ReseaRch papeR ReseaRch papeR OncoImmunology 2:1, e22664; January 2013; © 2013 Landes Bioscience Autologous lysate-pulsed dendritic cell vaccination followed by adoptive transfer of vaccine-primed ex vivo co-stimulated T cells in recurrent ovarian cancer Lana e. Kandalaft,1,† Daniel J. powell Jr.,1,† cheryl L. chiang,1 Janos Tanyi,1 sarah Kim,1 Marnix Bosch,5 Kathy Montone,4 Rosemarie Mick,3 Bruce L. Levine,4 Drew a. Torigian,2 carl h. June4 and George coukos1,* 1Ovarian cancer Research center; University of pennsylvania school of Medicine; philadelphia, pa Usa; 2Department of Radiology; University of pennsylvania school of Medicine; philadelphia, pa Usa; 3Department of Biostatistics and epidemiology; University of pennsylvania school of Medicine; philadelphia, pa Usa; 4Department of pathology; University of pennsylvania school of Medicine; philadelphia, pa Usa; 5Northwest Biotherapeutics; Bethesda, MD Usa †These authors equally contributed to this work. Keywords: adoptive T-cell transfer, autologous immunotherapy, DC vaccines Novel strategies for the therapy of recurrent ovarian cancer are warranted. We report a study of a combinatorial approach encompassing dendritic cell (Dc)-based autologous whole tumor vaccination and anti-angiogenesis therapy, followed by the adoptive transfer of autologous vaccine-primed cD3/cD28-co-stimulated lymphocytes. Recurrent ovarian cancer patients for whom tumor lysate was available from prior cytoreductive surgery underwent conditioning with intravenous bevacizumab and oral metronomic cyclophosphamide, sequentially followed by (1) bevacizumab plus vaccination with Dcs pulsed with autologous tumor cell lysate supernatants, (2) lymphodepletion and (3) transfer of 5 × 109 autologous vaccine-primed T-cells in combination with the vaccine. Feasibility, safety as well as immunological and clinical efficacy were evaluated. six subjects received this vaccination. Therapy was feasible, well tolerated, and elicited antitumor immune responses in four subjects, who also experienced clinical benefits. Of these, three patients with residual measurable disease received outpatient lymphodepletion and adoptive T-cell transfer, which was well tolerated and resulted in a durable reduction of circulating regulatory T cells and increased cD8+ lymphocyte counts. The vaccine-induced restoration of antitumor immunity was achieved in two subjects, who also demonstrated clinical benefits, including one complete response. Our findings indicate that combinatorial cellular immunotherapy for the treatment of recurrent ovarian cancer is well tolerated and warrants further investigation. several modifications of this approach can be envisioned to optimize immunological and clinical outcomes. Introduction Ovarian cancers express numerous tumor-associated anti- gens,5–9 although it is unclear which of these can drive tumor The overall five-year survival of patients affected by advanced rejection. Molecular cancer vaccines have used peptide stage epithelial ovarian cancer is < 40%, highlighting the unmet sequences, full-length proteins, or nucleic sequences, to date need for alternative therapies.1 Recent studies have demon- exhibiting limited success. An alternative approach is consti- strated that ovarian cancer patients exhibit spontaneous antitu- tuted by vaccines based on whole tumor cell lysates, which have mor immune responses. Peripheral blood tumor-specific T-cell generally produced better clinical responses than highly spe- precursors have been detected in 50% of subjects bearing ovar- cific tumor vaccines in various types of cancers.10 In principle, ian cancer.2 Along similar lines, tumor-infiltrating lymphocytes whole tumor vaccines encompass all potential antigens of a spe- (TILs) have also been found in approximately 50% of ovarian cific tumor, including both major histocompatibility complex cancer patients, correlating with improved overall survival.3,4 (MHC) Class I and II-restricted epitopes and hence eliciting a This suggests that immunotherapy constitutes a meaningful multivalent CD8+ and CD4+ antitumor response.11,12 Although approach to improve disease outcome among ovarian cancer there are theoretical concerns that tumor lysates could dampen patients. the immunogenicity of dendritic cells (DCs), it has been shown *Correspondence to: George Coukos; Email: [email protected] Submitted: 09/14/12; Revised: 10/25/12; Accepted: 10/25/12 http://dx.doi.org/10.4161/onci.22664. Citation: Kandalaft LE, Powell Jr DJ, Chiang CL, Tanyi J, Kim S, Bosch M, et al. Autologous lysate-pulsed dendritic cell vaccination followed by adoptive transfer of vaccine-primed ex vivo co-stimulated T cells in recurrent ovarian cancer. OncoImmunology 2013; 2:e22664 www.landesbioscience.com OncoImmunology e22664-1 that DCs pulsed with tumor lysates can expand tumor-reactive exhausted (Fig. 1C). Interestingly, this subject had previously autologous T cells in vitro.2,13 However, significant improve- failed to respond to bevacizumab. Two additional exhibited pro- ments are required to increase the potency of such DC vaccines. gressive disease, according to RECIST 1.1. Immunomodulation of the host represents one effective means We measured the effect of therapy on the frequency of periph- to augment the efficacy of vaccines. Among various targets of this eral blood CD4+CD25+ FOXP3+ Tregs. Subjects had baseline approach, regulatory T cells (Tregs) are known to accumulate circulating Treg frequencies ranging from 4% to 10%, consis- in ovarian tumors, where they promote immune dysfunction.14 tent with previous reports of increased Tregs in cancer patients.20 Hence, the elimination of Tregs could be an important prerequi- We did not observe a reduction in circulating Tregs following site for successful anticancer vaccines. In addition, the tumor vas- the administration of bevacizumab and metronomic cyclo- culature, under the influence of the vascular endothelial growth phosphamide (Fig. 2A). However, a modest reduction in Tregs factor (VEGF) and the endothelin systems, blocks the access of was observed in all subjects post-vaccination (mean = 6.02% at T cells to tumors, implying that an attenuation of this barrier EOS vs. 7.26 at baseline; p = 0.03). could also enhance vaccine efficacy.15–17 Another approach to We detected T cells specific for the Her2 peptide in 689–697 augment the activity of anticancer vaccines is represented by the subject S-02 following vaccination, but neither at baseline nor stimulation of effector T cells. Indeed, cancer vaccines normally between vaccination and bevacizumab and metronomic cyclo- rely on endogenous low-avidity tumor-reactive T-cell precursors, phosphamide courses (Fig. 3A). We also quantified tumor-spe- whose activity could be significantly enhanced by the provision cific T cells in baseline, pre-vaccine, and post-vaccine peripheral of co-stimulatory signals.18,19 blood lymphocytes (PBLs) using an interferon γ (IFNγ) ELISpot. We report a pilot clinical study that tested the feasibility, safety We detected a significant increase in tumor-reactive T cells after as well as immunological and clinical outcomes of a combinatorial vaccination (but not after bevacizumab alone) in all four subjects cell-based immunotherapy that translated the concepts described who exhibited clinical benefits (p < 0.05), but found no signs of above into the clinic. In a first part of the study, we treated sub- an immune response in the two subjects who experienced disease jects bearing advanced ovarian cancer with commercially avail- progression (Fig. 3B). The frequency of tumor-specific T cells able drugs that suppress Treg functions and tumor angiogenesis, ranged between ~1:500 and ~1:5,000. then administered them with vaccines based on DCs pulsed with We then tested the induction of humoral responses by pro- autologous tumor cell lysate supernatants. Subjects who achieved tein array in the two subjects who exhibited T-cell and clinical at least stable disease after the vaccine but failed to exhibit a responses to the vaccine (S-01 and S-02). We detected IgG as complete remission were enrolled in the second part of the trial, well as IgM seropositivity to a number of proteins that was mark- involving lymphodepletion and adoptive transfer of autologous edly higher in post-vaccine than in pre-vaccine sera, indicating vaccine-primed CD3/CD28-co-stimulated T cells. Our findings not only a significant boost in general immunity but also a prim- indicate that this sequence of treatments is feasible, safe, and can ing of humoral responses (Fig. 3C). The targets recognized by yield clinical benefits to advanced ovarian cancer patients. circulating IgM and IgG obtained from these two patients are listed in Supplemental Tables 4 and 5. Results Lastly, we examined the expression of MHC Class I molecules and infiltration by CD3+ cells in tumor tissues harvested prior to UPCC-11807. Seven subjects (age range 46–69, mean 55) were vaccination. There was a positive correlation between the expres- enrolled in the UPCC-11807 trial (Table S1). One subject failed sion of MHC-I and the number of tumor-infiltrating lymphocytes to yield sufficient DCs and was not vaccinated. Vaccine products (TILs) (Spearman rank correlation = 0.84, p = 0.04). In addition, met release criteria in six subjects (Table S2). The yield of DCs there was an association between the abundance of TILs and immu- weakly correlated with monocyte counts in the apheresis product nological or clinical responses to vaccine, although this did not (Fig. S1). All 6 subjects completed bevacizumab and metronomic reach the threshold for statistical significance (p = 0.06) (Fig. 3D). cyclophosphamide courses as well as vaccine doses. A total of 22 UPCC-10808. Following the completion of UPCC-11807 intradermal vaccinations were administered. All vaccines were trial, three of the four subjects (S-01, -02 and -03) who had at well tolerated and there were no grade > 2 toxicities. The most least SD after vaccination but did not achieve complete remission, frequent adverse event was grade 1 or 2 hypertension (3 occur- were eligible for and were enrolled in UPCC-10808. Post-vaccine rences), which we attributed to bevacizumab (Table S3). elutriated T cells expanded efficiently in response to CD3/CD28 Two subjects exhibited partial responses (PR) according to beads (Fig. 4A). Lymphocytes from patient S-01, S-02 and S-03 Response Evaluation Criteria In Solid Tumors (RECIST) 1.1 underwent ~29-, ~48 and ~31-fold expansion, respectively, by day (Fig. 1A and B). Interestingly, one of these subjects progressed on 11 of culture, and were harvested for infusion thereafter. Post- bevacizumab and metronomic cyclophosphamide prior to receiv- expansion T cells retained tumor-reactive clones, as documented ing the vaccine, but exhibited a PR afterwards. Two other sub- by the reactivity to HLA-A2-restricted HER2/neu peptides or to jects exhibited stable disease (SD). One of these subjects entered shared epitopes from SKOV3 cells (Fig. 4B). the trial with no evidence of disease (NED) after tertiary debulk- The infusion of 5 × 109 T cells after lymphodepleting chemo- ing surgery, remained in remission until end of study (EOS), and therapy was well tolerated, the most frequent toxicity being myelo- continued to receive vaccine boosts every 4 weeks combined with suppression (Table S3). Absolute lymphocyte counts (ALCs) bevacizumab, remaining disease-free for 14 mo, until vaccine was recovered to pre-treatment levels by day 2–7. Absolute neutrophil e22664-2 OncoImmunology Volume 2 Issue 1 Figure 1. Radiological response in Upcc-11807. (A) Radiological assessments were performed by cT (cT) at enrollment or baseline (BL), and at the end of study (eOs). Two subjects also had evaluation after completing bevacizumab and metronomic cyclophosphamide and before starting vaccine (pre- vac). Volumes of all individual tumor metastases are shown. estimated tumor volumes were calculated based on the formula V = ½ s * ½ s * L, where L is the longest and s is the shortest tumor diameter. Tumors were measured on the cT section that had the biggest tumor dimensions. subject (s)-01 and -02 achieved partial response. s-02 demonstrated disease progression on bevacizumab and metronomic cyclophosphamide, although her lesions regressed after vaccine administration. s-03 had overall bulky disease, while s-04 had stable no evidence of disease. subjects s-05 and s-06 exhibited disease progression. (B) example of a left para-aortic lymph node regressing in s-01 post-vaccination cT (right) relative to pre-vaccination cT (left). (C) s-04 experienced remission inversion with progression-free survival (pFs3) being twice as long as pFs2. www.landesbioscience.com OncoImmunology e22664-3 Figure 2. Lymphocyte changes during Upcc-11807 and Upcc-10808. (A) cD4+cD25+FOXp3+ regulatory T cell (Treg) frequency during therapy. No Treg reduction was seen following bevacizumab and metronominc cyclophosphamide. a mild (statistically significant) reduction in Treg frequency was seen after vaccine plus bevacizumab relative to pre-vaccine. Two out of three subjects exhibited marked and sustained reduction in circulating Tregs after T -cell transfer. (B) circulating cD4:cD8 T-cell ratio as measured at baseline and at the end of the study (eOs). Longitudinal assessment of tumor- reactive T cells by (IFNγ) eLIspot in s-01, s-02 and s-03 during Upcc-10808. counts (ANCs) recovered to baseline levels in all subjects by day CR and SD, respectively, but were not detectable in S-02, who 7 (Fig. 4C). At the EOS, patient S-01, who exhibited a PR during experienced disease progression. UPCC-11807, achieved a complete response (CR). Patient S-02, who also exhibited a PR during UPCC-11807, progressed, while Discussion patient S-03, achieving a SD in UPCC-11807, remained stable. We followed T-cell subsets in the peripheral blood upon the In this study, we demonstrated that whole tumor cell-based vac- T-cell transfer. The frequency of circulating Tregs decreased sig- cination, using lysate-pulsed autologous DCs in combination with nificantly in patients S-01 and S-02 but not in S-03 (Fig. 2A). In bevacizumab and metronomic cyclophosphamide, followed by addition, following adoptive T-cell transfer, reconstituted PBLs autologous adoptive T-cell therapy using vaccine-primed CD3/ were skewed toward CD8+ T-cells, the CD4:CD8 ratio being CD28-co-stimulated whole PBLs, is feasible and well tolerated by decreased 2–4-fold (Fig. 2B). Finally, we tested whether tumor- advanced recurrent ovarian cancer patients. Four of six patients reactive T cells would reconstitute in vivo after adoptive trans- (66%) achieved clinical benefits with the combination of beva- fer. We quantified these cells by IFNγ ELISpot using autologous cizumab, metronomic cyclophosphamide and the vaccine, which DCs pulsed with autologous tumor lysate as the antigen-pre- included two objective PRs and two SDs. One subject experiencing senting platform. At the time of apheresis, there were detectable SD had NED at study entry, and her post-vaccine remission lasted tumor-reactive T-cells in all subjects (0.5 to 2.6 spots per 1,000 for 14 mo in spite of a prior progression-free interval of 7 mo. These PBLs) (Fig. 2C). Tumor-reactive T cells were detectable after observations are suggestive of an effective therapy. Although one hematologic reconstitution in S-01 and S-03, who experienced cannot dissect the contribution of bevacizumab and metronomic e22664-4 OncoImmunology Volume 2 Issue 1 Figure 3. For figure legend, see page e22664-5. www.landesbioscience.com OncoImmunology e22664-5 Figure 3 (see previous page). Immune responses in Upcc-11807. (A and B) Thawed peripheral blood mononuclear cells (pBMcs) obtained at baseline (BL) and at the end of study (eOs) were co-incubated, in the absence of additional stimulation, with autologous Dcs pulsed with autologous tumor- cell lysates or control Dcs for ~18 h and evaluated by interferon γ (IFNγ) eLIspot. (A) Representative data of T-cell responses. Tumor-specific responses are not detectable among pBMcs collected prior to therapy or after two doses of bevacizumab and metronomic cyclophosphamide, demonstrating the requirement for vaccine to elicit antitumor immunity. s-02 was hLa-a0201, allowing for the detection of immune responses to heR2 (h2N)- or hTeRT-derived epitopes. a response is seen against heR2 . (B) summary of vaccine-induced T cell responses. a positive response was detected in 689–697 s-01, -02, -03 and -04. (C) humoral responses induced by the vaccine. sera from s-01 and s-02 were profiled on protoarray® human protein Microarrays v. 4.1, containing more than 8,000 human proteins. Increased IgG and IgM seropositivity is detected against human proteins post-vaccine as compared with pre-vaccine. Y-axes indicate the number of proteins recognized by serum antibodies. (D) cD3+ tumor-infiltrating T-cells (top) and Mhc class I expression levels (bottom) in tumors at BL. (E) scoring system. Figure 4. hematopoietic reconstitution in Upcc-10808. (A) Lymphocytes purified from peripheral blood mononuclear cells (pBMcs) by elutriation were stimulated and expanded for ~11 d using Dynal microbeads coated with anti-cD3 and anti-cD28 antibodies. expansion of three subjects and two normal donors is shown. a population doubling level (pDL) average of 5.14 is seen for three subjects with ovarian cancer. (B) expanded T cells from s-02 were tested by interferon γ (IFNγ) eLIspot for cross-reactivity against non-transduced or hLa-a2-transduced sKOV3 cells, T2 cells pulsed with heR2/neu 369 or 689 peptides or unpulsed T2 cells, autologous dendritic cells (Dcs) pulsed with sKOV3-cell lysates or unpulsed Dcs, or media (T cells only). prior to assessments, T cells were incubated for 7 d with autologous Dcs pulsed with sKOV3-cell lysates prepared either by freeze-thawing (FTL) or UV B (UVB) irradiation. Results are means of the number of IFNγ spots per 10 T cells ± seM of triplicates (p < 0.001; paired student’s t-test) (C) absolute lymphocyte count (aLc), white blood cells (WBc) and absolute neutrophil count (aNc) in subjects receiving adoptive T-cell transfer. s-02 (black) and s-03 (gray) received pegfilgrastim on the day of T-cell transfer. e22664-6 OncoImmunology Volume 2 Issue 1 cyclophosphamide to the clinical response in this small pilot study, population, and the tolerability of outpatient lymphodepletion it is worth noting that one of the subjects exhibiting a PR and the followed by T-cell transfer. The hematologic recovery was rapid 14-mo remission were observed in subjects who had previously and resulted in two important changes in circulating T cells: failed this chemotherapy, indicating that the observed clinical ben- (1) it reduced the relative frequency of Tregs over total CD4+ efits could be (at least in part) attributed to the elicitation of an cells, and (2) it decreased the CD4 to CD8 ratio by 2–4-fold. In antitumor immune response. In support of this hypothesis, clini- two subjects, we documented a restoration of antitumor immu- cal responses closely correlated with the detection of cellular and nity and a durable decline of circulating Tregs, which was associ- humoral immune response against tumor antigen upon vaccina- ated with clinical benefit (one subject achieved CR, while the tion. Along similar lines, the absence of immune responses to the other one—with bulky disease—was stable at the EOS). In the vaccine correlated with clinical progression. third subject, adoptive T-cell transfer did not restore antitumor All subjects exhibited a dampened T-cell response to the immunity, and disease progression was observed. Interestingly, diphtheria carrier protein CRM , a potent xeno-neoantigen of Tregs returned to pre-treatment levels in this patient. 197 Prevnar™, which was given along with the first vaccination to In summary, we treated a population of patients affected by monitor immune responsiveness. Interestingly, myeloma patients recurrent ovarian cancer (who have few effective therapeutic have previously been shown to exhibit robust T-cell responses to options) and obtained an overall clinical benefit of 50%, including CRM , suggesting that ovarian cancer patients may be char- one CR and one remission that lasted 14 mo. The latter patient was 197 acterized by a profound level of systemic immunosuppression. subsequently enrolled in a second vaccine study, using improved Nevertheless, DC vaccination induced an immune response lysate-pulsed DCs13 without bevacizumab, and remained in remis- against whole tumor-cell lysates and specific immune responses sion for one additional year (not shown). Importantly, this patient against peptides of known tumor-associated antigens such as and another who achieved PR had previously failed bevacizumab HER2. Furthermore, we detected increased IgM seropositivity and metronomic cyclophosphamide, indicating that therapeutic post-vaccine, indicating effective de novo B-cell priming. success cannot be attributed solely to anti-angiogenic therapy, at The frequency of tumor-specific T cells elicited by our vaccine least in this setting. Although these data are considered encourag- was quite low (< 1 tumor reactive T cell per 500 PBLs). Under ing, a number of opportunities may be pursued to optimize the these conditions, it is quite encouraging to see some clinical bene- efficacy of this approach. Importantly, we observed a marked sys- fits. It is possible that the observed benefits were due to a synergistic temic immunosuppression, which was also reported in patients effect of vaccine with anti-angiogenesis therapy. Tumor vascula- with ovarian cancer in remission receiving vaccines.27 Hence, ture not only directly supports increased tumor growth, but also therapeutic maneuvers that effectively reduce Tregs, activate innate can also provide a barrier to the extravasation of T cells into the immunity and stimulate antigen presentation (e.g., TLR agonists), tumor.15,21 Anti-VEGF therapy normalizes tumor vasculature and enhance T-cell activation (e.g., IL-2, IL-7) or block inhibitory increases tumor infiltration by T cells, and in the mouse this has checkpoints (e.g., anti-CTLA-4 and anti-PD-1 anitbodies) could been shown to improve the outcome of adoptive immunotherapy.22 markedly enhance immune activation and vaccine efficacy. In Metronomic cyclophosphamide also suppresses tumor angiogen- addition, we used supernatants from freeze-thawed tumor lysates. esis and reduces the number of circulating endothelial progenitor Alternative ways to prepare lysates may be preferable. Furthermore, cells in subjects with metastatic cancer.23 Cyclophosphamide also we used DCs that were not matured with any exogenous stimuli. has a dose-dependent immunomodulatory effect.24 Metronomic Although lysate pulsing can induce DC maturation, additional cyclophosphamide has been reported to decrease Tregs in sub- stimulation with exogenous cytokines and TLR agonists could sig- jects with advanced cancer, restore T-cell and NK-cell effector nificantly enhance vaccine potency. Whether the adoptive transfer functions,25 and enhance the efficacy of anticancer vaccines and of vaccine-primed co-stimulated T-cells is an effective way to boost anti-HER2 targeted therapy.26 Interestingly, we did not observe the efficacy of cancer vaccines still remains an important question. a decrease in circulating Tregs frequency after bevacizumab and In our study, the only CR was observed in the one patient who metronomic cyclophosphamide, as previously reported.25 Perhaps had high numbers of reactive T-cells in the T cell preparation and this reflects the fact that we employed a comparatively lower dose whose ALC was 0 on the day of transfer. Adoptive T-cell therapy of cyclophosphamide. Alternatively, the lack of Treg depletion may could be improved in the future by an intensification of lymphode- not necessarily preclude a positive effect of metronomic cyclophos- pleting regimens as well as by an enrichment of the preparation phamide on the immunogenicity of DC vaccines. with tumor-reactive T-cells. The depletion of Tregs from the apher- We sought to enhance antitumor immune responses generated esis product and systemic administration of cytokines in support by the vaccine by lymphodepletion followed by the adoptive trans- of T cells following transfer could further enhance the efficacy of fer of post-vaccine ex vivo co-stimulated T cells. The theoretical therapy. Future trials are required to address these issues. advantages of this approach mainly lie in the co-stimulatory sig- nals delivered to T cells during ex vivo CD3/CD28 bead-driven Materials and Methods expansion and in the host conditioning through lymphodeple- tion. We used this approach in patients who developed antitumor Overall design and subjects. We performed two linked open-label immune responses to the vaccine, but did not achieve a CR in the pilot studies in subjects with recurrent ovarian cancer. The first first part of our study. Our pilot study demonstrates the feasibility (UPCC-11807) assessed the feasibility, safety and immunological as of ex vivo T-cell expansion even in this heavily pretreated patient well as clinical effects of a DC-based autologous whole-tumor lysate www.landesbioscience.com OncoImmunology e22664-7 Figure 5. (A and B) clinical design of Upcc-11807 (A) and Upcc-10808 (B). vaccination in combination with anti-angiogenesis therapy. The 5 × 109 ex vivo CD3/CD28-co-stimulated T-cells on day 0; and second (UPCC-10808) tested the adoptive transfer of autologous DC vaccine boost on day 2 (Fig. 5B). All subjects were managed vaccine-primed CD3/CD28-co-stimulated lymphocytes. Subjects as outpatients and received antibiotic and antifungal prophylaxis. completing UPCC-11807 had the option of enrolling immediately EOS was set to day 70. into UPCC-10808, if study eligibility criteria were met. Cell manufacturing. Vaccine was manufactured at Cognate All eligible subjects were ≥ 18 y old; had recurrent advanced- Bioservices as described in Supplemental Materials and elsewhere.28 stage ovarian cancer; had undergone prior cytoreductive surgery DCs were cryopreserved at -140°C and shipped to the University of for recurrent disease, with sufficient tumor harvested for obtain- Pennsylvania on dry ice. T cells were manufactured at the Cell and ing a lysate; had undergone physician’s choice post-operative Vaccine Production Facility, University of Pennsylvania, as detailed chemotherapy; had residual tumors at study entry ≤ 4 cm; and in Supplemental Materials and administered fresh. had a baseline Eastern Cooperative Oncology Group perfor- Evaluation. Subjects were evaluated every two weeks in mance status < 2. Enrollment was allowed ≥ 4 weeks after sur- UPCC-11807; and twice a week until hematologic recovery, and gery or ≥ 3 weeks after any post-operative chemotherapy. Both weekly thereafter, in UPCC-10808. Safety was determined using studies (NCT01312376 and NCT00603460) were approved the NCI Common Terminology Criteria for Adverse Events by the Food and Drug Administration and the University of (CTCAE) version 3.0. Subjects underwent CT at enrollment and Pennsylvania Institutional Review Board. Both studies are now at EOS. Clinical response was determined using RECIST 1.1. closed for accrual. Clinical benefit was defined as CR, PR or SD. UPCC-11807. Subjects underwent leukapheresis on day -35 Immunological assessments. Immunological assessments were to -29, followed by at least two cycles of intravenous bevacizumab performed at the time of enrollment (baseline), after completion (10 mg/kg, on days -28 and -14) and oral metronomic cyclophos- of bevacizumab and metronomic cyclophosphamide (pre-vaccine), phamide (50 mg daily for 7 d after bevacizumab) (Fig. 5A), fol- and at EOS in UPCC-11807; and at enrollment, before lymphode- lowed by three doses of intradermal DCs (~5–10 × 106) every pletion, before T-cell infusion, and at EOS in UPCC-10808. T-cell two weeks in combination with intravenous bevacizumab. EOS responses to tumor antigens were assessed by interferon γ (IFNγ) was set to day 42. If subjects demonstrated clinical benefit by ELISpot assays following incubation of peripheral blood lympho- RECIST 1.1 but still had measurable disease (i.e., PR or SD), cytes (PBLs) with autologous DCs that were either left untreated then they were eligible to enroll in protocol UPCC-10808. or pulsed with autologous tumor cell lysates. CD8+ responses to UPCC-10808. Subjects underwent apheresis on day -13, fol- HER2/neu were also measured by IFNγ ELISpot in HLA- 369–377 lowed by outpatient non-myeloablating lymphodepleting chemo- A*0201 subjects. CD4+CD25+ FOXP3+ Tregs were quantified therapy with intravenous cyclophosphamide (300 mg/m2/day) by flow cytometry. Serum IgG and IgM were assessed using the and fludarabine (30 mg/m2/day) on days (-3 to -15); infusion of ProtoArray® Human Protein Microarrays v. 4.1 (Invitrogen). e22664-8 OncoImmunology Volume 2 Issue 1 We assessed pre-vaccine tumor-infiltrating CD3+ T cells and Disclosure of Potential Conflicts of Interest tumor MHC Class I expression levels by immunohistochemistry. No potential conflicts of interest were disclosed. Hematologic recovery was followed with blood cell analysis every 1–3 d after chemotherapy, until blood populations returned to Acknowledgments normal values. Details are reported in Supplemental Materials. We thank our patients for participating in this protocol. This Statistical analysis. Data were analyzed by paired Student’s study was supported by National Cancer Institute Ovarian t-test, and p values < 0.05 were considered statistically significant. SPORE grant P01-CA83638, National Institution of Health R01 Pearson’s correlation was employed to test the correlation between grant FD003520-02, and the Ovarian Cancer Immunotherapy two interval-scaled variables, while Spearman’s rank correlation Initiative. was employed to test the correlation between two ordinal-scaled variables. The non-parametric Wilcoxon rank sum test was used Supplemental Materials to compare immunologic outcomes (e.g., CD3+ TILs) between Supplemental materials may be found here: clinical responders and non-responders. These analyses were con- http://www.landesbioscience.com/journals/oncoimmunology/ ducted in SPSS 19 (SPSS, Inc.). article/22664/ References 11. Toes RE, Schoenberger SP, van der Voort EI, Offringa 20. Audia S, Nicolas A, Cathelin D, Larmonier N, Ferrand R, Melief CJ. CD40-CD40Ligand interactions and C, Foucher P, et al. Increase of CD4+ CD25+ regula- 1. Coukos G, Conejo-Garcia JR, Roden RB, Wu TC. their role in cytotoxic T lymphocyte priming and tory T cells in the peripheral blood of patients with Immunotherapy for gynaecological malignancies. Expert anti-tumor immunity. Semin Immunol 1998; 10:443- metastatic carcinoma: a Phase I clinical trial using Opin Biol Ther 2005; 5:1193-210; PMID:16120050; 8; PMID:9826577; http://dx.doi.org/10.1006/ cyclophosphamide and immunotherapy to eliminate http://dx.doi.org/10.1517/14712598.5.9.1193. smim.1998.0147. CD4+ CD25+ T lymphocytes. Clin Exp Immunol 2. Schlienger K, Chu CS, Woo EY, Rivers PM, Toll AJ, 12. Zajac AJ, Murali-Krishna K, Blattman JN, Ahmed R. 2007; 150:523-30; PMID:17956583; http://dx.doi. Hudson B, et al. TRANCE- and CD40 ligand-matured Therapeutic vaccination against chronic viral infection: org/10.1111/j.1365-2249.2007.03521.x. dendritic cells reveal MHC class I-restricted T cells the importance of cooperation between CD4+ and 21. Mazanet MM, Hughes CC. B7-H1 is expressed specific for autologous tumor in late-stage ovarian CD8+ T cells. Curr Opin Immunol 1998; 10:444-9; by human endothelial cells and suppresses T cell cancer patients. Clin Cancer Res 2003; 9:1517-27; PMID:9722921; http://dx.doi.org/10.1016/S0952- cytokine synthesis. J Immunol 2002; 169:3581-8; PMID:12684428. 7915(98)80119-2. PMID:12244148. 3. Zhang L, Conejo-Garcia JR, Katsaros D, Gimotty PA, 13. Chiang CL, Maier DA, Kandalaft LE, Brennan AL, 22. Shrimali RK, Yu Z, Theoret MR, Chinnasamy D, Massobrio M, Regnani G, et al. Intratumoral T cells, Lanitis E, Ye Q, et al. Optimizing parameters for Restifo NP, Rosenberg SA. Antiangiogenic agents can recurrence, and survival in epithelial ovarian cancer. clinical-scale production of high IL-12 secreting den- increase lymphocyte infiltration into tumor and enhance N Engl J Med 2003; 348:203-13; PMID:12529460; dritic cells pulsed with oxidized whole tumor cell lysate. the effectiveness of adoptive immunotherapy of can- http://dx.doi.org/10.1056/NEJMoa020177. J Transl Med 2011; 9:198; PMID:22082029; http:// cer. Cancer Res 2010; 70:6171-80; PMID:20631075; 4. Sato E, Olson SH, Ahn J, Bundy B, Nishikawa H, dx.doi.org/10.1186/1479-5876-9-198. http://dx.doi.org/10.1158/0008-5472.CAN-10-0153. Qian F, et al. Intraepithelial CD8+ tumor-infiltrating 14. Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, 23. Alfaro C, Perez-Gracia JL, Suarez N, Rodriguez J, lymphocytes and a high CD8+/regulatory T cell ratio Mottram P, et al. Specific recruitment of regulatory T Fernandez de Sanmamed M, Sangro B, et al. Pilot clini- are associated with favorable prognosis in ovarian cells in ovarian carcinoma fosters immune privilege and cal trial of type 1 dendritic cells loaded with autologous cancer. Proc Natl Acad Sci U S A 2005; 102:18538- predicts reduced survival. Nat Med 2004; 10:942-9; tumor lysates combined with GM-CSF, pegylated IFN, 43; PMID:16344461; http://dx.doi.org/10.1073/ PMID:15322536; http://dx.doi.org/10.1038/nm1093. and cyclophosphamide for metastatic cancer patients. pnas.0509182102. 15. Buckanovich RJ, Facciabene A, Kim S, Benencia F, J Immunol 2011; 187:6130-42; PMID:22048768; 5. Hellstrom I, Raycraft J, Kanan S, Sardesai NY, Verch Sasaroli D, Balint K, et al. Endothelin B receptor medi- http://dx.doi.org/10.4049/jimmunol.1102209. T, Yang Y, et al. Mesothelin variant 1 is released from ates the endothelial barrier to T cell homing to tumors 24. Greten TF, Ormandy LA, Fikuart A, Höchst B, tumor cells as a diagnostic marker. Cancer Epidemiol and disables immune therapy. Nat Med 2008; 14:28-36; Henschen S, Hörning M, et al. Low-dose cyclo- Biomarkers Prev 2006; 15:1014-20; PMID:16702385; PMID:18157142; http://dx.doi.org/10.1038/nm1699. phosphamide treatment impairs regulatory T cells http://dx.doi.org/10.1158/1055-9965.EPI-05-0334. 16. Kandalaft LE, Facciabene A, Buckanovich RJ, Coukos and unmasks AFP-specific CD4+ T-cell responses 6. Hellström I, Goodman G, Pullman J, Yang Y, Hellström G. Endothelin B receptor, a new target in cancer in patients with advanced HCC. J Immunother KE. Overexpression of HER-2 in ovarian carcinomas. immune therapy. Clin Cancer Res 2009; 15:4521-8; 2010; 33:211-8; PMID:20139774; http://dx.doi. Cancer Res 2001; 61:2420-3; PMID:11289108. PMID:19567593; http://dx.doi.org/10.1158/1078- org/10.1097/CJI.0b013e3181bb499f. 7. Odunsi K, Qian F, Matsuzaki J, Mhawech-Fauceglia 0432.CCR-08-0543. 25. Ghiringhelli F, Menard C, Puig PE, Ladoire S, Roux P, Andrews C, Hoffman EW, et al. Vaccination with 17. Manning EA, Ullman JG, Leatherman JM, Asquith S, Martin F, et al. Metronomic cyclophosphamide an NY-ESO-1 peptide of HLA class I/II specifici- JM, Hansen TR, Armstrong TD, et al. A vascu- regimen selectively depletes CD4+CD25+ regulatory T ties induces integrated humoral and T cell responses lar endothelial growth factor receptor-2 inhibitor cells and restores T and NK effector functions in end in ovarian cancer. Proc Natl Acad Sci U S A 2007; enhances antitumor immunity through an immune- stage cancer patients. Cancer Immunol Immunother 104:12837-42; PMID:17652518; http://dx.doi. based mechanism. Clin Cancer Res 2007; 13:3951-9; 2007; 56:641-8; PMID:16960692; http://dx.doi. org/10.1073/pnas.0703342104. PMID:17606729; http://dx.doi.org/10.1158/1078- org/10.1007/s00262-006-0225-8. 8. Vonderheide RH. Telomerase as a universal tumor- 0432.CCR-07-0374. 26. Emens LA, Asquith JM, Leatherman JM, Kobrin BJ, associated antigen for cancer immunotherapy. 18. Carpenito C, Milone MC, Hassan R, Simonet JC, Lakhal Petrik S, Laiko M, et al. Timed sequential treatment Oncogene 2002; 21:674-9; PMID:11850795; http:// M, Suhoski MM, et al. Control of large, established with cyclophosphamide, doxorubicin, and an allogene- dx.doi.org/10.1038/sj.onc.1205074. tumor xenografts with genetically retargeted human T ic granulocyte-macrophage colony-stimulating factor- 9. Luby TM, Cole G, Baker L, Kornher JS, Ramstedt U, cells containing CD28 and CD137 domains. Proc Natl secreting breast tumor vaccine: a chemotherapy dose- Hedley ML. Repeated immunization with plasmid DNA Acad Sci U S A 2009; 106:3360-5; PMID:19211796; ranging factorial study of safety and immune activa- formulated in poly(lactide-co-glycolide) microparticles is http://dx.doi.org/10.1073/pnas.0813101106. tion. J Clin Oncol 2009; 27:5911-8; PMID:19805669; well tolerated and stimulates durable T cell responses to 19. Lanitis E, Poussin M, Hagemann IS, Coukos G, http://dx.doi.org/10.1200/JCO.2009.23.3494. the tumor-associated antigen cytochrome P450 1B1. Sandaltzopoulos R, Scholler N, et al. Redirected 27. Chu CS, Boyer J, Schullery DS, Gimotty PA, Clin Immunol 2004; 112:45-53; PMID:15207781; Antitumor Activity of Primary Human Lymphocytes Gamerman V, Bender J, et al. Phase I/II random- http://dx.doi.org/10.1016/j.clim.2004.04.002. Transduced With a Fully Human Anti-mesothelin ized trial of dendritic cell vaccination with or with- 10. Neller MA, López JA, Schmidt CW. Antigens for can- Chimeric Receptor. Mol Ther 2012; 20:633-43; out cyclophosphamide for consolidation therapy of cer immunotherapy. Semin Immunol 2008; 20:286- PMID:22127019. advanced ovarian cancer in first or second remis- 95; PMID:18951039; http://dx.doi.org/10.1016/j. sion. Cancer Immunol Immunother 2012; 61:629-41; smim.2008.09.006. PMID:22021066; http://dx.doi.org/10.1007/s00262- 011-1081-8. www.landesbioscience.com OncoImmunology e22664-9 28. Liau LM, Prins RM, Kiertscher SM, Odesa SK, Kremen TJ, Giovannone AJ, et al. Dendritic cell vaccination in glioblastoma patients induces systemic and intracranial T-cell responses modulated by the local central nervous system tumor microenvironment. Clin Cancer Res 2005; 11:5515-25; PMID:16061868; http://dx.doi.org/10.1158/1078-0432.CCR-05-0464. e22664-10 OncoImmunology Volume 2 Issue 1