Supplementary Figure S1. Amivantamab shows antitumor effect and suppresses the expression of EGFR Exon20ins mutant in Ba/F3 cells overexpressing the indicated EGFR Exon20ins mutations.

Amivantamab significantly inhibited cell growth and suppressed EGFR Exon20ins mutant compared to

IgG1 control. A, Viability of the Ba/F3 cells was determined via CellTiter-Glo. Amivantamab or IgG1 was

treated for 72 hours. Data are presented as averages ± SD of triplicate independent experiments. *P <

0.0001; Student's t-test. B, Ba/F3 cells overexpressing the indicated EGFR Exon20ins mutations were

treated with amivantamab for 72 hours at the indicated concentrations. Immunoblot analysis was

performed for EGFR expressions after amivantamab treatment. C, Immunoblotting analysis was

performed for EGFR expressions after amivantamab treatment for 72 hours. D, Immunoblotting analysis

was performed for EGFR, AKT, ERK, and S6 expression after osimertinib or gefitinib treatment for 72

hours at the indicated concentrations in Ba/F3 cells overexpressing EGFR D770delinsGY or

H773_V774insH mutations.

Supplementary Figure S2. Sanger sequencing data depicting EGFR Exon20ins mutations and TP53 mutation in PDC and PDO models

Sanger sequencing data depicting the mutations on EGFR gene in the indicated patient-derived cell lines

and patient-derived organoids. A, DFCI-127 (P772_H773insPNP), B, DFCI-58 (H773_V774insNPH), C, YU-1163 (S768_D770dup on EGFR and R280T on TP53), D, Pyrosequencing data depicting the R280T

mutation (96% of mutant allele frequency) on TP53 gene in YU-1163. E, Knockdown of TP53 (50 nM) for

48 hours in YU-1163 pretreated with amivantamab (1 mg/ml) for 72 hours. Representative images of

triplicate experiments. Sanger sequencing data depicting the mutations on EGFR gene in the indicated

patient-derived organoids. F, YUO-036 (A767_V769dup), G, YUO-029 (S768_D770dup).

Supplementary Figure S3. Amivantamab strongly promotes internalization of EGFR in PDC cells expressing EGFR Exon20ins mutation.

Amivantamab-induced redistribution of EGFR in YU-1163 PDC. Immunofluorescence staining for EGFR

(green) in a panel of YU-1163-treated with 0.1 mg/ml IgG1 control or 0.1 mg/ml amivantamab for 72 hours

Supplementary Figure S4. Amivantamab reduces tumor burden in Ba/F3 cells and PDCs with EGFR Exon20ins xenograft models

A-C, Antitumor effects of amivantamab in Ba/F3 cells overexpressing V769_D770insASV or D770_N771insSVD-bearing NOG mice (n = 5/group). Mice were treated with amivantamab or vehicle twice per week i.p. injections dosing with 30 mg/kg. Data represent the mean ± SEM (n = 5/group). *P < 0.0001 vs. vehicle control; B, Waterfall plots showing the best response as a percentage change in tumor volume taken on the last day of treatment in the xenograft mice. C, Tumor lysates of vehicle- or amivantamab-treated Ba/F3 cells xenograft mice were harvested and subjected to immunoblotting for p-EGFR (Y1068), EGFR, p-cMET, and cMET. D, In BALB/c nude mice bearing PDCs with EGFR Exon20ins mutation (S768_D770dup) xenograft and E, NOG mice bearing Ba/F3 cells overexpressing D770_N771insSVD xenograft were administered with 30 mg/kg amivantamab, vehicle twice per week i.p. injections, or 5 mg/kg poziotinib, Q.D. Data represent the mean ± SEM (n = 5/group). Mice-treated with 5 mg/kg poziotinib suddenly died before 6 days after drug treatment. F-H, Severe skin problems and body weight loss of BALB/c nude mice treated with poziotinib (5 and 10 mg/kg) compared to amivantamab (30 mg/kg). Representative photographs of BALB/c nude mice treated with vehicle, 30 mg/kg amivantamab, or 5 mg/kg or 10 mg/kg poziotinib for 7 days. The body weight in poziotinib-treated group was decreased compared to that of 30 mg/kg amivantamab.

Supplementary Figure S5. Amivantamab has a superior antitumor effect compared to cetuximab

A, Antitumor effects of amivantamab or cetuximab in YU-1163 tumor-bearing BALB/c nude mice. Mice were treated with vehicle, amivantamab, or cetuximab, twice per week, i.p. injections dosing with 30 mg/kg. Data represent the mean ± SEM (n = 5/group). *P < 0.0001 vs. cetuximab at the same dose. Mice were treated with combination treatment of 30 mg/kg amivantamab twice per week i.p. and 10 mg/kg anti-CD16/32 antibody once weeckly i.p. Data represent the mean ± SEM (n = 3/group). *P < 0.0001 vs. amivantamab plus anti-CD16/32 antibody treatment. B, Immunohistochemical staining for mF4/80 (macrophages) and mNKp46 (NK cells) of tumor sections in YU-1163-bearing BALB/c nude mice models following vehicle or 30 mg/kg amivantamab treatment.

Supplementary Figure S6. Amivantamab shows ADCC activity in a PDX model

Tumor volume in YHIM-1029 PDX-bearing BALB/c nude mice models treated with vehicle, 10 mg/kg

amivantamab twice per week i.p. or combination treatment of 10 mg/kg amivantamab twice per week i.p.

and 10 mg/kg anti-CD16/32 antibody once weeckly i.p. Data represent the mean ± SEM (n = 5/group). *P

< 0.0001 vs. Amivantamab plus anti-CD16/32 antibody treatment.