TargetedT-cellTherapyinStageIVBreastCancer: A Phase I...

Cancer Therapy: Clinical

TargetedT-cell Therapy in Stage IVBreastCancer:A Phase I Clinical TrialLawrence G. Lum1,2,3, Archana Thakur1, Zaid Al-Kadhimi1,2, Gerald A. Colvin4,Francis J. Cummings5, Robert D. Legare6, Don S. Dizon7, Nicola Kouttab8, Abby Maizel6,William Colaiace9, Qin Liu10, and Ritesh Rathore5

Abstract

Purpose: This study reports a phase I immunotherapy trial in23 women with metastatic breast cancer consisting of eightinfusions of anti-CD3� anti-HER2 bispecific antibody (HER2Bi)armed anti-CD3–activated T cells (ATC) in combination withlow-dose IL-2 and granulocyte-macrophage colony-stimulatingfactor to determine safety, maximum tolerated dose (MTD),technical feasibility, T-cell trafficking, immune responses, timeto progression, and overall survival (OS).

Experimental Design: ATC were expanded from leukapheresisproduct using IL2 and anti-CD3monoclonal antibody and armedwith HER2Bi. In 3þ3 dose escalation design, groups of 3 patientsreceived 5, 10, 20, or 40 � 109 armed ATC (aATC) per infusion.

Results: There were no dose-limiting toxicities and the MTDwas not defined. It was technically feasible to grow 160 � 109

ATC from a single leukapheresis. aATC persisted in the bloodfor weeks and trafficked to tumors. Infusions of aATC inducedanti-breast cancer responses and increases in immunokines. At14.5 weeks after enrollment, 13 of 22 (59.1%) evaluablepatients had stable disease and 9 of 22 (40.9%) had progressivedisease. The median OS was 36.2 months for all patients, 57.4months for HER2 3þ patients, and 27.4 months for HER20–2þ patients.

Conclusions: Targeting HER2þ and HER2� tumors with aATCinfusions induced antitumor responses, increases in Th1 cyto-kines, and IL12 serum levels that suggest that aATC infusionsvaccinated patients against their own tumors. These results pro-vide a strong rationale for conducting phase II trials. ClinCancer Res;21(10); 2305–14. �2015 AACR.

IntroductionIn women who present with localized breast cancer, approx-

imately 10% develop metastatic breast cancer (MBC) in 5 years.Although most patients experience objective responses to che-motherapy or hormonal therapies, progression is inevitable(1–3). Overexpression of HER2/neu (HER2) in breast, ovarian,lung, gastric, head andneck, and prostate cancersmakes it an idealtarget for antitumor agents (4, 5). Furthermore, recent studiessuggest that the anti-HER2 reagents may be effective againstHER2þ cancer stem like cells in tumors that areHER2negative (6).

For patientswithprogressiveHER2þMBC,HER2-targeted agentssuch as trastuzumab, pertuzumab, trastuzumab–maytansine,(7–10) lapatinib, neratinib, and afatinib (11–13) have improvedprogression-free survival (PFS). However, these agents are noteffective for patients with MBC with HER2-negative disease. Non-toxic targeted approaches are needed for these patients.

Activated T cells (ATC) armed with anti-CD3 � anti-HER2bispecific antibody (HER2Bi) exhibit high levels of specific cyto-toxicity directed at both high and low HER2-expressing breastcancer cell lines (14). Arming ATCwithHER2Bi redirects the non-MHC–restricted cytotoxicity of ATC toHER2-specific targets (14).HER2Bi-armed ATC (aATC) repeatedly kill, proliferate, andrelease Th1 cytokines, RANTES, and MIP-1a when coculturedwithHER2-negative cell lines (15). Inmurine studies, infusions ofaATC completely prevented tumor development in coinjectionassays and inhibited established HER2þ PC-3 tumors in SCID/Beige mice (16, 17).

In this study, we used combination immunotherapy consistingof HER2Bi aATC infusions, IL2, and granulocyte macrophagecolony-stimulating factor (GM-CSF). GM-CSF was empiricallychosen because it is known as a potent immune adjuvantand approved for human use. Our data show that aATC infusionswere safe and feasible, persist in patients' blood, and inducecytotoxic responses to breast cancer cells and elevations of serumimmunokines.

Materials and MethodsClinical protocol

PatientswithMBCwere enrolled inphase I clinical trial at RogerWilliams Hospital in Providence (RWH; Providence, RI) andBarbara Ann Karmanos Cancer Institute (KCI), Wayne State

1Department of Oncology, Wayne State University and KarmanosCancer Institute, Detroit, Michigan. 2Department of Medicine,WayneState University and Karmanos Cancer Institute, Detroit, Michigan.3Department of Immunology and Microbiology,Wayne State Univer-sity and Karmanos Cancer Institute, Detroit, Michigan. 4South CountyHospital,Wakefield,Rhode Island. 5DivisionofHematologyandOncol-ogy, Department of Medicine, Roger Williams Hospital, Providence,Rhode Island. 6Women & Infants Hospital, Providence, Rhode Island.7Massachusetts General Hospital Cancer Center, Department of Med-icine,HarvardMedical School, Boston,Massachusetts. 8DepartmentofPathology, Roger Williams Medical Center, Providence, Rhode Island.9Department of Nuclear Medicine, Roger Williams Medical Center,Providence, Rhode Island. 10Department of Medicine,Wistar Institute,Philadelphia, Pennsylvania.

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

Corresponding Authors: Lawrence G. Lum, Wayne State University and Kar-manos Cancer Institute, 4100 John R. St., Detroit, MI 48201. Phone: 313-576-8326; Fax: 313-576-8939; E-mail: [email protected]; and Archana Thakur,E-mail: [email protected]

doi: 10.1158/1078-0432.CCR-14-2280

�2015 American Association for Cancer Research.

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University (WSU, Detroit, MI), between May 2001 and August2010. The protocol was reviewed and approved by protocolreview committees, institutional Human Investigational Com-mittees at RWH and WSU, and the FDA. RWH 01-351-46 andWSU 2006-130 (This trial was registered at http://www.clinical-trials.gov, NCT00027807) were monitored by RWMC and KCIdata safety monitoring committees, respectively. All patientssigned informed consent before enrollment.

Production of clinical HER2BiTrastuzumab (Herceptin, Genentech) was heteroconjugated to

anti-CD3 (OKT3, Centocor, Ortho-Biotech) to produce HER2Biunder cGMP conditions (14).

Phase I clinical trial designThe primary endpoint was to determine the safety and maxi-

mum tolerated dose (MTD) of aATC in a standard 3 þ 3 doseescalation trial with dose levels of 5, 10, 20, and 40 billion aATCper infusion (2 infusions/week for 4 weeks) for total doses of 40,80, 160, and 320� 109 aATC. aATCwere given with IL2 (300,000IU/m2/day) and GM-CSF (250 mg/m2/twice weekly) beginning3 days before the first infusion and ending 1 week after the lastaATC infusion. Fig. 1A shows the treatment schema. Tumorevaluations were performed 14.5 weeks after chemotherapy orhormonal therapy (time for lymphoid recovery, ATC production,4.5 for weeks after immunotherapy, and 4 weeks of observation).All patients withMBC (HER2 0–3þ) whomet enrollment criteriawere eligible.

Eligibility criteriaWomen 18 years of age or older with histologically documen-

ted metastatic infiltrating ductal or lobular breast carcinoma with0–3þHer2 expression, Karnofsky score of�70%,ECOG0–2, andlife expectancy of >3 months with good organ function wereeligible (See Supplementary Information). Women with no mea-surable disease were eligible if the tumor ormetastatic diseasewasremoved or successfully treated before enrollment in the study.No serious medical or psychiatric illness that prevents informed

consent or intensive treatment were allowed.Minor changes fromthese guidelines would have been allowed at the discretion of theattending team under special circumstances. The reasons forexceptions would have been documented. HER2/neu, estrogen,and progesterone receptor positivity were recorded. Details ofpatient characteristics are presented in Supplementary Table S1.

Leukopheresis, T-cell expansion, and production of aATCATC were produced as described (18). After 10–14 days, ATC

were harvested and armed with 50 ng of HER2Bi/106 ATC, andcryopreserved (18). Aliquots were tested for bacteria and fungus,endotoxin, mycoplasma, phenotype, and cytotoxicity.

Dose modification and toxicity scoring (NCI Toxicity Criteria,v2, June 1, 1999).

Patients were accrued to each dose level based on the dose-escalation schema (Supplementary Table S2). If there was onepatient with persistent grade 3 nonhematologic toxicity or grade4 toxicitywas encountered in thefirst 3 patients or 2 out of thefirst6 patients, the dose would not have been escalated. Patients withgrade 4 nonhematologic toxicity were removed from protocol. Ascardiac toxicity associatedwithHerceptin treatmentwas a concern(19), patients were removed if aATC infusions decreased theejection fraction from the baseline MUGA by >10%. Treatmentwas held for persistent grade 3 toxicity until toxicity decreased to <grade 2. If grade 3 toxicity occurred again, subsequent doses ofaATC were washed to eliminate DMSO. If toxicity persisted, thenthe next dose of aATC was resumed at a 50% reduction. If thetoxicities continued at the reduced dose of aATC, the IL2would bestopped and the aATC infusions continued at the reduced dose. Ifgrade 3 toxicity occurred again, the ATC infusions would bestopped. Toxicities were assessed for 7 days after each infusionand weekly for unresolved toxicities.

Non-MHC–restricted cytotoxicitySpecific cytotoxicity was performed with fresh PBMC from

patients (14) using SK-BR-3 (breast specific target) and K562 cells(NK cell target) in 51Cr release assay (20). 51Cr release and IFNgEliSpot assays were used to assess immediate cytotoxicity of freshendogenous PBMC directed at breast "tumor antigens" to imme-diately lyse tumor targets or T cells ability to secrete IFNg withoutin vitro restimulation. The cytotoxicity or IFNg EliSpots exhibitedby PBMC would represent the development of endogenousimmune responses to unknown tumor associated antigens onSK-BR-3 targets.

Cytokine profilesSerum cytokines were detected by multiplex cytokine array as

described previously (18) using the Bio-Plex system (Bio-RadLaboratory).

ImmunohistochemistryTissues samples were sectioned, deparaffinized, stained, with

hematoxylin and eosin and characterized for tumor content by apathologist. Adjacent sections were stained with anti-CD3 todetect T cells using the Catalyzed Signal Amplification (CSAPeroxidase System, DAKO) after target retrieval and endogenousbiotin/avidin and peroxidase quenching with the CSA AncillarySystem (DAKO). Anti-CD3 antibody (1 mg/mL) was diluted inbackground reducing components (CSA Ancillary System) andincubated with tissue samples for 30 minutes at room tempera-ture. Primary antibody was detected by incubating for 15minutes

Translational Relevance

Infusions of anti-HER2 � anti-CD3 bispecific antibody(HER2Bi) armed activated T cells (aATC) are feasible, safe,and did not cause dose-limiting toxicities. Our phase I clinicaltrial using HER2Bi aATC to target HER2� and HER2þ meta-static breast cancer in combination with IL2 and granulocytemacrophage colony-stimulating factor stabilized disease in 5women (5/22 evaluable patients, 22.7%) at 14.5 weeks. Giventhe number of patients is small, themedianOSof 36.2monthsfor all patients, 57.4 months for HER2 3þ patients, and 27.4months for HER2 0–2þ patients is encouraging. aATC infu-sions induced significant increases in the CTL activity follow-ing immunotherapy that argues for the development of breastcancer–specific endogenous immune responses. aATC infu-sions also polarized the immune system to a Th1/Type 1cytokine profile with remarkable increases in IL12 production.These results provide the rationale for the design of phase IIclinical trials in solid tumors.

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Clin Cancer Res; 21(10) May 15, 2015 Clinical Cancer Research2306




with biotinylated goat anti-mouse immunoglobulins, and thesignal was amplified and visualized by diaminobenzidine pre-cipitation at the antigen site. In parallel, adjacent sections werestained with biotinylated goat anti-mouse IgG2a followed bystreptavidin-FITC to detect the goat anti-mouse-IgG2a. Imagesacquired using fluorescent filters were overlayed upon imagesacquired by light microscopy creating composite images to eval-uate colocalization of staining.

Detection of aATC in patientsGoat anti-mouse IgG2a directed at the OKT3 part of the BiAb

was used to detect aATC in the peripheral bloodbyflow cytometryand in biopsy or surgical samples by immunohistochemistry.

Statistical analysisThe primary endpoint of the study was to determine safety and

MTD of aATC. The secondary endpoints were to assess responserates [complete response (CR), stable disease (SD), partialresponse (PR), and no evidence of disease (NED), time to pro-gression (TTP), andOS. TTP andOSweremeasured from the dateof enrollment. Kaplan–Meier estimates were performed for OS

and TTP. Descriptive statistical analyses were performed forimmune monitoring using Prism (GraphPad, Version 5.0).

ResultsPatient characteristics

Table 1 and Supplementary Table S1 (Supplemental Informa-tion) show the clinical characteristics, prior therapy, number oflines, aATC doses, sites of metastases, disease status at enrollmentand at 14.5 weeks, OS for 23 women, and TTP for 22 women.Median age was 48 years (range: 31–68 years). All of theHER2 3þpatients except one (patient #16) received Herceptin� (patient#16was randomized to receive no trastuzumabonCALTB49909).All patients received chemotherapy except one (patient #8). Onepatient (patient #12) was treated twice but counted once for doseescalation and survival analysis. One patient had T cells expandedbut was not treated due to disease progression (23 of 24 enrolledwere treated).

Phenotype and cytotoxicity of aATCOne patient underwent a second leukapheresis to obtain the

T cells necessary to meet the required dose and one patientunderwent a second leukopheresis to regrow product due to

Figure 1.A, treatment schema showsleukapheresis to obtain T cells forexpansion. HER2Bi armed ATC (aATC)were administered twice weekly for 4consecutive weeks. All patientsreceived SQ IL2 (300,000 IU/m2/day)and GM-CSF (250 mg/m2/twiceweekly), beginning 3 days before thefirst aATC infusion and ending 1 weekafter the last aATC infusion. Immunetesting was performed at indicatedtime points after aATC infusions. B, aPET/CT of a partial responder in theHER2-negative group who had twowell-defined liver metastases (2.5� 1.7cm and 2.5 � 1.3 cm, as shown inBefore). Reimaging afterimmunotherapy showed regression ofthe two lesions after 6 months (After).C, Kaplan–Meier curve for entire group(All), HER2 (3þ) positive, andHER2 (0–2þ) negative patients (left). Onepatient with an unknown HER2 statuswas analyzed with the HER2� group.Right, Kaplan–Meier curve for time toprogression for entire group, HER2(3þ) positive and HER2 (0–2þ)negative groups.

Targeted T-cell Therapy in Metastatic Breast Cancer Patients

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contamination or failure of aATC to mediate cytotoxicity. In theharvest product, the mean percent (� SD) viability was 91.88% �5.9% and the mean proportion [95% confidence interval (CI)] ofCD3,CD4, andCD8 cellswere 86.7 (79.9–93.6), 52.4 (44.6–60.2),and 34.6% (26.9–42.3), respectively. The ATC products wereenriched for CD4þ cells; greater than 90.8% of the T cells wereCD45ROþ after expansion. ATCs derived from the patients withMBC armed with Her2Bi exhibited a mean (� SD) specific cyto-toxicity of 59.3%� 11.9% (range, 12.1%–90.7%) at an E:T of 25:1directed at HER2þ SKBR3 targets by 51Cr release cytotoxicity assaywas significantly greater (P < 0.0001) than that exhibited bypatients' unarmed ATC (3.0% � 2.8%) before immunotherapy(Supplementary Table S3). Therewas an inverse correlation (Spear-man r ¼ �0.5642 with a P < 0.02) between in vitro cytotoxicity ofpatients' Her2Bi-ATC and proportion of CD4 cells in the expansionproduct. This was consistent with our report that enhanced in vitro

specific cytotoxicityof armedATCwashighest inCD8þATC, lowestin CD4þ ATC and intermediate with unfractionated T cells.

Phase I evaluation of MTDThe highest dose level completed was 20 � 109 aATC per

infusion (160� 109 total dose of aATC). We accrued one patientat the dose level of 40 � 109 aATC per infusion (320 � 109 totaldose), but it was not technically feasible to achieve the 320� 109

total dose with a single leukapheresis. The technically feasibledose was 160 � 109, and the MTD was not reached.

Phase I evaluation of toxicitiesThe most frequent side effect (SE) was grade 3 chills. Grade 3

headaches emerged as the second most common SE. Table 2shows the frequency of side effects as a function of dose level (NCIImmunotherapy Protocol Toxicity Table). By episode per infu-sion, the incidence of chills was 8.6%, 20.8%, and 43.1% at doselevels 1, 2, and 3, respectively. The incidence of headaches was3.1%, 8.3%, and 19.6% at dose levels, 1, 2, and 3, respectively. Allpatients with grade 3 chills responded tomeperidine. Patient #13at dose level 3 experienced a grade 4 headache and hypertensionand was removed from the study after 3 infusions (65.7 � 109

total aATC). The patient had developed a subdural hematomathat was evacuated without neurologic deficits or complications.Three additional patients were added to dose level 3 without anyDLTs. One patient achieved dose level 4 of 40 billion/infusiondose for a total of 320 billion. One patient (#2) died of digoxintoxicity related congestive heart failure and the autopsy showedno myocardial T-cell infiltrates. Patients #8 and #14 were admit-ted for management of hypotension, nausea, vomiting, anddehydration; there infusions were resumed and completed afterresolution of their SEs. There were no DLTs attributed to aATC.

Clinical responsesTwenty two of 23 patients were clinically evaluable at 14.5

weeks. Patient #2who died of digoxin toxicity was NED at time ofdeath. Although she was not evaluable for response, she wasincluded in the survival analysis. In the evaluable patients at 14.5weeks, onepatient hadNED,onepatient had aPR, 11patients had

Table 2. Toxicity evaluation at the dose levels 1 and 2 based on NCI toxicity criteria, v2

Number ofpatientsaffected(% at doselevel 1)

Total # ofepisodes bygrade at dose

level 1



level 2



level 3

Number ofPatientsaffected(% at doseLevel 4)

Total # ofepisodes bygrade at

dose level 4Category Adverse reaction 1 2 3 4 1 2 3 4 1 2 3 4 1 2 3 4

General disorder Chills 5 (62.5) 0 2 26 0 4 (66.7) 0 0 10 0 6 (75) 0 0 24 0 1 (100) 0 1 0 0Fever 2 (25) 5 5 0 0 3 (50) 6 3 0 0 2 (25) 0 4 0 0 1 (100) 1 0 0 0Malaise 2 (33.3) 8 0 0 0 1 (12.5) 0 1 0 0 1 (100) 6 1 1 0Pain 2 (33.3) 3 2 0 1Back pain 0 (0) 0 0 0 0 0 (0) 0 0 0 0 3 (37.5) 0 3 1 0Weight gain 1 (100) 1 0 0 0

Blood pressure Hypotension 1 (12.5) 0 1 0 0 2 (33.3) 0 4 0 0 2 (25) 0 3 0 0 1 (100) 1 0 0 0Hypertension 1 (12.5) 0 1 0 0 0 (0) 0 0 0 0 2 (25) 4 0 0 1

Cardiac Atrial rhythm 1 (12.5) 2 0 0 0Tachycardia 1 (16.7) 2 0 0 0 1 (12.5) 1 0 0 0

Gastrointestinal Nausea/vomiting 3 (37.5) 4 0 0 0 2 (33.3) 4 1 2 0 3 (37.5) 4 1 0 0 1 (100) 0 0 2 0Diarrhea 1 (16.7) 2 0 0 0

Pulmonary Dyspnea 0 (0) 0 0 0 0 0 (0) 0 0 0 0 1 (12.5) 0 1 0 0Neurological Headache 2 (25) 0 1 5 0 3 (50) 0 0 3 0 6 (75) 0 1 8 1 1 (100) 0 1 0 0Allergy Nasal congestion 1 (12.5) 1 0 0 0

Table 1. Patient characteristics

Patient data N (%)

Age, y<50 14 (60.09)�50 9 (39.1)

Cancer stageIV 23 (100)

Performance status (ECOG)0 18 (78.3)1 5 (21.7)2 0 (0)

ER/PR statusPositive 15 (65.2)Negative 7 (30.4)Unknown 1 (4.3)

HER2/neu status0 11 (47.8)1þ 2 (8.7)2þ 2 (8.7)3þ 7 (30.4)Unknown 1 (4.3)

Prior treatment with Herceptin�

Yes 7 (30.4)No 16 (69.6)

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SD, and 9 patients had PD. Patient #9 (ERþ, PR�, HER2�) whowas progressing on letrozole developed a PR after aATC treatmentthat continued beyond 7 months. She had two well-defined livermetastases on a PET/CT scan (2.5 � 1.7 cm and 2.5 � 1.3 cm asshown in Fig. 1B, "before") that decreased in size after immuno-therapy (Fig. 1B, "after"). The sum of longest diameters wasdecreased by 30% at 14.5 weeks and by >70% at 7 months. At14.5weeks, 59.1% (13/22) of patients had SDor better (NED, PR,or SD) and 40.9% (9/22) of patients had PD. Five of 22 (22.7%)patients with PD before immunotherapy achieved SD after aATCinfusions.

Overall survival and time to progressionSupplementary Table S1 shows the disease status (most

patientswith visceral disease) before therapy, status at 14.5weeks,TTP, and OS. Fig. 1C, left shows the Kaplan–Meier curve. Themedian OS for 23 patients is 36.2 months, 57.4 months forthe HER2 3þ group, and 27.4 months for the HER2 0–2þgroup. Fig. 1C, right shows the Kaplan–Meier curve for 22 patientswhowere evaluable for TTP. Themedian TTP after enrollmentwas4.2 months for the entire group, 7.9 months for the HER2 3þgroup, and 3.7months for theHER2 0–2þ group. SupplementaryTable S4 shows clinical responses in MBC patients who had SD at14.5 weeks compared with patients who had PD. The median OSis 40 and 57.9 months for the HER2 0–2þ and HER2 3þ patientswith SD, respectively, and 21.3 and 36.6 months for the HER2 0–2þ and HER2 3þ patients with PD, respectively. The proportion

of patients (Supplementary Table S5) who had SD or better was56.5% and the proportion of patients with PD was 39.1% at14.5 weeks (n ¼ 23). Five of 7 (71.4%) HER2 3þ patients hadSD or better disease, whereas 7 of 14 (50.0%) HER2 0–2þpatients had SD or better disease. Patient #14 with an unknownHER2 status had SD (included in the HER2 0–2þ group in theKaplan–Meier).

Immune responsesNon-MHC–restricted cytotoxic T-cell responses and IFNg eliSpots.Cytotoxicity exhibited by PBMC without stimulation directedat SK-BR-3 and K562 targets was performed to evaluate thedevelopment of immune responses by endogenous lympho-cytes. Figure 2A shows anti-SK-BR-3 cytotoxicity mediated byfresh PBMCof 12 patients alongwith their correspondingHER2BiaATC dose levels, OS, and TTP. There were no correlations foundbetween aATC dose andOS (r2¼ 0.0732), aATC dose and TTP (r2

¼ 0.3011), cytotoxicity and OS (r2¼ 0.0932), or cytotoxicity andTTP (r2 ¼ 0.0862). Figure 2B (top left) shows anti-SK-BR-3cytotoxicity before immunotherapy (preimmunotherapy), dur-ing (between infusion #4 and #5) immunotherapy (midimmu-notherapy) and 4weeks postimmunotherapy. Cytotoxicity direct-ed at SK-BR-3 increased significantly (P < 0.003) during immu-notherapy compared with preimmunotherapy levels. The topright panel of Fig. 2B shows the increased anti-K562 (NK activity)responses at midimmunotherapy and postimmunotherapy com-paredwith preimmunotherapy, but it was not significantly higher

Figure 2.A, shows the bar graph of cytotoxicitymediated by fresh PBMC of individualpatients (n ¼ 12) against SK-BR-3and their corresponding OS, TTP, andHER2Bi armed ATC doses.B, the enhanced cytotoxicity byPBMC directed at SK-BR-3 andK562 at preimmunotherapy,midimmunotherapy (between infusion#4 and #5), and postimmunotherapy(4 weeks after completion ofimmunotherapy; left). Bottom, T-cellIFNg EliSpots directed at SK-BR-3 andK562 targets at preimmunotherapy,midimmunotherapy, andpostimmunotherapy.






(P ¼ 0.07) at any time point. In parallel, PBMC from selectedpatients were directly stimulated with SK-BR-3 for IFNg EliSpots.Bottom left panel shows increased IFNg EliSpots at midimmu-notherapy (P < 0.04) and postimmunotherapy (P < 0.03) com-pared with the preimmunotherapy time point. These data showthat aATC infusions induced both specific anti-SK-BR-3 andinnate endogenous immune responses.

Antitumor immunokines. Serum cytokine levels were tested inpreimmunotherapy serum (preimmunotherapy), after aATCinfusion #4 (midimmunotherapy) and after completion ofaATC infusions (1 week after completion of all infusions,postimmunotherapy) to determine whether immunotherapyinduced changes in serum profiles (n ¼ 13). There were sig-nificant increases in IL12 (P < 0.0005), IL2 (P < 0.002), GM-CSF(P < 0.05), and IL10 (P < 0.01) levels during aATC infusions(midimmunotherapy) compared with baseline (preimmu-

notherapy). The same trend was seen for IFNg and TNFa, butthere were no statistical differences (Fig. 3). The botttom rightpanel of Fig. 3 shows the mean Th1/Th2 ratio ¼ [IL2 þ IFNg]/[IL4 þ IL10] at the pre-, mid-, and postimmunotherapy timepoints.

Tumormarkers. In 14of 22 evaluable patients, 4 had a reduction inCEA (2 of 4 had a >50% decrease and 2 of 4 had a 15%–50%decrease), 5 had a reduction in CA 27.29 (2 of 5 had a >50%decrease and 3 of 5 had a 15%–50% decrease), and 5 had areduction in soluble serum HER2 receptor (2 of 5 had >50%decrease and 3 of 5 had a 15%–50% decrease) levels (Supplemen-tary Table S6).

Kinetics and survival of infused aATC. Phenotyping of PBMC forIgG2aþ cells at pre- and postinfusion time points showed tran-sient increase in IgG2aþ cells up to 50% of the circulating T cells

Figure 3.Shows profile of serum cytokines. Analysis ofserum samples (n ¼ 13) at preimmunotherapy(preimmunotherapy), after aATC infusions #4(midimmunotherapy), and postimmunotherapy(4 weeks after completion of immunotherapy).Armed ATC infusions (immunotherapy)show increase in IFNg , IL2, IL12, TNFa,GM-CSF, and IL10 levels during aATC infusions(midimmunotherapy). Bottom right, the graphshows the mean ratio of Th1/Th2 ¼ [IL2þIFNg]/[IL4þIL10] at pre-, mid- andpostimmunotherapy time points.

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after 2- to 4-hour postinfusion in one patient (Fig. 4A, top left).Phenotyping in 4 patients at postinfusion #1, 5, 8, and 1 weekpostinfusion showed accumulation andpersistence of aATCup to1 week after the last infusion in dose level 1 patients (Fig. 4A, topright).

Trafficking and clearance of aATC. Infusions of 111In-labeled aATClocalized to the lungs, liver, and spleen, and eventually bonemarrow within 4 hours of injection in a HER2� patient (Fig. 4B,left). However, the 111In-labeled aATC could not be seen in thelung metastasis of a patient who was HER2�. After 24 hours,aATC had cleared from the lungs but persisted in the bonemarrow, liver, and spleen for up to 4 days after infusion. Serialmeasurements of 111In-labeled aATC (250 � 106) in wholeblood showed that about 50% of aATC cleared from the blood

within approximately 30 minutes but remained positive forradioactivity (1.1% of the initial concentration) up to 4 daysafter infusion (Fig. 4B, right).

Localization of aATC to tumors. Formalin-fixed, paraffin-embed-ded samples were prepared from a chest wall nodule excision anda sternal tumor biopsy and stained for IgG2a on aATC. IgG2aþ

aATC could be detected at 1 week (left) and 1 month (right) aftertreatment, respectively (Fig. 4C).

Antibody responses tomousemonoclonal antibody.Patient seraweretested for human anti-mouse antibodies (HAMA) directed atmurine IgG2a (OKT3). Human antibody responses to mouseIgG2a (OKT3) were very low (<10 ng/mL) and were not clinicallysignificant (Fig. 4D).

Figure 4.A, the proportion of circulating CD3þ cells and IgG2aþ cells in sequential samples from 1 patient stained with anti-human CD3 and anti-mouse IgG2aþ to quantitatethe proportion of HER2Bi armedATC in the PBMCby flowcytometry (left panel). The proportion of circulating PBL from4patients thatwere stainedwith anti-mouseIgG2a and quantitated by flow cytometry at preinfusion #1 (Inf #1), preinfusion #5 (Inf #5), preinfusion #8 (Inf #8), and 1 week after the 8th infusion (1-week post;right). B, HER2Bi-armed ATC localized to the bone marrow, lung, liver, and spleen within 4 hours of injection (left). By 24 hours, armed ATC had clearedthe lungs but persisted in the bone marrow, liver, and spleen for up to 96 hours (4 days) postinfusion. Blood was sampled and counted to determine theclearance of armed ATC from the blood. The clearance of 111In-labeled aATC (right). Heparinized blood (1 mL aliquots) were drawn at 0, 0.7, 1, 6, 8, 27, and 96 hoursafter infusion and counted for gamma irradiation. Results are presented as% radioactivity in serum (cpm) of injected dose. C, the sternal tumor biopsy 1 week(left) and 1 month (right) postimmunotherapy showing poorly differentiated mammary ductal carcinoma. Composite immunofluorescence staining detectedHER2Bi bispecific antibody using anti-mouse IgG-FITC overlaying IHC for detection of T cells using anti-CD3. HER2Bi detection colocalized with T cellsinfiltrating connective tissue surrounding nests of tumor cells. D, the HAMA response before each aATC infusion and post infusions at indicated time points.HAMA antibody response (n ¼ 11) could not be detected more than 10 ng/mL at any time point.






DiscussionThe results from this phase I trial of HER2Bi aATC infusions in

women with MBC are clinically encouraging. Multiple infusionsof aATC in combination with IL-2 and GM-CSF were safe andtechnically feasible with persistence of the infused aATC in thecirculation for 1 week. The median OS is 36.2 months for all 23patients (22 evaluable and1nonevaluable¼23), 57.4months forthe HER2 3þ patients, and 27.4 months for the HER2 0–2þpatients. Immunotherapy induced endogenous cytotoxic T-celland immunokine responses that persisted up to 4 months (15).

A highlight of available therapies in patients with progressivedisease (regardless of HER2 status) after treatment with anthra-cyclines and taxanes shows OS ranging from 4 to 18.1 months(21–26). Median OS for MBC was 18.6 months after first-linecapecitabine therapy, between 5 and 15months after second-linetherapy, and 8 months after third-line therapy (21–26). ForHER2� locally advanced or MBC patients, sorafenib in combi-nationwith capecitabine forfirst-line therapy resulted in amedianOS of 22.2 months (27). Second-line bevacizumab containingtherapy for TNBC (RIBBON-2) showed an OS of 17.9 months(28), and combination of cetuximab with cisplatin resulted in anOS of 12.9 months (29). Use of several third-generation aroma-tase inhibitors given after tamoxifen failure as first- or second-linetherapy in postmenopausal women with MBC reported OS up to26–28 months (30).

In this phase I study,most patientswere treatedwith�3 lines oftherapy (14/23, 61%) and many had visceral disease (17/23,74%; Supplementary Table S1). Infusions of aATC stabilizeddisease in 5 women (5/22, 22.7%) including a very good partialremission in patient #9. There were no DLTs observed. The majorside effects were chills, fever, headache, fatigue, and hypotension.Cytokine "flurries" were observed but not life-threatening cyto-kine "storm". Several patients had their aATC washed to reduceside effects, but no one had their dose of aATC reduced. Threepatients were hospitalized for cell-based toxicities, resolved theirside effects, and completed immunotherapy without recurrentDLTs. Only patient #13 stopped therapy due to a subduralhematoma most likely due to hypertension, and patient #2 diedof digoxin toxicity not related to aATC infusions. The remainingpatients received their infusions as outpatients, and there havebeen over 115 patients to date who have received armed ATCinfusionswithoutDLTs. Therewere 15patientswho received anti-CD3 � anti-CD20 aATC after high-dose chemotherapy and stemcell transplantation without DLTs (18, 31).

The development of CTL and IFNg Elispots directed at SK-BR-3provides immunologic evidence for the development of anendogenous cellular immune response. Consistent with our pre-viously reported data (15), these data also show persistence ofCTL up to 4 months after aATC infusions. High anti-SK-BR-3cytotoxicity levels cannot be attributed to the infused aATC as theywould make up only 1% of the endogenous lymphocyte popu-lation after dilution (�1 � 1012 cells). Furthermore, our earlieraATC depletion experiment showed that endogenous cells haddeveloped cytotoxicity directed at SK-BR-3 targets (15).

Increases in serum Th1 cytokine levels leading to high Th1/Th2ratios and increase in IL12 that developed mid immunotherapyand persisted for weeks after immunotherapy show that theimmunologic milieu and tumor microenvironment were shiftedtowards an antitumor environment. These results are corroborat-ed by our recent study showing that aATC targeting the triple-

negative cell lineMDA-MB-231 inMatrigel not only inhibited thegrowth of tumor cells but also inhibited the growth of immunesuppressor cells (32) and generated a Th1 cytokine rich micro-environment. These preclinical and clinical findings support theconcept of in situ vaccination with infusions of aATC.

The expansion of T cells resulted in >90% of the T cellsbecoming memory phenotype of CR45ROþwith more than 50%CD4þ T cells. HER2Bi aATC showed cytotoxicity to SK-BR-3 withconsistent increases in cytotoxicity as the proportion of CD8þ Tcells increased in the product.

There are major differences between chimeric antibodyreceptors (CAR) transduced anti-CD3/anti-CD28 activated Tcells (CART) and our approach of using the anti-CD3/IL2activated T cells armed with bispecific antibodies. CARTs rap-idly expand and develop an antitumor effect upon tumorengagement. On the other hand, armed ATC mediate imme-diate cytotoxicity, undergo short-term proliferation, and releaseTh1 cytokines/chemokines in the tumor microenvironment(15). The repeated infusions of armed ATC may overcome thetumor immunosuppressive factors to recruit endogenousimmune cells leading to in situ vaccination. Treating solidtumors with CAR or armed ATC approaches remains a chal-lenge due to tumor microenvironmental factors.

In summary, aATC were not only feasible and safe but alsoinduced endogenous cytotoxicity and cytokine responses inwom-en with MBC with a possible survival benefit. Our findings showthat cellular immune responses develop and may augmentimmune based killing of tumors even in patients who are pro-gressing. These results provide the rationale for thedesignof phaseII clinical trials using armed activated T cells in solid tumors.

Disclosure of Potential Conflicts of InterestL.G. Lum is a founder of and has ownership interest (including patents) in

Transtarget, Inc. D.S. Dizon is an employee of UpToDate. No potential conflictsof interest were disclosed by the other authors.

Authors' ContributionsConception and design: L.G. Lum, F.J. Cummings, R.D. Legare, R. RathoreDevelopment of methodology: A. Thakur, L.G. Lum, F.J. Cummings,D.S. Dizon, N. Kouttab, A. MaizelAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Thakur, L.G. Lum, Z. Al-Kadhimi, G.A. Colvin,F.J. Cummings, R.D. Legare, D.S. Dizon, R. RathoreAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Thakur, L.G. Lum, F.J. Cummings, A. Maizel,Q. LiuWriting, review, and/or revision of the manuscript: A. Thakur, L.G. Lum,Z. Al-Kadhimi, G.A. Colvin, F.J. Cummings, D.S. Dizon, N. Kouttab, Q. Liu,R. RathoreAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): A. Thakur, L.G. Lum, N. Kouttab, A. Maizel,R. RathoreStudy supervision: A. Thakur, L.G. Lum, Z. Al-Kadhimi, G.A. Colvin,D.S. Dizon, A. Maizel, R. RathoreOther (imaging interpretation): W. Colaiace

AcknowledgmentsThe authors thank the nurse clinical coordinators: Wendy Young, Annette

Olson, Lori Hall, Janet McIntyre, Patricia Steele, and KarenMyers who providedscheduling, informational, and emotional support to the women who partic-ipated in this clinical trial. The immunotherapy team acknowledges the specialefforts of all of the members of the Immunotherapy Program at Roger WilliamsHospital and the BMT/Immunotherapy Program at KCI that have providedsupport and infrastructure for the compassionate care of the women withMBC.

Lum et al.





The authors also appreciate the careful reading and suggestions made by Drs.Abhinav Deol and Ulka Vaishampayan.

Grant SupportThis study was supported by the National Cancer Institute of the NIH

under award numbers CA092344 (to L.G. Lum) and CA140314 (to L.G.Lum). The Microscopy, Imaging and Cytometry Resources Core is supported,in part, by NIH Center grant P30CA22453 to The Karmanos Cancer Institute,Wayne State University and the Perinatology Research Branch of the Nation-

al Institutes of Child Health and Development, Wayne State University. Thestudies were also supported by the Young Family Foundation and theRaymond Neag Foundation.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 12, 2014; revised January 14, 2015; accepted January 19,2015; published OnlineFirst February 16, 2015.

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Lum et al.




2015;21:2305-2314. Published OnlineFirst February 16, 2015.Clin Cancer Res Lawrence G. Lum, Archana Thakur, Zaid Al-Kadhimi, et al. Clinical TrialTargeted T-cell Therapy in Stage IV Breast Cancer: A Phase I

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