Intradermal Vaccinations With RNA Coding for TAA Generate CD8 + and CD4 + Immune Responses and Induce Clinical Benefit in Vaccinated Patients

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1 originl rticle The Americn Society of Gene & Cell Therpy Intrderml Vccintions With RNA Coding for TAA Generte CD8 + nd CD4 + Immune Responses nd Induce Clinicl Benefit in Vccinted Ptients Susnne M Rittig 1, Mik Hentschel 1, Ktrin J Weimer 1, Annkristin Heine 2, Mrtin R Muller 1, Wolfrm Brugger 3, Mrius S Horger 4, Olg Mksimovic 4, Arnulf Stenzl 5, Ingmr Hoerr 6, Hns-Georg Rmmensee 7, Tobis AW Holderried 2, Lothr Knz 1, Steve Pscolo 8 nd Peter Brossrt 1,2 1 Deprtment of Hemtology, Oncology, Rheumtology nd Immunology, Eberhrd-Krls-University of Tuebingen, Tuebingen, Germny; 2 Deprtment of Hemtology nd Oncology, University of Bonn, Bonn, Germny; 3 Deprtment of Hemtology nd Oncology, Villingen-Schwenningen, Germny; 4 Deprtment of Rdiology, Eberhrd-Krls-University of Tuebingen, Tuebingen, Germny; 5 Deprtment of Urology, Eberhrd-Krls-University of Tuebingen, Tuebingen, Germny; 6 Curevc GmbH, Tuebingen, Germny; 7 Deprtment of Immunology, Eberhrd-Krls-University of Tuebingen, Tuebingen, Germny; 8 Deprtment of Oncology, University of Zurich, Zurich, Switzerlnd The im of this phse I/II nonrndomized tril ws to ssess fesibility, sfety s well s immunologicl nd clinicl responses of mrna-bsed vccintion in ptients with stge IV renl cell cncer using grnulocyte-mcrophge colony stimulting fctor (GM-CSF) s djuvnt. Intrderml injections of in vitro trnscribed nked mrna, which ws generted using plsmids coding for the tumor-ssocited ntigens mucin 1(MUC1), crcinoembryonic (CEA), humn epiderml growth fctor receptor 2 (Her-2/neu), telomerse, survivin, nd melnom-ssocited ntigen 1 (MAGE-A1) were performed in 3 enrolled ptients. In the first 14 ptients (cohort A) vccintions were dministered on dys, 14, 28, nd 42 (2 µg/ntigen) while in the consecutive 16 ptients (cohort B) n intensified protocol consisting of injections t dys 3, 7 1, 28, nd 42 (5 µg/ntigen) ws used. In both cohorts, fter this induction period, vccintions were repeted monthly until tumor progression nlyzed by Response Evlution Criteri In Solid Tumors criteri (RECIST). Vccintions were well tolerted with no severe side effects nd induced clinicl responses [six stble diseses (SD) nd one prtil response in cohort A nd nine SD in cohort B]. In cohort A, 35.7% survived 4 yers (medin survivl 24 months) compred to 31.25% in cohort B (medin survivl 29 months). Induction of CD4 + nd CD8 + T cell responses ws shown for severl tumor-ssocited ntigens (TAA) using interferon-γ (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) nd Cr-relese ssys. Received 1 My 21; ccepted 29 November 21; published online 28 December 21. doi:1.138/mt Introduction Renl cell crcinom (RCC) is reltively rre tumor ccounting for 2 3% of mlignncies in dults. When dignosed t erly stge of disese, surgicl resection cn be curtive. However, ~3% of ptients develop metstses resulting in 5-yer survivl rte of <1%. 1 Despite the use of the recently introduced trgeted therpies, mngement of dvnced RCC remins chllenging. The fct tht RCC cn evoke n immune response tht occsionlly results in spontneous remissions, the detection of dendriticnd T-cells in RCC tissues, 2,3 nd dvnces in tumor immunology hve stimulted the development of vccintion strtegies for RCC ptients. Within the lst yers, severl tumor-ssocited ntigens (TAA) expressed in RCC nd recognized by cytotoxic T lymphocytes (CTL) hve been defined by using expression cloning, reverse immunology pproches, or the ppliction of DNA micro-rry technologies. 4,5 Recent reports from severl phse I/ II trils hve shown encourging results nd furthermore demonstrted the sfety of specific immuno Numerous studies hve shown tht dendritic cells (DC) trnsfected with mrna coding for TAA or whole tumor RNA re ble to induce potent ntigen- nd tumor-specific T cell responses This technology ws pioneered by E. Gilbo nd lter confirmed by severl other groups. As promising lterntive to vccintion with trnsfected DC, direct intrderml ppliction of nked mrna ws demonstrted to be effective in the expression of the encoded protein 26,27 nd the subsequent genertion of ntigen-specific T cell responses in severl mouse models We conducted phse I/II tril to investigte fesibility, sfety, nd immunologicl responses of nked mrna-bsed vccintion djuvnted with grnulocytemcrophge colony stimulting fctor (GM-CSF) in dvnced RCC ptients. Results Ptient chrcteristics nd study design Between August 23 nd November 25, 3 ptients ged yers were enrolled in the study. Intrderml injections of in vitro trnscribed nked mrna, which ws generted using plsmids coding for the tumor-ssocited ntigens mucin 1 (MUC1), S.M.R. nd M.H. hve contributed eqully to this work. S.P. nd P.B. shre equl seniorship. Correspondence: Peter Brossrt, Medizinische Klinik III, University of Bonn Medicl Center, Wilhemstrße 35-37; Bonn 53111, Germny. E-mil: peter.brossrt@ukb.uni-bonn.de 99 vol. 19 no. 5, my 211

2 The Americn Society of Gene & Cell Therpy Immuno With Nked RNA in Renl Cncer Cohort A Dy, 1 14, 15 28, 29 42, 43 7, 71 Vccine GM-CSF X X X X X Cohort B Dy, 1, 2, 3 7, 8, 9, 1 28, 29 42, 43 7, 71 IFN γ Spots Muc 1.1 Muc 1.2 Surv 1 Surv 2 CEA MAGE Before After 4 Ptient number 1 cohort A After 8 After 12 After 16 After 19 Vccine GM-CSF X X X X X X X X X Figure 1 Study design (injection schedule). Vccintions were performed on dys, 14, 28, nd 42 in cohort A nd on dys 3, 7 1, 28, nd 42 in cohort B (mrked by the rrow). Vccintions were repeted monthly until tumor progression. On the dy following mrna-injection, grnulocyte-mcrophge colony stimulting fctor (GM-CSF) ws pplied subcutneously (mrked by x ). crcinoembryonic (CEA), humn epiderml growth fctor receptor 2 (Her-2/neu), telomerse, survivin nd melnom-ssocited ntigen 1 (MAGE-1) were performed. In the first 14 ptients (cohort A), vccintions were dministered on dys, 14, 28, nd 42 (2 µg/ntigen) wheres in the consecutive 16 ptients (cohort B) n intensified protocol consisting of injections t dys 3, 7 1, 28, nd 42 (5 µg/ntigen) ws used. In both cohorts, fter this induction period, vccintions were repeted monthly until tumor progression nlyzed by Response Evlution Criteri In Solid Tumors (RECIST) criteri. An overview of the injection schedules is given in Figure 1. Three ptients in cohort A nd five in cohort B were femle. The medin ge in cohort A ws 64.5 compred to 64 yers in cohort B (Tble 1). All 3 ptients hd received prior, including surgery (ll ptients), interferon-α (five ptients in cohort A, one ptient in cohort B), nd rdio (one ptient in cohort A nd five ptients in cohort B). 19 ptients (9 in cohort A, 1 in cohort B) hd surgery only. Histology of renl cell crcinom ws mostly tht of cler cell crcinom or ppillry crcinom nd metsttic disese ws present t numerous ntomic sites including lung, lymph nodes, kidney, hert, bone, peritoneum, protid- nd, drenl glnd, pncres, nd retroperitoneum (Tble 1). According to Motzer risk criteri (MSKCC risk model), 32 four ptients in cohort A were t fvorble risk (no risk fctors) nd 1 hd one or two risk fctors (intermedite risk). In cohort B, seven ptients were t fvorble risk (no risk fctors) the remining nine ptients hd n intermedite risk. The following risk fctors were used, s ssessed by Motzer: low Krnofsky index (<8%), high lctte dehydrogense-levels (>1.5 upper norml level), low hemoglobin level, corrected clcium >1 mg/dl, bsence of prior nephrectomy. No risk fctors: fvorble risk; one or two risk fctors: intermedite risk; three or more risk fctors: poor risk. Sfety nd toxicity The toxicity ssocited with our vccintion ws miniml, lwys reversible nd minly restricted to swelling, redness, nd itching t the injection site of GM-CSF, fever 38 C nd hedche on the dy of GM-CSF dministrtion (no grd III or IV toxicity). b IFN γ Spots c IFN γ Spots Muc 1.1 Muc 1.2 Surv 1 Surv 2 CEA MAGE Before Muc 1.1 Muc 1.2 Surv 1 Surv 2 CEA MAGE Before After 8 Ptient number 11 cohort B After 9 After 4 After 11 Ptient number 16 cohort B After 8 After 17 After 2 After 12 Figure 2 Genertion of immunologicl responses upon vccintion with in vitro trnscribed mrna; exemplry interferon-γ (IFNγ) enzyme-linked immunosorbent spot (ELISpot) ssys: course of severl tumor-ssocited ntigen (TAA). Autologous peripherl blood mononucler cells from humn leukocyte ntigen (HLA)-A2 + ptients were pulsed with the peptides MUC 1.1, MUC 1.2, MAGE, CEA, survivin 1 nd 2 deduced from TAA used in the vccine nd used s stimultors. ( c) Representtive ELISpot ssys of severl different T cell response ptterns of three ptients. Smples from prevccintion nd representtive smples fter one or more vccintions were evluted simultneously. CEA, crcinoembryonic; MAGE, melnom-ssocited ntigen 1; MUC 1, mucin 1. One ptient developed n llergic rection to GM-CSF. No utoimmune phenomen were observed. Immunologicl responses The induction of TAA-specific T cell responses in vivo fter mrna injections ws ssessed by performing interferon-γ (IFNγ) enzyme-linked immunosorbent spot (ELISpot) nd cytotoxic T cell ssys. The vccintion-induced TAA-specific CTL responses in humn leukocyte ntigen (HLA)-A2 positive ptients were determined by nlysis of IFN-γ production of T lymphocytes fter stimultion with the cognte peptide epitope derived from used TAA in vitro. An immune response to peptides ws considered significnt when stimultion index of 2 s compred to the number of spots vccintion ws detected t lest t two different time points fter vccintions for two or more TAAderived peptides. In ELISpot ssys, ntigen-specific T cell induction ws observed for severl TAA in 8 out of 1 tested ptients, in Moleculr Therpy vol. 19 no. 5 my

3 Immuno With Nked RNA in Renl Cncer The Americn Society of Gene & Cell Therpy Tble 1 Ptient chrcteristics Ptient number Age (yers) Previous Site of metstsis Motzer risk group Cohort A 1 72 Surgery Lung Fvorble risk 2 36 Surgery Ovry, lymph nodes Intermedite risk 3 64 Surgery Bone Intermedite risk 4 79 Surgery Lymph nodes, lung Intermedite risk 5 71 Surgery Lung, liver, lymph nodes, pncres Intermedite risk 6 63 Surgery, IFN Lymph nodes, lung, retroperitoneum, bone Intermedite risk 7 61 Surgery Lymph nodes, lung Intermedite risk 8 65 Surgery, IFN Lymph nodes, lung Fvorble risk 9 6 Surgery Lymph nodes, lung Intermedite risk 1 73 Surgery, IFN Lung Intermedite risk Surgery, IFN Lung, liver Intermedite risk Surgery Lymph nodes, lung Fvorble risk Surgery, IFN, RT Lymph nodes, bone Intermedite risk Surgery Lymph nodes, drenl glnd, lung Fvorble risk Cohort B 1 64 Surgery, RT Bone, lung, lymph nodes, retroperitoneum Fvorble risk 2 6 Surgery Lung, hert Intermedite risk 3 44 Surgery Lung Intermedite risk 4 57 Surgery Lymph nodes, lung Intermedite risk 5 64 Surgery Peritoneum Intermedite risk 6 63 Surgery Lymph nodes, lung, drenl glnd Intermedite risk 7 68 Surgery Protid glnd, thyroid glnd, drenl glnd, lymph nodes, bone Fvorble risk 8 63 Surgery, RT Bone, lung, kidney, lymph nodes Fvorble risk 9 73 Surgery, RT Lung, bone Intermedite risk 1 56 Surgery Lymph nodes; lung Intermedite risk Surgery Lymph nodes Fvorble risk Surgery; IFN Lymph nodes Fvorble risk Surgery, RT Lung, bone, kidney Intermedite risk Surgery, RT Lung, bone Intermedite risk Surgery Lung, kidney Fvorble risk Surgery Lymph nodes, lung, pncres, kidney Fvorble risk No risk fctors: fvorble risk; one or two risk fctors: intermedite risk; three or more risk fctors: poor risk. IFN, interferon-α; RT, rdio. MSKCC risk model consisting of the following five risk fctors: low Krnofsky index (<8%), high lctte dehydrogense-levels (>1.5 upper norml level), low hemoglobin level, corrected clcium >1 mg/dl, bsence of prior nephrectomy. some ptients lredy fter the first four vccintions (1/1 tested ptient in rm A nd 7/9 tested ptients in rm B). In ptient number, three of rm B ELISpot ssy ws performed only t two time points ( vccintion nd fter 4th vccintion). A stimultion index 2 could be shown lredy fter four vccintions, but by reson of missing 2nd timepoint we defined this immunologicl response s negtive. Exemplry ELISpot ssys of CD8 + T cells rective to the TAA MUC1, survivin, CEA, nd MAGE re shown in Figure 2. As shown in Figure 3 immune responses ginst the epitopes MUC 1.2 nd survivin 1 were pronounced in ELISpot ssys s compred to the other (MUC 1.1 nd survivin 2) ntigenic peptides deduced from these tumor-ssocited ntigens indicting immunodominnce of the epitopes MUC 1.2 nd survivin 1. Figure 3 demonstrtes in detil the exemplry course of responses ginst HLA-A2 binding MUC 1 nd survivin peptides in three ptients. Due to lck of blood smples, we could perform ELISpot ssys using HLA-A2 binding peptides only for one of the four HLA-A2 positive ptients in rm A, but for ll nine HLA-A2 positive ptients in rm B (Supplementry Figures S1 nd S2). For the detection of CD4 + T cell responses CD4 + T lymphocytes were isolted by mgnetic ctivted cell sorting technology nd stimulted with utologous DC electroported with the TAA-mRNA-mix s described bove. The immune response to TAA used for vccintions ws nlyzed in IFN-γ ELISpot ssys nd ws considered significnt when stimultion index of 2 compred to the spot numbers vccintion ws detected vol. 19 no. 5 my 211

4 The Americn Society of Gene & Cell Therpy Immuno With Nked RNA in Renl Cncer : MUC-1 b: Survivin Survivin Pt. number 1 cohort A MUC Pt. number 1 cohort A fter 16. fter 16. fter 19. fter 19. fter 16. fter 16. fter 19. fter 19. Survivin Pt. number 15 cohort B MUC Pt. number 15 cohort B MUC Pt. number 16 cohort B 12 1 Survivin Pt. number 16 cohort B fter 5. fter 5. fter 9. fter fter 5. fter 5. fter 9. fter 9. c: CD4 + T cell responses IFN Y Spots CD4 + Pt. number 6 Cohort A fter 2. IFN Y Spots CD4 + Pt. number 4 Cohort B fter 9. IFN Y Spots CD4 + Pt. number 7 Cohort B fter 1. Figure 3 Genertion of immunologicl responses upon vccintion with in vitro trnscribed mrna. Exemplry enzyme-linked immunosorbent spot (ELISpot) ssys. (,b) Immunodominnt ntigens. Of ll used ntigens, immune responses for the epitopes MUC 1.2 nd survivin 1 were pronounced in ELISpot ssys indicting tht these epitopes re immunodominnt. Detiled course of immune responses re displyed for the epitopes () MUC 1.1 nd 1.2 (b) nd survivin 1 nd 2 in three ptients. (c) Results obtined for CD4 + cells. Autologous CD4 + T cells were isolted by mgnetic ctivted cell sorting technology nd stimulted with dendritic cells electroported with mixture of RNA coding for tumor-ssocited ntigen used in the vccine. Enhnced green fluorescent protein (EGFP)-RNA served s negtive control, counted spots in EGFP vils were subtrcted. MUC 1, mucin 1. t lest t one time point fter vccintion. Vccine-induced CD4 + T cell responses re shown in Figure 3c for three ptients. Responses were detected in four of eight tested ptients using IFN-γ ELISpot ssys. In order to nlyze the cytotoxic ctivity of vccine-induced T cells, peripherl blood leukocytes from vccinted HLA-A2 positive ptients were restimulted with utologous DC electroported with the mix of mrna coding for the used TAA. The lytic ctivity ws determined in chromium-relese ssys ginst utologous DC pulsed with HLA-A2-binding peptides derived from the TAA or HLA-A2-positive tumor cell lines expressing the ntigens. In ddition, DC electroported with the mrna-mix coding for the TAA were included s trgets in these ssys. As negtive control, DC trnsfected with enhnced green fluorescent protein (EGFP)-coding mrna were used. Dt nd results from two ptients re presented in Figure 4. CTL obtined fter one in vitro restimultion efficiently lysed trget cells pulsed with the ntigenic peptides (MUC1, survivin, MAGE, CEA), s well s DC electroported with mixture of RNA coding for TAA. Lysis ws shown to be ntigen-specific since no lysis ws detected when trget cells were loded with n irrelevnt peptide derived from HIV or when electroported with EGFP-RNA. We could demonstrte Moleculr Therpy vol. 19 no. 5 my

5 Immuno With Nked RNA in Renl Cncer The Americn Society of Gene & Cell Therpy Ptient number 11, cohort B DC + RNA-Mix DC + Flu-RNA b Ptient number 11, cohort B DC + MUC 1.2 peptide % Specific lysis DC + EGFP-RNA K562 A498 SKOV Cki-2 % Specific lysis DC + Survivin1 peptide DC + MAGE peptide DC + CEA peptide DC + HIV peptide E.T E.T c 9 Ptient number 8 cohort B d 9 Ptient number 8 cohort B DC + MUC 1.1 peptide % Specific lysis DC + RNA-Mix DC + Flu-RNA DC+ EGFP-RNA A498 Cki-2 K562 SKOV % Specific lysis 6 DC + Survivin1 peptide 5 DC + CEA peptide 4 3 DC + MAGE peptide 2 DC + HIV peptide E.T E.T Figure 4 Lytic ctivity of vccine-induced T lymphocytes. Cytotoxic T lymphocytes obtined fter in vitro restimultion efficiently lysed trget cells [dendritic cells (DC)] pulsed with the ntigenic peptides (MUC1, survivin, MAGE, CEA), s well s DC electroported with mixture of RNA coding for tumor-ssocited ntigen (TAA). Lysis ws shown to be ntigen-specific since no lysis ws detected when trget cells were loded with the peptide derived from HIV or when electroported with enhnced green fluorescent protein (EGFP) RNA. In ddition, the humn leukocyte ntigen (HLA)-A2 positive tumor cell line A498 [renl cell crcinom (RCC)], s well s HLA-A2 negtive control cell lines like SK-OV-3 cells (ovrin cncer) nd CKi-2 (RCC) were included demonstrting HLA restricted lysis of CTL. K562 [chronic myelogenous leukemi (CML) in blst crisis] ws used to exclude nturl killer-cell medited lysis. Dt from two ptients re presented (,b: ptient number 11 nd c,d: ptient number 8, cohort B). CEA, crcinoembryonic; MAGE, melnom-ssocited ntigen 1; MUC 1, mucin 1. ntigen-specific lysis of trget cells in chromium-relese ssys in 8 out of 1 tested ptients. In order to nlyze the possible genertion of ntibody responses we included the mrna coding for heptitis-b-surfce (HBS) ntigen in the experimentl vccine. However, we were not ble to determine ny seroconversion in ny of the vccinted ptients s ll remined nti-hbs negtive (centrl lbortory fcilities t the University of Tuebingen, dt not shown). Clinicl responses to mrna vccintions With the exception of one ptient, ll of the 3 ptients initilly enrolled in the study completed the first prt of the vccintion schedule the first stging time point (7 weeks fter study entry; 4 injections in cohort A nd 1 injections in cohort B). Seven ptients in cohort A were still on study tretment fter the first four vccintions due to objective response (n = 1) or stbiliztion of disese (n = 6) with the number of vccintions rnging from 7 to 32 (Tble 2). Nine of sixteen ptients in cohort B chieved stble disese fter 7 weeks of tretment nd received >1 vccintions, rnging from 12 to 22 vccintions (three ptients received 2 or more vccintions). Tretment of one ptient in cohort B could not be continued due to descent in generl condition. This ptient survived only 2 months fter the first vccintion. During dministrtion of the mrna formultion, there ws tumor regression or stbiliztion lsting for >3 months (defined s clinicl benefit) in 7 of 14 ptients in cohort A nd in 9 of 16 ptients in cohort B. Prtil response ws confirmed in ptient number six in cohort A nd ws observed s shrinking of cervicl nd medistinl lymph nodes s well s pulmonry metstses (Figure 5). In one ptient in cohort B (ptient number five), the need for bdominl prcentesis, tht hd to be performed every other dy due to refrctory scites nd extended bdominl tumor sites, diminished during the first vccintions. This ptient remined free of prcentesis for >3 months nd the scites completely resolved in line with decline of the tumor mrker CA-125 nd regression of bdominl tumor sites (Figure 5b). Dt of clinicl outcome re presented in Tble 2. The longest durtion of response for one ptient with prtil response ws 31 months in cohort A. In cohort B, the durtion of response ws 12+ months t most t the lst follow-up due to lter initition of this cohort. Medin durtion of response for ll responders ws 9.5 months. Medin progression free survivl ws 2 months in cohort A, 4 months in cohort B nd 4 months for ll ptients together. 35.7% vol. 19 no. 5 my 211

6 The Americn Society of Gene & Cell Therpy Immuno With Nked RNA in Renl Cncer Tble 2 Clinicl responses Ptient number Course vccintion Response Durtion of response Number of vccintions Sttus Survivl fter 1. vccintion (months) Cohort A 1 PD SD 13 2 DOD 32 2 PD PD 4 DOD 6 3 PD PD 4 DOD 24 4 PD PD 4 DOD 17 5 PD SD 1 13 AWD 85 6 PD PR AWD 75 7 SD PD 4 DOD 3 8 PD SD 16 2 AWD 78 9 PD PD 4 DOD 48 1 PD SD 7 9 DOD 8 11 PD PD 4 DOD 9 12 PD PD 4 DOD 8 13 PD SD 4 7 DOD PD SD AWD 8 Cohort B 1 PD SD 4 13 DOD 33 2 PD SD 4 12 DOD 8 3 PD PD 1 AWD 62 4 PD PD 11 DOD 15 5 PD SD 6 12 DOD 27 6 PD SD 8 17 DOD 41 7 PD PD 11 AWD 71 8 PD SD 9 17 DOD 57 9 PD PD 1 DOD 8 1 PD DIGC b 8 DOD 2 11 PD SD AWD PD SD 12 2 DOD PD PD 1 DOD SD PD 1 DOD PD SD 5 14 DOD PD SD AWD 68 AWD, live with disese; DOD, died of disese; PD, progressive disese; PR, prtil response; SD, stble disese. End of follow-up October 21. b Descent in generl condition. (5/14) of ptients in cohort A showed survivl of 4 yers or more fter the first vccintion (rnge of months, medin survivl 24 months). Four of fourteen (28.6%) were still live t the lst follow-up (October 21) with 5-yer survivl rte of 21.4% in this cohort. In cohort B, 31.25% (5/16) of ptients were live 4 yers fter study entry with survivl rnging from 48 to 71 months. Medin survivl ws 29 months. Four of sixteen ptients were live t follow-up in October 21 nd 25% hd survived >5 yers (Tble 2). Kpln Meier plots for overll survivl () nd progression free survivl (b) of the two cohorts re presented in Figure 6. While there ws no significnt difference in the overll survivl between cohort A nd cohort B, significnt improvement of the overll survivl ws found when compring ptients cliniclly responding to the tretment t the first stging time point with nonresponders (P <.2) (Figure 6c). Immunologicl nd clinicl responses correlted in most tested ptients, though we could not perform sttisticl nlysis, becuse not ll immunologicl ssys could be performed in ll ptients. This ws due to HLA-type nd lck of blood smples. Tken together, 12 ptients showed immunologicl responses in one or more of performed ssys. Five ptients showed no immunologicl response. Furthermore in two ptients immunologicl responses could be detected in one ssy (ELISpot) but not in performed chromium-relese ssys. As mentioned bove in one further ptient ELISpot ssy ws performed only t Moleculr Therpy vol. 19 no. 5 my

7 Immuno With Nked RNA in Renl Cncer The Americn Society of Gene & Cell Therpy August 16th 24 b CT scn ptient number 6 cohort A Tumor mrker of ptient number 5 cohort B First vc Months lter two time points ( vccintion nd fter four vccintions) due to lck of blood smples. A stimultion index >2 could be detected lredy fter four vccintions, but due to missing 2nd time point, we defined this immunologicl response s negtive. Tken together medin survivl ws 4.5 months in the 12 ptients showing immunologicl response versus 14 months in the 5 ptients showing no response in immunologicl ssys (Supplementry Tble S1). Discussion The discovery of new TAA nd the development of vrious vccintion methods led to the evlution of multiple experimentl immunotherpies in RCC ptients. Severl studies hve proven fesibility nd sfety 6,7,9 of these vccines nd hve proven to result in the genertion of ntigen-specific immune responses. Direct injection of nked mrna ws demonstrted to elicit immune response ginst the encoded ntigens A clinicl ppliction of nked mrna in melnom ptients ws recently published by Weide et l. Totl utologous tumor RNA ws extrcted, reversely trnscribed, nd dministered intrdermlly. The tretment ws shown to be sfe nd fesible but lcked cler clinicl effectiveness. 33 We used nked mrna molecules in our vccintion study since nturlly trnsient nd cytosoliclly ctive mrna molecules re considered to be possibly sfer nd more potent lterntive to Lst vc C125 (U/ml) Figure 5 Clinicl responses in vccinted ptients. () Computed tomogrphy (CT) scn of ptient number 6 (cohort A) with pulmonry metstses. Size of mettses diminished fter 4 months of vccintion. (b) Course of tumor mrkers under vccintion (ptient number 5, cohort B) The rrow mrks the first nd the lst injection. The need of prcentesis diminished during vccintion. % Survivl b % Survivl c % Survivl Overll survivl in months cohort A vs. cohort B 1 9 Cohort A 8 Cohort B OS in months Progression free survivl in months cohort A vs. cohort B Cohort A Cohort B PFS in months Overll survivl in responder vs. non-responder in months Non-responder Responder OS in months Figure 6 Kpln Meier grphs: survivl dt. () Overll survivl in months of ptients in cohort A versus cohort B. Log-rnk test showing no significnt differences in overll survivl (P =.85). Medin survivl cohort A: 24 months, cohort B: 29 months. (b) Progression free survivl in months of ptients in cohort A versus cohort B. Log rnk test showing no significnt differences in progression free survivl (P =.33). (c) Overll survivl in clinicl responders versus nonresponders. Ptients who hd shown objective response or t lest stble disese t the first stging time point were defined s clinicl responders (P =.2). Medin survivl nonresponder: 14 months, responder: 41 months. PFS, progression free survivl; OS, overll survivl. DNA for gene vccintion. Ptients suffering from metsttic RCC were repetedly injected intrdermlly with nked mrna coding for the TAA MUC1, CEA, Her-2/neu, telomerse, survivin- nd melnom-ssocited ntigen 1 (MAGE-A1). RNA pplictions vol. 19 no. 5 my 211

8 The Americn Society of Gene & Cell Therpy Immuno With Nked RNA in Renl Cncer were fesible, sfe nd well tolerted with no relevnt side effects. Even though we were ble to detect T cell responses cpble of recognizing nd lyzing trget cells expressing the used tumor-ssocited ntigens tht represent self-ntigens we did not observe ny side effects with regrd to utoimmune rections or orgn toxicity. This is in line with the results from our previous vccintion studies 8,34,35 nd studies performed by others. 6,7,33 The only so fr reported self-rections were documented in ptients with mlignnt melnom where vccine-induced vitiligo ws described. Clinicl responses [one prtil response nd six stble diseses (SD)] were chieved in 7 of 14 ptients in cohort A nd 9 of 16 ptients hd SD in the intensified cohort B, with medin survivl of 24 months in cohort A nd 29 months in cohort B, respectively. Survivl correlted with the MSKCC risk model nd showed promising results under vccintion in this smll group of ptients. Ptients without risk fctors survived 8 8 months (1-yer survivl rte 9.9%, medin survivl: 57 months). The expected 1 yer survivl rte for these ptients is 83% with suspected medin survivl of 2 months ccording to the MSKCC risk model. For ptients with intermedite risk our tretment resulted in 1-yer survivl rte of 63% (expected ccording to MSKCC risk model: 58%) nd medin survivl of 15 months compred to n expected medin survivl of 1 months. 32 Currently, 8 of our 3 ptients re still live (October 21). As frequently observed in former studies, immunologicl responses were detected in lmost ll ptients with clinicl response or stbiliztion of disese. It is of specil interest nd importnce tht T cell responses elicited in ptients upon vccintions lsted for severl months during tretment nd were directed ginst severl epitopes derived from defined ntigen demonstrting tht this pproch genertes polyclonl immune responses with brod specificity ginst multiple ntigens nd T cell epitopes. In ddition, our study convincingly shows tht mixture of severl mrna cn be sfely nd efficiently pplied in order to generte specific ntitumor immunity tht consists of both CD4 + nd CD8 + effector cells. We did not specificlly ddress the induction of regultory T cells in our study. However, the CD4 + cells detected nd expnded in our ptients secreted IFN-γ, thus excluding their regultory function nd indicting helper T cells 1 (Th-1) phenotype. In order to nlyze the possible genertion of ntibody responses, we included the RNA coding for HBS ntigen. However, we were not ble to determine ny seroconversion in vccinted ptients s ll remined nti-hbs ntigen negtive. A correltion between immunologicl responses nd survivl ws found in most of the tested ptients, though this observtion hs limited expressiveness, becuse we could not perform ll ssys in every ptient. In this study, we demonstrte tht specific immuno using intrderml injections of nked in vitro trnscribed mrna coding for severl TAA cn elicit immunologicl responses nd result in clinicl benefit in ptients with metsttic RCC. Messenger RNA ppliction llows trgeting multiple TAA nd epitopes independent of ptients HLA-type. Thus, it represents sfe nd verstile vccintion strtegy in the context of nticncer immunotherpies. Mterils nd Methods Ptient chrcteristics nd clinicl protocol. This is nonrndomized phse I/II tril using nked mrna trnscribed from DNA plsmids nd coding for MUC1, CEA, Her-2/neu, telomerse, survivin, nd MAGE-A1 tumor ntigens, s well s influenz-mtrix-m1 protein (IMP) nd heptitis-b-surfce ntigen (HBS ntigen) serving s potentil controls. The primry end point ws to ssess fesibility nd sfety. The secondry end points were the nlysis of immunologicl responses nd potentil ntitumor ctivity. Ptients with histologiclly confirmed dvnced RCC fter ccomplishing surgery, rdition or IFN-α or without prior were included in the study (Tble 1). Except for two ptients ll hd progressive disese (Tble 2). Inclusion criteri were bidimensionlly mesurble lesions, willingness, nd bility to give informed consent, ged yers, 6 weeks intervl to lst chemo/immuno nd/or rdition, bilirubin <2 mg/dl, cretinine <2 mg/dl, nd Krnofsky score >7%. Exclusion criteri were chemo or intke of immune-modulting drugs like corticosteroids within 6 weeks onset of vccintion, severe hert disese (NYHA 3), neurologicl or psychitric disorders, brin metstses, pregnncy, underge ptients or inbility to give informed consent, nd history of secondry mlignncies. The protocol ws pproved by the locl institutionl ethics committee t Eberhrd-Krls-University Tuebingen, Tuebingen, Germny. Written informed consent ws given by ech ptient prior to study inclusion. The Declrtion of Helsinki protocols were followed. Preceding the first vccintion, ll ptients underwent n extended clinicl evlution including physicl exmintion, hemtologicl nd biochemicl prmeters, nd computed tomogrphy scns. Messenger RNA were trnscribed from plsmids nd coded for MUC1, CEA, Her-2/neu, telomerse, survivin, nd MAGE-A1 tumor ntigens s well s IMP nd HBS s potentil controls (in collbortion with CureVc, Tuebingen, Germny). 2 µg (cohort A) or 5 µg (cohort B) of coding nked RNA per ntigen dissolved in 15 µl phosphte-buffered sline s injection solution were injected intrdermlly t two different sites. This ws not rndomized tril. In the first group of ptients vccintions were performed on dys, 14, 28, nd 42 (cohort A). In the following ptients, vccintion ws pplied in n intensified mnner on dys 3, 7 1, 28, nd 42 (cohort B). Bsed on previous results from niml studies 3 on the dy following RNA vccintion, GM-CSF 1 µg/ m 2 (cohort A) nd 25 µg (cohort B) ws dministered subcutneously t one of the injection sites of RNA. Injection sites were the lower bdomen or upper thigh nd sites nd locliztions could be chnged during tretment. Restging ws performed round week 7. Further restging ws performed every 6 8 weeks ccording to RECIST criteri. In cse of tumor regression or stble disese, vccintion ws repeted monthly until tumor progression (Figure 1). According to the pproch used (intrderml ppliction of RNA nd subcutneous ppliction of GM-CSF) we ssumed typicl rte of risk for locl nd systemic infection s well s possible llergic rection. Furthermore, we expected n inflmmtory skin rection t the injection site consisting of erythem, pruritus, nd swelling. Systemic flu-like symptoms such s mylgi, hedche, or fever were ssumed minly due to ppliction of the djuvnts. Every dverse event ws to be documented t every visit ccording to its chrcter, time point, intensity, nd durtion of ppernce s well s probble cuslity (Common Toxicity Criteri version 2.). Serious dverse events were to be immeditely reported to the principle investigtor nd locl institutionl ethics committee. In cse of deth due to side effects ptients should be utopsied. Cell isoltion nd cultures. Ptient-derived peripherl blood mononucler cells (PBMC) were isolted by FICOLL-Pque (Life Technologies, Grnd Islnd, NY) density grdient centrifugtion of 5-ml heprinized blood. Genertion nd chrcteriztion of ptients monocyte-derived dendritic cells ws performed s described previously. 34,36 The following cell lines were used: A498 (RCC, HLA-A2 +, Americn Type Culture Collection, Mnsss, VA), SK-OV-3 cells (ovrin cncer, HLA-A2-, kindly provided by O.J. Finn, University of Pittsburgh, Pittsburgh, PA), CKi-2 (RCC, HLA-A2-, kindly provided by collegues of Eberhrd-Krls-University Tuebingen), nd K562 [chronic myelogenous leukemi (CML) in blst Moleculr Therpy vol. 19 no. 5 my

9 Immuno With Nked RNA in Renl Cncer The Americn Society of Gene & Cell Therpy crisis; Americn Type Culture Collection]. Morphology of ll used cell lines ws checked by microscope once or twice week during culturing periods t both high nd low density. HLA-typing ws performed for indicted cell lines. Furthermore used cell lines hd been tested for severl trnscription fctors s well s tumor-ssocited ntigens in our lbortory. 8,37 39 Peptides. The HLA-A2 binding peptides derived from (sequence STAPPVHNV), (sequence LLLLTVLTV), survivin 1 (sequence ELTLGEFLKL), survivin 2 (sequence TLPPAWQPFL), CEA 8 (sequence YLSGANLNL), MAGE-A1 (sequence KVLEYVIKV), VMT-1_IAPUE (influenz mtrix protein, sequence: GILGFVFTL) were synthesized using stndrd F-moc chemistry on peptide synthesizer (432A; Applied Biosystems, Weiterstdt, Germny) nd nlyzed by reverse-phse highperformnce liquid chromtogrphy nd mss spectrometry ( syfpeithi.de). Peptides were kindly provided by Prof Stefn Stevnovic, Deprtment for Immunology, Eberhrd-Krls-University Tuebingen. ELISpot ssys. To nlyze vccine-induced expnsion of T cells specific for the used TAA-RNA, we performed IFN-γ ELISpot ssys in HLA-A2 positive ptients s described previously. 8,38 In brief, peripherl blood ws obtined the first vccintion nd t vrious time points during the vccintion period. PBMC were isolted by FICOLL grdient centrifugtion nd were cryopreserved. After completion of tretment, smples from prevccintion nd fter one nd more pplictions of the vccine were evluted simultneously. PBMC obtined the first vccintion nd PBMC from individul time points during tretment were incubted in 24-well pltes with HLA-A2-binding peptides derived from the TAA used in the vccine or dimethyl sulfoxide (used for peptide dilution, negtive control) for 7 dys t 37 C. One peptide ws used per well in concentrtion of 5 µg/ ml. For the redout, utologous PBMC were pulsed with the corresponding peptide nd used s stimultors in ELISpot ssys. Ech smple ws tested in duplicte. Spots obtined in dimethyl sulfoxide wells (without peptide) were considered s bckground ctivity nd subtrcted from vlues obtined with the tested epitopes. Phorbol myristte cette (PMA) nd ionomycin were used s positive controls. For CD4 + ELISpot ssys, CD4 + cells were sorted from isolted PBMC by mgnetic ctivted cell sorting technology ccording to the mnufcturers instructions. Autologous DC were generted from by monocyte plstic-dherence from PBMC s previously described. 4 DC were electroported with the mrna-mix (s described below) consisting of ll TAA were used s stimultors for the nlysis of vccine-induced CD4 + T lymphocyte responses ex vivo. Electroportion of DC ws performed s described previously. 15 The physicl prmeters used for electroportion were s follows: voltge of 3 V, cpcitnce of 15 µf, resistnce of 1,54 Ω, nd pulse time of 231 ms. After electroportion, cells were trnsferred immeditely into RP1 medium. DC nd CD4 + T cells from different time points were incubted in ELISpot ssys for 4 hours t 37 C. Ech smple ws tested in duplicte. DC electroported with EGFP-RNA served s negtive control nd the spots of these T cell responses were considered s bckground ctivity nd subtrcted from vlues obtined with the RNA-mix. Stndrd 51Cr-relese ssy. To nlyze the lytic ctivity of the induced CTL, utologous PBMC were restimulted twice with utologous DC, electroported with the mrna mixture consisting of ll five mrna coding for TAA used in the vccine, nd the lytic ctivity ws determined in stndrd 51 Cr-relese ssy. This ssy ws performed with some modifictions s described previously. 34 Following trgets were used: A498 (RCC, HLA-A2 + ), SK-OV-3 cells (ovrin cncer, HLA-A2 ), nd CKi-2 (RCC, HLA-A2 ), peptide-pulsed utologous DC or utologous DC electroported with RNA coding for the specific ntigens nd utologous DC electroported with mixture of TAA RNA. Enhnced green fluorescent protein (EGFP)-coding mrna served s negtive control. The physicl prmeters used for electroportion were s described bove. After electroportion, cells were trnsferred immeditely into RP1 medium supplemented with the cytokines GM-CSF (1 ng/ml) nd IL-4 (2 ng/ml) nd returned to the incubtor. Genertion of mrna by in vitro trnscription. EGFP-coding in vitro trnscript ws synthesized from the plsmid psp64 Poly(A) EGFP-2 (generously provided by V.F.I. Vn Tendeloo, Antwerp, Belgium) s described previously. 14,15 The genertion of mrna used in the ssys ws performed by CureVc s recently described. 3,41 Sttistics. For sttisticl nlyses, we used Jmp (ver 7). Survivl dt were bsed on informtion given by ptients, fmily members, or the fmily doctor. For survivl nlyses, we used the Kpln Meier method including evlution of medin nd men survivl. To compre survivl functions, we used log-rnk test. SUPPLEMENTARY MATERIALS Figure S1. Sctter plots of ELISpots. Figure S2. ELISpot dt. Tble S1. Immunologicl ssys. ACKNOWLEDGMENTS Potentil conflicts of interest: I.H.: Employment nd Ledership Position t CureVc GmbH, Role Held: Mnging Director, Stock Ownership: CureVc GmbH. We thnk Sylvi Stephn (1), Bruni Schuster (1), Solveig Decke (1), Rente Dreher (1) nd Corinn Jeth (1) for excellent technicl ssistnce. S.M.R. ws supported by the Europen Socil Fund in Bden-Württemberg. The work ws performed in Tuebingen, Germny. REFERENCES 1. Motzer, RJ, Bnder, NH nd Nnus, DM (1996). Renl-cell crcinom. N Engl J Med 335: Belldegrun, A, Muul, LM nd Rosenberg, SA (1988). 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