amivantamab (jnj-61186372), an fc enhanced egfr/ cmet … · (essen biosciences; catalog no. 4476)...

MOLECULAR CANCER THERAPEUTICS | LARGE MOLECULE THERAPEUTICS

Amivantamab (JNJ-61186372), an Fc Enhanced EGFR/cMet Bispecific Antibody, Induces ReceptorDownmodulation and Antitumor Activity by Monocyte/Macrophage Trogocytosis A C

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, and Sheri L. Moores

ABSTRACT◥

Smallmolecule inhibitors targetingmutant EGFR are standard ofcare in non–small cell lung cancer (NSCLC), but acquired resistanceinvariably develops through mutations in EGFR or through acti-vation of compensatory pathways such as cMet. Amivantamab(JNJ-61186372) is an anti-EGFR and anti-cMet bispecific low fucoseantibodywith enhanced Fc function designed to treat tumors drivenby activated EGFR and/or cMet signaling. Potent in vivo antitumorefficacy is observed upon amivantamab treatment of human tumorxenograft models driven by mutant activated EGFR, and thisactivity is associated with receptor downregulation. Despite theserobust antitumor responses in vivo, limited antiproliferative effectsand EGFR/cMet receptor downregulation by amivantamab were

observed in vitro. Interestingly, in vitro addition of isolated humanimmune cells notably enhanced amivantamab-mediated EGFR andcMet downregulation, leading to antibody dose-dependent cancercell killing. Through a comprehensive assessment of the Fc-mediated effector functions, we demonstrate that monocytesand/or macrophages, through trogocytosis, are necessary and suf-ficient for Fc interaction-mediated EGFR/cMet downmodulationand are required for in vivo antitumor efficacy. Collectively, ourfindings represent a novel Fc-dependent macrophage-mediatedantitumor mechanism of amivantamab and highlight trogocytosisas an important mechanism of action to exploit in designing newantibody-based cancer therapies.

IntroductionTreatment of non–small cell lung cancer (NSCLC) was transformed

upon identification and targeting of primary activating mutations inthe kinase domain of EGFR, which occur in 10% to 40% of patientswith lung cancer. Patients with activating EGFRmutations are initiallyresponsive to first- and second-generation EGFR tyrosine kinaseinhibitor (TKI) treatment. However, this efficacy is short-lived, andacquired resistance develops through secondarymutations in EGFR aswell as upregulation of other pathways, such as cMet (1–3). Osimer-tinib, a covalent third-generation EGFR TKI, is used for the treatmentof EGFR mutant NSCLC patients with acquired resistance to priorEGFR TKI therapy due to T790M mutation, and has recently becomethe standard of care front-line therapy (4). However, similar to thefirst-generation TKIs, acquired resistance to osimertinib is alsoobserved through additional mutations in EGFR (e.g., C797S) oractivation of the cMet pathway (5–7). Currently, there are no approvedtherapies for patients resistant to third-generation TKIs, highlightingthe significant unmet need for this population.

To address this unmet medical need, we developed amivantamab(JNJ-61186372), a fully human IgG1-based bispecific antibody target-

ing both EGFR and cMet (US Patent no. US9593164B2; ref. 8). Thisbispecific antibody binds EGFR agnostic of the diverse primary andacquired resistance mutations, potentially increasing the therapeuticreach of this antibody. Amivantamab is currently in clinical develop-ment, and consistentwith its preclinical profile (9), preliminary clinicaldata demonstrates that amivantamab treatment leads to NSCLCregression 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 METamplification (10, 11).

Amivantamab was produced in engineered CHO cell lines thatincorporate low levels of fucose, to generate a bispecific antibody withenhanced binding to Fc gamma receptor (FcgR)-IIIa/CD16a onimmune cells (12). Binding of huIgG1 antibodies to FcgRs can mediatetarget cell elimination through immune effector cell functions such asantibody-dependent cellular cytotoxicity (ADCC), antibody-dependentcellular phagocytosis (ADCP), and complement-dependent cytotoxicity(CDC; ref. 13). ADCC and ADCP are triggered when IgG1 antibodiesbind to cell surface antigens, subsequently clustering their Fc domainsto engage with FcgRs on natural killer (NK) cells (ADCC) and macro-phages (ADCP; ref. 14). CDC is activated through interaction of thecomplement component C1q with clustered antibodies bound to targetcells (13). Tumor-targeted antibodies such as rituximab (anti-CD20antibody), cetuximab (anti-EGFR antibody), and trastuzumab (anti-HER2 antibody) are known to exploit these three FcgR-mediatedmechanisms (15). Trogocytosis (antibody-dependent cellular trogocy-tosis, ADCT) is another important consequence of Fc–FcgR interaction,and is described as tumor-targeted antibody-mediated transfer ofmembrane fragments and ligands from tumor cells to effector cellssuch as monocytes, macrophages, and neutrophils (16). Unlike phago-cytosis, trogocytosis is underappreciated as a mechanism of actioncontributing to tumor cell death.

Janssen Research & Development, Spring House, Pennsylvania.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Smruthi Vijayaraghavan, Janssen Research & Develop-ment, 1400 McKean Road, Spring House, PA 19477. Phone: 215-628-5121; E-mail:[email protected]

Mol Cancer Ther 2020;19:2044–56

doi: 10.1158/1535-7163.MCT-20-0071

�2020 American Association for Cancer Research.

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Published OnlineFirst August 3, 2020; DOI: 10.1158/1535-7163.MCT-20-0071

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In previous studies we have shown that amivantamab can induceADCC and ADCP, in addition to Fc-independent EGFR and cMetsignal inhibition (9, 12). Further, in an in vivo study in an EGFRmutant cMet-activated tumor model, we showed that treatment withFc-silent IgG2s version of amivantamab elicits reduced tumor growthinhibition (TGI) compared with amivantamab (Fc-active version),suggesting that the Fc-mediated effector function of amivantamab isessential for its maximal antitumor efficacy (12). In addition, theIgG2s version of amivantamab exhibited reduced downregulation ofEGFR, cMet, and their respective signaling components in vivo,suggesting the requirement of amivantamab Fc interaction for recep-tor and signaling downmodulation activity.

Here we report that trogocytosis is a dominantmechanism of actionfor the preclinical in vitro and in vivo antitumor activity of amivanta-mab. We performed a comprehensive analysis of the Fc-mediatedeffector functions to dissect the key immune cell types underlying theFc-dependent mechanisms of amivantamab. This work led to theidentification of Fc-mediated monocyte and macrophage interactionsand associated trogocytosis as key mediators of receptor downmodu-lation and antibody-mediated cancer cell killing. Together, theseresults point to the importance of trogocytosis as a mechanism ofcancer cell death, which can be leveraged in the design of futureantibody-based therapies.

Materials and MethodsProliferation and apoptosis assays

For Incucyte-based proliferation and apoptosis assays, H1975 orSNU5 cell lines were infected with NucLight Red lentiviral reagent(Essen Biosciences; catalog no. 4476) and selected with 1 mg/mL or4 mg/mL puromycin to generate H1975 and SNU5 NucRed cells,respectively. NucRed target cells were plated at 5,000 cells/well andPBMCs (HemaCare) were added at an E:T (effector:target) ratio of10:1. Annexin-V Green reagent (Essen Biosciences; catalog no. 4642)was added, followed by therapeutic antibodies and scanned (4 images/well/scan) with IncucyteS3 every 4 hours. Total NucRed area (mm2/well) shows target cell viability and Total Green NucRed area (mm2/well) shows target cell apoptosis. Data were visualized with Spotfireand dose–response curves were generated using GraphPad Prism.

Simple Western assayH1975 or SNU5 target cells were plated (100,000 cells/well) in six-

well plates and PBMCs were added at an E:T ratio of 10:1 or individualimmune cells (NK cells, monocytes, or macrophages) at E:T ratio of5:1. Therapeutic antibodies were added and incubated for 48 hours.Protein lysates were extracted using RIPA lysis buffer (Thermo FisherScientific; catalog no. 89901) with 1-tab PhosSTOP (Sigma; catalog no.4906837001) and cOmplete Protease Inhibitor (Sigma; catalog no.04693159001). Protein concentration was determined using PierceBCAProteinAssayKit (Thermo Fisher Scientific; catalog no. 23227) asper manufacturer's protocol. Capillary-based electrophoresis was per-formed using PeggySue 12 to 230 kDa separation module (ProteinSimple; catalog no. SM-S001), anti-rabbit detection module (cat-alog no. DM-001), and anti-mouse antibody (catalog no. 042-205)as per the manufacturer's protocol. Briefly, protein lysates werediluted to 0.2 mg/mL and denatured at 95�C for 5 minutes. Primaryantibodies were diluted: EGFR (CST, 2646) at 1:50; phosphorylatedEGFR (pEGFR; R&D Systems, AF1095) at 1:50; cMet (CST, 3148)at 1:50; pMet (CST, 3077) at 1:50, and b-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 eight cycles. Data were analyzed using Compass for SWsoftware.

Confocal imaging trogocytosis assayDifferentiated macrophages were harvested and plated onto Cell-

Carrier96 ultra plates at 1,00,000 cells/well (Perkin-Elmer; catalog no.6055302) overnight. For assays with adhered target cells, H1975NucLight Red cells were plated at 20,000/well, then 4 hours latertreated with labeled Ab cocktail for 1 hour at 4�C. For assays withnonadhered target cells, target cells were stained with AF647-labeledamivantamab or control Abs. Labeling antibody cocktail containedanti-CD11b (BD Biosciences; catalog no. 557701), anti-CD14 (BDBiosciences; catalog no. 562689), and 1:8,000 Hoechst33342 (Biotium;catalog no. 40046). Images were obtained at 11-minute intervals on aPerkin-Elmer Phenix Opera using 60� water-immersion objectiveand analyzed using Columbus.

In vivo studiesH1975 cells were subcutaneously implanted into 6- to 8-week-old

female BALB/c nudemice (Charles River Laboratories). When tumorsreached an average of 72 � 8.7 mm3, intraperitoneal anti-mCSF-1Rantibody (400 mg/mouse) was administered three times weekly, begin-ning 6 days prior to compound dosing to facilitate macrophagedepletion. At day 5 (average tumor volume ¼ 102 � 36.6 mm3), micewere treated twice weekly by intraperitoneal dosing with 10 mg/kghuIgG1 isotype control, amivantamab, or EGFR/cMet IgG2s. SNU5cells were subcutaneously implanted into 7- to 8-week-old femaleCB17/SCID mice (HFK Bio-Technology Co. Ltd.). When tumorsreached an average of 155� 21.4 mm3, mice were treated twice weeklywith intraperitoneal PBS or 5 mg/kg amivantamab or EGFR/cMetIgG2s, for 3 weeks. For both studies, tumor measurements andbody weights were recorded twice weekly. TGI was calculated on thefinal day where >80% control mice remained on study, using thecalculation [1 � (T/C)] � 100. All in vivo experiments were done inaccordance with the Johnson and Johnson Institutional Animal Careand Use Committee and the Guide for Care and Use of LaboratoryAnimals.

ResultsAmivantamab antiproliferative and apoptotic activities requireFc binding to immune cells

We previously demonstrated that amivantamab (17) can inducein vivo EGFR and cMet receptor downregulation and tumor cellkilling, but such activity was not observed in cell culture (9, 12). Toassess the in vitro role of amivantamab Fc interactions in tumor cellkilling, the antiproliferative and apoptotic activities were evaluated inthe presence or absence of human immune cells (PBMCs) againstH1975 cells, a NSCLC cell line harboring mutant EGFR (L858R/T790M) and wild-type cMet (17). In the absence of PBMCs, amivan-tamab treatment did not inhibit H1975 cell proliferation or induceapoptosis (Fig. 1A and B; Supplementary Figs. S1A and S1B). How-ever, upon addition of PBMCs, amivantamab induced a dose-dependent decrease in tumor cell viability (Fig. 1A), with maximalantiproliferative effect observed at 72 hours (IC50 ¼ 0.0054 mg/mL,Fig. 1B, left) and beyond (Supplementary Figs. S1A and S1C).Amivantamab treatment also resulted in a dose-dependent increasein apoptosis in the presence of PBMCs (IC50¼ 0.0004 mg/mL, Fig. 1B,right), and this antiproliferative or apoptotic activity was not observedupon treatment with a nontargeting huIgG1 isotype control (Fig. 1B;Supplementary Figs. S1B and S1C). Similar results (proliferation

Amivantamab Induces EGFR/cMet Downmodulation by Trogocytosis

AACRJournals.org Mol Cancer Ther; 19(10) October 2020 2045




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Amivantamab-Fc interaction with immune cells enhanced in vitro cancer cell growth inhibition and apoptosis. A, Representative images depicting dose-dependenteffect 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 curvesmeasuring H1975 cell viability (AUC of NucRed area/well) after 72 hours and apoptosis (AUC of Annexinþ NucRed area/well) after 48 hours upon treatment withhuIgG1 isotype control (Isotype), amivantamab, or EGFR/cMet-IgG2s (IgG2s), in the 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) after 72 hours and apoptosis (AUC of Annexinþ NucRed area/well) after 24 hours upontreatment with huIgG1 isotype control (Isotype), amivantamab, or EGFR/cMet-IgG2s (IgG2s), in the presence or absence of PBMCs (E:T ratio of 10:1) from donor #3.D,Dose–response curvesmeasuringH1975 cell viability (AUCof NucRed area/well) after 72 hours and apoptosis (AUCof AnnexinþNucRed area/well) after 48 hoursupon treatmentwith amivantamabor cetuximab in the presence or absence of PBMCs (E:T ratio of 10:1) fromdonor #6.E,Dose–response curvesmeasuring SNU5 cellviability (Total NucRed area/well) after 48 hours upon treatment with amivantamab or cetuximab in the presence or absence of PBMCs (E:T ratio of 10:1) from donor#3. All data represented as mean � SEM within each treatment group.

Vijayaraghavan et al.

Mol Cancer Ther; 19(10) October 2020 MOLECULAR CANCER THERAPEUTICS2046




inhibition IC50 ¼ 0.01 mg/mL; apoptosis IC50 ¼ 0.002 mg/mL) wereobtained using PBMCs from a different donor (Supplementary Figs.S2A and S2B). To confirm the contribution of Fc engagement, theEGFR and cMet binding regions of amivantamabwere engineered intoan Fc effector silent molecule by replacing the wild-type IgG1 with aneffector silent IgG2s framework (9). In presence of PBMCs, treatmentwith EGFR/cMet-IgG2s resulted in no or minimal effects on H1975target cell viability and apoptosis (IC50¼ 12.1 mg/mL and IC50¼ 0.142mg/mL, respectively; Fig. 1B; Supplementary Figs. S1B, S2A, and S2B),suggesting that these antiproliferative and apoptotic activities were Fc-interaction dependent.

The antiproliferative (IC50¼ 0.0013mg/mL) and apoptotic activitiesof amivantamab were also observed against a MET amplified gastriccarcinoma cell line, SNU5, and were dependent on the presence ofPBMCs (Fig. 1C; Supplementary Fig. S2C). Similar to H1975 cells, noeffects on proliferation and apoptosis were observed upon treatmentwith huIgG1 isotype control or EGFR/cMet-IgG2s controls (Fig. 1C).The effects of amivantamab were then compared with that of cetux-imab (18), a bivalent chimeric anti-EGFR IgG1 antibody inH1975 andSNU5 cell lines. Although cetuximab was able to induce a dose-dependent antiproliferative effect (IC50 ¼ 0.51 mg/mL) and apoptosis(IC50 ¼ 0.56 mg/mL) in presence of PBMCs, the magnitude of thePBMC-dependent activities was inferior to that of amivantamab(Fig. 1D). Similar results were observed in SNU5 cell line, whereamivantamab was more potent compared with cetuximab (Fig. 1E).

Collectively, these studies demonstrated that amivantamab Fcinteraction 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 itsimpact on EGFR/cMet signaling, in comparison with cetuximab.ADCC-mediated cell lysis was measured using a short-term Euro-pium release assay in H1975 cells treated with huIgG1 isotypecontrol, amivantamab, cetuximab, or Fc silent EGFR/cMet-IgG2sin presence of PBMCs. Although amivantamab induced dose-dependent ADCC (68% max lysis), no ADCC lysis was observedwith huIgG1 isotype control or EGFR/cMet-IgG2s treatments,showing the contribution of Fc engagement in this process(Fig. 2A). Cetuximab induced dose-dependent ADCC (39% maxlysis) but to a lesser extent than amivantamab (Fig. 2A). Similarresults were obtained using PBMCs from a different donor (Sup-plementary Fig. S3A). Next, we demonstrated that similar levels ofADCC lysis was measured from isolated NK cells compared withPBMCs (Supplementary Fig. S3B), suggesting that NK cells accountfor amivantamab-induced ADCC. No ADCC was measured fromisolated monocytes (Supplementary Fig. S3B).

CDC activity triggered by the activation of the complementcomponent C1q was then assessed. First, we demonstrated thatamivantamab, cetuximab, rituximab, and huIgG1 isotype controlbind to C1q in a dose-dependent manner, whereas no measurablebinding was observed by the Fc silent EGFR/cMet-IgG2s (Supple-mentary Fig. S3C), suggesting that the C1q binding is Fc-structurespecific. We next investigated CDC-mediated target cell lysis ofNSCLC cell lines, H1975 (Fig. 2B) and H292 (SupplementaryFig. S3D) and showed that no measurable CDC activity wasobserved upon amivantamab or EGFR/cMet-IgG2s treatment. Aspositive control, rituximab (bivalent anti-CD20 antibody) added toCD20-positive Daudi cells (19) showed dose-dependent CDC activ-

ity (Fig. 2B). This demonstrated that amivantamab does not induceCDC against tested NSCLC cell lines.

Interaction of the Fc region of therapeutic antibodies with FcgRs onimmune cells is known to induce secretion of cytokines and chemokines(ADCR; ref. 20). A 71-plex MSD cytokine panel was utilized to assesschemokines and cytokines secreted upon treatmentwithhuIgG1 isotypecontrol, amivantamab, cetuximab, or Fc silent EGFR/cMet-IgG2s in thepresence or absence of PBMCs for 4 or 72 hours. We observed distinctdifferences in several secreted cytokines in a treatment- and timepoint-dependent manners (Fig. 2C; Supplementary Figs. S3E and S3F).Further analysis focusingoncytokineswith>1.5-folddifferencebetweentreatments showed that at 4 hours (Fig. 2D) and 72 hours post-treatment (Supplementary Fig. S3G), 13 and 7 cytokines, respectively,were upregulated upon amivantamab treatment comparedwith huIgG1isotype control or EGFR/cMet-IgG2s controls in the co-culture ofH1975þPBMCs. Fold induction of most of these cytokines were lowerfor cetuximab compared with amivantamab (Fig. 2D; SupplementaryFig. S3G).

We next interrogated the contribution of immune cell subtypes(PBMCs vs. NK cells, monocytes ormacrophages from same donor) inthe secretion of these cytokines. Heatmap analysis revealed distinctchanges in cytokine expression patterns, with chemotactic cytokines(CC chemokines) being the most frequently upregulated family uponamivantamab treatment (Fig. 2E; Supplementary Fig. S4A). CCchemokines are known to function as chemo-attractants for innateimmune cells like monocytes and macrophages (21–23). Intriguingly,upregulation of these chemokines upon amivantamab treatment wasspecific to PBMCs,monocytes, andmacrophages but not seenwithNKcells (Fig. 2E). For example, amivantamab treatment resulted in apotent increase in macrophage inflammatory protein (MIP)-1b (com-pared with huIgG1 isotype control) in the presence of PBMCs,monocytes, or M1 and M2 macrophages but no change was observedwith NK cells (Fig. 2E). Although cetuximab induced several of thesecytokines, the fold upregulation was lower compared with that ofamivantamab, suggesting that amivantamab is more potent thancetuximab in inducing CC chemokines (Fig. 2E).

Because EGFR and/or cMet signaling is key to the growth of thesetumor cells, we also investigated whether amivantamab or cetux-imab Fc interaction with immune cells had an effect on EGFR andcMet protein levels and downstream signaling. To examine this,H1975 cells were treated with huIgG1 isotype control, amivanta-mab, cetuximab, or Fc silent EGFR/cMet-IgG2s in the absence orpresence of PBMCs. Treatment with amivantamab or cetuximabalone (in the absence of PBMCs) only showed marginal down-regulation of EGFR and phosphorylated EGFR (pEGFR; pY1173)proteins (Fig. 2F). Compared with EGFR/pEGFR levels, amivanta-mab treatment (no PBMCs) showed a greater downregulation ofcMet protein (Fig. 2F), which may be attributed to the high-affinitycMet binding arm (9). pMet was below reliably measurable levels inH1975 cells and hence not depicted in this and subsequent analysis.Interestingly, the addition of PBMCs markedly potentiated ami-vantamab-mediated downregulation of EGFR, pEGFR, and cMet by44%, 54%, and 52%, respectively (Fig. 2F; densitometry in Supple-mentary Fig. S4B), whereas treatment with the huIgG1 isotypecontrol or EGFR/cMet-IgG2s in the absence or presence of PBMCshad no apparent effect (Fig. 2F). Although presence of immunecells enhanced cetuximab-induced downregulation of EGFR andpEGFR by 23% and 32%, respectively (Supplementary Fig. S4B), theeffect was less pronounced in comparison to that observed withamivantamab (Supplementary Fig. S4C). Cetuximab treatment didnot show an appreciable effect on cMet levels.






Taken together, these results demonstrate that amivantamab Fcinteraction with immune cells induces ADCC, stimulates productionofmacrophage-related chemokines, and enhances downmodulation ofEGFR and cMet, and is more potent than cetuximab in these assays.

Amivantamab Fc interaction induced EGFR and cMetdownmodulation is driven by monocyte composition

We next assessed the contribution of different immune cells atmediating EGFR and cMet downmodulation. PBMCs from seven

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Amivantamab–Fc interaction induces ADCC, ADCR, and EGFR/cMet downmodulation but not CDC. A, BATDA-loaded H1975 cells were treated for 2 hours withhuIgG1 isotype control (Isotype), amivantamab, cetuximab, or EGFR/cMet-IgG2s (IgG2s) atmultiple concentrations in the presence of PBMCs fromdonor #4 at an E:T ratio of 25:1 andADCC lysismeasured by Europium release.B,Dose–response rate of CDC lysismeasured by lactate dehydrogenase (LDH) levels fromH1975 targetcells after 2 hours of treatmentwith amivantamabor EGFR/cMet-IgG2s (IgG2s) in the presence of 5%baby rabbit serum. TheCD-20 antibody, rituximab, was used asa positive control in CD20 expressingDaudi cell line. All data represented asmean�SEMwithin each treatment group.C,Heatmapof log-transformedAUCvalues ofcytokines from the 71-plex MSD analysis of PBMCs alone, H1975 cells alone, or H1975 þ PBMCs (E:T ¼ 10:1) treated with huIgG1 isotype control (Isotype),amivantamab, cetuximab, or EGFR/cMet-IgG2s (IgG2s) for 4 hours. D, Bar graphs representing the relative change (measured in C) over huIgG1 isotype control(Isotype) upon treatment with amivantamab, cetuximab, or EGFR/cMet-IgG2s (IgG2s) antibodies for 4 hours. Graphs were limited to cytokines with greater than1.5� increase for amivantamab treatment compared with isotype. E, Heat map of log-transformed AUC values of cytokines from the 23-plex MSD analysis of H1975cells treated with multiple concentrations of isotype, amivantamab, cetuximab, or EGFR/cMet-IgG2s (IgG2s) for 4 hours in the presence of PBMCs (E:T ¼ 10:1) orindividual immune cells (E:T¼ 5:1), namely NK cells, monocytes, or M1 orM2cmacrophages. Heatmapwas limited to cytokineswith greater than 1.5� increase for theH1975þPBMCs condition. F, Western blot analysis (PeggySue capillary electrophoresis) of EGFR, pEGFR, cMet, and loading control actin performed following48 hours treatment of H1975 cells with 10 mg/mL of huIgG1 isotype control (Isotype), amivantamab, or cetuximab in the presence or absence of PBMCs (E:T ¼ 10:1)from donor #3.






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 moderatedownmodulation of cMet protein levels. Although the addition ofPBMCs enhanced the downmodulation of EGFR, pEGFR and cMetcompared with huIgG1 isotype control for most donors, the extentof this effect varied among donors (Fig. 3A; densitometry inSupplementary Fig. S5A). PBMCs from some donors (#3, #4, and#6) showed a substantial effect in potentiating EGFR and cMetdownmodulation, whereas PBMCs from other donors (#2, #5, and#7) had little to no effect. PBMCs from additional donors weresimilarly tested, and considerable variability in amivantamab-

mediated downmodulation was observed (Supplementary Figs.S5B and S5C).

To examine the role of amivantamab–Fc interaction in EGFR/cMetdownmodulation, H1975 cells were treated with huIgG1 isotypecontrol, amivantamab, or Fc silent EGFR/cMet-IgG2s in the presenceof PBMCs from selected donor #6 (Fig. 3B). Addition of PBMCsmarkedly potentiated amivantamab-mediated downregulation ofEGFR, pEGFR, and cMet by 46%, 67%, and 44%, respectively(Fig. 3B; densitometry in Supplementary Fig. S6A), whereas treatmentwith the huIgG1 isotype control or EGFR/cMet-IgG2s in the absenceor presence of PBMCs had no apparent effect, suggesting that it isspecific to the amivantamab–Fc interaction. Similar results wereobtained with donor #3 (Supplementary Fig. S6B). We confirmed this

0 5 10 15 20-100

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Figure 3.

Amivantamab–Fc interaction with immune cells enhances amivantamab induced EGFR/cMet downmodulation and correlates with monocyte composition.A, Western blot analysis (PeggySue capillary electrophoresis) of EGFR, pEGFR, cMet, and loading control actin performed following 48 hours treatment ofH1975 cells with 10 mg/mL of huIgG1 isotype control (Isotype) or amivantamab (Ami) in the presence of PBMCs from seven different donors. Western blotanalysis (PeggySue) of EGFR, pEGFR, cMet, pMet, and loading control actin performed following 48 hours treatment with 10 mg/mL of huIgG1 isotype control(Isotype), amivantamab (Ami), or EGFR/cMet-IgG2s (IgG2s) in the presence or absence of PBMCs from donor #6 against H1975 cancer cells (B) and donor #3against SNU5 cancer cells (C). D, Correlation between the percentage of NK cells, B cells, and T cells within PBMC sample from each donor and the percentagechange in EGFR inhibition upon amivantamab treatment in the presence of PBMCs. E, Correlation between the percentage of monocytes within the PBMCsamples and percentage changes in EGFR, pEGFR (Y1173), and cMet inhibition upon amivantamab treatment in presence of PBMCs. All data represented asmean � SEM within each treatment group.






effect of Fc engagement on EGFR and cMet expression in the MET-amplified SNU5 cell line (Fig. 3C). Similar to H1975 cells, presence ofPBMCs enhanced the ability of amivantamab to downregulate EGFRand cMet signaling, resulting in 79%, 89%, 87%, and 90% reduction inlevels of EGFR, pEGFR, cMet, and pMet proteins, respectively, and nomeasurable changewas seenwith the huIgG1 isotype control or EGFR/cMet-IgG2s control (Supplementary Fig. S6C).

To determine which immune cells are responsible for Fc-dependentdownmodulation, we compared percentages of key immune cellpopulations (NK cells, monocytes, B cells, and T cells) within PBMCs(Supplementary Fig. S7A) for each donorwith the observed differencesin their downmodulation effects. No correlationwas observed betweenNK, B, or T cells and the ability of the PBMCs to potentiate EGFRdownmodulation (Fig. 3D; Supplementary Fig. S7B). However, acorrelationwas observed between relativemonocyte populationwithinthe PBMCs and the downmodulation of EGFR (R2 ¼ 0.51), pEGFR(R2¼ 0.55), and cMet (R2¼ 0.48) protein levels (Fig. 3E). This findingsuggests that monocytes and their relative level within the PBMCsamples determine the extent of amivantamab-mediated EGFR andcMet downregulation.

Amivantamab Fc interaction with monocytes inducestrogocytosis and is required for EGFR and cMetdownmodulation

To confirm direct monocyte involvement in Fc interaction-mediated signal downregulation, NK cells or monocytes were depletedfrom PBMCs (Fig. 4A) and tested for amivantamab-mediated down-modulation of EGFR and cMet pathways. Consistent with previousresults, nondepleted PBMCs enhanced amivantamab-mediated down-regulation of EGFR, pEGFR, and cMet in H1975 cells (Fig. 4B;densitometry in Supplementary Fig. S8A). Although depletion of NKcells only had a marginal effect, depletion of monocytes effectivelyreversed the ability of PBMCs to potentiate amivantamab-mediatedreceptor downmodulation (Fig. 4B). As expected, no effect was seenwith EGFR/cMet-IgG2s treatment confirming that Fc interaction isrequired for EGFR/cMet downmodulation. Similar data were obtainedby depleting PBMCs of NK cells and monocytes from a second donor(Supplementary Figs. S8B–S8D).

To further confirm the role of monocytes, NK cell and monocyteswere isolated from the same PBMC donor (Supplementary Fig. S9A)and assessed for amivantamab-mediated downregulation of EGFR,pEGFR, and cMet proteins (Fig. 4C). Although the control (non-isolated) PBMC sample enhanced receptor downmodulation, NK cellsalone did not demonstrate a strong effect. However, isolated mono-cytes substantially enhanced the ability of amivantamab to down-modulate EGFR, pEGFR, and cMet proteins to a similar extent to thatof PBMCþ amivantamab control (Fig. 4C; densitometry in Supple-mentary Fig. S9B).

On the basis of this novel finding that monocytes enhance ami-vantamab-mediated downmodulation of EGFR and cMet proteins, weassessed if this downmodulation occurs through trogocytosis, an Fceffector function that mediates transfer of cell-surface proteins fromtumor to effector cells. To visualize amivantamab-induced monocytetrogocytosis, time-lapse microscopy was performed in H1975 targetcells opsonized with AF647-labeled antibodies (huIgG1 isotype con-trol, amivantamab, or Fc-silent EGFR/cMet-IgG2s). Monocytes werevisualized with AF488-labeled CD11b/CD14 antibody cocktail (green)and Hoechst stain for nuclei (blue) and H1975 target cells wereidentified by their NucLight Redþ nuclei (orange). When co-cultured with opsonized target cells, distinct transfer of labeled ami-vantamab antibody but not labeled huIgG1 isotype control or EGFR/

cMet-IgG2s antibodies into monocytes was observed (Fig. 4D; Sup-plementary Fig. S9C; Supplementary Movie M1), demonstrating thatamivantamab induced monocyte-dependent trogocytosis.

Collectively, these results demonstrate that monocytes mediatetrogocytosis and drive amivantamab–Fc interaction mediated down-modulation of EGFR and cMet receptors.

Amivantamab Fc interaction with macrophages inducestrogocytosis leading to EGFR and cMet downmodulation

Solid tumors, particularly NSCLC, have been reported to contain ahigh level of tumor-associatedmacrophages (TAM; ref. 24). Hence, weexamined if M1 and M2 macrophages are capable of amivantamabtrogocytosis similar to monocytes using time-lapse microscopy inH1975 target cells opsonized with AF647-labeled antibodies (huIgG1isotype control, amivantamab, or EGFR/cMet-IgG2s). Macrophageswere visualized with AF488-labeled CD11b/CD14 antibody cocktail(green), Hoechst stain for nuclei (blue), and H1975 target cells wereidentified by their NucLight Redþ nuclei (orange). Upon co-culturewith target cells opsonized with labeled amivantamab, a distinctaccumulation of AF647þ puncta (amivantamab) was observed withinthe M1 (Fig. 5A; Supplementary Movie M2) and M2c macrophages(Fig. 5B; Supplementary Movie M3), whereas no accumulation wasseen with labeled huIgG1 isotype control or EGFR/cMet-IgG2streatment (Fig. 5A and B; Supplementary Figs. S10A and S10B). Noappreciable phagocytosis was observed visually in the presence of M1or M2c macrophages in these microscopy assays, indicating thatmacrophage-mediated trogocytosis is the predominant mechanismmeasured.

To better simulate antibody interactions within the tumor, wetreated a co-culture of M1 or M2c macrophages and H1975 targetcells with AF647-labeled antibodies (huIgG1 isotype control, amivan-tamab, or EGFR/cMet-IgG2s) and monitored trogocytosis by time-lapse microscopy. As expected, huIgG1 isotype control antibodybound only toM1 andM2cmacrophages, whereas EGFR/cMet-IgG2sbound only to target cells, and amivantamab bound to both target cellsand macrophages (Supplementary Fig. S10C), thus confirming thebinding specificity of each antibody. Under these conditions (co-culture), amivantamab-mediated trogocytosis was readily observed,measured by a distinct transfer of labeled amivantamab into macro-phages, but no trogocytosis was observed with huIgG1 isotype controlor EGFR/cMet-IgG2s antibodies (Supplementary Fig. S10D; Supple-mentary Movies M4 and M5).

Finally, to examine if macrophage-mediated trogocytosis leads toreceptor downmodulation, monocytes were polarized into M1, M2a,or M2c macrophages, and their ability to potentiate amivantamab-mediated EGFR/cMet downmodulation was assessed. Compared withtreatment with amivantamab alone (nomacrophages), the presence ofM1 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 Supple-mentary Fig. S11). HuIgG1 isotype control or EGFR/cMet-IgG2streatments had no effect, suggesting that the signal downmodulationrequired Fc interaction.

Collectively, these results reveal trogocytosis as a novel Fc effectorfunction for amivantamab in the presence of macrophages, leading toEGFR/cMet receptor downmodulation.

Fc interaction and macrophages are required for amivantamabin vivo antitumor efficacy

The role and relevance of Fc/FcgR interactions in vivowas evaluatedusing H1975 and SNU5 cell line xenograft models. Mice harboring






H1975 or SNU5 tumors were treated with huIgG1 isotype control,amivantamab, or Fc silent EGFR/cMet-IgG2s antibodies for 3 weeks(Supplementary Figs. S12A and S12D). In the H1975 model, TGI wassuperior upon treatment with amivantamab (TGI ¼ 75%; ��, P <0.0001) compared with that of EGFR/cMet-IgG2s (TGI ¼ 30%;��, P < 0.0016; Fig. 6A; Supplementary Fig. S12C shows individualmice). Similar results were observed in the SNU-5 model, whereamivantamab treatment effectively reduced tumor growth (TGI ¼96%; ��, P < 0.0001) compared with EGFR/cMet-IgG2s treatment(TGI ¼ �17%; ns, P ¼ 0.53; Fig. 6B; Supplementary Fig. S12F shows

individual mice). None of the antibody treatments resulted in loss ofmouse body weight in either tumormodels (Supplementary Figs. S12Band S12E).

We previously demonstrated, using an HGF-expressing H1975 xeno-graft tumormodel, that Fc silent EGFR/cMet-IgG2s only showed partialeffect on EGFR/cMet signaling compared with amivantamab, suggestinga role for Fc-mediated signal downregulation in vivo (12). To extend thisobservation to the MET-amplified SNU5 model in vivo, tumors werecollected from the in vivo efficacy study (Fig. 6B) and changes in total andphospho-EGFR and cMet protein levels were measured. Similar to

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Monocytes mediated amivantamab Fc interaction induced trogocytosis and EGFR and cMet downmodulation. A, Contour plots from multicolor flow cytometryanalysis showing the composition of NK cells and monocytes within PBMCs (donor #3) upon CD56- and CD14-positive selection, respectively. B, Western blotanalysis (PeggySue capillary electrophoresis) of EGFR, pEGFR, cMet, and actin (loading control) performedonH1975 cell lysates following48 hours treatmentwith 10mg/mL of huIgG1 isotype control (Isotype), amivantamab (Ami), or EGFR/cMet-IgG2s (IgG2s) in the presence or absence of intact PBMCs, NK cell–depleted, ormonocyte (mono)–depleted PBMCs from donor #3. C,Western blot analysis (PeggySue) of EGFR, pEGFR, cMet, and loading control actin performed on H1975 celllysates following48hours treatmentwith 10mg/mLof huIgG1 isotype control (Isotype), amivantamab (Ami) or EGFR/cMet-IgG2s (IgG2s) in thepresenceor absenceof intact PBMCs, isolated NK cells, and isolated monocytes from donor #3. D, Representative images from high-content confocal microscopy of monocytes labeledwith 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-IgG2s (IgG2s) antibodies. White arrows depict trogocytosis events measured by transfer of AF647-labeled antibody from target cells tomonocytes. Yellow arrows point to one such event across time. Scale bar, 20 mm.






in vitro results (Fig. 3C), comparedwith vehicle treatment, amivantamabtreatment showed significant downregulation of EGFR, pEGFR, cMet,and pMet proteins but EGFR/cMet-IgG2s treatment only had marginaleffects, suggesting that Fc interaction is required for in vivo signaldownmodulation (Fig. 6C; quantification in Fig. 6C and SupplementaryFig. S13A). Thus, these in vivo studies demonstrate that amivantamab Fcinteraction is essential for its antitumor efficacy and in vivo EGFR/cMetsignal downmodulation.

The role of macrophages in amivantamab in vivo efficacy was nextassessed using anti-CSF1R–mediated depletion of TAMs in the H1975xenograft study. As expected, treatment with anti-CSF1R antibody

showed significant (��, P < 0.0001) reduction in TAMs (�2%)compared with untreated tumors (11%–15%; Fig. 6D). Animals werethen treated with huIgG1 isotype control or amivantamab for 3 weeks(Supplementary Fig. S13B) with no loss in mouse body weight (Sup-plementary Fig. S13C). As shown previously, treatment with amivan-tamab showed significantly higher antitumor efficacy comparedwith the isotype (��, P < 0.0001) or EGFR/cMet-IgG2s treatment(��, P¼ 0.004) in non-anti-CSF1R treated tumors (Fig. 6E). Strikingly,depletion of TAMs (anti-CSF1R-treated) significantly reduced amivan-tamab TGI from 72.8% to 38.5% (�, P¼ 0.014; Fig. 6E; SupplementaryFig. S13D shows individual mice), suggesting that macrophages play a

M1 Macrophages (Cd11b/CD14) H1975-Nuclight RedHoechst

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Figure 5.

AmivantamabFc interaction inducesmacrophage-trogocytosis andEGFRandcMetdownmodulation.Representative images fromhigh-content confocalmicroscopyofM1 (A) orM2 (B)macrophages labeledwithAF488-CD11b, AF488-CD14, andHoechst (nuclei) in co-culture (E:T ratioof 5:1)withH1975NucLightRed cells opsonizedwithAF647-labeled amivantamabor EGFR/cMet-IgG2s (IgG2s) antibodies.White arrows depict trogocytosis eventsmeasured by transfer of AF647 labeled antibody fromtarget cells tomacrophages. Yellow arrows point to one such event across time. Scale bar, 20 mm.Western blot analysis (PeggySue capillary electrophoresis) of EGFR,pEGFR, cMet, and actin (loading control) performedonH1975 cell lysates following48hours treatmentwith 10mg/mLof huIgG1 isotype control (Isotype), amivantamab(Ami), or EGFR/cMet-IgG2s (IgG2s) in the presence or absence of M1 macrophages (C), M2a macrophages (D), or M2c macrophages (E).






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Amivantamab–Fc interaction with macrophages are required for in vivo antitumor efficacy. A, Tumor volumes of subcutaneously injected H1975 cell line xenografttumors treatedwith 10mg/kg huIgG1 isotype control (Isotype), amivantamab, or EGFR/cMet-IgG2s (IgG2s; n¼ 10 per group) for 3weeks BIW. %TGI was calculatedat day 24, and P values were calculated using two-way ANOVA (�� , P < 0.0021; �� , P < 0.0001). B, Tumor volumes of subcutaneously injected SNU5 cell linexenograft tumors treated with vehicle (PBS), 5 mg/kg amivantamab (Ami), or EGFR/cMet-IgG2s (IgG2s; n¼ 8 per group) for 3 weeks BIW. %TGI was calculated atday34, andP valueswere calculated using two-wayANOVA (�� ,P<0.0001).C,Western blot analysis of SNU5xenograft tumors harvested 24hours after twodoses(treated as described inB)measuring protein levels of EGFR, pEGFR, cMet, pMet, or loading control GAPDH. Densitometrymeasurements for EGFR and cMet proteinwere normalized to loading control GAPDH. P values were calculated using one-way ANOVA (�� , P < 0.05; �� , P < 0.005). D, Multicolor flow cytometry analysis ofH1975 tumor samples isolated 24hours after twodoses of 10mg/kghuIgG1 isotype control (Isotype), amivantamab (Ami), or EGFR/cMet-IgG2s (IgG2s) treatment (n¼ 5 mice/treatment) to assess the percentage of macrophages (CD45þ CD11bþ Ly6G� Ly6C� F4/80þ) within the tumor post-depletion with anti-mouse CSF1Rantibody. E, Tumor volumes of subcutaneously injected H1975 cell line xenograft tumors treated with 10 mg/kg huIgG1 isotype control (Isotype), amivantamab, orEGFR/cMet-IgG2s (IgG2s; n ¼ 10 mice per group) for 3 weeks BIW in combination with anti-mouse CSF1R or its isotype control three times weekly to depletemacrophages. %TGI was calculated on day 21, and P values were calculated using one-way ANOVA (� , P < 0.005). F, Figure illustrating the multiple Fc-dependentand Fc-independent mechanisms of action contributing to amivantamab antitumor activity. All data represented as mean � SEM within each treatment group.






key role in mediating amivantamab antitumor efficacy. No significantdifference was observed between vehicle and vehicleþanti-CSF1Rtreatment (P ¼ 0.17).

Collectively, these findings demonstrate that amivantamab–Fcinteraction with macrophages and monocytes induces trogocytosis,which is a dominant mechanism underlying the antitumor efficacy ofamivantamab.

DiscussionIn this study, we demonstrated that in addition to the known Fc-

independent mechanisms (inhibition of ligand binding and liganddriven signaling; refs. 9, 12), the EGFR/cMet bispecific antibody,amivantamab, exerts antitumor efficacy through multiple Fc-dependent mechanisms (Fig. 6F). These mechanisms are initiatedthrough interaction of the Fc domain of the antibody with Fcgreceptors present on immune cells. Binding and activation of Fcgreceptors onNK cells led toADCC,while binding and activation of Fcgreceptors on monocytes and macrophages led to cytokine productionand trogocytosis. Trogocytosis caused downmodulation of EGFR andcMet receptors and their downstream signaling, which is required forproliferation of these cancer cells.

Although we demonstrated in vitroADCC lysis with amivantamab,we believe that this effect is minimal compared with that exerted bymonocytes and macrophages. ADCC lysis is a rapid mechanismoccurring at early timepoints (�2 to 4 hours; ref. 25), whereas 48 hoursand more were required to reach the maximal amivantamab in vitroantiproliferative effect in presence of PBMCs (SupplementaryFig. S1C). These suggest that the short-term ADCC is not sufficientlycontributing to the overall long-term activity of amivantamab and thatother effector functions (such as trogocytosis) contribute to additionalcytotoxic activity over time. Although we did not observe CDC activityin the cell lines evaluated, NSCLC cell lines are known to expresscomplement inhibitory proteins, CD46, CD55, and CD59 (26), sug-gesting that amivantamabmight trigger CDCactivity in other cell linesor tumor types not expressing these complement inhibitory proteins.Finally, we reported previously that amivantamab induces ADCPin vitro (9), however under the conditions tested in this study (confocalmicroscopy-based macrophage trogocytosis assay), minimal to noADCP was observed. Collectively, as depicted in Fig. 6F, our datasuggest that amivantamab antitumoral activity occurs through mul-tiple Fc-independent and Fc-dependent mediated mechanisms ofaction 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-dependenttrogocytosis and cytokine release. Because the content and diversity ofimmune cells within the tumor microenvironment (TME) variesamong patients, this may influence the response to amivantamabtreatment.

We showed that most cytokines upregulated by amivantamabbelong to the family of chemotactic cytokines called chemokines,specifically CC chemokines (21, 23). CC chemokines comprises oftwo key subfamilies, monocyte chemoattract protein (MCP) andMIP, which are both known to function as chemo-attractants forinnate immune cells such as monocytes and macrophages (22, 27).MCP-1 (CCL2) and MIP1b (CCL4) have been reported to inducerecruitment of monocytes and macrophages into the TME ofNSCLC (22, 28). Although these cytokines could attract immunecells to the TME, their production requires an initial tumor cell-immune cell contact mediated by amivantamab. So, we hypothesizethat trogocytosis is the initial and dominant mechanism of ami-

vantamab leading to downmodulation of EGFR and cMet, andpotentially fueling additional recruitment of monocytes or macro-phages by cytokine production.

In contrast to most other therapeutic antibodies in the clinic,amivantamab was designed and engineered with a low fucose back-bone, which enhances its binding to FcgRIIIa (12), present onNK cells,monocytes, and macrophages. This increased binding to FcgRIIIaenhanced induction of Fc effector functions in comparison to other(normal fucose) huIgG1 antibodies such as cetuximab and trastuzu-mab, and could also enable amivantamab to out-compete naturallycirculating IgG antibodies for FcgR binding, as shown preclinicallywith other enhanced Fc antibodies (29, 30). Given the predominance ofEGFR-drivenNSCLC tumors, it is confounding that cetuximab did notmeet clinical endpoints inNSCLC (31).We hypothesize that this couldresult from several factors that amivantamab can overcome. Pri-marily, cetuximab only targets EGFR and it is known that cancercells can quickly adapt to therapy by activating alternate pathways,the most common of which is cMet (32). In addition, the bivalentnature of the high-affinity EGFR arm in cetuximab results in on-target off tumor toxicity in the clinic (31). Studies have shown thatbispecificity of an antibody can increase tumor selectivity viasimultaneous targeting of two cancer antigens and varying antigenaffinity can drive selectivity towards cells expressing both antigenscompared with single antigen expressing normal cells (33–35).Thus, we hypothesize that the presence of the high-affinity cMettargeting arm (Kd ¼ 40 pmol/L), low-affinity EGFR targeting arm(Kd ¼ 1.4 nmol/L) combined with the high-affinity FcgRIIIabinding (low fucose) Fc portion could account for the improvedselectivity (36) and efficacy, and lowered toxicity of amivantamab incomparison with cetuximab.

Previously, trogocytosis was described as a mechanism of resistancefor antibodies such as rituximab (16, 37). In this context, the receptor isstripped from the cell surface by monocytes/macrophages, preventingoccurrence of NK cell-mediated functions towards the no-longeravailable targeting receptor. However, in this study, we demonstrateda novel role for trogocytosis (ADCT) as a crucial inducer of Fc-dependent receptor downmodulation and antitumor effects. Wehypothesize that the dichotomy of trogocytosis’ role (mediator ofresistance vs. antitumor effects) might be driven by the dependency ofthe tumor cells on the trogocytosis-targeted receptor. Although anti-tumor effects via trogocytosis have been suggested for cetuximab andtrastuzumab (30, 38), our work confirmed such amechanism of actionfor amivantamab and presents comprehensive data with primaryimmune cells (monocytes, M1 and M2 macrophages), demonstratingthe induction of trogocytosis leading to the downmodulation of thetarget receptors, EGFR and cMet. It is interesting to note thattrogocytosis was observed for both M1 and M2 macrophage popula-tions, suggesting this mechanism may also be operative in moreimmunosuppressive TMEs.

Therapeutic targeting of EGFR and cMet with an antibody suchas amivantamab with multiple and enhanced Fc mechanism(s) ofaction can provide future options to combat diverse types of cancer-driving mutations and drug-acquired resistance for patients. Inaddition to targeting a conserved region of an oncogenic receptor,which broadens the targeted population, the selectivity of bispecificantibodies like amivantamab, could offer a reduced potential fortoxicity, making combination therapy with additional agents bettertolerated. Our findings demonstrate that the Fc-dependent activityof amivantamab is driven by macrophage- and monocyte-mediatedtrogocytosis, a dominant mechanism of antibody-directed receptordownregulation and tumor cell killing. These findings highlight the






potential of trogocytosis as therapeutic modality but also provideinsights to guide patient selection and combination strategies foramivantamab.

Disclosure of Potential Conflicts of InterestS. Vijayaraghavan reports a patent for bispecific EGFR/cMet antibodymechanism

of action (planned). J. Sendecki is an employee of and holds shares and stock optionsin Janssen R&D. M.V. Lorenzi is an employee of and a shareholder in JanssenPharmaceuticals outside the submitted work. S.L. Moores reports a patent forUS9593164 - bispecific EGFR/c-Met antibodies issued, a patent for US9580508 -bispecific EGFR/c-Met antibodies issued, a patent forUS9695242 - bispecific EGFR/c-Met antibodies issued, a patent for U.S. patent application no. 16/697,249 - bispecificEGFR/cMet antibodies pending, and a patent for bispecific EGFR/cMet antibodymechanism of action (planned). No potential conflicts of interest were disclosed bythe other authors.

Authors’ ContributionsS. Vijayaraghavan: Conceptualization, software, formal analysis, supervision,

investigation, visualization, methodology, writing-original draft, projectadministration, writing-review and editing. L. Lipfert: Formal analysis, investigationand methodology. K. Chevalier: Formal analysis, investigation and methodology.B.S. Bushey: Formal analysis, investigation and methodology. B. Henley: Formalanalysis, validation, investigation and methodology. R. Lenhart: Software, formal

analysis, investigation and methodology. J. Sendecki: Software and formal analysis.M. Beqiri: Investigation and methodology. H.J. Millar: Conceptualization, supervision,investigation and methodology. K. Packman: Conceptualization, supervision andmethodology. M.V. Lorenzi: Conceptualization, supervision, writing-original draft,writing-review and editing. S. Laquerre: Conceptualization, supervision, writing-original draft, writing-review and editing. S.L. Moores: Conceptualization, resources,supervision, validation, writing-original draft, writing-review and editing.

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

generation was performed by Pete Buckley, Janssen Biotherapeutics. Assistance withperforming in vivo experiments was provided by David Walker, Janssen R&D, LLC.Technical guidance for the confocal microscopy–based trogocytosis assay wasprovided by Edward Keough, Janssen Biotherapeutics. Writing assistance wasprovided by Ramji Narayanan (SIRO Clinpharm Pvt Ltd.), funded by Janssen GlobalServices, LLC, and additional editorial support was provided by Tracy Cao (JanssenGlobal Services, LLC).

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

Received January 31, 2020; revised April 6, 2020; accepted July 27, 2020;published first August 3, 2020.

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2020;19:2044-2056. Published OnlineFirst August 3, 2020.Mol Cancer Ther Smruthi Vijayaraghavan, Lorraine Lipfert, Kristen Chevalier, et al. Antitumor Activity by Monocyte/Macrophage TrogocytosisBispecific Antibody, Induces Receptor Downmodulation and Amivantamab (JNJ-61186372), an Fc Enhanced EGFR/cMet

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