immunotherapies have shown promising results in cancer types previously hard to cure , such as melanoma,1 , 2 , 3 non - small cell lung cancer , and renal cell carcinoma .
in addition to checkpoint - inhibiting antibodies , patient - derived t  cells are a potent approach because they can be re - targeted against tumors ex  vivo , or they can be infused to the patient without modifications , when extracted from the tumor biopsy .
one advantage of using biopsy - derived polyclonal tumor - infiltrating lymphocytes ( tils ) is the presence of neoantigen - specific clones 
, til infusion requires high - dose preconditioning to eradicate suppressive immune cell subsets from the tumor microenvironment and postconditioning with high - dose systemic interleukin ( il)-2 , both often causing severe toxicities.6 , 8 instead of systemic administration of cytokines like il-2 , it could be more attractive to deliver them locally with gene therapy vectors , such as viruses.9 , 10 in particular , tumor - targeted replication - competent viruses ( i.e. , oncolytic viruses ) enable a thousand - fold amplification of transgene expression , restricted to tumor tissue . with regard to immunotherapy , oncolytic
adenovirus constitutes a personalized cancer vaccine generated for each patient in  situ , due to release of tumor - associated antigens . of note ,
virus - mediated danger signaling helps the immune system to recognize tumor cells , and immunostimulatory cytokines further boost this effect.3 , 13 , 14 we have shown that the most promising t  cell - stimulating factors , in the context of adoptive cell therapy , are il-2 and tumor necrosis factor alpha ( tnf-).15 , 16 , 17 regarding prior knowledge about the recombinant proteins , il-2 has been widely used in treating malignant melanoma and renal cell carcinoma and it stimulates t  cell proliferation and differentiation.9 , 19 , 20 like il-2 , tnf- can activate immune cells , but it also induces antitumor inflammation and the production of other cytokines and chemokines.22 , 23 moreover , it directly causes cancer cell necrosis and apoptosis . in this study , we constructed and characterized new oncolytic adenoviruses built on a backbone of ad5/3-e2f - d24 ( oad ) carrying human il-2 ( hil2 ) , tnf- , or both .
two modifications render virus replication tumor specific : an e2f promoter and a 24-base pair ( bp ) deletion in the constant region 2 of e1a , make the viruses selective for cells defective in the retinoblastoma / p16 pathway  including most tumor cells.24 , 25 in addition , the ad5/3 chimeric capsid featuring the ad3 knob but ad5 shaft and tail has demonstrated improved cancer cell transduction as well as antitumor efficacy .
importantly , safety of this configuration in humans has also been established.13 , 27 , 28 based on the results in an immunocompetent syrian hamster model of pancreatic cancer , these viruses emerged as strong candidates for stimulating the immune system in tumors locally , with the specific application of enabling effective and safe til therapy .
ad5/3-e2f - d24-htnfa - ires - hil2 ( tilt-123 ) rose as the leading candidate for human translation .
oncolytic adenoviruses were constructed to feature a backbone carrying serotype 5 ( ad5 ) nucleic acid with an ad3 fiber knob .
in addition , a 24-bp deletion ( d24 ) in the rb - binding region of adenoviral e1a together with the e2f promoter was established to direct the replication to rb - deficient cancer cells .
the transgenes were inserted into the e3 region , to replace some superfluous adenoviral open reading frames , which links expression to virus replication ( figure  1a ) .
all cytokine - armed adenovirus constructs were able to kill a panel of human cancer cell lines with similar efficacy as the virus without transgenes ( figures 1b and s1 ) .
importantly , when virus was combined with hapt1-targeting tils , the cell - killing effect was significantly increased ( figure  1c ) .
both human and hamster cells were able to produce biologically active human il-2 as well as tnf- in  vitro when infected with armed viruses ( figures 2a , 2b , and s2 ) .
importantly , local production of cytokines was observed with all three armed viruses in  vivo while systemic levels remained undetectable ( figure  2c ) , highlighting the feasibility of the technology from a safety perspective . to establish an optimal virus dose ,
immunocompromised mice bearing orthotopic human ovarian tumors ( skov3-luc ) received three different doses of ad5/3-e2f - d24-htnfa - ires - hil2 .
the best efficacy was achieved with the highest dose of 1   10 viral particles ( vps ) , which was significantly different compared with the untreated control group ( p  = 0.0085 ) as well as with the lowest dose of 1   10 vps ( p  = 0.0287 ) on day 18 ( figure  3a ) . when skov3-luc tumors were treated with ad5/3-e2f - d24-htnfa - ires - hil2 and control viruses , all viruses had similar antitumor efficacy ( figures 3b and 3c ) , suggesting that adenovirus replication rates in  vivo were comparable despite the inclusion of transgenes .
encouraged by the ex  vivo results ( figure  1c ) , the ability of cytokine - armed viruses to enhance til therapy was investigated in immunocompetent syrian hamsters ( figure  4 ) . the unarmed virus and
tils had only moderate antitumor effects when administered alone , but a significant improvement in efficacy was observed when they were combined ( p  = 0.002 ) ( figure  4a ) .
the armed viruses had tremendous efficacy even as single agents ( figures 4b4d ) , but the percentage of cured animals was higher in groups receiving tils and virus compared with the virus - only groups ( p  = 0.034 ; figure  4e ) . in fact , cured hamsters treated with the combination of ad5/3-e2f - d24-htnfa - ires - hil2 and til therapy comprised 100% ( figure  4e ) . 
the experiment was repeated with a reduced virus dose with similar results in efficacy ( figure  4f ) .
the cured animals from the second experiment stayed tumor free for the follow - up period of 3  months . to investigate immunological effects of the armed viruses , cells from tumors , spleens , and tumor - draining lymph nodes
natural killer ( nk ) cell marker gm1 and t  cell markers cd8 and cd4 were more frequent in tumors treated with il-2 , whereas the cytokine combination was the only treatment capable of increasing the level of major histocompatibility complex class ii ( mhc ii ) and decreasing the mac-2 expression in tumors ( figures 5a5e ) .
differences in the cell composition in spleens and lymph nodes were minor ( figure  s3 ) .
interestingly , splenocytes exhibited greater cell proliferation ex  vivo if the animals had been treated with armed viruses ( figure  5f ) .
to estimate whether the viral treatment established tumor - specific immunity , cured hamsters were re - challenged with the same cancer cells as previously ( hapt1 ) . as a control , different types of cancer cells ( ddt1-mf2 )
the animals that had previously been cured with cytokine - coding viruses rejected hapt1 , whereas the animal treated with unarmed virus had a stable condition .
the number of animals in these groups differs , because the curative potential of the unarmed virus was more limited .
ddt1-mf2 tumor growth in cured hamsters was comparable to growth in naive animals , indicating the induction of tumor - specific antitumor immunity .
treatment - related changes in tissue structures of the heart , lung , liver , and kidney were undetectable ( supplemental materials and methods ) .
meanwhile , spleens collected from all treatment groups showed mild and minimal lymphocyte hyperplasia , slightly expanded white pulp , and a mildly increased number of heterophils in the marginal zone or red pulp .
there were no differences in the severity of the changes between any of the treatment groups including mock and other controls , suggesting a lack of systemic effects linked to the transgenes as predicted by low serum concentrations ( figure  2c ) .
immuno - oncology has made some clinical breakthroughs over the years , but , currently , a minority of patients respond and single - agent treatment modalities seldom lead to lasting remissions.1 , 2 , 3 , 4 thus , the utility of immunotherapy has been established on a proof - of - concept level , but much work remains to help the majority of patients with currently incurable cancer .
checkpoint - inhibiting antibodies have received much attention due to their ability to downregulate immunosuppression , but they can not generate new immunity . by contrast , new immune reactions can be achieved with adoptive cell therapies and oncolytic immunotherapy , thus being complementary to the former .
however , adoptive cell therapy of non - melanoma solid tumors has proved clinically unimpressive results , because the tumor microenvironment is able to anergize the cell graft.16 , 30 this effect can be countered with the biological phenomena resulting from adenoviral oncolysis , and it can be optimized with tnf- and il-2.16 , 31 we previously studied the effects of these cytokines with recombinant cytokines and with replication - defective vectors coding for murine cytokines.15 , 16 , 17 here , we constructed clinically applicable oncolytic adenoviruses coding for human il-2 and tnf- and used them to boost adoptive cell transfer .
the viruses were capable of infecting and lysing a variety of human and syrian hamster cancer cell lines .
after intratumoral virus administration , high cytokine concentrations were achieved in target tissue , while blood levels remained undetectable .
in addition , signs of toxicity were absent in histopathological evaluation of all major organs .
taking into consideration the potential toxicity of high systemic cytokine levels,8 , 32 the results suggest a safer approach for cytokine delivery . moreover
, local delivery of il-2 could replace the need for high - dose il-2 administration often included in clinical t  cell therapy protocols , although detailed studies are needed to validate the concept . to investigate the efficacy of the viruses on human cancer cells in  vivo
, we established an orthotopic ovarian carcinoma model in immunocompromised scid mice . despite the replication competence of the virus ,
the problem of virus infiltration into the tumor is well known ; thus , intratumoral administration represents a more efficient way of delivering the virus .
in addition , this model develops resistance to oncolytic adenovirus , seen as a reduction in antitumor efficacy at later time points .
the mechanism of resistance was previously shown to be related to the induction of interferon pathways .
significant differences in the efficacy between the viruses were absent , which was expected because the cytokines have few effects in an immunocompromised model lacking a complete immune system .
cytokine - armed oncolytic viruses are potential enhancers of t  cells and t  cell - based therapies.14 , 35 , 36 to investigate the synergy between our viruses and tils , we chose syrian hamsters as an immunocompetent animal model .
syrian hamsters provide an interesting model for studying oncolytic adenoviruses , since human adenovirus is capable of replicating in hamsters , unlike in mice .
conveniently , human il-2 and human tnf- are also bioactive in hamsters , which is evident from the hamster experiment where the unarmed virus had only moderate antitumor effects as compared with the human cytokine - coding viruses that share the same backbone construct .
furthermore , we have developed a method to extract and expand tils from established hamster tumors , despite the limited availability of hamster - specific reagents .
unfortunately , specific expansion of , for example , cd8 t  cells is unfeasible and the til pool depends on the population present in the tumor at the time of collection . even in  vitro ,
the synergistic effect of combining the viruses and tils was evident . in  vivo ,
the combination of unarmed virus and tils led to improvement in efficacy . with regard to the cytokine - armed viruses ,
good efficacy was seen with single - agent treatment in two individual experiments . however , the combination of armed virus and til therapy resulted in the highest frequency of complete responses , as confirmed by histopathological analysis of the tumors . of note ,
all  tumors in hamsters treated with ad5/3-e2f - d24-htnfa - ires - hil2  and tils were cured , suggesting that inclusion of adenovirally delivered immunostimulatory cytokines contributes to the curative efficacy of til therapy .
immunocompetent mouse models have revealed that combining adenovirus - delivered tnf- and il-2 with adoptive t  cell transfer decreases immunosuppressive characteristics of the tumor microenvironment and increases the number of active cytotoxic t  cells in melanoma tumors.15 , 17 moreover , mouse studies have revealed that the danger signals and immunostimulation caused by adenovirus and cytokines can result in repertoire expansion by polyclonal amplification of many classes of antitumor t  cells while t  cell exhaustion seems to be thwarted.15 , 16 , 17 mouse tumor models are useful for immunological studies because many reagents are available .
however , human adenovirus does not replicate productively in mouse tissues ; therefore , the hamster model has some advantages in this regard .
there are only a few hamster - specific or cross - reactive antibodies , and only limited characterization of the immune cell subsets in the tumor is possible .
nevertheless , we saw decreased presence of macrophage marker mac-2 in the hamster tumors , whereas the frequency of gm1 ( mostly nk ) cells as well as cd4 and cd8 t  cells was increased following intratumoral treatment with the cytokine - coding viruses .
observed from immunocompetent mice that both cd8 and cd4 cells are essential for antitumor efficacy resulting from the combination of tnf- and il-2 .
il-2 induces both regulatory and helper t  cells ; however , in this study , the nature of cd4 cells could not be specified .
interestingly , the observed upregulation of mhc ii ( a marker for antigen - presenting cells ) might indicate that the combination of both cytokines enables efficient tumor recognition by cytotoxic cd4 t  cells,39 , 41 thus contributing to the overall efficacy .
in addition , the presence of tnf- stimulates the expression of il-6 , a cytokine needed for helper t  cell differentiation.40 , 42 taken together , although systemic il-2 may cause stimulation of regulatory t  cells , which may not necessarily be true for local il-2 , the possibility of the cd4 population also containing cytotoxic and helper t  cells can not be excluded .
in addition to immunological changes seen in the tumor microenvironment , further benefit from the cytokines was seen on the systemic level .
because the spleen serves as an indicator for the common status of the immune system , we investigated the proliferative capability of the splenocytes derived from the treated animals ex  vivo .
interestingly , the splenocytes from the cytokine - treated animals showed increased proliferative capability compared with the controls , indicating increased adaptive cellular response .
the same effect has  been seen in a study with oncolytic vaccinia virus coding for granulocyte - macrophage colony - stimulating factor .
moreover , adenovirally encoded cytokines evidently induced the formation of immunological memory , typically mediated by t  cells .
currently , because there are no anti - hamster antibodies available , the presence of memory - type t  cells could not be verified .
the animals treated with armed viruses resisted tumor recurrence unlike the animal treated with the virus without the cytokines , as also seen with other oncolytic viruses.10 , 43 , 44 in conclusion , we provide evidence that oncolytic adenoviruses coding for human il-2 and tnf- appear safe in immunocompetent hamsters . in addition to direct oncolytic effects and attractive immunological effects , these viruses seem useful for enabling successful til therapy of human solid tumors . whereas the virus itself shows potent antitumor efficacy , the cytokines are useful for induction of t  cells in the tumor and for immunological memory responses .
the preclinical data reported here allowed tilt biotherapeutics to initiate a human trial studying the utility of ad5/3-e2f - d24-tnfa - ires - hil2 in patients with advanced cancer receiving til therapy .
all cell lines were purchased from american type culture collection ( atcc ) unless otherwise stated . human lung adenocarcinoma a549 , human pancreatic cancer panc1 , human melanoma sk - mel-28 , human ovarian carcinoma ovcar-3 , mouse fibroblast l929 , and mouse t lymphocyte cell line ctll-2 were utilized in in  vitro assays .
negrin , stanford medical school ) , hamster leiomyosarcoma ddt1-mf2 ( a kind gift from dr .
william wold ) , and hamster pancreatic cancer hapt1 ( dsmz ) were used both in  vitro and in  vivo .
all cell lines were maintained in rpmi 1640 or dmem supplemented with 10% fetal bovine  serum ( fbs ) , 2  mm l - glutamine , 100  u / ml penicillin , and 100  g / ml streptomycin ( all from sigma - aldrich ) and cultured at  + 37c and 5% co2 .
the  transgenes in ad5/3-e2f - d24-hil2 ( oad.il2 ) , ad5/3-e2f - d24-htnfa ( oad.tnfa ) , and ad5/3-e2f - d24-htnfa - ires - hil2 ( oad.tnfa-il2 , also known as tilt-123 ) were placed into the e3 region and they were generated with the bacterial artificial chromosome ( bac)-recombineering strategy based on the selection marker  galk adapted from warming et  al . , ruzsics et  al . , and mck - husl et  al .
plasmids were propagated in electromax dh5-e competent cells ( thermo fisher scientific / invitrogen ) and the virus genomes were released from bacs with paci restriction enzyme ( thermo scientific ) .
the genomes were transfected into a549 cells with lipofectamine 2000 reagent ( invitrogen ) according to the manufacturer s instructions .
vp concentration was determined with optical density 260 ( od260 ) reading and infectious units by the tissue culture infectious dose ( tcid50 ) assay .
the functionality of the viruses was confirmed by infecting human and hamster cancer cell lines and measuring cell viability with the mts assay by adding 10% celltiter 96 aqueous one solution ( promega ) for the cells and reading the absorbance at 490  nm after 2  hr .
a549 , skov3-luc , hapt1 , and ddt1-mf2 cells were infected for 72  hr .
human il-2 and tnf- were detected from cell supernatants using the bd cytometric bead array human soluble protein master buffer kit together with human il-2 and tnf- flex sets ( bd ) according to the manufacturer s instructions .
the beads were detected with a bd accuri flow cytometer and the results were analyzed with fcap array software ( version 3.0.1 ; bd biosciences ) .
biological activity of the cytokines was confirmed with il-2-dependent ctll-2 cells or tnf--sensitive l929 cells .
ctll-2 cells were cultured with filtered supernatants or recombinant human il-2 ( r&d systems ) 10  ng / ml at  + 37c and 5% co2 for 72  hr . for tnf- activity experiments
, l929 cells were incubated with cell growth media supernatants or 0.5  ng / ml recombinant human tnf- ( r&d systems ) together with 2  g / ml actinomycin d for 24  hr . in both assays ,
was examined by injecting established hapt1 tumors with 1   10 vps 48  hr before collection .
human il-2 and tnf- levels in serum and in homogenized tumors were quantified with a cytometric bead array and normalized to total protein content .
subcutaneous hapt1 tumors established on syrian hamsters ( mesocricetus auratus , hsdhan : aura ; envigo ) were allowed to develop for 10  days .
tumor fragments ( 13  mm ) were cultured in g - rex10 ( wilson wolf ) in the presence of 3,000 iu / ml human il-2 ( peprotech ) .
half of the medium was replaced with fresh medium containing 1  g / ml concanavalin a ( con a ) ( sigma - aldrich ) after 5  days and every other day thereafter until day 10 .
fresh tils were employed in animal experiments or in an ex  vivo killing assay , where hapt1 cells were infected with 5,000 vps per cell for 72  hr before adding 2.5   10 tils extracted from hapt1 tumors .
the experimental animal committee of the university of helsinki and the provincial government of southern finland approved the animal experiments performed in this study .
immunocompromised female cb-17 scid mice ( janvier labs ) , aged 46  weeks , received 5   10 skov3-luc cells intraperitoneally .
to investigate the effect of different dosing of the virus , the animals were divided to four groups ( n  = 3 ) and treated with ad5/3-e2f - d24-htnfa - ires - hil2 in concentrations of 1   10 , 1   10 , or 1   10 vps in 300  l pbs intraperitoneally .
animals were imaged once a week with an ivis 100 imaging system ( xenogen ) .
three milligrams of d - luciferin ( synchem ) in 100  l pbs was administered intraperitoneally 8  minutes before bioluminescent imaging as previously described . to explore the differences in efficacy of the viruses in  vivo , the mice were randomized into groups of five to seven mice and treated with 1   10 vps in 300  l pbs intraperitoneally or pbs alone once a week .
subcutaneous hapt1 tumors ( 2   10 cells per tumor ) were established in 5- to 6-week - old immunocompetent male syrian hamsters .
when the average tumor diameter reached 0.5  cm , the animals were randomized into groups of six to seven .
intratumoral injection of 1   10 vps in 50  l pbs or pbs alone was performed .
viral treatments were repeated on days 4 , 8 , 13 , and 19 . on day 2 , hamsters received intratumoral administration of either 4   10 hapt1-derived tils in 50  l plain rpmi or media only .
twenty - five days after the treatments began , the animals were euthanized and tumors and selected organs were collected to evaluate the histopathological characteristics and immune cell subsets present .
the experiment was repeated with reduced virus dosing ( 1   10 vps on days 1 and 8) .
cured animals were re - challenged with the same tumors ( hapt1 ) or immunologically distinct tumors ( ddt1-mf2 ) ( 4   10 cells / tumor ) after a 2-week rest period .
tumor growth was followed for 18  days until ddt1-mf2 tumors reached the maximum tolerated diameter ( 2  cm ) .
cd8b , cd4 , and mhc class ii cells were detected from hamster tumors , spleens , and lymph nodes as described by siurala et  al .
polyclonal anti - asialo - gm1 af488 antibody ( ebioscience ) was used for detecting natural killer cells and a subset of monocytes / macrophages ( 0.5  g per reaction ) and anti - human / mouse galectin-3 ( mac-2 ) pe ( ebioscience ) for detecting macrophages and dendritic cells ( 0.2  g per reaction ) .
in addition , hamster splenocytes from each treatment group were pooled and assessed for proliferation after 72  hr of cultivation .
tumor samples along with tissue samples from the hamster heart , lung , liver , spleen , and kidney were collected for pathological evaluation .
samples were fixed in 10% formalin for 48  hr and stored in 70% ethanol until histological processing .
sections at 4-m thickness were cut from paraffin blocks and the slides were stained with hematoxylin and eosin .
differences between groups were estimated with the two - tailed student s t test , non - parametric mann - whitney test , or anova with graphpad prism software ( version 6.05 ) .
ibm ssps statistics software ( version 22.0.0.1 ) was utilized when analyzing log - transformed tumor volume data from hamster experiment using a linear mixed - effects model and for analyzing the curative effect of tils with the wilcoxon signed - rank test .
, a.e . , and a.k . provided materials for the experiments that were performed by r.h .
r.h . has been supported by the university of helsinki doctoral programme in clinical research .
received grants from helsinki university central hospital ( huch ) research funds , the sigrid juselius foundation , biocentrum helsinki , biocenter finland , and finnish cancer organizations , and he is a jane and aatos erkko professor of oncology at the university of helsinki .
in addition , a.h . is a shareholder in targovax asa and an employee and a shareholder in tilt biotherapeutics , ltd .
 adoptive cell therapy holds much promise in the treatment of cancer but results in solid tumors have been modest . 
 the notable exception is tumor - infiltrating lymphocyte ( til ) therapy of melanoma , but this approach only works with high - dose preconditioning chemotherapy and systemic interleukin ( il)-2 postconditioning , both of which are associated with toxicities . to improve and broaden the applicability of adoptive cell transfer , we constructed oncolytic adenoviruses coding for human il-2 ( hil2 ) , tumor necrosis factor alpha ( tnf- ) , or both . 
 the viruses showed potent antitumor efficacy against human tumors in immunocompromised severe combined immunodeficiency ( scid ) mice . in immunocompetent 
 syrian hamsters , we combined the viruses with til transfer and were able to cure 100% of the animals . 
 cured animals were protected against tumor re - challenge , indicating a memory response . 
 arming with il-2 and tnf- increased the frequency of both cd4 + and cd8 + tils in  vivo and augmented splenocyte proliferation ex  vivo , suggesting that the cytokines were important for t  cell persistence and proliferation . 
 cytokine expression was limited to tumors and treatment - related signs of systemic toxicity were absent , suggesting safety . 
 to conclude , cytokine - armed oncolytic adenoviruses enhanced adoptive cell therapy by favorable alteration of the tumor microenvironment . 
 a clinical trial is in progress to study the utility of ad5/3-e2f - d24-htnfa - ires - hil2 ( tilt-123 ) in human patients with cancer . 
Introduction
Results
Discussion
Materials and Methods
Author Contributions
Conflicts of Interest