A Novel Bispecific Antibody Targeting EGFR and cMet that...

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A Novel Bispecific Antibody Targeting EGFR and cMet is Effective Against EGFR Inhibitor-

Resistant Lung Tumors

Sheri L. Moores1*, Mark L. Chiu1*, Barbara S. Bushey1, Kristen Chevalier1, Leopoldo Luistro1,

Keri Dorn1, Randall J. Brezski1, Peter Haytko1, Thomas Kelly1, Sheng-Jiun Wu1, Pauline L. Martin1,

Joost Neijssen2, Paul W.H.I. Parren2, Janine Schuurman2, Ricardo M. Attar1, Sylvie Laquerre1,

Matthew V. Lorenzi1, and G. Mark Anderson1

1Janssen Research and Development, Spring House, PA; 2Genmab, Utrecht, the Netherlands

*Contributed equally to this work

Running title: EGFR/cMet bispecific Ab in EGFR TKI resistant lung tumors

Keywords: EGFR, cMet, bispecific antibody, ADCC, NSCLC

Corresponding author: Sheri L. Moores, Oncology Discovery, Janssen R&D, 1400 McKean Road, Spring House, PA 19477. Phone: 215-628-5740; E-mail: [email protected]

Disclosure of Potential Conflicts of Interest: S.L.Moores, M.L.Chiu, B.S.Bushey, K.Chevalier, L.Luistro, K.Dorn, R.J.Brezski, P.Haytko, T.Kelly, S.Wu, R.Attar, S.Laquerre, M.V.Lorenzi, and G.M.Anderson are or have been employees of Janssen R&D. J.Neijssen, P.Parren, and J.Schuurman are employees of Genmab. The authors disclose no additional financial support.

Word count = 5998; Total number of figures and tables = 7.

ABSTRACT

Non-small cell lung cancers (NSCLCs) with activating EGFR mutations become resistant to

tyrosine kinase inhibitors (TKIs), often through second-site mutations in EGFR (T790M) and/or

activation of the cMet pathway. We engineered a bispecific EGFR-cMet antibody (JNJ-

61186372) with multiple mechanisms of action to inhibit primary/secondary EGFR mutations

and the cMet pathway. JNJ-61186372 blocked ligand-induced phosphorylation of EGFR and

cMet and inhibited phospho-ERK and phospho-AKT more potently than the combination of

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single receptor-binding antibodies. In NSCLC tumor models driven by EGFR and/or cMet, JNJ-

61186372 treatment resulted in tumor regression through inhibition of signaling/receptor

downmodulation and Fc-driven effector interactions. Complete and durable regression of

human lung xenograft tumors was observed with the combination of JNJ-61186372 and a third

generation EGFR TKI. Interestingly, treatment of cynomolgus monkeys with JNJ-61186372

resulted in no major toxicities, including absence of skin rash observed with other EGFR-

directed agents. These results highlight the differentiated potential of JNJ-61186372 to inhibit

the spectrum of mutations driving EGFR TKI resistance in NSCLC.

INTRODUCTION

Non-small cell lung cancer (NSCLC) is frequently driven by activating mutations in the kinase

domain of the EGF receptor (EGFR), occurring most commonly as in-frame deletions in exon 19

and L858R exon 21 mutations. Most patients initially respond to first generation EGFR tyrosine

kinase inhibitors (TKIs) such as gefitinib and erlotinib, but the clinical benefits are not durable.

Drug resistance limits the response to a mean duration of <1 year (1, 2). In addition to the

T790M secondary mutation in EGFR (~50% of resistant cases; 1, 3-6) that reduces potency of

reversible TKIs (7), resistant tumors may also develop activation of the cMet pathway, through

MET gene amplification, increased cMet expression, and/or increased expression of the cMet

ligand, hepatocyte growth factor/HGF (5, 6, 8, 9). Stimulation of the cMet pathway provides an

alternate mechanism to bypass the TKI block of EGFR and facilitate the survival of cancer cells.

These two mechanisms can also occur simultaneously in EGFR TKI-resistant NSCLC patients (3,

6, 10, 11).




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Because of the signaling cross-talk between EGFR and cMet, inhibition of both receptors in

combination may limit compensatory pathway activation and improve overall efficacy. A novel

bispecific antibody platform was used to produce JNJ-61186372, an antibody that binds EGFR

with one Fab arm and cMet with the other Fab arm (12, 13). We have optimized JNJ-61186372

to engage multiple mechanisms of action. First, we demonstrated the dual inhibition of both

EGFR and cMet signaling, by blocking ligand-induced activation and by inducing receptor

degradation. In addition, high levels of EGFR and cMet on the surface of tumor cells allows for

targeting of these cells for destruction by immune effector cells, through Fc-dependent effector

mechanisms, such as antibody-dependent cellular cytotoxicity (ADCC). JNJ-61186372 is

produced by an engineered CHO cell line defective for protein fucosylation. The human

FcγRIIIa, critical for ADCC, binds antibodies with low core-fucosylation more tightly and

consequently mediates more potent and effective ADCC killing of cancer cells (14). Thus, the

low level core fucosylation in the JNJ-61186372 molecule translates to an enhanced level of

ADCC activity compared to the same fully fucosylated molecule.

JNJ-61186372 was demonstrated to employ multiple mechanisms to inhibit tumors with

primary EGFR activating mutations, tumors with the T790M second-site resistance mutation in

EGFR, and tumors with activation of the cMet pathway. Furthermore, the combination of JNJ-

61186372 and a third generation EGFR TKI (AZD9291) resulted in complete and durable

regression of tumors. Interestingly, treatment of cynomolgus monkeys with JNJ-61186372

resulted in no toxicities, including absence of skin rash observed with other EGFR-directed

agents. This profile of preclinical data supports the development of JNJ-61186372 in patients

with lung cancer and other malignancies associated with aberrant EGFR and cMet signaling.




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MATERIALS AND METHODS

Preparation of parental monoclonal antibodies (mAbs)

The EGFR arm of JNJ-61186372 is derived from zalutumumab that has a conformational epitope

on EGFR domain III, which overlaps with the EGF ligand binding site (15) but is different from

the epitopes of cetuximab (16) and panitumumab (17). The cMet binding arm of JNJ-61186372

has an epitope that blocks HGF ligand binding but is distinct from the onartuzumab epitope

(18). Parental mAbs used to generate JNJ-61186372 were prepared from CHO cell lines that

generate mAbs with low levels of core fucosylation. Antibody expression and purification is

described in detail elsewhere (19).

Controlled Fab-arm Exchange (cFAE) to generate bispecific Abs

Bispecific human IgG1 Abs were produced from the two purified bivalent parental antibodies,

each with the respective single complementary mutation: K409R or F405L (12). cFAE was

performed as described (12), with a 5% excess of anti-EGFR –F405L. Identical conditions for

preparation and characterizations were used to generate the normal fucose (EGFR-cMet) and

JNJ-61186372-IgG2σ Abs, and monovalent EGFR and cMet Abs. The EGFR monovalent antibody

consists of an EGFR-binding arm and a non-binding arm (either anti-HIV-gp120 or anti-RSV-F;

which did not bind the cell lines used); the cMet monovalent antibody consists of a cMet-

binding arm and similar aforementioned non-binding arm.

Competitive ligand binding




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EGFR-ECD-Fc (R&D Systems; 200ng/well) was coated on HighBind plate (Meso Scale Discovery

(MSD)) for 2h (all steps performed at room temperature (RT)). MSD Blocker A buffer (5%,

150μL/well) was added and incubated for 2h. Plates were washed 3x with 0.1M HEPES buffer,

pH 7.4, then the mixture of the MSD fluorescence dye-labeled Hu-EGF with different

competitors was added. Labeled EGF (50nM) was incubated with antibodies (1nM to 4μM),

then added to wells (25μL mixture). After 2h, plates were washed as above. MSD Read Buffer T

was diluted with distilled water (4-fold), dispensed (150μL/well), and analyzed with a SECTOR

Imager 6000.

PolyHistidine mAb050 (R&D Systems; 50ng/well) was coated on MSD HighBind plate for 2h.

Blocking and washing steps were as above, followed by capturing 30μL (10μg/mL) of cMet-ECD-

Fc (R&D Systems). After 1h, plates were washed as above. After the mixture of biotinylated HGF

with different antibodies was added to plates, labeled HGF (10nM), was incubated with

antibodies (1nM to 2μM), then added to wells (25μL mixture). After 1h incubation, plates were

washed and then MSD fluorescence dye-labeled Streptavidin, 25μL (10nM), was added to each

well and incubated for 1h. Plates were washed, read, and analyzed as above.

Cell culture and HGF-engineered cell line

For these studies, seven NSCLC tumor cell lines (NCI-H292, HCC827, NCI-H1975, NCI-H3255,

HCC4006, NCI-H820, and NCI-H1993) were selected to reflect patient diversity in the mutational

status of EGFR and gene copy number of MET. The tumor cell lines were obtained (2011-2013)

from the National Cancer Institute (H3255) or American Type Culture Collection (all others). Cell

lines were authenticated using short tandem repeat assay and either used immediately or




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banked, with all experiments occurring within 2 months of resuscitation. The mutations in each

cell line were confirmed using a custom somatic mutation array (SABiosciences). Cells were

cultured in 150cm2 tissue culture flasks under standard culture conditions (37°C, 5% CO2, 95%

humidity) and grown in RPMI 1640 Medium + GlutaMAX™ + 25mM HEPES (Life Technologies),

10% heat inactivated Fetal Bovine Serum (Life Technologies), 0.1mM Non-Essential Amino Acids

(Life Technologies), and 1mM Sodium Pyruvate (Life Technologies). Subconfluent cell

monolayers were passaged using 0.25%-w/v Trypsin (Life Technologies).

The H1975-HGF cell line was created by transducing H1975 cells with HGF lentivirus (generated

using human HGF plasmid and packaging kit, Genecopoeia) to express the human HGF gene.

H1975 cells were plated at one million cells in a 100mm2 dish in RPMI media. After infection,

cells were selected using 2μg/mL puromycin (Invitrogen); 20pg/mL HGF (MSD assay) was

secreted (5000 cells/well) over 72h incubation.

Receptor phosphorylation assays

Receptor phosphorylation assays were performed as described, with cells plated at 6.5-10K

cells/well (19). The signals were not corrected for total levels of receptor. Data was plotted as

relative ECL (RCL) signal versus the logarithm of antibody concentration. IC50 values were

calculated in GraphPad Prism 5 (GraphPad Software, Inc.) using a four-parameter logistic (4PL)

model.

ERK and AKT phosphorylation assays




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Cells were grown in RPMI growth medium supplemented with 7.5ng/mL HGF then lysed after

30m (pERK assay) or 1h (pAkt assay) treatment with the antibodies. Phospho-ERK

(Thr202/Tyr204; Thr185/Tyr187) levels and phospho-AKT (S473) levels were measured using

MSD assays. The signals were not corrected for total levels of ERK or Akt. Relative ECL (RCL)

signal was recorded and data plotted as with receptor phosphorylation assays, with statistical

analysis using GraphPad Prism 6. Extra sum-of-squares F-test was used to compare treatments.

Preparation/analysis of tumor lysates

Mice bearing established H1975-HGF tumors were treated with a single dose of JNJ-61186372

(1, 5, or 20mg/kg) or vehicle control (PBS). 72h post-dose, tumors were harvested, flash frozen

in liquid nitrogen, and lysed in ice cold RIPA buffer (Thermo Scientific) containing 2X HALT/EDTA

protease and phosphatase inhibitor cocktail (Thermo), 50mM NaF, 2mM Sodium

Orthovanadate (activated), and 1mM PMSF using a FastPrep-24 homogenizer (MP Biomedicals).

Lysates were cleared by centrifugation and protein concentrations were determined by BCA

protein assay (Pierce). Protein samples (50μg) were resolved by SDS-PAGE and transferred to

nitrocellulose membranes. Membranes were blocked in Odyssey blocking buffer (LI-COR) for 1h

at RT and incubated with the appropriate primary antibodies overnight at 4oC: anti-EGFR (EGF-

R2; Santa Cruz Biotechnology), anti-Met (L41G3; Cell Signaling Technology), anti-actin (Santa

Cruz Biotechnology), and anti-GAPDH (Cell Signaling Technology). Bands were detected with

anti-mouse IRDye680 (LI-COR) or anti-rabbit IRDye800 (LI-COR) and imaged using an Odyssey

Infrared Imaging System (LI-COR). Average total protein quantitated from Western blots,

relative to loading control (GAPDH or actin), was graphed and statistical analysis performed




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using GraphPad Prism 6. Total cMet data were analyzed using a two-sided, unpaired, equal

variance t-test, and total EGFR data were analyzed using a two-sided, unpaired t-test with

Welch’s Correction for unequal variance. Tumor lysates were examined using MSD assays for

phospho-proteins (p-EGFR, p-cMet, p-ERK, or p-AKT), as described above. Ordinary one-way

ANOVA and two-sided, unpaired Dunnett’s multiple comparisons tests on log-transformed MSD

signal values were used to compare treatment groups using GraphPad Prism 6. Multiplicity-

adjusted p-values were reported.

ADCC assays

Human peripheral blood mononuclear cells (PBMCs) were isolated from normal donor

leukopaks (Biological Specialty Corporation, Colmar, PA) for use as effector cells and were

cryopreserved. The PBMCs from donors with FcgRIIIa 158V/F (20) were used in ADCC assays

(21, 22).

In vivo tumor xenograft models

All of the procedures relating to animal care, handling, and treatment were performed

according to the guidelines approved by Institutional Animal Care and Use Committee (IACUC),

or under the Animals in Scientific Procedures Act 1986 (Precos, Belton UK). For the H1975 and

H1975-HGF models, 5 x 106 cells were injected subcutaneously into the flanks (0.2mL/mouse) of

female nude mice (CD-1 NU/NU, 4-6 weeks old, Charles River Laboratories, Wilmington, MA).

Studies with HCC827 and HCC827-ER1 were performed at Precos. Nude mice (MF1 NU/NU, 5-8




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weeks old, Harlan, Teklad, UK) were injected subcutaneously with 1 x 107 cells (0.2mL/mouse).

The LU1235 and LU1868 patient-derived xenograft (PDX) models were performed by Crown

Biosciences, Beijing, China. PDX tumor fragments, harvested from donor mice, were inoculated

into female nude mice (BALB/c, 8-10 weeks old, Beijing HFK Bio-Technology Co). Animals were

treated with compounds when tumors reached a mean tumor size of 120-170mm3. Percentage

tumor growth inhibition (%TGI) was defined as the difference between the control-treated

group MTV and the test compound-treated group MTV, expressed as a percentage of the MTV

of the control-treated group. The statistical analysis of one H1975-HGF study (Figure 4), was

performed comparing treatment groups to control by two-way ANOVA and Bonferroni multiple

comparisons tests (GraphPad Prism). The statistical analysis of HCC827, HCC827-ER1, LU1235,

LU1868, H1975, and H1975-HGF (Figure 7) studies, was performed on the log-transformed

tumor volume data and a Tukey-Kramer p value adjustment for multiplicity (SAS).

RESULTS

Bispecific antibody binding to EGFR and cMet prevents ligand binding

Parental monospecific bivalent antibodies (F405L for anti-EGFR; K409R for anti-cMet) were

expressed separately in cell lines incorporating low levels (<9%) of fucose to enhance ADCC.

The bispecific EGFR-cMet antibody (Ab), prepared using controlled Fab-arm exchange with

>95% yield (12, 13) (Figure 1a), was designated JNJ-61186372. After purification with




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hydrophobic interaction chromatography, JNJ-61186372 was confirmed to be >99% bispecific

Ab with <0.2% parental monoclonal Abs (mAbs).

JNJ-61186372 binding to the extracellular domains (ECD) of EGFR and cMet was confirmed

using surface plasmon resonance methods (19). JNJ-61186372 has an affinity (KD) of 1.4nM for

EGFR-ECD and a KD=40pM for cMet-ECD. Using similar methods, JNJ-61186372 was shown to

bind both EGFR-ECD and cMet-ECD simultaneously (19).

JNJ-61186372 blocked the ligand binding of each receptor in a dose-dependent manner (Figure

1b). JNJ-61186372 inhibited ruthenium-labeled EGF binding to EGFR-ECD with an IC50=10nM

and inhibited biotinylated-labeled HGF binding to cMet-ECD with an IC50=30nM. These results

demonstrated that JNJ-61186372 bound to both EGFR and cMet with high affinity, and

furthermore inhibited ligand binding to each receptor with similar potency compared to the

parental monospecific bivalent antibodies.

JNJ-61186372 inhibits receptor phosphorylation of EGFR and cMet

Human lung cancer cell lines, representing different EGFR and cMet genotypes that often occur

in NSCLC patients, were selected to evaluate the cellular activity of JNJ-61186372

(Supplemental Table S1). These included cell lines with EGFR primary activating mutations,

EGFR T790M mutation (confers resistance to EGFR TKIs), either alone or with MET gene

amplification; and cell lines with wild-type (WT) EGFR and WT levels of cMet. In these cell lines,




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JNJ-61186372 inhibited EGF-induced phosphorylation of EGFR and HGF-induced

phosphorylation of cMet in a dose-dependent manner (Figure 2A) with activities (IC50) below

100nM (Figure 2B). The level of phosphorylated EGFR in the untreated H1975 cells (EGFR

mutations L858R; T790M) was higher than that of H292 cells (EGFR WT), due to the activating

mutation in EGFR. Addition of EGF to H1975 cells resulted in a smaller induction of

phosphorylated EGFR compared to the magnitude of induction observed with H292 cells.

Nevertheless, JNJ-61186372 inhibited phosphorylation of EGFR in the EGF-stimulated H1975

cells, reducing the phosphorylation of EGFR to levels below the untreated condition.

Enhanced inhibition of downstream signaling with JNJ-61186372

To determine whether dual inhibition of receptor phosphorylation had downstream signaling

consequences, the inhibition of phospho-ERK and phospho-AKT by JNJ-61186372 was assessed

in the same panel of cell lines (Figure 3a). JNJ-61186372 demonstrated potent inhibition of

both phospho-ERK and phospho-AKT, with IC50 values in the low or sub-nanomolar range for

most cell lines tested. However, in cell lines with MET gene amplification (H820 and H1993),

JNJ-61186372 demonstrated either weak or no inhibition of phospho-ERK or phospho-AKT.

Single pathway inhibition (EGFR or cMet) by monovalent antibodies was also assessed and

compared to that of JNJ-61186372 (Figure 3b-e). In H292 cells, neither treatment with the

monovalent EGFR or monovalent cMet antibody caused complete inhibition of pERK, whereas

the combination of the two monovalent antibodies caused complete inhibition of pERK with an

IC50=35nM (Figure 3b). This result demonstrated that inhibition of both EGFR and cMet was

necessary for full inhibition of pERK in this cell line. Interestingly in H292 cells, treatment with




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JNJ-61186372 resulted in complete inhibition of pERK, but with a lower IC50=0.64nM; a 55-fold

increase in potency (p<0.0001) for JNJ-61186372 compared to that of the combination of

monovalent antibodies. Also in H292 cells, the monovalent cMet antibody was as effective as

the combination of monovalent antibodies at inhibiting pAKT, suggesting that pAKT was more

dependent on cMet signaling compared to EGFR signaling in this cell line (Figure 3c).

Nevertheless, JNJ-61186372 was 179-fold more potent for inhibition of pAKT (p<0.0001)

compared to the combination of monovalent antibodies, suggesting that the bispecific

molecule had additional biological effects not present in the combination of monovalent

antibodies. Similarly there was more potent inhibition of pERK and pAKT in H1975 cells using

JNJ-61186372 (65-fold greater inhibition of pERK (p<0.0001); 75-fold greater inhibition of pAKT

(p<0.0001) than the combination of monovalent antibodies (Figure 3d and 3e).

JNJ-61186372 caused tumor cell lysis through enhanced effector function

We hypothesized that high levels of EGFR and cMet on the surface of tumor cells may allow for

selective immune effector cell-targeting through ADCC. JNJ-61186372 was engineered to have

low levels of core fucose, which allows for tighter binding to human FcγRIIIa on NK cells, thus

resulting in a more potent and effective ADCC killing of target cells (14).

The ADCC in vitro activity of JNJ-61186372 was tested using H292 and H1975 cells with human

peripheral blood mononuclear cells (PBMCs) as the source of NK cells (Figure 4a). For

comparison, an analog of JNJ-61186372, but produced with normal fucose levels, and

cetuximab (anti-EGFR mAb) were assessed in the same assay. The low fucose JNJ-61186372 was

more potent and demonstrated increased levels of tumor cell lysis compared to the EGFR-cMet




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bispecific antibody with normal fucose levels. Similar results were observed using additional

lung cancer cell lines, including those with either WT or mutant EGFR, and WT or amplified MET

(Supplemental Table S2). The ADCC capacity of the low fucose JNJ-61186372 was similar to the

ADCC capacity of cetuximab. However, there were some cases where JNJ-61186372

demonstrated slightly improved ADCC activity in potency (EC50) and/or efficacy (% maximal

lysis).

JNJ-61186372 inhibited tumor growth in the H1975-HGF xenograft model

The H1975 cell line carries both a primary mutation in EGFR (L858R) and a second-site mutation

in EGFR (T790M) that confers resistance to small molecule EGFR TKIs such as erlotinib. In

addition, this line was engineered to express human HGF (H1975-HGF) so the human cMet on

the tumor cells would be activated in the mouse host (mouse HGF does not effectively activate

human cMet, 23). H1975-HGF cells were implanted into immune-compromised mice to assess

in vivo efficacy of JNJ-61186372 on xenograft tumors. In addition, the contribution of effector

function to efficacy in vivo was evaluated.

After subcutaneous tumors were established, the mice were treated with vehicle control (PBS),

JNJ-61186372, or an antibody with the same EGFR and cMet binding arms but with an IgG2σ

framework (JNJ-61186372-IgG2σ). Antibodies with an IgG2σ framework have less binding to Fc

receptors of effector cells (such as NK cells and macrophages), but maintain similar

pharmacokinetics to IgG1 antibodies, so any loss of activity observed with JNJ-61186372-IgG2σ

antibody should represent the contribution of the effector-mediated function of JNJ-61186372

(24).




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As shown in Figure 4b, JNJ-61186372 inhibited tumor growth by 80% compared to vehicle-

treated animals at day 32 (p<0.0001). Interestingly, JNJ-61186372-IgG2σ antibody (Fc silent

version) inhibited tumor growth by 47% (p=0.0232), but was not as effective as JNJ-61186372

with the FcR-binding framework. These results demonstrate efficacy of JNJ-61186372 in the

H1975-HGF model, with EGFR mutations (L858R; T790M) and cMet activation through

autocrine expression of HGF. In addition, without effector function, the inhibition of EGFR and

cMet alone reduced tumor growth relative to the vehicle control, but the addition of the

effector function (IgG1 framework) is necessary for complete JNJ-61186372 in vivo activity.

JNJ-61186372 induced receptor downmodulation in vivo

To further understand function of JNJ-61186372 in vivo, mice bearing H1975-HGF tumors were

treated with either PBS vehicle control or JNJ-61186372 at 1, 5, and 20mg/kg. Tumor lysates

from mice treated with 20mg/kg JNJ-61186372 and those treated with vehicle were analyzed

for total EGFR and cMet levels by Western blot (Figure 5a). The average total protein levels of

both EGFR and cMet were significantly decreased following antibody treatment compared to

those of vehicle treated tumors by 76% (p<0.0001) and 61% (p=0.0007), respectively. Not

surprisingly, because levels of total receptor were significantly reduced (Figure 5a and b),

phospho-EGFR and phospho-Met levels were also significantly reduced at all doses tested

compared to that of vehicle treated animals (Figure 5c). Phospho-AKT was significantly reduced

at 5 and 20mg/kg antibody treatment, but levels of phospho-ERK were not significantly

decreased in this experiment. These results demonstrated downmodulation of both receptor




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targets and downstream signaling in the tumor by JNJ-61186372 treatment; thus illustrating an

additional anti-tumor growth mechanism of action.

JNJ-61186372 is effective in EGFR mutant xenograft models, with or without cMet activation

JNJ-61186372 was evaluated in a pair of human lung adenocarcinoma HCC827 xenograft

models, both with a primary EGFR mutation (exon 19 deletion), but one with MET amplification

(clone of parental HCC827; originally selected to have resistance to erlotinib; HCC827-ER1). In

mice with HCC827 tumors, treatment with either erlotinib or JNJ-61186372 fully suppressed

tumor growth during the treatment period (28 days) and to approximately 50 days post-

treatment initiation (Figure 6a). Both JNJ-61186372 and erlotinib inhibited tumor growth by

89% compared to vehicle treated animals at day 45 (both p<0.05). In mice with HCC827-ER1

tumors, JNJ-61186372 fully suppressed tumor growth, whereas erlotinib was only partially

effective during treatment (days 7-31) and mean tumor volume increased at a similar rate as

vehicle treated tumors following cessation of treatment (Figure 6b). However, in animals

treated with JNJ-61186372, mean tumor volume slightly increased in 5 weeks following

cessation of treatment. At day 37, erlotinib inhibited tumor growth by 63% (p<0.05), while JNJ-

61186372 inhibited tumor growth by 92% (p<0.05), compared to vehicle-treated animals.

These results suggested that in tumors with both EGFR mutations and MET amplification, dual

inhibition of both EGFR and cMet was necessary for complete inhibition of tumor growth.

JNJ-61186372 is effective in patient derived tumor (PDX) models with EGFR mutations

We tested the bispecific EGFR-cMet antibody efficacy in patient-derived xenograft (PDX)

models with EGFR mutations. For these studies we used a version of JNJ-61186372 prepared




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with normal fucose levels. LU1235 PDX model is derived from a human lung primary

adenocarcinoma tumor with a primary activating EGFR mutation (exon 19 deletion). In these

tumors, treatments with erlotinib and JNJ-61186372 were both effective at regressing tumor

volumes, but JNJ-61186372 continued to suppress tumor growth following cessation of

treatment (Figure 6c). These results demonstrated that in tumors with a primary activating

mutation in EGFR, the bispecific EGFR-cMet antibody led to a more durable response than

erlotinib treatment. The human lung adenocarcinoma LU1868 PDX model contains both a

primary activating mutation in EGFR (L858R) and the second-site mutation in EGFR (T790M,

known to cause resistance to erlotinib treatment). As expected in this model, erlotinib was not

effective (Figure 6d). However, JNJ-61186372 treatment caused regression of the LU1868

tumors in a durable manner for up to 2 months following cessation of treatment. JNJ-61186372

was effective in PDX models with either primary activating mutations in EGFR alone, and with

the addition of T790M EGFR TKI-resistance mutation.

Combination of JNJ-61186372 and third generation EGFR TKI resulted in durable tumor

regression

Third generation EGFR TKIs, such as AZD9291 and CO-1686, preferentially inhibit mutant-

activated EGFR compared to WT EGFR. To assess whether the combination of a third generation

EGFR TKI and JNJ-61186372 would have benefit, we tested AZD9291 and JNJ-61186372 as

single agents and in combination in the parental H1975 model (Figure 7A) and the engineered

H1975-HGF model (Figure 7B). During the treatment period, AZD9291 inhibited tumor growth




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in both parental H1975 (TGI=100%) and H1975-HGF (TGI=94%) models, p<0.05 for both.

However, AZD9291-treated tumors increased in volume after treatment cessation in the

H1975-HGF model. During the treatment period, JNJ-61186372 was effective in both models

(TGI=79% and 90%, respectively, p<0.05 for both). The combination treatment with JNJ-

61186372 and AZD9291 was more effective than either single treatment in the H1975-HGF

model. After 3 weeks of treatment, tumors were monitored for more than a month and no

measurable tumors were observed in the combination groups in either tumor model. In a

second study using the H1975-HGF model (Figure 7C, D, E) tumors were grown to a larger size

(average of 500 mm3) before treatment initiation. Again, tumors completely regressed with the

combination treatment and no tumor growth was observed for up to 85 days of treatment.

Crizotinib, a small molecule cMet/ALK inhibitor, showed no efficacy in either model

(Supplemental Figure S3). Altogether, these results demonstrated that combination of a third

generation EGFR TKI and JNJ-61186372 cause regression of tumors in a sustained and durable

manner.

Absence of EGFR-mediated skin rash after repeat administration of JNJ-61186372

Both non-GLP and GLP safety studies were performed in cynomolgus monkeys. Animals were

given JNJ-61186372 intravenously at doses up to 120mg/kg once/week for up to 5 weeks. No

toxicities were observed in either study, including no skin or gut adverse events that are

commonly observed with other anti-EGFR agents (25). Notably, JNJ-61186372 recovered in

serum from cynomolgus monkeys treated with JNJ-61186372 was fully functional in inhibition

of phospho-EGFR and phospho-Met in vitro using H292 human cancer cell line (Supplemental




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Figure S4). Our preliminary data suggested that this lack of skin toxicity may be attributable to a

lower single arm binding affinity for purified EGFR protein compared to cetuximab (JNJ-

61186372 KD=1.4nM (19); cetuximab KD=0.4nM in same assay format). This lower affinity then

translates into less potent binding to cells expressing low levels of EGFR and cMet, such as

primary human dermal fibroblasts. We found that JNJ-61186372 bound to primary human

dermal fibroblasts with an EC50=2.0nM, while cetuximab bound 20-fold tighter, with an

EC50=0.1nM. Binding to normal tissues is also likely enhanced by the bivalent nature of

cetuximab, compared to the monovalent EGFR binding of JNJ-61186372.

DISCUSSION

Extensive evidence has demonstrated that the EGFR and cMet signaling pathways are partially

compensatory and mediate cross resistance to inhibitors of either pathway (4, 8, 9). Standard of

care treatment for lung cancer patients with activating mutations in EGFR involves small

molecule EGFR TKIs such as erlotinib or gefitinib. These therapies, though often initially very

effective, ultimately fail due to resistance mediated by second site EGFR mutations such as

T790M or activation of bypass pathways, such as the cMet pathway (4, 9, 10, 26). Second and

third generation EGFR TKIs such as afatinib, AZD9291, and CO-1686 have produced exciting

clinical results, but resistance to therapy remains a prominent challenge. Resistance

mechanisms to third generation EGFR TKIs are still being defined in patient samples, but an

emerging frequent mechanism is mutation of EGFR (C797S) (27). Evidence of MET amplification

in lung tumor cells from a patient treated with AZD9291 has also been reported, suggesting

that activation of the cMet pathway is clinically relevant for the third generation EGFR TKIs (28).




19

To address these resistance issues we have created a bispecific antibody, JNJ-61186372, that

recognizes both EGFR and cMet. We have demonstrated that JNJ-61186372 inhibits tumor

growth and progression by three distinct mechanisms. Two of these mechanisms involve

inhibition of EGFR and cMet signaling; first by inhibition of ligand-induced activation via

blocking ligand binding to each receptor; and second by receptor inactivation via degradation.

The third mechanism utilizes Fc effector-mediated killing of EGFR- and cMet-expressing tumor

cells by ADCC. Through these mechanisms of action, JNJ-61186372 had activity in multiple

xenograft models containing diverse EGFR mutations (exon 19 deletion, L858R, T790M). Given

that JNJ-61186372 recognizes the extracellular region of EGFR, this bispecific antibody has the

potential to be active in tumors with C797S mutation (located intracellularly) as well.

Additionally, JNJ-61186372 was active in models with cMet pathway activation, either by

autocrine ligand production or through MET gene amplification. JNJ-61186372 was also active

in a model with WT EGFR and autocrine HGF production (Supplemental Figure S5). Collectively,

the unique mechanisms of action of JNJ-61186372 cover the spectrum of EGFR mutations and

also inhibit the key pathways driving EGFR TKI-acquired resistances.

Combination therapies may be necessary to fully suppress the EGFR pathway and prevent

resistance from occurring through multiple mechanisms. The combination of afatinib and

cetuximab was shown to cause near complete tumor regression in mice with L858R/T790M

erlotinib-resistant tumors (29) by depleting phosphorylated and total EGFR more completely

than either single agent alone. Combining JNJ-61186372 with third generation EGFR TKIs might

have the advantage, in addition to simultaneous inhibition of the cMet pathway, of more

complete inhibition of the EGFR pathway through the ligand inhibition and receptor




20

degradation mechanisms. Addition of the immune effector-based mechanisms of JNJ-61186372

may also attack tumor cells resistant due to EGFR- and cMet-independent mechanisms. We

have shown here that the combination of JNJ-61186372 and AZD9291 was very effective at

inhibiting tumor growth in xenograft models with EGFR mutations and cMet activation.

Furthermore, in contrast to monotherapy, treatment with the combination prevented tumor

regrowth in all animals tested. In patients, tumor heterogeneity likely contributes to resistance;

recent observations suggest that T790M-positive and T790M-WT coexist in some lung cancers

with acquired resistance to initial EGFR TKI treatment (28, 30).Combination therapy with JNJ-

61186372 may delay or prevent the emergence of resistance, with activity on clonal

populations with varying EGFR mutations within the same tumor, more complete suppression

of the EGFR pathway, and inhibition of cMet, a key bypass pathway.

Recently, advances in protein engineering have made the production of bispecific biologics,

such as JNJ-61186372, more practical. One potential advantage of the bispecific configuration is

the significantly improved inhibition of downstream signaling when both targets are present on

the same tumor cell. This property may be explained by an avidity effect in which cells

expressing both targets engage both arms of the bispecific (cross-arm binding) with increased

apparent affinity as compared to those expressing only one target or otherwise engaging a

single Fab arm. We have described correlations between binding affinity, receptor density, and

receptor phosphorylation with JNJ-61186372 (19). Further, we have shown that JNJ-61186372

was more effective than the combination therapy of anti-EGFR and anti-cMet monovalent

antibodies in decreasing tumor growth in the H1975-HGF model (31).




21

This bispecific structure may also confer unexpected properties on JNJ-61186372. In contrast

with other EGFR-targeted agents (25), we did not observe EGFR-mediated skin toxicity with JNJ-

61186372 in cynomolgus monkeys, typically a critical limitation of this class of agents, even at

doses up to 120 mg/kg. We hypothesize that the lack of skin rash in cynomolgus monkeys

treated with JNJ-61186372 is linked to weaker, monovalent-binding of JNJ-61186372 to EGFR

compared to stronger binding of antibodies interacting bivalently with EGFR (e.g., cetuximab,

panitumumab) to normal skin and gut cells. Indeed, we found a 20-fold difference in affinity

between JNJ-61186372 and cetuximab in binding to normal human dermal fibroblasts. Because

normal tissues express low levels of cMet, the cMet-binding arm of JNJ-61186372 is not

engaged, thus resulting in overall weaker binding to skin and gut cells, along with lower

inhibition of EGFR in these normal tissues. In contrast in tumor cells, where high levels of EGFR

and cMet are co-expressed, both arms of JNJ-61186372 will be engaged and stronger binding

and functional inhibition is achieved. Alternatively, species differences in binding and activity

could contribute to the observed lack of skin toxicity. The potential lower toxicity profile of JNJ-

61186372 may also be an advantage in combination therapies, where efficacy may be limited

by toxicity issues, such as with the combination of afatinib and cetuximab (32).

We have demonstrated three distinct mechanisms of action for JNJ-61186372 that modulate

both EGFR and cMet signaling axes. The unique structure of JNJ-61186372 enables attack of

tumor cells with multiple mechanisms in a single agent, resulting in durable anti-tumor effects

in preclinical models resistant to other therapeutic approaches. These results support testing in

human clinical trials, as monotherapy in selected patients and in combination with other agents

addressing the same pathways, in lung cancer and other malignancies.




22

ACKNOWLEDGMENTS

The authors acknowledge the contributions of Amy Wong, Yi Tang, Diana Chin, Hillary Quinn,

Frank McCabe, Jeff Nemeth, Kerry Brosnan, and Eva Emmell to the in vivo studies in this report.

The authors thank Jeroen Lammerts van Bueren and Aran Labrijn for expert advice and helpful

discussions.

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28

FIGURE LEGENDS

Figure 1. Structure of JNJ-61186372 and inhibition of ligand binding. A, schematic showing the

cFAE method used to generate the bispecific antibody binding cMet and EGFR. B, JNJ-61186372

and parental EGFR antibody inhibited EGF binding to EGFR-Fc protein (left); JNJ-61186372 and

parental cMet antibody inhibited HGF binding to cMet-Fc protein (right). NF=no fit returned by

GraphPad Prism.

Figure 2. JNJ-61186372 inhibited ligand-induced phosphorylation of both EGFR and cMet in

lung cancer cell lines. A, H292 and H1975 cells were serum-starved, then stimulated with EGF

(left) or HGF (right) in the presence of JNJ-61186372. Levels of phospho-EGFR (left) or

phospho-cMet (right) were detected (RCL=relative chemiluminescence), with three biological

replicates at each point. Mean RCL values are plotted ±SEM. B, Summary IC50 data for a panel

of lung cancer cell lines for JNJ-61186372 inhibition of phospho-EGFR (light bars) and phospho-

cMet (dark bars) using MSD assays.

Figure 3. JNJ-61186372 inhibited phospho-ERK and phospho-AKT in lung cancer cell lines. A,

Summary IC50 data for a panel of cell lines for JNJ-61186372 inhibition of phospho-ERK (light

bars) and phospho-AKT (dark bars) using MSD assays. B-E; H292 cells (B, C) and H1975 cells (D,

E) were treated with antibodies and phospho-ERK (B, D) and phospho-AKT (D, E) measured

using the MSD assay (RCL=relative chemiluminescence), with three biological replicates at each

point. Mean RCL values are plotted ±SEM.




29

Figure 4. Fc effector function of JNJ-61186372 was enhanced in vitro and important for efficacy

in vivo. A, ADCC assays were performed using H292 (top) and H1975 (bottom) cells. Low fucose

JNJ-61186372 was compared to normal fucose EGFR x cMet antibody, cetuximab, and IgG1

control antibody. B, H1975-HGF model was used to compare JNJ-61186372 to JNJ-61186372-

IgG2σ for efficacy. Antibodies were dosed at 10mg/kg; 2x/week i.p. (10 animals/group).

Figure 5. Decreased EGFR and cMet protein, phospho-EGFR, phospho-cMet, and phospho-AKT

levels in H1975-HGF tumors from mice treated with JNJ-61186372. A, Mice with H1975-HGF

tumors were treated with a single dose of JNJ-61186372 (20mg/kg) or vehicle control (PBS).

Tumor lysates were examined by Western blot for total levels of EGFR, cMet, or actin/GAPDH

(loading controls). B, Average total protein quantitated from Western blots, relative to loading

controls. C, Mice with H1975-HGF tumors were treated with a single dose of JNJ-61186372 (1,

5, or 20mg/kg) or vehicle control (PBS). Tumor lysates examined using MSD assays for

phospho-EGFR, phospho-cMet, phospho-ERK, or phospho-AKT.

Figure 6. JNJ-61186372 was efficacious in EGFR mutant models, with or without cMet

activation. JNJ-61186372 (10mg/kg, 2x/week i.p.) was tested for efficacy in A, HCC827 model,

and B, HCC827-ER1 model (9 animals/group). JNJ-61186372 was efficacious in PDX models with

EGFR mutations: exon 19 deletion (C) and L858R; T790M (D). The JNJ-61186372 used in C and D

was an earlier preparation with normal fucose levels and residual parental bivalent EGFR

antibody remaining (12%). Erlotinib was dosed at 25 or 50mg/kg, daily p.o.; 9 or 10

animals/group.




30

Figure 7. JNJ-61186372 in combination with AZD9291 was effective and durable in H1975 and

H1975-HGF xenograft models with and without cMet pathway activation. A, H1975 model; B,

H1975-HGF model; C, D, E results of a different study in H1975-HGF starting with larger tumors

(~500mm3) and plotting individual tumor volumes over time. In A and B, JNJ-61186372 dosed at

10mg/kg, 2x/week i.p.; AZD9291 dosed at 30mg/kg, daily p.o. Note that in A, the curves for

AZD9291 and for JNJ-61186372+AZD9291 are superimposed. In C and E, JNJ-61186372 was

dosed at 10mg/kg, 2x/week i.p. In D and E, AZD9291 was dosed at 5mg/kg, daily p.o. For C, D,

and E, dosing was continuous throughout the study.




Figure 1

A

B HGF Binding EGF Binding

cMet EGFR JNJ-61186372




Figure 2

A

B

pMet

H292

H1975

pEGFR

0.1

1

10

100

1000

IC50

(nM

)

EGFR MET

IC5

0 (

nM

)




Figure 3 A

B pERK pAKT C

D

H292

H1975

EGFR MET

No

t ac

tive

N

ot

acti

ve

E

IC5

0 (

nM

)




Figure 4

H1975-HGF (EGFR L858R; T790M)

1

A B

PBS vehicle

JNJ-61186372-IgG2σ

JNJ-61186372




Figure 5

A B

C

JNJ-61186372 (mg/kg)

pAKT

pMet pEGFR

JNJ-61186372 (mg/kg) JNJ-61186372 (mg/kg)

JNJ-61186372 (mg/kg)

pERK




Figure 6

1

HCC827-ER1 (EGFR ex19del; MET amp)

PBS Vehicle

Erlotinib

HCC827 (EGFR ex19del)

PBS Vehicle

JNJ-61186372 Erlotinib

Days Days

A B

LU1235 (EGFR: ex19 del) LU1868 (EGFR: L858R; T790M)

Erlotinib

Erlotinib

0 20 40 60 800

500

1000

1500

2000

2500

Dosing phase

Me

an t

um

ou

r vo

lum

e /

mm

3

0 20 40 600

500

1000

1500

2000

2500

Dosing phase

Me

an t

um

ou

r vo

lum

e /

mm

3 PBS Vehicle

Days Days

C D

Me

an T

um

or

Vo

lum

e (

mm

3)

+ SE

M

Me

an T

um

or

Vo

lum

e (

mm

3)

+ SE

M

Me

an T

um

or

Vo

lum

e (

mm

3)

+ SE

M

Me

an T

um

or

Vo

lum

e (

mm

3)

+ SE

M

JNJ-61186372

JNJ-61186372 JNJ-61186372

PBS Vehicle




Figure 7

A

B

Vehicle JNJ-61186372

AZD9291

AZD9291 + JNJ-61186372

H1975 (EGFR L858R; T790M)

H1975-HGF (EGFR L858R; T790M; HGF autocrine)

Me

an T

um

or

Vo

lum

e (

mm

3)

+ SE

M

Me

an T

um

or

Vo

lum

e (

mm

3)

+ SE

M

Vehicle

JNJ-61186372

AZD9291

AZD9291 + JNJ-61186372

C

D

E Days

Days




Published OnlineFirst May 23, 2016.Cancer Res Sheri L Moores, Mark > Chiu, Barbara S Bushey, et al. Effective Against EGFR Inhibitor- Resistant Lung TumorsA Novel Bispecific Antibody Targeting EGFR and cMet that is

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