Amivantamab (JNJ-61186372), an Fc Enhanced EGFR/Cmet Bispecific

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Author Manuscript Published OnlineFirst on August 3, 2020; DOI: 10.1158/1535-7163.MCT-20-0071 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

TITLE PAGE

Type: Research Article

Target journal: Molecular Cancer Therapeutics

Title (164 characters): Amivantamab (JNJ-61186372), An Fc Enhanced EGFR/cMet Bispecific

Antibody Induces Receptor Downmodulation And Anti-tumor Activity By

Monocyte/Macrophage Trogocytosis

Authors: Smruthi Vijayaraghavan*, Lorraine Lipfert, Kristen Chevalier, Barbara S. Bushey,

Benjamin Henley, Ryan Lenhart, Jocelyn Sendecki, Marilda Beqiri, Hillary J. Millar, Kathryn

Packman, Matthew V. Lorenzi, Sylvie Laquerre, Sheri L. Moores

Affiliations: Janssen Research & Development, Spring House, PA, USA

Running title (60/60 characters): Amivantamab Induces EGFR/cMet Downmodulation By

Trogocytosis

Keywords: Amivantamab, Bispecific antibody therapeutics, Trogocytosis, Fc effector functions,

JNJ-61186372

Downloaded from mct.aacrjournals.org on September 26, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 3, 2020; DOI: 10.1158/1535-7163.MCT-20-0071 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

*Corresponding Author:

Smruthi Vijayaraghavan

1400 McKean Road

Spring House, PA 19477

Tel: +1 (215) 628-5121

Email: [email protected]

Conflict of Interest: All authors are full-time employees of Janssen Research & Development,

LLC

Funding: The study was funded by Janssen Research & Development, LLC

Abstract: 198/250 words

Main text: 5141 of 5000 words

Reference: 38 of 50

Main Figures: 6 of 6 figures

Supplementary Figures: 13

ABSTRACT (198/250)

Small molecule inhibitors targeting mutant epidermal growth factor receptor (EGFR) are

standard of care in non-small cell lung cancer (NSCLC), but acquired resistance invariably

develops through mutations in EGFR or through activation of compensatory pathways such as

cMet. Amivantamab (JNJ-61186372) is an anti-EGFR and anti-cMet bispecific low fucose antibody with enhanced Fc function designed to treat tumors driven by activated EGFR and/or cMet signaling. Potent in vivo anti-tumor efficacy is observed upon amivantamab treatment of

human tumor xenograft models driven by mutant activated EGFR and this activity is associated

with receptor downregulation. Despite these robust anti-tumor responses in vivo, limited anti-

proliferative effects and EGFR/cMet receptor downregulation by amivantamab were observed in

vitro. Interestingly, in vitro addition of isolated human immune cells notably enhanced

amivantamab-mediated EGFR and cMet downregulation leading to antibody dose-dependent

cancer cell killing. Through a comprehensive assessment of the Fc-mediated effector functions,

we demonstrate that monocytes and/or macrophages, through trogocytosis, are necessary and

sufficient for Fc interaction-mediated EGFR/cMet downmodulation and are required for in vivo

anti-tumor efficacy. Collectively, our findings represent a novel Fc-dependent macrophage-

mediated anti-tumor mechanism of amivantamab and highlight trogocytosis as an important

mechanism of action to exploit in designing new antibody-based cancer therapies.

INTRODUCTION

Treatment of non-small cell lung cancer (NSCLC) was transformed upon identification and

targeting of primary activating mutations in the kinase domain of epidermal growth factor

receptor (EGFR) which occur in 10-40% of lung cancer patients. Patients with activating EGFR

mutations are initially responsive to first- and second-generation EGFR tyrosine kinase inhibitor

(TKI) treatment. However, this efficacy is short-lived, and acquired resistance develops through

secondary mutations in EGFR as well as upregulation of other pathways, such as cMet (1-3).

Osimertinib, a covalent third-generation EGFR TKI, is used for the treatment of EGFR mutant

NSCLC patients with acquired resistance to prior EGFR TKI therapy due to T790M mutation, and has recently become the standard of care front line therapy (4). However, similar to the first

generation TKIs, acquired resistance to osimertinib is also observed through additional mutations

in EGFR (e.g.C797S) or activation of the cMet pathway (5-7). Currently, there are no approved

therapies for patients resistant to third generation TKIs, highlighting the significant unmet need

for this population.

To address this unmet medical need, we developed amivantamab (JNJ-61186372), a fully human

IgG1-based bispecific antibody targeting both EGFR and cMet (8) (US Patent#US9593164B2).

This bispecific antibody binds EGFR agnostic of the diverse primary and acquired resistance mutations, potentially increasing the therapeutic reach of this antibody. Amivantamab is currently in clinical development, and consistent with its preclinical profile (9), preliminary

clinical data demonstrates that amivantamab treatment leads to NSCLC regression across EGFR

driver mutations, including exon 19 deletion, L858R mutation, Exon20 insertions, EGFR-TKI

resistance mutations (T790M, C797S), as well as against drug-resistance due to MET

amplification (10,11).

Amivantamab was produced in engineered CHO cell lines that incorporate low levels of fucose,

to generate a bispecific antibody with enhanced binding to Fc gamma receptor (FcR)-IIIa/

CD16a on immune cells (12). Binding of huIgG1 antibodies to FcγRs can mediate target cell

elimination through immune effector cell functions such as antibody-dependent cellular

cytotoxicity(ADCC), antibody-dependent cellular phagocytosis(ADCP) and complement-

dependent cytotoxicity(CDC) (13). ADCC and ADCP are triggered when IgG1 antibodies bind

to cell surface antigens, subsequently clustering their Fc domains to engage with FcγRs on natural killer (NK) cells (ADCC) and macrophages (ADCP)(14). CDC is activated through

interaction of the complement component C1q with clustered antibodies bound to target cells

(13). Tumor-targeted antibodies such as rituximab (anti-CD20 antibody), cetuximab (anti-EGFR

antibody), and trastuzumab (anti-HER2 antibody), are known to exploit these three FcγR-

mediated mechanisms (15). Trogocytosis (ADCT - antibody-dependent cellular trogocytosis) is

another important consequence of Fc-FcγR interaction, and is described as tumor-targeted

antibody mediated transfer of membrane fragments and ligands from tumor cells to effector cells

such as monocytes, macrophages and neutrophils (16). Unlike phagocytosis, trogocytosis is underappreciated as a mechanism of action contributing to tumor cell death.

In previous studies we have shown that amivantamab can induce ADCC and ADCP, in addition

to Fc-independent EGFR/cMet signal inhibition (9,12). Further, in an in vivo study in an EGFR

mutant cMet activated tumor model, we showed that treatment with Fc-silent IgG2σ version of

amivantamab elicits reduced tumor growth inhibition compared to amivantamab (Fc-active

version), suggesting that the Fc-mediated effector function of amivantamab is essential for its

maximal anti-tumor efficacy (12). Additionally, the IgG2σ version of amivantamab exhibited reduced downregulation of EGFR, cMet, and their respective signaling components in vivo, suggesting the requirement of amivantamab Fc interaction for receptor and signaling downmodulation activity.

Here we report that trogocytosis is a dominant mechanism of action for the preclinical in vitro and in vivo anti-tumor activity of amivantamab. We performed a comprehensive analysis of the

Fc-mediated effector functions to dissect the key immune cell types underlying the Fc-dependent mechanisms of amivantamab. This work led to the identification of Fc-mediated monocyte and

macrophage interactions and associated trogocytosis as key mediators of receptor

downmodulation and antibody-mediated cancer cell killing. Together, these results point to the

importance of trogocytosis as a mechanism of cancer cell death, which can be leveraged in the

design of future antibody-based therapies.

MATERIALS AND METHODS

Proliferation and Apoptosis Assays

For Incucyte-based proliferation and apoptosis assays, H1975 or SNU5 cell lines were infected

with NucLight Red lentiviral reagent (Essen Biosciences; Cat#4476,) and selected with 1µg/ml

puromycin to generate H1975 and SNU5 NucRed cells. NucRed target cells were plated at 5,000

cells/well and PBMCs (HemaCare) were added at an E:T (effector:target) ratio of 10:1. Annexin-

V Green reagent (Essen Biosciences; Cat#4642,) was added, followed by therapeutic antibodies

and scanned (4 images/well/scan) with IncucyteS3 every 4 hours. Total NucRed Area(µm2/well) shows target cell viability and Total Green NucRed Area (µm2/well) shows target cell apoptosis.

Data was visualized with Spotfire and dose-response curves were generated using GraphPad

Prism.

Simple Western Assay

H1975 or SNU5 target cells were plated (100,000 cells/well) in 6-well plates and PBMCs were added at an E:T ratio of 10:1 or individual immune cells (NK cells, Monocytes or Macrophages) at E:T ratio of 5:1. Therapeutic antibodies were added and incubated for 48hrs. Protein lysates were extracted using RIPA lysis buffer (ThermoFisher; Cat#89901) with 1-tab PhosSTOP

(Sigma; Cat#4906837001) and cOmplete™ Protease Inhibitor (Sigma; Cat#04693159001).

Protein concentration was determined using Pierce BCA Protein assay kit (ThermoFisher;

Cat#23227) as per manufacturer’s protocol. Capillary based electrophoresis was performed using

PeggySue 12-230kDa Separation module (Protein Simple; Cat#SM-S001), anti-rabbit detection module (Cat#DM-001) and anti-mouse antibody (Cat#042-205) as per the manufacturer’s protocol. Briefly, protein lysates were diluted to 0.2 mg/mL and denatured at 95˚C for 5 min.

Primary antibodies were diluted: EGFR (CST#2646) at 1:50; phosphorylated EGFR

(pEGFR;R&D Systems#AF1095) at 1:50; cMet (CST#3148) at 1:50; pMet (CST#3077) at 1:50 and ß-Actin (CST#4947) at 1:100. Ladder, samples, primary and secondary antibodies, separation and stacking matrices were added to the 384-well Peggy Sue plate and run for 8 cycles. Data was analyzed using Compass for SW software.

Confocal Imaging Trogocytosis Assay

Differentiated macrophages were harvested and plated onto CellCarrier96 ultra plates at 1,00,000

cells/well (Perkin-Elmer; Cat#6055302) overnight. For assays with adhered target cells, H1975

NucLight Red cells were plated at 20,000/well, then 4 hours later treated with labeled Ab

cocktail for 1 hour at 4°C. For assays with non-adhered target cells, target cells were stained with

AF647-labeled amivantamab or control Abs. Labeling antibody cocktail contained anti-CD11b

(BD; Cat#557701), anti-CD14 (BD; Cat#562689), and 1:8000 Hoechst33342 (Biotium;

Cat#40046). Images were obtained at 11-minute intervals on a Perkin-Elmer Phenix Opera using

60x water-immersion objective and analyzed using Columbus.

In vivo studies

H1975 cells were subcutaneously implanted into 6-8-week-old female BALB/c nude mice

(Charles River Laboratories). When tumors reached an average of 72±8.7mm3, intraperitoneal

anti-mCSF-1R antibody (400µg/mouse) was administered thrice weekly, beginning five days

prior to compound dosing to facilitate macrophage depletion. At day 5 (average tumor volume =

102±36.6mm3), mice were treated twice weekly by intraperitoneal dosing with 10 mg/kg isotype, amivantamab, or EGFR/cMet IgG2σ. SNU5 cells were subcutaneously implanted into 7-8 week

old female CB17/SCID mice (HFK Bio-Technology Co.Ltd., China). When tumors reached an

average of 155±21.4mm3, mice were treated twice weekly with intraperitoneal PBS or 5 mg/kg

amivantamab or EGFR/cMet IgG2σ, for three weeks. For both studies, tumor measurements and

body weights were recorded twice weekly. Tumor growth inhibition (TGI) was calculated on the

final day where >80% control mice remained on study, using the calculation [1-(T/C)]*100. All

in vivo experiments were done in accordance with the Johnson and Johnson Institutional Animal

Care and Use Committee and the Guide for Care and Use of Laboratory Animals.

RESULTS

Amivantamab Anti-proliferative and Apoptotic Activities Require Fc Binding to Immune

Cells.

We previously demonstrated that amivantamab (17) can induce in vivo EGFR and cMet receptor

downregulation and tumor cell killing, but such activity was not observed in cell culture (9,12).

To assess the in vitro role of amivantamab Fc interactions in tumor cell killing, the anti- proliferative and apoptotic activities were evaluated in the presence or absence of human immune cells (PBMCs) against H1975 cells, a NSCLC cell line harboring mutant EGFR

(L858R/T790M) and wild-type cMet (17). In the absence of PBMCs, amivantamab treatment did not inhibit H1975 cell proliferation or induce apoptosis (Fig. 1A, 1B; Supplementary Fig. S1A,

S1B). However, upon addition of PBMCs, amivantamab induced a dose-dependent decrease in

tumor cell viability (Fig. 1A), with maximal anti-proliferative effect observed at 72 hours

(IC50=0.0054µg/mL, Fig. 1B left panel) and beyond (Supplementary Fig. S1A, S1C).

Amivantamab treatment also resulted in a dose-dependent increase in apoptosis in the presence

of PBMCs (IC50=0.0004µg/mL, Fig. 1B right panel), and this anti-proliferative or apoptotic

activity was not observed upon treatment with a non-targeting huIgG1 isotype control (Fig 1B;

Supplementary Fig. S1B-C). Similar results (proliferation inhibition IC50=0.01µg/mL; apoptosis

IC50=0.002µg/mL) were obtained using PBMCs from a different donor (Supplementary Fig.

S2A, S2B). To confirm the contribution of Fc engagement, the EGFR and cMet binding regions

of amivantamab were engineered into an Fc effector silent molecule by replacing the wild-type

IgG1 with an effector silent IgG2σ framework (9). In presence of PBMCs, treatment with

EGFR/cMet-IgG2σ resulted in no or minimal effects on H1975 target cell viability and apoptosis

(IC50=12.1µg/mL and IC50=0.142µg/mL, respectively) (Fig. 1B; Supplementary Fig. S1B, S2A and S2B), suggesting that these anti-proliferative and apoptotic activities were Fc-interaction dependent.

The anti-proliferative (IC50 = 0.0013µg/mL) and apoptotic (IC50 = 0.014µg/mL) activities of amivantamab were also observed against a MET amplified gastric carcinoma cell line, SNU5, and were dependent on the presence of PBMCs (Fig. 1C; Supplementary Fig. S2C). Similar to

H1975 cells, no effects on proliferation and apoptosis were observed upon treatment with isotype or EGFR/cMet-IgG2σ controls (Fig. 1C). The effects of amivantamab were then compared to that of cetuximab (18), a bivalent chimeric anti-EGFR IgG1 antibody in H1975 and SNU5 cell lines. While cetuximab was able to induce a dose-dependent anti-proliferative effect

(IC50=0.51µg/mL) and apoptosis (IC50=0.56µg/mL) in presence of PBMCs, the magnitude of the

PBMC-dependent activities was inferior to that of amivantamab (Fig. 1D). Similar results were observed in SNU5 cell line, where amivantamab was more potent compared to cetuximab (Fig.

1E).

Collectively, these studies demonstrated that amivantamab Fc interaction with immune cells is essential for its in vitro anti-proliferative effects.

Amivantamab Fc Interaction with Immune Cells Induces ADCC, ADCR and EGFR/cMet

Downmodulation

We next interrogated the different immune effector mechanisms (ADCC, CDC, ADCR, ADCT)

induced by amivantamab and its impact on EGFR/cMet signaling, in comparison with

cetuximab. ADCC-mediated cell lysis was measured using a short-term Europium release assay

in H1975 cells treated with isotype control, amivantamab, cetuximab or Fc silent EGFR/cMet-

IgG2σ in presence of PBMCs. While amivantamab induced dose-dependent ADCC (68% max

lysis), no ADCC lysis was observed with isotype control or EGFR/cMet-IgG2σ treatments, showing the contribution of Fc engagement in this process (Fig. 2A). Cetuximab induced dose- dependent ADCC (39% max lysis) but to a lesser extent than amivantamab (Fig. 2A). Similar results were obtained using PBMCs from a different donor (Supplementary Fig. S3A). Next, we

demonstrated that similar levels of ADCC lysis was measured from isolated NK cells compared

to PBMCs (Supplementary Fig. S3B), suggesting that NK cells account for amivantamab -

induced ADCC. No ADCC was measured from isolated monocytes (Supplementary Fig. S3B).

CDC activity triggered by the activation of the complement component C1q was then assessed.

First, we demonstrated that amivantamab, cetuximab, rituximab and isotype bind to C1q in a

dose-dependent manner, while no measurable binding was observed by the Fc silent

EGFR/cMet-IgG2σ (Supplementary Fig S3C), suggesting that the C1q binding is Fc-structure

specific. We next investigated CDC-mediated target cell lysis of NSCLC cell lines, H1975 (Fig

2B) and H292 (Supplementary Fig S3D) and showed that no measurable CDC activity was

observed upon amivantamab or EGFR/cMet-IgG2σ treatment. As positive control, rituximab

(bivalent anti-CD20 antibody) added to CD20 positive Daudi cells (19), showed dose-dependent

CDC activity (Fig 2B). This demonstrated that amivantamab does not induce CDC against tested

NSCLC cell lines.

Interaction of the Fc region of therapeutic antibodies with FcR on immune cells is known to

induce secretion of cytokines and chemokines (ADCR) (20). A 71-plex MSD cytokine panel was

utilized to assess chemokines and cytokines secreted upon treatment with isotype control,

amivantamab, cetuximab or Fc silent EGFR/cMet-IgG2σ in the presence or absence of PBMCs

for 4 or 72 hours. We observed distinct differences in several secreted cytokines in a treatment-

and timepoint-dependent manners (Fig 2C; Supplementary Fig S3E, S3F). Further analysis

focusing on cytokines with >1.5-fold difference between treatments showed that at 4h (Fig 2D)

and 72h post-treatment (Supplementary Fig S3G), 13 and 7 cytokines, respectively, were

upregulated upon amivantamab treatment compared to isotype or EGFR/cMet-IgG2σ controls in

the co-culture of H1975+PBMCs. Fold induction of most of these cytokines were lower for

cetuximab compared to amivantamab (Fig 2D; Supplementary Fig S3G).

We next interrogated the contribution of immune cell subtypes (PBMCs vs NK cells, monocytes

or macrophages from same donor) in the secretion of these cytokines. Heatmap analysis revealed distinct changes in cytokine expression patterns, with chemotactic cytokines (CC chemokines) being the most frequently upregulated family upon amivantamab treatment (Fig 2E;

Supplementary Fig S4A). CC chemokines are known to function as chemo-attractants for innate immune cells like monocytes and macrophages (21-23). Intriguingly, upregulation of these

chemokines upon amivantamab treatment was specific to PBMCs, monocytes and macrophages

but not seen with NK cells (Fig 2E). For example, amivantamab treatment resulted in a potent

increase in macrophage inflammatory protein (MIP)-1 (compared to isotype) in the presence of

PBMCs, monocytes or M1 and M2 macrophages but no change was observed with NK cells (Fig

2E). While cetuximab induced several of these cytokines, the fold upregulation was lower compared to that of amivantamab, suggesting that amivantamab is more potent than cetuximab in inducing CC chemokines (Fig 2E).

Because EGFR and/or cMet signaling is key to the growth of these tumor cells, we also investigated whether amivantamab or cetuximab Fc interaction with immune cells had an effect on EGFR/cMet protein levels and downstream signaling. To examine this, H1975 cells were treated with isotype control, amivantamab, cetuximab or Fc silent EGFR/cMet-IgG2σ in the absence or presence of PBMCs. Treatment with amivantamab or cetuximab alone (in the absence of PBMCs) only showed marginal downregulation of EGFR and phosphorylated EGFR (pEGFR; pY1173) proteins (Fig. 2F). Compared to EGFR/pEGFR levels, amivantamab treatment (no

PBMCs) showed a greater downregulation of cMet protein (Fig. 2F), which may be attributed to the higher affinity cMet binding arm (9). pMet was below reliably measurable levels in H1975 cells and hence not depicted in this and subsequent analysis. Interestingly, the addition of

PBMCs markedly potentiated amivantamab-mediated downregulation of EGFR, pEGFR, and cMet by 44%, 54% and 52%, respectively (Fig. 2F; densitometry in Supplementary Fig S4B), while treatment with the isotype control or EGFR/cMet-IgG2σ in the absence or presence of

PBMCs had no apparent effect (Fig 2F). While presence of immune cells enhanced cetuximab- induced downregulation of EGFR and pEGFR by 23% and 32%, respectively (Supplementary

Fig S4B), the effect was less pronounced in comparison to that observed with amivantamab

(Supplementary Fig S4C). Cetuximab treatment did not show an appreciable effect on cMet

levels.

Taken together, these results demonstrate that amivantamab Fc interaction with immune cells

induces ADCC, stimulates production of macrophage-related chemokines and enhances

downmodulation of EGFR and cMet, and is more potent than cetuximab in these assays.

Amivantamab Fc Interaction Induced EGFR/cMet Downmodulation is Driven by

Monocyte Composition

We next assessed the contribution of different immune cells at mediating EGFR/cMet

downmodulation. PBMCs from seven donors were tested for their ability to potentiate

amivantamab-mediated downregulation of EGFR, cMet, and pEGFR protein levels (Fig. 3A).

Treatment with amivantamab, in the absence of PBMCs, showed marginal downregulation of

EGFR, pEGFR and moderate downmodulation of cMet protein levels. While the addition of

PBMCs enhanced the downmodulation of EGFR, pEGFR and cMet compared to isotype control

for most donors, the extent of this effect varied among donors (Fig 3A; densitometry in

Supplementary Fig S5A). PBMCs from some donors (#3, #4 and #6) showed a substantial effect

in potentiating EGFR/cMet downmodulation, while PBMCs from other donors (#2, #5 and #7)

had little to no effect. PBMCs from additional donors were similarly tested, and considerable

variability in amivantamab-mediated downmodulation was observed (Supplementary Fig S5B

and S5C).

To examine the role of amivantamab-Fc interaction in EGFR/cMet downmodulation, H1975 cells were treated with isotype control, amivantamab, or Fc silent EGFR/cMet-IgG2σ in the

presence of PBMCs from selected donor#6 (Fig 3B). Addition of PBMCs markedly potentiated

amivantamab-mediated downregulation of EGFR, pEGFR, and cMet by 46%, 67% and 57%,

respectively (Fig 3B; densitometry in Supplementary Fig S6A), while treatment with the isotype

control or EGFR/cMet-IgG2σ in the absence or presence of PBMCs had no apparent effect,

suggesting that it is specific to the amivantamab Fc interaction. Similar results were obtained

with donor#3 (Supplementary Fig S6B). We confirmed this effect of Fc engagement on EGFR

and cMet signaling in the MET-amplified SNU5 cell line (Fig 3C). Similar to H1975 cells,

presence of PBMCs enhanced the ability of amivantamab to downregulate EGFR/cMet

signaling, resulting in 79%, 89%, 87% and 90% reduction in levels of EGFR, pEGFR, cMet and

pMet proteins, respectively, and no measurable change was seen with the isotype or

EGFR/cMet-IgG2σ controls (Supplementary Fig S6C).

To determine which immune cells are responsible for Fc-dependent downmodulation, we

compared percentages of key immune cell populations (NK cells, monocytes, B cells, and T

cells) within PBMCs (Supplementary Fig S7A) for each donor with the observed differences in

their downmodulation effects. No correlation was observed between NK, B, or T cells and the

ability of the PBMCs to potentiate EGFR downmodulation (Fig 3D; Supplementary Fig. S7B).

However, a correlation was observed between relative monocyte population within the PBMCs

and the downmodulation of EGFR (R2=0.51), pEGFR (R2=0.55), and cMet (R2=0.48) protein levels (Fig 3E). This finding suggests that monocytes and their relative level within the PBMC

samples determine the extent of amivantamab -mediated EGFR/cMet downregulation.

Amivantamab Fc Interaction with Monocytes Induces Trogocytosis and is Required for

EGFR and cMet Downmodulation

To confirm direct monocyte involvement in Fc interaction-mediated signal downregulation, NK

cells or monocytes were depleted from PBMCs (Fig 4A) and tested for amivantamab-mediated

downmodulation of EGFR/cMet pathways. Consistent with previous results, non-depleted

PBMCs enhanced amivantamab-mediated downregulation of EGFR, pEGFR, and cMet in

H1975 cells (Fig 4B; densitometry in Supplementary Fig. S8A). While depletion of NK cells

only had a marginal effect, depletion of monocytes effectively reversed the ability of PBMCs to

potentiate amivantamab-mediated signal downmodulation (Fig 4B). As expected, no effect was

seen with EGFR/cMet-IgG2σ treatment confirming that Fc interaction is required for

EGFR/cMet downmodulation. Similar data were obtained by depleting PBMCs of NK cells and

monocytes from a second donor (Supplementary Fig. S8B – S8D).

To further confirm the role of monocytes, NK cell and monocytes were isolated from the same

PBMC donor (Supplementary Fig S9A) and assessed for amivantamab-mediated downregulation of EGFR, pEGFR, and cMet proteins (Fig 4C). While the control (non-isolated) PBMC sample enhanced receptor downmodulation, NK cells alone did not demonstrate a strong effect.

However, isolated monocytes substantially enhanced the ability of amivantamab to downmodulate EGFR, pEGFR, and cMet proteins to a similar extent to that of PBMC+ amivantamab control (Fig 4C; densitometry in Supplementary Fig S9B).

Based on this novel finding that monocytes enhance amivantamab mediated downmodulation of

EGFR and cMet proteins, we assessed if this downmodulation occurs through trogocytosis, an Fc effector function that mediates transfer of cell-surface proteins from tumor to effector cells. To visualize amivantamab-induced monocyte trogocytosis, time-lapse microscopy was performed in

H1975 target cells opsonized with AF647-labeled antibodies (isotype, amivantamab or Fc-silent

EGFR/cMet-IgG2σ). Monocytes were visualized with AF488-labeled CD11b/CD14 antibody cocktail (green) and Hoechst stain for nuclei (blue) and H1975 target cells were identified by their NucLight Red+ nuclei (orange). When co-cultured with opsonized target cells, distinct transfer of labeled amivantamab antibody but not labeled isotype or EGFR/cMet-IgG2σ antibodies into monocytes was observed (Fig. 4D; Supplementary Fig S9C; Supplementary

Movie M1), demonstrating that amivantamab induced monocyte-dependent trogocytosis.

Collectively, these results demonstrate that monocytes mediate trogocytosis and drive amivantamab Fc-interaction mediated downmodulation of EGFR and cMet receptors.

Amivantamab Fc Interaction with Macrophages Induces Trogocytosis Leading to

EGFR/cMet Downmodulation

Solid tumors, particularly NSCLC, have been reported to contain a high level of tumor- associated macrophages (TAMs) (24). Hence, we examined if M1 and M2 macrophages are capable of amivantamab trogocytosis similar to monocytes using time-lapse microscopy in

H1975 target cells opsonized with AF647-labeled antibodies (isotype, amivantamab or

EGFR/cMet-IgG2σ). Macrophages were visualized with AF488-labeled CD11b/CD14 antibody cocktail (green), Hoechst stain for nuclei (blue), and H1975 target cells were identified by their

NucLight Red+ nuclei (orange). Upon co-culture with target cells opsonized with labeled amivantamab, a distinct accumulation of AF647+ puncta (amivantamab) was observed within the

M1 (Fig. 5A; Supplementary Movie M2) and M2c macrophages (Fig. 5B; Supplementary Movie

M3), while no accumulation was seen with labeled isotype or EGFR/cMet-IgG2σ treatment (Fig.

5A, 5B; Supplementary Fig S10A, S10B). No appreciable phagocytosis was observed visually in the presence of M1 or M2c macrophages in these microscopy assays, indicating that macrophage-mediated trogocytosis is the predominant mechanism measured.

To better simulate antibody interactions within the tumor, we treated a co-culture of M1 or M2c macrophages and H1975 target cells with AF647-labeled antibodies (isotype, amivantamab or

EGFR/cMet-IgG2σ) and monitored trogocytosis by time-lapse microscopy. As expected, isotype control antibody bound only to M1 and M2c macrophages, while EGFR/cMet-IgG2σ bound only to target cells, and amivantamab bound to both target cells and macrophages (Supplementary Fig

S10C), thus confirming the binding specificity of each antibody. Under these conditions (co- culture), amivantamab-mediated trogocytosis was readily observed, measured by a distinct

transfer of labeled amivantamab into macrophages, but no trogocytosis was observed with

isotype or EGFR/cMet-IgG2σ antibodies (Supplementary Fig S10D; Supplementary Movie M4,

M5).

Finally, to examine if macrophage-mediated trogocytosis leads to receptor downmodulation,

monocytes were polarized into M1, M2a and M2c macrophages, and their ability to potentiate

amivantamab-mediated EGFR/cMet downmodulation was assessed. Compared to treatment with

amivantamab alone (no macrophages), the presence of M1 or either subtype of M2 macrophages

examined (M2a, M2c), notably enhanced amivantamab-mediated downregulation of EGFR,

pEGFR and cMet proteins levels (Fig. 5C-E; quantitation in Supplementary Fig. S11). Isotype

control or EGFR/cMet-IgG2σ treatments had no effect, suggesting that the signal

downmodulation required Fc interaction.

Collectively, these results reveal trogocytosis as a novel Fc effector function for amivantamab in

the presence of macrophages, leading to EGFR/cMet receptor downmodulation.

Fc interaction and Macrophages are Required for Amivantamab in vivo Anti-tumor

Efficacy

The role and relevance of Fc/FcR interactions in vivo was evaluated using H1975 and SNU5

cell line xenograft models. Mice harboring H1975 or SNU5 tumors were treated with isotype

control, amivantamab or Fc silent EGFR/cMet-IgG2σ antibodies for 3 weeks (Supplementary

Fig. S12A, S12D). In the H1975 model, tumor growth inhibition (TGI) was superior upon

treatment with amivantamab (TGI=75%; ****,p< 0.0001) compared to that of EGFR/cMet-

IgG2σ (TGI=30%; **,p< 0.0016) (Fig 6A; Supplementary Fig S12C shows individual mice).

Similar results were observed in the SNU-5 model, where amivantamab treatment effectively

reduced tumor growth (TGI=96%; ****,p< 0.0001) compared to EGFR/cMet-IgG2σ treatment

(TGI=-17%; ns,p=0.53) (Fig 6B; Supplementary Fig S12F shows individual mice). None of the antibody treatments resulted in loss of mouse body weight in either tumor models

(Supplementary Fig S12B, S12E).

We previously demonstrated, using an HGF-expressing H1975 xenograft tumor model, that Fc

silent EGFR/cMet-IgG2σ only showed partial effect on EGFR/cMet signaling compared to amivantamab, suggesting a role for Fc-mediated signal downregulation in vivo (12). To extend

this observation to the MET-amplified SNU5 model in vivo, tumors were collected from the in

vivo efficacy study (Fig 6B) and changes in total and phospho-EGFR and cMet protein levels

were measured. Similar to in vitro results (Fig 3C), compared to vehicle treatment, amivantamab treatment showed significant downregulation of EGFR, pEGFR, cMet and pMet proteins but

EGFR/cMet-IgG2σ treatment only had marginal effects, suggesting that Fc interaction is required for in vivo signal downmodulation (Fig 6C; quantification in Fig 6C and Supplementary

Fig S13A). Thus, these in vivo studies demonstrate that amivantamab Fc interaction is essential

for its anti-tumor efficacy and in vivo EGFR/cMet signal downmodulation.

The role of macrophages in amivantamab in vivo efficacy was next assessed using anti-CSF1R

mediated depletion of TAMs in the H1975 xenograft study. As expected, treatment with anti-

CSF1R antibody showed significant (**,p<0.002) reduction in TAMs (~2%) compared to

untreated tumors (11-15%) (Fig 6D). Animals were then treated with isotype control or

amivantamab for 3 weeks (Supplementary Fig S13B) with no loss in mouse body weight

(Supplementary Fig S13C). As shown previously, treatment with amivantamab showed

significantly higher anti-tumor efficacy compared to the isotype (****,p<0.0001) or

EGFR/cMet-IgG2σ treatment (**,p=0.004) in non-anti-CSF1R treated tumors (Fig 6E).

Strikingly, depletion of TAMs (anti-CSF1R-treated) significantly reduced amivantamab TGI

from 72.8% to 38.5% (**,p<0.01) (Fig 6E; Supplementary Fig S13D shows individual mice),

suggesting that macrophages play a key role in mediating amivantamab anti-tumor efficacy. No

significant difference was observed between vehicle and vehicle+anti-CSF1R treatment

(p=0.17).

Collectively, these findings demonstrate that amivantamab Fc-interaction with macrophages and

monocytes induces trogocytosis which is a dominant mechanism underlying the anti-tumor efficacy of amivantamab.

DISCUSSION

In this study, we demonstrated that in addition to the known Fc-independent mechanisms

(inhibition of ligand binding and ligand driven signaling (9,12)), the EGFR/cMet bispecific

antibody, amivantamab, exerts anti-tumor efficacy through multiple Fc-dependent mechanisms

(Fig 6F). These mechanisms are initiated through interaction of the Fc domain of the antibody with Fcγ receptors present on immune cells. Binding and activation of Fcγ receptors on NK cells

led to ADCC, while binding and activation of Fcγ receptors on monocytes and macrophages led to cytokine production and trogocytosis. Trogocytosis caused downmodulation of EGFR and

cMet receptors and their downstream signaling, which is required for proliferation of these

cancer cells.

Although we demonstrated in vitro ADCC lysis with amivantamab, we believe that this effect is

minimal compared to that exerted by monocytes and macrophages. ADCC lysis is a rapid

mechanism occurring at early timepoints (~2 to 4hrs (25)), while 48 hours and more were

required to reach the maximal amivantamab in vitro anti-proliferative effect in presence of

PBMCs (Supplementary Fig S1C). This suggests that the short-term ADCC is not sufficiently

contributing to the overall long-term activity of amivantamab and that other effector functions

(such as trogocytosis) contribute to additional cytotoxic activity over time. While we did not

observe complement dependent cytotoxicity (CDC) activity in the cell lines evaluated, NSCLC

cell lines are known to express complement inhibitory proteins, CD46, CD55, and CD59 (26),

suggesting that amivantamab might trigger CDC activity in other cell lines or tumor types not

expressing these complement inhibitory proteins. Finally, we reported previously that

amivantamab induces ADCP in vitro (9), however, under the conditions tested in this study

(confocal microscopy-based macrophage trogocytosis assay), minimal to no ADCP was

observed. Collectively, as depicted in Fig 6F, our data suggest that amivantamab anti-tumoral

activity occurs through multiple Fc-independent and Fc-dependent mediated mechanisms of action linked to EGFR and cMet binding on cancer cells. Fc-dependent amivantamab mechanism of action comprises NK-dependent ADCC as well as monocyte- and macrophage-dependent trogocytosis and cytokine release. Since the content and diversity of immune cells within the tumor microenvironment varies among patients, this may influence the response to amivantamab treatment.

We showed that most cytokines upregulated by amivantamab belong to the family of chemotactic cytokines called chemokines, specifically CC chemokines (21,23). CC chemokines comprises of two key subfamilies, monocyte chemoattract protein (MCP) and macrophage inflammatory protein (MIP), which are both known to function as chemo-attractants for innate immune cells such as monocytes and macrophages (22,27). MCP-1 (CCL2) and MIP1 (CCL4) have been reported to induce recruitment of monocytes and macrophages into the tumor microenvironment (TME) of NSCLC (22,28). While these cytokines could attract immune cells to the TME, their production requires an initial tumor cell-immune cell contact mediated by amivantamab. So, we hypothesize that trogocytosis is the initial and dominant mechanism of amivantamab leading to downmodulation of EGFR and cMet, and potentially fueling additional recruitment of monocytes or macrophages by cytokine production.

In contrast to most other therapeutic antibodies in the clinic, amivantamab was designed and engineered with a low fucose backbone, which enhances its binding to FcRIIIa (12), present on

NK cells, monocytes and macrophages. This increased binding to FcRIIIa enhanced induction of Fc effector functions in comparison to other (normal fucose) huIgG1 antibodies such as cetuximab and trastuzumab, and could also enable amivantamab to out-compete naturally circulating IgG antibodies for FcR binding, as shown pre-clinically with other enhanced Fc antibodies (29,30). Given the predominance of EGFR-driven NSCLC tumors, it is confounding that cetuximab did not meet clinical endpoints in NSCLC (31). We hypothesize that this could result from several factors that amivantamab can overcome. Primarily, cetuximab only targets

EGFR and it is known that cancer cells can quickly adapt to therapy by activating alternate

pathways, the most common of which is cMet (32). Additionally, the bivalent nature of the high affinity EGFR arm in cetuximab results in on-target off tumor toxicity in the clinic (31). Studies have shown that bispecificity of an antibody can increase tumor selectivity via simultaneous targeting of two cancer antigens and varying antigen affinity can drive selectivity towards cells expressing both antigens compared to single antigen expressing normal cells (33-35). Thus, we hypothesize that the presence of the high affinity cMet targeting arm (Kd=40pmol/L), lower affinity EGFR targeting arm (Kd=1.4nmol/L) combined with the higher affinity FcRIIIa binding (low fucose) Fc portion could account for the improved selectivity (36) and efficacy, and lowered toxicity of amivantamab in comparison with cetuximab.

Previously, trogocytosis was described as a mechanism of resistance for antibodies such as rituximab (16,37). In this context, the receptor is stripped from the cell surface by monocytes/macrophages, preventing occurrence of NK cell-mediated functions towards the no- longer available targeting receptor. However, in this study, we demonstrated a novel role for trogocytosis (ADCT) as a crucial inducer of Fc-dependent receptor downmodulation and anti- tumor effects. We hypothesize that the dichotomy of trogocytosis’ role (mediator of resistance vs anti-tumor effects) might be driven by the dependency of the tumor cells on the trogocytosis- targeted receptor. While anti-tumor effects via trogocytosis have been suggested for cetuximab and trastuzumab (30,38), our work confirmed such a mechanism of action for amivantamab and presents comprehensive data with primary immune cells (monocytes, M1 and M2 macrophages), demonstrating the induction of trogocytosis leading to the downmodulation of the target receptors, EGFR and cMet. It is interesting to note that trogocytosis was observed for both M1

and M2 macrophage populations, suggesting this mechanism may also be operative in more

immunosuppressive TMEs.

Therapeutic targeting of EGFR and cMet with an antibody such as amivantamab with multiple

and enhanced Fc mechanism(s) of action can provide future options to combat diverse types of

cancer-driving mutations and drug-acquired resistance for patients. In addition to targeting a

conserved region of an oncogenic receptor, which broadens the targeted population, the

selectivity of bispecific antibodies like amivantamab, could offer a reduced potential for toxicity,

making combination therapy with additional agents better tolerated. Our findings demonstrate

that the Fc-dependent activity of amivantamab is driven by macrophage- and monocyte-mediated

trogocytosis, a dominant mechanism of antibody-directed receptor downregulation and tumor cell killing. These findings highlight the potential of trogocytosis as therapeutic modality but also provide insights to guide patient selection and combination strategies for amivantamab.

ACKNOWLEDGMENTS

The study was funded by Janssen Research & Development, LLC. Antibody generation

performed by Pete Buckley, Janssen Biotherapeutics. Assistance with performing in vivo

experiments was provided by David Walker, Janssen R&D, LLC. Technical guidance for the

confocal microscopy based trogocytosis assay was provided by Edward Keough, Janssen

Biotherapeutics. Writing assistance was provided by Ramji Narayanan (SIRO Clinpharm Pvt

Ltd, Thane, India), funded by Janssen Global Services, LLC and additional editorial support was

provided by Tracy Cao (Janssen Global Services, LLC, NJ).

AUTHORSHIP CONTRIBUTION

Participated in research design: Smruthi Vijayaraghavan, Benjamin Henley, Ryan Lenhart,

Hillary J. Millar, Kathryn Packman, Sheri Moores, Sylvie Laquerre, Matthew Lorenzi.

Conducted experiments: Smruthi Vijayaraghavan, Lorraine Lipfert, Kristen Chevalier, Barbara

Bushey, Benjamin Henley, Ryan Lenhart, Marilda Beqiri.

Performed data analysis and data interpretation: Smruthi Vijayaraghavan, Kristen Chevalier,

Barbara Bushey, Benjamin Henley, Ryan Lenhart, Jocelyn Sendecki, Sheri Moores, Sylvie

Laquerre, Matthew Lorenzi.

Wrote or contributed to the writing of the manuscript: Smruthi Vijayaraghavan, Sheri

Moores, Sylvie Laquerre, Matthew Lorenzi.

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MAIN FIGURE LEGENDS

Figure 1. Amivantamab Fc interaction with immune cells enhanced in vitro cancer cell

growth inhibition and apoptosis: A) Representative images depicting dose-dependent effect of amivantamab on viability of NucLight Red labeled H1975 cells in the presence or absence of

PBMCs at E:T ratio of 10:1 at 72 hours. B) Dose-response curves measuring H1975 cell viability

(AUC of NucRed area/well) post 72 hours and apoptosis (AUC of Annexin+ NucRed area/well)

post 48 hours upon treatment with isotype control, amivantamab or EGFR/cMet-IgG2σ (IgG2σ), in presence or absence of PBMCs (E:T ratio of 10:1) from donor #6. C) Dose-response curves

measuring SNU5 cell viability (AUC of NucRed area/well) post 72 hours and apoptosis (AUC of

Annexin+ NucRed area/well) post 24 hours upon treatment with isotype control, amivantamab or

EGFR/cMet-IgG2σ (IgG2σ), in presence or absence of PBMCs (E:T ratio of 10:1) from donor

#3. D) Dose-response curves measuring H1975 cell viability (AUC of NucRed area/well) post 72

hours and apoptosis (AUC of Annexin+ NucRed area/well) post 48 hours upon treatment with

amivantamab or cetuximab, in presence or absence of PBMCs (E:T ratio of 10:1) from donor #6.

E) Dose-response curves measuring SNU5 cell viability (Total NucRed area/well) post 48 hours

treatment with amivantamab or cetuximab, in presence or absence of PBMCs (E:T ratio of 10:1)

from donor #3.

Figure 2. Amivantamab Fc interaction induces ADCC, ADCR and EGFR/cMet

downmodulation but not CDC: A) BATDA-loaded H1975 cells were treated for 2 hours with

isotype, amivantamab, cetuximab or EGFR/cMet-IgG2σ (IgG2σ) at multiple concentrations in

presence of PBMCs from donor #4 at an E:T ratio of 25:1 and ADCC lysis measured by

Europium release. B) Dose response of CDC lysis measured by LDH (Lactate dehydrogenase)

levels from H1975 target cells after 2 hours of treatment with amivantamab or EGFR/cMet-

IgG2σ (IgG2σ) in presence of 5% baby rabbit serum. CD-20 antibody, Rituximab, was used as a

positive control in CD20 expressing Daudi cell line. C) Heat map of log-transformed AUC

values of cytokines from the 71-plex MSD analysis of PBMCs alone, H1975 cells alone or

H1975 + PBMCs (E:T=10:1) treated with isotype control, amivantamab, cetuximab or

EGFR/cMet-IgG2σ (IgG2σ) for 4 hours. D) Bar graphs representing the relative change

(measured in panel C) over isotype control upon treatment with amivantamab, cetuximab or

EGFR/cMet-IgG2σ (IgG2σ) antibodies for 4 hours. Graphs were limited to cytokines with

greater than 1.5X increase for amivantamab treatment compared to isotype. E) Heat map of log-

transformed AUC values of cytokines from the 23-plex MSD analysis of H1975 cells treated

with multiple concentrations of isotype, amivantamab, cetuximab or EGFR/cMet-IgG2σ (IgG2σ)

for 4 hours in presence of PBMCs (E:T=10:1) or individual immune cells (E:T=5:1), namely NK

cells, monocytes, M1 or M2c macrophages. Heat map was limited to cytokines with greater than

1.5X increase for in the H1975+PBMCs condition. F) Western blot (PeggySue capillary

electrophoresis) of EGFR, pEGFR, cMet and loading control actin performed following 48 hours treatment of H1975 cells with 10g/ml of isotype, amivantamab or cetuximab in presence or

absence of PBMCs (E:T=10:1) from Donor#3.

Figure 3. Amivantamab Fc interaction with immune cells enhances amivantamab induced

EGFR/cMet downmodulation and correlates with monocyte composition: A) Western blot

(PeggySue capillary electrophoresis) of EGFR, pEGFR, cMet and loading control actin

performed following 48 hours treatment of H1975 cells with 10g/ml of Isotype or amivantamab

(Ami) in presence of PBMCs from seven different donors. Western blot (PeggySue) of EGFR,

pEGFR, cMet, pMet and loading control actin performed following 48 hours treatment with

10g/ml of Isotype control, amivantamab (Ami) or EGFR/cMet-IgG2σ (IgG2σ) in presence or

absence of PBMCs from B) donor #6 against H1975 cancer cells and C) donor #3 against SNU5

cancer cells. D) Correlation between the percentage (%) of NK cells, B cells and T cells within

PBMC sample from each donor and the percentage (%) change in EGFR inhibition upon

amivantamab treatment in the presence of PBMCs. E) Correlation between the percentage (%) of

monocytes within the PBMC samples and percent (%) changes in EGFR, pEGFR (Y1173) and

cMet inhibition upon amivantamab treatment in presence of PBMCs.

Figure 4. Monocytes mediated amivantamab Fc interaction induced trogocytosis and

EGFR/cMet downmodulation: A) Contour plots from multi-color flow cytometry analysis

showing the composition of NK cells and monocytes within PBMCs (Donor #3) upon CD56 and

CD14 positive selection respectively. B) Western Blot (PeggySue capillary electrophoresis) of

EGFR, pEGFR, cMet and actin (loading control) performed on H1975 cell lysates following 48

hours treatment with 10g/ml of isotype, amivantamab (Ami) or EGFR/cMet-IgG2σ (IgG2σ) in

presence or absence of intact PBMCs, NK cell depleted or monocyte (mono) depleted PBMCs

from donor #3. C) Western Blot (PeggySue) of EGFR, pEGFR, cMet and loading control actin

performed on H1975 cell lysates following 48 hours treatment with 10g/ml of isotype,

amivantamab (Ami) or EGFR/cMet-IgG2σ (IgG2σ) in the presence or absence of intact PBMCs, isolated NK cells and isolated monocytes from donor #3. D) Representative images from high-

content confocal microscopy of monocytes labeled with AF488-CD11b, AF488-CD14 and

Hoechst (nuclei) in co-culture (E:T ratio of 5:1) with H1975 NucLight Red cells opsonized with

AF647-labeled amivantamab (Ami) or EGFR/cMet-IgG2σ (IgG2σ) antibodies. White arrows

depict trogocytosis events measured by transfer of AF647 labeled antibody from target cells to

monocytes. Yellow arrows point to one such event across time. Scale bar = 20µm.

Figure 5. Amivantamab Fc interaction induces macrophage-trogocytosis and EGFR/cMet

downmodulation: Representative images from high-content confocal microscopy of A) M1 or

B) M2 Macrophages labeled with AF488-CS11b, AF488-CD14 and Hoechst (nuclei) in co-

culture (E:T ratio of 5:1) with H1975 NucLight Red cells opsonized with AF647-labeled

amivantamab or EGFR/cMet-IgG2σ (IgG2σ) antibodies. White arrows depict trogocytosis events

measured by transfer of AF647 labeled antibody from target cells to macrophages. Yellow

arrows point to one such event across time. Scale bar = 20µm. Western Blot (PeggySue capillary

electrophoresis) of EGFR, pEGFR, cMet and actin (loading control) performed on H1975 cell

lysates following 48 hours treatment with 10g/ml of isotype, amivantamab (Ami) or

EGFR/cMet-IgG2σ (IgG2σ) in the presence or absence of C) M1 macrophages (M1) , D) M2a

macrophages (M2a) or E) M2c macrophages (M2c)

Figure 6. Amivantamab Fc interaction with macrophages are required for in vivo anti-

tumor efficacy: A) Tumor volumes of subcutaneously injected H1975 cell line xenograft tumors treated with 10mg/kg isotype control, amivantamab or EGFR/cMet-IgG2σ (IgG2σ) (n=8 per

group) for 3 weeks BIW. %TGI was calculated at day 24 and p-values were calculated using

2Way ANOVA; ** = p<0.0021, **** = p<0.0001. B) Tumor volumes of subcutaneously

injected SNU5 cell line xenograft tumors treated with Vehicle (PBS), 5mg/kg amivantamab

(Ami) or EGFR/cMet-IgG2σ (IgG2σ) (n=8 per group) for 3 weeks BIW. %TGI was calculated at day 34 and p-values were calculated using 2Way ANOVA; **** = p<0.0001. All data

represented as Mean ± S.E.M within each treatment group. C) Western blot analysis of SNU5 xenograft tumors harvested 24hrs after two doses (treated as described in B) measuring protein

levels of EGFR, pEGFR, cMet, pMet or loading control GAPDH. Densitometry measurements

for EGFR and cMet protein were normalized to loading control GAPDH. p-values were

calculated using OneWay ANOVA; ** = p<0.05, ***= p<0.005. D) Multi-color flow cytometry

analysis of H1975 tumor samples isolated 24hrs after two doses of 10mg/kg isotype,

amivantamab (Ami) or EGFR/cMet-IgG2σ (IgG2σ) treatment (n=5 mice/treatment) to assess the

percentage (%) of macrophages (CD45+ CD11b+ Ly6G- Ly6C- F4/80+) within the tumor post

depletion with anti-mouse CSF1R antibody. E) Tumor volumes of subcutaneously injected

H1975 cell line xenograft tumors treated with 10mg/kg isotype, amivantamab, or EGFR/cMet-

IgG2σ (IgG2σ) (n=8 mice per group) for 3 weeks BIW in combination with anti-mouse CSF1R

or its isotype control thrice weekly to deplete macrophages. %TGI was calculated on day 21 and

p-values were calculated using 2Way ANOVA; **, p value < 0.001. F) Cartoon illustrating the

multiple Fc-dependent and Fc-independent mechanisms of action contributing to amivantamab

anti-tumor activity.

A B 80 150 IsotySe PCetXxL aE 60 $PLYDQWDPDE 100 'DXGL5LtX[LPaE IgG2 ı + $PLYDQWDPDE 40 +, gG2ı

50 20 % Maximal Lysis (ADCC) Lysis Maximal % % Target cell lysis (CDC) 0 0 -4 -2 0 2 0.001 0.01 0.1 1 10 100 Log treatment conc. (µg/ml) Treatment conc. (µg/ml)

C D Count 0 5 10 15 20

1 2 3 4 5 log(AUC) 4 +3%0&,VRW\SH

+3%0&$PLYDQWDPDE 3 $PLYDQWDPDE

+3%0&&HWX[LPDE e)

+3%0&,J*ı \S IgG2ı +,VRW\SH 2 CetX[LPaE +$PLYDQWDPDE +,J*ı 1 3%0&,VRW\SH (relative to Isot to (relative

3%0&$PLYDQWDPDE change fold Relative 0 3%0&,J*ı MIF ,/í ,/í ,/í ,/í ,/í ,/í ,/íȕ ,3í ,)1íȖ 7$5& ,í7$& )/7/ 71)íĮ Eotaxin 0&3í 0&3í 0&3í 0&3í ,/í5$ 0í&6) *52íĮ 0,3íȕ <./í 6')íĮ (1$í

,)1íĮ 2a IP-10 9(*)í$ IFN- *0í&6) ,/íS TARC (RWD[LQí MCP-3 IL-1RA MCP-1 TNF- GRO- Fractalkine MIP-1 ENA-78

E F CC Count CXC 0 2 4 6 0.2 0.4 0.6 0.8 1 ,QWHUOHXNLQ Percentile

+3%0&,VRW\SH Iso +3%0&$PLYDQWDPDE $PLYDQWDPDE +3%0&&HWX[LPDE Cetuximab +3%0&,J*ı PBMC +1.,VRW\SH +1.$PLYDQWDPDE +1.&HWX[LPDE EGFR +1.,J*ı +0RQRF\WH,VRW\SH +0RQRF\WH$PLYDQWDPDE +0RQRF\WH&HWX[LPDE +0RQRF\WH,J*ı pEGFR +00DFURSKDJH,VRW\SH +00DFURSKDJH$PLYDQWDPDE +00DFURSKDJH,J*ı +0&0DFURSKDJH,VRW\SH cMet +0&0DFURSKDJH$PLYDQWDPDE +0&0DFURSKDJH,J*ı Actin ,3 ,/,5$ MCP-2 0&3 0&3 0,3ȕ 0,3Į

A B C PBMC Donor # Isotype Isotype $PL $PL Iso $PL IgG2ı PBMC 1 2 3 4 5 6 7 IgG2ı PBMC PBMC

EGFR EGFR EGFR

pEGFR pEGFR pEGFR

cMet cMet cMet pMet

Actin Actin Actin

D 100 100 100

50 50 50

0 0 0

-50 -50 -50 % EGFR inhibtion EGFR % % EGFR inhibtion EGFR % % EGFR inhibtion EGFR % R2 = 0.06 R2 = 0.0079 R2 = 0.45 -100 -100 -100 0 5 10 15 20 10 12 14 16 18 0 20 40 60 80 % of NK cells % of B cells % of T cells E

100 100 100

50 50 50

0 0 0

-50 -50 -50 % Met inhibtion Met % % EGFR inhibtion EGFR % R2 = 0.5088 inhibtion pEGFR % R2 = 0.55 R2 = 0.475 -100 -100 -100 0 10 20 30 0 10 20 30 0 10 20 30 % of Monocytes % of Monocytes % of Monocytes

A B NK Mono PBMCs depleted depleted

5 5 10 NK cells (CD56+ cells) 10 NK cells (CD56+ cells) Isotype 13.9% 3.02%

4 4 $PL 10 10 IgGı

3 3 Immune cells 10 10 CD56 (Qdot 705) CD56 (Qdot 705) 0 0

3 3 EGFR -10 -10

-103 0 103 104 105 -103 0 103 104 105 CD3 (Qdot 605) CD3 (Qdot 605)

250K 250K pEGFR Monocytes Monocytes 200K (CD14+ cells) 200K (CD14+ cells) CD14-cells 42.6% CD14-cells 0.055% 53.0% 98.7% 150K 150K SSC-A SSC-A 100K 100K FMet

50K 50K

0 0 3 3 4 5 3 3 4 5 Actin -10 0 10 10 10 -10 0 10 10 10 CD14 (PacBlue) CD14 (PacBlue)

D C $PL (No green)T=0 $PL IgG2ı PBMCs NK cells Monocytes Isotype $PL IgGı Immune cells

EGFR T=55

pEGFR

FMet T=77

Actin

T=99

Hoechst Monocytes (Cd11b/CD14) H1975-Nuclight Red

A T=11 T=44 T=77 T=110 $PLYDQWDPDE ,J*ı

Hoechst M1 Macrophages (Cd11b/CD14) H1975-Nuclight Red

B T=11 T=44 T=77 T=110 $PLYDQWDPDE ,J*ı

Hoechst M2 Macrophages (Cd11b/CD14) H1975-Nuclight Red

C Isotype D Isotype E Isotype $PL $PL $PL IgGı IgGı IgGı Macs Macs Macs

EGFR EGFR EGFR

pEGFR pEGFR pEGFR

cMet cMet cMet

Actin Actin Actin

A 2000 B 2000 Isotype Control Vehicle PBS ns -17% TGI ± SEM) ± SEM) $PLYDQWDPDE $PLYDQWDPDE 3 3 IgG2 IgG2ı 1500 ı 1500

30% TGI 1000 1000 **

500 500 **** 75% TGI **** 96% TGI

0 (mm Volume Tumor SNU5 Mean 0 Mean H1975 Tumor Volume (mm Volume Tumor H1975 Mean 0 5 10 15 20 25 10 15 20 25 30 35 Days post tumor implantation Days post tumor implantation

C PBS $PL EGFR cMet IgG2ı 0.3 0.8 ** EGFR ** ** 0.6 *** 0.2 pEGFR 0.4

cMet 0.1 0.2 (Normalized to GAPDH) (Normalized to GAPDH) Mean Normalized Signal Mean Normalized Signal pMet 0.0 0.0

IgG2ı IgG2ı GAPDH YDQWDPDE ehicle PBS YDQWDPDE ehicle PBS V V $PL $PL

D F fects 25 ** nt ef nde epe d Trogocytos -d P an is 20 FC DC A on an radati d Enha 15 Deg nc or nal Downmod ed pt t Sig ulat ce Me ion 10 e /c R FR G E Trogocytosis F C 5 - in E d Macrophage e Monocyte D G p 0 F e o R n

(% F4/80+ cells of single cells) w d / Tumor associated Macrophages n c e m M n $PL $PL t ,J*ı C o e e t f C d f NK e u S D Cells c l i t g a s Isotype Control Isotype Control A t n i CSF1R Tx o a E n l 1500 ADCR - Cytokine NSCLC Release Tumor Isotype Control ± SEM)

3 $PLYDQWDPDE IgG2 1000 ı Isotype Control + CSF1R cMET-binding Į EGFR-binding $PLYDQWDPDE + ĮCSF1R 29% TG, EGFR 38% TG, Fc Region cMet JNJ-61186372 Fc Receptor 500 ADCR TUMOR CELL 73% TG, KILLING ** Mean Tumor VolumeMean Tumor (mm

0 0 5 10 15 20 ĮCSF1R Tx

$PLYDQWDPDETx Days post tumor implantation

Amivantamab (JNJ-61186372), An Fc Enhanced EGFR/cMet Bispecific Antibody Induces Receptor Downmodulation And Anti-tumor Activity By Monocyte/Macrophage Trogocytosis

Smruthi Vijayaraghavan, Lorraine Lipfert, Kristen Chevalier, et al.

Mol Cancer Ther Published OnlineFirst August 3, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1535-7163.MCT-20-0071

Supplementary Access the most recent supplemental material at: Material http://mct.aacrjournals.org/content/suppl/2020/08/01/1535-7163.MCT-20-0071.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet been Manuscript edited.

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