a live-attenuated listeria vaccine (anz-100) and a live ......cancer therapy: clinical a...

Cancer Therapy: Clinical

A Live-Attenuated Listeria Vaccine (ANZ-100) and aLive-Attenuated Listeria Vaccine Expressing Mesothelin(CRS-207) for Advanced Cancers: Phase I Studies of Safetyand Immune Induction

Dung T. Le1, Dirk G. Brockstedt4, Ran Nir-Paz5, Johannes Hampl6, Shruti Mathur6, John Nemunaitis7,Daniel H. Sterman8, Raffit Hassan9, Eric Lutz1, Bentley Moyer6, Martin Giedlin6, Jana-Lynn Louis1,Elizabeth A. Sugar1,2, Alice Pons1, Andrea L. Cox1,3, Jordana Levine3, Aimee Luck Murphy4, Peter Illei1,Thomas W. Dubensky Jr6, Joseph E. Eiden6, Elizabeth M. Jaffee1, and Daniel A. Laheru1

AbstractPurpose: Listeriamonocytogenes (Lm)-based vaccines stimulate both innate and adaptive immunity. ANZ-

100 is a live-attenuated Lm strain (Lm DactA/DinlB). Uptake by phagocytes in the liver results in localinflammatory responses and activation and recruitment of natural killer (NK) andT cells, in associationwith

increased survival of mice bearing hepatic metastases. The Lm DactA/DinlB strain, engineered to expresshuman mesothelin (CRS-207), a tumor-associated antigen expressed by a variety of tumors, induces

mesothelin-specific T-cell responses against mesothelin-expressing murine tumors. These two phase I

studies test ANZ-100 and CRS-207 in subjects with liver metastases and mesothelin-expressing cancers,

respectively.

Experimental Design: A single intravenous injection of ANZ-100 was evaluated in a dose escalation

study in subjects with liver metastases. Nine subjects received 1� 106, 3� 107, or 3 � 108 colony-formingunits (cfu). CRS-207 was evaluated in a dose-escalation study in subjects with mesothelioma, lung,

pancreatic, or ovarian cancers. Seventeen subjects received up to 4 doses of 1 � 108, 3 � 108, 1 � 109,or 1 � 1010 cfu.Results: A single infusion of ANZ-100 was well tolerated to the maximum planned dose. Adverse events

included transient laboratory abnormalities and symptoms associated with cytokine release. Multiple

infusions of CRS-207 were well tolerated up to 1 � 109 cfu, the determined maximum tolerated dose.Immune activation was observed for both ANZ-100 and CRS-207 as measured by serum cytokine/

chemokine levels and NK cell activation. In the CRS-207 study, listeriolysin O and mesothelin-specific

T-cell responses were detected and 37% of subjects lived �15 months.Conclusions: ANZ-100 and CRS-207 administration was safe and resulted in immune activation.

Clin Cancer Res; 18(3); 858–68. �2011 AACR.

Introduction

Cancer vaccines aim to induce immunity specific toprotein antigens that are differentially expressed by cancercells relative to thenormal cells fromwhich they are derived.Through its network of specialized antigen-presenting andeffector cells, the immune system has the ability to becomeactivated to recognize and lyse cancer cells. Current vaccinestrategies aim to provide a series of signals that activate andmature dendritic cells (DC) for efficient antigen processingand presentation which in turn activate effector cells of theadaptive immune response. Listeria monocytogenes (Lm)-based vaccine vectors directly target and activate DCs invivo, but in addition, take advantage of the capability ofimmunogenic infectious vectors to stimulate both adaptive

Authors' Affiliations: 1The Sidney Kimmel Cancer Center, the Skip ViraghClinical Pancreatic Cancer Center, and the SolGoldmanPancreaticCancerCenter at Johns Hopkins; 2Bloomberg School of Public Health at JohnsHopkins; 3Johns Hopkins Department of Medicine, Baltimore, Maryland;4Aduro BioTech, Berkeley, California; 5Hadassah Hebrew-University Med-ical Center, Jerusalem, Israel; 6Cerus Corporation, Concord, California;7Mary Crowley Cancer Center, Dallas, Texas; 8Abramson Cancer Center atthe University of Pennsylvania, Philadelphia, Pennsylvania; and 9NationalCancer Institute, Bethesda, Maryland

Note: Supplementary data for this article are available at Clinical CancerResearch Online (http://clincancerres.aacrjournals.org/).

Corresponding Author: Dung T. Le, Sidney Kimmel Cancer Center atJohns Hopkins University School of Medicine, 1650 Orleans Street, Room407, Baltimore, MD 21231. Phone: 443-287-0002; Fax: 410-614-8216;E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-11-2121

�2011 American Association for Cancer Research.

ClinicalCancer

Research

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and innate immune responses. Lm is an intracellular bac-terium that has access to both class I and II antigen-proces-sing pathways. Lm provides a potent stimulation of innateimmunity and also stimulates an adaptive immuneresponse through recruitment and activation of CD4þ andCD8þ T cells specific for encoded heterologous antigens(1–4). The ability of Lm to stimulate adaptive immunity ismainly based on its intracellular lifecycle and the ability totarget DCs in vivo (4). ANZ-100 is a live-attenuated double-deleted Lm strain (LADD; Lm DactA/DinlB). This strain hasdeletions of 2 virulence genes, actA and internalin B (InlB).These virulence determinants facilitate cell-to-cell spreadand invasion of nonphagocytic cells, and their combineddeletion results in 1,000-fold attenuation when comparedwith wild-type Lm (5). However, uptake of ANZ-100 byphagocytic cells in the liver and spleen is retained and resultsin a local proinflammatory cytokine response resulting inactivation and recruitment of both innate and adaptiveeffector cells. This immune response results in delay intumor growth and increased survival ofmice bearing hepat-ic metastases (6, 7). Importantly, multiple doses of Lmfurther extend survival.The LADD strain has also been engineered to express

humanmesothelin and the resulting strain has been termedCRS-207 (Lm-mesothelin). CRS-207 has been shown toefficiently deliver mesothelin antigen into both class I andII antigen-processing pathways. Mesothelin is a tumor-associated antigen present on normal mesothelial cells andhighly expressed by many human tumor types, includingmesotheliomas, pancreatic adenocarcinomas (PDA), non–small cell lung cancers (NSCLC), and ovarian cancers (8–16). This expression profile, combined with limited expres-sion on the surface of normal tissues, makes mesothelin anattractive target for active tumor-specific immunotherapy.Support formesothelin as a T-cell target comes from studies

showing a correlation between positive clinical outcomesand the induction of mesothelin-specific cellular immunityin subjects with PDA following vaccination with an irradi-ated allogeneic whole-cell vaccine encoding granulocytemacrophage colony-stimulating factor (GM-CSF). In aphase I study, a dose-dependent systemic antitumorresponse was reported to be associated with anti-mesothe-lin CD8þ T-cell responses (17, 18). In subsequent studies,the induction of mesothelin-specific T cells as well as theincreased post-vaccination diversity and avidity of the T-cellrepertoire were shown to be associated with improveddisease-free (DFS) and overall survival (OS; refs. 19, 20).Furthermore, CRS-207 mediates the induction of mesothe-lin-specific T-cell responses that correlatewith tumor regres-sions of mesothelin-expressing murine tumors (unpub-lished data).

A single intravenous dose of ANZ-100 underwent eval-uation in a phase I dose escalation study of safety andtolerability in adults with carcinoma and liver metastases(NCT00327652). A total of 9 subjects received single-doseinfusions at 3 dose levels [1� 106, 3� 107, 3� 108 colony-forming units (cfu)]. Subsequently, CRS-207 underwentevaluation in a phase I, open-label, multiple dose, dose-escalation study in subjects with mesothelioma, NSCLC,PDA, or ovarian cancer (NCT00585845). Seventeen sub-jects were enrolled into 4 cohorts (1� 108, 3� 108, 1� 109,and 1� 1010 cfu). Here, we report the safety, shedding andclearance data, clinical activity, and the induction of immu-nologic responses to both ANZ-100 and CRS-207.

Subjects and Methods

Construction of ANZ-100 and CRS-207ANZ-100 (LADD; Lm DactA/DinlB), Lm strain CERS

382.20, was constructed by deletion of the actA and inlBgenes from the Streptomycin-resistant wild-type strain DP-L4056. Using standard techniques, deletions of actA andinlB were made by homologous recombination of themutant alleles into the wild-type chromosome (21). Dele-tion mutations were confirmed by PCR.

CRS-207, Lm strain hMeso38, was constructed by theaddition of a mesothelin expression cassette into the CERS382.20 strain. The same homologous recombinationapproach used to delete inlBwas used to insert themesothe-lin antigen. Mesothelin is expressed as an ActA fusionprotein under the transcriptional control of the actA pro-moter. The actA promoter is strongly induced in host cells,resulting in efficient production of the heterologous anti-gen. All genomicmodifications were confirmed by PCR andDNA sequencing, and the attenuated phenotype of bothstrains were showed in vivo by LD50 (tested inCD-1, C57BL/6, and BALB/cmice: LD50 of 8.0� 107, 1.2� 108, and 8.4�107 cfu, respectively, compared with 3.0 � 104 cfu of thewild-type Lm strain) and clearance in liver and spleen ofmice and in vitro by infectivity and intracellular growthkinetics. Clinical grade material of ANZ-100 and CRS-207 was manufactured at the Waisman Clinical BioManu-facturing Facility.

Translational Relevance

Listeria monocytogenes (Lm)-based vaccine vectors canstimulate both innate and adaptive immune responses.In preclinical studies, administration of Lm vaccinesresults in enhanced tumor-specific immune responses,delayed tumor growth, and improved survival. Further-more,Lm canbemodified to encodeheterologous tumorantigens resulting in recruitment and activationof tumorantigen–specific T cells. In these two first-in-humanphase I clinical studies in patients with advanced cancer,ANZ-100 [a live-attenuated Lm strain (Lm DactA/DinlB)]and CRS-207 (the Lm DactA/DinlB strain engineered toexpress human mesothelin), were well tolerated withencouraging dose-dependent evidence of immune acti-vation. These results provide valuable insight into thesafety and dosing of this new vaccine approach that willadvance the further development of Lm vaccines asanticancer agents for multiple tumor types.

Live-Attenuated Listeria Vaccines for Advanced Cancer

www.aacrjournals.org Clin Cancer Res; 18(3) February 1, 2012 859




Study designNine subjects were enrolled into the ANZ-100 study at

Johns Hopkins University (JHU), Baltimore, MD, andMaryCrowley Cancer Center, Dallas, TX, between October 9,2006, and January 7, 2008. The primary objective of theANZ-100 study was to determine the maximum tolerateddose (MTD) of a single dose of ANZ-100 in subjects withcarcinoma and liver metastases. Using a standard 3 þ 3design, eligible subjects received a single 2-hour intrave-nous infusion of ANZ-100 (22).

Seventeen subjects were enrolled into the CRS-207 studyat JHU; the National Cancer Institute (NCI), Bethesda, MD;the University of Pennsylvania, Philadelphia, PA; Hadas-sah-Hebrew University Medical Center, Jerusalem, Israel;and at Mary Crowley Cancer Center between December 13,2007, and January 5, 2009. The primary objective of theCRS-207 studywas to determine theMTDofmultiple dosesof CRS-207 in subjects with malignancies known to expressmesothelin. Secondary objectives included assessing safety,biodistribution and clearance of Lm, immunologic end-points, and antitumor activity. Using a 3 þ 3 design,sequential cohorts of 3 to 6 subjects received up to 4 dosesof CRS-207 administered 3 weeks apart.

These multi-institutional, first-in-human, phase I, dose-escalation studies were reviewed and approved by localInstitutional Review Boards, Institutional Biosafety Com-mittees, the U.S. Food and Drug Administration, and theNIH Recombinant DNA Advisory Committee. All partici-pating subjects signed informed consent.

Subject selectionIn the ANZ-100 study, eligible subjects had treatment-

refractory carcinoma and hepatic metastases. In the CRS-207 study, eligible subjects had treatment-refractory meso-thelioma, PDA, NSCLC or ovarian cancer. For both studies,main eligibility criteria included: no cancer therapy for 4weeks, age�18 years old, a life expectancy of�12weeks, anEasternCooperativeOncologyGroupperformance status of0 to 1 or Karnofsky performance status of 80% to 100%,adequate organ function, no ongoing infections, history ofbrain metastases, or history of autoimmunity. Concurrentantineoplastic therapies, history of listeriosis or vaccinationwith a Lm-based vaccine, known allergy to both penicillinand sulfa, and artificial implants (except biliary stents) werenot permitted. Subjects who were HIV, HTLV-1, HCV, orHBV positive were excluded.

Procedures and treatmentTests were conducted for baseline toxicity (complete

blood counts and chemistry profile) and tumor assessment(computerized tomography scan). The intervention anddata collection schedules are shown in Supplementary Figs.S1 (ANZ-100) and S2 (CRS-207).

Nine subjects [6 with colorectal cancer (CRC), 2 withPDA, 1 with melanoma] with treatment-refractory carci-noma and liver metastases received a single-dose 2-hourinfusion at 1 of 3 dose levels (1� 106, 3� 107, 3� 108 cfu)of ANZ-100 (Table 1). Subjects were observed in an

in-patient facility for 5 days and evaluated for toxicity ondays 6, 9, 16, and 28.

Seven subjects with PDA, 5 with mesothelioma, 3 withNSCLC, and 2 with ovarian cancer received up to 4 intra-venous infusions of CRS-207 in 21-day intervals and wereobserved for toxicities in an in-patient facility for 24 to 48hours (Table 1). Subjects were evaluated for toxicity on days4 and 7 in the clinic and by phone on day 14. Following thefinal administration, subjects were evaluated by phone 21days after dosing and returned for a final clinic visit on the28th day after the final dose (day 91).

A 10-day course of oral amoxicillin or trimethoprim/sulfamethoxazole in penicillin-allergic subjects was initiat-ed 6 to 7days following the subject’s last dose. Subjectswererestaged radiographically [Response Evaluation Criteria inSolid Tumors (RECIST) 1.0] at days 28 and 91 (CRS-207study only). Subjects with progressive disease at day 28 onthe CRS-207 study were allowed to continue on study ifclinically stable.

AssessmentsToxicities. Adverse events were graded using the NCI

Common Terminology Criteria for Adverse Events(CTCAE) v3.0. Initially, a dose-limiting toxicity (DLT)was defined as the occurrence of any NCI CTCAE (Version3.0) �grade III that were determined to be possibly orprobably related to the agent, during the 28 days after thefirst dose. For individuals who had alanine aminotrans-ferase (ALT), aspartate aminotransferase (AST), or alka-line phosphatase elevations � grade I severity at studyentry, a DLT was defined as enzyme elevations >5� upperlimit of normal (ULN) that were determined to be relatedand persisted for more than 7 days. For individuals whohad ALT, AST, or alkaline phosphatase levels that wereabove 2.5 � ULN to 3.5 � ULN at study entry, a DLT wasdefined as enzyme elevations above 10 � ULN that weredetermined to be related and persisted for more than 7days. Early initiation of antibiotics coincident with iso-lation of Lm from a sterile body site, other than blood(e.g., CSF, joint fluid) was considered a DLT. DLT criteriawere modified several times during the CRS-207 study. Inaddition to the liver enzyme criteria mentioned, a DLTwas defined as a treatment-related � grade III laboratoryabnormality lasting for more than 48 hours, fever above40�C lasting greater than 24 hours, hypotension unre-sponsive to intravenous fluids, and grade IV lymphocytedecreases that persisted for more than 4 days. The MTDwas the highest dose at which no more than 1 of 6subjects experienced a DLT.

Shedding and clearance. Specimens were obtained forculture to assess the Lm distribution and clearance. Witheach administration of ANZ-100, blood, urine, stool, andsputum specimens were cultured at baseline, 6 hours, days1–5, days 8, 16, and 28. An additional blood culture wastaken at 2 hours. With each administration of CRS-207,urine and stool cultures were obtained at baseline, 4 hours,days 1, 4, and 7. Blood cultures were obtained at baseline, 4hours, days 1 and 4.

Le et al.

Clin Cancer Res; 18(3) February 1, 2012 Clinical Cancer Research860




Immunohistochemistry. Mesothelin expression on thetumor was not required for CRS-207 study entry, butimmunohistochemistry (IHC) was conducted on availablearchived tissue. IHC was conducted using a monoclonalantibody (2C6) on a Leica BondMax autostainer. The stain-ing intensity and extent were scored. The percentage oftumor cells showingmembranous staining (predominantlyluminal) were evaluated for intensity (0, none; 1þ, weakthin; 2þ, moderate; and 3þ, strong and thick membranousstaining).

Immunologic assessmentsMultiplexed serum chemokine assay. Chemokines were

detected using a custom Cytokine Bead Array (BD) usingfrozen serum samples collected before, 4 hours, and 24hours postinfusion with ANZ-100 and at baseline and at 24hours after CRS-207. The array was specific for interleukin(IL)-6, IL-8, IL-9, IL-10, IFN-g–induced protein 10 (IP-10),lymphotoxin-a (LT-a), monocyte chemotactic protein-1(MCP-1), macrophage inflammatory protein-1a (MIP-1a),

andMIP-1b. Upon collection of all samples within a patientcohort, samples were tested by a blinded operator. Dataare presented as change in fold concentration becausepredose chemokine concentration in sera varied consider-ably between subjects.

Multiplexed serum cytokine assay. Cytokines weredetected using the Meso Scale Discovery (MSD) platformusing frozen serum samples collected before, 4 hours, and24 hours postinfusionwith ANZ-100 or CRS-207. ANZ-100samples were tested using a 9-plex proinflammatory kit(GM-CSF, IFN-g , IL-1b, IL-2, IL-6, IL-8, IL-10, IL-12p70,TNF-a) and CRS-207 samples were tested using a 7-plex kit(IFN-g , IL-1b, IL-6, IL-8, IL-10, IL-12p70, TNF-a). Uponcollection of all samples within a patient cohort, sampleswere tested by a blinded operator. Results are expressed asmean � SD.

Lymphocyte counts. For the ANZ-100 study, a BDFACSCalibur was used to determine absolute numbersof lymphocyte subpopulations after applying BD Mul-titest CD3/CD16, CD56/CD45/CD19 with Trucount,

Table 1. Patient characteristics

ID Age (sex) Cancer type Dose level, cfu ECOG PS Prior therapies Mesothelin IHCa No. of doses

ANZ-10001-002 57 (F) Pancreatic 106 0 2 NA 102-003 53 (M) Colorectal 106 0 2 NA 102-006 69 (M) Colorectal 106 1 5 NA 101-005 62 (M) Pancreatic 3 � 107 0 2 NA 102-054 66 (M) Melanoma 3 � 107 0 4 NA 102-058 71 (M) Colorectal 3 � 107 0 4 NA 101-008 49 (F) Colorectal 3 � 108 0 4 NA 102-062 57 (M) Colorectal 3 � 108 0 5 NA 102-064 60 (M) Colorectal 3 � 108 0 5 NA 1

CRS-207001-001 65 (M) Pancreatic 108 0 5 50%, 2–3þ 4001-002 61 (F) Pancreatic 108 0 4 NE 4003-001 65 (F) Mesothelioma 108 0 2 25%, 1–2þ 4002-001 64 (F) Ovarian 108 1 8 NE 2002-002 60 (M) Mesothelioma 108 1 1 NE 2004-001 55 (M) Mesothelioma 108 0 4 NE 3005-002 56 (F) NSCLC 3 � 108 0 4 70%, 2–3þ 1001-004 68 (F) Pancreatic 3 � 108 0 1 NE 3001-005 60 (M) Pancreatic 3 � 108 1 1 75%, 2–3þ 2005-003 79 (F) Pancreatic 3 � 108 0 0 80%, 2–3þ 2004-002 59 (M) Pancreatic 109 1 4 NE 4005-001 56 (F) NSCLC 109 0 5 50%, 2þ 4001-003 61 (M) Pancreatic 109 0 2 NE 4002-003 72 (F) NSCLC 109 1 5 100%, 3þ 4002-004 75 (M) Mesothelioma 109 1 4 NE 2003-002 52 (F) Ovarian 109 1 10 NE 4003-003 40 (M) Mesothelioma 1010 1 2 NE 1

Abbreviations: ECOG, Eastern Cooperative Oncology Group; NA, not applicable; NE, not evaluable; PS, performance status.aMesothelin IHC: the percentage of tumor cells showing membranous staining were evaluated for intensity (0, no staining; 1þ, weakstaining; 2þ, moderate staining; 3þ, strong staining).






and BD Multitest CD3/CD8/CD45/CD4 with Trucountreagent cocktails.

Determination of cellular activation. For the ANZ-100study, a BD FACSCalibur was used to determine the expres-sion level of CD38 on natural killer (NK) cells in bloodusing CD3-FITC, CD38-PE, CD4-PerCp, CD16-APC, CD56APC, and IgG Isotype-PE. CD38 expression levels wereanalyzed by a blinded operator and reported as a ratio ofpeak CD38 MESF value between 48 and 96 hours postdos-ing and predose value. A histogram is also presented forsubjects 001–005.

Detection of antigen-specific T cells. Peripheral bloodmononuclear cells (PBMC) were prepared within 4 to 6hours after collection and cryopreserved at each clinical site.

Detection of listeriolysin O–specific T cells by IFN-gELISPOT. PBMCs were analyzed using a 2-step IFN-gELISPOT assay inwhich first autologousDCswere obtainedthrough in vitro culture (23). Monocytes were isolated fromPBMCs using a 1-hour adherence step. Nonadherent cellswere collected and cryopreserved. Adherent cells were cul-tured for 2 days in DC induction medium (RPMI with 1%autologous plasma and GM-CSF and IL-4) and then acocktail containing IL-1b, IL-6, and prostaglandin E2-a(24). After 2 days of culture, mature DCs were harvested,counted, and added at a 1:10 ratio to 2 � 105 nonadherentthawed PBMCs. Antigen was added as pools of 15-merpeptides whose sequences overlap by 11 amino acids andcover the entire sequence of the listeriolysin O (LLO), a 130amino acid protein (25, 26). The CEF pool was used as apositive control. The CEF is a pool of 32 peptides of definedCD8þ epitopes against cytomegalovirus (CMV), Epstein-Barr virus (EBV), and influenza (27). Cells were cultured for24 hours in RPMI containing 10% human AB serum beforethey were washed and spots visualized using BD Bios-ciences’ Human IFN-g ELISPOT and AEC substrate kits.Spots were enumerated in an ELISPOT reader (CellularTechnology, Ltd.) and analyzed using a software package(Immunospot software v. 3.6). T-cell responses to LLOwereconsidered positive when specific T-cell frequencies were�1 in 105 PBMCs and increased by at least 2-fold comparedwith baseline.

Detection of mesothelin-specific T cells by IFN-gELISPOT. The methodology for the synthesis of peptides,ELISA assay for identifying reactive mesothelin peptides,and ELISPOT assays have previously been described (18–20). Samples were tested from subjects with HLA-A1, A2,A3, and A24 alleles if pre- and posttreatment samples wereavailable. T-cell responses to mesothelin were consideredpositivewhen the frequencyof specific responseswere�1 in105 CD8þ peripheral blood lymphocytes (PBL) above thecontrol sample and increased by at least 2-fold comparedwith baseline. Themaximal response to a single best peptideis reported.

Statistical considerationsThe main objectives of these studies were to determine

the MTD of ANZ-100 or CRS-207 in subjects with cancer. Astandard 3þ3designwasused for dose-escalation (22). The

incidence of toxicities is summarized by cohort. Exploratoryanalyses included evaluation of RECIST response, OS, cyto-kine/chemokine responses, immune cell phenotyping, andT-cell responses. The NK cell and lymphocyte values beforeand after treatment are plotted for each individual. Log-linear models are used to compare fold-upregulation andinduction between dose levels. For analysis incorporatingmultiple time points, linear mixed-effects models are usedto account for the within-individual repeated measure-ments. The numbers of individuals with LLO-specific andmesothelin-specific T-cell responses were tabulated. Thesurvival is documented for each individual and individualswith survival �15 months are considered "long-term" sur-vivors. The relationship between disease and immunologiccharacteristics are explored by tabulating the number oflong-term survivors in different subcategories.

Results

Subject characteristicsSubject characteristics for both studies are shown in Table

1. For ANZ-100, 6 subjects with CRC, 2 with PDA, and 1withmelanoma received a single dose of either 106, 3� 107,or 3 � 108 cfu of Lm. Their median age was 60 (range, 49–71). The median number of prior therapies was 4. For CRS-207, 5 subjects with mesothelioma, 7 with PDA, 3 withNSCLC, and 2 with ovarian cancer received at least oneinfusion of CRS-207 (6 subjects at 1� 108, 4 subjects at 3�108, 6 subjects at 1 � 109, and 1 subject at 1 � 1010 cfu).Their median age was 61 (range, 40–79). The mediannumber of prior therapies was 4.

Treatment-related toxicities and DLT eventsA detailed description of treatment related grade �II

toxicities for both studies is provided in SupplementaryTable S1. ANZ-100 was well tolerated at all dose levels. Themost frequent adverse events of any grade were transientgrade �III lymphopenia (9 patients, 100%), grade �IIIhyperglycemia (8 patients, 89%), hypophosphatemia (5patients, 56%), and fever (7 patients, 78%). No DLTs wereobserved and ANZ-100 was well tolerated up to the max-imum planned dose.

CRS-207 was also well tolerated. The most frequentadverse events of any grade were transient lymphopenia(17 patients, 100%), hypophosphatemia (6 patients, 35%),transaminitis (7 patients, 41%), fever (9 patients, 53%),chills/rigors (9 patients, 53%), nausea (9 patients, 53%),fatigue (6 patients, 35%), and hypotension (6 patients,35%). All of these adverse events were grade �II except fortransient �grade III lymphopenia and hypophosphatemia,one grade III transaminitis, and one grade III fever. The firstdose cohort received 1 � 108 cfu of CRS-207 at 3-weekintervals for 4 doses. Two adverse events occurred in sub-jects dosed in cohort 1 (1 � 108 cfu) which met the initialprotocol-defined criteria of DLTs. One subject experiencedtransient grade III hypophosphatemia 4 hours followingthe second infusion of CRS-207. A second subject experi-enced a grade III temperature approximately 22 hours after

Le et al.





receiving the first infusion. The subject was treated withacetaminophen and temperature returned to baseline with-in 24 hours. DLT criteria were amended to allow for tran-sient grade III hypophosphatemia and fever. After transientgrade IV lymphopenia was identified in the 1� 109 cohort,grade IV lymphopenia was considered as a DLT only if itpersisted for more than 4 days. Following dosing of 3subjects in cohort 2 (1 � 109 cfu) without reaching a DLT,one subject was dosed in cohort 3 at 1� 1010 cfu dose. Thissubject experienced a grade II cytokine release syndromerequiring aggressive fluid resuscitation. Because of thenature of the event, this was considered a DLT and thedose level was considered too toxic for further recruitment.The MTD was determined to be 1 � 109 cfu. To bettercharacterize toxicities, additional subjects were enrolledinto cohort 2 (1 � 109 cfu, n ¼ 3 þ 3 ¼ 6). Manageablehypotension and modest elevations in liver function tests(LFT) were observed. An intermediate dose of 3 � 108 cfuwas also added. Four subjects were enrolled into this cohortbefore the study was terminated. Manageable hypotensionand transient LFT elevations (3 of 4 subjects) was alsoobserved at the new dose level. One subject had elevationsup to 11 � ULN after a second dose in the absence of anychanges in bilirubin. The LFTs improved without interven-tion. The subject was not redosed.In general, subjects treated at all dose levels experienced

symptoms that might be expected from a cytokine release–like syndrome from a bacteremia. While not all subjectsexperienced the same events, a constellation of symptomswas common. Lmwas administered intravenously over a 2-hour period. Subjects typically had a temperature peak at 2to 4 hours, sometimes associated with rigors, nausea, head-aches, dehydration, and dry mouth. Mild hypotension wasself-correcting or corrected with intravenous fluids. Themost consistent laboratory abnormalities were transient,self-correcting electrolyte abnormalities, and lymphopeniaswith nadirs at 4 hours postinfusion. The degree of lympho-penia was dose-dependent and the most significant hypo-tension occurred at the highest dose level. The transient,self-correcting nature suggests that these abnormalities arethe result of electrolytes and lymphocytes transiently shift-ing out of the blood compartment. Overall review of thesafety data from the trial did not identify any significanttoxicity with Lm or Lm-mesothelin that was not reversible orunexpected from either previous studies in cynomologusmonkeys or based on mechanism of action.

Shedding and clearanceANZ-100 was not detected in the blood, stool, urine, or

sputumspecimens collected at any timepoint. Lm suspectedto be CRS-207 was detected in blood cultures of 4 subjects.In 2 of the subjects, the cultures were negative by 24 hours.An ovarian cancer subject who received a dose of 1� 108 cfuhad positive cultures at 4 and 24 hours after the firstintravenous infusion. Subsequent blood cultures 4 daysafter dosing were negative. Blood cultures taken after hersecond infusion were negative. Lm suspected to be CRS-207was detected in blood cultures of a mesothelioma subject

receiving 1 � 109 cfu at day 4 after the second intravenousinfusion. Subsequent cultures were negative. All remainingblood, stool, or urine specimens collected throughout thestudy for all subjects were negative.

ImmunohistochemistryMesothelin membranous staining was detected in all 7

available archived samples for subjects enrolled into theCRS-207 study. The extent of staining is listed in Table 1.

PBMC phenotype analysis after ANZ-100administration

Peripheral blood was analyzed prior to treatment withANZ-100, daily for 5 days following treatment, and thenweekly for 1month. Immune activation was determined byphenotypic analysis of NK cells (CD3�CD16/56þ). Inter-estingly, there was a transient reduction in peripheral lym-phocyte andNK cell numbers (Fig. 1A and B) that reached anadir at day 2 following treatment, suggesting the possibil-ity that ANZ-100–induced activation results in lymphocyteandNK cellmargination from the peripheral blood to othercompartments. A significant upregulation of the activationmarker CD38 was noted on NK cells for all dose levels(P¼ 0.0008; Fig. 1C). There appears to be a dose-dependenttrend but it was not statistically significant (P ¼ 0.1238).This level increased at the 96-hour time point as shown forone patient (Fig. 1D).

Cytokine/chemokine induction after ANZ-100 andCRS-207 administration

Serum samples were collected prior to and at 2 hours, 6hours, and daily for 5 days following ANZ-100 adminis-tration and analyzed for the presence of MCP-1, MIP-1a,MIP-1b, LT-a, IP-10, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-10, IL-12p70, IFN-g , and TNF-a. At the highest doselevel of 3� 108 cfu, a significant induction of cytokines andchemokines such as MCP-1 and MIP-1b was observed(P ¼ 0.0006 and 0.0002, respectively; Fig. 2A and B). Theresponse peaked at 2 hours after the completion of the2-hour intravenous ANZ-100 infusion and returned tobaseline within 48 to 72 hours. Subjects at the highest doselevel also had a consistent induction of the TH1 cytokinesIFN-g and IL-12p70 (Fig. 2C and D). The upregulation ofthe CD38 activation marker suggests biologic activity ofANZ-100 at doses as low as 1 � 106 cfu. However, a moreconsistent induction of proinflammatory cytokines andchemokines was observed in subjects receiving the higherdose level of 3 � 108 cfu.

In the CRS-207 study, chemokine induction was notedfor all dose levels including the starting dose level of 1� 108cfu. For MCP-1, MIP-1b, and IP-10, the pattern was con-sistently elevated for all dose levels (P¼ 0.0050, P < 0.0001,and P < 0.0001, respectively) and did not vary significantlyacross time points (Fig. 3A–C). The degree of upregulationis less than that observed in the ANZ-100 study, whichmaybedue to the fact that the levelswere taken at 24hours ratherthan at the expected 2-hour postinfusion peak (Fig. 3A–C).The induction of IL-10 (P ¼ 0.049), IL-12p70 (P ¼ 0.100),






IL-6 (P ¼ 0.059), and TNF-a (P ¼ 0.092) are higher in the1 � 109 cfu dose level than in the 3 � 108 dose level (Fig.3D). There was no significant difference in the inductionlevels for IFN-g (P¼ 0.375) or IL-8 (P¼ 0.171). The data forthe single subject with a dose of 1� 1010 cfu are included inthe plot for reference.

Detection of LLO-specific and mesothelin-specificT-cell responses after CRS-207 administration

LLO-specific T-cell responses were analyzed in 8 subjectswith viable samples pretreatment and after the second andfourth infusion of CRS-207 (Fig. 4A). Final T-cell responsesare reported. Six of the subjects were positive for vaccine-induced Lm-specific responses. The CEF-specific responsesare provided in Supplementary Fig. S3. In the CRS-207study, mesothelin-specific CD8þ T-cell responses wereinduced in 6 of the 10 evaluable subjects (Fig. 4B).

Efficacy and survivalWhile the CRS-207 study enrolled subjects with multiple

disease types andwas not powered to assess survival, 37%ofthis phase I patient population survived for �15 months,with 3 subjects alive as ofOctober 14, 2010 (Table 2).Of the6 long-term survivors, 3 had PDA, 2 had NSCLC, and 1 hadmesothelioma. These 6 subjects had prior immunotherapyor subsequent local radiation. Five of 6 subjects received all4 doses of CRS-207 and all 5 evaluable subjects showedvaccine-induced Lm-specific responses. One subject hadbeen discontinued from study after 1 dose because of aprotocol violation and samples were not collected forimmunologic evaluation. In addition, 4 of the 5 evaluablesubjects among the long-term survivors had stable diseaseby RECIST at day 91 (end of study). Eight of 8 evaluablesubjects in the group that survived

�15 months developed LLO responses. One additionalsubject lived �15 months but was not tested because aposttreatment sample was not collected. Thus, the induc-tion of LLO-specific T-cell responses may serve as a bio-marker of immunocompetency in future studies. In thissmall subset of 10 subjects withmultiple histologic types ofcancer, the induction of mesothelin-specific responses didnot correlate with survival. The induction of mesothelin-specific T-cell responses as amarker of response to CRS-207requires further investigation in a larger study of morehomogenous subjects. These data provide the rationale forfurther evaluation of this Lm-mesothelin vaccine in a phaseII study.

Discussion

These data from the phase I studies of ANZ-100 and CRS-207 Lm vaccines support the following conclusions. First,both vaccines are safe and tolerable in subjects withadvanced, treatment-refractory cancers at immune activat-ing doses. Second, there is a dose-dependent augmentationof systemic cytokine and chemokine responses that mayserve as biomarkers of Lm vaccine bioactivity. Finally, a

tumor antigen–modified Lm can induce tumor antigen–specific T-cell responses in subjectswith advanced cancer. Assuch, Lm vaccine responses require further evaluation as acandidate biomarker of improved clinical outcomes.

These studies support that an attenuated bacteria can begiven safely to subjects with advanced cancer with transientside effects. This is inmarked contrast tomany conventionaloptions in which the toxicities can be cumulative andimpairment in quality of life have to be weighed againstpotential benefit. Defining the tolerability of these con-structs as single agents lays important groundwork forfuture studies in which these vaccines will be used incombinations. There are a number of unpublished preclin-ical studies testing Lm vaccines in combination with eitherother vaccine constructs or immune-modifying agentswhich show enhanced efficacy of the combination. A clin-ical trial has recently opened to enrollment testing CRS-207in combination with an allogeneic GM-CSF–secreting PDAvaccine in subjects with advanced PDA. The study concept isbased on mouse models which show that the combinationof the GM-CSF and Lm-based vaccines in a heterologousprime/boost regimen results in the induction of antigen-specific T-cell responses of greater magnitude than either

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agent alone and correlates with superior antitumorresponses. Interestingly, all 3 PDA subjects on the CRS-207 study who lived �15 months had received prior GM-CSF vaccine therapy.

With both biologic and targeted agents, dose selectioncan be complex as the usual drug development philosophyof using the MTDmay not be relevant. The maximum dosemay not be the most biologically effective dose. There does

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Figure 3. CRS-207 induction ofchemokines/cytokines is presentat all dose levels tested. A, MCP-1chemokine expression presentedby cohort dose level after dose 1and 2. B, MIP-1>b chemokineexpression presented by cohortdose level after dose 1 and 2. C,IPhemokine expression presentedby cohort dose level after dose 1and 2. D, cytokine expression 24hours after first dose of CRS-resented by cohort dose level. Thedata for the single subject with adose of 1� 1010 cfu are included inthe plot for reference.<

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Figure 4. CRS-207 induces both LLO-specific and mesothelin-specific T-cell responses. A, LLO-specific T-cell responses were analyzed using IFN-gELISPOT. B, mesothelin-specific T-cell responses were analyzed using IFN-g ELISPOT. �, T-cell responses were considered positive when the frequency ofspecific responses were �1 in 105 PBMCs (LLO ELISPOT) or CD8þ PBL (mesothelin ELISPOT) and increased by at least 2-fold compared with baseline.Final T-cell responses are reported. The maximal response to a single best peptide is reported. The boxed patient identification numbers representsubjects who survived �15 months.

Le et al.





appear to be a dose-dependent augmentation of cytokineand chemokine responses. However, it remains unknownwhether there is a dose-dependent induction of T-cellresponses. Importantly, these studies show not only safetybut also immune activity in the range of doses selected fortesting. Of note, a phase I study of a different Lm-basedvaccine has been reported (28). This study evaluatedLm-LLO-E7, a live-attenuated Lm that secretes the HPV-E16 E7 antigen fused to LLO, in subjects with previouslytreated cervical carcinoma and reported a similar adverseevent profile and similar dose range for the MTD.The transient transaminitis cases were expected on the

basis of mechanism of action and preclinical studies.Another example of transaminitis in the context of immu-notherapy is the flares in chronic hepatitis B patientsinduced by Peg-IFN-a-2b (29). Interestingly, host-induced flares which were followed by HBV DNAdecreases were highly associated with response. Theseflares are thought to be due to the stimulatory effect ofIFN, which is capable of increasing T-cell cytolytic activityand NK cell function. Likewise, with Lm-based therapies,the transaminitis is likely to be inflammatory in natureand not necessarily a negative finding. This will be mon-itored in future studies.With the recent approval of Provenge for the treatment of

metastatic castrate-resistant prostate cancer, there is mixedenthusiasm and continued skepticism about vaccination asa treatment for cancer. Provenge has been shown to prolong

survival without evidence of appreciable RECIST responseor prolongation of time to progression (30). In addition,another recently approved immunotherapy, ipilimumab,an antagonist antibody to cytotoxic T-lymphocyte–associ-ated-4 (CTLA-4), has also shown a survival benefit inmelanoma despite 5.7% to 10.9% response rates (31). Inthese studies, some subjects show increases in tumor vol-ume before a delayed response and therefore response rateis likely to underestimate the activity of these agents. There-fore, OS is currently the best endpoint to evaluate immu-notherapeutic agents in advanced cancer.While the survivaldata presented here are only hypothesis generating, it isprovocative.

Conclusions

In summary, Lm-based vaccines, ANZ-100 and CRS-207,arewell tolerated in subjects with advanced cancers. There isencouraging evidence of immune activation and potentialclinical benefit, thus warranting further clinical studies.

Disclosure of Potential Conflicts of Interest

Under licensing agreements betweenAduroBioTech, Inc. (Aduro) and theJHU, E.M. Jaffee and JHU have the potential to receive royalties received onsales of products/technology described in this article. The terms of thisarrangement are being managed by JHU in accordance with its conflict ofinterest policies. D.G. Brockstedt, A.L. Murphy, and T.W. Dubensky haveownership interests in Aduro. T.W. Dubensky is a member of the scientificadvisory board of Aduro. J.E. Eiden has ownership interests in Cerus

Table 2. CRS-207 clinical and immune response summary

ID Cancer Dose level LLO response Mesothelin response RECIST Survival, mo Prior immunotherapy

�15 mo OS001-001 PDA 108 Yes No PD 15 GVAX001-002 PDA 108 Yes NE SD 30þ GVAX003-001 Mesothelioma 108 Yes Yes SD 29þ IFN-b gene transfera005-002 NSCLC 3 � 108 NE NE NE 23 No004-002 PDA 109 Yes No SD 17 GVAX005-001 NSCLC 109 Yes Yes SD 26þ No

Corporation. P. Illei is a consultant for Leica Microsytem. No potentialconflicts of interests were disclosed by other authors.

Grant Support

D.T. Le has received American Society of Clinical Oncology CareerDevelopment Award and NIH/GI SPORE (2P50 CA062924). E. Lutz hasreceived AACR-FNAB Fellows Grant for Translational Pancreatic CancerResearch and Anti-Cancer Drug Development Fellowship (NIH/NCI T32CA009243). E.M. Jaffee has obtained grant from NIH/GI SPORE (2P50

CA062924). D.A. Laheru has obtained support from NIH/GI SPORE (2P50CA062924), NCI K23 CA093566-01A1, and The Viragh Family Foundation.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received August 22, 2011; revised November 11, 2011; acceptedNovember 26, 2011; published OnlineFirst December 6, 2011.

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2012;18:858-868. Published OnlineFirst December 6, 2011.Clin Cancer Res Dung T. Le, Dirk G. Brockstedt, Ran Nir-Paz, et al. Cancers: Phase I Studies of Safety and Immune InductionListeria Vaccine Expressing Mesothelin (CRS-207) for Advanced A Live-Attenuated Listeria Vaccine (ANZ-100) and a Live-Attenuated

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