High-Dose Sirolimus and Immune-Selective Pentostatin Plus

Published OnlineFirst June 12, 2015; DOI: 10.1158/1078-0432.CCR-15-0340

Cancer Therapy: Clinical Clinical Cancer Research High-Dose Sirolimus and Immune-Selective Pentostatin plus Cyclophosphamide Conditioning Yields Stable Mixed Chimerism and Insufﬁcient Graft-versus-Tumor Responses Miriam E. Mossoba1, David C. Halverson1, Roger Kurlander2, Bazetta Blacklock Schuver1, Ashley Carpenter1, Brenna Hansen1, Seth M. Steinberg3, Syed Abbas Ali1, Nishant Tageja1, Frances T. Hakim1, Juan Gea-Banacloche1, Claude Sportes4, Nancy M. Hardy5, Dennis D. Hickstein1, Steven Z. Pavletic1, Hanh Khuu6, Marianna Sabatini6, David Stroncek6, Bruce L. Levine7, Carl H. June7, Jacopo Mariotti8, Olivier Rixe9, Antonio Tito Fojo10, Michael R. Bishop11, Ronald E. Gress1, and Daniel H. Fowler1

Abstract

Purpose: We hypothesized that lymphoid-selective host con- (days 0, 14, and 45 posttransplant), and sirolimus was discon- ditioning and subsequent adoptive transfer of sirolimus-resistant tinued early (day 60 posttransplant). allogeneic T cells (T-Rapa), when combined with high-dose Results: PC conditioning depleted host T cells without sirolimus drug therapy in vivo, would safely achieve antitumor neutropenia or infection and facilitated donor engraftment effects while avoiding GVHD. (10 of 10 cases). High-dose sirolimus therapy inhibited Experimental Design: Patients (n ¼ 10) with metastatic renal multiple T-Rapa DLI, as evidenced by stable mixed donor/ cell carcinoma (RCC) were accrued because this disease is rela- host chimerism. No antitumor responses were detected by tively refractory to high-dose conditioning yet may respond to RECISTcriteriaandnosigniﬁcant classical acute GVHD was high-dose sirolimus. A 21-day outpatient regimen of weekly observed. pentostatin (P; 4 mg/m2/dose) combined with daily, dose-adjust- Conclusions: Immune-selective PC conditioning represents a ed cyclophosphamide (C; 200 mg/d) was designed to deplete new approach to safely achieve alloengraftment without neutro- and suppress host T cells. After PC conditioning, patients received penia. However, allogeneic T cells generated ex vivo in sirolimus matched sibling, T-cell–replete peripheral blood stem cell are not resistant to the tolerance-inducing effects of in vivo sir- allografts, and high-dose sirolimus (serum trough target, 20–30 olimus drug therapy, thereby cautioning against use of this ng/mL). To augment graft-versus-tumor (GVT) effects, multiple intervention in patients with refractory cancer. Clin Cancer Res; T-Rapa donor lymphocyte infusions (DLI) were administered 21(19); 4312–20. 2015 AACR.

Introduction graft-versus-tumor (GVT) effects. Reduced intensity conditioning decreases toxicity but promotes mixed chimerism (1) and graft Allogeneic hematopoietic cell transplantation (HCT) is limited rejection or tumor progression (2). Intensiﬁed post-HCT immune by conditioning toxicity, graft rejection, GVHD, and insufﬁcient suppression or graft T-cell depletion reduces GVHD but can decrease GVT effects (3); furthermore, donor lymphocyte infusion (DLI) using unmanipulated T cells can mediate GVT effects but 1Experimental Transplantation and Immunology Branch, Center for Cancer Research, NIH, Bethesda, Maryland. 2Department of Labora- also mediate GVHD (4). To address these limitations, we con- tory Medicine, Clinical Center, NIH, Bethesda, Maryland. 3Biostatistics ducted a pilot clinical trial of immune-selective conditioning, in and Data Management, NCI, NIH, Bethesda, Maryland. 4Georgia vivo 5 sirolimus GVHD prophylaxis, and multiple infusions of DLI Regents University Cancer Center, Augusta, Georgia. University of ex vivo Maryland Greenbaum Cancer Center, Baltimore, Maryland. 6Depart- products manufactured in sirolimus. ment of Transfusion Medicine, Clinical Center, NIH, Bethesda, Mary- Conditioning varies from myeloablative to reduced intensity to 7 land. University of Pennsylvania, Abramson Family Cancer Research nonmyeloablative (5). However, an ultra–low-intensity platform Center, Philadelphia, Pennsylvania. 8Fondazione IRCCS Instituto Nazionale dei Tumori, Milan, Italy. 9University of New Mexico Cancer that avoids any neutropenia has not been fully explored. Stem cell Center, Albuquerque, New Mexico. 10Genitourinary Malignancies engraftment may not be fully reliant on conditioning-induced 11 Branch, Center for Cancer Research, NIH, Bethesda, Maryland. The "marrow space" (6); we thus reasoned that nonneutropenic University of Chicago School of Medicine, Chicago, Illinois. conditioning would permit stem cell engraftment if immunologic Corresponding Author: Daniel H. Fowler, NIH, 10 Center Drive, CRC, 3-EAST rejection was prevented. Conditioning also reduces tumor bur- Labs, 3-3330. Bethesda, MD 20892. Phone: 301-435-8641; Fax: 301-480-4354; den, but this rationale is weakened in chemotherapy refractory E-mail: [email protected] settings. Finally, because conditioning reduces graft rejection, it doi: 10.1158/1078-0432.CCR-15-0340 should be directed toward host T cells that primarily mediate 2015 American Association for Cancer Research. rejection (7). An immune-selective regimen that avoided

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conditioning (26) or with calcineurin inhibitors (27). However, Translational Relevance campath plus high-dose sirolimus (target, 30 ng/mL) after non- Graft rejection, GVHD, and insufficient graft-versus-tumor myeloablative conditioning was safe and effective for GVHD (GVT) effects each limit the therapeutic application of alloge- prevention (28). We thus reasoned that high-dose, single-agent neic hematopoietic cell transplantation (HCT), particularly for sirolimus would be safe after immune selective PC conditioning solid tumors such as metastatic renal cell carcinoma. This trial and represent adequate GVHD prophylaxis after T-cell–replete used three interventions to address these limitations: (i) lym- transplant and multiple T-Rapa DLI. phocyte-selective host conditioning using pentostatin plus We evaluated this therapy in refractory metastatic renal cell dose-adjusted cyclophosphamide (PC regimen), (ii) high- carcinoma (RCC), thereby providing a rationale for lymphocyte- dose sirolimus therapy, and (iii) augmentation of T-cell– specific conditioning intended for rejection abrogation rather replete allogeneic HSCT with additional donor lymphocyte than tumor reduction. Metastatic RCC is susceptible to an allo- þ infusions (DLI) consisting of CD4 T cells manufactured ex geneic GVT effect that is generally associated with GVHD, as vivo in sirolimus (T-Rapa cells). The PC regimen represents a described by the National Heart Lung and Blood Institute (29) new approach to prevent graft rejection, as it selectively mod- and a summation report from 21 European countries (30). ulated host T cells without neutropenia or infection and safely However, GVT effects were not observed in a separate study permitted alloengraftment. High-dose sirolimus effectively (31). Thus, more robust methods of harnessing GVT effects and prevented GVHD but also inhibited multiple T-Rapa DLI, modulating GVHD are required in patients with RCC. Finally, thereby offsetting any potential beneficial direct antitumor metastatic RCC can respond to temsirolimus (32), thus suggesting effect of sirolimus. These results caution against use of high- that high-dose sirolimus might yield an antitumor benefit. dose sirolimus during allogeneic T-cell therapy in patients with refractory cancer. Materials and Methods Clinical trial design and implementation The trial (schema, Fig. 1; ClinicalTrials.gov, NCT00923845) neutropenia would yield direct safety advantages and also reduce was approved by the National Cancer Institute (NCI) institutional conditioning-related induction of GVHD (8). review board and implemented according to an Investigational We used pentostatin plus daily cyclophosphamide (Cy) con- New Drug Application accepted by FDA. All subjects provided ditioning that was personalized on the basis of efficacy [absolute informed consent before participation. On-study dates ranged lymphocyte count (ALC) reduction] and safety [absolute neutro- from June 2008 to June 2010. Subjects were enrolled on the basis phil count (ANC) preservation]. Pentostatin was used as trans- of age (18–75 years), presence of metastatic RCC [any histology; plant conditioning with neutropenia-inducing doses of total active central nervous system (CNS) disease excluded], availabil- body irradiation (9, 10) or busulfan (11). Pentostatin plus bolus ity of an eligible 6 of 6 HLA-matched sibling donor, history of dose Cy (600 mg/m2) caused neutropenia (12); as such, we used nephrectomy, 1 prior systemic therapy, life expectancy 3 low-dose Cy (200 mg/d) and allowed Cy reductions depending months, Karnofsky score 80, and adequate organ function. upon achievement of ALC reduction targets. In a murine model, Patients received a 21-day course of pentostatin (Nipent, Hospira; pentostatin plus daily Cy (PC) depleted and suppressed T cells intravenous infusion on days 1, 8, and 15; 4 mg/m2/dose, and prevented marrow allograft rejection more effectively than adjusted for renal insufficiency) and oral Cy (200 mg flat dose/ fludarabine plus Cy (13). PC therapy also prevented murine day). The protocol stated that the goal of PC therapy was to reduce sensitization to a foreign immunotoxin (14); in the clinic, PC the ALC (<200 cells/mL) while preserving the ANC (>1,000 cells/ therapy modulated immunity without neutropenia and facilitat- mL). Cy was reduced or held if ALC reduction goals were met. ed immunotoxin therapy of mesothelioma (15). Specifically, Cy was reduced to 100 mg/d if ALC values on days 8, We used rapamycin-resistant T cells (T-Rapa) for DLI therapy. 11, 15, or 18 were 251–499, 201–399, 151–299, or 101–199, Unmanipulated DLI mediate GVHD and have limited efficacy for respectively. Cy was held if ALC values on days 8, 11, 15, or 18 treating malignancy (16) or promoting engraftment (17). Costi- were 250, 200, 150, 100, respectively. Cy was also to be mulated DLI products have previously been evaluated (18); reduced for ANC values < 1,000; however, this never occurred. recently, we evaluated DLI composed of T-Rapa cells (19). Murine After PC conditioning ("day 2"), patients started sirolimus (16 mg þ CD4 T-Rapa cells were apoptosis-resistant, prevented rejection, loading dose; then, 4 mg/d), with further dosing adjusted to achieve and were effective against GVHD (20–22). In the previous trial, 20 to 30 ng/mL trough levels through day 60 post-HCT. On day 0, one T-Rapa infusion was associated with conversion of mixed patients received a mobilized, T-cell–replete allograft (>3 106 þ chimerism toward full donor elements; a low rate of acute GVHD; CD34 /kg) and the first infusion of T-Rapa cells (2.5 107 cells/kg), and GVT effects against lymphoid malignancy. In the current trial, which were generated by a 12-day or 6-day culture, as previously we used T-cell–replete allografts augmented with T-Rapa DLI at described (19, 33); furthermore, T-Rapa cells (2.5 107 cells/kg) days 0, 14, and 45 post-HCT. were administered at days 14 and 45 post-HCT. GVHD prophylaxis typically consists of a calcineurin inhibitor The primary study objective was to determine whether this plus a second agent (23). Previously, we used cyclosporine plus transplant approach could safely achieve clinical regression of short-course, standard-dose sirolimus through d14 post-HCT. metastatic RCC [goal of ruling out a 20% overall partial response Sirolimus inhibits mTOR and thereby has an antitumor mecha- (PR)/complete response (CR) rate in favor of a 40% PR/CR rate]; nism, including mediation anti-lymphoma effects post-HCT using a Simon 2-stage design, the occurrence of 3 responses in (24). Because the degree of mTOR inhibition correlates with the first 12 evaluable patients would be required to proceed to the sirolimus concentration (25), higher dosing may optimize anti- second stage of study design. Organ toxicity was evaluated by NCI tumor effects. Sirolimus has substantial toxicity after intensive Common Toxicity Criteria (version 2.0). GVHD was evaluated

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Lymphoid-selective conditioning In vivo and ex vivo sirolimus

Pentostatin (4 mg/m2; d 1, 8, and 15) High-dose sirolimus (serum target: 20–30 ng/mL)

2– 6+ 0 1234567 89101112131415161718192021 0 + 14 + 45 HSCT + T-Rapa T-Rapa Cyclosphosphamide (200 mg/day; d 1 to 21) T-Rapa

Figure 1. Clinical trial of ex vivo and in vivo sirolimus after immune-selective PC conditioning. Prior to allogeneic HCT, patients received a 21-day course of pentostatin (4 mg/m2 given on days 1, 8, and 15) combined with cyclophosphamide [200 mg/d, days 1–21; dose-adjusted for efﬁcacy (reduction in ALC) and toxicity (reduction in ANC)]. After PC conditioning, patients were started on high-dose sirolimus (serum trough levels of 20–30 ng/mL through day 60 posttransplant). The T-cell–replete allograft was augmented with an infusion of ex vivo manufactured donor rapamycin-resistant T cells (T-Rapa), which were also administered at days 14 and 45 posttransplant.

using acute (34) and chronic grading (35); acute GVHD onset Results within the first 100 days post-HCT was considered classical acute Patient characteristics (34), whereas onset after day 100 was considered late acute (35). Table 1 describes characteristics of the 10 enrolled patients (6 Disease responses were evaluated by computed tomographic male and 4 female; median age, 55 years; range, 40–69). Histol- measurements and according to the RECIST Committee criteria. ogy was exclusively or primarily clear cell (7 of 10 cases); 2 Alloengraftment was monitored using variable N-terminal repeat patients had collecting duct carcinoma and 1 patient had papillary PCR assays on total peripheral blood mononuclear cell (PBMC), carcinoma with a clear cell component. Median number of prior enriched T cells (CD3-selected), or enriched myeloid cells (CD15- therapies was 3.5 (range, 3–5); all patients had nephrectomy of selected). the primary tumor. Median number of metastatic sites was 3.5 – Immune monitoring (range, 3 9). Plasma IL7 and IL15 levels were measured by bioplex assay (EMD Millipore). Cytokine assays were performed on cryopre- Immune-selective pentostatin plus cyclophosphamide regimen served peripheral blood samples taken pre- and post-condition- The 21-day PC regimen was safely administered in 10 of 10 ing and at various points post-HCT. As described (19), cells were cases:therewerenoinfectionsandnograde2orgreater thawed and stimulated with anti-CD3/28 beads, and resultant toxicities attributable to the chemotherapy. Each patient supernatants were tested for cytokine content by multiplex assay received the planned 3 doses of pentostatin. Only 2 patients (EMD Millipore); flow cytometry for transcription factor and received the planned 200 mg/d dose of Cy for the entire 21-day differentiation marker expression was also performed as previ- interval (total Cy, 4,200 mg); the range of total Cy dosing was ously described. The statistical significance of the absolute differ- 1,400 to 4,200 mg (median, 1,900). Relative to precondition- ence or the relative difference from the baseline (or earlier) values ing, PC therapy reduced the median ALC (all values, cells/mL) was determined using a Wilcoxon signed rank test. Whether the from 1,156 (range, 375–2,415) to 160 (range, 40–400); the absolute difference or relative difference was selected for evalu- majority of ALC reduction was achieved in the first week (Fig. ation depended on which one was less dependent on the baseline 2a, left). The PC regimen did not reduce the ANC (no single values. All P values are reported without formal adjustment for value < 1,000 cells/mL; Fig. 2A, right) and did not reduce red þ multiple comparisons. In view of the number of tests performed, cells or platelets (not shown; no transfusions required). CD4 T only P < 0.01 would be interpretable as being associated with cells were markedly depleted, with reduction in median CD4 þ significant differences, whereas P values between 0.01 and 0.05 from 503 (range, 124–1,497) to 23 (range, 54–107); CD8 T would suggest strong trends. cells were reduced from a median of 239 (range, 56–770) to 45

Table 1. Patient characteristics Prior therapy Metastatic sites UPN Age/Sex Tumor type No. Types No. Organs 01 63/M Clear cell 3 IFNa, sunitinib, temsirolimus 3 Lung, lymph nodes, brain 02 69/F Clear cell (þglandular) 3 Sorafenib, sunitinib, ixabepilone 3 Lung, lymph nodes, bone 03 54/F Clear cell (þsarcomatoid) 4 Sunitinib, temsirolimus, ixabepilone, XRT 3 Lung, renal, soft tissue 04 63/M Clear cell 4 Avastin þ erlotinib þ imatinib, sunitinib, 9 Pleura, lung, bone, lymph nodes, everolimus, IL2 renal, heart, mesentery, pancreas, adrenal 05 65/F Clear cell (þpapillary) 5 Carboplatin [C] þ taxol [T], CT þ avastin, 3 Lung, lymph nodes, renal temsirolimus, sorafenib, ixabepilone 06 55/M Collecting duct 3 Sunitinib, XRT everolimus 3 Lung, mediastinum, retroperitoneum 07 50/F Clear cell 3 Ixabepilone, XRT, sunitinib 6 Lung, lymph nodes, brain, bone, skin, thyroid 08 55/M Papillary (þclear cell) 4 Radiofrequency ablation, sunitinib, XRT, 4 Lung, lymph nodes, bone, soft tissue everolimus 09 40/M Collecting duct 4 Cisplatin þ gemcitabine, CT, sorafenib 5 Bone, lung, liver, lymph nodes, chest wall þ erlotinib, XRT 10 52/M Clear cell 3 Sorafenib, temsirolimus, XRT 4 Lung, lymph nodes, bone, liver

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Figure 2. The PC regimen depletes and suppresses host lymphocytes. A, ALC (left) and ANC (right) are shown for each patient before start of the PC regimen and then at 6 time points performed on a biweekly basis over the 21-day treatment interval. The crossbar at each measurement indicates the median value. B, absolute number of CD4þ T-cell subsets (left) and CD8þ T-cell subsets (right) was determined before and after the 21-day PC regimen [subsets including na€ve, central memory, effector memory (EM), and T effector memory expressing CD45RA (TEMRA)]. Values shown are mean values SEM. C, T cells collected before and after administration of the PC regimen were costimulated for 24 hours; the resultant supernatant was tested for cytokine content (results expressed as pg/mL per 1 106 cells/mL per 24 hours). D, plasma was collected before the PC regimen (day 21) and after the PC regimen (day 3) and tested for IL7 and IL15 content by Luminex assay (results in pg/mL).

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(range, 12–131). Absolute numbers of na€ve, central memory, þ þ and effector memory subsets for CD4 and CD8 Tcellswere all significantly reduced after PC therapy (Fig. 2B). B cells were essentially eliminated [median CD19 cells reduced from 56 (range, 20–176) to 1 (range, 0–7)]. Natural killer cells were relatively resistant [median NK cell reduction from 151 (range, 60–269) to 64 (22–485)]. In murine models, the PC regimen was immunosuppressive, as defined by reduced cytokine secretion in residual, post-therapy T cells (13). Pre-PC T cells responded to co-stimulation with secretion of IFNg, TNFa, granulocyte macrophage colony-stimulating factor (GM-CSF), IL4, IL5, IL10, and IL13, often at levels greater than 100 pg/mL (n ¼ 17 measurements were above this value; Fig. 2C); in each of these cases (17 of 17), post-PC T cells had a reduction in the cytokine measurement. In clinical trials using Flu/Cy conditioning, the T-cell homeostatic cytokines IL7 and IL15 increased to median values (at the time of transplant) of 37 and 57 pg/mL, respectively (36, 37). In contrast, using PC conditioning, there was no statistically significant increase in IL7 and IL15 levels (Fig. 2D).

Stable mixed chimerism despite multiple T-Rapa cell DLI Median percent donor T-cell chimerism at days 7, 14, 28, 45, and 60 post-HCT were 61 (range, 7–77), 72 (range, 9–91), 74 (range, 21–88), and 77 (range, 26–95), respectively (Fig. 3A). Transplantation of T-cell–replete allografts plus the first infusion of T-Rapa cells thus yielded immediate donor T-cell engraftment; however, chimerism values remained relatively constant during sirolimus therapy despite of multiple T-Rapa DLI. After discontinuation of sirolimus at day 60 post-HCT, donor T-cell chimerism increased to a median value of 92% (range, 21–100). The PC regimen yielded limited donor myeloid engraftment (Fig. 3B): Figure 3. median donor myeloid chimerism at days 7 and 14 post-HCT Establishment and maintenance of split, mixed chimerism. Percent donor were each 0% (ranges, 0–1 and 0–27). Median percent donor chimerism was determined for each patient by VNTR assay at the indicated time points for purified T cells (A) and for purified myeloid cells (B). C, myeloid chimerism increased modestly during sirolimus therapy, percentage of CD4þ T-cell subsets (left) and CD8þ T-cell subsets (right) was with values at days 28, 45, and 60 post-HCT of 13 (range, 4–50), determined by flow cytometry at the indicated posttransplant time points 18 (range, 11–67), and 26 (range, 13–76). After discontinuation [subsets including na€ve, central memory, effector memory (EM), and T of sirolimus at day 60 post-HCT, donor myeloid chimerism effector memory expressing CD45RA (TEMRA)]. increased to a median of 61% (range, 16–100). The two patients with the lowest T-cell chimerism through day 100 post-HCT also therapy (Fig. 4, right). Upon ex vivo co-stimulation, post-HCT T had the lowest myeloid cell chimerism; both of these patients cells secreted both T 1 (IFNg, TNFa, GM-CSF) and T 2 (IL4, IL5, received each of the planned T-Rapa DLI plus additional unman- H H IL10, IL13) cytokines, with the magnitude of cytokine secretion ipulated DLI. þ being similar at days 14, 60, and 100 post-HCT (not shown). In experimental models, rapamycin therapy increased CD8 T- cell memory responses (38). Given these data, we measured na€ve, þ þ Clinical outcome central memory, and effector memory CD4 and CD8 T cell Clinical results are summarized in Table 2. No partial or subsets during and after sirolimus therapy: these subsets did not complete clinical responses were observed by RECIST criteria in differ substantially when comparing the days 14, 60, and 100 the first 10 evaluable patients; because the primary objective was post-HCT time points (Fig. 3c). not achieved, further accrual to the protocol was stopped at this point. T-cell phenotype post-HCT Eight of 10 patients received each of the 3 planned T-Rapa DLI; Previously, T-Rapa DLI increased TH1- and TH2-type responses one patient each received either 1 or 2 doses of T-Rapa cells by transcription factor analysis and cytokine secretion assays (19). because of poor performance status or inability to tolerate sir- Similar to this initial trial, we found a preponderance of GATA- olimus. Six of 10 patients received additional, unmanipulated DLI þ þ þ þ 3 CD4 T cells relative to T-bet CD4 T cells at day 14 post-HCT to treat progressive disease (PD; median day of unmanipulated (TH2-type > TH1-type); however, these values did not increase DLI therapy was day 87 post-HCT; range, 76–607 days). In terms with additional T-Rapa DLI and did not increase after sirolimus of adverse events through day 100 post-HCT, there was no discontinuation (Fig. 4). In experimental models, rapamycin engraftment syndrome or other serious adverse events attribut- prevention of acute GVHD increased regulatory T cells (39); in able to T-Rapa cell therapy. High-dose sirolimus was generally þ þ our trial, FoxP3 CD4 T cells did not increase during sirolimus well-tolerated; one patient needed to discontinue planned

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Figure 4. þ Determination of posttransplant T-cell phenotype. CD4 T cells were evaluated by intracellular ﬂow cytometry for expression of transcription factors for TH2, TH1, or regulatory T-cell subsets (GATA-3, left; T-bet, middle; FoxP3, right). Time points evaluated were before initiation of the PC regimen (day 21) and at the indicated posttransplant times.

sirolimus therapy (due to exacerbation of renal insufficiency compartment, as evidenced by absence of neutropenia and related to hypercalcemia). One patient required red cell transfu- extremely low initial levels of donor myeloid cell engraftment. sion during sirolimus therapy; transplant-associated microangio- Single-agent, high-dose sirolimus was sufficient for acute GVHD pathy did not occur. Four patients required topical corticosteroid prophylaxis despite of T-cell–replete transplantation and multiple therapy for sirolimus-related oral ulcers (grade 2 toxicity); there T-Rapa DLI. High-dose sirolimus yielded stable mixed T-cell were no other gastrointestinal toxicities. Two patients each had chimerism and a stable posttransplant T-cell cytokine phenotype. catheter-related thrombosis or catheter-related line infection. One However, high-dose sirolimus did not control tumor progression. patient each had bacteremia attributable to an intestinal metas- In sum, these results have identified a novel platform for establish- tasis, bacterial pneumonia, or viral infection (BK cystitis). ing stable mixed chimerism, yet on the other hand, caution Classical acute GVHD of grade II or higher did not occur. Four of against use of high-dose sirolimus in conjunction with allogeneic 6 patients developed late acute GVHD (at days 115, 124, 163, and T-cell therapy. 284 post-HCT); 2 cases involved skin only and 2 involved the gut. The PC regimen is an immune-selective method of nonmye- One of 4 evaluable patients developed chronic GVHD, which was loablative transplantation that resides at the far range of this limited to vulvovaginal involvement. By day 60 post-HCT, despite discipline (toward nonmyelosuppressive transplantation). The high-dose sirolimus therapy, 4 of 10 patients had PD by RECIST PC regimen was immune-depleting and immunosuppressive, as criteria; 5 additional patients developed PD by day 100 posttrans- residual host T cells were not capable of high-level cytokine plant. One patient continues to have stable disease more than 4 secretion. This result mirrors our findings in a murine model of years' posttransplant (collecting duct carcinoma). Each of the 9 graft rejection (13), where pentostatin was more immunosup- protocol deaths were primarily attributable to progressive RCC; pressive than fludarabine; it is thus possible that the clinical trial one case was additionally attributable to late acute gut GVHD and results described here may be relatively unique to pentostatin (i.e., CMV enteritis that occurred after multiple unmanipulated DLI. not necessarily interchangeable with other purine analogues). The þ PC regimen differentially depleted immune subsets, with CD4 T þ Discussion cells and B cells more sensitive than CD8 T cells; the majority of Pentostatin plus low-dose, dose-adjusted Cy yielded sufficient lymphocytes remaining after PC therapy were NK cells. Also, the € host T-cell depletion and suppression to permit prompt donor T- na ve T-cell subset, which in murine models mediate increased cell engraftment. The PC regimen preserved the host myeloid alloreactivity (40), was nearly eliminated. Finally, in contrast to

Table 2. Transplantation outcome GVHD Summary of DLI therapy Acute Tumor staging UPN No. of DLI Speciﬁcs s g l Gr Late acute Chronic Day þ28 Day þ60 Day þ100 Overall outcome 01 3 T-Rapa series 0 0 0 0 G N.E. SD SD PD Death (d131) RCC 02 4 T-Rapa series þ standard DLI (1) 1 0 0 I None V-V SD SD PD Death (d740) RCC 03 3 T-Rapa series 0 0 0 0 N.E. N.E. SD PD PD Death (d134) RCC 04 7 T-Rapa series þ standard DLI (4) 0 0 0 0 S, G, L None SD SD PD Death (d303) infection, GVHD, RCC 05 5 T-Rapa series þ standard DLI (2) 0 0 0 0 N.E. N.E. SD SD PD Death (d127) RCC 06 4 T-Rapa series þ standard DLI (1) 0 0 0 0 S None SD SD SD Alive (d þ1,591) 07 2 T-Rapa #1, #2 0 0 0 0 S N.E. SD PD PD Death (d165) RCC 08 5 T-Rapa series þ standard DLI (2) 0 0 0 0 None None SD SD PD Death (d300) RCC 09 4 T-Rapa series þ standard DLI (1) 0 0 0 0 N.E. N.E. SD PD PD Death (d143) RCC 10 1 T-Rapa #1 2 0 0 I N.E. N.E. SD PD NA Death (d78) RCC NOTE: T-Rapa Series indicates patient received T-Rapa cells at day 0 of transplant and at days 14 and 45 posttransplant. Standard DLI indicates donor lymphocyte infusion consisting of unmanipulated donor T cells. Abbreviations: N.A., not applicable; N.E., not evaluable; S, G, L, skin, gut, or liver acute GVHD; V-V, vulvar vaginal chronic GVHD; SD, stable disease; RCC, disease active at time of death (SD or PD). aPatients UPN07 and UPN10 did not receive all T-Rapa infusions due to poor performance status.

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previous immune-ablative regimens (36, 37) and other nonmye- conditioning was likely counterproductive, as donor APC con- loablative conditioning (41), PC therapy did not significantly tribute to GVT effects in experimental models (50). Finally, increase IL7 or IL15 levels. Given that some studies have associ- previous clinical trials have indicated that GVT effects against ated IL7 and IL15 levels with acute GVHD risk (36, 42), this RCC are associated with clinical GVHD (29, 30). characteristic may have contributed to the nominal GVHD and The overall transplant approach we evaluated was not suitable stable mixed T-cell chimerism. for therapy of refractory cancer but may be advantageous for The mixed chimerism was split in terms of T cell versus myeloid therapy of nonmalignant disease. These results provide a cau- populations. The paucity of donor myeloid engraftment early tionary note in terms of combining adoptive T-cell therapy with post-HCT likely represents the lowest values reported to date and high-dose sirolimus therapy, which did not prevent tumor pro- demonstrates the relative lack of myeloid toxicity from the PC gression and limited posttransplant T-cell effects. Our future regimen. Nonetheless, stem cell engraftment was secured, as each investigations will continue to incorporate ultra–low-intensity patient had a gradual increase in donor myeloid components; this conditioning such as the PC regimen as a safe platform for apparent competitive advantage of donor stem cells was likely evaluation of novel T-cell therapy products. However, such plat- generated through a GVH reaction that was largely subclinical. forms will incorporate calcineurin inhibitors as GVHD prophy- Although this pattern of alloengraftment was not conducive to laxis to avoid the in vivo tolerizing effects of sirolimus therapy. refractory cancer therapy, split mixed chimerism favoring donor T-cell elements may be favorable for therapy of T-cell–based Disclosure of Potential Conflicts of Interest immune deficiencies. No potential conflicts of interest were disclosed. Several factors likely contributed to the low rate and severity of acute GVHD. First, the PC regimen did not increase GVHD- Authors' Contributions provoking T-cell homeostatic cytokines. Second, the lymphocyte Conception and design: S.M. Steinberg, J. Gea-Banacloche, C. Sportes, specificity of the PC regimen may have constrained host inflam- J. Mariotti, A.T. Fojo, M.R. Bishop, R.E. Gress, D.H. Fowler mation, which is known to potentiate GVHD (8). Third, mixed Development of methodology: M.E. Mossoba, D. Stroncek, B.L. Levine, chimerism itself limits clinical acute GVHD (43). Experimental C.H. June, M.R. Bishop, D.H. Fowler Acquisition of data (provided animals, acquired and managed patients, GVHD is restricted by host T cells (44) and promoted by donor provided facilities, etc.): M.E. Mossoba, D.C. Halverson, R. Kurlander, antigen-presenting cells (45); because the PC regimen preserved B.B. Schuver, B. Hansen, F.T. Hakim, J. Gea-Banacloche, C. Sportes, N.M. Hardy, some component of host T cells and severely limited donor S.Z. Pavletic, M. Sabatini, D. Stroncek, J. Mariotti, O. Rixe, A.T. Fojo, myeloid engraftment, the pattern of split mixed chimerism gen- M.R. Bishop, R.E. Gress, D.H. Fowler erated may have been particularly protective against GVHD. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, Fourth, the T-Rapa DLI product expresses a balanced pattern of computational analysis): M.E. Mossoba, D.C. Halverson, S.M. Steinberg, N. Tageja, S.Z. Pavletic, M.R. Bishop, D.H. Fowler T 1- and T 2-type cytokines and previously yielded a low rate of H H Writing, review, and/or revision of the manuscript: M.E. Mossoba, acute GVHD (19). Finally, high-dose sirolimus can protect against D.C. Halverson, R. Kurlander, A. Carpenter, S.M. Steinberg, S.A. Ali, N. Tageja, acute GVHD (28). In experimental models, rapamycin anti- F.T. Hakim, C. Sportes, S.Z. Pavletic, B.L. Levine, O. Rixe, M.R. Bishop, GVHD effects occurred by TH1 cytokine inhibition (46) and D.H. Fowler regulatory T-cell promotion (39); because we found low levels Administrative, technical, or material support (i.e., reporting or organizing þ þ þ data, constructing databases): M.E. Mossoba, B. Hansen, N. Tageja, J. Gea- of T-bet and FoxP3 CD4 T cells post-HCT, inhibition of TH1 Banacloche, D.D. Hickstein, H. Khuu, D. Stroncek, R.E. Gress, D.H. Fowler cells may have been largely operational in this trial. Given these Study supervision: B.B. Schuver, A. Carpenter, A.T. Fojo, D.H. Fowler collective results, similar to our findings in an experimental model (47), we conclude that ex vivo manufactured T-Rapa cells are not in vivo Acknowledgments resistant to rapamycin therapy. Special recognition goes to Vicki Fellowes for her efforts in T-Rapa cell Several factors likely contributed to the lack of GVT effects. First, manufacturing; Daniele Avila, Amanda Urban, Jennifer Mann, and Tiffani responses against RCC after allogeneic HCT preferentially occur in Taylor of the NCI-ETIB for their excellence and dedication in the care of protocol patients with less than 3 metastatic sites (48); our study was patients; Rashmika Patel of the NCI-CCR for her protocol support efforts; the composed of heavily pretreated patients (median, 3.5 prior regi- NIH Clinical Center and Department of Nursing; and to the Medical Oncology mens) with extensive metastatic burden (median, 3.5 sites). Fellows at the NCI. Second, our hypothesis that high-dose sirolimus would limit tumor progression was not confirmed, as 9 of 10 patients had Grant Support PD during or shortly after sirolimus therapy. Third, high-dose This work was supported by the Center for Cancer Research, NCI. The costs of publication of this article were defrayed in part by the payment of sirolimus was counterproductive due to promotion of stable page charges. This article must therefore be hereby marked advertisement in mixed chimerism (which was seen in a previous study of high- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. dose sirolimus; ref. 28) and due to inhibition of TH1-type cells (which are necessary for murine GVT effects against RCC cells; Received February 19, 2015; revised May 14, 2015; accepted May 26, 2015; ref. 49). Fourth, the low donor myeloid engraftment after PC published OnlineFirst June 12, 2015.

References 1. Baron F, Baker JE, Storb R, Gooley TA, Sandmaier BM, Maris MB, et al. 2. Kahl C, Storer BE, Sandmaier BM, Mielcarek M, Maris MB, Blume KG, et al. Kinetics of engraftment in patients with hematologic malignancies given Relapse risk in patients with malignant diseases given allogeneic hemato- allogeneic hematopoietic cell transplantation after nonmyeloablative con- poietic cell transplantation after nonmyeloablative conditioning. Blood ditioning. Blood 2004;104:2254–62. 2007;110:2744–8.

4318 Clin Cancer Res; 21(19) October 1, 2015 Clinical Cancer Research

High-Dose Sirolimus and GVT Effects

3. Pavletic SZ, Kumar S, Mohty M, de Lima M, Foran JM, Pasquini M, et al. NCI 21. Mariotti J, Foley J, Jung U, Borenstein T, Kantardzic N, Han S, et al. Ex vivo first international workshop on the biology, prevention, and treatment of rapamycin generates apoptosis-resistant donor Th2 cells that persist in vivo relapse after allogeneic hematopoietic stem cell transplantation: report and prevent hemopoietic stem cell graft rejection. J Immunol 2008;180: from the committee on the epidemiology and natural history of relapse 89–105. following allogeneic cell transplantation. Biol Blood Marrow Transplant 22. Mariotti J, Foley J, Ryan K, Buxhoeveden N, Kapoor V, Amarnath S, et al. 2010;16:871–90. Graft rejection as a Th1-type process amenable to regulation by donor Th2- 4. Frey NV, Porter DL. Graft-versus-host disease after donor leukocyte infu- type cells through an interleukin-4/STAT6 pathway. Blood 2008;112: sions: presentation and management. Best Pract Res Clin Haematol 4765–75. 2008;21:205–22. 23. Ram R, Storb R. Pharmacologic prophylaxis regimens for acute graft- 5. Gill S, Porter DL. Reduced-intensity hematopoietic stem cell transplants for versus-host disease: past, present and future. Leuk Lymphoma 2013;54: malignancies: harnessing the graft-versus-tumor effect. Annu Rev Med 1591–601. 2013;64:101–17. 24. Armand P, Gannamaneni S, Kim HT, Cutler CS, Ho VT, Koreth J, et al. 6. Fujisaki J, Wu J, Carlson AL, Silberstein L, Putheti P, Larocca R, et al. In vivo Improved survival in lymphoma patients receiving sirolimus for graft- imaging of Treg cells providing immune privilege to the haematopoietic versus-host disease prophylaxis after allogeneic hematopoietic stem-cell stem-cell niche. Nature 2011;474:216–9. transplantation with reduced-intensity conditioning. J Clin Oncol 7. Schwartz E, Lapidot T, Gozes D, Singer TS, Reisner Y. Abrogation of bone 2008;26:5767–74. marrow allograft resistance in mice by increased total body irradiation 25. Hartmann B, Schmid G, Graeb C, Bruns CJ, Fischereder M, Jauch KW, et al. correlates with eradication of host clonable T cells and alloreactive cyto- Biochemical monitoring of mTOR inhibitor-based immunosuppression toxic precursors. J Immunol 1987;138:460–5. following kidney transplantation: a novel approach for tailored immuno- 8. Jankovic D, Ganesan J, Bscheider M, Stickel N, Weber FC, Guarda G, et al. suppressive therapy. Kidney Int 2005;68:2593–8. The Nlrp3 inflammasome regulates acute graft-versus-host disease. J Exp 26. Cutler C, Stevenson K, Kim HT, Richardson P, Ho VT, Linden E, et al. Med 2013;210:1899–910. Sirolimus is associated with veno-occlusive disease of the liver after 9. Pavletic SZ, Bociek RG, Foran JM, Rubocki RJ, Kuszynski CA, Wisecarver JL, myeloablative allogeneic stem cell transplantation. Blood 2008;112: et al. Lymphodepleting effects and safety of pentostatin for nonmyeloa- 4425–31. blative allogeneic stem-cell transplantation. Transplantation 2003;76: 27. Ho VT, Cutler C, Carter S, Martin P, Adams R, Horowitz M, et al. Blood and 877–81. marrow transplant clinical trials network toxicity committee consensus 10. Miller KB, Roberts TF, Chan G, Schenkein DP, Lawrence D, Sprague K, et al. summary: thrombotic microangiopathy after hematopoietic stem cell A novel reduced intensity regimen for allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow Transplant 2005;11:571–5. transplantation associated with a reduced incidence of graft-versus-host 28. Hsieh MM, Kang EM, Fitzhugh CD, Link MB, Bolan CD, Kurlander R, et al. disease. Bone Marrow Transplant 2004;33:881–9. Allogeneic hematopoietic stem-cell transplantation for sickle cell disease. 11. Kharfan-Dabaja MA, Anasetti C, Fernandez HF, Perkins J, Ochoa-Bayona N Engl J Med 2009;361:2309–17. JL, Pidala J, et al. Phase II study of CD4þ-guided pentostatin lymphode- 29. Childs R, Chernoff A, Contentin N, Bahceci E, Schrump D, Leitman S, et al. pletion and pharmacokinetically targeted busulfan as conditioning for Regression of metastatic renal-cell carcinoma after nonmyeloablative hematopoietic cell allografting. Biol Blood Marrow Transplant 2013;19: allogeneic peripheral-blood stem-cell transplantation. N Engl J Med 1087–93. 2000;343:750–8. 12. Kay NE, Geyer SM, Call TG, Shanafelt TD, Zent CS, Jelinek DF, et al. 30. Barkholt L, Bregni M, Remberger M, Blaise D, Peccatori J, Massenkeil G, Combination chemoimmunotherapy with pentostatin, cyclophospha- et al. Allogeneic haematopoietic stem cell transplantation for metastatic mide, and rituximab shows significant clinical activity with low accom- renal carcinoma in Europe. Ann Oncol 2006;17:1134–40. panying toxicity in previously untreated B chronic lymphocytic leukemia. 31. Rini BI, Halabi S, Barrier R, Margolin KA, Avigan D, Logan T, et al. Adoptive Blood 2007;109:405–11. immunotherapy by allogeneic stem cell transplantation for metastatic 13. Mariotti J, Taylor J, Massey PR, Ryan K, Foley J, Buxhoeveden N, et al. The renal cell carcinoma: a CALGB intergroup phase II study. Biol Blood pentostatin plus cyclophosphamide nonmyeloablative regimen induces Marrow Transplant 2006;12:778–85. durable host T cell functional deficits and prevents murine marrow 32. Hudes G, Carducci M, Tomczak P, Dutcher J, Figlin R, Kapoor A, et al. allograft rejection. Biol Blood Marrow Transplant 2011;17:620–31. Temsirolimus, interferon alfa, or both for advanced renal-cell carcinoma. 14. Mossoba ME, Onda M, Taylor J, Massey PR, Treadwell S, Sharon E, et al. N Engl J Med 2007;356:2271–81. Pentostatin plus cyclophosphamide safely and effectively prevents 33.CastielloL,MossobaM,ViterboA,SabatinoM,FellowesV,FoleyJE, immunotoxin immunogenicity in murine hosts. Clin Cancer Res 2011; et al. Differential gene expression profile of first-generation and second- 17:3697–705. generation rapamycin-resistant allogeneic T cells. Cytotherapy 2013;15: 15. Hassan R, Miller AC, Sharon E, Thomas A, Reynolds JC, Ling A, et al. Major 598–609. cancer regressions in mesothelioma after treatment with an anti-mesothe- 34. Przepiorka D, Weisdorf D, Martin P, Klingemann HG, Beatty P, Hows J, lin immunotoxin and immune suppression. Sci Transl Med 2013;5: et al.1994 Consensus Conference on Acute GVHD Grading. Bone Marrow 208ra147. Transplant 1995;15:825–8. 16. van den Brink MR, Porter DL, Giralt S, Lu SX, Jenq RR, Hanash A, et al. 35. Filipovich AH, Weisdorf D, Pavletic S, Socie G, Wingard JR, Lee SJ, et al. Relapse after allogeneic hematopoietic cell therapy. Biol Blood Marrow National Institutes of Health consensus development project on criteria for Transplant 2010;16:S138–45. clinical trials in chronic graft-versus-host disease: I. Diagnosis and staging 17. Bethge WA, Hegenbart U, Stuart MJ, Storer BE, Maris MB, Flowers ME, et al. working group report. Biol Blood Marrow Transplant 2005;11:945–56. Adoptive immunotherapy with donor lymphocyte infusions after alloge- 36. Dean RM, Fry T, Mackall C, Steinberg SM, Hakim F, Fowler D, et al. neic hematopoietic cell transplantation following nonmyeloablative con- Association of serum interleukin-7 levels with the development of acute ditioning. Blood 2004;103:790–5. graft-versus-host disease. J Clin Oncol 2008;26:5735–41. 18. Kumar AJ, Hexner EO, Frey NV, Luger SM, Loren AW, Reshef R, et al. Pilot 37. Boyiadzis M, Memon S, Carson J, Allen K, Szczepanski MJ, Vance BA, et al. study of prophylactic ex vivo costimulated donor leukocyte infusion after Up-regulation of NK cell activating receptors following allogeneic hemato- reduced-intensity conditioned allogeneic stem cell transplantation. Biol poietic stem cell transplantation under a lymphodepleting reduced inten- Blood Marrow Transplant 2013;19:1094–101. sity regimen is associated with elevated IL-15 levels. Biol Blood Marrow 19. Fowler DH, Mossoba ME, Steinberg SM, Halverson DC, Stroncek D, Khuu Transplant 2008;14:290–300. HM, et al. Phase 2 clinical trial of rapamycin-resistant donor CD4þ Th2/ 38. Araki K, Turner AP, Shaffer VO, Gangappa S, Keller SA, Bachmann MF, et al. Th1 (T-Rapa) cells after low-intensity allogeneic hematopoietic cell trans- mTOR regulates memory CD8 T-cell differentiation. Nature 2009;460: plantation. Blood 2013;121:2864–74. 108–12. 20. Foley JE, Mariotti J, Ryan K, Eckhaus M, Fowler DH. Th2 cell therapy of 39. Zeiser R, Leveson-Gower DB, Zambricki EA, Kambham N, Beilhack A, Loh J, established acute graft-versus-host disease requires IL-4 and IL-10 and is et al. Differential impact of mammalian target of rapamycin inhibition on abrogated by IL-2 or host-type antigen-presenting cells. Biol Blood Marrow CD4þCD25þFoxp3þ regulatory T cells compared with conventional Transplant 2008;14:959–72. CD4þ T cells. Blood 2008;111:453–62.

www.aacrjournals.org Clin Cancer Res; 21(19) October 1, 2015 4319

Mossoba et al.

40. Anderson BE, McNiff J, Yan J, Doyle H, Mamula M, Shlomchik MJ, et al. costimulatory molecules in murine chronic graft-versus-host disease: Memory CD4þ T cells do not induce graft-versus-host disease. J Clin Invest requirements depend on target organ. Blood 2005;105:2227–34. 2003;112:101–8. 46. Blazar BR, Taylor PA, Panoskaltsis-Mortari A, Vallera DA. Rapamycin 41. De Bock M, Fillet M, Hannon M, Seidel L, Merville MP, Gothot A, et al. inhibits the generation of graft-versus-host disease- and graft-versus-leu- Kinetics of IL-7 and IL-15 levels after allogeneic peripheral blood stem cell kemia-causing T cells by interfering with the production of Th1 or Th1 transplantation following nonmyeloablative conditioning. PLoS One cytotoxic cytokines. J Immunol 1998;160:5355–65. 2013;8:e55876. 47. Jung U, Foley JE, Erdmann AA, Toda Y, Borenstein T, Mariotti J, et al. Ex vivo 42. Thiant S, Yakoub-Agha I, Magro L, Trauet J, Coiteux V, Jouet JP, et al. Plasma rapamycin generates Th1/Tc1 or Th2/Tc2 Effector T cells with enhanced in levels of IL-7 and IL-15 in the ﬁrst month after myeloablative BMT are vivo function and differential sensitivity to post-transplant rapamycin predictive biomarkers of both acute GVHD and relapse. Bone Marrow therapy. Biol Blood Marrow Transplant 2006;12:905–18. Transplant 2010;45:1546–52. 48. Nakayama K, Tannir NM, Liu P, Wathen JK, Cheng YC, Champlin RE, et al. 43. Nikolousis E, Robinson S, Nagra S, Brookes C, Kinsella F, Tauro S, et al. Natural history of metastatic renal cell carcinoma in patients who under- Post-transplant T cell chimerism predicts graft versus host disease but went consultation for allogeneic hematopoietic stem cell transplantation. not disease relapse in patients undergoing an alemtuzumab based Biol Blood Marrow Transplant 2007;13:975–85. reduced intensity conditioned allogeneic transplant. Leuk Res 2013;37: 49. Ramirez-Montagut T, Chow A, Kochman AA, Smith OM, Suh D, Sindhi H, 561–5. et al. IFN-gamma and Fas ligand are required for graft-versus-tumor activity 44. Blazar BR, Lees CJ, Martin PJ, Noelle RJ, Kwon B, Murphy W, et al. Host T against renal cell carcinoma in the absence of lethal graft-versus-host cells resist graft-versus-host disease mediated by donor leukocyte infu- disease. J Immunol 2007;179:1669–80. sions. J Immunol 2000;165:4901–9. 50. Reddy P, Maeda Y, Liu C, Krijanovski OI, Korngold R, Ferrara JL. A crucial 45. Anderson BE, McNiff JM, Jain D, Blazar BR, Shlomchik WD, Shlomchik MJ. role for antigen-presenting cells and alloantigen expression in graft-versus- Distinct roles for donor- and host-derived antigen-presenting cells and leukemia responses. Nat Med 2005;11:1244–9.

4320 Clin Cancer Res; 21(19) October 1, 2015 Clinical Cancer Research

High-Dose Sirolimus and Immune-Selective Pentostatin plus Cyclophosphamide Conditioning Yields Stable Mixed Chimerism and Insufficient Graft-versus-Tumor Responses

Miriam E. Mossoba, David C. Halverson, Roger Kurlander, et al.

Clin Cancer Res 2015;21:4312-4320. Published OnlineFirst June 12, 2015.

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