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Seminars in Immunology 28 (2016) 10–21

Contents lists available at ScienceDirect

Seminars in Immunology

j our na l ho me pa ge: www.elsev ier .com/ locate /ysmim

CR-engineered T cells to treat tumors: Seeing but not touching?

eno Debetsa,∗, Emmanuel Donnadieub,c,d,e, Salem Chouaib f, George Coukosg

Laboratory of Tumor Immunology, Department of Medical Oncology, Erasmus MC Cancer Institute, Rotterdam, The NetherlandsInserm U1016, Institut Cochin, Paris, FranceCNRS UMR8104, Paris, FranceUniversité Paris Descartes, Paris, FranceEquipe Labellisée Ligue Contre le Cancer, Paris, FranceINSERM U1186, Equipe Labellisée Ligue Contre le Cancer, Gustave Roussy Campus, Villejuif, FranceDepartment of Oncology, University Hospital of Lausanne (CHUV), University of Lausanne, Switzerland

r t i c l e i n f o

rticle history:eceived 23 February 2016eceived in revised form 2 March 2016ccepted 4 March 2016vailable online 17 March 2016

a b s t r a c t

Adoptive transfer of T cells gene-engineered with T cell receptors (TCRs) has proven its feasibility andtherapeutic potential in the treatment of malignant tumors. To ensure further clinical development of TCRgene therapy, it is necessary to accurately select TCRs that demonstrate antigen-selective responses thatare restricted to tumor cells and, at the same time, include strategies that restore or enhance the entry,migration and local accumulation of T cells in tumor tissues. Here, we present the current standing of TCR-engineered T cell therapy, discuss and propose procedures to select TCRs as well as strategies to sensitize

eywords:ntigen recognition

mmune evasion cell cell receptor cell trafficking

the tumor to T cell trafficking, and provide a rationale for combination therapies with TCR-engineered Tcells.

© 2016 Elsevier Ltd. All rights reserved.

umor micro-environment

Abbreviations: 5-AZA, 5-azacytidine; bFGF, basic fibroblast growth factor; BORIS,rother of regulator of imprinted sites; CAF, cancer-associated fibroblast; CAR,himeric antigen receptor; CCL, chemokine (CC motif) ligand; CDR, complemen-ary determining region; CEA, carcinoembryonic antigen; CR, complete response;TLA-4, cytotoxic T lymphocyte antigen-4; CXCL, chemokine (CXC motif) lig-nd; DZNep, 3-deazaneplanocin A; ECM, extracellular matrix; ETBR, endothelin Beceptor; FAP-�, fibroblast activated protein-�; gp100, glycoprotein 100; HERV,uman endogenous retrovirus; HIF, hypoxia inducible factor; HLA, human leuko-yte antigen; HPV-E6, human papillomavirus-E6; ICAM-1, intercellular adhesionolecule-1; IDO1, indoleamine 2,3-dioxygenase; IFN, interferon; IL, interleukin;

AGE-1, L antigen family member-1; LOX, lysyl oxidase; MAGE, melanoma-ssociated antigen; MART-I, melanoma antigen recognized by T cells-I; MDSC,yeloid derived suppressive cells; MHC, major histocompatibility complex; NFAT,

uclear factor of activated T cells; NF-�B, nuclear factor-kappa B; nr, none reported;SG, non-obese diabetic, severe combined immunodeficiency, interleukin-2 recep-

or gamma-deficient; NY-ESO-1, New York esophageal squamous cell carcinomantigen-1; OR, objective response; PD-1, programmed cell death-1; PD-L, pro-rammed death-ligand; PI3K, phosphoinositide 3 kinase; PGE2, prostaglandin E2;R, partial response; PTEN, phosphatase and tensin homolog; R, receptor; RT-CR, reverse transcriptase-polymerase chain reaction; TCR, T cell receptor; TGF-�,ransforming growth factor-�; Th17, T helper-type 17 cells; TME, tumor micro-nvironment; TNF-�, tumor necrosis factor-�; Treg, regulatory T cells; VCAM-1,ascular adhesion molecule-1; VEGF, vascular endothelial growth factor∗ Corresponding author at: Laboratory of Tumor Immunology, Erasmus MC Cancer

nstitute, Office Be-430b, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands.E-mail address: j.debets@erasmusmc.nl (R. Debets).

ttp://dx.doi.org/10.1016/j.smim.2016.03.002044-5323/© 2016 Elsevier Ltd. All rights reserved.

1. Therapy with TCR-engineered T cells: setting therequirements

Therapy with TCR-engineered T cells entails the transfer ofautologous T cells following genetic introduction of a T cell recep-tor (TCR) to treat patients with cancer or other diseases. TCR�and � genes are envisioned as “off the shelf” reagents to confertumor reactivity to patients whose tumor expresses the appro-priate antigen. Clinical activities that have been reported withTCR-engineered T cells are summarized in Table 1, and at leasta dozen other trials are currently open and actively recruitingpatients (www.clinicaltrials.gov).

Most clinical TCRs tested so far were Human Leukocyte Antigen(HLA-)A1 and A2-restricted and directed against either differen-tiation, onco-fetal or cancer germline antigens. Collectively, thesetrials have demonstrated significant clinical responses in patientswith metastatic melanoma, colorectal carcinoma and synovial sar-coma (see Table 1 for references). Currently, clinical outcomesare confronted by treatment-related toxicities and a transientnature of tumor regression. Treatment-related toxicities are caused

either by a target antigen that is (besides tumor cells) expressedon healthy cells (on-target toxicity) or by a TCR that recognizes(besides target antigen) a highly similar antigen expressed byhealthy cells (off-target toxicity). Examples of on-target toxicity

dx.doi.org/10.1016/j.smim.2016.03.002http://www.sciencedirect.com/science/journal/10445323http://www.elsevier.com/locate/ysmimhttp://crossmark.crossref.org/dialog/?doi=10.1016/j.smim.2016.03.002&domain=pdfmailto:j.debets@erasmusmc.nlhttp://www.clinicaltrials.govhttp://www.clinicaltrials.govhttp://www.clinicaltrials.govdx.doi.org/10.1016/j.smim.2016.03.002

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Table 1Overview of clinical TCR T cell trials.

Antigen (epitope) Source forTCR

TCR genemodifications (TCRcode)

Malignancy No. ofpatients

OR (%) PR CR Immunerelated adverseeffects

References

MART-I/HLA-2 (AAG) DMF4T cellclone

No Melanoma 17 2 (12) 2 0 nr [143]

MART-I/HLA-2 (AAG) DMF5T cellclone

No Melanoma 20 6 (30) 6 0 Destruction ofnormalmelanocytes,anterioruveitis, hearingloss

[144]

MART-I/HLA-A2 (ELA) 1D3T cellclone

TCR-Cmurinization,incorporation ofadditional cyteineresidue, and codonoptimization

Melanoma 1a Lethal cardiactoxicity

[145]

gp100/HLA-2 (KTW) Immunizedmice

No Melanoma 16 3 (19) 2 1 Destruction ofnormalmelanocytes,anterioruveitis, hearingloss

[144]

CEA/HLA-A2 (IMI) Immunizedmice

CDR3� mutation(S112T)

Colon carcinoma 3 1 (33) 1 0 Severeinflammatorycolitis

[146]

NY-ESO-1, LAGE-1/HLA-A2(SLL)

1G4T cell clone CDR3� mutations(1G4-�95:LY)

Melanoma 20 11 (55) 7 4 nr [147,148]Synovial cell sarcoma 18 11 (61) 10 1

NY-ESO-1, LAGE-1/HLA-A2 (SLL) 1G4T cellclone

CDR mutations(clone 259)b

Multiple Myeloma 20 18 (90) 4 14c nr [149]

MAGE-A3, A9/HLA-A2 (KVA) Immunizedmice

CDR3� mutations(A118T)

Melanoma 9 5 (56) 3 2 Changes inmental status,coma anddeath (n = 2)

[69]

MAGE-A3/HLA-A1 (EVD) nr CDR2� mutations (a3a) Melanoma 1 0 (0) 0 0 Lethal cardiactoxicity

[150]

Multiple myeloma 1 0 (0) 0 0 Lethal cardiactoxicity

See abbreviations.a Study has been amended and re-opened, but at moment of writing results are not published yet.b NY-ESO-1 c259 T cells have also been used to treat patients with synovial sarcoma and ovarian carcinoma, but at moment of writing results are not published yet.c CR includes nearly and stringent CR.

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nclude severe inflammation of skin, eyes, ears (melanoma anti-en recognized by T cells (MART-)I/HLA-A2; glycoprotein (gp)00/HLA-A2) and colon (carcinoembryonic antigen (CEA)/HLA-2). Examples of off-target toxicity include neurological toxicities

melanoma-associated antigen (MAGE-)A3, A9/HLA-A2 TCR mostikely recognized MAGE-A12/HLA-A2 epitope), and cardiac toxi-ities (MAGE-A3/HLA-A1 TCR recognized Titin/HLA-A1 epitope).n addition to mentioned toxicities, in some trials T-cell medi-ted inflammation or cytokine release syndrome has been notedMART-I/HLA-A2, other epitope as above; and New York esophagealquamous cell carcinoma antigen (NY-ESO-) 1, L antigen familyember (LAGE-) 1/HLA-A2).Anti-tumor responses may lead to transient tumor regression

ut are not sustainable in the majority of patients. Escape ofntigen-negative tumor variants following T cell-dependent elim-nation of antigen-positive cancer cells, i.e., immune editing of theumor, is not necessarily a driving mechanism in such scenariosf tumor progression or recurrence. Early studies from the Lud-ig Institute for Cancer Research, Brussels, demonstrated that inatients receiving MAGE-A1 targeting immunotherapy, progress-

ng melanoma lesions contain MAGE-A1-positive, immunogenicancer cells [1]. In extension to these studies, it was observedhat mouse melanoma that has become resistant to T cell treat-

ent showed continued expression of the cognate antigen and,hen re-transplanted on to mice, regained sensitivity to T cell-

reatment [2]. These studies point to the possible involvementf tumor micro-environment (TME) mechanisms contributingowards tumor progression and resistance to T cell therapy. In fact,tromal cells surrounding tumor cells may play a detrimental rolen limiting T cell trafficking towards and within tumors and localctivation of T cells ([3,4], and studies mentioned in Section 3.1).

Important and timely requirements advance the clinical devel-pment of TCR-engineered T cells include the selection ofumor-specific TCRs as well as strategies to enhance the success-ul engraftment of T cells in tumors. In this review, we will addressoth requirements in more detail, and propose directions for futurerials with TCR-engineered T cells.

. Selection of T cell receptors

.1. Antigen first

The presence of antigen is the prime reason for T cells to accu-ulate and become activated in tumors. Candidate target antigens

or T cell treatment should preferably fulfill the following criteria:e selectively expressed in tumors and not in normal tissues (tumorpecificity); be related to oncogenesis (tumor addiction); and be ableo evoke a T cell response (immunogenicity).

.1.1. Antigens with selective expression in tumorsConsidering the potency of T cell therapy, selective expression

f antigens in tumors is one of the most important criteria for tar-et antigens. Candidate antigens that demonstrate tumor-selectivexpression include neo-antigens, oncoviral and cancer germlinentigens. Neo-antigens are derived from somatic alterations, andhe higher the mutational load, the higher the frequency of suchntigens within cancers. Identification of neo-antigens requireshole genome or exome sequencing to identify tumor-specificutations, RNA sequencing to examine expression, and prediction

lgorithms to determine whether a neo-epitope will be presentedy major histocompatibility complex (MHC) and recognized by T

ells [5–7]. Viral antigens, although mostly cleared following annti-virus immune response, can, in case of retroviruses, be incor-orated in the human genome and become reactivated in a numberf tumors [8]. Such endogenous viruses, often genetically compro-

unology 28 (2016) 10–21

mised and not able anymore to produce a virus, may provide asource of non-self antigens. In example, human endogenous retro-virus (HERV-)E is expressed in the majority of renal cell cancersbut not in healthy tissue and is able to evoke cellular and humoralimmune responses [8]. MAGEs and NY-ESO-1 represent examplesof the third group of antigens, cancer germline antigens, that showgene-expression that is de-repressed in tumor cells yet strictlyrepressed in normal cells. Gene de-repression coincides with his-tone acetylation, DNA de-methylation, dissociation of polycombproteins, and binding of the transcription factor brother of reg-ulator of imprinted sites (BORIS) at the promotor site of cancergermline antigens [9]. BORIS is a mammalian CCCTC-binding fac-tor paralog, absent in normal cells, and which acts on opening andactivation of DNA chromatin in tumor cells [10,11]. Another lineof evidence that certain cancer germline antigens do not induceon-target toxicities comes from studies in which induction of sig-nificant numbers of MAGE-specific T cells results in clinical benefitwithout detectable toxicity [12]. In this communication, we explic-itly recommend, irrespectively of choice target antigen, to test theantigen’s absence from a large panel of healthy organs by reversetranscription-polymerase chain reaction (RT-PCR) and/or immunehistochemistry as illustrated in Fig. 1A.

2.1.2. Antigens related to oncogenesisThe targeting of antigens that provide tumors with a growth

advantage is less likely to result in escape and progression ofantigen-negative tumor cells. With respect to neo-antigens, muta-tions related to oncogenic pathways may represent perfect T celltargets. In fact, adoptive T cell transfer studies revealed that sig-nificant tumor regression and, in some cases, durable responsesin patients with melanoma or cholangiocarcinoma were accompa-nied by T cell reactivity against mutated antigens related to cellularproliferation [13,14]. Realistically, however, true driver mutationscover only a fraction of the total number of mutations (estimatedat most at 15%), and only a small fraction of mutations consti-tutes predicted T cell epitopes (about 1%) [15]. In addition, ofthese only a negligible fraction is shared between patients [16].For oncoviral antigens, it is important to note that more than 10%of human cancers is caused by viruses, such as Epstein-Barr virus,human papillomaviruses (HPV), and human T cell lymphotropicvirus-1 [17]. Following the initial infection, viral products maycontribute to oncogenesis, which over a period of several yearsto decades may result in the formation of virally-induced tumors[8,17]. A number of cancer germline antigens has been associ-ated with advanced stages of various cancers and unfavourablepatient prognoses [18]. Interestingly, MAGE proteins have beenreported to interact and enhance ubiquitin ligases thereby sup-pressing p53-dependent apoptosis [19–21]. Also, these proteinscan cause fibronectin-controlled increase in tumor cell prolifera-tion and metastasis [22], and induce epithelial-to-mesenchymaltransition [23].

2.1.3. Antigens that are sufficiently immunogenicSetting aside the importance of the above two criteria, antigens

should be able to evoke a T cell response. The immunogenicityof neo-antigens has been elegantly revealed by genomic studiesin patients with melanoma or lung cancer treated with check-point inhibitors, which argued that clinical benefit correlates tomutational load [24,25]. Also, in microsatellite-instable colorec-tal cancer the density of intra-tumoral CD8 T cell infiltration wasfound to correlate to the number of frameshift mutations [26]. Infact, neo-antigens, by many tumor immunologists, are considered a

primary source of immunogenic tumor antigens. However, cautionshould be exercised when generalizing such principles. First, notin all human cancers, CD8 T cell density correlates to mutationalload, and, second, in preclinical mouse models not all predicted

R. Debets et al. / Seminars in Immunology 28 (2016) 10–21 13

Fig. 1. Testing on-target and off-target recognition of a TCR transgene.A TCR transgene is obtained, and in some cases enhanced, to recognize and bind a tumor epitope of interest (upper part of figure). Importantly, therapeutic use of TCRtransgenes warrants stringent safety tests, which should cover two angles: on-target and off-target recognition. (A) Recognition of the cognate peptide (i.e., on-target,depicted in purple) outside tumor tissue can be assessed by ‘a healthy tissue scan’ using quantitative PCRs on cDNAs derived from a large set of different healthy tissues (asshown in Figure). Alternatively, in situ stainings (when antibody is available) on healthy tissues could be employed. (B) Recognition of a highly similar peptide (i.e., off-target,d detaila y testh

nttroccatcba

oicestiomaidipariae

epicted in purple/blue) can be assessed via various methods, see Section 2.2 for lanine replacements at every single position (as shown in figure) represents a keuman proteins containing this motif.

eo-antigens produce an effective T cell response [27] nor is muta-ional load a prerequisite for checkpoint inhibitors to unleash aumor-specific T cell response [28]. The immunogenicity of oncovi-al antigens is generally high. A well-known example is HPV-E6ncoprotein, of which HLA-A2-restricted epitopes clearly evoke a Tell response. In fact, a TCR derived from T cells present in anal can-er from a patient who experienced a prolonged disease-free periodfter surgery, enabled the generation of T cells that recognizedumor cells positive for HPV-E16 [29]. In contrast to viral antigens,ancer germline antigens generally provide lower immunogenicity,ut with additional strategies such immunogenicity can be lifted tollow the therapeutic targeting of such antigens (see below).

Recent studies demonstrate that the level of expression, theff-rate of the peptide epitope from the MHC complex, and thenvolvement of CD4 T cell epitopes represent key attributes thatontribute to a target’s immunogenicity. With respect to level ofxpression, there is the timely notion that epigenetic drugs canensitize tumors to T cell therapy [30,31]. Treatment with agentshat mediate demethylation and/or histone acetylation resultedn enhanced and more homogeneous intra-tumoral expressionf cancer germline antigens [32,33]. Interestingly, such therapiesay also up-regulate expression of endogenous retroviral antigens

long with an interferon signature, which contributes to tumormmunogenicity [34]. Notably, experiments in mouse models haveemonstrated that the gene-inducing potential of demethylat-

ng agents is restricted to tumors and not normal tissue [35]. Inatient studies, findings with epigenetic agents confirmed safetynd showed that these agents result in the induction of T cell

esponses directed against cancer germline antigens [36,37]. Strik-ngly, the combination of epigenetic drug treatment of tumor cellsnd the use of TCR-engineered T cells resulted in a drastic syn-rgy with respect to MAGE-C2-specifc T cell responses [38]. The

s. The testing of TCR-engineered T cells versus altered peptide ligands containing and allows the determination of the recognition motif and search and testing of

potency of T cell responses may be further governed by the offrate of peptide from the corresponding MHC molecule [39]. Longerbinding times of peptide result in improved presentation, possiblycross-presentation by stromal cells, and enhanced T cell responses.Finally, preclinical studies have suggested that the targeting ofCD4 T cell antigens contributes to the effector phase of anti-tumorresponses [40]. CD4 T cell responses, in particular T helper-type 17(Th17) responses, following immunization with three MHC classII-restricted antigens were accompanied by cures of tumor-bearingmice [41]. With respect to human antigens, it is interesting to notethat certain X-chromosome-linked cancer germline antigens arecoordinately expressed in tumor tissues [42], which may allow thesimultaneous targeting of such antigens.

2.2. T cell receptor second

Following the choice of target antigen, the next step is to obtainantigen-specific T cells and derive the corresponding TCRs. Nowa-days, with available molecular tools, one can genetically enhanceTCRs and improve expression and function of TCRs. Clinical expe-rience so far (Table 1), however, does warrant stringent safetyassessment of TCRs prior to their therapeutic testing of in patients.

2.2.1. Obtaining T cell receptors, to be tolerant or not?Procedures to obtain antigen-specific T cells can generally be

divided into those that rely on tolerant and those that rely onnon-tolerant repertoires of T cells. Tolerant repertoires, where dele-tion has occurred of T cells with an avidity outside the thymic

selection window, have been used to obtain T cell clones frompatients following successful TIL therapy (Table 1, MART-I-specificDMF4 and 5 clones) or peptide vaccination (MART-I-specific 1D3clone and NY-ESO-1-specific 1G4 clone). In addition, tolerant T cells

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an be obtained from in vitro systems using autologous dendriticells, a system that has demonstrated successes in the genera-ion of antigen-specific T cells. The use of non-tolerant repertoires,ith the rationale of allowing the generation of high-avidity T

ells, is exemplified by in vitro as well as in vivo systems. In vitro,LA-mismatched antigen-presenting cells [43] or artificial antigen-resenting cells positive for HLA-A2, loaded with a peptide of

nterest, and co-stimulatory ligands [44] can be co-cultured with cells and used to generate antigen-specific T cells. In vivo, miceransgenic for human HLA and immunized with human antigensave been used to generate T cells (Table 1, gp100-specific TCR).long the same lines, mice transgenic for human TCR and HLA-2 genes, with murine TCR and H2 genes being inactivated [45]ave been immunized and used as a source of TCRs. The latterystem yielded anti-MAGE-A1 and NY-ESO-1 TCRs with improvedunctional performance in vitro and in vivo compared to TCRserived from human donors [46]. After having obtained antigen-pecific T cells, these T cells can be cloned at the single cell levelnd sequences of the TCR� and � chains can be determined byolecular techniques such as 5′ RACE [47]. Recent developments

ircumvent the requirement to make single cell clones but ratherely on directly isolating single T cells and enhanced PCR meth-ds. Alternatively, methods have been developed to obtain correctCR�� pairs from larger populations of T cells present in peripherallood or tumor samples, which rely on emulsion PCR [48], cap-uring and indexing of genomic DNA-encoding TCR chains [49],r sequencing and pairing of TCR chains based on combinatoriallgorithms [50].

.2.2. Genetic enhancement of T cell receptorsGenetic engineering of TCRs can be used to enhance the

eceptor’s surface expression, ligand-binding affinity and signal-ng potency. Surface expression of TCRs can be improved through

ultiple strategies, some of which do not depend on gene engi-eering and are reviewed elsewhere [51–53]. Gene-engineeringf TCRs with the intent to improve pairing of the correspondingCR� and � chains generally results in improved surface expres-ion and is thoroughly reviewed in Ref. [54]. TCR affinities can benhanced to very high levels by rationally designed mutagenesis ofomplementary-determining region (CDR) loops [55] or phage dis-lay selection from libraries of CDR site-directed TCR mutants [56].linical reports may suggest that CDR mutations in TCRs directedgainst CEA/HLA-A2, MAGE-A3/HLA-A2 and MAGE-A3/HLA-A1,ut not NY-ESO-1/HLA-A2, were associated with patient toxici-ies (Table 1). Early studies already argued that high-affinity TCRsossess the ability to react against self-peptide MHC [57]. Struc-ural analysis of the interaction between affinity-enhanced TCRith either the cognate peptide MAGE-A3 or a cross-reactive

itin demonstrated that loss of antigen discrimination is governedy direct molecular mimicry and is difficult to restore by addi-ional TCR mutations [58]. In addition, affinity enhancement ofCRs may be confronted by a functional ceiling of TCR-mediatedesponses [59,60]. Studies with primary human T cells trans-uced with affinity-enhanced TCRs pointed to the existence of ahreshold off-rate (in this case between pMHC and TCR), belowhich T cells showed an enhanced expression of PD1 and became

unctionally compromised ([61], Govers et al., manuscript submit-ed). Lastly, in an effort to enhance T cell co-signaling, one canquip TCR transgenes with a cassette that harbors a co-stimulatoryolecule. Such a signaling cassette typically introduces accessory

nd co-stimulatory molecules to improve the function of T cellsxpressing the TCR transgene. It is noteworthy that clinical trials

sing chimeric antigen receptors (CARs) containing CD28 or CD137emonstrated significant objective responses in patients with B cell

eukemia [62–64]. Our laboratory has coupled two-chain TCR geneso a combination of CD28 and CD3 molecules, and the resulting

unology 28 (2016) 10–21

TCRs were shown to provide T cells with improved function in vitro[65] and prolonged peripheral persistence and anti-tumor activityin vivo [66]. Collectively, studies with gene-enhanced TCRs call forprudency and, if one chooses to proceed with such TCRs, stringentpreclinical assessment is highly recommended (see below).

2.2.3. Preclinical assessment of T cell receptorsTesting of TCRs, once introduced into T cells, starts with in vitro

analysis of the sensitivity of T cell responses towards the cognatepeptide. To this end, one generally performs peptide titrations usingtarget cells expressing the restrictive HLA allele and determinesthe effective concentration to induce a 50% effect (EC50 value) asa surrogate marker for T cell avidity. Next, TCRs are tested againsttumor cells natively expressing the antigen and HLA allele of inter-est. To test additional parameters, such as T cell trafficking to andwithin the tumor, T cell persistence and anti-tumor responses, itis recommended to use mouse models. Mice transgenic HLA-A2transplanted with a syngeneic tumor that is forced to express thehuman antigen of interest [2,46], or immune-deficient mice (NSG:non-obese diabetic (NOD), severe combined immunodeficiency(SCID), interleukin-2 receptor gamma-deficient) transplanted withhuman tumor that endogenously expresses the antigen and HLAallele of interest [67] are suitable models. As mentioned above, TCRsshould not move into clinical trials before stringent safety analy-ses. This is particularly true for TCRs from a non-tolerant repertoireand/or following gene enhancement. In silico and in vitro strate-gies to address off-target reactivities are highly recommended inthis respect [38,46,68]. We would like to recommend that suchtests include assessment of responsiveness towards: self peptides(for HLA-A2, described in Ref. [69]); sequence-related peptides asidentified by NCBI-BLAST; and altered peptide ligands containingamino acid replacements (mostly alanines) at every single position.In particular the latter test is relevant as it allows the determinationof the peptide recognition motif and identification of human pro-teins containing such a motif as illustrated in Fig. 1B. Various newTCRs against cancer germline antigens (see above-mentioned ref-erences) have passed these preclinical tests, and are now scheduledto be tested in patient trials.

3. Sensitizing tumors for adoptively transferred T cells

The paradigm-shifting realization that tumor heterogeneity isdetermined at both the cellular (tumor cells) as well as tissue level(TME) is important to understand the success (or lack thereof) ofadoptive T cell therapy. In addition to tumor cell heterogeneity, spa-tial information of immune cells in TME is important to improveour understanding of therapy responsiveness [70]. Notably, theabsence of immune effector cells, such as CD8 T cells, insidetumors and their confinement to the surrounding stroma has beenreported to provide worsened prognostic value in many tumortypes [71–73]. Intriguingly, tissues that harbor highly effective CD8T cells, such as allorejected kidney transplants, show a distinctivesignature of genes related to interferon (IFN) responses, T cell traf-ficking and T cell effector functions [74]. When such lessons aretranslated to tumors, it was found that high numbers of intra-tumoral CD8 T cells indeed coincide with elevated expression of Tcell trafficking molecules [75]. Importantly, current T cell therapies,including the checkpoint inhibitors anti-cytotoxic T lymphocyteassociated (CTLA-)4 and programmed cell death (PD-)1 antibodies,are ineffective in a significant portion of patients with melanoma,lung, renal and other cancers, which is in part related to a lack of

intra-tumoral trafficking of T cells [76,77].

In the second part of this review, we will focus on the barri-ers tumors impose towards effective trafficking and intra-tumoralaccumulation of T cells, and how such barriers can be successfully

R. Debets et al. / Seminars in Immunology 28 (2016) 10–21 15

Fig. 2. The tumor micro-environment efficiently evades CD8T cell trafficking and accumulation.The tumor micro-environment (TME) puts a brake on the trafficking and accumulation of intra-tumoral numbers of CD8 T cells, which represents a major means of immune-evasion of multiple tumor types. Mechanisms that limit CD8 T cell numbers within tumors can be functionally categorized in three main categories, namely those that limit:e of T a ally de

ta

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ntry of T cells (with contributions of chemokines and endothelial cells); migrationnd immune-suppressive cells and molecules). These three categories are schematicxplained in more detail in Section 3.1. See abbreviations.

argeted to enhance the therapeutic efficacy and applicability ofdoptive therapy with TCR-engineered T cells.

.1. The tumor micro-environment uses multiple barriers to keep cells out

The TME represents the molecular and cellular ecosystem sur-ounding the tumor cells, also termed stroma, including endothelialells, fibroblasts, immune cells, and their soluble products, many ofhich are signaling molecules (eicosanoids, cytokines) and extra-

ellular matrix (ECM) elements. Here, we will discuss three barriersowards trafficking and intra-tumoral accumulation of T cells,escribed below in more detail and schematically depicted in Fig. 2.

.1.1. Attraction and entry of T cells: chemokines and endothelialells

Endothelial cells govern the initial steps of T cell entry, a processhat in normal tissues is driven by inflammation. T cell recog-ition of chemokines (produced not only by endothelial cells,ut also other stromal as well as epithelial cells) is consideredritical for recruitment, whereas T cell recognition of adhesionolecules and co-stimulatory ligands expressed by endothelial

ells is considered critical for subsequent adhesion and trans-ndothelial movement. The contribution of chemokines, such as

hemokine (CXC motif) ligands (CXCL)9 and 10, as well as lig-nds for T cell integrins, such as intercellular adhesion moleculeICAM-)1 and vascular cell adhesion molecule (VCAM)1, to T cellntry has already been established in early studies [78]. Interest-

cells (extracellular matrix and fibroblasts); and local activation of T cells (hypoxiaepicted in this figure, with their cellular and molecular features listed in boxes, and

ingly, a recent study argues that the chemokine receptor CXCR3,the receptor for CXCL9 and 10, but not other chemokine receptors,uniquely functions to stabilize intravascular adhesion and extra-vasation of CD8 T cells [79]. In vivo imaging studies revealed thatT cells demonstrate early and preferential accumulation withinantigen-positive tumors [67,80]. Furthermore, sustained TCR- andCD28-mediated interactions appear necessary for T cells to accu-mulate and retain in antigen-positive tissue [81].

Strikingly, endothelial cells in growing tumors may adopt aphenotype that appears designed to keep CD8 T cells out. In exten-sion to findings in human melanoma [75], mouse melanoma thatrecurred following adoptive T cell therapy showed decreased infil-tration of CD8 T cells which coincided with decreased expressionof selected chemokines such as CXCL9 and 10 [2]. Chemokinesreported to recruit regulatory T cells (Treg), such as chemokine(CC motif) ligand (CCL)22 and CXCL12, were not differentiallyexpressed in melanoma tumors that recurred. Interestingly, selec-tive shut-down of T cell-attracting chemokines may be linked toepigenetic silencing [82] or disruptive post-translational modifica-tions, such as nitration or cleavage mediated by reactive nitrogenspecies or dipeptidylpeptidases, respectively [83,84]. The expres-sion of ICAM-1 and certain galectins, annexins and tetraspanins,with reported contributions to leukocyte trafficking and tumordevelopment [85,86] was also dramatically down-regulated in

recurred melanoma [2]. The lowered expression of ICAM-1 in tumorendothelial cells may be functionally linked to over-expression ofthe receptor for endothelin B (ETBR) [87], basic fibroblast growthfactor (bFGF) and vascular endothelial growth factor (VEGF) [88]

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n these cells. With respect to antigen and co-stimulation, tumorndothelial cells show little or no expression of MHC class I andncreased expression of the checkpoint inhibitor B7-H3 [89], whichurther limits T cell entry. In addition to limiting T cell entry,umor endothelial cells can inhibit the function or even kill CD8

cells. Molecules derived from tumor cells, often angiogenic andmmune-suppressive in nature, can induce expression of ligandsor inhibitory receptors (i.e., programmed death-ligand (PD-L)1nd 2) and the ligand to the death receptor Fas (FasL) [88]. FasLecomes expressed by endothelial cells upon exposure to VEGF,

nterleukin (IL-)10 and prostaglandin (PG)E2, and mediates killingf Fas-positive CD8 effector T cells, while CD4 Tregs resist FasL-nduced killing [90].

.1.2. Migration of T cells: extracellular matrix and fibroblastsOnce T cells have crossed endothelial cells, the positioning and

igration of T cells within tissues is normally (at least in part) con-rolled by chemokines, fibroblasts and ECM components, such asype I collagen. Remarkably, CD8 T cells in progressing tumors arearely in contact with tumor cells but instead are often enriched inhe tumor-surrounding stroma [91,92]. Studies provide evidencehat cancer-associated fibroblasts (CAFs) negatively regulate thebility of T cells to contact tumor cells. CAFs, although consid-red a heterogeneous cell population of multiple origins, oftenxpress the membrane protein fibroblast activation protein (FAP-� [93] and secrete excessive amounts of ECM components [94]. Inact, the functional relevance of CAFs came from studies in whichhese cells were depleted from pancreatic tumors, which allowedancer-specific T cells to rapidly control tumor growth [28,95]. Theeposition of a dense ECM has long been recognized to support can-er cell growth and metastatic dissemination [96]. Interestingly,n breast tumorigenesis it was observed that matrix cross-linkings accompanied by increased focal adhesion in stromal cells, andnhanced integrin and phosphoinositide 3 kinase (PI3K) activity ineighboring tumor cells [97]. There is now compelling evidenceemonstrating that dense collagen structures of solid tumors hin-er T cell migration and their ability to reach malignant cells. By

ooking at the dynamics of plated and endogenous CD8 T cells inuman lung and ovarian tumor slices, T cell positioning and migra-ion was found to be markedly reduced in matrix-rich stromalegions [92,98]. Notably, such dense matrix structures were oftenbserved around tumor islets, and a decrease of collagen fibersith collagenase resulted in an increase of the number of T cells

hat are in contact with tumor cells [92]. This intra-stromal trap-ing and prevention of T cells from contacting tumor cells has alsoeen observed in other cancers such as pancreatic carcinomas andelanomas [99–101].Apart from a key role played by ECM, CAFs may employ addi-

ional mechanisms in T cell evasion. This became evident from twoeports on preclinical models of pancreatic cancer, a hard-to-treatumor type characterized by a profound desmoplastic reaction and

limited number of intra-tumoral CD8 T cells. The first report pro-osed that absence of T cells in tumor islets was due to the presencef CAF-derived CXCL12 in cancer cell regions, which shielded cancerells from CD8 T cells [28]. The second report demonstrated that,hen treated with an agonist of Toll-like receptor 9, pancreatic

tellate cells (potential precursors of CAFs) secreted CCL3, whichecruited Tregs and induced proliferation of myeloid-derived sup-ressor cells (MDSCs) [102]. The latter two cell types have beeneported to hinder T cell accumulation in tumors [103,104]. In addi-

ion, a population of cells sharing markers with dendritic cells and

acrophages can arrest the motility and compromise the functionf intra-tumoral CD8 T cells, potentially through nonproductiveellular interactions at the tumor margins [105,106].

unology 28 (2016) 10–21

3.1.3. Local activation of T cells: hypoxia and immune-inhibitorycells and molecules

When T cells normally migrate into tissue, they can encounterhypoxic areas, where key biochemical and cellular pathwaysimportant for neo-vascularization and wound healing are activated.In many tumor types, hypoxia represents a prominent feature andsignificantly contributes to evasion of a CD8 T cell response, tumorprogression and metastasis [107]. In fact, Inhibition of hypoxia (viahypoxia-controlled production of soluble VEGF receptor (VEGFR)2 and neutralization of VEGF) has been shown to result in strongreduction of tumor burden and eradication of metastases in amouse melanoma model [108]. Hypoxia plays a crucial role in theangiogenic switch, modulating a large set of genes (among whichVEGF) mainly through the activation of the hypoxia inducible fac-tor (HIF) transcriptional complex. This angiogenic switch resultsin an abnormal tumor vasculature, which has been reported tocontribute to arrested T cell infiltration and is featured by ves-sels with irregular structures, insufficient pericyte coverage, aswell as enhanced leakiness, and interstitial pressure. Moreover,hypoxia was shown to induce the expression of Nanog, which wasobserved to result in decreased numbers of CD8 T cells via directup-regulated production of TGF-�1 by tumor cells [109]. In additionto the effects on intra-tumoral numbers of CD8 T cells, hypoxia wasalso demonstrated to adversely affect the maturation of dendriticcells [110], and enhance the number and activity of intra-tumoralimmune-suppressive cells. For instance, hypoxia-induced Nanogwas observed to enhance numbers of intra-tumoral Tregs andtumor-associated macrophages [109]. Also, the HIF pathway hasbeen reported to induce expression of CCL28, which preferentiallyrecruits Tregs [111], as well as to enhance the immune-suppressiveactivity of these cells by promoting the expression of their lineagetranscriptional regulator forkhead box P (FOXP) 3 [112] as well asto. With respect to MDSCs, hypoxia was found to enhance theirrecruitment [113] and selectively up-regulate the expression of PD-L1 on these cells (also on macrophages, dendritic cells and tumorcells) [114]. The up-regulated expression of PD-L1 is governed byHIF-1� binding to a hypoxia-response element in the PD-L1 prox-imal promoter. Once present, immune suppressor cells can reducenumbers of intra-tumoral CD8 T cells by limiting their infiltrationinto tumors or inhibiting their local proliferation by release of reac-tive nitrogen and oxygen species [115], and metabolic disturbancesdue to the expression of indoleamine 2,3-dioxygenase (IDO) andarginase [116,117].

Collectively, the above-mentioned studies clearly show thattrafficking towards and within tumors and local activation of CD8 Tcells is defective at multiple levels. In fact, when analyzing tumorsfor the evasion of numbers and function of CD8 T cells, one comesto the realization that it is difficult to have one without the other.Question remains whether there are tumor cell intrinsic changesthat initiate such T cell evasion. Although this type of researchis fairly new, we would like to highlight a few seminal observa-tions that point to the involvement of three signaling pathwaysthat may potentially be positioned upstream of T cell evasion.First, Thomas Gajewski and co-workers, using human melanomasamples and mouse melanoma models, demonstrated that acti-vation of tumor cell WNT signaling resulted in absence of CD8 Tcells, mediated via down-regulated expression of a chemokine anddown-regulated influx of dendritic cells [118]. Second, Patrick Hwuand colleagues showed, again using patient samples and mousemodels of melanoma, that loss of phosphatase and tensin homolog(PTEN) (and enhanced PI3K signaling) in tumor cells decreases T-cell trafficking into tumors [119]. Thirdly, Amer Beg and his team,

using human and mouse lung samples, observed that lowerednuclear factor-kappa B (NF-�B) activity in tumor cells was accom-panied by lowered T cell infiltration, mediated via down-regulated

R. Debets et al. / Seminars in Immunology 28 (2016) 10–21 17

Table 2Interventions to enhance trafficking and accumulation of CD8 T cells in tumors. Strategies or drugs to sensitize tumors for trafficking and intra-tumoral accumulation of CD8T cell are listed according to the immune-evasive mechanisms depicted in Figure 2, and are explained in more detail in text (see also abbreviations). Interventions underlinedhave already shown activity in patient studies and are considered candidate treatments for clinical testing in combination with adoptive transfer of TCR-engineered T cells.

T cell entry: chemokines and endothelial cells- Chemotherapeutic drugs (dacarbazine, temozolomide, and cisplatin) or epigenetic drugs (5′-AZA) to increase expression of chemokines [82,121]- Inhibitors of reactive nitrogen species or dipeptidylpeptidase to prevent inactivation of chemokines [83,84]- T cells gene-engineered with chemokine receptors [122–124]- Inhibitors of ETBR to restore expression of adhesion molecules by endothelial cells [87]- Blocking mAb against PD-L1 (MDX-1105) or B7-H3 to act against endothelial cell-mediated T cell inhibition [151]- Drugs that block prostaglandins (Aspirin) and antibodies that block IL-10RA, VEGF (Bevacizumab) or FasL to act against endothelial cell-mediated killing of

CD8 T cells [90]- T cells gene-engineered with co-stimulatory TCRs [65,66]

T cell migration: extracellular matrix and fibroblasts- Inhibition of collagen cross-linking enzymes (lysyl oxidase) [97]- Degradation of matrix fibers with (Pirfenidone) [126]- T cells gene-engineered with heparanase [127]- Inhibition of CAF-derived immune-suppressive chemokines, such as CXCL12 and CCL3 [28,102]

Local T cell activation: hypoxia and immune-suppressive cells and molecules- Blocking hypoxia, antibody against VEGF (Bevacizumab) or TNF fusion protein to normalize tumor vessel structure [132–134,152]- T cells gene-engineered with CAR directed against VEGFR2 or with NFAT-inducible IL-12 to remodel vessels [135,136]- Depleting antibodies directed against CTLA-4 and OX40, or inhibitors of IDO1 (1 methyl D tryptophan) to counteract Tregs [116,137]- Chemotherapy (Docetaxel) to deplete MDSCs [139]- Blocking antibody against TGF� (Fresolimumab) or T cells gene-engineered with dominant-negative receptor for TGF� to neutralize effects towards CD8 T

cell accumulation [141]ductioming o

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- Blocking antibody against PD-L1 (MDX-1105) to act against MDSC-derived pro- Innate agonists of IFN type I signaling to stimulate dendritic cells and cross-pri

xpression of chemokines [120]. These studies elegantly and con-incingly point to a link between oncogenic pathway disturbancesn tumor cells and T cell evasion (and not cancer cell proliferationer se), and may provide novel therapeutic angles to support T cellherapies.

.2. Strategies to sensitize tumors for T cell trafficking

An overview of drugs and strategies that restore trafficking andntra-tumor accumulation of T cells are described below and sum-

arized in Table 2.

.2.1. Attraction and entry of T cells: chemokines and endothelialells

Interventions that normalize the production of T cell-recruitinghemokines show great promise. Such drug interventions includehemotherapeutics, epigenetic drugs and drugs that modify post-ranslational processes. For instance, the chemotherapeutic drugsacarbazine, temozolomide, and cisplatin enhanced the expressionf CCL5, CXCL9 and 10 in human melanoma, which in turn cor-elated with improved tumor control [121]. The epigenetic drugs-deazaneplanocin A (DZNep: inhibitor of histone methylation)nd 5-azacytidine (5-AZA: inhibitor of DNA methylation) enhancexpression of CXCL9 and 10 in human ovarian cancers, increase CD8

cell infiltration, and improve the therapeutic efficacy of adop-ive T cell therapy in tumor-bearing NSG mice [82]. Drugs thatnhibit the local production of reactive nitrogen species (AT38) pre-ent nitration of the chemokine CCL2, whereas those (sitagliptin)hat inhibit CD26 dipeptidylpeptidase prevent cleavage of CXCL1083,84]. Both these drugs resulted in improved intra-tumoral Tell migration and function in tumor-bearing mice. In additiono the restoration of local chemokine production, some investiga-ors have gene-engineered T cells to express chemokine receptorsnd demonstrated enhanced trafficking towards and infiltration in

umors secreting the corresponding chemokine ligands [122–124].esides chemokines, other therapeutics target the inhibitory phe-otype of tumor endothelial cells. Inhibition of ETBR (using BQ-788)estored the expression of ICAM1 on endothelial cells and normal-

n of IL-10 [114]f CD8 T cells [142]

ized CD8 T cell infiltration [87]. Along the same lines, one could optto neutralize ligands for T cell inhibition (B7-H3) or death (FasL).Monoclonal antibodies to block the T cell inhibitory ligand PD-L1and its receptor PD-1 have demonstrated clear clinical successesin the treatment of advanced cancers, with beneficial outcome, atleast in part, relying on enhanced infiltration of CD8 T cells [76,77].Authors would like to advocate explorative studies to test block-ing T cell co-inhibitory molecules in combination with adoptivetransfer of T cells. Also with respect to T cell co-signaling, investi-gators have used gene-engineering approaches and generated TCRtransgenes that contain a co-stimulatory molecule (i.e., CD28) toconfer T cells with enhanced ability to infiltrate in tumors and anti-tumor activity [66]. Notably, this co-stimulatory TCR, when testedfor peptide fine specificity using altered peptide ligands (as exem-plified in Fig. 1B), showed no off-target recognition in vitro. Withrespect to inhibition of FasL or neutralization of its upstream induc-ers, it is interesting to note that the University of Pennsylvanmiahas started clinical trials to treat patients with ovarian cancer withthe VEGF antibody bevacizumab and the prostaglandin synthesisinhibitor aspirine in combination with dendritic cell vaccinationand low doses of the chemotherapeutic agent cyclophosphamide.

3.2.2. Migration of T cells: extracellular matrix and fibroblastsThe dense ECM network of tumors provides a valid target to

enhance T cell motility within tumors [125]. The lysyl oxidase (LOX)enzyme that cross-links collagen into fibers has been targeted in amurine model of mammary carcinoma using chemical inhibitorsor soluble antibodies, which resulted in decreased cancer progres-sion and metastasis [97]. It remains to be determined whether thereduced density of collagen fibers upon inhibition of LOX resultsin more T cells actively patrolling the tumor stroma in search oftumor antigens. Another example of a drug that can be consideredto target ECM is pirfenidone, an anti-fibrotic drug for the treatmentof idiopathic pulmonary fibrosis and known for its inhibiting effect

towards the production of pro-collagens [126]. Furthermore, in arecent study, T cells were engineered to express heparanase, anECM-degrading enzyme, and demonstrated enhanced infiltrationinto tumors and antitumor efficacy [127]. In addition to target-

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ng ECM directly, other avenues that have been explored are theargeting of CAFs, their products or tumor cells. Targeting of FAP--expressing CAFs is not recomended since FAP-� is expressed inarious healthy organs, such as muscle and bone marrow, and itsargeting may result in severe cachexia and anemia [128,129]. Tar-eting the products of CAFs, such as certain immune-suppressivehemokines, may be a safer strategy. The use of AMD3100, annhibitor of CXCR4 (the receptor for CXCL12) in a preclinical modelf pancreatic cancer, resulted in increased T cell accumulation andherapeutic responses [28], and warrants further investigations.nother strategy may be the use of Toll-like receptor 9 antago-ists to counteract the production of CCL3 by pancreatic stellateells [102,130]. Of further interest to the topic of this review is theecent observation that adoptive therapy with T cells engineeredo express a TCR directed against mesothelin-positive tumor cellslready shows beneficial effects in a preclinical model of pancreasancer despite excessive amounts of ECM [131]. However, in thistudy, TCR-engineered T cells become progressively less functionalnd may benefit from tumor pre-treatment with above-mentionedrugs that target ECM.

.2.3. Local activation of T cells: hypoxia and immune-inhibitoryells and molecules

Hypoxia and its effects on vascular changes can be counteractedith the drug inositol trispyrophosphate, which was observed to

nhibit endothelial cell PI3K signaling, and consequently enhancedxygen release and resulted in vessel normalization in models ofelanoma and breast carcinoma [132]. Other therapeutic means

o normalize tumor vasculature include antibodies directed againstEGF and vascular homing peptides conjugated to tumor necrosis

actor (TNF-)� [133,134]. TNF-�’s effect on vessel remodeling is,t least in part, mediated by macrophages and low doses of TNF-�ubstantially improved adoptive T-cell therapy [134]. Also, T cellsan be gene-engineered to express a CAR directed against VEGFR2nd target tumor endothelium [135]. In extension, T cells can beene-engineered to express IL-12 under the control of the nuclearactor of activated T cells (NFAT) promoter, which becomes acti-ated upon intra-tumoral encounter of cognate antigen and TCRignaling [136]. IL-12 production by intra-tumoral T cells facilitatedroduction of monocyte-derived TNF-� and enhanced the CD8 Tell response towards tumor. Along this principle, T cells have beenngineered to express other inducible cytokines and chemokineso explore their beneficial effects towards T cell trafficking andnti-tumor responses (Kunert and Chmielewski, Manuscript, sub-itted). Another effect of hypoxia that can be targeted is the

ecruitment and activation of immune-suppressive cells. Strate-ies to deplete or inactivate Tregs include combined intra-tumoralnjection of anti-CTLA-4 and OX40 antibodies or blocking IDO

ith 1 methyl D tryptophan [137,138]. Interestingly, inhibition ofDO prevents tryptophan depletion and enhances T cell infiltra-ion [116] and induces conversion from Treg to Th17 cells, whichurther helps to build an anti-tumor T cell response [138]. Withespect to MDSCs, docetaxel is able to deplete these cells and, whenombined with adoptive T cell therapy and dendritic cell vaccina-ion, results in enhanced anti-tumor responses [139]. In additiono depletion of immune-suppressive cells, one could also targetheir downstream products. For example, genetic introduction of

dominant-negative transforming growth factor (TGF-)� recep-or II in TCR-engineered T cells resulted in increased anti-tumor

cell responses in a spontaneous model of prostate cancer [140].iven that hypoxia-induced Nanog adversely affects numbers of

ntra-tumoral CD8 T cells, which is mediated by TGF-�, this may

rovide an additional rationale to use non TGF-�-responsive Tells in a therapeutic setting or use blocking antibodies directedgainst TGF-� [141]. Also, blockade of PD-L1 under hypoxic con-itions enhanced MDSC-mediated T-cell activation by attenuating

unology 28 (2016) 10–21

MDSC secretion of IL-6 and IL-10 [114]. Setting aside the neutraliza-tion of immune-suppressive cells or their products, another meanto enhance numbers of intra-tumoral CD8 T cells is to stimulatedendritic cells, the latter often functionally compromised as a con-sequence of hypoxia. In this respect, innate agonists of the IFN typeI response in dendritic cells may result in enhanced production ofCXCL9 and 10 chemokines, generation of CD103-positive dendriticcells and cross-priming of CD8 T cells [142].

Taken together, the above studies show the drug-ability of var-ious cells and molecules that are involved in CD8 T cell traffickingand accumulation in tumors, and advocate studies to combine suchtreatments with adoptive T cell therapy.

4. Conclusion & Perspective

Currently, therapy with TCR-engineered T cells would greatlybenefit from selection and assessment of a TCR that sees the cor-rect antigen, as well as sensitization of the TME to open gatewaysfor T cells to be able to touch tumor cells. With respect to selec-tion of TCR (and thus of an antigen), neo, oncoviral, and certaincancer germline antigens are considered as candidate target anti-gens. When opting to target one of these antigens, one needs toindividually test antigens with respect to absent expression in amultitude of healthy tissues to limit chances of on-target toxicity.Corresponding TCRs, of which retrieval can be greatly facilitatedby advancements in the isolation of paired TCR� and � sequences,need to be audited for anti-tumor effects in vitro and in vivo. Impor-tantly, TCR-mediated recognition of similar, but unrelated peptidesneeds be excluded to limit chances of off-target toxicity. This isparticularly relevant when TCRs are derived from non-tolerant set-tings and exposed to gene-enhancing strategies. To this end, it isstrongly recommended to determine the cognate peptide’s recog-nition motif and perform in silico analysis and preclinical tests toexclude recognition of proteins containing such a motif.

With respect to sensitisation of TME, defects in the traffickingand accumulation of CD8T cells in tumors follow common denom-inators and have been categorized according to entry of T cells;migration of T cells; and local activation of T cells. Strategies focuson the targeting of selective chemokines; the inhibitory phenotypeof tumor endothelial cells; dense ECM; the activated phenotypeof CAFs; hypoxia; and immune-suppressive cells and molecules.A combination of strategies that boost T cell trafficking, some ofwhich use gene-engineering techniques or make use of drugs withclinical precedent, shows great promise to enhance the anti-tumorefficacy and feasibility of adoptive therapy with TCR-engineered Tcells.

Acknowledgement

Authors would like to thank Andre Kunert for his help inpreparing the figures, and dr. Cor Lamers for critically reading thismansucript. Research of 1st author RD is funded by various orga-nizations, among which European Community (ATTACK2), 7th fw;305863; Dutch Cancer Society EMCR2014-7087, EMCR2015-7741and WUR2015-7734; and Erasmus MC.

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