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

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

MOLECULAR CANCER THERAPEUTICS | LARGE MOLECULE THERAPEUTICS

Amivantamab (JNJ-61186372), an Fc Enhanced EGFR/ cMet Bispeciﬁc Antibody, Induces Receptor Downmodulation 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 ◥ Small molecule inhibitors targeting mutant EGFR are standard of observed in vitro. Interestingly, in vitro addition of isolated human care in non–small cell lung cancer (NSCLC), but acquired resistance immune cells notably enhanced amivantamab-mediated EGFR and invariably develops through mutations in EGFR or through acti- cMet downregulation, leading to antibody dose-dependent cancer vation of compensatory pathways such as cMet. Amivantamab cell killing. Through a comprehensive assessment of the Fc- (JNJ-61186372) is an anti-EGFR and anti-cMet bispecific low fucose mediated effector functions, we demonstrate that monocytes antibody with enhanced Fc function designed to treat tumors driven and/or macrophages, through trogocytosis, are necessary and suf- by activated EGFR and/or cMet signaling. Potent in vivo antitumor ficient for Fc interaction-mediated EGFR/cMet downmodulation efficacy is observed upon amivantamab treatment of human tumor and are required for in vivo antitumor efficacy. Collectively, our xenograft models driven by mutant activated EGFR, and this findings represent a novel Fc-dependent macrophage-mediated activity is associated with receptor downregulation. Despite these antitumor mechanism of amivantamab and highlight trogocytosis robust antitumor responses in vivo, limited antiproliferative effects as an important mechanism of action to exploit in designing new and EGFR/cMet receptor downregulation by amivantamab were antibody-based cancer therapies.

Introduction ing both EGFR and cMet (US Patent no. US9593164B2; ref. 8). This bispecific antibody binds EGFR agnostic of the diverse primary and Treatment of non–small cell lung cancer (NSCLC) was transformed acquired resistance mutations, potentially increasing the therapeutic upon identification and targeting of primary activating mutations in reach of this antibody. Amivantamab is currently in clinical develop- the kinase domain of EGFR, which occur in 10% to 40% of patients ment, and consistent with its preclinical profile (9), preliminary clinical with lung cancer. Patients with activating EGFR mutations are initially data demonstrates that amivantamab treatment leads to NSCLC responsive to first- and second-generation EGFR tyrosine kinase regression across EGFR driver mutations, including exon 19 deletion, inhibitor (TKI) treatment. However, this efficacy is short-lived, and L858R mutation, Exon20 insertions, EGFR-TKI resistance mutations acquired resistance develops through secondary mutations in EGFR as (T790M, C797S), as well as against drug resistance due to MET well as upregulation of other pathways, such as cMet (1–3). Osimer- amplification (10, 11). tinib, a covalent third-generation EGFR TKI, is used for the treatment Amivantamab was produced in engineered CHO cell lines that of EGFR mutant NSCLC patients with acquired resistance to prior incorporate low levels of fucose, to generate a bispecific antibody with EGFR TKI therapy due to T790M mutation, and has recently become enhanced binding to Fc gamma receptor (FcgR)-IIIa/CD16a on the standard of care front-line therapy (4). However, similar to the immune cells (12). Binding of huIgG1 antibodies to FcgRs can mediate first-generation TKIs, acquired resistance to osimertinib is also target cell elimination through immune effector cell functions such as observed through additional mutations in EGFR (e.g., C797S) or antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent activation of the cMet pathway (5–7). Currently, there are no approved cellular phagocytosis (ADCP), and complement-dependent cytotoxicity therapies for patients resistant to third-generation TKIs, highlighting (CDC; ref. 13). ADCC and ADCP are triggered when IgG1 antibodies the significant unmet need for this population. bind to cell surface antigens, subsequently clustering their Fc domains To address this unmet medical need, we developed amivantamab to engage with FcgRs on natural killer (NK) cells (ADCC) and macro- (JNJ-61186372), a fully human IgG1-based bispecific antibody target- phages (ADCP; ref. 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 Janssen Research & Development, Spring House, Pennsylvania. antibody), cetuximab (anti-EGFR antibody), and trastuzumab (anti- Note: Supplementary data for this article are available at Molecular Cancer HER2 antibody) are known to exploit these three FcgR-mediated Therapeutics Online (http://mct.aacrjournals.org/). mechanisms (15). Trogocytosis (antibody-dependent cellular trogocy- Corresponding Author: Smruthi Vijayaraghavan, Janssen Research & Develop- tosis, ADCT) is another important consequence of Fc–FcgR interaction, ment, 1400 McKean Road, Spring House, PA 19477. Phone: 215-628-5121; E-mail: and is described as tumor-targeted antibody-mediated transfer of [email protected] membrane fragments and ligands from tumor cells to effector cells Mol Cancer Ther 2020;19:2044–56 such as monocytes, macrophages, and neutrophils (16). Unlike phago- doi: 10.1158/1535-7163.MCT-20-0071 cytosis, trogocytosis is underappreciated as a mechanism of action 2020 American Association for Cancer Research. contributing to tumor cell death.

AACRJournals.org | 2044

Amivantamab Induces EGFR/cMet Downmodulation by Trogocytosis

In previous studies we have shown that amivantamab can induce and run for eight cycles. Data were analyzed using Compass for SW ADCC and ADCP, in addition to Fc-independent EGFR and cMet software. signal inhibition (9, 12). Further, in an in vivo study in an EGFR mutant cMet-activated tumor model, we showed that treatment with Confocal imaging trogocytosis assay Fc-silent IgG2s version of amivantamab elicits reduced tumor growth Differentiated macrophages were harvested and plated onto Cell- inhibition (TGI) compared with amivantamab (Fc-active version), Carrier96 ultra plates at 1,00,000 cells/well (Perkin-Elmer; catalog no. suggesting that the Fc-mediated effector function of amivantamab is 6055302) overnight. For assays with adhered target cells, H1975 essential for its maximal antitumor efficacy (12). In addition, the NucLight Red cells were plated at 20,000/well, then 4 hours later IgG2s version of amivantamab exhibited reduced downregulation of treated with labeled Ab cocktail for 1 hour at 4C. For assays with EGFR, cMet, and their respective signaling components in vivo, nonadhered target cells, target cells were stained with AF647-labeled suggesting the requirement of amivantamab Fc interaction for recep- amivantamab or control Abs. Labeling antibody cocktail contained tor and signaling downmodulation activity. anti-CD11b (BD Biosciences; catalog no. 557701), anti-CD14 (BD Here we report that trogocytosis is a dominant mechanism of action Biosciences; catalog no. 562689), and 1:8,000 Hoechst33342 (Biotium; for the preclinical in vitro and in vivo antitumor activity of amivanta- catalog no. 40046). Images were obtained at 11-minute intervals on a mab. We performed a comprehensive analysis of the Fc-mediated Perkin-Elmer Phenix Opera using 60 water-immersion objective effector functions to dissect the key immune cell types underlying the and analyzed using Columbus. Fc-dependent mechanisms of amivantamab. This work led to the identification of Fc-mediated monocyte and macrophage interactions In vivo studies and associated trogocytosis as key mediators of receptor downmodu- H1975 cells were subcutaneously implanted into 6- to 8-week-old lation and antibody-mediated cancer cell killing. Together, these female BALB/c nude mice (Charles River Laboratories). When tumors results point to the importance of trogocytosis as a mechanism of reached an average of 72 8.7 mm3, intraperitoneal anti-mCSF-1R cancer cell death, which can be leveraged in the design of future antibody (400 mg/mouse) was administered three times weekly, begin- antibody-based therapies. ning 6 days prior to compound dosing to facilitate macrophage depletion. At day 5 (average tumor volume ¼ 102 36.6 mm3), mice were treated twice weekly by intraperitoneal dosing with 10 mg/kg Materials and Methods huIgG1 isotype control, amivantamab, or EGFR/cMet IgG2s. SNU5 Proliferation and apoptosis assays cells were subcutaneously implanted into 7- to 8-week-old female For Incucyte-based proliferation and apoptosis assays, H1975 or CB17/SCID mice (HFK Bio-Technology Co. Ltd.). When tumors SNU5 cell lines were infected with NucLight Red lentiviral reagent reached an average of 155 21.4 mm3, mice were treated twice weekly (Essen Biosciences; catalog no. 4476) and selected with 1 mg/mL or with intraperitoneal PBS or 5 mg/kg amivantamab or EGFR/cMet 4 mg/mL puromycin to generate H1975 and SNU5 NucRed cells, IgG2s, for 3 weeks. For both studies, tumor measurements and respectively. NucRed target cells were plated at 5,000 cells/well and body weights were recorded twice weekly. TGI was calculated on the PBMCs (HemaCare) were added at an E:T (effector:target) ratio of final day where >80% control mice remained on study, using the 10:1. Annexin-V Green reagent (Essen Biosciences; catalog no. 4642) calculation [1 (T/C)] 100. All in vivo experiments were done in was added, followed by therapeutic antibodies and scanned (4 images/ accordance with the Johnson and Johnson Institutional Animal Care well/scan) with IncucyteS3 every 4 hours. Total NucRed area (mm2/ and Use Committee and the Guide for Care and Use of Laboratory well) shows target cell viability and Total Green NucRed area (mm2/ Animals. well) shows target cell apoptosis. Data were visualized with Spotfire – and dose response curves were generated using GraphPad Prism. Results Simple Western assay Amivantamab antiproliferative and apoptotic activities require H1975 or SNU5 target cells were plated (100,000 cells/well) in six- Fc binding to immune cells well plates and PBMCs were added at an E:T ratio of 10:1 or individual We previously demonstrated that amivantamab (17) can induce immune cells (NK cells, monocytes, or macrophages) at E:T ratio of in vivo EGFR and cMet receptor downregulation and tumor cell 5:1. Therapeutic antibodies were added and incubated for 48 hours. killing, but such activity was not observed in cell culture (9, 12). To Protein lysates were extracted using RIPA lysis buffer (Thermo Fisher assess the in vitro role of amivantamab Fc interactions in tumor cell Scientific; catalog no. 89901) with 1-tab PhosSTOP (Sigma; catalog no. killing, the antiproliferative and apoptotic activities were evaluated in 4906837001) and cOmplete Protease Inhibitor (Sigma; catalog no. the presence or absence of human immune cells (PBMCs) against 04693159001). Protein concentration was determined using Pierce H1975 cells, a NSCLC cell line harboring mutant EGFR (L858R/ BCA Protein Assay Kit (Thermo Fisher Scientific; catalog no. 23227) as T790M) and wild-type cMet (17). In the absence of PBMCs, amivan- per manufacturer's protocol. Capillary-based electrophoresis was per- tamab treatment did not inhibit H1975 cell proliferation or induce formed using PeggySue 12 to 230 kDa separation module (Protein apoptosis (Fig. 1A and B; Supplementary Figs. S1A and S1B). How- Simple; catalog no. SM-S001), anti-rabbit detection module (cat- ever, upon addition of PBMCs, amivantamab induced a dose- alog no. DM-001), and anti-mouse antibody (catalog no. 042-205) dependent decrease in tumor cell viability (Fig. 1A), with maximal fl ¼ as per the manufacturer's protocol. Brie y, protein lysates were antiproliferative effect observed at 72 hours (IC50 0.0054 mg/mL, diluted to 0.2 mg/mL and denatured at 95C for 5 minutes. Primary Fig. 1B, left) and beyond (Supplementary Figs. S1A and S1C). antibodies were diluted: EGFR (CST, 2646) at 1:50; phosphorylated Amivantamab treatment also resulted in a dose-dependent increase ¼ EGFR (pEGFR; R&D Systems, AF1095) at 1:50; cMet (CST, 3148) in apoptosis in the presence of PBMCs (IC50 0.0004 mg/mL, Fig. 1B, at 1:50; pMet (CST, 3077) at 1:50, and b-actin (CST, 4947) at 1:100. right), and this antiproliferative or apoptotic activity was not observed Ladder, samples, primary and secondary antibodies, separation, upon treatment with a nontargeting huIgG1 isotype control (Fig. 1B; and stacking matrices were added to the 384-well Peggy Sue plate Supplementary Figs. S1B and S1C). Similar results (proliferation

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

Vijayaraghavan et al.

A treatment 50 g/mL 0 g/mL

+PBMC

B 40 250 H1975 - Isotype - no PBMC H1975 - Isotype - PBMC 200 20 H1975 - - no PBMC 0 100 H1975 - - PBMC -20 H1975 - IgG2 - no PBMC -40 0 H1975 - IgG2 - PBMC H1975 Apoptosis H1975 Proliferation -60 IC = 0.0054 μg/mL 50 IC50 = 0.0004 μg/mL (NucRed AUC Normalized to Cnt) (NucRed (Annexin AUC Normalized to Cnt) AUC Normalized to Cnt) (Annexin -100 IC50 = 12.1 μg/mL IC = 0.142 μg/mL -80 50 -6 -4 -202 -6 -4 -202 Log treatment conc. (μgμm/ L) goL tnemtaert .cnoc ( g/mL)

C 50 100 SNU5 - Isotype - no PBMC SNU5 - Isotype - PBMC

SNU5 - - no PBMC 0 50 SNU5 - - PBMC

SNU5 - IgG2 - no PBMC -50 SNU5 - IgG2 - PBMC SNU5 Apoptosis 0 SNU5 Proliferation

IC50 = 0.0013 μg/mL

IC = 0.035 μg/mL AUC Normalized to Cnt) (Annexin (NucRed AUC Normalized to Cnt) AUC Normalized to Cnt) (NucRed 50 -100 -6 -4 -202 -6 -4 -202 Log treatment conc. (μg/mL) Log treatment conc. (μg/mL) D 40 300 H1975 - Cet - no PBMC IC50 = 0.56 μg/mL IC = 0.0066 μg/mL H1975 - Cet - PBMC 20 50 200 H1975 - - no PBMC 0 H1975 - - PBMC -20 100

-40 H1975 Apoptosis

H1975 Proliferation 0 -60 IC50 = 0.51 μg/mL

(NucRed AUC Normalized to Cnt) IC = 0.0054 μg/mL 50 AUC Normalized to Cnt) (Annexin -80 -100 -6 -4 -20 2 -6 -4 -20 2 Log treatment conc. (μg/mL) Log treatment conc. (μg/mL)

E 50 - - no PBMC - - PBMC

0 - - no PBMC - - PBMC

-50 SNU5 Proliferation (NucRed Area Normalized to Cnt) Area Normalized to Cnt) (NucRed -100 -6 -4 -20 2 Log treatment conc. (μg/mL)

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) after 72 hours and apoptosis (AUC of Annexinþ NucRed area/well) after 48 hours upon treatment 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 #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 upon treatment 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 curves measuring H1975 cell viability (AUC of NucRed area/well) after 72 hours and apoptosis (AUC of Annexinþ 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 #6. E, Dose–response curves measuring SNU5 cell viability (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.

2046 Mol Cancer Ther; 19(10) October 2020 MOLECULAR CANCER THERAPEUTICS

Amivantamab Induces EGFR/cMet Downmodulation by Trogocytosis

¼ ¼ inhibition IC50 0.01 mg/mL; apoptosis IC50 0.002 mg/mL) were ity (Fig. 2B). This demonstrated that amivantamab does not induce obtained using PBMCs from a different donor (Supplementary Figs. CDC against tested NSCLC cell lines. S2A and S2B). To confirm the contribution of Fc engagement, the Interaction of the Fc region of therapeutic antibodies with FcgRs on EGFR and cMet binding regions of amivantamab were engineered into immune cells is known to induce secretion of cytokines and chemokines an Fc effector silent molecule by replacing the wild-type IgG1 with an (ADCR; ref. 20). A 71-plex MSD cytokine panel was utilized to assess effector silent IgG2s framework (9). In presence of PBMCs, treatment chemokines and cytokines secreted upon treatment with huIgG1 isotype with EGFR/cMet-IgG2s resulted in no or minimal effects on H1975 control, amivantamab, cetuximab, or Fc silent EGFR/cMet-IgG2s in the target cell viability and apoptosis (IC50 ¼ 12.1 mg/mL and IC50 ¼ 0.142 presence or absence of PBMCs for 4 or 72 hours. We observed distinct mg/mL, respectively; Fig. 1B; Supplementary Figs. S1B, S2A, and S2B), differences in several secreted cytokines in a treatment- and timepoint- suggesting that these antiproliferative and apoptotic activities were Fc- dependent manners (Fig. 2C; Supplementary Figs. S3E and S3F). interaction dependent. Further analysis focusing on cytokines with >1.5-fold difference between The antiproliferative (IC50 ¼ 0.0013 mg/mL) and apoptotic activities treatments showed that at 4 hours (Fig. 2D) and 72 hours post- of amivantamab were also observed against a MET amplified gastric treatment (Supplementary Fig. S3G), 13 and 7 cytokines, respectively, carcinoma cell line, SNU5, and were dependent on the presence of were upregulated upon amivantamab treatment compared with huIgG1 PBMCs (Fig. 1C; Supplementary Fig. S2C). Similar to H1975 cells, no isotype control or EGFR/cMet-IgG2s controls in the co-culture of effects on proliferation and apoptosis were observed upon treatment H1975þPBMCs. Fold induction of most of these cytokines were lower with huIgG1 isotype control or EGFR/cMet-IgG2s controls (Fig. 1C). for cetuximab compared with amivantamab (Fig. 2D; Supplementary The effects of amivantamab were then compared with that of cetux- Fig. S3G). imab (18), a bivalent chimeric anti-EGFR IgG1 antibody in H1975 and We next interrogated the contribution of immune cell subtypes SNU5 cell lines. Although cetuximab was able to induce a dose- (PBMCs vs. NK cells, monocytes or macrophages from same donor) in ¼ dependent antiproliferative effect (IC50 0.51 mg/mL) and apoptosis the secretion of these cytokines. Heatmap analysis revealed distinct ¼ (IC50 0.56 mg/mL) in presence of PBMCs, the magnitude of the changes in cytokine expression patterns, with chemotactic cytokines PBMC-dependent activities was inferior to that of amivantamab (CC chemokines) being the most frequently upregulated family upon (Fig. 1D). Similar results were observed in SNU5 cell line, where amivantamab treatment (Fig. 2E; Supplementary Fig. S4A). CC amivantamab was more potent compared with cetuximab (Fig. 1E). chemokines are known to function as chemo-attractants for innate Collectively, these studies demonstrated that amivantamab Fc immune cells like monocytes and macrophages (21–23). Intriguingly, interaction with immune cells is essential for its in vitro anti- upregulation of these chemokines upon amivantamab treatment was proliferative effects. specific to PBMCs, monocytes, and macrophages but not seen with NK cells (Fig. 2E). For example, amivantamab treatment resulted in a Amivantamab Fc interaction with immune cells induces ADCC, potent increase in macrophage inflammatory protein (MIP)-1b (com- ADCR, and EGFR/cMet downmodulation pared with huIgG1 isotype control) in the presence of PBMCs, We next interrogated the different immune effector mechanisms monocytes, or M1 and M2 macrophages but no change was observed (ADCC, CDC, ADCR, ADCT) induced by amivantamab and its with NK cells (Fig. 2E). Although cetuximab induced several of these impact on EGFR/cMet signaling, in comparison with cetuximab. cytokines, the fold upregulation was lower compared with that of ADCC-mediated cell lysis was measured using a short-term Euro- amivantamab, suggesting that amivantamab is more potent than pium release assay in H1975 cells treated with huIgG1 isotype cetuximab in inducing CC chemokines (Fig. 2E). control, amivantamab, cetuximab, or Fc silent EGFR/cMet-IgG2s Because EGFR and/or cMet signaling is key to the growth of these in presence of PBMCs. Although amivantamab induced dose- tumor cells, we also investigated whether amivantamab or cetux- dependent ADCC (68% max lysis), no ADCC lysis was observed imab Fc interaction with immune cells had an effect on EGFR and with huIgG1 isotype control or EGFR/cMet-IgG2s treatments, cMet protein levels and downstream signaling. To examine this, showing the contribution of Fc engagement in this process H1975 cells were treated with huIgG1 isotype control, amivanta- (Fig. 2A). Cetuximab induced dose-dependent ADCC (39% max mab, cetuximab, or Fc silent EGFR/cMet-IgG2s in the absence or lysis) but to a lesser extent than amivantamab (Fig. 2A). Similar presence of PBMCs. Treatment with amivantamab or cetuximab results were obtained using PBMCs from a different donor (Sup- alone (in the absence of PBMCs) only showed marginal down- plementary Fig. S3A). Next, we demonstrated that similar levels of regulation of EGFR and phosphorylated EGFR (pEGFR; pY1173) ADCC lysis was measured from isolated NK cells compared with proteins (Fig. 2F). Compared with EGFR/pEGFR levels, amivanta- PBMCs (Supplementary Fig. S3B), suggesting that NK cells account mab treatment (no PBMCs) showed a greater downregulation of for amivantamab-induced ADCC. No ADCC was measured from cMet protein (Fig. 2F), which may be attributed to the high-affinity isolated monocytes (Supplementary Fig. S3B). cMet binding arm (9). pMet was below reliably measurable levels in CDC activity triggered by the activation of the complement H1975 cells and hence not depicted in this and subsequent analysis. component C1q was then assessed. First, we demonstrated that Interestingly, the addition of PBMCs markedly potentiated ami- amivantamab, cetuximab, rituximab, and huIgG1 isotype control vantamab-mediated downregulation of EGFR, pEGFR, and cMet by bind to C1q in a dose-dependent manner, whereas no measurable 44%, 54%, and 52%, respectively (Fig. 2F; densitometry in Supple- binding was observed by the Fc silent EGFR/cMet-IgG2s (Supple- mentary Fig. S4B), whereas treatment with the huIgG1 isotype mentary Fig. S3C), suggesting that the C1q binding is Fc-structure control or EGFR/cMet-IgG2s in the absence or presence of PBMCs specific. We next investigated CDC-mediated target cell lysis of had no apparent effect (Fig. 2F). Although presence of immune NSCLC cell lines, H1975 (Fig. 2B) and H292 (Supplementary cells enhanced cetuximab-induced downregulation of EGFR and Fig.S3D)andshowedthatnomeasurableCDCactivitywas pEGFR by 23% and 32%, respectively (Supplementary Fig. S4B), the observed upon amivantamab or EGFR/cMet-IgG2s treatment. As effect was less pronounced in comparison to that observed with positive control, rituximab (bivalent anti-CD20 antibody) added to amivantamab (Supplementary Fig. S4C). Cetuximab treatment did CD20-positive Daudi cells (19) showed dose-dependent CDC activ- not show an appreciable effect on cMet levels.

AACRJournals.org Mol Cancer Ther; 19(10) October 2020 2047

Vijayaraghavan et al.

A 80 B 150 Isoty e Cet x a 60 100 t a IgG2 40 gG2

50 20 % Maximal lysis (ADCC) % Target cell lysis (CDC) 0 0 -4 -20 2 0.001 0.01 0.1 1 10 100 Log treatment conc. (μg/mL) Treatment conc. (μg/mL) C D Count 05101520

12345 Log (AUC) 4

3 e) IgG2 2 Cet a

1 (relative to Isot Relative fold change Relative fold change 0 2a MIF

Eotaxin IP-10 IFN- TARC MCP-3 IL-1RA MCP-1 TNF- GRO- Fractalkine MIP-1 ENA-78

CC E Count F CXC 0246

0.2 0.4 0.6 0.8 1 Percentile

Iso

Cetuximab PBMC

EGFR

pEGFR

cMet

Actin MCP-2

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 huIgG1 isotype control (Isotype), amivantamab, cetuximab, or EGFR/cMet-IgG2s (IgG2s) at multiple concentrations in the presence of PBMCs from donor #4 at an E: T ratio of 25:1 and ADCC lysis measured by Europium release. B, Dose–response rate of CDC lysis measured by lactate dehydrogenase (LDH) levels from H1975 target cells after 2 hours of treatment with amivantamab or EGFR/cMet-IgG2s (IgG2s) in the presence of 5% baby rabbit serum. The CD-20 antibody, rituximab, was used as a positive control in CD20 expressing Daudi cell line. All data represented as mean SEM within each treatment group. C, Heat map of log-transformed AUC values of cytokines 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 than 1.5 increase for amivantamab treatment compared with isotype. E, Heat map of log-transformed AUC values of cytokines from the 23-plex MSD analysis of H1975 cells 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) or individual immune cells (E:T ¼ 5:1), namely NK cells, monocytes, or M1 or M2c macrophages. Heat map was limited to cytokines with greater than 1.5 increase for the H1975þPBMCs condition. F, Western blot analysis (PeggySue capillary electrophoresis) of EGFR, pEGFR, cMet, and loading control actin performed following 48 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.

Taken together, these results demonstrate that amivantamab Fc Amivantamab Fc interaction induced EGFR and cMet interaction with immune cells induces ADCC, stimulates production downmodulation is driven by monocyte composition of macrophage-related chemokines, and enhances downmodulation of We next assessed the contribution of different immune cells at EGFR and cMet, and is more potent than cetuximab in these assays. mediating EGFR and cMet downmodulation. PBMCs from seven

2048 Mol Cancer Ther; 19(10) October 2020 MOLECULAR CANCER THERAPEUTICS

Amivantamab Induces EGFR/cMet Downmodulation by Trogocytosis

A PBMC Donor # B Isotype C Isotype Iso IgG2 PBMC 1234567 IgG2 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 inhibtion % EGFR inhibtion R2 = 0.06 R2 = 0.0079 R2 = 0.45 -100 -100 -100 0 5 10 15 20 10 12 14 16 18 020406080 % of NK cells % of B cells % of T cells E 100 100 100

50 50 50

0 0 0

-50 -50 -50 % Met inhibtion % EGFR inhibtion R2 = 0.5088 % pEGFR inhibtion R2 = 0.55 R2 = 0.475 -100 -100 -100 0102030 0102030 0102030 % of Monocytes % of Monocytes % of Monocytes

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 of H1975 cells with 10 mg/mL of huIgG1 isotype control (Isotype) or amivantamab (Ami) in the presence of PBMCs from seven different donors. Western blot analysis (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 #3 against 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 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 percentage changes in EGFR, pEGFR (Y1173), and cMet inhibition upon amivantamab treatment in presence of PBMCs. All data represented as mean SEM within each treatment group. donors were tested for their ability to potentiate amivantamab- mediated downmodulation was observed (Supplementary Figs. mediated downregulation of EGFR, cMet, and pEGFR protein levels S5B and S5C). (Fig. 3A). Treatment with amivantamab, in the absence of PBMCs, To examine the role of amivantamab–Fc interaction in EGFR/cMet showed marginal downregulation of EGFR, pEGFR, and moderate downmodulation, H1975 cells were treated with huIgG1 isotype downmodulation of cMet protein levels. Although the addition of control, amivantamab, or Fc silent EGFR/cMet-IgG2s in the presence PBMCs enhanced the downmodulation of EGFR, pEGFR and cMet of PBMCs from selected donor #6 (Fig. 3B). Addition of PBMCs compared with huIgG1 isotype control for most donors, the extent markedly potentiated amivantamab-mediated downregulation of of this effect varied among donors (Fig. 3A; densitometry in EGFR, pEGFR, and cMet by 46%, 67%, and 44%, respectively Supplementary Fig. S5A). PBMCs from some donors (#3, #4, and (Fig. 3B; densitometry in Supplementary Fig. S6A), whereas treatment #6) showed a substantial effect in potentiating EGFR and cMet with the huIgG1 isotype control or EGFR/cMet-IgG2s in the absence downmodulation, whereas PBMCs from other donors (#2, #5, and or presence of PBMCs had no apparent effect, suggesting that it is #7)hadlittletonoeffect.PBMCsfrom additional donors were speciﬁc to the amivantamab–Fc interaction. Similar results were similarly tested, and considerable variability in amivantamab- obtained with donor #3 (Supplementary Fig. S6B). We conﬁrmed this

AACRJournals.org Mol Cancer Ther; 19(10) October 2020 2049

Vijayaraghavan et al.

effect of Fc engagement on EGFR and cMet expression in the MET- cMet-IgG2s antibodies into monocytes was observed (Fig. 4D; Sup- amplified SNU5 cell line (Fig. 3C). Similar to H1975 cells, presence of plementary Fig. S9C; Supplementary Movie M1), demonstrating that PBMCs enhanced the ability of amivantamab to downregulate EGFR amivantamab induced monocyte-dependent trogocytosis. and cMet signaling, resulting in 79%, 89%, 87%, and 90% reduction in Collectively, these results demonstrate that monocytes mediate levels of EGFR, pEGFR, cMet, and pMet proteins, respectively, and no trogocytosis and drive amivantamab–Fc interaction mediated down- measurable change was seen with the huIgG1 isotype control or EGFR/ modulation of EGFR and cMet receptors. cMet-IgG2s control (Supplementary Fig. S6C). To determine which immune cells are responsible for Fc-dependent Amivantamab Fc interaction with macrophages induces downmodulation, we compared percentages of key immune cell trogocytosis leading to EGFR and cMet downmodulation populations (NK cells, monocytes, B cells, and T cells) within PBMCs Solid tumors, particularly NSCLC, have been reported to contain a (Supplementary Fig. S7A) for each donor with the observed differences high level of tumor-associated macrophages (TAM; ref. 24). Hence, we in their downmodulation effects. No correlation was observed between examined if M1 and M2 macrophages are capable of amivantamab NK, B, or T cells and the ability of the PBMCs to potentiate EGFR trogocytosis similar to monocytes using time-lapse microscopy in downmodulation (Fig. 3D; Supplementary Fig. S7B). However, a H1975 target cells opsonized with AF647-labeled antibodies (huIgG1 correlation was observed between relative monocyte population within isotype control, amivantamab, or EGFR/cMet-IgG2s). Macrophages the PBMCs and the downmodulation of EGFR (R2 ¼ 0.51), pEGFR were visualized with AF488-labeled CD11b/CD14 antibody cocktail (R2 ¼ 0.55), and cMet (R2 ¼ 0.48) protein levels (Fig. 3E). This finding (green), Hoechst stain for nuclei (blue), and H1975 target cells were suggests that monocytes and their relative level within the PBMC identified by their NucLight Redþ nuclei (orange). Upon co-culture samples determine the extent of amivantamab-mediated EGFR and with target cells opsonized with labeled amivantamab, a distinct cMet downregulation. accumulation of AF647þ puncta (amivantamab) was observed within the M1 (Fig. 5A; Supplementary Movie M2) and M2c macrophages Amivantamab Fc interaction with monocytes induces (Fig. 5B; Supplementary Movie M3), whereas no accumulation was trogocytosis and is required for EGFR and cMet seen with labeled huIgG1 isotype control or EGFR/cMet-IgG2s downmodulation treatment (Fig. 5A and B; Supplementary Figs. S10A and S10B). No To confirm direct monocyte involvement in Fc interaction- appreciable phagocytosis was observed visually in the presence of M1 mediated signal downregulation, NK cells or monocytes were depleted or M2c macrophages in these microscopy assays, indicating that from PBMCs (Fig. 4A) and tested for amivantamab-mediated down- macrophage-mediated trogocytosis is the predominant mechanism modulation of EGFR and cMet pathways. Consistent with previous measured. results, nondepleted PBMCs enhanced amivantamab-mediated down- To better simulate antibody interactions within the tumor, we regulation of EGFR, pEGFR, and cMet in H1975 cells (Fig. 4B; treated a co-culture of M1 or M2c macrophages and H1975 target densitometry in Supplementary Fig. S8A). Although depletion of NK cells with AF647-labeled antibodies (huIgG1 isotype control, amivan- cells only had a marginal effect, depletion of monocytes effectively tamab, or EGFR/cMet-IgG2s) and monitored trogocytosis by time- reversed the ability of PBMCs to potentiate amivantamab-mediated lapse microscopy. As expected, huIgG1 isotype control antibody receptor downmodulation (Fig. 4B). As expected, no effect was seen bound only to M1 and M2c macrophages, whereas EGFR/cMet-IgG2s with EGFR/cMet-IgG2s treatment confirming that Fc interaction is bound only to target cells, and amivantamab bound to both target cells required for EGFR/cMet downmodulation. Similar data were obtained and macrophages (Supplementary Fig. S10C), thus confirming the by depleting PBMCs of NK cells and monocytes from a second donor binding specificity of each antibody. Under these conditions (co- (Supplementary Figs. S8B–S8D). culture), amivantamab-mediated trogocytosis was readily observed, To further confirm the role of monocytes, NK cell and monocytes measured by a distinct transfer of labeled amivantamab into macro- were isolated from the same PBMC donor (Supplementary Fig. S9A) phages, but no trogocytosis was observed with huIgG1 isotype control and assessed for amivantamab-mediated downregulation of EGFR, or EGFR/cMet-IgG2s antibodies (Supplementary Fig. S10D; Supple- pEGFR, and cMet proteins (Fig. 4C). Although the control (non- mentary Movies M4 and M5). isolated) PBMC sample enhanced receptor downmodulation, NK cells Finally, to examine if macrophage-mediated trogocytosis leads to alone did not demonstrate a strong effect. However, isolated mono- receptor downmodulation, monocytes were polarized into M1, M2a, cytes substantially enhanced the ability of amivantamab to down- or M2c macrophages, and their ability to potentiate amivantamab- modulate EGFR, pEGFR, and cMet proteins to a similar extent to that mediated EGFR/cMet downmodulation was assessed. Compared with of PBMCþ amivantamab control (Fig. 4C; densitometry in Supple- treatment with amivantamab alone (no macrophages), the presence of mentary Fig. S9B). M1 or either subtype of M2 macrophages examined (M2a, M2c), On the basis of this novel finding that monocytes enhance ami- notably enhanced amivantamab-mediated downregulation of EGFR, vantamab-mediated downmodulation of EGFR and cMet proteins, we pEGFR, and cMet proteins levels (Fig. 5C–E; quantitation in Supple- assessed if this downmodulation occurs through trogocytosis, an Fc mentary Fig. S11). HuIgG1 isotype control or EGFR/cMet-IgG2s effector function that mediates transfer of cell-surface proteins from treatments had no effect, suggesting that the signal downmodulation tumor to effector cells. To visualize amivantamab-induced monocyte required Fc interaction. trogocytosis, time-lapse microscopy was performed in H1975 target Collectively, these results reveal trogocytosis as a novel Fc effector cells opsonized with AF647-labeled antibodies (huIgG1 isotype con- function for amivantamab in the presence of macrophages, leading to trol, amivantamab, or Fc-silent EGFR/cMet-IgG2s). Monocytes were EGFR/cMet receptor downmodulation. visualized with AF488-labeled CD11b/CD14 antibody cocktail (green) and Hoechst stain for nuclei (blue) and H1975 target cells were Fc interaction and macrophages are required for amivantamab identified by their NucLight Redþ nuclei (orange). When co- in vivo antitumor efficacy cultured with opsonized target cells, distinct transfer of labeled ami- The role and relevance of Fc/FcgR interactions in vivo was evaluated vantamab antibody but not labeled huIgG1 isotype control or EGFR/ using H1975 and SNU5 cell line xenograft models. Mice harboring

2050 Mol Cancer Ther; 19(10) October 2020 MOLECULAR CANCER THERAPEUTICS

Amivantamab Induces EGFR/cMet Downmodulation by Trogocytosis

NK Mono PBMCs depleted depleted

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

4 4 10 10 IgG

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

3 3 EGFR −10 −10

3 0-10 103 104 105 -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 Met

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)

(No green) IgG2 C PBMCs NK cells Monocytes D T = 0 Isotype

IgG Immune cells

EGFR T = 55

pEGFR

Met T = 77

Actin

T = 99

Hoechst Monocytes (Cd11b/CD14) H1975-Nuclight Red

Figure 4. Monocytes mediated amivantamab Fc interaction induced trogocytosis and EGFR and cMet downmodulation. A, Contour plots from multicolor 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 analysis (PeggySue capillary electrophoresis) of EGFR, pEGFR, cMet, and actin (loading control) performed on H1975 cell lysates 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 intact PBMCs, NK cell–depleted, or monocyte (mono)–depleted PBMCs from donor #3. C, Western blot analysis (PeggySue) of EGFR, pEGFR, cMet, and loading control actin performed on H1975 cell lysates 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 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-IgG2s (IgG2s) 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, 20 mm.

H1975 or SNU5 tumors were treated with huIgG1 isotype control, individual mice). None of the antibody treatments resulted in loss of amivantamab, or Fc silent EGFR/cMet-IgG2s antibodies for 3 weeks mouse body weight in either tumor models (Supplementary Figs. S12B (Supplementary Figs. S12A and S12D). In the H1975 model, TGI was and S12E). superior upon treatment with amivantamab (TGI ¼ 75%; , P < We previously demonstrated, using an HGF-expressing H1975 xeno- 0.0001) compared with that of EGFR/cMet-IgG2s (TGI ¼ 30%; graft tumor model, that Fc silent EGFR/cMet-IgG2s only showed partial , P < 0.0016; Fig. 6A; Supplementary Fig. S12C shows individual effect on EGFR/cMet signaling compared with amivantamab, suggesting mice). Similar results were observed in the SNU-5 model, where a role for Fc-mediated signal downregulation in vivo (12).Toextendthis amivantamab treatment effectively reduced tumor growth (TGI ¼ observation to the MET-ampliﬁed SNU5 model in vivo,tumorswere 96%; , P < 0.0001) compared with EGFR/cMet-IgG2s treatment collected from the in vivo efﬁcacy study (Fig. 6B) and changes in total and (TGI ¼17%; ns, P ¼ 0.53; Fig. 6B; Supplementary Fig. S12F shows phospho-EGFR and cMet protein levels were measured. Similar to

AACRJournals.org Mol Cancer Ther; 19(10) October 2020 2051

Vijayaraghavan et al.

A 11=T 44=T 77=T 011=T

Hoechst M1 Macrophages (Cd11b/CD14) H1975-Nuclight Red B 11=T 44=T 77=T 011=T

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

C Isotype D Isotype E Isotype

IgG IgG IgG Macs Macs Macs

EGFR EGFR EGFR

pEGFR pEGFR pEGFR

cMet cMet cMet

Actin Actin Actin

Figure 5. Amivantamab Fcinteraction induces macrophage-trogocytosis and EGFR and cMet downmodulation.Representative images from high-content confocal microscopy of M1 (A)orM2(B) macrophages 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 or EGFR/cMet-IgG2s (IgG2s) 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, 20 mm. Western blot analysis (PeggySue capillary electrophoresis) of EGFR, pEGFR, cMet, and actin (loading control) performed on H1975 cell lysates 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 M1 macrophages (C), M2a macrophages (D), or M2c macrophages (E).

in vitro results (Fig. 3C), compared with vehicle treatment, amivantamab showed significant (, P < 0.0001) reduction in TAMs (2%) treatment showed significant downregulation of EGFR, pEGFR, cMet, compared with untreated tumors (11%–15%; Fig. 6D). Animals were and pMet proteins but EGFR/cMet-IgG2s treatment only had marginal then treated with huIgG1 isotype control or amivantamab for 3 weeks effects, suggesting that Fc interaction is required for in vivo signal (Supplementary Fig. S13B) with no loss in mouse body weight (Sup- downmodulation (Fig. 6C; quantification in Fig. 6C and Supplementary plementary Fig. S13C). As shown previously, treatment with amivan- Fig. S13A). Thus, these in vivo studies demonstrate that amivantamab Fc tamab showed significantly higher antitumor efficacy compared interaction is essential for its antitumor efficacy and in vivo EGFR/cMet with the isotype (, P < 0.0001) or EGFR/cMet-IgG2s treatment signal downmodulation. (, P ¼ 0.004) in non-anti-CSF1R treated tumors (Fig. 6E). Strikingly, The role of macrophages in amivantamab in vivo efficacy was next depletion of TAMs (anti-CSF1R-treated) significantly reduced amivan- assessed using anti-CSF1R–mediated depletion of TAMs in the H1975 tamab TGI from 72.8% to 38.5% (, P ¼ 0.014; Fig. 6E; Supplementary xenograft study. As expected, treatment with anti-CSF1R antibody Fig. S13D shows individual mice), suggesting that macrophages play a

2052 Mol Cancer Ther; 19(10) October 2020 MOLECULAR CANCER THERAPEUTICS

Amivantamab Induces EGFR/cMet Downmodulation by Trogocytosis

A 2,000 B 2,000 Isotype control Vehicle PBS ns -17% TGI ± SEM) ± SEM) 3 3 IgG2 IgG2 1,500 1,500

30% TGI 1,000 1,000 **

500 500 **** 75% TGI **** 96% TGI 0 0 Mean SNU5 tumor volume (mm Mean H1975 tumor volume (mm 0 5 10 15 20 25 01 15 20 25 5303 Days post tumor implantation Days post tumor implantation C PBS RFGE teMc IgG2 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

IgG2 IgG2 GAPDH ehicle PBS ehicle PBS V V

DF 25 ***

TAMs 10

0 (% F4/80+ cells of single cells)

Isotype control Isotype control CSF1R Tx

E 1,500 Isotype control Amivantamab IgG2

± SEM) Isotype Control + αCSF1R 3 α 1,000 Amivantamab + CSF1R

29% 38.5%

500 *

72.8% Mean tumor volume (mm

-5 015 0152025 αCSF1R Tx Amivantamab Tx Days post tumor implantation

Figure 6. Amivantamab–Fc interaction with macrophages are required for in vivo antitumor efﬁcacy. A, Tumor volumes of subcutaneously injected H1975 cell line xenograft tumors treated with 10 mg/kg huIgG1 isotype control (Isotype), amivantamab, or EGFR/cMet-IgG2s (IgG2s; n ¼ 10 per group) for 3 weeks BIW. %TGI was calculated at 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 line xenograft tumors treated with vehicle (PBS), 5 mg/kg amivantamab (Ami), or EGFR/cMet-IgG2s (IgG2s; n ¼ 8 per group) for 3 weeks BIW. %TGI was calculated at day 34, and P values were calculated using two-way ANOVA (, P < 0.0001). C, Western blot analysis of SNU5 xenograft tumors harvested 24 hours 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 one-way ANOVA (, P < 0.05; , P < 0.005). D, Multicolor ﬂow cytometry analysis of H1975 tumor samples isolated 24 hours after two doses of 10 mg/kg huIgG1 isotype control (Isotype), amivantamab (Ami), or EGFR/cMet-IgG2s (IgG2s) treatment (n ¼ 5 mice/treatment) to assess the percentage of macrophages (CD45þ CD11bþ Ly6G Ly6C F4/80þ) within the tumor post-depletion with anti-mouse CSF1R antibody. E, Tumor volumes of subcutaneously injected H1975 cell line xenograft tumors treated with 10 mg/kg huIgG1 isotype control (Isotype), amivantamab, or EGFR/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 deplete macrophages. %TGI was calculated on day 21, and P values were calculated using one-way ANOVA (, P < 0.005). F, Figure illustrating the multiple Fc-dependent and Fc-independent mechanisms of action contributing to amivantamab antitumor activity. All data represented as mean SEM within each treatment group.

AACRJournals.org Mol Cancer Ther; 19(10) October 2020 2053

Vijayaraghavan et al.

key role in mediating amivantamab antitumor efficacy. No significant vantamab leading to downmodulation of EGFR and cMet, and difference was observed between vehicle and vehicleþanti-CSF1R potentially fueling additional recruitment of monocytes or macro- treatment (P ¼ 0.17). phages by cytokine production. Collectively, these findings demonstrate that amivantamab–Fc In contrast to most other therapeutic antibodies in the clinic, interaction with macrophages and monocytes induces trogocytosis, amivantamab was designed and engineered with a low fucose back- which is a dominant mechanism underlying the antitumor efficacy of bone, which enhances its binding to FcgRIIIa (12), present on NK cells, amivantamab. monocytes, and macrophages. This increased binding to FcgRIIIa enhanced induction of Fc effector functions in comparison to other (normal fucose) huIgG1 antibodies such as cetuximab and trastuzu- Discussion mab, and could also enable amivantamab to out-compete naturally In this study, we demonstrated that in addition to the known Fc- circulating IgG antibodies for FcgR binding, as shown preclinically independent mechanisms (inhibition of ligand binding and ligand with other enhanced Fc antibodies (29, 30). Given the predominance of driven signaling; refs. 9, 12), the EGFR/cMet bispecific antibody, EGFR-driven NSCLC tumors, it is confounding that cetuximab did not amivantamab, exerts antitumor efficacy through multiple Fc- meet clinical endpoints in NSCLC (31). We hypothesize that this could dependent mechanisms (Fig. 6F). These mechanisms are initiated result from several factors that amivantamab can overcome. Pri- through interaction of the Fc domain of the antibody with Fcg marily, cetuximab only targets EGFR and it is known that cancer receptors present on immune cells. Binding and activation of Fcg cells can quickly adapt to therapy by activating alternate pathways, receptors on NK cells led to ADCC, while binding and activation of Fcg the most common of which is cMet (32). In addition, the bivalent receptors on monocytes and macrophages led to cytokine production nature of the high-affinity EGFR arm in cetuximab results in on- and trogocytosis. Trogocytosis caused downmodulation of EGFR and target off tumor toxicity in the clinic (31). Studies have shown that cMet receptors and their downstream signaling, which is required for bispecificity of an antibody can increase tumor selectivity via proliferation of these cancer cells. simultaneous targeting of two cancer antigens and varying antigen Although we demonstrated in vitro ADCC lysis with amivantamab, affinity can drive selectivity towards cells expressing both antigens we believe that this effect is minimal compared with that exerted by compared with single antigen expressing normal cells (33–35). monocytes and macrophages. ADCC lysis is a rapid mechanism Thus,wehypothesizethatthepresenceofthehigh-affinity cMet K ¼ fi occurring at early timepoints ( 2 to 4 hours; ref. 25), whereas 48 hours targeting arm ( d 40 pmol/L), low-af nity EGFR targeting arm in vitro K ¼ fi and more were required to reach the maximal amivantamab ( d 1.4 nmol/L) combined with the high-af nity FcgRIIIa antiproliferative effect in presence of PBMCs (Supplementary binding (low fucose) Fc portion could account for the improved Fig. S1C). These suggest that the short-term ADCC is not sufficiently selectivity (36) and efficacy, and lowered toxicity of amivantamab in contributing to the overall long-term activity of amivantamab and that comparison with cetuximab. other effector functions (such as trogocytosis) contribute to additional Previously, trogocytosis was described as a mechanism of resistance cytotoxic activity over time. Although we did not observe CDC activity for antibodies such as rituximab (16, 37). In this context, the receptor is in the cell lines evaluated, NSCLC cell lines are known to express stripped from the cell surface by monocytes/macrophages, preventing complement inhibitory proteins, CD46, CD55, and CD59 (26), sug- occurrence of NK cell-mediated functions towards the no-longer gesting that amivantamab might trigger CDC activity in other cell lines available targeting receptor. However, in this study, we demonstrated or tumor types not expressing these complement inhibitory proteins. a novel role for trogocytosis (ADCT) as a crucial inducer of Fc- Finally, we reported previously that amivantamab induces ADCP dependent receptor downmodulation and antitumor effects. We in vitro (9), however under the conditions tested in this study (confocal hypothesize that the dichotomy of trogocytosis’ role (mediator of microscopy-based macrophage trogocytosis assay), minimal to no resistance vs. antitumor effects) might be driven by the dependency of ADCP was observed. Collectively, as depicted in Fig. 6F, our data the tumor cells on the trogocytosis-targeted receptor. Although anti- suggest that amivantamab antitumoral activity occurs through mul- tumor effects via trogocytosis have been suggested for cetuximab and tiple Fc-independent and Fc-dependent mediated mechanisms of trastuzumab (30, 38), our work confirmed such a mechanism of action action linked to EGFR and cMet binding on cancer cells. Fc- for amivantamab and presents comprehensive data with primary dependent amivantamab mechanism of action comprises NK- immune cells (monocytes, M1 and M2 macrophages), demonstrating dependent ADCC as well as monocyte- and macrophage-dependent the induction of trogocytosis leading to the downmodulation of the trogocytosis and cytokine release. Because the content and diversity of target receptors, EGFR and cMet. It is interesting to note that immune cells within the tumor microenvironment (TME) varies trogocytosis was observed for both M1 and M2 macrophage popula- among patients, this may influence the response to amivantamab tions, suggesting this mechanism may also be operative in more treatment. immunosuppressive TMEs. We showed that most cytokines upregulated by amivantamab Therapeutic targeting of EGFR and cMet with an antibody such belong to the family of chemotactic cytokines called chemokines, as amivantamab with multiple and enhanced Fc mechanism(s) of specifically CC chemokines (21, 23). CC chemokines comprises of action can provide future options to combat diverse types of cancer- two key subfamilies, monocyte chemoattract protein (MCP) and driving mutations and drug-acquired resistance for patients. In MIP, which are both known to function as chemo-attractants for addition to targeting a conserved region of an oncogenic receptor, innate immune cells such as monocytes and macrophages (22, 27). which broadens the targeted population, the selectivity of bispecific MCP-1 (CCL2) and MIP1b (CCL4) have been reported to induce antibodies like amivantamab, could offer a reduced potential for recruitment of monocytes and macrophages into the TME of toxicity, making combination therapy with additional agents better NSCLC (22, 28). Although these cytokines could attract immune tolerated. Our findings demonstrate that the Fc-dependent activity cells to the TME, their production requires an initial tumor cell- of amivantamab is driven by macrophage- and monocyte-mediated immune cell contact mediated by amivantamab. So, we hypothesize trogocytosis, a dominant mechanism of antibody-directed receptor that trogocytosis is the initial and dominant mechanism of ami- downregulation and tumor cell killing. These findings highlight the

2054 Mol Cancer Ther; 19(10) October 2020 MOLECULAR CANCER THERAPEUTICS

Amivantamab Induces EGFR/cMet Downmodulation by Trogocytosis

potential of trogocytosis as therapeutic modality but also provide analysis, investigation and methodology. J. Sendecki: Software and formal analysis. insights to guide patient selection and combination strategies for M. Beqiri: Investigation and methodology. H.J. Millar: Conceptualization, supervision, amivantamab. investigation and methodology. K. Packman: Conceptualization, supervision and methodology. M.V. Lorenzi: Conceptualization, supervision, writing-original draft, writing-review and editing. S. Laquerre: Conceptualization, supervision, writing- Disclosure of Potential Conflicts of Interest original draft, writing-review and editing. S.L. Moores: Conceptualization, resources, S. Vijayaraghavan reports a patent for bispecific EGFR/cMet antibody mechanism supervision, validation, writing-original draft, writing-review and editing. of action (planned). J. Sendecki is an employee of and holds shares and stock options in Janssen R&D. M.V. Lorenzi is an employee of and a shareholder in Janssen Acknowledgments Pharmaceuticals outside the submitted work. S.L. Moores reports a patent for The study was funded by Janssen Research & Development, LLC. Antibody US9593164 - bispecific EGFR/c-Met antibodies issued, a patent for US9580508 - generation was performed by Pete Buckley, Janssen Biotherapeutics. Assistance with bispecific EGFR/c-Met antibodies issued, a patent for US9695242 - bispecific EGFR/c- performing in vivo experiments was provided by David Walker, Janssen R&D, LLC. Met antibodies issued, a patent for U.S. patent application no. 16/697,249 - bispecific Technical guidance for the confocal microscopy–based trogocytosis assay was EGFR/cMet antibodies pending, and a patent for bispecific EGFR/cMet antibody provided by Edward Keough, Janssen Biotherapeutics. Writing assistance was mechanism of action (planned). No potential conflicts of interest were disclosed by provided by Ramji Narayanan (SIRO Clinpharm Pvt Ltd.), funded by Janssen Global the other authors. Services, LLC, and additional editorial support was provided by Tracy Cao (Janssen Global Services, LLC). Authors’ Contributions S. Vijayaraghavan: Conceptualization, software, formal analysis, supervision, The costs of publication of this article were defrayed in part by the payment of page advertisement investigation, visualization, methodology, writing-original draft, project charges. This article must therefore be hereby marked in accordance administration, writing-review and editing. L. Lipfert: Formal analysis, investigation with 18 U.S.C. Section 1734 solely to indicate this fact. and methodology. K. Chevalier: Formal analysis, investigation and methodology. B.S. Bushey: Formal analysis, investigation and methodology. B. Henley: Formal Received January 31, 2020; revised April 6, 2020; accepted July 27, 2020; analysis, validation, investigation and methodology. R. Lenhart: Software, formal published first August 3, 2020.

References 1. Sequist LV, Yang JC, Yamamoto N, O'Byrne K, Hirsh V, Mok T, et al. 13. Stewart R, Hammond SA, Oberst M, Wilkinson RW. The role of Fc gamma Phase III study of afatinib or cisplatin plus pemetrexed in patients with receptors in the activity of immunomodulatory antibodies for cancer. metastatic lung adenocarcinoma with EGFR mutations. J Clin Oncol 2013; J Immunother Cancer 2014;2:29. 31:3327–34. 14. Rajasekaran N, Chester C, Yonezawa A, Zhao X, Kohrt HE. Enhancement of 2. Park K, Tan EH, O'Byrne K, Zhang L, Boyer M, Mok T, et al. Afatinib versus antibody-dependent cell mediated cytotoxicity: a new era in cancer treatment. gefitinib as first-line treatment of patients with EGFR mutation-positive non- Immunotargets Ther 2015;4:91–100. small-cell lung cancer (LUX-Lung 7): a phase 2B, open-label, randomised 15. Clynes RA, Towers TL, Presta LG, Ravetch JV. Inhibitory Fc receptors modulate controlled trial. Lancet Oncol 2016;17:577–89. in vivo cytotoxicity against tumor targets. Nat Med 2000;6:443–6. 3. Kobayashi S, Boggon TJ, Dayaram T, Janne PA, Kocher O, Meyerson M, et al. 16. Taylor RP, Lindorfer MA. Fcgamma-receptor-mediated trogocytosis impacts EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J mAb-based therapies: historical precedence and recent developments. Blood Med 2005;352:786–92. 2015;125:762–6. 4. Soria JC, Ohe Y, Vansteenkiste J, Reungwetwattana T, Chewaskulyong B, Lee 17. Jarantow SW, Bushey BS, Pardinas JR, Boakye K, Lacy ER, Sanders R, et al. KH, et al. Osimertinib in untreated EGFR-mutated advanced non-small-cell Impact of cell-surface antigen expression on target engagement and function of lung cancer. N Engl J Med 2018;378:113–25. an epidermal growth factor receptor x c-MET bispecific antibody. J Biol Chem 5. Turke AB, Zejnullahu K, Wu YL, Song Y, Dias-Santagata D, Lifshits E, et al. 2015;290:24689–704. Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC. 18. Ayoub D, Jabs W, Resemann A, Evers W, Evans C, Main L, et al. Correct primary Cancer Cell 2010;17:77–88. structure assessment and extensive glyco-profiling of cetuximab by a combina- 6. Yano S, Yamada T, Takeuchi S, Tachibana K, Minami Y, Yatabe Y, et al. tion of intact, middle-up, middle-down and bottom-up ESI and MALDI mass Hepatocyte growth factor expression in EGFR mutant lung cancer with intrinsic spectrometry techniques. mAbs 2013;5:699–710. and acquired resistance to tyrosine kinase inhibitors in a Japanese cohort. 19. Li B, Zhao L, Guo H, Wang C, Zhang X, Wu L, et al. Characterization of a J Thorac Oncol 2011;6:2011–7. rituximab variant with potent antitumor activity against rituximab-resistant B- 7. Grabe T, Lategahn J, Rauh D. C797S resistance: the undruggable EGFR cell lymphoma. Blood 2009;114:5007–15. mutation in non-small cell lung cancer? ACS Med Chem Lett 2018;9: 20. Kinder M, Greenplate AR, Strohl WR, Jordan RE, Brezski RJ. An Fc engineering 779–82. approach that modulates antibody-dependent cytokine release without altering 8.ChiuSM,NeijssenJ,ParrenP,SchuurmanJ.JanssenBiotechInc, cell-killing functions. mAbs 2015;7:494–504. assignee. Bispecific EGFR/c-Met antibodies. United States US9593164 21. Graves DT, Jiang Y. Chemokines, a family of chemotactic cytokines. Crit Rev 2013: Example #10. Oral Biol Med 1995;6:109–18. 9. Moores SL, Chiu ML, Bushey BS, Chevalier K, Luistro L, Dorn K, et al. A novel 22. Uguccioni M, D'Apuzzo M, Loetscher M, Dewald B, Baggiolini M. Actions of the bispecific antibody targeting EGFR and cMet is effective against EGFR inhibitor- chemotactic cytokines MCP-1, MCP-2, MCP-3, RANTES, MIP-1 alpha and resistant lung tumors. Cancer Res 2016;76:3942–53. MIP-1 beta on human monocytes. Eur J Immunol 1995;25:64–8. 10. Cho BC, Lee JS, Han JY, Cho EK, Haura E, Lee KH, et al. JNJ-61186372 (JNJ-372), 23. Balkwill F. Cancer and the chemokine network. Nat Rev Cancer 2004;4:540–50. an EGFR-cMET bispecific antibody, in advanced non-small cell lung cancer 24. Zheng P, Luo Q, Wang W, Li J, Wang T, Wang P, et al. Tumor-associated (NSCLC): an update on phase 1 results. [poster 1497p]. Presented at the 43rd. macrophages-derived exosomes promote the migration of gastric cancer cells by Annual Congress of the European Society for Medical Oncology, 19–23 October transfer of functional apolipoprotein E. Cell Death Dis 2018;9:434. 2018, Munich, Germany. 25. Toth G, Szollosi J, Vereb G. Quantitating ADCC against adherent cells: imped- 11. Haura EB, Cho BC, Lee JS, Han JY, Lee KH, Sanborn RE, et al. JNJ-61186372 ance-based detection is superior to release, membrane permeability, or caspase (JNJ-372), an EGFR-cMet bispecific antibody, in EGFR-driven advanced activation assays in resolving antibody dose response. Cytometry A 2017;91: non-small cell lung cancer (NSCLC). J Clin Oncol 2019;37:9009. 1021–9. 12. Grugan KD, Dorn K, Jarantow SW, Bushey BS, Pardinas JR, Laquerre S, et al. Fc- 26. Varsano S, Rashkovsky L, Shapiro H, Ophir D, Mark-Bentankur T. Human lung mediated activity of EGFR x c-Met bispecific antibody JNJ-61186372 enhanced cancer cell lines express cell membrane complement inhibitory proteins and are killing of lung cancer cells. mAbs 2017;9:114–26. extremely resistant to complement-mediated lysis; a comparison with normal

AACRJournals.org Mol Cancer Ther; 19(10) October 2020 2055

Vijayaraghavan et al.

human respiratory epithelium in vitro, and an insight into mechanism(s) of 32. Passiglia F, Van Der Steen N, Raez L, Pauwels P, Gil-Bazo I, Santos E, et al. The resistance. Clin Exp Immunol 1998;113:173–82. role of cMet in non-small cell lung cancer resistant to EGFR-inhibitors: did we 27. Loetscher P, Seitz M, Clark-Lewis I, Baggiolini M, Moser B. Monocyte chemo- really find the target? Curr Drug Targets 2014;15:1284–92. tactic proteins MCP-1, MCP-2, and MCP-3 are major attractants for human 33. Sellmann C, Doerner A, Knuehl C, Rasche N, Sood V, Krah S, et al. Balancing CD4þ and CD8þ T lymphocytes. FASEB J 1994;8:1055–60. selectivity and efficacy of bispecific epidermal growth factor receptor (EGFR) x c- 28. Arenberg DA, Keane MP, DiGiovine B, Kunkel SL, Strom SR, Burdick MD, et al. MET antibodies and antibody-drug conjugates. J Biol Chem 2016;291:25106–19. Macrophage infiltration in human non-small-cell lung cancer: the role of CC 34. Mazor Y, Hansen A, Yang C, Chowdhury PS, Wang J, Stephens G, et al. Insights chemokines. Cancer Immunol Immunother 2000;49:63–70. into the molecular basis of a bispecific antibody's target selectivity. mAbs 2015;7: 29. Gerdes CA, Nicolini VG, Herter S, van Puijenbroek E, Lang S, Roemmele M, et al. 461–9. GA201 (RG7160): a novel, humanized, glycoengineered anti-EGFR antibody 35. Mazor Y, Oganesyan V, Yang C, Hansen A, Wang J, Liu H, et al. Improving target with enhanced ADCC and superior in vivo efficacy compared with cetuximab. cell specificity using a novel monovalent bispecific IgG design. mAbs 2015;7: Clin Cancer Res 2013;19:1126–38. 377–89. 30. Velmurugan R, Challa DK, Ram S, Ober RJ, Ward ES. Macrophage-mediated 36. ZhengS, Moores S, Jarantow S, Pardinas J,Chiu M,Zhou H, et al. Cross-armbinding trogocytosis leads to death of antibody-opsonized tumor cells. Mol Cancer Ther efficiency of an EGFR x c-Met bispecific antibody. mAbs 2016;8:551–61. 2016;15:1879–89. 37. Pham T, Mero P, Booth JW. Dynamics of macrophage trogocytosis of rituximab- 31. Lynch TJ, Patel T, Dreisbach L, McCleod M, Heim WJ, Hermann RC, et al. coated B cells. PLoS One 2011;6:e14498. Cetuximab and first-line taxane/carboplatin chemotherapy in advanced non- 38. Matlung HL, Babes L, Zhao XW, van Houdt M, Treffers LW, van Rees DJ, et al. small-cell lung cancer: results of the randomized multicenter phase III trial Neutrophils kill antibody-opsonized cancer cells by trogoptosis. Cell Rep 2018; BMS099. J Clin Oncol 2010;28:911–7. 23:3946–59.

2056 Mol Cancer Ther; 19(10) October 2020 MOLECULAR CANCER THERAPEUTICS

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

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

Mol Cancer Ther 2020;19:2044-2056. 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

Cited articles This article cites 36 articles, 10 of which you can access for free at: http://mct.aacrjournals.org/content/19/10/2044.full#ref-list-1

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at Subscriptions [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://mct.aacrjournals.org/content/19/10/2044. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.