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Keywords:

  • T cell;
  • TCR ;
  • MHC-multimer;
  • cancer exome;
  • immunotherapy;
  • gene therapy

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Understanding intratumoral TCR repertoires
  5. Utilizing intratumoral TCR repertoires
  6. Concluding remarks
  7. Acknowledgements
  8. References

The infiltration of human tumors by T cells is a common phenomenon, and over the past decades, it has become increasingly clear that the nature of such intratumoral T-cell populations can predict disease course. Furthermore, intratumoral T cells have been utilized therapeutically in clinical studies of adoptive T-cell therapy. In this review, we describe how novel methods that are either based on T-cell receptor (TCR) sequencing or on cancer exome analysis allow the analysis of the tumor reactivity and antigen-specificity of the intratumoral TCR repertoire with unprecedented detail. Furthermore, we discuss studies that have started to utilize these techniques to probe the link between cancer exomes and the intratumoral TCR pool. Based on the observation that both the cancer epitope repertoire and intratumoral TCR repertoire appear highly individual, we outline strategies, such as ‘autologous TCR gene therapy’, that exploit the tumor-resident TCR repertoire for the development of personalized immunotherapy.

This article is part of a series of reviews covering Adoptive Immunotherapy for Cancer appearing in Volume 257 of Immunological Reviews.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Understanding intratumoral TCR repertoires
  5. Utilizing intratumoral TCR repertoires
  6. Concluding remarks
  7. Acknowledgements
  8. References

Cancer is a genetic disease. The accumulation of genetic mutations within tumor cells leads to changes in their proteome, and it is this change which controls the process of cellular transformation. By the same token, through this process of genetic mutation, a ‘cancer anti-genome’ [1] is generated that may be recognized by T cells. The spectrum of epitopes that forms the cancer anti-genome includes peptides from genes that are aberrantly expressed within tumor cells, but also the ‘neo-antigens’ that arise as a direct consequence of somatic mutations within tumor cells. While human tumor types display clear mutational signatures that reflect the underlying mutational process [2], the specific mutations found in any pair of tumors of a given histology are in most cases largely distinct [3]. As such, even tumors of the same histological origin do exhibit highly diverse cancer anti-genomes.

The diversity of human tumors that is seen at the genetic level is paralleled by a strong diversity in the T-cell infiltrates within the tumor lesions. This variability not only concerns the magnitude of the T-cell infiltrate, but also the ratio of different T-cell subsets and their location within the tumor (summarized in [4]). An important unsolved issue here is which factors determine the nature of the T-cell infiltrate in human tumors. Tumor-intrinsic factors that are likely to play a role include the size of the cancer anti-genome and the pro- or anti-inflammatory consequences of the specific mutations within an individual tumor. Tumor-extrinsic factors that could conceivably play a role include patient genotype, possibly including the HLA haplotype, and perhaps even microbiome.

The magnitude and composition of the intratumoral T-cell infiltrate shows a clear correlation with clinical prognosis in a number of human tumor types (summarized in [4]). In particular a fierce infiltration of CD8+ T cells has been shown to correlate with an improved clinical outcome in a number of tumor types. For other T-cell subsets (e.g. Tregs), the data obtained thus far are more ambiguous, possibly in part because of methodological differences.

While the observed correlation between the extent of CD8+ T-cell infiltration (or CD8+ T cell/Treg ratio) and clinical prognosis has often been interpreted as evidence for a role of these CD8+ T cells in tumor control, the evidence is obviously indirect. Indeed, with melanoma as a notable exception [5-7], the evidence that the intratumoral T-cell repertoire is commonly tumor reactive in human cancers is thus far lacking.

On a related note, the analyses of the intratumoral T-cell infiltrates that have thus far been carried out have largely disregarded the T-cell receptor (TCR) repertoire that is expressed by these T cells. The reactivity of the TCRs expressed by intratumoral T cells will critically determine their capacity to interact with other cells in the tumor microenvironment, and because of this, it seems plausible that the TCR repertoire found in intratumoral T-cell subsets may form a prognostic signature or predictive signature in cancer (immuno-) therapies. As such, analysis of the characteristics of the intratumoral TCR repertoire with respect to its (i) diversity; (ii) degree of tumor-reactivity; and (iii) antigen specificity will likely be informative. Moreover, an understanding of how these factors are modulated by immunotherapeutic intervention may provide important insights into the mechanism of action of such therapies.

In this review, we discuss the potential value of new technologies and tools for the analysis of intratumoral TCR repertoires. Furthermore, we provide a perspective on how these recent technological advancements may be exploited to utilize intratumoral TCR repertoires for personalized immunotherapies.

Understanding intratumoral TCR repertoires

  1. Top of page
  2. Summary
  3. Introduction
  4. Understanding intratumoral TCR repertoires
  5. Utilizing intratumoral TCR repertoires
  6. Concluding remarks
  7. Acknowledgements
  8. References

Diversity of the intratumoral TCR repertoire

In contrast to TCRα alleles, which in approximately 20% of all T cells are both functionally recombined [8], TCRβ alleles are subjected to strict allelic exclusion. Because of this, TCR β-chain usage can be used as a straightforward means to analyze the diversity of TCR repertoires. Traditionally, such analyses have been performed by flow cytometry using TCR Vβ-segment specific antibodies [9], or by CDR3 size spectratyping [10]. However, the resolution of these approaches is only modest, as TCR clonotypes using the same TCR Vβ-segment or with the same CDR3 length cannot be distinguished. With the development of next generation sequencing (NGS), techniques have been developed that can reveal the nucleotide sequences of all TCRβ CDR3 sequences present within a given T-cell population. Because of the immense read depth that can be achieved, NGS of TCR repertoires allows the quantitative detection of even low-frequency TCR sequences [note that proper filtering to exclude sequence errors [11] is required]. Furthermore, because of the high diversity of the TCRβ CDR3 repertoire, the sequences obtained will in most cases represent individual TCR clonotypes (a noted exception are TCRs sharing a common/public TCR β-chain but distinct TCR α-chains). NGS sequencing of TCRβ CDR3 has first been used in a study that analyzed the TCR distribution among various T-cell compartments in a healthy individual [12]. More recently, the technology has also been implemented to analyze TCR repertoires in disease settings, such as TCR reconstitution upon allogeneic hematopoietic stem cell transplantation [13].

The TCR repertoire diversity of different T-cell subsets (e.g. CD8+ effector T cells) found within different human tumors has not been studied systematically thus far. As exceptions, Sherwood et al. [14] assessed the TCR diversity of tumor-infiltrating lymphocytes (TILs) derived from colorectal cancer and compared it to the TCR repertoire of mucosa-infiltrating T cells. This study revealed that the TCR repertoire diversity in colorectal cancer TILs is more restricted compared with TCR repertoires found among mucosal T cells and variable between patients [14]. In another study, the same group analyzed the TCR repertoire of TILs in ovarian cancer, showing them to be largely distinct from circulating T cells [15]. It will be interesting to assess whether the steady-state diversity of intratumoral TCR repertoires correlates with clinical prognosis, and, more importantly, whether (changes in) TCR repertoire diversity among intratumoral T cells may be a predictive marker for a clinical response following immunotherapeutic interventions, such as TIL therapy or T-cell checkpoint blockade. Given the rapid progress in the development of high-throughput sequencing technologies, the use of TCRβ CDR3-sequencing for patient selection would seem a realistic option with regards to time and financial requirements.

Tumor reactivity of the intratumoral TCR repertoire

The analysis of TCR repertoire diversity by bulk TCR gene sequencing reveals the total number of TCRs present in a T-cell population and the extent of clonal dominance within such populations. As outlined above, such data may prove valuable in the context of biomarker identification. Furthermore, because the TCRβ sequences obtained function as genetic barcodes, such information may also be used to describe kinship between different intratumoral T-cell subsets [16]. As a downside, this method does not identify the TCRαβ pairs of individual T cells, implying that the information cannot be utilized to analyze or reconstruct the tumor reactivity or antigen specificity of the intratumoral TCR pool.

Understanding which fraction of intratumoral T cells is reactive to tumor cells and which determinants these cells recognize is of obvious interest. Thus far, the tumor-reactivity of the intratumoral T-cell pool has primarily been assessed in two ways. First, the ability of bulk tumor-resident T cells to recognize HLA-matched allogeneic or (preferably) autologous tumor has been studied in functional assays from the 1980s until today, primarily for melanoma [17-19]. These assays give a straightforward overview of the functional capacity of the T cells on a population level and this type of analysis has inspired the development of TIL therapy [20, 21]. Second, in several studies, T-cell clones generated from intratumoral T cells have been utilized to obtain TCR genes that could subsequently be shown to confer tumor reactivity after TCR gene transfer [22-24]. Furthermore, TCRs obtained in this fashion have been utilized in the first clinical studies of TCR gene therapy [25, 26].

As a downside to these approaches, both these strategies will not capture the activity of T cells that do carry a tumor-reactive TCR but have been rendered anergic. Furthermore, since these studies commonly utilized in vitro expanded T-cell material, they are restricted to those T cells that can expand in vitro, thereby likely resulting in a significant bias or even precluding analysis altogether (e.g. for tumor types for which such T-cell expansion cannot be achieved). Therefore, while these studies have provided ample evidence for the presence of tumor-reactivity within the intratumoral pool of T cells, by their nature they cannot provide an unbiased enumeration of the ‘true’ fraction of tumor-reactive T cells within human tumors.

To obtain a better understanding of the tumor reactivity within intratumoral TCR repertoires, it would be of value to determine the frequency of tumor-reactive TCRs among different intratumoral T-cell subsets without a requirement for (prolonged) in vitro expansion. Toward this goal, we propose to isolate large libraries of TCRαβ gene pairs directly from intratumoral T cells. Identified TCR pairs can then be introduced in peripheral blood lymphocytes by gene transfer, to allow assessment of autologous tumor recognition independent of the parental T-cell phenotype. Such analyses could be focused on intratumoral CD8+ T cells but may also be of interest for the heterogeneous population of intratumoral CD4+ T-cell subsets, for which studies on tumor-reactivity are particularly limited at this moment. This type of autologous TCR gene transfer experiments would provide insights into several aspects of the intratumoral TCR repertoire. First, in analogy to the analysis of TCR repertoire diversity (see above), the frequency of tumor-reactive TCRs may represent a prognostic or predictive clinical marker. Second and somewhat related, these analyses would establish whether the previously described correlation between tumor infiltrating lymphocyte numbers and clinical prognosis can be explained by the tumor-recognition capacity of the tumor-resident TCR repertoire. Finally, such studies would make it is feasible to explore whether the frequency or diversity of tumor-reactive TCRs found in intratumoral TCR repertoires correlates with the size of the cancer anti-genome, as for instance reflected by the mutational load of tumors.

The isolation of the large TCR libraries that are required for such experiments has become a realistic option following the development of a number of strategies to identify TCRαβ gene pairs [27-29]. First, the sequencing of cDNA generated from single T cells has proven a viable strategy to identify TCRαβ pairs [27, 28]. To date, these strategies have not been utilized to dissect intratumoral TCR repertoires, but this will likely prove feasible. In addition, our laboratory has developed an independent method to infer TCRαβ sequences directly from the genome (rather than RNA) of T cells that has already been utilized to recreate TCR pairs from intratumoral T cells. This approach, called ‘TCR gene capture’ utilizes an RNA-bait library targeting the TCRαβ and TCRβ loci to specifically select and sequence genomic fragments encoding the TCR sequences of T cells [29] (Fig. 1).

image

Figure 1. Schematic representation of T-cell receptor (TCR) gene capture approach. Genomic DNA is extracted from (oligo)clonal T-cell populations of interest, the DNA is sheared into small fragments (average length 500 basepairs). Using an RNA-bait library targeting all functional TCR V- and J-segments on both the TCRα and TCRβ loci, all DNA fragments encoding TCR sequences are selected and subsequently analyzed by paired-end Illumina sequencing. Using bioinformatic tools [63], TCR CDR3 sequences are identified in the resulting sequencing data.

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TCR gene capture has proven a versatile tool to analyze TCR repertoires within intratumoral CD8+ T cells. First, with the goal to identify TCRs that may be used clinically, we have utilized the technique to assemble TCR libraries from single T cells expanded in vitro for a short period. Using MHC-multimer selected T cells as input, this resulted in the identification of a large panel of TCRs against shared tumor-antigens (we isolated 21 different TCRs against nine distinct Cancer/Germline antigens). In addition, we isolated a library of 19 different tumor-reactive TCRs from a library of clonal tumor-reactive T-cell populations from a melanoma patient without further knowledge of their antigen specificity. The latter data illustrate that the tumor-reactive TCR repertoire of intratumoral CD8+ T cells can be broad and that it is feasible to rapidly assemble a library of patient-specific tumor-reactive TCRs. Second, since TCR gene capture supplies quantitative read counts for all TCR α- and β-sequences within the sample, TCRαβ pairs can be directly identified within oligoclonal T-cell populations through matching of TCR α- and β-sequences that occur with similar frequency. For polyclonal T-cell populations, such ‘frequency-based matching’ will be precluded by the occurrence of multiple TCR clonotypes at the same frequency. However, we have successfully unraveled the TCR repertoire of several intratumoral CD8+ T-cell populations specific for shared melanoma antigens (Meloe-1, MAGE-A1, MAGE-10, TAG-1, LAGE-1) by this approach. In these experiments, in which bulk MHC-multimer positive T-cell populations from TILs were used as input for TCR gene capture, the TCR repertoire of the tumor antigen-specific T-cell populations analyzed commonly was restricted to only 1–5 TCRs. Comparison of the TCR diversity within individual tumor antigen-specific T-cell populations and within the entire tumor-reactive T-cell pool may in future studies perhaps be used to provide a first estimate of the breadth of the antigen repertoire that is recognized.

Using TCR gene capture, it has also proven feasible to identify dominant TCRs within the intratumoral tumor-reactive T-cell population without knowledge of their antigen specificity. Specifically, profiling of the TCR repertoire of three TCRVβ subpopulations among intratumoral, tumor-reactive CD8+ T cells showed that the TCR repertoire in each of these subpopulations was markedly restricted: the two most abundant TCRs comprised at least 75% of all functional TCRαβ CDR3 sequences. This recognition allowed the straightforward identification of tumor-reactive TCRαβ pairs.

Both the recently developed single cell-based approaches and the TCR gene capture strategy described above will be of substantially value to increase our understanding of the intratumoral TCR repertoire in human cancers. Nevertheless, the development of technologies that can sample the repertoire of intratumoral TCRαβ pairs at even greater depth (e.g. revealing the identity of many thousands of TCRαβ pairs) remains an important goal. The recent description of an emulsion-PCR based approach to identify TCRαβ pairs from single cells that are contained within droplets is of interest [30]. While further technical developments will clearly be required to allow an unbiased analysis of TCR repertoires with this type of technology, it does offers the potential to advance capacities for (intratumoral) TCR profiling well beyond the currently available medium-throughput approaches.

Antigen specificity of the intratumoral TCR repertoire

In particular for intratumoral T cells in melanoma, convincing evidence for tumor reactivity is provided by the clinical responses seen in patients that are treated with autologous TIL products. Furthermore, the fact that such clinical responses have also been observed in patients treated with CD8+ enriched TIL cell products [31] maps at least part of the clinically relevant tumor reactivity toward the cytotoxic T-cell subset. At present, our understanding of the critical cancer regression antigens in TIL therapy is limited. A better understanding of the role of different antigens in the clinical responses in patients receiving TIL therapy (or other non-antigen directed immunotherapies, such as blockade of CTLA-4 or PD-1) would possibly enable the ‘engineering’ of anti-tumor immunity in the large group of patients who do not benefit from current immunotherapies. Because of this, a large effort has been made over past few years to reveal the antigen-specificity of tumor-specific CD8+ T-cell response in patients receiving immunotherapy.

As demonstrated by pioneering work by Altman and Davis [32], fluorescently labeled multimeric pMHC-complexes can be used to detect antigen-specific T cells with high sensitivity and independent of their functional capacities. However, the large-scale screens that are required to assess the antigen specificity of the intratumoral T-cell pool by MHC multimer-based analyses have only become feasible with two subsequent developments: (i) the generation of a high-throughput pipeline to obtain the pMHC collections required for such monitoring and (ii) the design of experimental approaches for multiplexed analysis that allows comprehensive screens to be performed with clinically realistic amounts of biological material.

To obtain very large collections of pMHC-complexes in a high throughput fashion, we have developed (and now routinely use) a peptide exchange technology in which collections of pMHC complexes of interest can be generated in a 1 h procedure [33, 34]. This approach is based on the use of pMHC-complexes that carry a peptide ligand that cleaves itself upon UV exposure, and by exposing such conditional pMHC complexes to UV light in the presence of peptide ligands of interest. Peptide exchange technology is now available for around two dozen HLA class I alleles ([33-35], M. Toebes and L. van Dijk personal communication), also through work from the Grotenbreg lab [36]. As a side note, recent work demonstrates that MHC multimer-based detection of antigen-specific T cells is highly sensitive to minor sequence variation between HLA subtypes (M. M. van Buuren, F. E. Dijkgraaf, C. Linnemann, M. Toebes, C. X. L. Chang, J. Y. Mok, M. Nguyen, W. J. E. van Esch, P. Kvistborg, G. M. Grotenbreg and T. N. M. Schumacher, manuscript submitted). The requirement for proper matching between patient HLA alleles and HLA tools used for immunomonitoring that is revealed by these data will make it essential to further expand the HLA-based toolkit in the years to come.

To allow the analysis of T-cell reactivity against many (potential) tumor antigens in MHC-multimer-based screens, both our group and the Davis laboratory have developed the concept of combinatorial MHC-multimer staining [37, 38]. In this approach, each pMHC complex is coupled to a unique combination of fluorochromes or more recently lanthanides (see below). The use of such coding schemes then allows one to define the pMHC specificity of individual T cells/TCRs by the combinatorial code that they bind. In addition to offering the possibility of multiplexed analysis, this approach also markedly increases the sensitivity of pMHC-based T-cell detection, enabling the unambiguous detection of low-frequency antigen-specific T-cell populations [38]. The fluorochrome-based combinatorial coding schemes that to date have been used to analyze tumor-specific T-cell responses allow analysis of around 30 T-cell responses within a single sample. Furthermore, as an interesting extension of this approach, Newell and Davis recently demonstrated that the use of combinatorial coding schemes in MHC multimer mass cytometry can allow multiplexed analysis at an even higher complexity [39].

In recent studies, the combination of UV-mediated peptide exchange and combinatorial coding has been used to study the T cell/TCR repertoire in TIL products used for adoptive T-cell therapy of melanoma patients [40, 41]. In work that analyzed T-cell reactivity against a panel of around 150 shared melanoma-associated tumor antigens (TAAs), reactivity patterns in more than 50 TIL products were analyzed. While this work was primarily restricted to the HLA-A2 allele – few shared TAA are known for most other HLA class I alleles – several important conclusions can be drawn from these studies. First, in every TIL culture, an essentially unique pattern of antigen reactivities was found. Furthermore, for the few antigen-TIL product combinations for which this was examined, expression of a given melanocyte differentiation antigen/cancer-germline antigen was generally accompanied by the presence of T cells specific for this epitope, suggesting that tumor antigen expression may in most cases be noted by the immune system (note that a larger data set will certainly be required to test this notion in a rigorous manner). These T-cell monitoring data extend the concept of ‘tumor heterogeneity’ – described above for tumor genomes and for T-cell infiltrates – to the TCR specificities of the intratumoral CD8+ T-cell pool. A second important observation made in these studies has been that the frequency of antigen-specific T cells that were detected for the shared TAA used in these studies was very low (median of less than 1% for all HLA-A2-restricted T-cell responses detected per TIL product). Even taking into account the fact that T-cell reactivity against the five other possible HLA alleles was not measured, these data suggest that reactivity against shared TAA may only explain part of the composition of clinically used TIL products. As a first explanation for this discrepancy, the fraction of non-tumor reactive ‘bystander’ TCR specificities in TIL products may in many cases be high. As a second explanation, a large fraction of the tumor-specific TCRs within the intratumoral repertoire may recognize highly patient-specific antigens. Early evidence for a (perhaps small) contribution by bystander cells has been obtained by the detection of low frequencies of CMV and EBV-specific T cells in TIL products. However, the contribution of bystander cells may be addressed in a more definitive manner by the isolation of large TCR libraries from the intratumoral TCR repertoire and their subsequent characterization with regards to tumor-reactivity (see section ‘Tumor-reactivity of the intratumoral TCR repertoire’). With respect to the second possibility, the particularly high mutational load of melanoma and other tumors, such as lung cancer, raises the question whether the intratumoral T-cell repertoire could contain a variety of TCRs that recognize neo-antigens derived from tumor-specific mutations, thereby forming a highly personalized, tumor-reactive TCR repertoire.

Recent and exciting studies in animal models by the Sahin and Schreiber groups [42, 43] have demonstrated that cancer exome sequencing data may be utilized to analyze T-cell reactivities against neo-antigens formed by tumor-specific mutations. The ability to dissect T-cell reactivity against neo-antigens on the basis of human cancer exome data has now also been demonstrated by others and us. Rosenberg et al. [44] have utilized cancer exome sequencing data and recognition of target cells loaded with putative neo-antigens by autologous CD8+ TILs to uncover neo-antigen-specific T-cell reactivity within TIL products. On average, two T-cell responses against neo-antigens were identified in the three patients analyzed. As neo-antigen-specific T-cell reactivity was only analyzed for some of the HLA alleles expressed by these patients, these data suggest that TILs may potentially recognize a series of patient-specific antigens. However, as this study focused on patients that experienced a particularly strong clinical response upon TIL therapy, it is possible that broad neo-antigen-specific T-cell reactivity may not be invariably present in melanoma, and it will be important to extend these studies to address this issue. In parallel work, our group has provided proof of concept for the combination of cancer exome sequencing and MHC-multimer technologies to reveal T-cell responses against patient-specific neo-antigens arising from genomic mutations [45]. Comparison of whole exome sequencing data of a melanoma tumor with that of autologous healthy tissue revealed more than 1000 non-synonymous changes resulting in altered open reading frames. These data were then combined with RNA-expression data to predict potential neo-epitopes for four of the HLA class I alleles expressed by this patient. When TILs from this patient were then screened with a library of MHC-multimers containing these putative T-cell epitopes, two neo-antigen-specific T-cell responses were identified: a low-frequency response against a mutated peptide of ZNF462 gene (0.003% of CD8+ T cells) and a dominant response (3.3% of CD8+ T cells) against a neo-antigen derived from the ATR DNA damage response gene. The magnitude of the T-cell response against the ATR neo-antigen was of a considerably higher magnitude than virtually all of the T-cell responses against shared antigens seen in our previous analyses of TILs [40, 41]. While more data are certainly required, these data suggest that (some) T-cell responses against neo-antigens may perhaps be of a higher magnitude than T-cell responses against shared (self) antigens, due to their foreign nature. Comparison of the tumor recognition potential of neo-antigen and shared antigen-specific T cells will also be of importance to address the relative importance of the two antigen classes.

Analysis of peripheral blood samples pre- and post-treatment with anti-CTLA-4 showed an approximately fivefold increase in the frequency of ATR-specific CD8+ T cells upon treatment. This increase in frequency, which coincided with a partial tumor regression within this patient, is consistent with the possibility that the T-cell response against the mutated ATR peptide may have been therapeutically meaningful, but the evidence is obviously indirect. By the same token, Lu et al. [46] used cDNA library screening to reveal a T-cell response against a mutated peptide of PPP1R3B in a metastatic melanoma patient, and could show that a long-term T-cell response against this epitope was present in this patient who experienced a durable complete response after TIL therapy.

The recent studies that have started to link cancer exome data to tumor-specific T-cell responses will likely still only sketch a fraction of the patient-specific intratumoral TCR repertoire. As a first issue, imperfection in epitope predictions will lead investigators to miss neo-antigens, in particular for the less well-studied HLA alleles. Furthermore, in addition to neo-antigens that arise as a consequence of single nucleotide variants or insertions/deletions in known open reading frames, it is highly likely that T-cell epitopes arising from alternative translation events [47, 48] will also form part of the patient-specific cancer anti-genome. As precedent for the latter, minor histocompatibility antigens, which bear some similarity with neo-antigens in solid tumors (both generally originate from single nucleotide differences), can also be derived from alternative open reading frames [49, 50].

A number of developments can be foreseen that will make the identification of patient-specific antigens recognized by the intratumoral TCR repertoire more efficient. First, an increase in the quality/coverage of sequence data and in particular the quality of epitope predictions will facilitate the identification of neo-antigens. With respect to the latter, especially for the less common HLA-A and -B alleles and for the HLA-C alleles, the quality of prediction algorithms may readily be increased by the generation of more input data. As a less biased approach (that altogether avoids the need to predict T-cell epitopes), systems that allow the efficient expression of the entire set of tumor-specific mutations could be valuable, in particular to identify T-cell epitopes from non-canonical sources (e.g. alternative translation events). Finally, the moment it becomes feasible to (roughly) predict the epitope recognized by a TCR on the basis of TCR sequence data (at present a far removed goal), the combination of intratumoral TCR sequence data and cancer exome data could form a strategy to identify patient-specific antigens in a manner that is altogether independent of immunological analyses.

Utilizing intratumoral TCR repertoires

  1. Top of page
  2. Summary
  3. Introduction
  4. Understanding intratumoral TCR repertoires
  5. Utilizing intratumoral TCR repertoires
  6. Concluding remarks
  7. Acknowledgements
  8. References

Both TIL therapy and T-cell checkpoint blockade with anti-CTLA4 or anti-PD1 mAbs induce tumor regression in only a fraction of patients [51-53]. One possible reason for therapy failure could be that in many patients the intratumoral TCR repertoire lacks the capacity to recognize any tumor-expressed antigens. However, in particular for tumors with a high mutational load, such as melanoma, smoking-associated lung cancer, and esophageal cancer [2], the numerous changes in protein-sequences should be expected to lead to T-cell epitopes for which thymus-induced tolerance did not occur. The notion that TCR reactivity against neo-epitopes in such tumors with a high mutational load may be present (perhaps at a low level) but does not suffice for tumor control raises the question whether such TCR repertoires could be exploited by other therapeutic strategies.

The majority of mutations found in melanoma and other tumors with a high-mutational load are ‘passenger-mutations’ that are unrelated to the cellular transformation process [54]. As such, the vast majority of potential neo-epitopes in these cancers are patient-specific. In addition, for the small fraction of mutations that do occur at an appreciable frequency, the polymorphism in HLA-alleles forms a second variable between tumors. For these two reasons, at best a few neo-antigen/HLA combinations will be sufficiently common within the human population to be targeted by off-the-shelf approaches. Consequently, the therapeutic utilization of mutation-induced tumor antigens is likely to benefit primarily from the development of more personalized treatment strategies. Such personalized treatment strategies could either rely on active immunization (i.e. vaccination) or on passive immunization (i.e. adoptive therapy), and two potential approaches that involve the latter, the use of the patient-specific anti-genome in adoptive therapy, are discussed below.

Antigen-specific TIL therapy?

The T cells present in currently used TIL products are not selected to any substantial extent. Depending of the protocol used, the cell product is either formed by those T cells present in the tumor that were able to grow out in vitro, or – in case multiple TIL cultures are initiated that are then tested for tumor recognition – those T cells that stochastically were present in the culture that showed the highest degree of tumor recognition. As a first potential step forward, T-cell products may be generated that are derived from those T cells that were enriched on the basis of autologous tumor recognition. This approach can likely be implemented quite readily in clinical protocols and does not rely on any knowledge on the antigen(s) recognized. An implicit assumption in such a process is that T cells with in vitro tumor recognition capacity differ little amongst themselves in their tumoricidal potential under in vivo conditions. As an alternative approach, with the increasing ability to identify (patient-specific) tumor antigens recognized by intratumoral T cells, it may become feasible to develop antigen-defined TIL products. In such a strategy, T cells specific for antigens of interest could be enriched by standard MHC multimer or MHC streptamer technology, the latter offering the advantage of reversibility [55]. Such an approach could be argued to offer increased safety compared with current TIL therapy protocols since selected antigen-specificities will form the majority of the cell product infused into the patient. However, in view of the modest T-cell-mediated toxicity in current TIL therapy, the gain here is at best small. More importantly, the anti-tumor effects of a selected TIL product may potentially be enhanced through a number of mechanisms: depletion of (non-reactive) passenger cells, removal of suppressive T-cell subsets, and increased frequency of highly tumor-reactive T cells [56]. As a potential downside, at the current state of technology development, many T-cell responses within TIL products that are reactive with neo-antigens or other relevant antigens are still not identified. As such, the generation of antigen-specific TIL products will result in a narrowing of the tumor-specific T-cell repertoire, and in particular for highly heterogeneous tumors, such a narrowing could conceivably increase the likelihood of tumor escape. Preclinical studies in which the ability of poly-specific or oligo-specific T-cell products to control growth of autologous tumor is examined will be useful to address these issues.

Autologous TCR gene therapy?

The clinical use of TIL therapy is restricted to those few tumor types for which TILs can reliably be grown. In addition, the cells that are obtained are highly differentiated, which is likely to limit the in vivo activity of the cells following transfer. TCR gene therapy offers the possibility to overcome the limitations associated with TIL therapy in terms of expansion potential and functional properties of the T-cell product that is given. Thus far, TCR gene therapy has been used exclusively to target tumor-antigens that are shared between large subgroups of patients. Here, we would like to propose the concept of autologous TCR gene therapy: the transfer of a set of autologous tumor-reactive TCRs into ‘young’ T cells, to effectively target the patient-specific cancer anti-genome (Fig. 2).

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Figure 2. Concept of autologous T-cell receptor (TCR) gene transfer. After excision of a tumor lesion, tumor-reactive intratumoral T cells are identified. Tumor-reactive TCR genes are obtained from such tumor-reactive intratumoral T cells and subsequently utilized for TCR gene transfer into autologous peripheral blood lymphocytes. The resulting tumor-reactive T-cell populations are briefly expanded in vitro and then re-infused into the patient.

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The isolation and transfer of a numbers of TCRs on a patient-specific basis in a clinically relevant time frame would have appeared unrealistic a few years ago. However, technical advances make such an approach an increasingly feasible goal now. First, the high-throughput isolation of patient-specific TCRs has become possible with the above-described methods to rapidly identify TCRαβ pairs [27-29]. Second, while the production of clinical-grade retrovirus or lentivirus is involved with regards to both time and finances, alternative non-viral gene transfer systems can now be used for the stable genetic modification of T cells. Transposon-based approaches, such as the hyperactive Sleeping Beauty transposase [57], have been shown to form suitable systems for T-cell engineering [58]. Furthermore, ongoing work suggests that it is feasible to further develop these systems, to reduce transposon related cytotoxicity (A. Hollenstein and R. Mezzadra, manuscript in preparation) and increase the percentage of gene-modified T cells within the cell product (R. Mezzadra, manuscript in preparation).

For such autologous TCR gene therapy, we consider the simultaneous use of multiple TCRs important to minimize the likelihood of tumor escape, and there is in our view no technical reason why TCR gene therapy should be restricted to a single specificity. The collection of tumor-reactive TCRs that could be used for autologous TCR gene therapy may be generated in a number of different ways. First, a set of TCRs that are reactive with neo-antigens may be created using the tools outlined in this review. Analogous to antigen-specific TIL therapy, this will form a highly safe therapeutic approach since only antigens that are solely expressed within tumor tissue are being targeted.

Second, one could consider the identification of large numbers of autologous tumor-reactive TCRs and their direct use for autologous TCR gene therapy, without knowledge of their antigen specificity. In this way a broad, tumor-reactive TCR repertoire can be utilized that is not restricted by our still limited understanding of the cancer anti-genome. With regards to safety considerations, we note that any TCR transferred in this way will have undergone prior thymic selection in the same individual. Furthermore, with the safety record of transfer of autologous TIL transfer established, there is no a priori reason to consider autologous tumor-derived TCR transfer overly risky. Finally, to prevent toxicity due to the ‘accidental’ engineering of strong T-cell responses against potentially unsafe targets (e.g. some of the MAGE antigens) [59, 60], a ‘safety-switch’ may be co-introduced into the TCR-modified T cells to control their in vivo function [61, 62].

Concluding remarks

  1. Top of page
  2. Summary
  3. Introduction
  4. Understanding intratumoral TCR repertoires
  5. Utilizing intratumoral TCR repertoires
  6. Concluding remarks
  7. Acknowledgements
  8. References

Although it is evident that intratumoral T cells can have the potential to recognize tumor cells, the extent of this tumor recognition and more importantly the specific antigens recognized by intratumoral T cells remain still largely unknown, in particular outside of melanoma. Novel methods for the isolation of TCR genes, allowing the rapid isolation of large TCR libraries from intratumoral T cells, will offer the possibility to assess the antigen-specificity of intratumoral TCRs independent from original (commonly limited) primary material. Furthermore, the availability of such TCR gene libraries may facilitate efforts to locate target epitopes within the cancer anti-genome. The first exploration of these tools suggests that T-cell reactivities against patient-specific, mutated antigens may form an important ingredient of the anti-tumor-potential of the intratumoral TCR repertoire.

Gaining an understanding of the crucial antigen-specificities for cancer regression within intratumoral TCR repertoires could allow the field to improve adoptive T-cell therapies in two ways. First, it may allow the selection/generation of antigen-specific TILs, thereby enabling antigen-directed TIL therapy. Second, it may allow the targeting of the patient-specific cancer anti-genome by autologous TCR gene therapy. Finally, a better understanding of the intratumoral TCR repertoire will also serve to guide the development of active immunotherapies that aim to increase the activity of the tumor-resident T-cell pool.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Understanding intratumoral TCR repertoires
  5. Utilizing intratumoral TCR repertoires
  6. Concluding remarks
  7. Acknowledgements
  8. References

C. L. is a fellow in the PhD Fellowship Program of Boehringer Ingelheim Fonds – Foundation for Basic Research in Biomedicine. T. N. M. S receives grant support from the Dutch Cancer Society (NKI 2009-4282), the FP7 ITN ATTRACT program, and the K. G. Jebsen Center for Cancer Immunotherapy. We thank G. M. Bendle, P. Kvistborg, and M. M. van Buuren for critical discussions. The authors declare no competing financial interests.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Understanding intratumoral TCR repertoires
  5. Utilizing intratumoral TCR repertoires
  6. Concluding remarks
  7. Acknowledgements
  8. References