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Clinical Trials: Immunotherapy

Phase I Immunotherapy Trial with Two ChimericHER-2 B-Cell Peptide Vaccines Emulsified inMontanide ISA 720VG and Nor-MDP Adjuvant inPatients with Advanced Solid TumorsTanios Bekaii-Saab1, Robert Wesolowski2,3, Daniel H. Ahn1, Christina Wu4,Amir Mortazavi2,3, Maryam Lustberg2,3, Bhuvaneswari Ramaswamy2,3,Jeffrey Fowler5, Lai Wei3,6, Jay Overholser3,5, and Pravin T.P. Kaumaya3,5

Abstract

Purpose: This first-in-human phase I study (NCT01417546) evaluated the safety profile, optimal immunologic/biological dose (OID/OBD), and immunogenicity of thecombination of two peptide B-cell epitope vaccines engi-neered to represent the trastuzumab- and pertuzumab-binding sites. Although trastuzumab and pertuzumabhave been approved for clinical use, patients oftendevelop resistance to these therapies. We have advanceda new paradigm in immunotherapy that focuses onhumoral responses based on conformational B-cell epi-tope vaccines.

Patients and Methods: The vaccine is comprised oftwo chimeric HER-2 B-cell peptide vaccines incorporatinga "promiscuous T-cell epitope." Patients were immunizedwith the vaccine constructs emulsified with nor-muramyl-dipeptide adjuvant in a water-in-oil Montanide ISA 720VG

vehicle. Eligible patientswithmetastatic and/or recurrent solidtumors received three inoculations every 3 weeks.

Results: Forty-nine patientswith amedian of 4 prior lines ofchemotherapy received at least 1 vaccination. Twenty-eightpatients completed the 3 vaccination regimens. Six patientsreceived 1 six-month boost after the regimen, and one patientreceived 7 six-month boosts. No serious adverse reactions ordose-limiting toxicities were observed. The vaccine was welltolerated with dose level 2 as the recommended phase IIdose. The most common related toxicity in all patients wasinjection-site reactions (24%). Two patients had a partialresponse, 14 had stable disease, and 19 had progressive disease.

Conclusions: The study vaccine is safe, exhibits antitu-mor activity, and shows preliminary indication that peptidevaccination may avoid therapeutic resistance and offer apromising alternative to monoclonal antibody therapies.

IntroductionHER-2 is a transmembrane receptor that is overexpressed in

multiple epithelial tumors, including subsets of breast, gastro-esophageal, esophageal, endometrial, uterine, ovarian, colorectal,and lung cancers (1–6). HER-2 is associated with more aggressiveforms of cancer (7), an increased risk of metastasis, increasedtumor invasion, and decreased overall survival (8, 9). Therefore,HER-2 is a key therapeutic target in several cancers. Trastuzumab(Herceptin; Genentech) was the first humanized monoclonal

antibody targeting HER-2 in combination with chemotherapyto be approved for clinical use in patients with metastatic HER-2–overexpressing breast cancer (10–17). Despite the benefitobserved from trastuzumab, approximately one third ofpatients with metastatic, HER-2–positive breast cancer experi-ence primary resistance (18), and most responding patientseventually develop acquired resistance within 1 year of thera-py (19). Since 2007, four additional HER-2–targeted therapies(lapatinib, neratinib, pertuzumab, and T-DM1) have beenapproved by the FDA for the treatment of breast cancer. Studiesinvestigating novel agents and combination therapies withanti–HER-2–directed therapy are also under investigation forsolid tumor malignancies. A recent phase III clinical trialshowed that the addition of pertuzumab, a recombinanthumanized monoclonal antibody that blocks the heterodimer-ization of HER-2 with other HER family members, to docetaxeland trastuzumab in patients with untreated HER-2–overexpres-sing breast cancer, resulted in improvement in progression-freeand overall survival from 12.4 to 18.5 months and from 40.8 to56.5 months, respectively (20–22).

To date, most HER-2 peptide cancer vaccine strategieshave sought to induce a cellular antigen–specific T-cellresponse (23, 24). CD8þ and CD4þ T-cell vaccines are humanleukocyte antigen (HLA)–restricted, which limits their universalapplicability, and therefore, they may need to be tailored to thespecific subtype of cancer and tumor antigen expression level to

1Department of Internal Medicine, MayoClinic, Phoenix, Arizona. 2Department ofInternal Medicine, Division of Medical Oncology, The Ohio State University,Columbus, Ohio. 3Arthur G. James Cancer Hospital/Comprehensive CancerCenter, Columbus, Ohio. 4Department of Hematology and Medical Oncology,Emory University School of Medicine, Atlanta, Georgia. 5Department of Obstet-rics and Gynecology, The Ohio State University, Columbus, Ohio. 6Center forBiostatistics, The Ohio State University, Columbus, Ohio.

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

Corresponding Author: Pravin T.P. Kaumaya, Ohio State University, Suite 316,420W. 12th Avenue, Columbus, OH 43210. Phone: 614-292-7028; Fax: 614-688-8586; E-mail: [email protected]

Clin Cancer Res 2019;25:3495–507

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achievemaximumeffectiveness. Approaches aimed at stimulatinghumoral immunity specific to HER-2 may provide an advantageover T-cell–directed therapies. For instance, in contrast to T-cellreceptor/antigen interactions, epitopes tomonoclonal antibodiesare not required to be presented on specific isoforms of majorhistocompatibility complex (MHC), a process that can beimpaired in some cancers (25). In addition, inducing B-cell–mediated immunity could enable the host to generate antibodiespotentially capable of functioning as "endogenous" trastuzumaband pertuzumab. However, unlike passive chimeric antibodytherapy which is expensive, associated with clinically significanttoxicities, and requires repeated treatment, a peptide vaccine hasthe potential to result in sustained humoral immunity.

We have advanced a new paradigm in immunotherapy thatfocuses on humoral responses based on conformational B-cellepitope vaccines. These novel platforms elicit high-affinityantipeptide antibodies against tumors that help circumventintrinsic drug resistance. Importantly, this is hypothesized toprovide durable treatment effects due to immunologic mem-ory (26). We have previously identified the first generation ofHER-2 B-cell peptide epitopes (628–647) and (316–339)through computer immunogenicity algorithms and extensivein vitro and in vivo preclinical studies (27, 28). In a recentlypublished phase I clinical trial, we showed that the combina-tion of the two chimeric HER-2 vaccines in patients withmetastatic solid tumors was safe, demonstrated activity (diseasecontrol rate of 24%), and elicited HER-2–specific humoralresponses in 62.5% of patients (29).

Our research group has developed two novel B-cell epitope–specific vaccines consisting of epitopes derived from the extra-cellular domain of the HER-2/neu molecule that are bindingsites of trastuzumab and pertuzumab. Using the X-ray struc-tures of the HER-2–trastuzumab and HER-2–pertuzumab com-plexes (30–32), we have rationally designed the trastuzumab-binding epitope (597–626) and the pertuzumab-binding epi-tope (266–296; refs. 33, 34). A series of six conformationalpeptides spanning residues 563–626 (trastuzumab-binding

site) and 266–333 (pertuzumab-binding site) were engineered,synthesized, and characterized to mimic the trastuzumab- andpertuzumab-binding sites and were tested for immunogenicityin mice and rabbits (33, 34). The highest affinity and titerantibody responses were seen with epitope 266–296 located indomain II (pertuzumab-binding site) and 597–626 located indomain IV (trastuzumab-binding site). Thus, the vaccine wasexpected to stimulate patients' immune systems to elicit apolyclonal antibody response to HER-2 and in particular tosimulate trastuzumab and pertuzumab.

Our translational studies show that active immunotherapywith our peptide vaccine led to the generation of HER-2/neu–specific B lymphocytes that had the ability to inhibit HER-2signaling while inducing potent antitumor humoral immuneresponse against HER-2–positive cells, as confirmed in priorpreclinical in vitro and in vivo experiments (26, 28, 35). Thefindings demonstrated that these peptides were recognized bytrastuzumab and pertuzumab, elicited a strong sustained humor-al and cell-mediated immunity, interfered with HER-2 signaling,and led to antitumor effects in preclinical models (33, 34).Additionally, these peptides inhibited multiple signaling path-ways, including HER-2–specific inhibition of cellular pro-liferation and cytoplasmic receptor domain phosphorylation.The peptide antibodies mediated antibody-dependent cellularcytotoxicity (ADCC). These vaccines had statistically reducedtumor onset in two transplantable tumor models (FVB/n andBALB/c) and led to significant reduction in tumor developmentin two transgenic mouse tumor models (BALB-neuT andVEGFþ/�Neu2–5þ/�; refs. 33, 34). Lastly, these 2 vaccines werecapable of generating antibodies that exhibited propertiessimilar to trastuzumab and pertuzumab, validating their usein this phase I clinical trial.

Overall, immunotherapy using cancer vaccines is an excitingand rapidly evolving field in oncology that leverages patients'immune systems to target cancer. Chimeric B-cell epitope peptidevaccines incorporating a "promiscuous" T-cell epitope offer anattractive immunotherapeutic option in the treatment of cancer,with considerable advantages in their safety, ease ofmanufacture,and administration. Additional advantages of B-cell cancervaccines are exquisite specificity, the potential for a durable treat-ment effect due to immunologic memory, and universal coveragebypassing HLA restriction. Herein, we report the results fromthe first-in-human, dose escalation portion of the phase I studytesting the combination of two peptide B-cell epitope vaccines,MVF-HER-2 (597–626) and MVF-HER-2 (266–296), incorporat-ing a promiscuous measles virus (MVF) T-cell epitope.

Patients and MethodsObjectives

The primary objectives were to assess the safety and clinicaltoxicity of immunization, determine the optimum immunologic/biological dose (OID/OBD) of combination HER-2 vaccines,measure both humoral and cellular immune responses, includingthe specificity, class and kinetics of anti–HER-2 peptide, andevaluate whether the combination of HER-2 vaccines demon-strates therapeutic benefit, provides synergistic and/or additiveeffects, and to enumerate mechanisms of action. Secondaryobjectives were to collect and analyze postimmune sera andperipheral blood cells for an additional 6 months following thelast injection and document clinical responses.

Translational Relevance

This is the first-in-human dose escalation study of thecombination of two HER-2 B-cell peptide vaccines to safelydeliver curative and transformative cancer immunotherapiesto advanced cancer patients.We have created and established anovel chimeric B-cell peptide vaccine eliciting antibodies withhigh immunogenicity that binds with high specificity to nativehuman HER-2, demonstrates promising antitumor activity,and shows preliminary indication that peptide vaccinationmay avoid therapeutic resistance and offer a promising safealternative to monoclonal antibody therapies. Continuousdevelopment of the vaccine is ongoing in a phase II trial atthe suggested optimum biological dose in a less heavilypretreated patient population in breast and/or gastrointestinalmalignancies with HER-2/EGFR overexpression. B-cell cancervaccines have the advantage of producing specific immuneresponses that can potentially induce memory B- and T-cellresponses. We need to await the results of a future randomizedstudy before definitive conclusions about the vaccine's efficacycan be made.

Bekaii-Saab et al.

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PatientsEligible patients were required to have the following: (i) met-

astatic, incurable solid tumor malignancy, at least 3 weeks pastany prior surgery, cytotoxic chemotherapy, other immunotherapy,hormonal therapy, or radiotherapy; (ii) adequate end organ func-tion; and (iii) Eastern Cooperative Oncology Group performancestatus of 0–2. Subjects with history of properly treated and stablebrainmetastases were also eligible as long as there was no evidenceof CNS progression within 3 months prior to the first dose of thestudy vaccine. Patients were excluded if they had a significantconcurrent illness; left ventricular ejection fractionof<50%, uncon-trolled or severe cardiac disease; active viral hepatitis, HIV, or otheractive infections requiring use of antibiotics; active autoimmunedisease; or corticosteroid requirement. Baseline disease assessmentwas performed by physical examination, medical history, chestX-ray, and computed tomography (CT) scans.

The clinical trial was conducted under an investigational newdrug application (IND# 14633, PKaumaya) approved by theFDA. The study protocol (2010C0075) was approved by TheOhio State University Cancer Institutional Review Board. Allpatients gave written informed consent prior to participationin the study (NCT01376505). The study was conducted inaccordance with ethical principles founded in The CommonRule, the Belmont Report, Good Clinical Practice guidelines,and applicable local laws.

Peptide selection, manufacturing, and vaccine preparationThe rationale, selection, design, synthesis, and character-

ization of the two peptide constructs were originally describedby the Kaumaya laboratory (33, 34). The GMP peptides werepurchased from Peptisynthia and acquired by the SolwayGroup.N-acetyl-glucosamine-3yl-acetyl-L-alanyl-D-isoglutamine(nor-MDP) was purchased from Peninsula Labs. The GMP pep-tides met all the FDA and U.S. Pharmacopeia requirements forsterility (i.e., bacterial/fungal), endotoxins, and potency. The bulkpeptides were supplied to University of Iowa Pharmaceuticalsmanufacturing facility (Iowa City, Iowa) for sterile vialing in 3mglots. Endotoxin levels of these peptides were tested and deter-mined to be within acceptable levels as Good ManufacturingPractice (GMP) grade. The vehicle Montanide ISA 720 was pur-chased from SEPPIC, and it had an approval certificate of analysesfor toxicity, emulsifying property, and sterility. The immunoge-nicity of each individual peptide and combination was verified inpairs of New Zealand rabbits. The combination vaccine was pre-pared by mixing the two chimeric MVF-B-cell epitope peptidesthat correspond to amino acid sequences 266–296 (LHCPALV-TYNTDTFESMPNPEGRYTPGASCV: pertuzumab-binding site)and 597–626 (VARCPSGVKPDLSYMPIWKFPDEEGACQPL: tras-tuzumab-binding site). The chimeric constructs were synthesizedwith theMVF sequenceKLLSLIKGVIVHRLEGVEat theN-terminusvia a linker consisting of GPSL. On the day of vaccination for eachdose level, the appropriate peptide concentration was preparedby dissolving with n-MDP (N-acetyl-glucosamine-3yl-acetyl-L-alanyl-D-isoglutamine) adjuvant (0.025 mg) in a total volumeof 1.0 mL. This solution was emulsified with 1.0 mL saline-oilphase vehicle (Montanide ISA 720; SEPPIC Inc., a subsidiary ofAir Liquid group, Paris, France).

Immunization scheduleAll eligible patients underwent a skin test prior to the first dose

of the study vaccine. Approximately 0.1 mL of each peptide was

injected intradermally in two separate areas of the forearm.Patients were subsequently examined for delayed-type hypersen-sitivity to the test dose approximately 20 minutes after adminis-tration (>20 mm erythema or development of a wheal). Patientswho did not develop hypersensitivity reaction to the test dosewere to receive 3 intramuscular vaccinations, each given 21 daysapart (days 1, 22, and 43). The vaccine was injected into thegluteus maximus muscle, with subsequent injections given in themuscle contralateral to the prior vaccine administration site. Nopremedications were required but treating physicians had anoption of ordering them per their discretion. Following threedoses, patients with responding or stable disease had an option ofreturning to receive booster vaccinations at 6-month intervals.

Study design and treatmentThe dose escalation part of this clinical trial used a 3 þ 3

schema. Initially, 3 patients were to be treated at each dose level(DL) and observed for a minimum of 4 weeks. If 0–1 patientsexperienced a dose-limiting toxicity (DLT), an additional 3patients were entered at that DL and were observed for a mini-mumof 4 weeks. Dose escalation could proceed only after at least6 evaluable patients in a cohortwere observed for aminimumof 4weeks, and if only 0–1 of the 6 patients in that DL experienced aDLT. If 2 or more of the 6 patients at a DL experienced a DLT,additional enrollment at that DL was terminated, and the pre-vious DL was defined as the maximum tolerable dose (MTD).

At least 6 patients at eachDLwere required to receive a total of 3consecutive inoculations of the combination vaccines at 3-weekintervals in order to better evaluate for the OID/OBD. The DLswere 1.0 mg (DL1), 1.5 mg (DL2), 2.0 mg (DL3), and 2.5 mg(DL4) of each peptide. Each dose contained 0.025 mg of n-MDP.

Definition of DLTAll toxicities were graded based on Common Terminology

Criteria for Adverse Events (CTCAE) version 4.0. The DLT periodwas defined as the first 4 weeks after the first administration of thestudy vaccine. In order to be evaluable for DLTs, patients wererequired to receive at least 2 doses of the study vaccine. DLTs wereadverse events that were at least possibly related to study therapyanddefined as follows: (i) Any grade 3or greater toxicity includingflu-like symptoms; (ii) any grade 3–4 neutropenia lasting morethan 5 days or accompanied by � grade 2 fever, or any grade4 thrombocytopenia; (iii) clinical inability (due to toxicity) tostart next cycle of treatment within 3 weeks of planned start date;(iv) any grade 3 injection-site reaction, defined as an abscessformation or cellulitis requiring antibiotics or surgical proceduresuch as incision and drainage or injection-site reaction severeenough to require narcotic analgesia. Any grade 2 allergic reactionof asymptomatic bronchospasm or generalized urticaria resultedin cessation of vaccination in that individual and was consideredto be dose-limiting only if serious allergic reactions have alsobeen reported at that dose cohort.

Assessment of toxicity and responsePhysical exam was performed at baseline and prior to each

vaccine administration and on day 71 (4 weeks after third immu-nization). Laboratory studies such as complete blood countswith differential, complete metabolic panel and urinalysis werealso obtained. Cardiac function was assessed by history andphysical exam and by Multigated Acquisition Scan (MUGA) atbaseline and prior to booster vaccinations. Troponin I levels

Phase I Immunotherapy Trial with Two B-cell Vaccines

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were checked prior to and following every vaccination tomonitor for theoretical potential cardiotoxic effect of the vac-cine and to ensure that vaccination did not cause cardiotoxicity.

Tumor response was measured by radiologic assessment withCT or magnetic resonance imaging (same measure was usedserially) pretreatment, day 71 and subsequently at the 6 monthsboost. Radiologic assessment was required for patients removedfrom study, unless patients withdrew further participation.Response was measured according to Response EvaluationCriteria in Solid Tumors (RECIST) version 1.1 criteria.

Procurement of patient plasmaBlood was drawn from patients using standard phlebotomy

techniques. Peripheral blood for humoral and antitumorimmune correlative studies was drawn from study patientsinto heparinized tubes prior to (40 mL) and 4 hours after(10 mL) each vaccination as well as on day 71. Blood sampleswere also obtained monthly for patients who received a6 months booster vaccine to monitor levels of antibodies intheir blood samples.

Cell lines, antibodies, and proteinCell culture medium, fetal calf serum, and supplements were

purchased from Invitrogen Life Technologies. The human breasttumor cell line BT-474 (ATCC) was maintained according to thesupplier's guidelines, and all tests were done within 6 months ofreceipt. ATCC authenticates cell lines by observations of recoveryalong with morphologic appearance, testing viability by trypanblue exclusion. Isoenzymology and/or Cytochrome C subunitI (COI) by PCR is performed to confirm species of samples.Human cell lines are tested by STR analyses to determine identity.Lines are also tested for Mycoplasma contamination by PCR.AG825, a selective HER-2/neu kinase inhibitor (Calbiochem),human recombinant HER-2 protein (100 mg, Acrobiosystem andHerceptin (Genentech) was used as a control.

Patient sera purificationThree milliliters crude sera 1:1 diluted with Protein A/G

Binding Buffer (Thermo Scientific) was applied to equilibratedprotein A/G column with 10 mL of Binding Buffer followed bywash with 20 mL of Binding Buffer. Elution buffer (10 mL;Thermo Scientific) was used to elute bound antibodies andseparately collect initial and last 1 mL eluate and middle 8 mLeluate. Eluate (8 mL) was then concentrated and exchanged withphosphate-buffered saline (PBS), and the final volume wasadjusted to 3 mL. Protein concentration was determined with acalibration factor 1.5 using Bradford reagent.

Enzyme-linked immunosorbent assayLevels of anti–HER-2 antibody were determined prior to

immunization, during immunizations, and monthly after thethird and last immunizations. Although the induction of anti-body against the immunizing peptide is an important obser-vation relative to the approach in general, the key determina-tion is whether the immunogen elicits an antibody that bindsHER-2 protein. Because tissue distribution is essential foractivity, it is also important to determine whether the antibodyclass elicited is IgG. The presence of antibodies specific for theHER-2 peptide vaccine in patient serum was directly assessedby using enzyme-linked immunosorbent assay (ELISA), asdescribed previously (27).

Isotyping antibody identification of patients vaccinated withpeptide-based HER-2 vaccine

To identify what subclass of IgG antibodies patients weregenerating against the HER-2 peptides, we used an isotypingELISA. Plates were washed with PBT and incubated at roomtemperature for 1 hour with (i) mouse anti-human antibodiesof different isotypes (anti-IgA, anti-IgD, anti-IgE, anti-IgG, andanti-IgM; Southern Biotech), (ii) anti-human isotyping IgG(type 1, 2, 3, and 4) antibodies conjugated to horseradish per-oxidase (HRP). Absorbance at 410 nm was read in an ELISAreader. The percentage of isotype antibodies in sera was repre-sented by their respective absorbance relative to the total absor-bance by all 5 isotype antibodies. For IgG subtype analysis,mouse anti-human antibodies of different IgG subtypes (anti-IgG1, anti-IgG2, anti-IgG3, and anti-IgG4) acted as the probe forbound sera antibodies

Flow cytometryImmunofluorescence staining of cells to measure binding

of human antibodies after immunization was evaluated as pre-viously described (29). Human HER-2–overexpressing breastcancer BT-474 (1 � 106) cells were incubated with patient serumin 100 mL of 2% FCS/0.1% NaN3 in PBS for 2 hours at 4�C.Preimmune sera were used as a negative control, and trastuzu-mab was used as a positive control. Unbound antibodies wereremoved with PBS, and the cells were incubated with fluoresceinisothiocyanate–conjugated antihuman antibody for 30 minutesat 4�C in 100 mL of 2% FCS in PBS. Cells were washed in PBS andwere fixed in 1% formaldehyde before they were analyzed byCoulter ELITE flow cytometer (Coulter). A total of 10,000 cellswere gated by light-scatter assessment before single-parameterhistograms were drawn and smoothed.

MTT cell proliferation assayThe proliferation assay was performed as previously described

(33, 34) with BT-474 cells (2 � 104 per well) in 96-well, flat-bottom plates overnight. Inhibition percentage was calculated aspreviously described, and trastuzumab was used as a positive con-trol while preimmune sera served as negative controls (29).

HER-2 receptor phosphorylation assayThe HER-2 phosphorylation assay was performed by using

BT-474 cells (1 � 106 per well), as described previously in6-well plates, which were incubated at 37�C overnight (33, 34).Supernatants were collected, and protein concentration of eachsample was measured by Coomassie plus protein assay reagentkit and lysates were stored at �80�C. Phosphorylation wasdetermined as previously described (34) by using the Duoset ICfor human phosphor-ErbB2 (R&D Systems) according to themanufacturer's directions.

ADCCADCC was measured by using the bioluminescence nonradio-

active cytotoxicity assay (aCella-TOX kit; Cell Technology)according to the manufacturer's recommendations as previouslydescribed (29). Briefly, BT-474 target cells (1 � 104/well) wereplaced in a 96-well plate, and patient antibodies (50 mg) andtrastuzumab (50 mg) as positive control were added to the wellscontaining the BT-474 target cells. The plate was incubated at37�C for 15 minutes to allow opsonization of patient antibodiesto occur. Human peripheral blood mononuclear cells (hPBMC

Bekaii-Saab et al.





from Red Cross) as effector cells were then added to the wells atthree different effector-to-target (E:T) ratios (20:1, 10:1, and 5:1)and the plate incubated at 37�C for 3 hours. Normal human IgGsserved as negative controls. Lytic agent (10 mL) was added to thecontrol wells for maximum lysis and incubated for 15 minutes atroom temperature followed by the addition of 100 mL of theEnzyme Assay reagent containing G3P to all wells. The detectionreagent was added, and the plate was immediately read using anilluminometer.

Caspase activity assay for apoptosisApoptosis was measured using the caspase activity assay

(Caspase-GLO; Promega) per manufacturer's instructions as pre-viously described (36). Apoptosis was evaluated by measuringthe amount of luminescence (as readout of caspase activity)using a luminometer. The percentage of increased release ofcaspases was calculated using the formula (ODUNTREATED �ODTREATED)/ODUNTREATED � 100. All experimental treat-ments were performed in triplicate.

Assessment of optimum immunologic/biological doseFor the purpose of this study, the OID/OBD was defined as the

dose of the vaccine that is capable of inducing strong immuno-genic response in 5 of 6 treated patients in the given DL. Strongimmunogenic response was defined when the optical density(ELISA) was consistently greater than 1.5 across the various timepoints after the third vaccination; that is, where the observedimmunogenicity (antibody titers) reaches a maximum response(titers have plateaued). Analysis of antitumor responses providedadditional criteria for defining the OID/OBD.

Statistical analysisSummary statistics were calculated for patient demographics

and clinical characteristics. The maximum grade for each type oftoxicity was recorded for each patient, and frequency tables wereprovided. P values < 0.05 were considered statistically significant.All analyses were conducted in SAS version 9.4 (SAS Institute).

ResultsPatients and demographics

Between July 2011 and February 2016, 56 patients werescreened. Forty-nine patients were determined to be eligible forthe study and received at least 1 dose of study vaccine (N ¼ 8, 9,20, 12 in cohorts 1–4, respectively; see Supplementary Table S1).The study population consisted of subjects with a wide range ofdifferent malignancies (see Supplementary Table S2 for demo-graphics). Themedian age of patients was 59 (range, 35–81), witha majority of patients having received �4 prior lines of therapy(see Table 1).

Study treatmentWe utilized the cohorts-of-3 rule (see Materials and Methods)

to simultaneously evaluate tolerability and evidence of biologicalactivity at each DL (37). Twenty-eight of the 49 patients (57%)completed the required 3 vaccinations (N¼ 5, 5, 10, 8 in cohorts1–4, respectively). Of those, 6 patients received 1 booster at 6months due to clinical benefit (Table 1), with 1 patient withparotid cancer in DL2 receiving a total of 7 booster vaccinations.He subsequently developed pulmonary nodules which weresuspicious for disease progression and was removed from the

study after receiving study treatment for over 3 years. Commonreasons for discontinuation of therapy in the evaluable patientswere disease progression, grade 3 injection-site reaction, andpatient or physician preference. Patients (N ¼ 21) who did notreceive the required 3 vaccinations were not evaluable for DLTs.The percentage of patients who discontinued due to diseaseprogression includes patients who received any number of thevaccinations (including patientwhohad less than3vaccinations).

Safety and tolerabilityThe vaccine was well tolerated overall, with minimal or no

toxicities in most patients. No DLTs were observed. The mostcommon toxicities at least possibly related to study treatmentwere injection-site reactions (grade 1–2 in 24% of patients).Grade 2 buttock hematoma developed at the site of the injectionin 1 (2%) patient. Additionally, 2% of patients developed grade 2systemic allergic reaction (mild hypotension and diaphoresis).Grade 1 rash also developed in 2% of patients. Common adverseevents that were considered at least possibly related to studytherapy are listed in Table 2.

Response to treatmentFigure 1A displays radiographic response rates. The waterfall

plot illustrates the maximum percentage of tumor reductionfor target lesions in patients with measurable disease,and Fig. 1B shows the swimmers plot of time to response andtime to off study for all 49 patients. Progressive disease (PD) atthe time of the first restaging scans developed in 19 (54%)evaluable patients. Partial response (PR) as the best responsewas observed in 2 (6%) evaluable patients. Stable disease (SD)was observed in 14 (40%) of evaluable patients (Supple-mentary Table S3). We have utilized standard RECIST 1.1criteria to define PR, PD, and SD (38).

Antibody response to peptide vaccine and recombinantHER-2 protein

Antibody responses to the combination vaccines were assessedby ELISA against MVF-HER-2 (266–296), MVF-HER-2 (597–626),and recombinant HER-2 protein. Wemonitored patients' immuneresponses over the course of the trial (data not shown). Patientsera were purified using protein A/G column. Figure 2 showsindividual patient purified antibodies in cohort 2 at 4 weeks afterthird vaccination (5 patients in this cohort completed the 3 vacci-nation regimen). As shown in Fig. 2, panel 7, similar antibodyresponses were elicited in cohort 2 patients (2A–F) against theindividual vaccine immunogen and recombinant HER-2 in therange of 0.3–0.4 absorbance. Two patients (2A and 2F) had anincreased response to the 597–626 epitope. Overall, cohort 2 pati-ents had more durable responses. Antibody responses in the othercohorts were variable: in cohort 1, the antibody responses werequite low; in cohort 3, there were 3 patients who responded withreasonable titers, 2 did not respond, and2 hadmoderate response;in cohort 4 at the highest dose, 1 patient had high response andthe others had low responses (Supplementary Table S4).

Reactivity of peptide antibodies with the native HER-2 receptorAntibodies elicited by peptide vaccination are effective only

if they recognize the native receptor. Direct binding of purifiedpatient antibodies to the native HER-2 receptor was evaluatedby immunofluorescence staining and cytofluorimetric analy-sis (FACS) using HER-2–overexpressing cells BT-474. As






shown in Fig. 2 (panels 1–6), antibodies on day 71 (3Yþ4weeks) for all 6 patients in cohort 2 (2A–2F) showed significantfluorescent shift in binding relative to normal human IgG.Preimmune antibodies were also used as negative control (datanot shown). This observation is similar to the positive control,trastuzumab, although the fluorescent strength is weaker thanthe latter. This result demonstrated that patient antibodyraised against the vaccine can recognize HER-2 receptor–overexpressed tumor cells. Variable results were obtained forcohorts 1, 3, and 4 with less pronounced fluorescent shifts(Supplementary Figs. S1–S3).

Patient 2C received seven 6 months booster vaccinationsPatient 2C traveled to Columbus, Ohio, from Seattle,

Washington, to receive his first vaccine on May 30, 2012, andtwo boosters at 3 weeks apart. The patient returned every6 months through December 10, 2015, to receive the 7 boostervaccinations, spanning 3.5 years. Prior to enrollment in ourstudy, this patient was diagnosed with T2N2bM0 poorly dif-ferentiated carcinoma of the right parotid gland and under-went radical parotidectomy and selective right neck dissection.The surgical pathology revealed 25 lymph nodes positivefor metastatic disease. Interestingly, the tumor also had

Table 1. Summary of patients by cohort

ID# Cohort# of priortherapies Primary cancer

Vaccinations(# of cycles þ# of boosts)

HER-2/EGFRstatus

Previous treatmenttype

Bestresponse

01-01 1 7 Colon 1 �ve/�ve MC PD01-02 2 Anal 2 Unknown MC PD01-03 1 Cartilage 3 þ1 1A/�ve/�ve MC SD01-04 7 Lung 3 þ1 1B/�ve/�ve MC SD01-05 1 Breast 3 þ1 1C/þve/þve MC þ trastuzumab PR01-06 3 Bladder 3 1D/Unknown MC PD01-07 11 Ovarian 3 1E/�ve/þve MC PR01-08 4 Colon 2 1F/�ve/þve MC SD02-01 2 3 Anal 1 Unknown MC PD02-02 5 Colon 2 �ve/þve MC PD02-03 3 Bladder 1 �ve/þve MC SD02-04 2 Anus 3 þ1 2A/�ve/þve MC SD02-05 3 Colon 2 2B/�ve/þve MC PD02-06 0 Parotid 3 þ7 2C/þve Radiation only NE02-07 3 Colon 3 þ1 2D/�ve/þve MC SD02-08 3 Breast 3 2E/þve/þve MC þ trastuzumab PD02-09 8 Colon 3 2F/Unknown MC SD03-01 3 5 Rectal 3 3A/�ve MC SD03-02 5 Breast 2 þve MC þ trastuzumab NE03-03 2 Colon 3 Unknown MC PD03-04 4 Colon 3 �ve/þve MC SD03-05 5 Ovarian 1 Unknown MC NE03-06 5 Lung 2 Unknown MC SD03-07 4 Lung 2 Unknown MC SD03-08 4 Peritoneal 3 3B/þve/�ve MC þ trastuzumab; Perjeta SD03-09 2 Ovarian 2 �ve MC NE03-10 8 Colon 3 3C/Unknown MC PD03-11 5 Cervical 2 Unknown MC NE03-13 4 Colon 2 Unknown MC PD03-14 5 Lung 3 Not tested MC NE03-15 3 Esophageal 1 �ve MC PD03-16 8 Ovarian 2 �ve/þve Trastuzumab NE03-18 2 Colon 3 Unknown MC NE03-19 6 Rectal 3 3D/�ve MC SD03-20 7 Breast 3 3E/þve MC þ cetuximab SD03-24 7 Rectal 3 �ve/�ve MC PD03-26 5 Breast 2 þve MC/trastuzumab; Perjeta/Kadcyla NE04-01 4 3 Breast 3 þve MC þ trastuzumab; Perjeta PD04-02 2 Ovarian 1 unknown MC NE04-03 3 Esophageal 3 4A/þve/U MC þ trastuzumab PD04-04 5 Ovarian 3 4B/�ve MC PD04-06 7 Ovarian 2 Unknown MC NE04-07 1 Lung 3 4D/U/�ve MC PD04-08 9 Ovarian 3 U MC NE04-10 8 Colon 3 4E/�ve/�ve MC PD04-12 4 Rectum 2 4C/�ve MC PD04-13 1 Breast 3 Unknown MC PD04-14 4 Rectal 2 Unknown MC NE04-15 1 Breast 3 4F/�ve MC NE

NOTE: Evaluable patients who completed all three vaccinations are highlighted in bold.Abbreviations: MC, multiple chemotherapy treatments; NE, nonevaluable; PD, progressive disease; PR, partial response; SD, stable disease;�ve¼ negative;þve¼positive. We have utilized standard RECIST 1.1 criteria to define PR, PD, and SD (37).

Bekaii-Saab et al.





"patchy foci" of 3þ Her2/neu staining based on IHC on abackground of equivocal 2þ positivity. Recurrence was dis-covered in the right axillary lymph nodes about 2 years later

and the patient underwent right axillary lymph node dis-section, which revealed that four of 46 lymph nodes werepositive. He was subsequently enrolled in our study. The excep-tional response in this patient may also be due to the factthat this patient did not receive any chemotherapy prior toenrollment. Thus, his immune system was very much intactand likely more responsive to vaccine treatment. This is animportant fact as the vaccine therapy should be more effectivein such patients.

Figure 3F shows the immune responses of purified antibo-dies at various intervals. Significant antibody levels weredetected to recombinant HER-2, MVF-HER-2 (266–296), andMVF-HER-2 (597–626) after the first boost (3 weeks aftersecond vaccination, 2Yþ3) and subsequent 6 months boost.These results demonstrate that patient 2C was able to elicitantipeptide antibodies that recognized the recombinant HER-2protein. In parallel studies, immunofluorescence staining andfluorescence cytofluorimetric analysis were used to confirm therelative binding affinities of the patient sample antibodies(Fig. 3A–E) using BT-474 cancer cells.

Vaccine elicits predominantly IgG (IgG3 and IgG4)antipeptide antibodies

Patients in cohort 2 elicited predominantly antibodies ofthe IgG isotypes (Fig. 4A and B) to both vaccines MVF-HER-2(266–296) and MVF-HER-2 (597–626). The amount of IgA,

Table 2. Treatment-related toxicities in all 49 patients

Toxicity (number of patients %) Grades 1–2 Grades 3–4 Total

Lymph node pain 1 (2%) 0 (0%) 1 (2%)Fatigue 4 (8%) 0 (0%) 4 (8%)Fever 2 (4%) 0 (0%) 2 (4%)Flu-like symptoms 1 (2%) 0 (0%) 1 (2%)Injection-site reaction 12 (24%) 0 (0%) 12 (24%)Allergic reaction 1 (2%) 0 (0%) 1 (2%)Alanine transaminase elevation 1 (2%) 0 (0%) 1 (2%)Alkaline phosphatase elevation 1 (2%) 0 (0%) 1 (2%)Lymphopenia 2 (4%) 0 (0%) 2 (4%)Leukopenia 1 (2%) 0 (0%) 1 (2%)Hypoalbuminemia 2 (4%) 0 (0%) 2 (4%)Hyponatremia 2 (4%) 0 (0%) 2 (4%)Hypophosphatemia 0 (0%) 1 (2%) 1 (2%)Buttock pain 1 (2%) 0 (0%) 1 (2%)Myalgia 2 (4%) 0 (0%) 2 (4%)Dry skin 1 (2%) 0 (0%) 1 (2%)Pain of skin 1 (2%) 0 (0%) 1 (2%)Pruritus 1 (2%) 0 (0%) 1 (2%)Maculopapular rash 1 (2%) 0 (0%) 1 (2%)Skin and subcutaneous tissue disorder 3 (6%) 0 (0%) 3 (6%)Skin ulceration 1 (2%) 0 (0%) 1 (2%)Hematoma 1 (2%) 0 (0%) 1 (2%)

Figure 1.

Response rates for patients by doselevel. A, Radiographic response.The waterfall plot illustrates themaximum percentage of tumorreduction for target lesions inpatients with evaluable disease fortumor response. Each barrepresents an individual patient.The red dashed line represents thecutoff for disease progression, andthe black dashed line represents thecutoff for partial responsemeasured by RECIST 1.1. B, Theswimmers plot of time to responseand time to off study for all patients.Duration of response (months) totreatment for the two patients whoachieved partial response. Eachindividual bar represents onepatient. Red triangle indicatespatients who achieved a partialresponse. PD, progressive disease;PR, partial response; SD, stabledisease.






IgD, and IgE was minimal; however there were some 20%IgM antibodies. The elicited IgG were further subtyped (Fig. 5Cand D) into mostly IgG3 with variable amounts of IgG1,IgG2, and IgG4. Similar profiles were also obtained for cohorts1, 3, and 4 (see Supplementary Table S5A and S5B). Thesestudies indicate that isotype switching occurred during theprocess of vaccination and boosting with activation of theT cells.

Patient vaccine antibodies cause enhanced antiproliferativeeffects

The effects of patient antibodies were tested using BT-474 in thepresence of native HER-2 ligand, heregulin (HRG). Figure 5Ashows that each of the patient sera in cohort 2 was able to inhibitproliferation of the HER-2–overexpressing cells BT-474 as com-pared with preimmune sera. The level of inhibition was compa-rable to trastuzumab, suggesting that our vaccine antibodies wereequally effective. Results for cohorts 1, 3, and 4 are shown inSupplementary Table S6A.

Patient vaccine antibodies inhibit HER-2 phosphorylationPhosphorylation of HER-2 plays a critical role in signaling

pathway response to ligand binding. The main mode of actionof pertuzumab is the interruption of HER-2/neu dimerizationwith other members of the ErbB receptor family. To determinewhether our peptide antibodies disrupted dimerization and

phosphorylation of the receptor cytoplasmic tyrosine kinaseregions, we used a phospho-HER-2 ELISA. Sequence 266–296represents a sequence that mimics the binding site of pertuzu-mab that has been shown to disrupt ligand-dependent receptorcomplexes independent of HER-2 expression. To evaluate ifpatient sera containing HER-2–specific antibodies were able tofunction as dimerization inhibitors, we used a total phospho-HER-2 ELISA. BT-474 cells were treated with patient serumantibodies, and HRG was used to activate HER-3 before celllysates were captured with an anti–HER-2 monoclonal anti-body and were probed with a phospho-HER-2 antibody. Allpatients at DL2 lowered the concentration of the phospho-tyrosine on BT-474 cells by 25% to 35% compared with theHER-2 phosphorylation inhibitor AG825, which was used asthe positive control. Overall, as shown in Fig. 5B, our datasuggest that patient antibodies inhibited receptor phosphory-lation. Results for cohorts 1, 3, and 4 are shown in Supple-mentary Table S6B.

Patient antibodies induce apoptosisTargeting apoptotic regulatory pathways in cancer is a pro-

mising strategy for therapeutic agents. We next evaluatedwhether patients' vaccine antibodies following vaccinationwere capable of inducing apoptosis of cancer cells by caspaseactivity assay. Using breast cancer cells BT-474 treated withpatient antibodies as inhibitors, we found that each of the

Figure 2.

Cohort 2 antibody binding analyses. Panels 1–6, Flow cytometry analysis of cohort 2 patient antibodies binding to BT-474 with overexpressed HER-2 (cohort 2patients 2A–2F) after the third vaccination. BT-474 cells (5� 105) were incubated with 500 mg/mL patient antibodies, 500 mg/mL control antibody (normalhuman IgG), and 50 mg/mL commercial human anti-HER2 (trastuzumab) in staining buffer (PBSþ 0.05% BSAþ 0.2% NaN3), respectively, at 4�C for 2 hours andthen with 1.6 mg/mL Alexa Fluor 488 antihuman IgG at 4�C for 45 minutes. The stained cells were analyzed on an FACS Caliber machine. Panel 7, Binding ofcohort 2 antibodies to MVF-HER-2 (266–296; blue), MVF-HER-2 (597–626; orange), and recombinant HER-2 (yellow) assessed using ELISA. Antibodies purifiedvia protein A/G column from sera before or after immunization were diluted to 20 mg/mLwith PBS/HS and incubated with immobilized MVF-HER-2 vaccines andrecombinant HER-2. Bound antibodies were probed using HRP-conjugated anti-human IgG. IgG, normal human IgG.

Bekaii-Saab et al.





patient antibodies (3Yþ4; 4 weeks after third vaccine) demon-strated significant increases in the amount of caspase-3 and -7activity in treated cells as compared with the negative controls(preimmune sera; Fig. 5C). Vaccine antibodies increased cas-pase release more than 100-fold, clearly indicative of increasedcell death similar to trastuzumab. Apoptosis results for cohorts1, 3, and 4 are shown in Supplementary Table S6C.

Patients' vaccine antibodies induce ADCC of cancer cellsOne major mechanism of immunologic action of antibodies

is to induce ADCC of cancer cells. It has been well documentedthat in vivo, the Fc portions of Abs can be of foremost impor-tance for efficacy against tumor targets (39). We determined theability of the patient antibodies to mediate ADCC in vitro usingBT-474 breast cancer cells as targets and peripheral bloodmononuclear cells (PBMC) from normal human donors aseffector cells using a bioluminescence cytotoxicity assay kit(aCella-Tox) as previously described (29). All patient anti-bodies demonstrated increased tumor cell lysis (40%–45% at20:1 E:T) in a dose-dependent manner similar to trastuzumab(Fig. 5D). Normal human IgG was used as negative control.ADCC results for cohorts 1, 3, and 4 are shown in Supplemen-tary Table S6D.

DiscussionThis phase I clinical trial evaluated the safety, tolerability,

toxicity, and immunogenicity of a combination of two peptidevaccines, as well as the OID/OBD and the MTD. The vaccinecombination consisted of two B-cell HER-2 epitopes: HER-2(266–296) andHER-2 (597–626) fused to aMeasles Virus Fusionprotein (MVF) "promiscuous" T helper cell epitope. The peptideswere combined with a potent adjuvant nor-muramyl dipeptide(n-MDP) and emulsified in Montanide ISA 720. Three vaccina-tions given at 3-week intervals with the chimeric peptides at fourdifferent doses were well tolerated without any severe systemicadverse events, with the most common adverse events consistingof grade 1–2 injection-site reactions.DL2was selected as theOID/OBD given its immunogenicity, ability to inhibit proliferationand phosphorylation of the chimeric vaccines, and its biologicalproperties such as ADCC and apoptosis.

The vaccine generated sustained humoral response elicitingantibodies that recognized the HER-2 receptor in the majority ofresponding patients. Of note, the clinical benefit was observedacross all DLs, with few patients showing evidence of sustainabledisease control (as shown in Fig. 1). Also of note, these patientswere a heavily pretreated cohort that had been exposed to an

Figure 3.

Patient 2C antibody binding analyses. A–E, Flow cytometry of patient 2C antibodies by multiple boosts binding to BT-474 with overexpressed HER-2.BT-474 cells (5 � 105) were incubated with 500 mg/mL patient antibodies (blue), 500 mg/mL control antibody (normal human IgG; red), and50 mg/mL commercial human anti-HER2 (trastuzumab; yellow) in staining buffer (PBS þ 0.05% BSA þ 0.2% NaN3), respectively, at 4�C for 2 hoursand then with 1.6 mg/mL Alexa Fluor 488 anti-human IgG at 4�C for 45 minutes. The stained cells were analyzed on an FACS Caliber machine. F,ELISA purified 2C antibodies binding to rhHER-2, MVF-HER-2 (266–296), and MVF-HER-2(597–626). Pre, preimmunization; control, normal human IgG.1Yþ3w, blood drawn 3 weeks after the first immunization. 2Yþ3w, blood drawn 3 weeks after the second immunization. Months (mts) and years (yr)denote the time of drawing blood after first immunization.






average of 4 prior therapies. There was preliminary evidence ofclinical activity in 6 patients (21.4%) who received 6 monthsboost, including 1 patient who received seven 6 months boostervaccinations over 3.5 years without evidence of resistance totherapy. The number of patients who received 6 months boosts,including the one patient who received seven 6-month boots withour vaccine treatment, highlights the great potential of thistherapy for circumventing the intrinsic drug resistance that limitscurrently available cancer therapies. The promising preliminaryresults suggest an important potential benefit of this vaccinecompared with humanized monoclonal antibodies in whichmost patients develop secondary resistance. One needs to exercisecaution when interpreting the efficacy results in this trial becauseof limited patient numbers and phase I study design which isfocused on establishing safety and best dose of the vaccine forfurther clinical development.

In order to elicit high-affinity antibodies (a prerequisite for aneffective outbred vaccine), two criteria are essential: (i) the con-formational epitopes must mimic the tertiary structure of theantigen; (ii) the peptides must include a "promiscuous" T-cellepitope such as one from theMVF to ensure high immunogenicityandmaintenance of sustained immune response by activating theTh2 helper T-lymphocyte subset. We also combined our vaccinewith a potent adjuvant n-MDP and emulsified in Montanide ISA720 (SEPPIC, Inc.) to improve antigen presentation by formationof the depot of the vaccine peptides at the site of injection. Incontrast to Incomplete Freund's adjuvant, this solution is biode-gradable and less cytotoxic (40, 41). Previous studies have shownthat trastuzumab blocks tumor growth through reducing down-streamsignaling, inhibiting angiogenesis, and increasing immuneactivity, primarily ADCC (19). Constitutive PI3K/Akt activationthrough PTEN downregulation or PIK3CA hyperactivating muta-tions significantly abrogates response to trastuzumab (42, 43).

The lack of effective ADCC immune response also promotestrastuzumab resistance (44–46).

The study vaccine bypasses the disadvantages of passive immu-notherapy with monoclonal antibodies, i.e., high cost and theneed for repeated treatments that can result in serious toxicities,such as hypersensitivity reactions, cardiomyopathy, and, in rarecases, pneumonitis. Our peptide vaccine was not associated withan increased risk of cardiomyopathy, despite sustained produc-tion of endogenous, fully human antibodies that function verysimilarly to trastuzumab and pertuzumab. Peptide cancer vac-cines are an attractive therapeutic option as they are safe and easilymanufactured and administered. Additional advantages of pep-tide cancer vaccines are exquisite specificity, low toxicity, and thepotential for a durable treatment effect due to immunologicmemory. In addition, our vaccine avoided the limitations ofT-cell–specific cancer vaccines that require specific MHC isoformsin order to bind the peptide.

The trial had limitations that should be addressed. First, it was asingle institutional study in a heterogeneous group of cancerpatients, many of whom were heavily pretreated. Such patientstend to have poor ability to mount an effective antitumor immu-nity because of tumor- and treatment-induced immunosuppres-sion. Although we found that such patients were still able togenerate a sustained humoral response to the vaccine, patientswho are less heavily pretreated or have a lower tumor burdenmayhave been able to mount a more effective, sustained immuneresponse to the vaccine, potentially resulting in a greater observedclinical benefit. Notably, the patient who received multiple boos-ters did not receive any chemotherapy prior to enrollment. Thus,his immune systemwas very much intact and likely more respon-sive to vaccine treatment. This is an important fact as the vaccinetherapy should bemore effective in such patients. Additionally, itis possible that somepatientswho received 3ormore vaccinations

Figure 4.

Cohort 2 antibody isotype and IgGsubtype identification.A and B,Antibody isotype identification ofcohort 2 patient sera by ELISA.After incubation of sera (1:16) withimmobilized MVF-HER-2 (266–296)(A) and MVF-HER-2 (597–626) (B),isotypes of bound antibodies wereidentified using HRP-conjugateddifferent secondary antibodies. Thepercentage of isotype antibodies insera was represented by theirrespective absorbance relative tothe total absorbance by all fiveisotype antibodies. C and D, IgGsubtype identification of cohort 2patient sera by ELISA. Afterincubation of sera (1:16) withimmobilized MVF-HER-2 (266–296;C) and MVF-HER-2 (597–626; D),IgG subtypes of bound antibodieswere probed using HRP-conjugateddifferent secondary antibodies. Thepercentage of isotype antibodies insera was represented by theirrespective absorbance relative tothe total absorbance by all fourisotype antibodies.

Bekaii-Saab et al.





may have more indolent malignancy and therefore receive morevaccinations before progression based on RECIST 1.1 criteriacompared with patients who completed fewer than 3 vaccina-tions.However, the phase I studies are not designed to test efficacyof experimental therapy but to establish safety profile and bestdose of the therapy. This trial also suffered from lack of random-ization,making it difficult to ascertain whether the vaccine is trulyeffective, but randomization is not commonly used in phase Itrials. Despite these limitations, our trial shows at least a signal ofpromising antitumor activity of the vaccine. We need to await theresults of a future randomized study before definitive conclusionsabout the vaccine's efficacy can be made.

Lastly, a limitation for the study is the absence of consistentmeasurement of HER-2 overexpression in patients enrolled in thedose escalation part. This is partly because the phase I trial wasopened to all patients irrespective of whether they overexpressedHER-2. Because this was a first-in-human trial focused on estab-lishing the safety profile and recommended phase II dose of thevaccine, we did not collect information about HER-2 expressionin this portion of the study. We therefore cannot make any

conclusions that overexpression of the target protein was respon-sible for efficacy in the responding patients.

In conclusion, we demonstrate that the combination of HER-2vaccines is well tolerated and able to generate sustained anti–HER-2 immune response. The most common toxicity was aninjection-site reaction. A majority of the patient antibodies thatwere generated in response to the vaccine showed potentantitumor activity and defense mechanisms (induction ofADCC and apoptosis, inhibition of proliferation and phosphor-ylation). There were limited but few very meaningful clinicalresponses in this heavily pretreated patient population with aheterogeneous group malignancies. Given the initial promise,continuous development of the vaccine is ongoing at thesuggested OBD in a less heavily pretreated patient populationin breast and/or gastrointestinal malignancies (gastroesopha-geal and colorectal) with HER-2/EGFR overexpression.

Disclosure of Potential Conflicts of InterestD.H. Ahn is a consultant/advisoryboardmember forCelltrion, Eisai, Cardinal

Health, Lexicon, Exelixis, and Astellas. M. Lustberg is a consultant/advisory

Figure 5.

Effects of cohort 2 purified antibodies on proliferation, phosphorylation, apoptosis, and antibody-dependent cell cytotoxicity. A, BT-474 (10,000 cells perwell) were treated for 72 hours before addition of MTT. After extraction with lysis buffer, plates were incubated overnight at 37�C and were read on anELISA plate reader at 570 nm. Results shown are an average of three different experiments with each treatment performed in triplicates for dose level 2.Cells were treated with medium alone or with preimmunized, postimmunized antibodies, and control, respectively. B, BT-474 (1 million cells per well) in6-well plate were incubated with patient antibodies and cells lysed in RIPA lysis buffer and then, spun at 13,000 � g and supernatants collected.Phosphorylation was determined by Duoset IC (R&D Systems) for total human phospho-HER-2 according to the manufacturer's directions. Results shownare an average of three different experiments with each treatment performed in triplicates for dose level 2. C, BT-474 breast cancer cells were treatedwith the vaccine antibodies, and caspase activity was measured after treatment and results show the activity levels after treatment. Data show averageof three different experiments following treatment with patient antibodies from dose level 2. D, Cohort 2 patients purified antibodies cause ADCC.BT-474 target cells were incubated with different amounts of effector cells (PBMC) after treatment with 100 mg patient antibodies 2A–2F in dose level 2.Trastuzumab was used as positive control, and normal human IgG was used as the negative control. Results shown are an average of three differentexperiments with each treatment performed in triplicates for dose level 2.






boardmember for PledPharma. B. Ramaswamy is a consultant/advisory boardmember for Pfizer. Nopotential conflicts of interest were disclosed by the otherauthors.

Authors' ContributionsConception and design: T. Bekaii-Saab, L. Wei, P.T.P. KaumayaDevelopment of methodology: P.T.P. KaumayaAcquisition of data (provided animals, acquired and managedpatients, provided facilities, etc.): T. Bekaii-Saab, R. Wesolowski, C. Wu,A. Mortazavi, M. Lustberg, B. Ramaswamy, J. Fowler, J. Overholser,P.T.P. KaumayaAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): T. Bekaii-Saab, R. Wesolowski, D.H. Ahn,A. Mortazavi, L. Wei, P.T.P. KaumayaWriting, review, and/or revision of the manuscript: T. Bekaii-Saab,R. Wesolowski, D.H. Ahn, C. Wu, A. Mortazavi, M. Lustberg, B. Ramaswamy,J. Fowler, L. Wei, P.T.P. KaumayaAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): T. Bekaii-Saab, L. Wei, P.T.P. Kaumaya

Study supervision:T. Bekaii-Saab, R.Wesolowski, A.Mortazavi, B. Ramaswamy,L. Wei, P.T.P. KaumayaOther (enrolling patients, managing patients, collecting and analyzing theclinical data): A. Mortazavi

AcknowledgmentsThe authors would like to thank all the patients who participated in the trial

and their families, as well as the OSU James clinical site. The authors are gratefulfor the assistance of Stephanie Fortier for manuscript preparation. This studywas funded by NIH/NCI R01 CA CA84356 to P.T.P. Kaumaya and NIH R21CA13508 to P.T.P. Kaumaya.

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 December 7, 2018; revised January 18, 2019; accepted February 21,2019; published first February 25, 2019.

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2019;25:3495-3507. Published OnlineFirst February 25, 2019.Clin Cancer Res Tanios Bekaii-Saab, Robert Wesolowski, Daniel H. Ahn, et al. Adjuvant in Patients with Advanced Solid TumorsPeptide Vaccines Emulsified in Montanide ISA 720VG and Nor-MDP Phase I Immunotherapy Trial with Two Chimeric HER-2 B-Cell

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