PHASE I STUDIES

A phase I study of the human monoclonal anti-NRP1antibody MNRP1685A in patients with advanced solid tumors

Colin D. Weekes & Muralidhar Beeram & Anthony W. Tolcher &

Kyriakos P. Papadopoulos & Lia Gore & Priti Hegde & Yan Xin & Ron Yu &

L. Mason Shih & Hong Xiang & Rainer K. Brachmann & Amita Patnaik

Received: 15 October 2013 /Accepted: 3 February 2014# Springer Science+Business Media New York 2014

Summary The human monoclonal antibody MNRP1685Atargets the VEGF binding domain of neuropilin-1 (NRP1), amulti-domain receptor necessary for neural development andblood vessel maturation. In nonclinical studies, MNRP1685Aprevents vascular maturation by keeping blood vessels in animmature, highly VEGF-dependent state. We explored thesafety and tolerability of MNRP1685A in patients with ad-vanced solid tumors. Patients were treated with MNRP1685Agiven intravenously every 3 weeks using a 3+3 dose-escalation design with 7 dose-escalation cohorts. Twenty-fourof 35 patients (69 %) experienced drug-related adverse events(AEs) of infusion-related reaction on the day of MNRP1685Aadministration. With premedication including dexamethasone,infusions were well-tolerated with main symptoms of pruritusand rash. Outside the day of infusion, most common (≥ 2patients) related AEs were fatigue (17 %), pruritus (9 %), my-algia and thrombocytopenia (both 6 %) (all were Grade 1–2).MNRP1685A-related Grade ≥ 3 AEs consisted of one dose-limiting toxicity of Grade 3 upper gastrointestinal bleeding and

one related Grade 3 thrombocytopenia, coinciding with unrelat-ed Grade 3 fungemia and duodenal obstruction. MNRP1685Ashowed nonlinear PK with more-than-dose proportional in-creases in exposure, consistent with broad target expression.Transient platelet count reductions (≥ 30 % from predose) wereobserved in 56 % of evaluable patients. Nine patients were onstudy for ≥ 4 cycles, one colorectal cancer patient for one year.MNRP1685A was generally well-tolerated. The primaryMNRP1685A-related AE was infusion-related reaction, whichwere attenuated by premedication including dexamethasone.Transient platelet count reductions were frequent but did notimpact MNRP1685A dosing.

Keywords Neuropilin-1 .MNRP1685A . VEGF .

Clinical trial

Introduction

Neuropilin-1 (NRP1) is a multi-functional receptor that con-tributes to the development of both the nervous and vascularsystems [1]. During embryogenesis, NRP1 binds thesemaphorin 3A ligand in the central nervous system (CNS),acting with plexin co-receptors to regulate axon guidance [2].Concurrently, NRP1 binds members of the vascular endothe-lial growth factor (VEGF) ligand family, including VEGF andPlacental Growth Factor 2 (PlGF), to mediate angiogenesis[3–5]. Loss of NRP1 function results in vascular remodelingand branching defects [6–8]. NRP1 has also been reported as adirect tumor target, possibly regulating tumor cell survival andproliferation [8–10].

MNRP1685A (Genentech, Inc.) is a first-in-class, phage-derived, high-affinity recombinant human IgG1 monoclonalantibody that targets NRP1 [11, 12]. MNRP1685A blocksbinding of VEGF to the b1b2 domain of NRP1 on vascular

Electronic supplementary material The online version of this article(doi:10.1007/s10637-014-0071-z) contains supplementary material,which is available to authorized users.

C. D. Weekes (*) : L. GoreUniversity of Colorado School of Medicine and DevelopmentalTherapeutics Program, University of Colorado Cancer Center,Mail Stop 8117, RC1 South, Rm 8123, 12801 E. 17th Avenue,Aurora, CO 80045, USAe-mail: colin.weekes@ucdenver.edu

M. Beeram :A. W. Tolcher :K. P. Papadopoulos :A. PatnaikSouth Texas Accelerated Research Therapeutics, San Antonio,TX, USA

P. Hegde :Y. Xin :R. Yu : L. M. Shih :H. Xiang : R. K. BrachmannGenentech Research and Early Development, South San Francisco,CA, USA

Invest New DrugsDOI 10.1007/s10637-014-0071-z

endothelial cells to reduce angiogenesis and vascularremodeling. MNRP1685A does not block binding ofsemaphorin 3A to NRP1, thereby leaving neuronal guid-ance intact [13]. In xenograft tumor models, single-agent anti-NRP1 resulted in immature intratumoralblood vessel formation with scarce coverage by supportcells, including pericytes. Combining anti-NRP1 withanti-VEGF treatment resulted in synergistic inhibitionof intratumoral vessel formation to promote tumorgrowth inhibition [14], supporting a role for NRP1blockade in causing increased survival dependency onVEGF.

A single-agent Phase Ia dose escalation study was initiatedin patients with solid tumors to study safety, pharmacokinetics(PK), pharmacodynamics (PD) and to evaluate early signs ofefficacy of MNRP1685A administered intravenously.

Materials and methods

Study participants were recruited by 2 centers in the US.Institutional review boards at each study site approved theprotocol (ClinicalTrials.gov identifier NCT00747734), and allsubjects provided written informed consent.

Patients

Eligible patients were male or female, ≥ 18 years of age, withhistologically or cytologically documented, incurable, locallyadvanced, or metastatic solid malignancy that had progressedon, or failed to respond to, at least one prior regimen, and anECOG performance status of 0 or 1. Patients had to haveevaluable disease per RECIST (version 1.0). Prostate andovarian cancer patients were eligible if PSA and CA-125,respectively, met certain elevation criteria. Key exclusioncriteria included inadequate hematologic and organ function;primary CNS malignancy or untreated/active CNS metasta-ses; and any condition causing bevacizumab ineligibility.

Study design

This study was designed to evaluate the safety, tolerability,PK, and PD of MNRP1685A utilizing a 3+3 dose-escalationdesign. Seven dose levels (2, 5, 10, 15, 20, 30, and 40 mg/kg)were evaluated to determine the maximum tolerated dose(MTD) or the maximum administered dose (MAD) (Fig. 1a).

Assessments

Safety Safety was assessed at each visit and 30 days after thelast dose. The DLT assessment window was 21 days. DLTswere defined as NCI-CTCAE (version 3.0) Grade ≥ 3 non-hematologic or non-hepatic major organ AE, any Grade ≥ 3

hepatic transaminases or alkaline phosphatase, Grade ≥ 4thrombocytopenia or neutropenia of a certain time period,Grade ≥ 3 febrile neutropenia and bevacizumab-relatedGrade ≥ 1 or ≥ 2 AEs. If a DLT occurred in 1 patient, then 3additional patients were to be enrolled to the specified cohort;dose escalation was allowed if no further DLT occurred. IfDLT occurred in ≥ 2 patients, then the next lower dose levelwas to be evaluated to define the MTD.

To characterize infusion-related reactions (IRRs), plasmasamples from 19 patients were assayed for 89 chemokines,cytokines, and growth factors using a multiplex Luminex-based ELISA assay (Rules Based Medicine, Austin, TX).

Pharmacokinetics PK serum samples were obtained predoseand post dose at 30 min, 2, 5, and 24 h, as well as Days 4, 8,11, and 15 of Cycle 1. Cycle 2 PK sampling was performedpredose and at 30 min and at Days 8 and 15. PK sampling wasrestricted to Day 1 for all other cycles and at study terminationvisit. MNRP1685A concentration was determined using avalidated ELISA assay with a lower limit of quantificationof 75 ng/mL [11, 12]. PK parameters were derived from non-compartmental analysis (NCA; WinNonlin v5.2.1, PharsigntCorp.,Mountain View, CA) using the complete Cycle 1 serumconcentration-time profile of MNRP1685A from 29 patient.

Serum anti-therapeutic antibody (ATA) samples were col-lected at Day 1 of each cycle (predose), and at study termina-tion. ATA samples were analyzed using a validated bridgingantibody immunoassay [11].

Clinical activity Disease status was evaluated at screeningand every other cycle starting with Cycle 2 using RECIST1.0. The same radiographic procedure used to define baselinemeasurable lesions was used throughout the study for eachpatient.

Statistical analysis

The sample size for this trial (planned enrollment of approx-imately 21–48 patients) was based on the dose-escalationrules. All pat ients who received any amount ofMNRP1685A were included in the analyses for safety andclinical activity. Verbatim descriptions of treatment-emergentAEs were mapped to MedDRA (Medical Dictionary forRegulatory Activities) terminology thesaurus terms (version13.1) and graded according to the NCI-CTCAE (version 3.0).AEs that were assessed by the investigator to be related to thestudy drug and occurred during or after MNRP1685A infu-sion on day of infusion were deemed infusion-related reac-tions. Clinical activity of MNRP1685Awas assessed throughbest overall response and duration of stable disease, whichwas defined as the time from the first dose of MNRP1685A tothe last tumor assessment date on which the patient wasknown to be disease progression-free.

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RESULTS

Study population

Thirty-five patients with a median age of 60 were enrolled tothe study (Table 1). Thirty patients (86 %) werediscontinued from the study because of progressive disease;3 patients (9 %) withdrew consent; and 2 patients (6 %)were discontinued due to AEs (one related Grade 3 uppergastrointestinal hemorrhage and one unrelated Grade 3 gas-trointestinal perforation).

Safety

Adverse events Minimal drug-related Grade 1 or 2 AEs wereobserved. Excluding days of infusion, fatigue was the mostcommon AE occurring in 6 of 35 patients (17 %) (Table 2);two drug-related Grade 3 AEs were observed: 1 DLT oftransient Grade 3 upper gastrointestinal tract hemorrhage(20 mg/kg cohort) and one episode of Grade 3 thrombocyto-penia (30 mg/kg cohort) that coincided with unrelated Grade 3

fungemia and duodenal obstruction. No drug-related Grade 4or 5 AEs were reported.

Thrombocytopenia was the most common hematologictoxicity associated with MNRP1685A administration; Grade1 (n=4), Grade 2 (n=6), and Grade 3 (n=2). Platelet reduc-tions of ≥ 30%were noted in 18 of 32 patients (56 %). Fifteenof these patients had normal baseline platelet counts (≥ 150×109 per liter). Reduced platelet counts remained above 150×109 per liter in 8 of these 15 patients. Reductions in plateletcounts occurred primarily in patients receiving at least10 mg/kg of MNRP1685A (Fig. 2). Three patients at the40 mg/kg dose level underwent additional scheduled plateletsampling on Days 2, 4 or 5, and 11. Two of the 3 patientsexperienced platelet nadir prior to day 8. Platelet recovery tobaseline typically occurred within the 3-week cycle(Supplemental Figure 1). There was no correlation betweenextent of reductions and baseline platelet counts orMNRP1685A PK. However, a slight trend between reductionsand Cmax and AUC for MNRP1685A was observed.Hematologic laboratory evaluation did not provide evidenceof MNRP1685A-induced thrombosis or coagulopathy.

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No dexamethasone premedication (n=13): dose cohorts 2, 5, and 10 mg/kg, 1 patient at 15 mg/kg, 2 patients at 20 mg/kg.

Dexamethasone premedication (n=22): dose cohorts 15 mg/kg (except for 1), 20 mg/kg (except for 2), 30 mg/kg and 40 mg/kg.

9 of 13 with infusion reaction 10 of 22 with infusion reaction55504540353025201510

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Fig. 1 a Study design schema. b Infusion-related symptoms with andwithout dexamethasone premedication (Cycle 1). Premedication in bothgroups (except for dose cohorts 2 and 5 mg/kg and 1 patient at 10mg/kg):Diphenhydramine, H2 receptor antagonist of choice, acetaminophen. c

Observed mean concentration-time profile of MNRP1685A after the firstdose administration. d Levels of MCP-1 over the first 24 h ofMNRP1685A (15 and 20mg/kg cohorts) administrationwith and withoutpremedication

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MNRP1685A-related infusion reactions Infusion-related reac-tions (IRRs) represented the primary AEs associated withMNRP1685 administration (Table 2). Twenty-four patients(69 %) experienced MRNRP1685A-related IRRs, predomi-nantly Grade 2 with no Grade 3–4 events according to NCI-CTCAE 4.0. The study was conducted utilizing NCI-CTCAE3.0 for which infusion reaction is not an AE term. A total of 99MNRP1685 infusions were administered resulting in 116 AEsduring the day of infusion; 55 (47 %) Grade 1, 59 (51 %)Grade 2 and 2 (2 %) Grade 3 AEs. Infusion interruptionsoccurred with 24 (24 %) infusions, 8 (8 %) had infusion ratereductions, 5 (5 %) were not completed. In general, infusion-related AEs decreased for individual patients with increasingnumber of prior MNRP1685A infusions.

Premedication with anti-histamine and anti-inflammatorymedications improved MNRP1685A tolerability. Since IRRswere observed upon initial dose levels in Cycle 1, 4 of 6 patientsat the 2 mg/kg and 5 mg/kg dose levels received premedicationwith diphenhydramine and acetaminophen, with or withoutibuprofen for MNRP1685A infusions beyond Cycle 1.

At the 10 mg/kg dose level, the first patient did not com-plete the first MNRP1685A infusion because of infusion-related symptoms (Grade 1 hypertension and pyrexia, Grade

2 hypoxia and chills). Subsequent patients (n=3) receivedpremedication with diphenhydramine and acetaminophen, oribuprofen prior to cycle 1 dosing.

The first patient at the 20 mg/kg dose level experiencedGrade 2 dizziness, hypotension, pruritus, and chills. The sec-ond patient had Grade 1 headache, Grade 2 dizziness, pyrexia,somnolence, balance disorder, and Grade 3 fatigue despitepremedication with diphenhydramine and acetaminophen.Enrollment to 20 mg/kg was temporarily held, and patientswere enrolled to an intermediate 15 mg/kg dose level to gainadditional experience in the management of IRRs. The firstpatient at 15 mg/kg was premedicated with diphenhydramine,acetaminophen, and ibuprofen, experienced Grade 1 hypoten-sion, and Grade 2 fatigue, headache, and chills. Therefore, allsubsequent patients were required to receive premedicationwith dexamethasone, diphenhydramine, and acetaminophenon the day of MNRP1685A infusion. Administration of oraldexamethasone the evening prior and an intravenous H2receptor antagonist the day of MNRP1685A infusion aspremedication were added at the 30 mg/kg dose level.

A total of 19 drug-related Grade 2 or 3 AEswere reported onthe day of infusion for 8 of 33 MNRP1685A infusions (24 %)without dexamethasone premedication. A total of 42 drug-related Grade 2 or 3 AEs were reported on the day of infusionfor 22 of 66 MNRP1685A infusions (33 %) with dexametha-sone premedication. However, dexamethasone premedicationmostly occurred at higher dose levels. MNRP1685A infusionsappeared to be overall better tolerated with dexamethasonepremedication. Whereas pyrexia, chills, headache, hypotensionand hypertension were the most common AEs on the day ofinfusion without dexamethasone premedication, pruritus, rash,hypotension, and tachycardia were most common after institu-tion of dexamethasone premedication (Fig. 1b).

Plasma samples were assayed for a total of 90 chemokines,cytokines and growth factors to better understand the mecha-nism of MNRP1685A IRRs. Twelve assayed proteins showedacute and transient increases after MNRP1685A infusion(Table 3, see Supplemental Table 2). Of these, premedicationwith dexamethasone led to an attenuated elevation in 6markers as demonstrated for the time points of both 2 and5 h after MNRP1685A infusion, including MCP-1 (Fig. 1c).Four markers, including tumor necrosis factor-α (TNF-α) andvon Willebrand Factor, were not altered by dexamethasonepremedication at the time points evaluated. Two markers,interleukin-1 receptor anatagonist (IL1-Ra) and IL-10,showed further elevation upon steroid administration.

Pharmacokinetics and pharmacodynamics

MNRP1685A displayed typical nonlinear PK in serum follow-ing intravenous infusions, with non-paralleled concentration-time profiles across doses (Fig. 1d) similar to previouslypublished interim results [11, 12]. At low concentrations

Table 1 Patient characteristics

No. of patients (%) All patients (N=35)

Age in years, median (range) 60 (41–78)

Gender

Male 17 (49)

Female 18 (51)

ECOG performance status

0 3 (9)

1 32 (91)

Number of prior systemic therapies, median (range) 4 (0–12)

Number of prior systemic therapies

≤ 2 12 (34)

3 or 4 8 (23)

≥ 5 15 (43)

Tumor type

Colorectal 9 (26)

Ovarian 4 (11)

Pancreas 3 (9)

Cervical 2 (6)

Kidney 2 (6)

Liver 2 (6)

Lung 2 (6)

Other 11 (31)

Other tumor types: one patient each for adenoid cystic, adrenal cortical,breast, endometrial, esophageal, duodenal, gastric, melanoma, neuroen-docrine, peritoneal, and prostate

ECOG Eastern Cooperative Oncology Group

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(below ~10 μg/mL), MNRP1685A concentrations droppedquickly due to significant target-mediated clearance.

Non-compartmental analysis (Supplemental Table 1)demonstrated a dose-proportional increase of meanCmax. Mean clearance decreased with increasing doseand appeared to be approaching a dose-independentmean value of 9–19 mL/day/kg at 15 mg/kg and above.As a result, mean AUC increased more than dose-proportionally across the dose range studied. Mean Vssranged from 42–91 mL/kg and was similar across doselevels. The observed low Vss, as for other monoclonalantibodies, is interpreted as MNRP1685A being largelyconfined to the vascular and interstitial spaces.

Plasma placental growth factor (PlGF) and circulatingNRP1 (cNRP1) were identified as biomarkers of NRP1pathway inhibition and target engagement, respectively[12].

Anti-MNRP1685A antibodies were detected in onepatient in the lowest dose cohort (2 mg/kg) after 6 cy-cles of treatment and in five patients at higher doses(10–30 mg/kg) with the earliest onset of 21 days postdose. The presence of ATAs did not appear to impactthe PK of MNRP1685A based on limited available data(Supplemental Tables 3 and 4).

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Table 2 Related AEs occurring in > 5 % of patients

All patients (N=35)

Event term All AEs (%) AEs on day ofinfusionb (%)

AEs outside dayof infusionc (%)

Grade 1d Grade 2d Grade 3d

Pruritusa 13 (37) 13 (37) 3 (9) 2 10 1

Fatigue 9 (26) 3 (9) 6 (17) 3 5 1

Chills 8 (23) 8 (23) 1 (3) 2 6 –

Hypotension 7 (20) 7 (20) – 1 6 –

Pyrexia 7 (20) 7 (20) 1 (3) 6 1 –

Rash 6 (17) 5 (14) 1 (3) 2 4 –

Dizziness 4 (11) 4 (11) – 1 3 –

Headache 4 (11) 4 (11) 1 (3) 2 2 –

Myalgia 4 (11) 2 (6) 2 (6) 2 2 –

Tachycardia 4 (11) 4 (11) – 3 1 –

Diarrhea 3 (9) 2 (6) 1 (3) 3 – –

Hypertension 3 (9) 3 (9) – 2 1 –

Urticaria 3 (9) 2 (6) 1 (3) – 3 –

Flushing 2 (6) 2 (6) – 1 1 –

Hypoxia 2 (6) 2 (6) – – 2 –

Nausea 2 (6) 2 (6) – 2 – –

Peripheral neuropathy 2 (6) 1 (3) 1 (3) 2 – –

Thrombocytopenia 2 (6) – 2 (6) – 1 1

a Includes “pruritus generalized”b Day of Infusion defined as occurring during or after MNRP1685A infusionc Outside day of infusion defined as occurring any other day or on day of MNRP1685A infusion prior to start of infusiond For patients with several occurrences of a specific AE, only the highest grade is counted

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Clinical activity

Evidence of clinical activity was modest with stable disease asthe best observed response. Eleven of 35 patients (31 %) hadstable disease for ≥ 2 cycles. Three patients (9 %) had stabledisease for ≥ 4 cycles. One patient with colorectal cancer wason study for nearly 1 year with stable disease (Fig. 3).

Discussion

Blockade of NRP1 activity represents a novel anti-angiogenicstrategy since it not only inhibits angiogenesis but also vas-cular remodeling, potentially by altering pericyte function.This Phase Ia study represents the first-in-human experiencewith the human anti-NRP1 antibody MNRP1685A.Administration of MNRP1685A was well-tolerated with pri-mary toxicities of infusion related reactions (IRR) and tran-sient platelet count reductions that were generally wellameliorated by the institution of a defined premedicationregimen that allowed continued dosing. Notably, neuropathywas not an observed MNRP1685A-related toxicity. This ob-servation is consistent with theMNRP1685A specificity to theb1b2 domain of NRP1 that regulates angiogenesis in contrastto the semaphorin 3A binding of the a1a2 domain that regu-lates neurogenesis [2].

Most drug-related AEs occurred within 24 h ofMNRP1685A infusions and were consistent with IRRs.Infusion-related AEs were dose-related as reflected in the

increasing use of premedication that ultimately included dexa-methasone. However, a clear dose response or threshold effectfor MNRP1685A-related IRRs could not be established. Thelack of clear dose response may be due to the fact that conductof the study required simultaneous modification of two vari-ables, dose escalation of MNRP1685A and empiric introduc-tion of dexamethasone premedication.

Therapeutic antibodies fall into several categories regardingIRRs. Rarely do antibodies cause severe IgE-mediated type Ihypersensitivity reactions (anaphylactic reactions). A signifi-cant number, including bevacizumab, are uncommonly asso-ciated with IRRs. One group of antibodies, exemplified bytrastuzumab, has been associated with IRRs without cytokineproduction. Data for MNRP1685A IRRs are consistent withantibodies that induce cytokine release syndrome [15].Cytokine release syndrome occurs with the first administrationand less frequently with subsequent administrations. AEs typ-ically show a decrease in severity with increased number ofadministrations and are manageable with appropriatepremedication. The clinical experience with MNRP1685A isconsistent with these attributes of cytokine release syndrome.

Cytokine release syndrome results in increased circulatingTNF-α and IFN-γ within 2 h, followed by increased IL-6 andIL-10 and on occasion IL-2 and IL-8. The experience withMNRP1685A administration demonstrates increased TNF-α,IL-6, IL-8, and IL-10. Prophylactic dexamethasone adminis-tration decreased the amplitude of MNRP1685A inducedcirculating cytokines with two exceptions. IL-10 and IL-1RA were elevated in association with dexamethasone

Table 3 Cytokine increases after MNRP1685A infusion and impact of dexamethasone premedication

Without dexamethasone premedication (pg/mL) With dexamethasone premedication (pg/mL)

Cytokine Baselinehealthydonors(n=20)

Medianbaseline(n=13)

Median[2 h]

Foldchange[2 h]

P-value Median[5 h]

Foldchange[5 h]

P-value Medianbaseline(n=6)

Median[2 h]

Foldchange[2 h]

P-value Median[5 h]

Foldchange[5 h]

P-value

Attenuation of cytokine increase with dexamethasone premedication

MCP-1 114.5 167 7,420 44.4 0.001 2,195 13.1 0.01 142 1,555 11.0 0.09 189 1.3 0.16

MIP-1b 101.4 208 7,050 33.9 0.003 2,135 10.3 0.0002 152.5 3,140 20.6 0.010 1065.5 7.0 0.06

IL-6 2.6 3.4 100 29.4 0.02 30.5 9.0 0.02 5.45 4.2 0.8 0.27 4.4 0.8 0.34

G-CSF 5.6 8.9 193.5 21.7 0.01 187 21.0 0.005 18.5 242 13.1 0.09 83.5 4.5 0.38

IL-8 5.5 23 148.5 6.5 0.02 127 5.5 0.004 38 186.5 4.9 0.04 70.5 1.9 0.10

MMP-9 26.7 264 1,285 4.9 0.003 1,052 4.0 0.002 528 1,480 2.8 0.14 765.5 1.4 0.55

Further cytokine increase with dexamethasone premedication

IL-1ra 54.3 141 2,470 17.5 0.02 2,370 16.8 0.04 120 7,195 60.0 0.10 2,595 21.6 0.43

IL-10 5.0 15.4 33 2.1 0.03 38.5 2.5 0.008 8.95 146.5 16.4 0.01 38.5 4.3 0.06

No significant change in pattern with dexamethasone premedication

TNF-a 5.3 6.85 36.5 5.3 0.04 27.5 4.0 0.009 8.3 41.5 5.0 0.03 26.5 3.2 0.20

vWF 19.5 14.1 44.5 3.2 0.09 38 2.7 0.06 19 41 2.2 0.07 46 2.4 0.57

TNF RII 3.6 7.1 19 2.7 0.0002 18 2.5 0.0005 8.55 23.5 2.7 0.02 20 2.3 0.49

MIP-1a 68.6 115.5 298.5 2.6 0.06 201 1.7 0.01 107.5 169 1.6 0.01 122 1.1 0.02

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administration. Interleukin-10 elevation has been demonstrat-ed to mediate immune tolerance [16, 17]. IL-1RA is a com-petitive inhibitor of IL-1 receptor binding, resulting in potentanti-inflammatory properties [18]. Dexamethasone inducedIL-10 and IL-1RAmay represent a mechanism of suppressingacute inflammation induced by MNRP1685A.

Hematologic toxicites consisted primarily of platelet countreductions 56 % of patients with evaluable post-infusion plate-let counts demonstrated a reduction of >30 % of pre-doseplatelet levels. Reductions did not result in MNRP1685A dosemodification or altered dose intensity. In general, resolution ofplatelet count reductions to normal or acceptable levels oc-curred with in the 3-week treatment cycle and did not precludethe next scheduled MNRP1685A administration. Platelet countreductions were not associated with an increased bleeding risk,although one DLT of gastrointestinal bleeding with unclearattribution to MNRP1685A was observed. Mechanistic evalu-ation of MNRP1685A-induced platelet reduction support amodel by which MNRP1685A can bind platelets, resulting inmodest platelet activation, leading to platelet clearance [19].There was no associated increased thrombosis risk.

The non-linear kinetics of MNRP1685Awas due to target-mediated clearance, consistent with the wide expression oftarget NRP1. Additional population modeling indicated thatMNRP1685A also has faster nonspecific clearance than typ-ical IgG1 monoclonal antibodies that is probably due tononsaturable off-target binding [11, 12]. The MTD was notdetermined in this study. The MAD was established as40 mg/kg administered once every 3 weeks. Assessment of

exploratory biomarkers of anti-angiogenesis showed thatdoses of 20 mg/kg or higher result in sustained elevation(longer than 15 days) of plasma PlGF [11, 12]. This observa-tion is consistent with MNRP1685A binding to the b1b2domain of NRP1 to alter tumor-related angiogenesis. Thecombined PK/PD analysis suggests that an MNRP1685Adose below the maximum administered dose (i.e., 25 mg/kgevery 3 weeks) is expected to achieve maximum target occu-pancy despite its fast nonspecific clearance.

Single-agent administration of MNRP1685A resulted inmodest evidence of clinical activity with no patients achievingcomplete or partial responses and about one quarter of patientsdemonstrating prolonged stable disease. The majority of thesepatients had either colorectal or ovarian cancer, two epithelialmalignancies for which anti-angiogenesis therapy, such asbevacizumab, has demonstrated clinical benefit [20, 21]. Themodest clinical single-agent activity seen here is consistentwith results from nonclinical efficacy models where single-agent anti-NRP1 showed some reduction in xenografted tu-mor growth, but where a profound additive effect was seenwhen combining anti-NRP1 with anti-VEGF therapy,resulting in altered blood vessel maturation and vascular re-modeling [14]. This observation may be a reflection of theobservation that NRPs are often the only VEGF-receptorsexpressed on tumor cells; thus, binding of VEGF bybevacizumab along with MNRP1685A binding to NRP1may totally inhibit VEGF-related tumorigenic effects [9, 22].This may be an important consideration in the further devel-opment of anti-NRP therapeutics.

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360Time (Days)

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BreastColorectal

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OvarianDuodenalPancreas

GastricAdrenal

MesotheliomaEndometrial

NeuroendocrineSarcomaOvarian

ColorectalRenal cellPancreas

ColorectalNSCLC

ColorectalColorectal

Renal PNETCervical

ColorectalEsophageal

CervicalPancreas

ColorectalColorectal

ProstateColorectalMelanoma

HepatocellularOvarian

Hepatocellular

DLT: G3 UGI bleed

G3 GI perforation

Subject decision to withdraw

Subject decision to withdraw

Subject decision to withdraw

C2: 10 mg/kg for IRR

C2: 15 mg/kg, C4: 10 mg/kg; dose reductions for IRR

Fig. 3 Days on study. Patients off study for progressive disease unless noted otherwise. AIR; Infusion-related Reaction; UGI, upper gastrointestinal

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In summary, MNRP1685 was well tolerated when admin-istered as a single agent. IRRs were manageable withpremedication including dexamethasone. Transient plateletcount reductions were observed but did not appreciably im-pact MNRP1685A dosing. MNRP1685A is predicted to de-pend on concurrent VEGF blockade for clinical efficacy. Toenable testing of this hypothesis in the clinic, a Phase Ib studyfor MNRP1685A in combination with bevacizumab in pa-tients with advanced solid tumors was pursued [23].

Acknowledgement We thank the patients who participated in the studyand their families. Medical writing assistance provided by Genentech, Inc.Research support This study was sponsored by Genentech, Inc.

Disclosure information CDW: advisor to and research support fromGenentech, Inc.

MB: research support from Genentech, Inc.AWT: advisor to and research support from Genentech, Inc.KP: nothing to discloseLG: nothing to disclosePH, YX, RY, LMS, HX, and RKB: employees of Genentech, Inc., and

shareholders of F. Hoffmann La Roche, Ltd.AP: advisor to and research support from Genentech, Inc.

Author contributions CDW: conception and design, collection andassembly of data, data analysis and interpretation, provision of studypatients

MB: collection and assembly of dataAWT: collection and assembly of data, provision of study patientsKP: collection and assembly of dataLG: data analysis and provision of study patientsPH: conception and design, data analysis and interpretationYX: data analysis and interpretationRY: data analysis and interpretationLMS: data analysis and interpretationHX: conception and design, data analysis and interpretationRKB: conception and design, collection and assembly of data, data

analysis and interpretationAP: conception and design, collection and assembly of data, data

analysis and interpretation, provision of study patientsAll authors participated in manuscript writing, and approved the final

version of the manuscript.

References

1. Tam SJ, Watts RJ (2010) Connecting vascular and nervous systemdevelopment: angiogenesis and the blood-brain barrier. Annu RevNeurosci 33:379–408

2. He Z, Tessier-Lavigne M (1997) Neuropilin is a receptor for theaxonal chemorepellent semaphorin III. Cell 90:739–51

3. Soker S, Takashima S, Quan H et al (1998) Neuropilin-1 is expressedby endothelial and tumor cells as an isoform-specific receptor forvascular endothelial growth factor. Cell 92:735–45

4. Kawasaki T,KitsukawaT, BekkuY (1999)A requirement for neuropilin-1 in embryonic vessel formation. Development 126:4895–902

5. Mamluk R, Gechtman Z, Kutcher ME, Gasiunas N, Gallagher J,Klagsbrun M (2002) Neuropilin-1 binds vascular endothelial growthfactor 165, placenta growth factor-2, and heparin via its b1b2 domain.J Biol Chem 277:24818–25

6. Takashima S, Kitakaze M, Asakura M et al (2002) Targeting of bothmouse neuropilin-1 and neuropilin-2 genes severely impairs devel-opmental yolk sac and embryonic angiogenesis. Proc Natl Acad SciU S A 99:3657–62

7. Gerhardt H, Ruhrberg C, Abramsson A, Fujisawa H, Shima D,Betsholtz C (2004) Neuropilin-1is required for endothelial tip cellguidance in the developing central nervous system. Dev Dyn 231:503–9

8. Grandclement C, Borg C (2011) Neuropilins: a new target for cancertherapy. Cancers 3:1899–1928

9. Bielenberg DR, Pettaway CA, Takashima S et al (2006) Neuropilinsin neoplasms: expression, regulation, and function. Exp Cell Res312:584–93

10. Beck B, Driessens G, Goossens S et al (2011) Avascular niche and aVEGF-Nrp1 loop regulate the initiation and stemness of skin tumors.Nature 478:399–403

11. Xin Y, Bai S, Damico-Beyer LA et al (2012) Anti-neuropilin-1(MRNP1685A): unexpected pharmacokinetic differences acrossspecies, from preclinical models to humans. Pharm Res 29:2512–21

12. Xin Y, Li J, Wu J, et al. (2012b) Pharmacokinetic and pharmacody-namic analysis of circulating biomarkers of anti-NRP1, a novel anti-angiogenesis agent, in two Phase I trials in patients with advancedsolid tumors, Clinical Cancer Research, in press.

13. Liang W-C, Dennis MS, Stawicki S et al (2007) Function blockingantibodies to neuropilin-1 generated from a designed human synthet-ic antibody phage library. J Mol Biol 366:815–29

14. Pan Q, Chanthery Y, Liang W-C (2007) Blocking neuropilin-1 func-tion has an additive effect with anti-VEGF to inhibit tumor growth.Cancer Cell 11:53–67

15. Bugelski PJ, Achuthanandam R, Capocasale RJ et al (2009)Monoclonal antibody-induced cytokine-release syndrome. ExpertRev Clin Immunol 5:499–521

16. Xia CQ, Peng R, Beato F, Clare-Salzler MJ (2005) Dexamethasoneinduces IL-10-producing monocyte-derived dendritic cells with du-rable immaturity. Scand J Immunol 62:45–54

17. Boks MA, Kager-Groenland JR, Haasjes MS, Zwaginga JJ, vanHam SM, ten Brinke A (2012) IL-10-generated tolerogenic den-dritic cells are optimal for functional regulatory T cell induction–a comparative study of human clinical-applicable DC. Clin Immunol142:332–42

18. Gabay C, Lamacchia C, Palmer G (2010) IL-1 pathways in inflam-mation and human diseases. Nat Rev Rheumatol 6:232–41

19. Darbonne WC, Du X, Dhawan P, et al (2011) Mechanism for plateletreduction in anti-neuropilin-1 (MNRP1685A)-treated phase I pa-tients. J Clin Oncol 29

20. Grothey A, Galanis E (2009) Targeting angiogenesis: progress withanti-VEGF treatment with large molecules. Nat Rev Clin Oncol 6:507–18

21. Raja FA, Chopra N, Ledermann JA (2010) Optimal first-line treat-ment in ovarian cancer. Ann Oncol 23(10):x118–x127

22. Pellet-Many C, Frankel P, Jia H (2008) Neuropilins: structure, func-tion and role in disease. Biochem J 411:211–26

23. Pataik A, LoRusso PM, Messersmith W (2013) A Phase Ib studyevaluating MNRP1685A, a fully human anti-NRP1 monoclonalantibody, in combination with bevacizumab and paclitaxel in patientswith advanced solid tumors. Cancer Chemother Pharmacol. (in press)

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