adhesion of t cells to endothelial cells facilitates ... · neurologic adverse events (nae) are...

CANCER RESEARCH | TRANSLATIONAL SCIENCE

Adhesion of T Cells to Endothelial Cells FacilitatesBlinatumomab-Associated Neurologic AdverseEvents A C

Matthias Klinger1, Gerhard Zugmaier1, Virginie N€agele1, Maria-Elisabeth Goebeler2, Christian Brandl1,Matthias Stelljes3, Hans Lassmann4, Arend von Stackelberg5, Ralf C. Bargou2, and Peter Kufer1

ABSTRACT◥

Blinatumomab, a CD19/CD3-bispecific T-cell engager (BiTE)immuno-oncology therapy for the treatment of B-cell malignancies,is associated with neurologic adverse events in a subgroupof patients. Here, we provide evidence for a two-step processfor the development of neurologic adverse events in response toblinatumomab: (i) blinatumomab induced B-cell–independentredistribution of peripheral T cells, including T-cell adhesionto blood vessel endothelium, endothelial activation, and T-celltransmigration into the perivascular space, where (ii) blinatumo-mab induced B-cell–dependent T-cell activation and cytokinerelease to potentially trigger neurologic adverse events. Evidencefor this process includes (i) the coincidence of T-cell redistributionand the early occurrence of most neurologic adverse events, (ii) T-cell transmigration through brain microvascular endothelium, (iii)

detection of T cells, B cells, and blinatumomab in cerebrospinalfluid, (iv) blinatumomab-induced T-cell rolling and adhesionto vascular endothelial cells in vitro, and (v) the ability of anti-adhesive agents to interfere with blinatumomab-induced interac-tions between T cells and vascular endothelial cells in vitro and inpatients. On the basis of these observations, we propose amodel thatcould be the basis of mitigation strategies for neurologic adverseevents associated with blinatumomab treatment and other T-celltherapies.

Significance: This study proposes T-cell adhesion to endothelialcells as a necessary but insufficient first step for development ofblinatumomab-associated neurologic adverse events and suggestsinterfering with adhesion as a mitigation approach.

IntroductionNeurologic adverse events (NAE) are common side effects of

therapies that exploit activated T cells to destroy malignant Bcells (1–6). Blinatumomab is a CD19/CD3-bispecific T-cell engager(BiTE) immuno-oncology therapy that activates patients’ own T cellsto target CD19þ B cells, leading to T-cell proliferation and B-cellapoptosis (7–9). Blinatumomab treatment has shown clinical efficacyin patients with both relapsed/refractory (r/r) B-cell precursor acutelymphoblastic leukemia (ALL; ref. 2) and indolent as well as aggressiver/r non-Hodgkin lymphoma (NHL; refs. 10, 11). Blinatumomab-associated grade�3 NAEs include encephalopathy, headache, alteredstate of consciousness, aphasia, ataxia, confusional state, nervoussystem disorder, tremor, neurotoxicity, and seizure (1, 3, 12). MostNAEs occur early during the first treatment cycle (i.e., 12–48 hoursafter infusion start or dose step) and are transient and fully revers-

ible (1). Grade �3 NAEs occurring in patients with r/r ALL receivingblinatumomab are managed by withholding blinatumomab untilresolution of NAEs to grade �1 and then restarting at the initial(lower) blinatumomab dose (13), as higher doses of blinatumomabcorrelate with a higher incidence of NAEs (10). In patients with r/rNHL, dose steps [i.e., starting at a low dose and then increasing tomultiple higher dose(s); refs. 10, 11], prophylactic steroids (10, 11), andpentosan polysulfate (PPS) coadministration (10) have been exploredas potential ways to reduce the incidence or severity of blinatumomab-associated NAEs. Dose steps have the potential to limit the efficacy ofblinatumomab in some patients as may high-dose steroids such asdexamethasone; however, a clear benefit of these two approaches tomitigate NAEs has yet to be proven (11). The effect of PPS, anantiadhesive agent, on blinatumomab-associated NAEs was evaluatedin a small subset of patients (n¼ 3) with r/r NHL at high risk of NAEs;no severe NAEs were observed in these patients who received PPSduring start of blinatumomab infusion and again during dose step (10).It was hypothesized that PPS may interfere with T-cell migration intothe central nervous system (CNS) and mitigate blinatumomab-associated NAEs. The purpose of this study is to determine themechanism(s) of blinatumomab-associated NAEs in patients withr/r ALL and r/r NHL, using a combination of in vitro T-cell rollingexperiments and in vivo pharmacodynamic/pharmacokinetic assays.

Materials and MethodsClinical studies and compassionate-use patients

Data from five clinical studies with blinatumomab were ana-lyzed: a phase I study and a phase II study in adults with r/rNHL (NCT00274742, NCT01741792; refs. 10, 11), a phase Ib/2 studyin pediatric patients with r/r ALL (NCT01471782; ref. 14),and two phase II studies in adults with r/r ALL (NCT01209286,NCT01466179; refs. 1, 13). Individual study designs and primary

1Amgen Research (Munich) GmbH, Munich, Germany. 2Comprehensive CancerCenter Mainfranken, University W€urzburg, W€urzburg, Germany. 3MedizinischeKlinik A, University M€unster, M€unster, Germany. 4Department of Neuroimmu-nology, Medical University of Vienna, Vienna, Austria. 5Department of Pediatrics,Division of Oncology and Hematology, Charit�e, Berlin, Germany.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

M. Klinger, G. Zugmaier, R.C. Bargou, and P. Kufer contributed equally to thisarticle.

Corresponding Author: Matthias Klinger, Amgen Research (Munich) GmbH,Staffelseestrasse 2, Munich, Bavaria 81477, Germany. Phone: 49-89-895277-530; E-mail: [email protected]

Cancer Res 2020;80:91–101

doi: 10.1158/0008-5472.CAN-19-1131

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results have been described in detail elsewhere (1, 10, 11, 13, 14)and are briefly summarized in Supplementary Information (Sup-plementary Table S1). Different target doses of blinatumomabwere used for ALL and NHL, with dose levels of 15 mg/m2/day(equivalent to 28 mg/day for an average adult) for ALL and 60 mg/m2/day (equivalent to 112 mg/day) for NHL. To mitigate adverseevents, especially cytokine release syndrome (CRS), step dosing ofblinatumomab is recommended (15). In addition to the clinicaltrials, a pediatric patient case from compassionate use of blina-tumomab is described (16). The patients selected for specificanalyses are summarized in Table 1.

Study protocols were approved by the independent ethics commit-tee of each institution, and all patients provided written informedconsent. Clinical studies were conducted in accordance with theprovisions of the Declaration of Helsinki and with Good ClinicalPractice guidelines.

Leukocyte analysisLymphocyte analysis was performed as described previous-

ly (17, 18). Briefly, peripheral blood mononuclear cells (PBMC)were isolated from blood samples of patients with r/r NHL(NCT00274742). PBMC samples were collected prior to start ofinfusion (day 1), and at 45 minutes, 2, 6, 24, 30, 48, and 168 hoursduring continuous intravenous (cIV) infusion of blinatumomab(week 1); and following dose step(s) [day 8 (and day 15)] at thesame time intervals [week 2 (and week 3)]. Cerebrospinal fluid(CSF) samples were taken from an adult with r/r NHL(NCT00274742) on day 2 after treatment discontinuation due toNAEs and a compassionate-use pediatric patient with r/r ALL onday 15 and day 43 of cIV infusion. PBMC and CSF samples wereanalyzed by flow cytometry on a FACSCanto II system (BDBiosciences). T and B cells were stained by dye-labeled antibodiesagainst CD3 (BD Biosciences) and CD19 (Agilent), respectively.T-cell adhesiveness was assessed by binding of a human intercellularadhesion molecule (ICAM)-1/Fc chimera protein (Bio-Techne) to theintermediate-affinity conformation of lymphocyte function-associatedantigen (LFA)-1 on T cells and subsequent detection by dye-labeledantibodies against human Fc (Dianova). Absolute numbers of lym-phocytes, monocytes, and platelets were determined by differentialblood counts.

Angiopoietin-2 assessmentAt time points described above (leukocyte analysis), plasma con-

centrations of angiopoietin (Ang)-2, an endothelial activation bio-marker, were measured for 10 patients with r/r NHL (NCT00274742)using the Human Angiopoietin-2 Quantikine ELISA Kit (Bio-Techne)according to the manufacturer's instructions. Briefly, 100 mL of assaydiluent followed by 50 mL of standard, control, or prediluted samplewere added to each well of a precoated microplate. After incubation atroom temperature for 2 hours and subsequent four-time washing, 200mL of conjugate were added. The plate was incubated at roomtemperature for 2 hours and washing steps were repeated before200 mL of substrate were added. After light-protected incubation atroom temperature for 30 minutes, 50 mL of stop solution were added.Finally, the plate was read within 30 minutes at 450/570 nm on aPowerWaveX Select instrument (BioTek).

HistopathologyIHC was performed with a biotin/avidin/peroxidase technique as

previously described in detail (19). Paraffin blocks of formaldehyde-fixed brain autopsy tissues from frontal cortex and white matter,cerebellum, and hippocampus were available for analysis. Serial par-affin sections were stained by IHC with human-specific antibodiesagainst T-cell markers CD3 (Thermo Fisher Scientific) and CD8(Agilent).

Pharmacokinetic assessmentBlinatumomab serum and CSF concentrations were quantified

using a validated T-cell activation assay as described previous-ly (14, 17). Briefly, Raji cells (human B-cell line) were incubatedwith HPB-ALL cells (human T-cell line) in the presence of serialblinatumomab dilutions in serum ranging from 200 ng/mL to3 pg/mL. A blank sample was included to measure backgroundsignal. To avoid any matrix effects of CSF on the assay, purified ratanti-blinatumomab antibodies were spiked into one half of the CSFsample to simulate a predose (i.e., blank) sample whereas the otherhalf remained unspiked. After incubation, T cells were labeled withmouse anti-human CD69 IgG1 FITC (BD Biosciences) and ana-lyzed on a FACSCanto II system (BD Biosciences). The activationmarker CD69 was expressed on T cells in a blinatumomab con-centration-dependent manner.

Table 1. Patient descriptions and selection criteria.

Description N Selection criteria Analyses

Adults with r/r NHL 10 Available Ang-2 measurements Leukocytes and Ang-2 concentrationsNCT00274742Fig. 1A–DAdult with r/r ALL 1 Recurring NAEs; available brain autopsy tissue HistopathologyNCT01209286Fig. 2APediatric patient with r/r ALL 1 No NAEs; available CSF during/after cIV infusion Lymphocyte and T-cell subpopulationsCompassionate useFig. 2BAdult with r/r NHL 1 Recurring NAEs; available CSF after NAEs Lymphocyte subpopulationsNCT00274742Fig. 2CAdults with r/r NHL 6 PPS coadministration (n ¼ 3); comparable blinatumomab

dosing regimens w/o peripheral B cellsaT-cell redistribution kinetics

NCT00274742Fig. 5

aB/T-cell ratio of patients with PPS coadministration: 90/1,319, 21/2,537, 4/332; of patients without PPS coadministration: 0/141; 0/524; 64/764.

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Multivariate analysesBecause of multiple correlating factors potentially leading to the

development of NAEs, data of blinatumomab-treated patients with r/rNHL or r/r ALL from various studies (NCT00274742, NCT01741792,NCT01209286,NCT01466179; n¼ 188 at data cutoff) or of the r/rALLsubset (n¼ 92 at data cutoff) were pooled and subjected tomultivariateanalyses to identify any associated or predictive risk factors. Covariatesassessed in the regression analysis were sex, age, B/T-cell ratio, diseaseseverity, steroid pretreatment, maximum dose received, cytokinelevels, and laboratory values (e.g., monocytes).

Endothelial cell lines and culturing of cells under hydrodynamicconditions

Human brain microvascular endothelial cells (HBMEC) were pur-chased from ScienCell Research Laboratories (#1000) and humanumbilical vein endothelial cells (HUVEC) were purchased fromPromoCell (#C-12200). Both cell lines were confirmed to be negativefor Mycoplasma by Venor GeM Advance Mycoplasma Detection Kit,and were not analyzed further for validation. Culturing of HBMECs orHUVECs under constant unidirectional flow conditions was doneusing the Ibidi Pump System (Ibidi) according to the manufacturer'sinstructions until passage 11. Briefly, HBMECs or HUVECs wereseeded into m-slide I0.4 Luer (Ibidi; Collagen IV or ibiTreat, respec-tively) and cultured at 37�C, 5% CO2 for 48 hours in RPMI1640medium (Biochrom; supplemented with Nu-Serum IV, FBS, MinimalEssential Medium, L-glutamine, sodium pyruvate, heparin, Endothe-lial Cell Growth Supplement) at shear stress of 5 dyn/cm2, or inendothelial cell growth medium (PromoCell; supplemented with FBS)at 10 dyn/cm2, respectively. Preincubation of endothelial cells with 200mg/mL PPS SP54 (bene-Arzneimittel) was initiated 24 hours after startof cell culturing under constant unidirectional flow conditions.

Isolation and preparation of T cells for rolling experimentsPBMCswere isolated from freshblood of healthydonors as described

previously (17, 18). Subsequently, T cells were purified from PBMCsusing the humanPan TCell Isolation Kit (Miltenyi Biotec) according tothemanufacturer's instructions. Preincubation of T cells with 50mg/mLminocycline (Triax Pharmaceuticals) or with 1 mg/mL natalizumab(Elan Pharma International) was done at 37�C, 5% CO2 for 2 hours inDPBS or for 10 minutes in RPMI1640, respectively. T cells wereresuspended in RPMI1640 medium before rolling experiments.

T-cell rolling and adhesion experimentsT-cell interactions with endothelial cells under hydrodynamic

conditions were visualized using an in vitro flow chamber system.This system consisted of the pump system connected to a m-slide I0.4

Luer containing confluently grown endothelial cells, a heating system,and a CO2 gas incubation unit (all Ibidi). This allowed application of aconstant unidirectional flow at physiologic conditions (37�C, 5%CO2)within the channel of the m-slide ensuring viability of both T cells andendothelial cells during long-term (�2 hours) assays. Rolling experi-ments utilized HBMECs, whereas firm adhesion was only measurableto HUVECs. T-cell rolling and adhesion were closely monitored usinga microscopic system consisting of an inverse microscope (Nikon), adigital camera (Hamamatsu Photonics), and an imaging software(NIS-Elements AR; Nikon). Experiments were run at a T-cell con-centration of 1� 106/mL�10 ng/mL blinatumomab, and additionally�200mg/mLPPS,�50 mg/mLminocycline, or�1mg/mL natalizumabat shear stress of 1 dyn/cm2 for up to 2 hours. Individual T-cell rollingvelocities and absolute numbers of adhering T cells were determinedby automatic ormanual tracking of single T cells at 0 and 45minutes of

continuous rolling on endothelial cells using the NIS-Elements ARsoftware. Subsequently, the mean T-cell rolling velocity (�SD) oftrackable T cells was calculated for each experimental condition.

IHCAfter T-cell rolling on endothelial cells, m-slides were fixed with 4%

paraformaldehyde solution (Sigma-Aldrich) and blocked with anavidin/biotin blocking reagent (Dianova) prior to immunostaining ofP-selectin, ICAM-1, and vascular cell adhesion molecule (VCAM)-1.P-selectin surface expression on endothelial cells was visualized bystaining with a mouse anti-human P-selectin IgG1 (15 mg/mL; Bio-Techne) and goat anti-mouse IgG secondary antibody, Alexa Fluor 488(1:100; Thermo Fisher Scientific). Staining of ICAM-1 was done withrabbit anti-human ICAM-1 IgG, biotin (10 mg/mL; Abcam) andstreptavidin, Cy3 (1:100; Dianova). VCAM-1 expression was assessedwith rabbit anti-human VCAM-1 IgG (5 mg/mL; Abcam) and goatanti-rabbit IgG secondary antibody, DyLight 350 (20 mg/mL; ThermoFisher Scientific). Stained HBMECs were analyzed by fluorescencemicroscopy using ultraviolet light, and image acquisitionwith theNIS-Elements AR software.

PPS coadministrationThree patients with r/r NHL (NCT00274742) and at high risk of

developing NAEs due to their low B/T-cell ratios (Table 1) receivedPPS coadministration at 100 mg by bolus intravenous infusion 1 to3 hours before start of blinatumomab infusion and dose step followedby intravenous perfusion of 300 mg/day for 48 hours thereafter (10).Three additional patients with r/r NHL from the same phase I studywith similar baseline characteristics and a comparable blinatumomabstep-dosing regimen but no PPS coadministration were selected as acomparator group based on the absence of peripheral B cells at start ofinfusion or dose step, which is a prerequisite to demonstrate an effect ofPPS on T-cell redistribution kinetics.

Statistical analysisStatistical interpretation of in vitro T-cell rolling velocity data was

performed by one-way ANOVA combined with Tukey post hoc testusing the Prism software (GraphPad Software). A P <0.05 wasregarded as statistically significant.

Data and material availabilityQualified researchersmay request data fromAmgen clinical studies.

Complete details are available at the following link: http://www.amgen.com/datasharing

ResultsT-cell redistribution coincides with endothelial activation

The pharmacodynamic effects of blinatumomab, more extensivelyanalyzed at start of infusion in a subset of 10 patients with r/r NHL(NCT00274742) were consistent with previous studies (Fig. 1;refs. 18, 20). Infusion of blinatumomab and modulation of itssteady-state serum concentration (i.e., dose step) induced rapid T-cell redistribution characterized by T cells disappearing from periph-eral blood within 2 to 6 hours, followed by their subsequent return tobaseline levels within the following 7 days (Fig. 1A). Coincident withT-cell disappearance, the adhesion molecule LFA-1 on T cells shiftedfrom a low to an intermediate affinity conformation (21) as evidencedby its increased binding to an ICAM-1/Fc chimera protein (Fig. 1B).Because this increased T-cell adhesiveness suggested interactions withblood vessel endothelium, we further investigated the release of Ang-2

Mitigation of Blinatumomab-Associated NAE

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by endothelial cells, a marker of endothelial cell activation. Ang-2 isstored in Weibel–Palade bodies within endothelial cells and is releasedintoperipheral blooduponactivationafter interactionswithT cells (22).Consistent with T-cell redistribution kinetics, Ang-2 serum concentra-tions increased markedly within 6 hours after start of infusion beforeslowly returning to baseline within the following 7 days (Fig. 1C).Moreover, monocytes (Fig. 1D) and platelets displayed redistributionpatterns similar to T cells, although they are not directly engaged byblinatumomab. Effects on redistribution and endothelial activationsimilar to those shown in Fig. 1A–D were observed at dose step(s)and in patients with minimal residual disease (MRD) or r/r ALL.

T cells transmigrate through brain microvascular endotheliumand are detectable in CSF

Histopathologic examination of brain sections and analyses of CSFfrom patients treated with blinatumomab identified T cells at the brainmicrovascular endothelium and in CSF (Fig. 2); moreover, blinatu-momab was detectable in CSF.

Histopathology of brain sections revealed CD3þ and CD8þ T cellsat the luminal surface of capillary endothelium (Fig. 2A) and in theunderlying perivascular or leptomeningeal space of a 24-year-oldpatient with r/r ALL (NCT01209286) who was treated at reduceddose of 5 mg/m2/day due to recurring NAEs in cycles 1 and 2.Blinatumomab infusion was permanently discontinued due to infec-

tion in cycle 3, and the patient died 4 days later due to a brain steminfarction caused by a fungal plaque.

A 17-year-old compassionate-use patient with r/r ALLwas treated ata dose of 15 mg/m2/day for 6 weeks without evidence of NAEs or anopen blood–CSF barrier, as monitored by CSF albumin concentration(day 15 and 43). Blinatumomab concentration in CSF was measuredand below the limit of detection throughout treatment.At day 15 (undercIV infusion), a low level of predominantly CD8þ T cells was found inCSF (Fig. 2B); no B or natural killer cells were observed in CSF. At day43 (immediately after end of infusion), a marked increase in T-cellcounts throughout all T-cell subpopulations was detected (Fig. 2B).

A 55-year-old patient with r/r NHL (NCT00274742) who wastreated at a dose of 60 mg/m2/day and permanently discontinuedblinatumomab infusion at day 2 of an additional treatment cycle due toNAEs had quantifiable blinatumomab of 20 pg/mL in CSF. FACSanalysis of CSF taken immediately after infusion termination revealednot only an increased white blood cell count of 6/mL, but also thepresence of CD3þ T cells and CD19þ B cells or B-cell debris (Fig. 2C).

Blinatumomab concentration in CSF was also systematically eval-uated in children and adolescents with r/r ALL treated with thestandard 5 to 15 mg/m2/day dose-step regimen (NCT01471782). Atday 15 (under cIV infusion), a mean (�SD) blinatumomab concen-tration in CSF of 18.2 (�26.2) pg/mL was measured; the mean (�SD)CSF/serum concentration ratio was 0.036 (�0.061). Although most of

Figure 1.

Leukocyte redistribution and endothelial activation at start of blinatumomab infusion in patients with r/r NHL. A, T-cell redistribution in peripheral blood. B,Conformational shift of LFA-1 on T cells from a low to an intermediate-affinity state (LFA-1þ). C, Ang-2 concentration in serum. D, Monocyte redistribution inperipheral blood. Mean values (þSD) of 4 to 10 corresponding patients with r/r NHL treated in study NCT00274742 are depicted. The value on study day 1 ispreinfusion.

Klinger et al.





these patients presented with none to only mild disturbance of theirblood–CSF barrier, a maximal blinatumomab concentration in CSF of94.0 pg/mL was detected in a patient with an open blood–CSF barrier.

A low peripheral B/T-cell ratio increases the risk of developingNAEs

The multivariate analyses identified the ratio of B cells to T cells inperipheral blood before the start of infusion (B/T-cell ratio) as themostconsistent risk factor for developing NAEs. In the full analysis set (n¼188), 74% (57/77) of patients with a B/T-cell ratio <1/8 developed

NAEs throughout blinatumomab treatment compared with 51% (56/111) of patients with a B/T-cell ratio�1/8 (P¼ 0.0001; SupplementaryTable S2). In the r/r ALL subset, 82% (23/28) of patients with a B/T-cellratio <1/8 had NAEs compared with 52% (33/64) of patients with a B/T-cell ratio�1/8 (P¼ 0.006; Supplementary Table S2). A low B/T-cellratio seems to be associatedwith a higher risk of developingNAEs earlyduring the first treatment cycle, whereas in subsequent cycles, the B/T-cell ratio seems to have less significance. Interestingly, the incidencerate of grade �3 NAEs was lower in patients with MRD ALL (13%;ref. 23) compared with patients with r/r NHL (30%; ref. 10), despite

Figure 2.

T-cell migration into the CNS duringblinatumomab infusion. A, Histopath-ologic examination of brain sectionstaken from a patient with r/r ALL(NCT01209286) who had died dueto a brain stem infarction causedby a fungal plaque. IHC staining of bothCD3þ and CD8þ T cells is shown.B, FACS analysis of CSF taken from acompassionate-use pediatric patientwith r/r ALL during blinatumomabinfusion at 15 mg/m2/day (day 15) andimmediately after end of infusion (day43). Lymphocyte and T-cell subpopu-lation counts are displayed. Therewas no evidence for an open blood–CSF barrier. Blinatumomab concentra-tion in CSF was below the limit ofdetection. C, FACS analysis of CSFtaken from a patient with r/r NHL(NCT00274742) immediately after dis-continuation of blinatumomab infusionat 60 mg/m2/day due to grade �3NAEs on day 2 of an additional treat-ment cycle. CD3þ T cells and CD19þ B-cell debris are depicted. There wasindication of a disturbed blood–CSFbarrier. White blood cell count in CSFwas 6,000/mL. Blinatumomab con-centration in CSF was 20 pg/mL.






very low peripheral B-cell counts before start of infusion in patientswith MRD ALL (18).

Blinatumomab reduces T-cell rolling velocity and promotes T-cell adhesion to vascular endothelial cells, leading toendothelial cell activation in vitro

Leukocyte extravasation is a complex multistep cascade of eventsincluding initial rolling on, subsequent firm adhesion to, and finaltransmigration through the blood vessel endothelial cell layer (24).Weestablished an in vitro flow chamber system mimicking capillaryhydrodynamic conditions, which allowed us to measure blinatumo-mab-mediated effects on T-cell rolling and adhesion to HBMECs and

HUVECs, respectively (in the absence of target B cells). Freshlyisolated T cells from healthy volunteers were circulated for 45 minuteson a monolayer of flow-cultivated HBMECs and T-cell rolling veloc-ities were measured. No difference in mean (�SD) T-cell rollingvelocities was seen between the beginning [344 (�58) mm/second]and end of the flow experiment [323 (�78) mm/second; Fig. 3A].When blinatumomab (10 ng/mL) was added to the flow chambersystem, the mean (�SD) T-cell rolling velocity after 45 minutes ofcontinuous circulation was significantly reduced to 209 (�40) mm/second (�1.5-fold reduction; Fig. 3A), suggesting increased T-cellinteractions with HBMECs. However, this effect was not immediate[342 (�69) mm/second at t ¼ 0 minute; Fig. 3A]. Moreover, the

Figure 3.

In vitro flow chamber systemmimicking blinatumomab-induced T-cell redistribution and endothelial activation.A,Mean (þSD) T-cell rolling velocities on HBMECs�blinatumomabafter 0minute and45minutes of continuous circulation are displayed. SignificancewasdeterminedbyANOVAcombinedwith Tukeypost hoc test andis denoted by number (N) of tracked T cells. �� , P <0.001; ns, nonsignificant, P�0.05.B, IHC staining of different adhesionmolecules on the surface of HBMECs after45 minutes of continuous T-cell rolling �blinatumomab.

Klinger et al.





Figure 4.

Interference of antiadhesive substanceswith blinatumomab-induced interactions between T cells and vascular endothelial cells in vitro.A,Mean (þSD) T-cell rollingvelocities on HBMECs�blinatumomab and�PPS after 45 minutes of continuous circulation are shown. B, IHC staining of P-selectin on the surface of HBMECs after45 minutes of continuous T-cell rolling � blinatumomab and �PPS. C, Mean (þSD) T-cell rolling velocities on HBMECs �blinatumomab and �minocycline after45 minutes of continuous circulation are displayed. D, Absolute numbers of T cells (within one image section) firmly adhering to HUVECs after 45 minutes ofcontinuous T-cell rolling�blinatumomab and�minocycline are shown. E,Mean (þSD) T-cell rolling velocities on HBMECs�blinatumomab and�natalizumab after45 minutes of continuous circulation are displayed. Significance was determined by ANOVA combined with Tukey post hoc test and is denoted by number (N) oftracked T cells. � , P < 0.05; �� , P < 0.01; �� , P < 0.001; ns, nonsignificant, P � 0.05.






addition of blinatumomab also increased the absolute number of Tcells adhering to HUVECs after 45 minutes of continuous circulationcompared with the adhesion of T cells to HUVECs in the absence ofblinatumomab. In addition, immunofluorescence analysis ofHBMECs after T-cell rolling in the presence of blinatumomab showedpronounced upregulation of endothelial adhesionmolecules: ICAM-1,P-selectin, and VCAM-1 (Fig. 3B). Thus, blinatumomab addition tothe in vitro flow chamber system reduced T-cell rolling velocity andincreased T-cell adhesion, coinciding with upregulation of relevantadhesion molecules on HBMECs.

Antiadhesive substances interfere with blinatumomab-inducedinteractions between T cells and vascular endothelial cellsin vitro

We analyzed the potential of substances with antiadhesive proper-ties to reverse the effects of blinatumomab on T-cell rolling andadhesion. HBMECs were preincubated with or without the semisyn-thetic heparinoid PPS, an inhibitor of P-selectin expressed on theluminal surface of endothelial cells (25). Addition of PPS to the rollingexperiments increased themean (�SD)T-cell rolling velocity of freshlyisolated T cells from 399 (�153) mm/second to 515 (�159) mm/second(Fig. 4A). Moreover, addition of PPS in the presence of blinatumomabresulted in a mean (�SD) T-cell rolling velocity of 465 (�116) mm/second (Fig. 4A), which was comparable with the mean T-cell rollingvelocity without blinatumomab. In line with these observations,immunofluorescence staining of HBMECs after T-cell rolling showedthat PPS treatment markedly decreased P-selectin signals comparedwith untreated cells with or without blinatumomab (Fig. 4B).

Another agent with potential antiadhesive properties is thetetracycline antibiotic minocycline, which interferes with LFA-1binding to ICAM-1 by chelating Ca2þ ions and by downregulatingLFA-1 on T cells (26). T cells preincubated with minocycline had asignificantly higher rolling velocity than untreated cells, demon-

strating minocycline as an effective antiadhesive agent (Fig. 4C). Asbefore, addition of blinatumomab markedly reduced the mean(�SD) T-cell rolling velocity [127 (�41) mm/second]; however,this attenuated effect was blocked when T cells were preincubatedwith minocycline [217 (�92) mm/second; Fig. 4C]. Addition ofminocycline also reversed the blinatumomab-induced increase offirm T-cell adhesion to HUVECs (Fig. 4D). A third compound withantiadhesive properties is the humanized mAb natalizumab, whichbinds to very late antigen (VLA)-4 on T cells and blocks VLA-4interaction with VCAM-1 on endothelial cells (27). Results com-parable with PPS and minocycline were obtained (Fig. 4E). Insummary, these three distinct antiadhesive agents prevented blina-tumomab-induced reduction of mean T-cell rolling velocities andadhesion to vascular endothelial cells in vitro.

T-cell redistribution kinetics in vivoThe initial T-cell kinetics differed for the 3 patients who received

PPS compared with those who did not (Fig. 5). In contrast tothe usually measured 20% to 60% decline in T-cell counts 45 minutesafter start of blinatumomab infusion or dose step in the absence of PPS(Fig. 5, top), T-cell counts remained at or above baseline levels at 45minutes in samples from the 3 patients treated with PPS (Fig. 5,bottom). T-cell redistribution was delayed in patients who receivedPPS. T-cell redistribution started 2 to 6 hours after start of infusion ordose step and was prolonged relative to patients without PPS coad-ministration. Interestingly, only one of the 3 patients who received PPScoadministration had moderate NAEs 48 hours after the blinatumo-mab dose step (when the PPS IV perfusion had been stopped) despitetheir low B/T-cell ratios (Table 1). These observations are consistentwith PPS transiently preventing T cells from entering the perivascularspace (PVS), where they could be activated by (rare) target B cells andtrigger NAEs through the release of cytokines and other proinflam-matory factors.

Figure 5.

Delayed T-cell redistribution kinetics inthe absence of peripheral blood target Bcells in three patients with r/r NHL(NCT00274742) who received PPScoadministration at start of blinatumo-mab infusion (5 mg/m2/day) and dosestep (60 mg/m2/day). Absolute T-cell(and B-cell) counts in peripheral bloodprior to, and 45 minutes, and 2 hoursafter either start of infusion or dose stepare plotted for the three patients withPPS coadministration (bottom). Exem-plary T-cell (and B-cell) kinetics of threepatients from the same study who didnot receive PPS coadministration arealso depicted at start of infusion or dosestep (top). Blinatumomab dose levelsafter start of infusion or dose step arematched with those shown for the threepatients with PPS coadministration.

Klinger et al.





DiscussionThe clinical and nonclinical data reported here build on previous

observations in patients with r/r NHL or r/r ALL and suggest that NAEsare linked to blinatumomab-inducedT-cell activity in theCNS.Changesof blinatumomab steady-state serum concentrations, such as at start ofinfusion and dose step, were associated with T-cell redistributioncharacterized by rapid T-cell disappearance from peripheral blood andsubsequent gradual return to baseline levels within 1 week. T-cellredistribution coincided with a conformational shift of LFA-1 on Tcells from a low to an intermediate-affinity state, as well as an increase inthe serum concentration of the endothelial activation marker Ang-2 (22). Although not directly engaged by blinatumomab, monocyte andplatelet redistribution were also observed.

In a single-patient case study, T-cell adhesion to the luminal surfaceof brain microvascular endothelium was observed followed by poten-tial T-cell transmigration into the PVS through an intact blood–CSFbarrier. These observations suggest that blinatumomabmight facilitateT-cell influx into CSF despite an intact blood–CSF barrier. One canhypothesize that massive leukocyte adhesion to blood vessel endothe-lial cells (including brain microvascular endothelium) might result intransient disturbance of microcirculation and subsequent transientfocal cerebral hypoxia (28), potentially contributing to the reversibleclinical symptoms of blinatumomab-associated NAEs. Blinatumomabwas detected in CSF of several patients with r/r ALL and of a patientwith r/r NHL. In addition, B-cell debris and an influx of (effector) Tcells were detected in CSF of two patients immediately after end ofinfusion; in one case, infusion was terminated due to a grade�3 NAE.However, we were unable to see a direct relationship between CSFlevels of blinatumomab and neurotoxicity due to small sample size andlow concentrations of blinatumomab. Similar to our observations withblinatumomab, higher numbers of CD19CART cells were seen in CSFof patients who experienced NAEs compared with those who didnot (29). Finally, a low B/T-cell ratio (i.e., a low B-cell count inperipheral blood and bone marrow) has been identified as a potentialrisk factor for the development of NAEs in patients with r/r NHL or r/rALL. The lower incidence of NAEs in children and adolescents with r/rALLmay be explained by the higher number of both peripheral B cellsand bone marrow blasts due to a required blast count of >25% beforestart of blinatumomab treatment. In contrast, the respective inclusioncriterion for adults with r/r ALL was >5% blasts in bone marrow. As

patients with MRD ALL had frequently received standard intrathecalchemotherapy prophylaxis before and throughout blinatumomabtreatment, which might have reduced or even eliminated residualtarget B cells in the brain, the risk of developing severe NAEs may alsohave been reduced irrespective of the B/T-cell ratio. In addition,patients with MRD ALL received blinatumomab as first- or second-line treatment, whereas patients with r/r NHLmay have had increasedresidual target B cells in the brain due to their advanced disease state.

On the basis of our pharmacodynamic and clinical observations, wepropose the following model for the development of blinatumomab-associated NAEs: (i) start of blinatumomab infusion or dose stepsincrease the adhesiveness of circulating T cells to blood vessel endo-thelium, including brain endothelium forming the blood–CSF barrier(Fig. 6A). (ii) Adhering T cells activate the endothelium and begin toextravasate into the PVS. The activated endothelium on its part attractsother circulating leukocytes, such as monocytes (30) and platelets(Fig. 6B). (iii) It is possible that in the absence of peripheral B cells (i.e.,low B/T-cell ratio) extravasated T cells first encounter (rare) target Bcells in the CNSwhere T cells get activated by blinatumomab to secretecytokines and chemokines triggering transient local neuroinflam-mation, including transmigration of monocytes (Fig. 6C). (iv)Although it is speculation, transmigration of monocytes, attractednon-T cells, and released factors into the CNS may disturb theblood–CSF barrier by enhancing transient local neuroinflamma-tion, leading to local micro-edema in the brain, and may aggravateNAEs (Fig. 6D; ref. 31). Interestingly, some aspects of our modelhave also been described in the context of CD19 CAR T-cell–associated neurotoxicity. Particularly, cytokine-induced CNS endo-thelial cell activation was implicated in the early pathophysiology ofNAEs, with the authors reporting disruption of the blood–brainbarrier and cerebral edema in patients with fatal neurotoxicity (32).Of note, no consistent pattern of blood–CSF barrier disruption orpost-baseline changes in CNS MRI have been observed with bli-natumomab. In another study of CD19 CAR T cells, intracranialedema and CAR T-cell influx into the CSF and cerebral CRS wereassociated with the development of NAEs (33). However, althoughCD19 CAR T-cell–associated neurotoxicity may be favored bysystemic CRS (32, 34), blinatumomab-associated NAEs occurindependent of cytokine serum concentrations, especially inpatients with r/r NHL where no CRS has been reported (10, 11). On

Figure 6.

Proposed pathogenetic model forthe development of blinatumomab-associated NAEs.






the basis of the B/T-cell ratio, we hypothesize that the location of initialT-cell activation and subsequent T-cell–mediated cytokine release (i.e.,peripheral blood vs. CSF) is more critical for the development ofblinatumomab-associated NAEs than maximum systemic cytokinelevels, which are relatively low compared with those observed withCAR T-cell therapies.

According to ourmodel, blinatumomab-induced T-cell adhesion tobrain microvascular endothelial cells is the first necessary but notsufficient step for the development of blinatumomab-associatedNAEs.With T cells largely disappearing from circulation within 2 to 6 hoursafter start of infusion and dose step(s), the onset of most NAEs shortlyfollowed this T-cell redistribution and coincided with peak T-celladhesiveness and endothelial activation. Thus, transiently interferingwith leukocyte adhesion at start of blinatumomab infusion and dosestep(s) seems to be a promising approach for mitigating NAEs. Ourresults with the in vitro flow chamber system are consistent withblinatumomab-induced pharmacodynamic effects in vivo (e.g., T-cellredistribution and endothelial activation); however, these findingsshould be replicated in patient-derived T cells. These pharmacody-namic findings support the proposed mode of action of PPS, and noapparent negative impact on either safety or efficacy of blinatumomabtreatment was found with PPS coadministration in a very limitedpatient sample. Thus, PPS coadministration with blinatumomabtreatment may be a beneficial mitigation strategy in patients with lowB/T-cell ratios or other NAE risk factors.

In conclusion, coadministration of antiadhesive agents with blina-tumomab to mitigate NAEs is the first mechanistic-based approach toreduce the risk of developing NAEs. Mitigation of this risk wouldprevent treatment interruptions and/or discontinuations, allow higher(starting) doses of blinatumomab, and thus increase efficacy forpatients with r/r NHL where NAEs (and not CRS) are the dose-limiting adverse events. Transient coadministration of antiadhesiveagents that interfere with T-cell adhesion to blood vessel endotheliumshould be explored in clinical studies as a potential mitigation strategyfor blinatumomab-associated NAEs to further improve the safety andefficacy of blinatumomab therapy.

Disclosure of Potential Conflicts of InterestM. Klinger is a principal scientist and has ownership interest (including patents)

in Amgen, Inc. G. Zugmaier is an executive director at Amgen, Inc. and hasownership interest (including patents) in 9688760, 20190300609, 20150071928,

8840888, 20140228316, 20140227272, 20130323247, 20130287778, 20130287774,20110262440, 20100112603, 7700299, 20190142846, 20070037228, 20190127465,10130638, 20170327581, 20170122947, 9486475, 20160208001, and 9192665.V. N€agele is a senior scientist and has ownership interest (including patents) inAmgen Inc. M.-E. Goebeler reports consulting or advisory roles for ROCHE PharmaAG,Novartis, andGemoab and received travel grants fromBMS, Pfizer, JanssenCilag,andGilead. C. Brandl is a principal scientist at Amgen, Inc.M. Stelljes has a consultingor advisory role at Pfizer, Jazz Pharmaceuticals, Gilead Sciences, MSD, and Amgen;has received honararia from Speakers' Bureau from Pfizer, Medac, MSD, and Incyte;reports receiving research funding from Pfizer (Inst); and reports travel, accommoda-tions, and expenses fromMedac andNeovii Biotech. A. von Stackelberg is an advisoryboardmember ofMorphosys andRoche; has received speakers bureau honoraria fromAmgen, Novartis, Shire, and Miltenyi; and is a consultant/advisory board member ofPfizer. R.C. Bargou has received speakers bureau honoraria from Amgen and hasownership interest (including patents) in patent blinatumomab. P. Kufer is anexecutive director at BiTE Technology and has ownership interest (including patents)in Amgen. No potential conflicts of interest were disclosed by the other authors.

Authors’ ContributionsConception and design: M. Klinger, G. Zugmaier, R.C. Bargou, P. KuferDevelopment of methodology: M. Klinger, V. N€agele, C. BrandlAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): M. Klinger, V. N€agele, M.-E. Goebeler, C. Brandl, M. Stelljes,H. Lassmann, A. von Stackelberg, R.C. BargouAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): M. Klinger, G. Zugmaier, V. N€agele, M.-E. Goebeler,C. Brandl, H. Lassmann, R.C. Bargou, P. KuferWriting, review, and/or revision of the manuscript: M. Klinger, G. Zugmaier,V. N€agele, M.-E. Goebeler, C. Brandl, M. Stelljes, H. Lassmann, A. von Stackelberg,R.C. Bargou, P. KuferStudy supervision: R.C. Bargou, P. Kufer

AcknowledgmentsThe authors would like to thank Prof Klaus-Michael M€uller, MD, Department of

Pathology, and Prof Tanja Kuhlmann, MD, Department of Neuropathology, Uni-versity Hospital M€unster, M€unster, Germany, for performing the initial brainautopsy. Editorial support was provided by Allison Saviano and Kathleen Raulin ofSephirus Communications Inc., funded by Amgen Inc., and Beatrice Chiang andJulie Gegner, employees of Amgen Inc. The study was funded by Amgen Research(Munich) GmbH and Amgen Inc.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 10, 2019; revised September 13, 2019; accepted October 23, 2019;published first October 29, 2019.

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2020;80:91-101. Published OnlineFirst October 29, 2019.Cancer Res Matthias Klinger, Gerhard Zugmaier, Virginie Nägele, et al. Blinatumomab-Associated Neurologic Adverse EventsAdhesion of T Cells to Endothelial Cells Facilitates

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