Recent advances in genetic engineering have enabled the delivery of clinical trials using patient T cells redirected to recognize tumor-associated antigens. The most dramatic results have been seen with T cells engineered to express a chimeric antigen receptor (CAR) specific for CD19, a differentiation antigen expressed in B cells and B lineage malignancies. We propose that antigen expression in nonmalignant cells may contribute to the efficacy of T-cell therapy by maintaining effector function and promoting memory. Although CAR recognition is limited to cell surface structures, T-cell receptors (TCRs) can recognize intracellular proteins. This not only expands the range of tumor-associated self-antigens that are amenable for T-cell therapy, but also allows TCR targeting of the cancer mutagenome. We will highlight biological bottlenecks that potentially limit mutation-specific T-cell therapy and may require high-avidity TCRs that are capable of activating effector function when the concentrations of mutant peptides are low. Unexpectedly, modified TCRs with artificially high affinities function poorly in response to low concentration of cognate peptide but pose an increased safety risk as they may respond optimally to cross-reactive peptides. Recent gene-editing tools, such as transcription activator–like effector nucleases and clustered regularly interspaced short palindromic repeats, provide a platform to delete endogenous TCR and HLA genes, which removes alloreactivity and decreases immunogenicity of third-party T cells. This represents an important step toward generic off-the-shelf T-cell products that may be used in the future for the treatment of large numbers of patients.

Rapid improvements of gene transfer technologies have provided a robust platform to redirect the specificity of primary T cells.1  This has overcome a major obstacle for targeted T-cell therapy, posed by the relatively low frequency of cancer-reactive T cells that are naturally present in patients or that can be induced by vaccination. Retro- and lentiviral gene transfer platforms have been developed to achieve the expression of cancer-reactive T-cell receptors (TCRs) and chimeric antigen receptors (CARs) in primary T cells, generating therapeutic cellular products with a high level of tumor specificity.2  There is now the opportunity to direct the therapeutic power of T-cell therapy toward defined cancer antigens and thus avoid the toxicity of donor lymphocyte infusion, which is caused by the alloreactivity of the polyclonal TCR repertoire of infused T cells.

TCRs recognize peptide fragments presented by HLA molecules.3  An evolutionary advantage of this mode of antigen recognition enables T cells to recognize and attack virus-infected cells, even when viral proteins are “hidden” inside cells and absent from the surface.4  Similarly, this mode of recognition renders intracellular cancer proteins susceptible for targeted attack by TCRs (Figure 1). For example, most cancer testis antigens are not expressed on the surface of tumor cells but are nevertheless efficiently targeted by TCRs, but not by CARs.5  Similarly, intracellular proteins such as Wilms tumor antigen-1 and minor histocompatibility antigens have been validated in preclinical experiments as attractive targets for the treatment of hematologic malignancies.6-9  TCR gene therapy trials targeting these antigens are currently open for recruitment of leukemia patients.

Figure 1

Peptide processing, HLA binding, and TCR recognition. The proteasome degrades proteins to produce peptide fragments, which are transported from the cytosol into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) complex. Inside the ER, peptides bind to HLA class I molecules, which are then transported to the cell surface where the HLA/peptide structure is recognized by TCRs. The peptide residues important for HLA binding are indicated in pink and yellow, and the mutated residue is indicated by a cross. Note that the proteasome may not cleave at the appropriate position and that the mutation-containing peptide may not be transported by TAP or fail to bind to HLA.

Figure 1

Peptide processing, HLA binding, and TCR recognition. The proteasome degrades proteins to produce peptide fragments, which are transported from the cytosol into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP) complex. Inside the ER, peptides bind to HLA class I molecules, which are then transported to the cell surface where the HLA/peptide structure is recognized by TCRs. The peptide residues important for HLA binding are indicated in pink and yellow, and the mutated residue is indicated by a cross. Note that the proteasome may not cleave at the appropriate position and that the mutation-containing peptide may not be transported by TAP or fail to bind to HLA.

Close modal

The TCR recognition is focused on short linear peptide epitopes presented by HLA class I molecules (9-10 amino acid peptides) and HLA class II molecules (15-18 amino acids).10,11  Although some of these peptide residues mediate HLA binding, other residues interact primarily with the complementary determining region 3 of the TCR, providing appropriate engagement required for T-cell activation.12  Both HLA binding and TCR interaction can be exquisitely sensitive to single amino acid substitutions, which is an important consideration for cancer immunology. Although patient T cells are tolerant to self-peptides derived from self-proteins, point mutations in tumor cells resulting in single amino acid changes can elicit robust T-cell responses.13-20  There are 2 mechanisms whereby point mutations can generate immunogenic epitopes to which patient T cells are not tolerant. First, mutations may generate novel TCR contact residues and thus produce immunogenic neoepitopes, or, alternatively, they may create novel HLA-binding residues resulting in the presentation of peptides in tumor cells that are absent in normal tissues. Because of recognition of linear peptide sequences, TCRs can potentially target the mutational landscape associated with cancer development, although, as discussed subsequently, cellular mechanisms of peptide production and transport impose considerable limitations. In contrast to TCRs, point mutations largely escape antibody recognition and thus CAR targeting because the vast majority of mutated proteins are intracellular and because antibodies are less effective in the specific recognition of point mutations in otherwise unaltered self-proteins.

Although the HLA-dependent antigen recognition gives TCRs access to target antigens that escape CAR recognition, it is also a major disadvantage because therapeutic TCR can only be used in a limited number of patients with the appropriate HLA alleles. To date, the vast majority of TCRs that are in clinical trials or in preclinical testing are restricted by HLA-A*0201, which is found in ∼45% of white people. Therapeutic TCRs restricted by 4 common HLA class I alleles (A*0201, A*0301, A*2402, and B*0702) would substantially extend the applicability of TCR gene therapy and cover >90% of the white population in the United States.21 

CARs recognize their antigen directly and can be used for the treatment of all malignancies bearing the relevant antigen irrespective of patients’ HLA genetics. CARs have the further advantage that they do not mispair with endogenous TCR chains, a mechanism that can create novel specificities in TCR gene-modified primary T cells. Although clinical trials have not yet demonstrated toxicity because of TCR mispairing, it is clear from murine model experiments that it can cause severe graft-versus-host disease (GVHD)–like pathology.22 

Overviews of the ongoing TCR gene therapy trials in hematologic malignancies and in solid tumors have been published recently.23-25  The first TCR gene therapy trial was performed in melanoma, using a TCR specific for the melanocyte-specific differentiation antigen MART-1.26  The results of all published trials to date showed that TCR gene-modified patient T cells can have clinical activity and reduce tumor burden. However, it is clear that therapeutic TCRs have not yet delivered the dramatic and lasting anticancer responses that have been seen with CARs targeting CD19 on chronic lymphocytic leukemia (CLL), acute lymphoblastic leukemia (ALL), and B-cell lymphoma.27-30  Clinical trials of NY-ESO-1 TCR gene therapy in melanoma and synovial cell carcinoma, and in multiple myeloma patients undergoing autologous stem cell transplantation, have shown clear antitumor activity.31,32  In the myeloma study, disease progression was correlated with loss of persistence of the gene-modified T cells or with loss of NY-ESO-1 expression in the myeloma cells, suggesting that immune editing allowed myeloma cells to escape T-cell attack. It is important to note that all completed and ongoing TCR gene therapy trials target tumor-associated antigens that are also expressed to various extents in normal tissues. The physiological expression of TCR-targeted antigens poses the risk of “on-target” immune pathology as has been observed with TCR-targeting of MART1, glycoprotein 100, and carcinoembryonic antigen.33-35  However, we need to consider that antigen-expression in normal tissues can also enhance the efficacy of adoptive therapy with engineered T cells.

To date, the highest level of clinical efficacy of any form of immunotherapy has been achieved with T cells engineered with CARs specific for CD19. In this case, control of B-cell malignancies is associated with the elimination of CD19-positive normal B cells and persistence of transferred T cells.30,36  It is not known from the human studies whether the continued elimination of B cells might trigger the sustained T-cell persistence. We would like to speculate that the interaction with normal B cells, which can provide T-cell costimulation and function as efficient antigen-presenting cells,37  might contribute to promoting the expansion and maintenance of the therapeutic T cells in vivo (Figure 2). Similar to CD19-targeted immunotherapy, successful immunotherapy of melanoma is associated with vitiligo caused by the T-cell recognition of tissue-specific antigens in normal melanocytes.38,39  Interestingly, studies in murine models have indeed provided evidence that the interaction with melanocytes provided T-cell stimulation and contributed to sustaining the memory potential of the therapeutic T cells.40 

Figure 2

Interaction of tumor-reactive T cells with normal tissues. Cells in normal tissues may be able to provide costimulation, which results in T-cell activation and expansion (right). The interaction with cells that do not provide costimulation might induce a state of “sleepy” T-cell anergy (left).

Figure 2

Interaction of tumor-reactive T cells with normal tissues. Cells in normal tissues may be able to provide costimulation, which results in T-cell activation and expansion (right). The interaction with cells that do not provide costimulation might induce a state of “sleepy” T-cell anergy (left).

Close modal

However, the expression of self-antigens in tissues and cell types that are unable to provide T-cell costimulation may result in the induction of anergy (Figure 2). Again, this was demonstrated in a murine model where adoptive transfer of TCR-engineered T cells specific for the MDM2 antigen resulted in the loss of T-cell function.41  It was shown that loss of T-cell function was driven by MDM2 antigen expression in nonhematopoietic tissues, whereas hematopoietic cells promoted T-cell function. The lack of costimulation and potential induction of anergy may be particularly relevant for T cells engineered with TCRs, whereas the engineering with CARs that contain costimulatory signaling domains may protect T cells from anergy induction.

For TCR gene therapy, it is therefore advisable to consider the expression pattern of therapeutic target antigens in the context of possible toxicity and to also take into account the impact on the modulation of T-cell function. We suggest that it may be desirable for tumor-associated self-antigens to be expressed in normal cells that are capable of T-cell stimulation and whose loss, as a consequence of T-cell attack, does not cause untreatable toxicity. The observation that antigen expression in the hematopoietic compartment can promote T-cell function suggests that lineage-specific antigens may be good targets for the treatment of hematologic malignancies. The haploidentical transplantation protocol that was recently used for adoptive transfer of nonengineered T cells could potentially provide an excellent platform to achieve the selective TCR targeting of leukemia.42  For example, it would be possible to engineer donor T cells with a TCR specific for a hematopoietic-specific antigen presented by an HLA class I allele that is expressed by the patient but not by the donor. In this case, the engineered T cells would selectively attack patient leukemia and hematopoietic cells but spare donor cells that lack the HLA molecule required for the recognition of the hematopoietic antigen.

Chronic myeloid leukemia (CML) is characterized by the breakpoint cluster region (BCR)/Abelson (ABL) translocations that often produce the same mutant protein in patients.43  The targeting of this mutant protein with small-molecule inhibitors such as imatinib was a first example of a highly disease-specific therapy resulting in CML control in the majority of patients. The BCR/ABL fusion protein also contains linear peptide sequences that are unique to CML and absent in all nonmalignant cells. Despite substantial efforts to exploit fusion peptides for vaccination and to identify fusion-specific T-cell responses, the clinical results have been disappointing, and clear evidence of fusion-specific T cells capable of recognizing and killing CML remains absent.44  This is puzzling, considering that mutation-specific responses by both CD4+ helper T cells and CD8+ cytotoxic T cells have now been clearly identified in patients with melanoma and gastrointestinal cancer.13,16,19,20  Why is it, therefore, that BCR/ABL “escapes” T-cell immunity when other mutations trigger robust T-cell responses?

There are a number of requirements that a peptide has to meet to function as an epitope for T-cell attack (Figure 1). The proteasome has to cleave the protein to generate the appropriate peptide, which then has to interact productively with the TAP complex for transport into the ER, and inside the ER, peptides have to display sufficient binding affinity for HLA molecules to compete successfully with the large number of normal self-peptides generated from the set of 10 000 to 15 000 proteins that are expressed in human cells.45  The following considerations illustrate that the majority of cancer mutations are predicted to be invisible to immune recognition. Although tumors such as lung cancer and melanoma harbor ∼30 000 somatic mutations, the protein-encoding exome represents 1% of the entire genome, indicating that ∼300 mutations occur in protein-coding sequences. Of the 300 DNA alterations, only the nonsynonymous mutations and frameshifts will change amino acids and create an altered protein. Proteasome-mediated cleavage of the protein needs to release the mutation-containing peptides with the correct C-terminal residues required for HLA binding (Figure 1).46  This, however, is often not the case, and it has been demonstrated that proteasome cleavage can instead destroy TCR-recognized peptide epitopes.47,48  The efficiency of peptide transport from the cytosol into the ER is the next rate-limiting step, and experiments have demonstrated that certain amino acids in peptides can greatly decrease transport efficacy.49  Finally, HLA binding typically requires preferred peptide residues to function as anchors that mediate HLA binding; usually only 2 of the 20 possible amino acids can mediate efficient anchor function.50  Together, these considerations provide some explanation of why BCR/ABL fusion peptides may fail efficient HLA presentation. It also explains published studies of the immunogenicity of the cancer mutagenome, which have shown that only 0.3% to 1.3% of mutated sequences induced CD8 T-cell responses and only 0.5% of mutated peptides induced detectable responses by CD4 T cells.13,16,19,20 

The frequency of mutations in hematologic malignancies is 10 to 20 times lower than melanoma and lung cancer. Multiple myeloma has ∼3000 mutations in the genome, whereas the frequency in acute myeloid leukemia, ALL, and CLL is ∼1500 to 2000 mutations.51  Despite this reduced mutation load, it is premature to conclude that neopeptides cannot function as targets for TCR gene therapy in hematologic malignancies. It is possible that the mutation-specific T cells detected in melanoma patients are directed against immune-dominant peptides that are presented at high levels. This would be similar to T-cell responses in viral infections that are often directed against dominant viral peptides. In this case, subdominant T-cell epitopes are detected after immunization with vaccines lacking the immune-dominant peptides.52  It is therefore possible that mutation-containing neopeptides that are inefficiently presented may serve as targets for TCR gene therapy, even when they do not stimulate T-cell responses in patients. The targeting of these subdominant neoepitopes would require high-avidity TCRs that can activate T-cell effector function at low peptide concentration. The immunization of mice transgenic for HLA and the human TCR gene repertoire, or the in vitro immunization of T cells from healthy donors, may provide a source for such high-avidity TCRs.5,53 

The affinity and the expression level of therapeutic TCRs are 2 key parameters that determine how much antigen is needed for the triggering T-cell effector function. Several engineering strategies have been employed to enhance the level of TCR expression on the T-cell surface. This includes codon optimization, introduction of an additional disulfide bond between the TCR chains, and the introduction of murine residues into the constant region domain.54-57  Despite these steps, there are still remarkable differences in in the expression levels achievable with TCRs that differ only in the variable region domains.58  We have performed extensive comparisons between poorly and strongly expressed human TCRs and have been able to identify key residues affecting the level of surface expression. Interestingly, these residues are outside the complementary determining regions of the variable domains and are therefore accessible to replacements without affecting T-cell specificity. Thus, the replacement of key residues in the framework of the variable region can improve TCR expression and enhance antigen-specific effector function.

An alternative strategy to enhance TCR expression is the provision of additional CD3 molecules in engineered T cells. We have shown that the transfer of TCR and CD3 genes into primary T cells enhanced TCR expression and improved recognition of low concentrations of antigen, which resulted in augmented tumor protection in vivo.59  However, additional CD3 also leads to enhanced expression of endogenous and mispaired TCR, thus increasing the risk of potential toxicity. The most effective strategy to prevent this risk is the disruption of endogenous TCR genes using zinc finger nucleases, transcription activator–like effector nucleases (TALENs), or clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 technologies.60 

Altering TCR affinity is another strategy that has been used to achieve T-cell stimulation by low antigen concentration. Unexpectedly, experiments have shown that high affinities that increase the TCR binding to HLA peptide from the “physiological” half-life of seconds into the range of minutes and hours result in impaired ability to trigger T-cell stimulation when the peptide concentration is low.61  As a consequence, T cells expressing modified TCRs with affinities similar to that of antibodies are efficiently stimulated by high doses of cognate peptide but fail to respond to low-dose peptide (Figure 3). At a low dose of cognate peptide, 1 HLA/peptide complex needs to stimulate multiple TCR by a mechanism of serial triggering, which is disrupted when the half-life of TCR binding to HLA peptide is too long.62  Therefore, artificially high affinity not only impairs response to low concentration of cognate peptide, but it also enhances the risk of optimal triggering by cross-reactive peptides (Figure 3). Compared with the binding of cognate antigen, cross-reactivity usually occurs at lower affinity and shorter half-life and may therefore fall into the optimal range for serial triggering required for activation at low peptide concentration. In other words, artificially affinity-matured TCR may be more effective in stimulating T-cell responses against cross-reactive peptides compared with cognate peptides. This might have contributed to the fatal toxicities that have been seen with an affinity-matured MAGE-3A TCR that cross-reacted against MAGE-A12 in the brain and against Titin expressed in heart tissue.63,64 

Figure 3

Risk of cross-reactivity by affinity-matured TCR. Engineered TCRs with artificially high affinity require high peptide concentration to stimulate T-cell responses (black titration curve indicates stimulation with cognate peptide). This is because of the long half-life of binding, which prevents sequential engagement of several TCRs with a single HLA/peptide ligand. Cross-reactive peptides are expected bind the same TCR with reduced affinity and binding half-life and may fall into the optimal range for sequential TCR engagement that is required for T-cell activation at low peptide concentration. Hence, the red titration curve indicates that the affinity-matured TCR is triggered by a lower concentration of cross-reactive peptide compared with cognate peptide (black curve).

Figure 3

Risk of cross-reactivity by affinity-matured TCR. Engineered TCRs with artificially high affinity require high peptide concentration to stimulate T-cell responses (black titration curve indicates stimulation with cognate peptide). This is because of the long half-life of binding, which prevents sequential engagement of several TCRs with a single HLA/peptide ligand. Cross-reactive peptides are expected bind the same TCR with reduced affinity and binding half-life and may fall into the optimal range for sequential TCR engagement that is required for T-cell activation at low peptide concentration. Hence, the red titration curve indicates that the affinity-matured TCR is triggered by a lower concentration of cross-reactive peptide compared with cognate peptide (black curve).

Close modal

There is increasing evidence for a key role of CD4+ T cells in cancer immunity. Data from melanoma trials have suggested that adoptive transfer of both CD4+ and CD8+ T-cell subsets provided efficient antitumor activity. More recently, the adoptive transfer of CD4+ T cells specific for a mutation present in cholangiocarcinoma resulted in impressive clinical responses.18  A side-by-side comparison of adoptive therapy with antigen-specific CD4+ or CD8+ T-cell subsets in a murine melanoma model showed that the former were more effective in eradicating established tumors.65  The separation of the function of CD4+ and CD8+ T-cell subsets into production of helper cytokines and delivery of cytotoxicity is no longer valid, as data in humans and mice have demonstrated efficient cytotoxicity mediated by CD4+ T cells.66 

These observations have provided a rationale for clinical trials of adoptive therapy with CD4+ T cells after allogeneic stem cell transplantation, and the results of these trials have shown that donor CD4+ T cells can mediate antileukemic effects with reduced risk of GVHD. However, the induction of HLA class II expression in nonhematopoietic tissues caused by cytomegalovirus reactivation was associated with GVHD.67 

TCR engineering offers the opportunity to transfer HLA class I–restricted TCRs into CD4+ T cells, thus redirecting their specificity against the same target epitopes that are normally recognized by CD8+ T cells.68,69  Consequently, activation of CD4+ T-cell function is no longer dependent on antigen-presentation by HLA class II, which is frequently absent in tumor cells. A possible disadvantage of this strategy is that HLA class I is widely expressed in most tissues, thus extending possible toxicities of therapy with redirected CD4+ T cells or impairing their functional potential as a consequence of interaction with cells and tissues that can induce T-cell anergy (Figure 2).

The first successful gene deletion in primary T cells was achieved using zinc finger nucleases to disrupt the endogenous TCR in engineered T cells expressing a therapeutic TCR.60  The more recently developed TALEN and CRISPR technologies are likely to increase the efficiency of gene editing in T cells. A recent study has used the TALEN platform to disrupt the expression of endogenous TCR and of CD52, which is recognized by alemtuzumab antibodies that are used to suppress immune rejection after allogeneic stem cell transplantation.70  Disruption of the endogenous TCR expression removed the ability of allogeneic T cells to cause GVHD, and the lack of CD52 made them resistant to alemtuzumab-mediated immune suppression. The TALEN-engineered third-party T cells were equipped with a CD19-specific CAR, enabling them to attack CD19-expressing ALL cells in an infant who had relapsed after allogeneic stem cell transplantation. This first example of combining lentiviral gene transfer with TALEN-mediated gene disruption demonstrates the feasibility of employing genetic engineering to add and remove genes with the goal to maximize the functional profile of therapeutic T cells (Figure 4). The design of third-party T cells as “off the shelf” therapeutics will require the complete removal of endogenous TCR to avoid GVHD pathology and also the removal of HLA class I and class II antigens to reduce immunogenicity and thus reduce the risk of rejection. The introduction of ligands for killer cell inhibitory receptors, such as nonclassical HLA-G, might reduce the risk of natural killer cell–mediated rejection triggered by the absence of classical HLA molecules.

Figure 4

Adding and deleting genes to enhance T-cell function. Retro- and lentiviral gene transfer can be used to redirect the specificity of T cells and the metabolic functions, response to chemokines, and cytokine secretion. Gene disruption technologies can be used to delete endogenous TCR genes, HLA genes, and genes involved in negative T-cell regulation. The indicated genes are simply examples, and the editing technologies can be applied to add or disrupt any gene involved in T-cell function.

Figure 4

Adding and deleting genes to enhance T-cell function. Retro- and lentiviral gene transfer can be used to redirect the specificity of T cells and the metabolic functions, response to chemokines, and cytokine secretion. Gene disruption technologies can be used to delete endogenous TCR genes, HLA genes, and genes involved in negative T-cell regulation. The indicated genes are simply examples, and the editing technologies can be applied to add or disrupt any gene involved in T-cell function.

Close modal

Optimal functional capability of engineered T cells may be achievable by the targeted disruption of genes involved in the inhibition of T-cell function and by providing transgenes that encode immune-enhancing molecules (Figure 4). Optimal function may be particularly important in solid cancer and lymphoma, where the tumor microenvironment may be low in amino acids, glucose, and oxygen and rich in immune-suppressive cytokines such as transforming growth factor β.71  Nutrient depletion and transforming growth factor β signaling can lead to the inhibition of mammalian target of rapamycin complex 1 (mTORC1) signaling and thus impair T-cell activation. Although small-molecule drugs such as rapamycin have been used to inhibit mTORC1 signaling, genetic strategies can be used to achieve enhanced mTORC1 function. For example, forced expression of Ras homolog enriched in brain in engineered T cells has led to the upregulation of mTORC1 signaling, which in an experimental murine tumor model has improved elimination of established tumors following adoptive T-cell therapy.72 

The delivery of immune stimulatory cytokines is another strategy that has been used to enhance the antitumor effect of engineered T cells. The most studied cytokine is interleukin 12 (IL-12), which was controlled by an NFAT promoter to achieve IL-12 production following TCR engagement.73  Such TCR-controlled expression can potentially overcome the substantial toxicity that is seen when IL-12 is given systemically. However, recent results of a clinical trial revealed toxic side effects of engineered T cells expressing IL-12 under the control of the NFAT promoter.73  Thus, the experience to date has shown that IL-12 is effective in enhancing antitumor activity of adoptive T-cell transfer, but cytokine-mediated toxicity remains a major challenge. The regulated expression of other cytokines and T-cell effector molecules may in the future enhance tumor protection in the absence of systemic side effects (Figure 4).

Exciting research developments combined with impressive clinical results have triggered enthusiasm for T-cell engineering in the academic, clinical, and commercial sectors. Although rapidly evolving tools enable complex genetic modifications to optimize T-cell function, the most important aspect remains the selection of the best target antigens. Although mutated cancer genes are conceptually attractive, they are rarely shared between cancers of the same tissue type, and they may not be presented effectively for TCR recognition. In contrast, a recent study of HLA-presented peptides in CLL revealed a group of 49 tumor-associated self-proteins that provide shared TCR epitopes in many patients with this type of malignancy.74  Therefore, TCRs for shared tumor-associated and linage-specific antigens remain attractive therapeutics for the treatment of hematologic malignancies.

The authors thank Duncan Gordon-Smith for assisting with the illustrations.

This work was supported by a program grant from Bloodwise and funding from the Medical Research Council, European Framework Program (EU-FP7), Biomedical Research Centre, Experimental Cancer Medicine Centre, and Wellcome Trust.

Contribution: H.J.S. and E.C.M. wrote the manuscript.

Conflict-of-interest disclosure: H.J.S. is scientific advisor and shareholder at Cell Medica. The remaining author declares no competing financial interests.

Correspondence: Hans J. Stauss, Institute of Immunity and Transplantation, Royal Free Campus, University College London, Rowland Hill St, London NW3 2PF, United Kingdom; e-mail: [email protected].

1
Maus
 
MV
Fraietta
 
JA
Levine
 
BL
Kalos
 
M
Zhao
 
Y
June
 
CH
Adoptive immunotherapy for cancer or viruses.
Annu Rev Immunol
2014
, vol. 
32
 (pg. 
189
-
225
)
2
June
 
CH
Riddell
 
SR
Schumacher
 
TN
Adoptive cellular therapy: a race to the finish line.
Sci Transl Med
2015
, vol. 
7
 
280
pg. 
280ps7
 
3
Bjorkman
 
PJ
Saper
 
MA
Samraoui
 
B
Bennett
 
WS
Strominger
 
JL
Wiley
 
DC
Structure of the human class I histocompatibility antigen, HLA-A2.
Nature
1987
, vol. 
329
 
6139
(pg. 
506
-
512
)
4
Townsend
 
AR
Rothbard
 
J
Gotch
 
FM
Bahadur
 
G
Wraith
 
D
McMichael
 
AJ
The epitopes of influenza nucleoprotein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides.
Cell
1986
, vol. 
44
 
6
(pg. 
959
-
968
)
5
Obenaus
 
M
Leitão
 
C
Leisegang
 
M
et al. 
Identification of human T-cell receptors with optimal affinity to cancer antigens using antigen-negative humanized mice.
Nat Biotechnol
2015
, vol. 
33
 
4
(pg. 
402
-
407
)
6
Schmitt
 
TM
Aggen
 
DH
Stromnes
 
IM
et al. 
Enhanced-affinity murine T-cell receptors for tumor/self-antigens can be safe in gene therapy despite surpassing the threshold for thymic selection.
Blood
2013
, vol. 
122
 
3
(pg. 
348
-
356
)
7
Tsuji
 
T
Yasukawa
 
M
Matsuzaki
 
J
et al. 
Generation of tumor-specific, HLA class I-restricted human Th1 and Tc1 cells by cell engineering with tumor peptide-specific T-cell receptor genes.
Blood
2005
, vol. 
106
 
2
(pg. 
470
-
476
)
8
van Loenen
 
MM
de Boer
 
R
Hagedoorn
 
RS
van Egmond
 
EH
Falkenburg
 
JH
Heemskerk
 
MH
Optimization of the HA-1-specific T-cell receptor for gene therapy of hematologic malignancies.
Haematologica
2011
, vol. 
96
 
3
(pg. 
477
-
481
)
9
Xue
 
SA
Gao
 
L
Hart
 
D
et al. 
Elimination of human leukemia cells in NOD/SCID mice by WT1-TCR gene-transduced human T cells.
Blood
2005
, vol. 
106
 
9
(pg. 
3062
-
3067
)
10
Falk
 
K
Rötzschke
 
O
Rammensee
 
HG
Cellular peptide composition governed by major histocompatibility complex class I molecules.
Nature
1990
, vol. 
348
 
6298
(pg. 
248
-
251
)
11
Hunt
 
DF
Michel
 
H
Dickinson
 
TA
et al. 
Peptides presented to the immune system by the murine class II major histocompatibility complex molecule I-Ad.
Science
1992
, vol. 
256
 
5065
(pg. 
1817
-
1820
)
12
Garcia
 
KC
Degano
 
M
Stanfield
 
RL
et al. 
An alphabeta T cell receptor structure at 2.5 A and its orientation in the TCR-MHC complex.
Science
1996
, vol. 
274
 
5285
(pg. 
209
-
219
)
13
Linnemann
 
C
van Buuren
 
MM
Bies
 
L
et al. 
High-throughput epitope discovery reveals frequent recognition of neo-antigens by CD4+ T cells in human melanoma.
Nat Med
2015
, vol. 
21
 
1
(pg. 
81
-
85
)
14
Lu
 
YC
Yao
 
X
Crystal
 
JS
et al. 
Efficient identification of mutated cancer antigens recognized by T cells associated with durable tumor regressions.
Clin Cancer Res
2014
, vol. 
20
 
13
(pg. 
3401
-
3410
)
15
Matsushita
 
H
Vesely
 
MD
Koboldt
 
DC
et al. 
Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting.
Nature
2012
, vol. 
482
 
7385
(pg. 
400
-
404
)
16
Robbins
 
PF
Lu
 
YC
El-Gamil
 
M
et al. 
Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells.
Nat Med
2013
, vol. 
19
 
6
(pg. 
747
-
752
)
17
Skipper
 
J
Stauss
 
HJ
Identification of two cytotoxic T lymphocyte-recognized epitopes in the Ras protein.
J Exp Med
1993
, vol. 
177
 
5
(pg. 
1493
-
1498
)
18
Tran
 
E
Turcotte
 
S
Gros
 
A
et al. 
Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer.
Science
2014
, vol. 
344
 
6184
(pg. 
641
-
645
)
19
van Rooij
 
N
van Buuren
 
MM
Philips
 
D
et al. 
Tumor exome analysis reveals neoantigen-specific T-cell reactivity in an ipilimumab-responsive melanoma.
J Clin Oncol
2013
, vol. 
31
 
32
(pg. 
e439
-
e442
)
20
Wick
 
DA
Webb
 
JR
Nielsen
 
JS
et al. 
Surveillance of the tumor mutanome by T cells during progression from primary to recurrent ovarian cancer.
Clin Cancer Res
2014
, vol. 
20
 
5
(pg. 
1125
-
1134
)
21
González-Galarza
 
FF
Takeshita
 
LY
Santos
 
EJ
et al. 
Allele frequency net 2015 update: new features for HLA epitopes, KIR and disease and HLA adverse drug reaction associations.
Nucleic Acids Res
2015
, vol. 
43
 
D1
(pg. 
D784
-
D788
)
22
Bendle
 
GM
Linnemann
 
C
Hooijkaas
 
AI
et al. 
Lethal graft-versus-host disease in mouse models of T cell receptor gene therapy.
Nat Med
2010
, vol. 
16
 
5
(pg. 
565
-
570
)
23
Duong
 
CP
Yong
 
CS
Kershaw
 
MH
Slaney
 
CY
Darcy
 
PK
Cancer immunotherapy utilizing gene-modified T cells: from the bench to the clinic.
Mol Immunol
2015
, vol. 
67
 
2, pt A
(pg. 
46
-
57
)
24
Karpanen
 
T
Olweus
 
J
T-cell receptor gene therapy--ready to go viral?
Mol Oncol
2015
, vol. 
9
 
10
(pg. 
2019
-
2042
)
25
Gilham
 
DE
Anderson
 
J
Bridgeman
 
JS
et al. 
Adoptive T-cell therapy for cancer in the United kingdom: a review of activity for the British Society of Gene and Cell Therapy annual meeting 2015.
Hum Gene Ther
2015
, vol. 
26
 
5
(pg. 
276
-
285
)
26
Morgan
 
RA
Dudley
 
ME
Wunderlich
 
JR
et al. 
Cancer regression in patients after transfer of genetically engineered lymphocytes.
Science
2006
, vol. 
314
 
5796
(pg. 
126
-
129
)
27
Brentjens
 
RJ
Davila
 
ML
Riviere
 
I
et al. 
CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapy-refractory acute lymphoblastic leukemia.
Sci Transl Med
2013
, vol. 
5
 
177
pg. 
177ra38
 
28
Kalos
 
M
Levine
 
BL
Porter
 
DL
et al. 
T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia.
Sci Transl Med
2011
, vol. 
3
 
95
pg. 
95ra73
 
29
Lee
 
DW
Kochenderfer
 
JN
Stetler-Stevenson
 
M
et al. 
T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial.
Lancet
2015
, vol. 
385
 
9967
(pg. 
517
-
528
)
30
Porter
 
DL
Levine
 
BL
Kalos
 
M
Bagg
 
A
June
 
CH
Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia.
N Engl J Med
2011
, vol. 
365
 
8
(pg. 
725
-
733
)
31
Rapoport
 
AP
Stadtmauer
 
EA
Binder-Scholl
 
GK
et al. 
NY-ESO-1-specific TCR-engineered T cells mediate sustained antigen-specific antitumor effects in myeloma.
Nat Med
2015
, vol. 
21
 
8
(pg. 
914
-
921
)
32
Robbins
 
PF
Morgan
 
RA
Feldman
 
SA
et al. 
Tumor regression in patients with metastatic synovial cell sarcoma and melanoma using genetically engineered lymphocytes reactive with NY-ESO-1.
J Clin Oncol
2011
, vol. 
29
 
7
(pg. 
917
-
924
)
33
Chodon
 
T
Comin-Anduix
 
B
Chmielowski
 
B
et al. 
Adoptive transfer of MART-1 T-cell receptor transgenic lymphocytes and dendritic cell vaccination in patients with metastatic melanoma.
Clin Cancer Res
2014
, vol. 
20
 
9
(pg. 
2457
-
2465
)
34
Johnson
 
LA
Morgan
 
RA
Dudley
 
ME
et al. 
Gene therapy with human and mouse T-cell receptors mediates cancer regression and targets normal tissues expressing cognate antigen.
Blood
2009
, vol. 
114
 
3
(pg. 
535
-
546
)
35
Parkhurst
 
MR
Joo
 
J
Riley
 
JP
et al. 
Characterization of genetically modified T-cell receptors that recognize the CEA:691-699 peptide in the context of HLA-A2.1 on human colorectal cancer cells.
Clin Cancer Res
2009
, vol. 
15
 
1
(pg. 
169
-
180
)
36
Maude
 
SL
Frey
 
N
Shaw
 
PA
et al. 
Chimeric antigen receptor T cells for sustained remissions in leukemia.
N Engl J Med
2014
, vol. 
371
 
16
(pg. 
1507
-
1517
)
37
Fuchs
 
EJ
Matzinger
 
P
B cells turn off virgin but not memory T cells.
Science
1992
, vol. 
258
 
5085
(pg. 
1156
-
1159
)
38
Teulings
 
HE
Limpens
 
J
Jansen
 
SN
et al. 
Vitiligo-like depigmentation in patients with stage III-IV melanoma receiving immunotherapy and its association with survival: a systematic review and meta-analysis.
J Clin Oncol
2015
, vol. 
33
 
7
(pg. 
773
-
781
)
39
Yeh
 
S
Karne
 
NK
Kerkar
 
SP
et al. 
Ocular and systemic autoimmunity after successful tumor-infiltrating lymphocyte immunotherapy for recurrent, metastatic melanoma.
Ophthalmology
2009
, vol. 
116
 
5
(pg. 
981
-
989
)
40
Byrne
 
KT
Côté
 
AL
Zhang
 
P
et al. 
Autoimmune melanocyte destruction is required for robust CD8+ memory T cell responses to mouse melanoma.
J Clin Invest
2011
, vol. 
121
 
5
(pg. 
1797
-
1809
)
41
Ghorashian
 
S
Veliça
 
P
Chua
 
I
et al. 
CD8 T cell tolerance to a tumor-associated self-antigen is reversed by CD4 T cells engineered to express the same T cell receptor.
J Immunol
2015
, vol. 
194
 
3
(pg. 
1080
-
1089
)
42
Martelli
 
MF
Di Ianni
 
M
Ruggeri
 
L
et al. 
HLA-haploidentical transplantation with regulatory and conventional T-cell adoptive immunotherapy prevents acute leukemia relapse.
Blood
2014
, vol. 
124
 
4
(pg. 
638
-
644
)
43
Pane
 
F
Intrieri
 
M
Quintarelli
 
C
Izzo
 
B
Muccioli
 
GC
Salvatore
 
F
BCR/ABL genes and leukemic phenotype: from molecular mechanisms to clinical correlations.
Oncogene
2002
, vol. 
21
 
56
(pg. 
8652
-
8667
)
44
Posthuma
 
EF
van Bergen
 
CA
Kester
 
MG
et al. 
Proteosomal degradation of BCR/ABL protein can generate an HLA-A*0301-restricted peptide, but high-avidity T cells recognizing this leukemia-specific antigen were not demonstrated.
Haematologica
2004
, vol. 
89
 
9
(pg. 
1062
-
1071
)
45
Ramsköld
 
D
Wang
 
ET
Burge
 
CB
Sandberg
 
R
An abundance of ubiquitously expressed genes revealed by tissue transcriptome sequence data.
PLOS Comput Biol
2009
, vol. 
5
 
12
pg. 
e1000598
 
46
Weimershaus
 
M
Evnouchidou
 
I
Saveanu
 
L
van Endert
 
P
Peptidases trimming MHC class I ligands.
Curr Opin Immunol
2013
, vol. 
25
 
1
(pg. 
90
-
96
)
47
Beekman
 
NJ
van Veelen
 
PA
van Hall
 
T
et al. 
Abrogation of CTL epitope processing by single amino acid substitution flanking the C-terminal proteasome cleavage site.
J Immunol
2000
, vol. 
164
 
4
(pg. 
1898
-
1905
)
48
Ossendorp
 
F
Eggers
 
M
Neisig
 
A
et al. 
A single residue exchange within a viral CTL epitope alters proteasome-mediated degradation resulting in lack of antigen presentation.
Immunity
1996
, vol. 
5
 
2
(pg. 
115
-
124
)
49
Momburg
 
F
Roelse
 
J
Howard
 
JC
Butcher
 
GW
Hämmerling
 
GJ
Neefjes
 
JJ
Selectivity of MHC-encoded peptide transporters from human, mouse and rat.
Nature
1994
, vol. 
367
 
6464
(pg. 
648
-
651
)
50
Falk
 
K
Rötzschke
 
O
Stevanović
 
S
Jung
 
G
Rammensee
 
HG
Allele-specific motifs revealed by sequencing of self-peptides eluted from MHC molecules.
Nature
1991
, vol. 
351
 
6324
(pg. 
290
-
296
)
51
Alexandrov
 
LB
Nik-Zainal
 
S
Wedge
 
DC
et al. 
Australian Pancreatic Cancer Genome Initiative; ICGC Breast Cancer Consortium; ICGC MMML-Seq Consortium; ICGC PedBrain
Signatures of mutational processes in human cancer [published correction appears in Nature. 2013;502(7470):258].
Nature
2013
, vol. 
500
 
7463
(pg. 
415
-
421
)
52
Rodriguez
 
F
Slifka
 
MK
Harkins
 
S
Whitton
 
JL
Two overlapping subdominant epitopes identified by DNA immunization induce protective CD8(+) T-cell populations with differing cytolytic activities.
J Virol
2001
, vol. 
75
 
16
(pg. 
7399
-
7409
)
53
Sadovnikova
 
E
Jopling
 
LA
Soo
 
KS
Stauss
 
HJ
Generation of human tumor-reactive cytotoxic T cells against peptides presented by non-self HLA class I molecules.
Eur J Immunol
1998
, vol. 
28
 
1
(pg. 
193
-
200
)
54
Cohen
 
CJ
Li
 
YF
El-Gamil
 
M
Robbins
 
PF
Rosenberg
 
SA
Morgan
 
RA
Enhanced antitumor activity of T cells engineered to express T-cell receptors with a second disulfide bond.
Cancer Res
2007
, vol. 
67
 
8
(pg. 
3898
-
3903
)
55
Cohen
 
CJ
Zhao
 
Y
Zheng
 
Z
Rosenberg
 
SA
Morgan
 
RA
Enhanced antitumor activity of murine-human hybrid T-cell receptor (TCR) in human lymphocytes is associated with improved pairing and TCR/CD3 stability.
Cancer Res
2006
, vol. 
66
 
17
(pg. 
8878
-
8886
)
56
Kuball
 
J
Dossett
 
ML
Wolfl
 
M
et al. 
Facilitating matched pairing and expression of TCR chains introduced into human T cells.
Blood
2007
, vol. 
109
 
6
(pg. 
2331
-
2338
)
57
Sommermeyer
 
D
Uckert
 
W
Minimal amino acid exchange in human TCR constant regions fosters improved function of TCR gene-modified T cells.
J Immunol
2010
, vol. 
184
 
11
(pg. 
6223
-
6231
)
58
Heemskerk
 
MH
Hagedoorn
 
RS
van der Hoorn
 
MA
et al. 
Efficiency of T-cell receptor expression in dual-specific T cells is controlled by the intrinsic qualities of the TCR chains within the TCR-CD3 complex.
Blood
2007
, vol. 
109
 
1
(pg. 
235
-
243
)
59
Ahmadi
 
M
King
 
JW
Xue
 
SA
et al. 
CD3 limits the efficacy of TCR gene therapy in vivo.
Blood
2011
, vol. 
118
 
13
(pg. 
3528
-
3537
)
60
Provasi
 
E
Genovese
 
P
Lombardo
 
A
et al. 
Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer.
Nat Med
2012
, vol. 
18
 
5
(pg. 
807
-
815
)
61
Thomas
 
S
Xue
 
SA
Bangham
 
CR
Jakobsen
 
BK
Morris
 
EC
Stauss
 
HJ
Human T cells expressing affinity-matured TCR display accelerated responses but fail to recognize low density of MHC-peptide antigen.
Blood
2011
, vol. 
118
 
2
(pg. 
319
-
329
)
62
Valitutti
 
S
Müller
 
S
Cella
 
M
Padovan
 
E
Lanzavecchia
 
A
Serial triggering of many T-cell receptors by a few peptide-MHC complexes.
Nature
1995
, vol. 
375
 
6527
(pg. 
148
-
151
)
63
Cameron
 
BJ
Gerry
 
AB
Dukes
 
J
et al. 
Identification of a Titin-derived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells.
Sci Transl Med
2013
, vol. 
5
 
197
pg. 
197ra103
 
64
Morgan
 
RA
Chinnasamy
 
N
Abate-Daga
 
D
et al. 
Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy.
J Immunother
2013
, vol. 
36
 
2
(pg. 
133
-
151
)
65
Kerkar
 
SP
Sanchez-Perez
 
L
Yang
 
S
et al. 
Genetic engineering of murine CD8+ and CD4+ T cells for preclinical adoptive immunotherapy studies.
J Immunother
2011
, vol. 
34
 
4
(pg. 
343
-
352
)
66
Mucida
 
D
Husain
 
MM
Muroi
 
S
et al. 
Transcriptional reprogramming of mature CD4⁺ helper T cells generates distinct MHC class II-restricted cytotoxic T lymphocytes.
Nat Immunol
2013
, vol. 
14
 
3
(pg. 
281
-
289
)
67
Stevanović
 
S
van Bergen
 
CA
van Luxemburg-Heijs
 
SA
et al. 
HLA class II upregulation during viral infection leads to HLA-DP-directed graft-versus-host disease after CD4+ donor lymphocyte infusion.
Blood
2013
, vol. 
122
 
11
(pg. 
1963
-
1973
)
68
Morris
 
EC
Tsallios
 
A
Bendle
 
GM
Xue
 
SA
Stauss
 
HJ
A critical role of T cell antigen receptor-transduced MHC class I-restricted helper T cells in tumor protection.
Proc Natl Acad Sci USA
2005
, vol. 
102
 
22
(pg. 
7934
-
7939
)
69
Xue
 
SA
Gao
 
L
Ahmadi
 
M
et al. 
Human MHC class I-restricted high avidity CD4(+) T cells generated by co-transfer of TCR and CD8 mediate efficient tumor rejection in vivo.
OncoImmunology
2013
, vol. 
2
 
1
pg. 
e22590
 
70
Qasim
 
W
Amrolia
 
PJ
Samarasinghe
 
S
et al. 
First clinical application of Talen engineered universal CAR19 T cells in B-ALL [abstract].
Blood
2015
, vol. 
126
 
23
 
Abstract 2046
71
Noy
 
R
Pollard
 
JW
Tumor-associated macrophages: from mechanisms to therapy [published correction appears in Immunity. 2014;41(5):866].
Immunity
2014
, vol. 
41
 
1
(pg. 
49
-
61
)
72
Veliça
 
P
Zech
 
M
Henson
 
S
et al. 
Genetic regulation of fate decisions in therapeutic T cells to enhance tumor protection and memory formation [published correction appears in Cancer Res. 2015;75(24):5402].
Cancer Res
2015
, vol. 
75
 
13
(pg. 
2641
-
2652
)
73
Zhang
 
L
Morgan
 
RA
Beane
 
JD
et al. 
Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding interleukin-12 for the immunotherapy of metastatic melanoma.
Clin Cancer Res
2015
, vol. 
21
 
10
(pg. 
2278
-
2288
)
74
Kowalewski
 
DJ
Schuster
 
H
Backert
 
L
et al. 
HLA ligandome analysis identifies the underlying specificities of spontaneous antileukemia immune responses in chronic lymphocytic leukemia (CLL) [published correction appears in Proc Natl Acad Sci USA. 2015;112(45):E6254-E6256 and E6258-E6260].
Proc Natl Acad Sci USA
2015
, vol. 
112
 
2
(pg. 
E166
-
E175
)
Sign in via your Institution