valuable discussions. TNS and NH are founders and stockholders of Neon Therapeutics. TNS is employee and stockholder of Kite Pharma.
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ScienceDirect Neoantigens encoded in the cancer genome Ton N Schumacher1 and Nir Hacohen2,3 Somatic mutations in the genome represent one of the major drivers of malignancy. However, non-synonymous mutations are also a source of mutated peptides that are presented by HLA molecules to induce protective CD4 and CD8 T cell responses. Consistent with this notion, the mutation burden of a tumor is correlated with local immunity as well as outcome of therapy and patient survival. Furthermore, neoantigen-specific T cells appear sufficient to control tumors prophylactically and therapeutically. While the role of neoantigens as a determinant of the foreignness of human cancers is now well established, major questions, including the relative importance of clonal vs subclonal neoantigens, and CD4 vs CD8 T cells, remain unanswered. We expect continued animal studies to address some of the open issues and ongoing clinical trials to establish the utility of therapeutic strategies to enhance neoantigenspecific T cell responses in human cancer. Addresses 1 Division of Immunology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX, Amsterdam, The Netherlands 2 Cancer Center and Center for Cancer Immunology, Massachusetts General Hospital, Boston, MA, USA 3 Broad Institute of Harvard and MIT, Cambridge, MA, USA Corresponding authors: Schumacher, Ton N ([email protected]) and Hacohen, Nir ([email protected])
Current Opinion in Immunology 2016, 41:98–103 This review comes from a themed issue on Special section: Cancer immunology: Genomics & biomarkers Edited by Ton N Schumacher and Nir Hacohen For a complete overview see the Issue and the Editorial Available online 9th August 2016 http://dx.doi.org/10.1016/j.coi.2016.07.005 0952-7915/# 2016 Elsevier Ltd. All rights reserved.
Introduction Antibodies against molecules such as PD-1 and CTLA-4 that block the inhibitory signals that T cells receive are revolutionizing cancer treatment, with clinical activity being observed across a range of tumor types [1]. It is evident however, that blockade of such inhibitory signals can only be of value if tumors do express antigens that are seen as foreign by T cells, and that can thereby give the ‘Signal 1’ that is essential for T cell activation. Put differently, the ‘foreignness’ of human cancers, as determined by the antigens they express, is one of the essential components of effective cancer immunotherapy [2]. As detailed further below, a large body of recent data suggests Current Opinion in Immunology 2016, 41:98–103
that the neoantigens that arise as a consequence of tumorspecific DNA alterations are an important driver of the currently used cancer immunotherapies. This realization has spurred a major push in both academia and pharma to develop technologies to monitor T cell responses against this class of antigens, and to enhance such T cell responses. In this review, we have chosen not to provide a comprehensive overview of the history of the field — a number of recent reviews have covered this area extremely well. Rather, we will focus on a handful of recent findings and open questions that we feel will in part determine the future development of the field. Before getting to this, we would like to outline our view on a few hotly debated topics in this research area. First, a number of processes such as aberrant RNA splicing or post-translational protein modifications have also been suggested to lead to the formation of epitopes that should be considered neo-antigens [3]. While some of these alterations may indeed not be present in healthy tissues (and T cell recognition of such antigens may contribute to tumor control, see next point below), currently available technologies do not allow us to accurately determine which of these antigens meet the criterion of being truly ‘‘new’’ relative to other tissues in the same individual. Thus, in the current review, we reserve the term ‘neoantigen’ to denote antigens that result from somatic mutations (not only point mutations, but all DNA alterations that lead to the production of novel protein sequence) encoded in the cancer genome or that are derived from foreign material, such as viral antigens. Second, the available data on cancer neoantigens do not exclude a role of T cell recognition of non-mutated self antigens in tumor control. As described below, there is now very substantial data arguing in favor of T cell recognition of neoantigens in the effects of clinically active immunotherapies. These data however do not address whether such neoantigen recognition is paralleled by clinically meaningful recognition of non-mutant self antigens. Rather, the full tumor specificity of neoantigens makes them attractive targets for therapeutic interventions regardless of whether tumor-associated self antigens play an additional role in the final tumor regression process.
Evidence that neoantigen load predicts intratumoral immunity, patient outcome, and response to immunotherapy If neoantigens are critical targets for protective immunity against tumors, a simple prediction is that the abundance of tumor neoantigens (the ‘neoantigen load’) would correlate with first, tumor-associated immune responses; second, overall patient survival irrespective of treatment www.sciencedirect.com
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strategy; and third, regression of tumors in response to immunotherapy. A steadily increasing number of studies have analyzed patient cohorts to test these three hypotheses. In these studies, both mutational burden and the number of neo-antigens predicted to arise from this mutational burden are generally analyzed. We note that these two factors are highly correlated, and there is presently little evidence to suggest that predicted neoantigen counts have superior value (something that could for instance be supported by comparing the predictive power of patient-matched versus randomly selected HLA alleles in neoantigen predictions). A series of studies has demonstrated a relationship between mutational burden/predicted neoantigen load and both the intratumoral immune infiltrate and patient survival. A long-term study of immunity in 619 CRC patients [4] in the absence of immunotherapies found that higher predicted neoantigen load was associated with lymphocytic infiltration of tumors and with disease-specific survival, even for microsatellite-stable tumors. Tumors with a high predicted neoantigen load were enriched in CD45RO+ T cells and harbored a high frequency of HLA mutations, suggestive of Darwinian selection by immune pressure. Consistent with these results, a high neoantigen load in endometrial cancer [5] was also associated with MSI status and elevated CD3+ and CD8+ T cell infiltrates. Similarly, predicted neoantigen load in ovarian cancer was associated with mutations in DNA repair pathways, increased expression of T cell cytotoxicity genes, increased CD3+ and CD8+ T cell infiltrates and improved overall survival. Specifically, tumors with BRCA1/2 mutations exhibited higher CD8+ T cell infiltrates, suggesting that this subclass of tumors may be more sensitive to checkpoint blockade therapy [6]. A study of renal clear cell cancer [7] showed that a combined score of neoantigen load and HLA expression was a predictor of overall survival in patients undergoing surgery and standard therapy. In contrast, a cytolytic T cell score (consisting of the GZMA and PRF1 genes) was not correlated or negatively correlated with outcome, perhaps reflecting immunosuppression. The importance of neoantigens was further assessed in a genetic analysis that showed that that the number of neoantigens predicted to bind autologous HLA Class I proteins in colorectal cancer and kidney clear cell cancer was significantly lower than expected by chance based on the synonymous mutation rate [9], providing the first genetic evidence for immune editing at the level of neoantigen loss. Interestingly, this study and a second study [8] also found that tumors with at least 150–200 non-synonymous (NS) somatic mutations are routinely associated with an elevated cytolytic T cell score [9], suggesting that with this level of DNA damage, foreignness of tumors due to the formation of neoantigens may be highly common. Based on the fact that generation of www.sciencedirect.com
neo-antigens will be a probabilistic process, lower numbers of NS mutations can still be associated with neoantigen-driven immune responses but at increasingly lower rates [10]. A series of studies in the past years have also addressed the relationship between mutational burden or predicted neoantigen load and outcome upon immunotherapeutic intervention. Early studies supported the hypothesis that the extent of DNA damage is correlated with response to therapy in melanoma patients treated with anti-CTLA4 [11], and NSCLC patients treated with anti-PD1 [12]. A more recent study also found predictive power for neoantigen load in anti-CTLA4-treated melanoma patients [13], albeit more weakly. Of note, this study did not observe an association with the class of tetrapeptide motifs in presumed neo-antigens that had in earlier work been proposed to be enriched in clinical responders to anti-CTLA4 [11]. This discrepancy may simply be explained by the lack of a proper validation cohort within the first report (see the correction to [11]). Related to this, the variability in peptide binding motifs of different HLA alleles also makes the occurrence of such motifs across HLA-disparate patients unlikely [14]. Most dramatically, comparison of the effects of PD-1 blockade in patients with mismatch repair proficient and deficient cancers has shown a strong association between clinical response and mismatch repair deficiency [15]. An analysis of advanced urothelial carcinoma patients treated with anti-PDL1 [16] found that higher predicted neoantigen load was predictive of response to therapy regardless of immune infiltrate. Finally, a study in melanoma patients treated with anti-PD1 [17] showed improved overall survival in patients with high-mutation load tumors, especially when combined with short-term response scores, which were independent of mutation load. The aggregation of these studies, some of which were re-analyzed in [18] (see below), thus demonstrates a robust positive relationship between predicted neoantigen load, immunity and overall survival. We should stay open to the possibility that the extent of DNA damage may also influence immune recognition through factors that are unrelated to neoantigen formation, such as detection of cellular stress by innate immune cells. In addition, it is unclear why neoantigen load is not in all cases correlated with T cell infiltration or with shortterm clinical responses [16,17]. One possibility is technical: either the relevant biopsy timepoints were not sampled in these studies, or the prediction of neoantigen targets is insufficiently accurate. As a second possibility, other antigens may also drive the strength of T cell infiltrates. Finally, the specific oncogenic pathways activated within a tumor can influence T cell priming and attraction capacity, thereby creating a barrier against immune control of intrinsically foreign tumors [19,20]. Current Opinion in Immunology 2016, 41:98–103
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Conceivably, such factors may be a significant determinant of short-term clinical response, with mutational burden more prominently determining the subsequent likelihood of outgrowth of antigen-loss variants (see also below). Future studies will need to identify the relative role of the different determinants of cancer–immune interaction (such as T cell priming capacity, local cytokine production, tumor-derived innate immune activation) that together with tumor foreignness determine the extent of T cell control [2].
The effect of genetic heterogeneity In the above studies, the relationship between genomic changes and both immunological properties and clinical outcome was analyzed without taking into account the fact that significant genetic heterogeneity is present within individual human tumors. Intratumoral heterogeneity (ITH) can be expected to significantly influence the outcome of immunotherapies, and recent work from the Swanton group is providing first insights into this [18]. In this work, survival of lung adenocarcinomas was shown to be better predicted by mutational burden when ITH was taken into account. Likewise, reanalysis of some of the earlier studies that examined the relationship between response to checkpoint blocking antibodies and mutational burden showed an increased predictive value when ITH was also assessed. Finally, T cell responses against (a small number of) clonal neoantigens were observed in this study, whereas no responses against non-clonal neoantigens were identified. If a predominant T cell reactivity against clonal neoantigens is confirmed in larger studies, it will be important to understand the mechanistic basis for this bias. Taking into account that protein production levels vary over orders of magnitude, presence of an antigen in either a subset of tumor cells or in all tumor cells seems unlikely to influence antigen load in a decisive way. Conceivably, T cell priming may be most efficient at an early phase during the oncogenic process, at which point clonal mutations will be more prevalent. In addition, an early T cell response against a clonal neoantigen may potentially suppress T cell responses against subsequently developing subclonal mutations through immunodominance. Importantly, even if T cell responses initially are primarily formed against clonal neoantigens, such mutations may become subclonal or lost subsequently. Prior work in mouse models has provided evidence that T cellrecognized neoantigens can be lost from a tumor cell population when exposed to T cell pressure [21]. Furthermore, recent work analyzing the fate of T cell recognized neoantigens in two melanoma patients has also provided evidence for significant dynamics within the human neoantigen landscape. Specifically, of the 6 T cell-recognized neoantigens that were followed longitudinally in these patients, 4 changed over time, either through altered RNA expression or through loss of the Current Opinion in Immunology 2016, 41:98–103
mutant allele [22]. The dynamics of the CD8+ T cellrecognized neoantigen landscape as seen in these analyses indicates that therapeutic targeting of multiple neoantigens will likely be essential to avoid escape from CD8+ T cell pressure. In summary, these recent data suggest that genetic heterogeneity will be of substantial importance in determining outcome upon immunotherapy. More data will be required to understand whether intratumoral heterogeneity will primarily influence patient outcome through its effect on T cell-recognized neoantigens [18], [22], or mostly will lead to therapy resistance by other mechanisms. By analogy with acquired resistance upon targeted therapies, we speculate that the escape pathways that will be identified in the coming years will be diverse.
The role of CD4s and CD8s Analysis of naturally occurring neoantigen specific T cell responses in patients with melanoma, and GI tract cancers has provided evidence for both CD8+ and CD4+ T cell recognition of neoantigens [23–25]. Within these studies, the number of T cell responses against both class I and class II restricted mutant epitopes appears roughly similar. A surprising observation has therefore been that neoantigen vaccine-induced T cell responses in a mouse model were heavily biased toward class II restricted epitopes, also when specifically aiming to induce CD8+ T cell responses [26]. This pronounced CD4+ T cell reactivity may not necessarily be detrimental, as the importance of CD4+ T cells in control of even HLA class II-negative tumors has been established long ago [27]. In addition, the clinical activity of neoantigen-specific CD4+ T cells was recently demonstrated by Rosenberg and colleagues [28]. Nevertheless, it will be important to understand the mechanistic explanation for this apparent discrepancy. As a first possible explanation, natural neoantigen-specific immune responses may only cover a small part of the HLA class II-presented neoantigen repertoire, and by vaccination this limitation is overcome. As a second, and less favorable, explanation, the uptake and processing of antigen by APCs within the tumor microenvironment may be fundamentally different from uptake and processing of vaccine antigens by APCs at a vaccination site, significantly restricting or altering the repertoire of class II-associated peptides, and many vaccine-elicited Class II-restricted T cell responses may thereby be functionally irrelevant. Clearly, only in the former model, the induction of broad CD4+ T cell responses by neoantigen vaccines should be seen as an unexpected asset. A second major surprise in the analysis of naturally occurring neoantigen specific T cell responses in patients has been that only a tiny fraction of predicted neoantigens leads to detectable T cell reactivity. Only part of this low ‘hit rate’ appears to be explained by suboptimal precision www.sciencedirect.com
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of the epitope prediction pipelines, suggesting that there may be tumor-presented neoantigens that do not lead to detectable natural immune responses. To address this question experimentally, Immune responses against series of predicted neoantigens were recently induced in T cell populations from healthy individuals that were matched for the relevant HLA allele. These experiments demonstrated the induction of CD8+ T cell reactivity against many additional neoantigens, including neoantigens that were endogenously presented by tumor cells [29], thereby providing evidence for the existence of a neglected neoantigen repertoire on human tumors that may be exploited therapeutically. It will be important to understand why some MHC-associated neoantigens present on tumor cells do not induce detectable T cell reactivity in patients. As a first explanation, in many human cancers T cell priming may be inefficient, for instance because of lack of sufficient inflammatory signals. In addition, immunodominance may prevent the broadening of T cell reactivity beyond a handful of epitopes. In both these scenarios, efforts to increase the breadth of neoantigen specific T cell responses by vaccination would appear an attractive strategy to circumvent the limitations of the endogenous T cell response. As alternative explanations, lack of detectable T cell reactivity against such neglected neoantigens could also reflect holes within the TCR repertoire of that patient, or could reflect prior tolerization of the relevant neoantigen reactive T cells. Should either of these models prove correct, only the supply of TCRs obtained from an exogenous source could be utilized to achieve reactivity against this class of neoantigens.
Therapeutic approaches to enhance neoantigen-specific T cell responses What kind of neoantigen-based therapies can be envisioned? In the majority of proposed pipelines, a set of target antigens is identified by sequencing tumor and normal tissue, and predicting which mutated epitopes, including unique and shared neoantigens, would be processed and bind the HLA alleles of the patient. Alternatively, as technology advances, mass spectrometry may be used to directly identify HLA-presented neoantigens in individual tumors (see review by Bassani-Sternberg in this issue). The resulting set of predicted or identified tumor-specific neoepitopes can then be used to create a personalized therapy using different strategies. In the first and simplest strategy, a personalized vaccine that combines the selected neoantigens with immunological adjuvant is manufactured and delivered to patients to induce CD4+ and CD8+ T cell responses. These antigens may be delivered in DNA, RNA or peptide form using either synthetic or viral formulations, or may be loaded on antigen-presenting cells. Several proof of principle studies in mice have demonstrated prophylactic and therapeutic protection against tumor growth of such vaccines, and synergy with T cell checkpoint blockade [30–33]. www.sciencedirect.com
Recently, a human trial that utilized DCs loaded with mutated peptides demonstrated induction of neoantigenspecific T cells in 3 patients, with selective recognition of mutated over native peptides and a diverse T cell receptor repertoire per antigen [34]. Of note, recognition of autologous tumor cells by such vaccine-induced T cell responses remains to be demonstrated, and recent work underscores the view that many predicted neoantigens that are immunogenic may not be relevant for tumor control [35]. We expect future trials to further optimize this class of personalized vaccines through selection of better adjuvants, antigen delivery systems, antigenpresenting cells and combination with checkpoint blockade and other therapies (e.g. [36]). In the second approach, T cells are removed from a patient, stimulated with neoantigens ex vivo and infused back into the patient. A clinical trial using adoptive T cell therapy recently demonstrated effective control of a tumor in a patient with metastatic cancer by infusion of neoantigen-specific CD4 T cells [28]. Furthermore, analysis of intratumoral T cell populations from additional patients with either melanoma or GI tract cancers has shown the presence of both CD4+ and CD8+ T cells against neoantigens in most patients [37,25,38,39]. In a third approach, neoantigenspecific TCRs may be identified from either patients [37,25] or healthy individuals [29], cloned and expressed in T cells, and these T cells could then be expanded and transferred into patients. Several challenges need to be addressed to make neoantigen-targeting therapies more practical. The first is the development of rapid and cost-effective GMP-compliant manufacturing processes for these personalized products. A classical vaccine formulation ‘antigen + adjuvant’ is likely to be the most economical, but its ability to mount sufficiently strong T cell responses remains to be established, and conceivably combination with checkpoint blockade therapy will prove critical. Delivery of ex vivo expanded or genetically engineered neoantigen specific T cells can likely be used to create very profound T cell responses, however, logistic hurdles are even more significant, and generation of a sufficiently diverse T cell response may be difficult. The second challenge is to predict the most immunogenic neoantigens per patient. While this problem has been addressed by numerous groups for several decades [40], more comprehensive analyses of endogenously presented peptides (see review by Bassani-Sternberg in this issue) and their immunogenicity [41,42,43,29] will without doubt lead to more reliable predictions.
Conclusion A role for tumor neoantigens in anti-tumor immunity is supported by at least four types of evidence: a sometimes high frequency of neoantigen-specific T cells in patients undergoing checkpoint blockade therapy and TIL therapy; a correlation between mutation load, T cell infiltration Current Opinion in Immunology 2016, 41:98–103
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and survival in several tumor types; the ability of neoantigen vaccines to induce tumor regression in mouse models; and the tumor control achieved upon infusion of neoantigen-specific T cells in a case report. Many questions remain open, including the importance of clonal vs. subclonal mutations; dominant vs. subdominant responses; highly vs. lowly expressed neoantigens and the potential accompanying role of tumor-associated self antigens. At present, many clinical trials are underway (see [44] for description of ongoing trials) and planned, and their success will in large part depend on our ability to address these questions.
Acknowledgements This work was supported by the Dutch Cancer Society Queen Wilhelmina Award NKI 2013-6122 and Dutch Cancer Society grant NKI 2012-5463 (to TNS), and the Blavatnik Family Foundation (to NH). We would like to thank E. Fritsch (Neon Therapeutics) for valuable discussions. TNS and NH are founders and stockholders of Neon Therapeutics. TNS is employee and stockholder of Kite Pharma.
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