溶瘤病毒联合CAR-T治疗肿瘤效果更好

1、 IntroductionAdoptive transfer of T cells genetically modified to express a CD19-specific chimericantigen receptor (CAR coupled with costimulatory endodomains has shown significantclinical impact in lymphoid leukemias (13. In contrast, the clinical relevance of thisapproach has been more limited for

2、 solid tumors (46.Insufficient migration of CAR-T cells to the tumor site and suboptimal persistence withinthe immune suppressive tumor environment are all critical factors that limit the impact of T-cell immunotherapies in solid tumors. Insufficient migration of tumor-specific T cells mayresult fro

3、m unfavorable chemokine gradients as tumor-specific T cells may lack theappropriate chemokine receptors for chemokines secreted by tumor cells(7. Alternatively,tumor cells or stromal cells produce chemokines that preferentially attract T cells withregulatory function rather than T cells with antitum

4、or activity(8, 9. Even if tumor-specific T cells reach the tumor environment, multiple mechanisms exploited by tumor cellsthemselves or by surrounding stromal cells can block an effective immune response. Among these mechanisms, downregulation of costimulatory molecules (CD28 and CD86 by tumorcells(

5、10, abundant TGF- production(11 and infiltration by regulatory T cells (Tregs(9significantly impair activation and proliferation of tumor-specific T cells, while over-expression of death receptor ligands by tumor cells including FasL and PD-L1 may directlyfavor premature apoptosis of T lymphocytes(1

6、2, 13.Engineering of CAR-T cells to include costimulatory endodomains can rescue T-cellactivation through the autocrine production of IL-2(1417, but this may still be insufficientto reverse the pro-anergic properties of the tumor environment(18, 19. CAR-T cells willalso need to be further modified n

7、ot only to migrate to the tumor site through co-expressionof specific chemokine receptors matching relevant tumor-secreted chemokines such as Gro-(20, CCL17(21 and CCL2(22, but also to specifically counter the local inhibitory milieu,for example due to TGF-(23 and Fas-L(24, and to make use of autocr

8、ine or administeredgrowth factors(2527. Since tumor cells likely use multiple inhibitory strategies, it ischallenging to construct vectors able to accommodate and express in T lymphocytes all theimmunomodulatory genes required to overcome tumor inhibition while increasing T-celltrafficking, survival

9、 and safety(28.To solve this complex task, we exploited OVs that selectively infect, lyse and replicate inmalignant cells whilst leaving non-malignant cells unaffected(29. OVs have sufficientcargo capacity to insert ectopic genes, and have produced clinical responses in patients withsolid tumors(30.

10、 We therefore armed an oncolytic adenovirus (Ad524 with thechemokine RANTES and the cytokine IL-15 to enhance the subsequent trafficking andsurvival of T cells expressing a tumor-directed CAR.Materials and MethodsTumor cell linesThe NB cell lines IMR-32, LAN-1, SKNLP, SK-N-SH, SH-SY5Y and the lung c

11、arcinomacell line A549 were obtained from the American Type Culture Collection, while NB cellNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscriptlines LAN-5 and CHLA-255 were gifts from Dr. LS. Metelitsa (Baylor College of Medicine,TX, and we verified that these lines retain th

12、e surface expression of the target antigen GD2.Cells were maintained in RPMI-1640 (IMR-32, LAN-1, LAN-5, SKNLP, SK-N-SH, andSH-SY5Y or IMDM (CHLA-255 and A549. All media was supplemented with 10% fetalbovine serum (FBS and 2mM L-glutamine (Invitrogen, with the exception of CHLA-255,which was supplem

13、ented with 20% FBS. Tumor cell lines were transduced with a gammaretroviral vector encoding enhanced green fluorescent protein (eGFP to obtain GFP positive tumor cells.Oncolytic virusOncolytic adenovirus Ad524 and non-replicable control adenovirus Ad5-Luc1 were kindly provided by Dr. Akseli Hemminki

14、 (University of Helsinki, Finland. Ad524.RANTES,using a 2A-like sequence(25.Flow cytometryThe following monoclonal antibodies conjugated with fluorochromes were used: Coxackie-adenovirus receptor, GD2, CD95 (Fas, CD80, CD86, CD40L, OX40L, CD25, CD69, IFN,CD3, granzyme B (BD Biosciences, TRAIL, TRAIL

15、-R1 and TRAIL-R2 (Biolegend.Expression of GD2.CAR by T cells was detected using a specific anti-idiotype, 1A7(TriGem, Titan, followed by staining with secondary antibody RAM-IgG1 (BDBiosciences. FACS data were collected with a FACSCalibur (Becton Dickinson andanalyzed using FloJo software version 9.

16、3 (Tree Star. For the caspase assay, CHLA-255cells were seeded in 24-well plates (1 105/well, infected with Ad524 or mock (100 vp/cell, and cultured for 4 days. Control and GD2.CAR-T cells (2.5 105/well were thenadded to the tumor cells. Active caspases in CHLA-255 cells were measured at 0, 2 and 4h

17、rs by FACS. The apoptotic cells were stained using Vybrant FAM Poly Caspases Assay Kit (Molecular Probes according to manufactures instructions. The frequency of earlyapoptotic cells was determined as percentage of FAM + (carboxyfluorescein group as areporter cells excluding CD3+ and PI + cells from

18、 the analysis(32.MTS assayCells were seeded into 96-well plates (104 cells per well, infected with or without viruses at the indicated doses and incubated at 37C for 96 hrs. Cell viability was analyzed by MTSassay according to manufactures instruction (CellTiter 96 AQueous One Solution CellProlifera

19、tion Assay, Promega. Values of virus-infected cells were normalized to those ofmock-infected cells (percent cell viability. The experiments were performed in triplicateand repeated three times.Retroviral production and CAR-T cell generationThe vectors encoding the GD2 specific CAR (GD2.CAR, incorpor

20、ating the CD28 andOX40 costimulatory endodomains, the fusion protein eGFP-firefly luciferase (FFluc, andthe methodology for the production of retroviral supernatant and CAR-T cells have been NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscriptdescribed previously(15, 16, 33. Fo

21、r the T-cell proliferation assay in vitro we used a firstgeneration GD2.CAR that lacks both CD28 and OX40 signaling domains.Co-culture experiments Tumor cells were seeded in 24-well plates (5 104/well for cytotoxicity assay and 1 105/well for T-cell proliferation assay, infected with Ad524 (50 100 v

22、p/cell and then cultured for 3 days. Control and GD2.CAR-T cells (3 104/well for cytotoxicity assay and 5 104/well for T-cell proliferation assay were then added and cultured for additional 3 days.Residual GFP + NB cells and T cells were then counted based on GFP and CD3 expression,respectively, usi

23、ng microbeads (CountBright Absolute Counting Beads, Invitrogen.Normalized residual tumor cells were calculated as 100 tumor cell counts with treatment/tumor cell counts without treatment (%.Confocal microscopic video imaging GFP-labeled CHLA-255 cells were seeded into 8-well chamber slide (Lab-TekII

24、, Thermo scientific (104 cells/well, infected with Ad524 (100 vp/cell and cultured for 3 days.Control and GD2.CAR-T cells were then added to the well (105 cells/well. GFP + NB cells stained with Annexin-V (Invitrogen were imaged using a spinning disk confocal microscope for 16 hrs. Imaging data were

25、 acquired and analyzed using Zen software (Zeiss.Migration assay Migration assays were conducted as previously described(21 with minor modifications using 5 m pore 24-well transwell plates (Corning Life Science. The percentage of migrating cells was calculated as follows: 100cell count of experiment

26、al sample cellcount of negative control / cell count of positive control cell count of negative control.ELISA and Milliplex assayTo measure the in vitro production of chemokines and cytokines, tumor cells were plated at5 105 cells/ml in 24-well plates and infected with viruses (50100 vp/cell. Supern

27、atantswere collected 72 hrs later and analyzed for the production of RANTES, MIP-1, MIP-1,MCP-1, IP-10, and IL-15. To measure the in vivo production of RANTES and IL-15, tumorand blood samples were collected 14 18 days after virus inoculation. Tumor homogenatesand serum were separated and finally as

28、sayed using specific ELISA kits (R&D Systems.Human IL-17F, GM-CSF, IFN, IL-10, CCL-20, IL-12p70, IL-13, IL-17, IL-22, IL-9,IL-1, IL-33, IL-2, IL-21, IL-4, IL-23, IL-5, IL-6, IL-25, IL-27, IL-31, TNF , TNF andIL-28, and mouse G-CSF, GM-CSF, IFN, IL-1, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9,IL-10, I

29、L-12p40, IL-12p70, IL-13, IL-15, IL-17 and TNF in the serum were measuredusing Milliplex assay kits (Millipore following manufactures protocols.NB xenograft animal modelTo assess antitumor effects and persistence of GD2.CAR-T cells, we used NOD.Cg-Prkdc scid Il2rg tm1Wjl /SzJ (NSG mice (Jackson Labo

30、ratory. All mouse experiments wereperformed in accordance with Baylor College of Medicine Animal Husbandry andInstitutional Animal Care and Use Committee guidelines. Mice were engrafted withNIH-PA Author ManuscriptNIH-PA Author ManuscriptNIH-PA Author ManuscriptCHLA-255 cells (3 106 s.c. and after 1

31、0 14 days, mice were inoculated i.t. with PBS orOVs (106 109 vp/tumor. Mice were then infused i.v. with either control or GD2.CAR-Tcells (107 cells 3 4 days after virus inoculation. Tumor volumes were monitored twice aweek by caliper measurement (volume = length width 2/2. To track the migration and

32、survival of GD2.CAR-T cells in vivo , control and GD2.CAR-T cells were labeled witheGFP.FFLuc(15. Biodistribution was assessed using the in vivo imaging system (Xenogenas previously described(15.ImmunohistochemistryTumor samples were fixed, processed and stained according to standard procedures. Wep

33、erformed Hematoxylin and Eosin staining and labeling of human T cells using polyclonalrabbit anti-human CD3 mAb (A0452, Dako. For detection we used Dako LSAB + System-HRP (K0679, Dako.Statistical analysisAnalysis of variance (ANOVA with Bonferroni correction and the 2-sided unpaired t testwere used

34、for comparison of 3 or more groups, or 2 groups, respectively as stated in thefigure legends. Mixed-model ANOVA was applied to compare tumor growth in differentgroups of mice. Survival curves were plotted using the Kaplan-Meier methods, and thedifferences in the survival between groups were assessed

35、 by log rank test. Data are presented as mean SD or SEM as stated in the figure legends. Statistical significance was defined atp0.05. Statistical analysis was performed with Prism 5 (GraphPad Software.ResultsCoxackie-adenovirus receptor is functionally expressed in neuroblastoma (NB cell lines but

36、not by GD2.CAR-T cellsTo determine if Ad524 and CAR-T cells could be combined without toxicity to Tlymphocytes in a NB tumor model, we first compared the expression of the Coxackie-adenovirus receptor in 7 NB cell lines and CAR-T cells specifically targeting the GD2antigen expressed by NB cells. As

37、shown in Fig. 1A, all NB cell lines expressed the virusreceptor as assessed by flow cytometry, while CAR-T cells did not. Incubation with theoncolytic adenovirus Ad524 induced cellular toxicity in 5/7 NB cell lines in a dose-dependent manner (Fig. 1B, while GD2.CAR-T cells were unaffected, even at t

38、he highestdose and for up to 7 days of culture (Fig. 1B. Thus Ad524 is cytopathic for NB cells butnot for GD2.CAR-T cells.Combined therapy of GD2.CAR-T cells with Ad524 enhances apoptosis of NB cells in vitroWe next explored whether combining Ad524 and GD2.CAR-T cells enhanced theelimination of NB c

39、ells in vitro in co-culture experiments. When CHLA-255 cells wereincubated with 75 vp/cell of Ad524 and a low ratio of GD2.CAR-T cells (Effector:Target =3:5, tumor cells were effectively eliminated by day 7 of culture compared to tumor cellsincubated with each agent alone. Residual tumor cells were

40、significantly decreased to lessthan 5% in the presence of Ad524 together with GD2.CAR-T cells, compared to 30% NIH-PA Author Manuscript NIH-PA Author ManuscriptNIH-PA Author Manuscript7% and 33% 7% in the presence of GD2.CAR-T cells (P=0.01 and Ad524 combinedwith control T cells (P=0.01 (Fig. 2A. Co

41、mparable results were obtained when two otherNB cell lines IMR-32 and SKNLP were used (Fig. 2A. To clarify the mechanism behindthe superior antitumor effects of the combined treatment, we explored whether Ad524potentiated the function of T cells by upregulating in infected tumor cells the GD2 antige

42、n,death receptors (Fas and TRAIL R1/R2 or costimulatory molecules and ligands (CD80,CD86, OX40L and CD40L. However, no upregulation of any of these molecules by NBcells was detected upon infection with Ad524 (Supplementary Fig. 1. We alsoinvestigated whether Ad524-infected NB cells could indirectly

43、increase the activation oreffector function of CAR-T cells. However, neither CD25, CD69, IFN , granzyme B norTRAIL was significantly upregulated by GD2.CAR-T cells exposed to Ad524-infected NBcells (Supplementary Fig. 2. By contrast, we found that Ad524-infected NB cellsunderwent more rapid apoptosi

44、s when exposed to GD2.CAR-T cells. As illustrated in Fig.2B and Supplementary Movie 1, we conducted sequential confocal microscope imaging ofGFP + CHLA-255 cells co-cultured with GD2.CAR-T cells. We found that NB cellspreincubated for 72 hrs with Ad524 became Annexin-V + within 4 hrs after exposure

45、toGD2.CAR-T cells, while non infected CHLA-255 cells exposed to GD2.CAR-T cellsrequired more than 16 hrs to bind detectable levels of Annexin-V. We quantified the activecaspases in NB cells and found in response to GD2.CAR-T cells a consistent increase in thepercentage of early apoptotic cells when

46、NB cells were preincubated with Ad524compared to NB cells that had not been exposed to Ad524 (FAM +/PI cells were 20% 2% vs.15% 1% at 2 hrs, P 0.05; 24% 2% vs.18% 2% at 4 hrs, P 0.05 (Fig.2C and2D. These results indicate that the caspase activity and apoptosis in NB cells are morepronounced after in

47、cubation with CAR-T cells if NB cells are infected with Ad524.Combined GD2.CAR-T cells and Ad524 have more robust antitumor activity in vivoWe examined whether the combination of GD2.CAR-T cells and Ad524 was also effectivein a NB xenograft mouse model. Mice engrafted subcutaneously (s.c. with the N

48、B cell lineCHLA-255 were inoculated intratumorally (i.t. with either PBS or Ad524 and theninfused intravenously (i.v. with either control or GD2.CAR-T cells (107 cells. Controlmice, inoculated with PBS and control T cells, had rapid tumor progression and wereeuthanized within 20 days (Supplementary

49、Fig. 3. When mice were inoculated with a highdose of Ad524 (109 vp, tumor growth was controlled irrespective of T-cell transfer (Fig.3A. However, when mice were inoculated with lower doses of Ad524, the addition ofCAR-T cells improved tumor control compared to Ad524 plus control T cells (P=0.04 in10

50、6 vp (Fig. 3A.We also found that GD2.CAR-T cells showed enhanced, although still transient, persistencein vivo at the tumor site when tumors were inoculated with Ad524. As shown in Fig.3Band 3C, FFluc-labeled GD2.CAR-T cells localized and persisted up to day 6 in Ad524-inoculated tumors compared to

51、PBS-inoculated tumors (1.8 105 0.63 105 p/s/cm 2/srvs. 1.5 103 0.2 103 p/s/cm 2/sr, P 0.01 and then became undetectable. These findingsraise the possibility that Ad524-infected NB cells may release T-cell chemoattractantfactors. However, measurement of the chemokines RANTES, MIP-1, MIP-1, MCP-1 andI

52、P-10 in the culture of NB cells infected with Ad524 showed no significant production of NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscriptthese cytokines before and after infection, with the exception of the SK-N-SH NB cell linethat spontaneously produced a high level of MCP-

53、1 (Supplementary Table 1. In vitromigration assays confirmed that supernatants collected from NB cells infected with Ad524did not promote T-cell migration, excluding the possibility that other unknown factors thatfavor T-cell migration are released by Ad524-infected NB cells (Fig. 3D. Thus thetransi

54、ent beneficial effects of the combined Ad524 and GD2.CAR-T cells support the needfor the engineering of Ad524 to increase the antitumor benefits.NB cells infected with Ad524 engineered to release RANTES and IL-15 produce functional amounts of both proteinsand prolong persistence of GD2.CAR-T cells a

55、t the tumor site (Fig. 4A, and demonstratedthat NB cells infected with this virus release RANTES (709 2247 pg/ml at 72 hrs andIL-15 (864 2131 pg/ml at 72 hrs in vitro (Fig. 4B. Both factors were functional because0.07 fold in mock; P=0.01, and vs. 0.55 0.04 fold in Ad524; P=0.005 of GD2.CAR-Tcells i

56、n vitro (Fig.4C and 4D without impairing the oncolytic property of the virus (Fteins.Mice engrafted with CHLA-255 cells were inoculated i.t. with either Ad524 orlabeled GD2.CAR-T cells (107 cells. As shown in Fig.5A and 5B, bioluminescence signalsof GD2.CAR-T cells were higher at the site of t

57、umors inoculated with1.0 107 p/s/cm 2/sr vs. 5.6 105 4.1 105 p/s/cm 2/sr at day 12, P 0.001. RANTES andcontrol T cells lacking CAR expression, indicating that antigen-specificity of T cells is alsonecessary for T-cell persistence at the tumor site (Fig. 5C. The increase in photon intensitycorrespond

58、ed to an increased numeric infiltration of human T cells within the tumor asdemonstrated by both flow cytometry and immunohistochemistry (Fig.5D and 5E.Quantification of RANTES and IL-15 in the sera and tumor biopsies confirmed these twoMoreover, both RANTES and IL-15 were predominantly detected at the tumor site (5890 1055 pg/ml and 1762