Brigatinib overcomes ALK resistance mechanisms preclinically Zhang et al Supplementary information

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1 Supplementary information The potent ALK inhibitor brigatinib (AP26113) overcomes mechanisms of resistance to first- and second-generation ALK inhibitors in preclinical models Sen Zhang, Rana Anjum, Rachel Squillace, Sara Nadworny, Tianjun Zhou, Jeff Keats, Yaoyu Ning, Scott D. Wardwell, David Miller, Youngchul Song, Lindsey Eichinger, Lauren Moran, Wei- Sheng Huang, Shuangying Liu, Dong Zou, Yihan Wang, Qurish Mohemmad, Hyun Gyung Jang, Emily Ye, Narayana Narasimhan, Frank Wang, Juan Miret, Xiaotian Zhu, Tim Clackson, David Dalgarno, William Shakespeare, and Victor M. Rivera ARIAD Pharmaceuticals, Inc, Cambridge, MA 1

2 Supplementary Methods: Supplementary information In vitro cellular assays related to insulin-like growth factor 1 receptor (IGF-1R), insulin receptor (INSR), and EGFR: To evaluate IGF-1R and INSR phosphorylation, HepG2 cells and H-4-II-E cells, respectively, were serum-starved for 18 h and then treated with increasing concentrations of compound for 30 min, followed by stimulation for 10 min with 10 nm IGF-1 and 100 nm insulin, respectively. Cells lysates were immunoblotted with p-igf-1r, p-insr, and actin antibodies (Cell Signaling Technology). To assess native EGFR phosphorylation, H358 (NSCLC) cells were serum-starved for 18 h, treated with increasing concentrations of inhibitor for 2 h, and then stimulated for 10 min with 50 ng/ml EGF. Cells lysates were subjected to an ELISA for p-egfr (Cell Signaling Technology). Crystallography: X-ray diffraction data were collected to 2.0 Å resolution at 100 K on a Rigaku rotating anode and R-AXIS IV++ imaging system. The data were indexed and scaled using the HKL2000 package (HKL Research, Inc.). The structure was determined by molecular replacement with CNX (Accelrys Inc.) and a search model of ALK (PDB code of 2XB7). The costructure was built in Quanta (Accelrys Inc.) and refined with CNX (Accelrys Inc.). After several cycles of refinement and model building on ALK protein, brigatinib was added and built into the electron density map. The final ALK-brigatinib co-structure was refined to 2 Å with an R-Value of and R-free of 0.269, respectively. Brigatinib is very well defined in the electron density map except the solubilizing group, i.e. the methyl piperazinyl piperidine, which is partially 2

3 disordered. In addition, the catalytic lysine (K1150) is also partially disordered despite its close proximity to the DMPO moiety of brigatinib. The ALK:brigatinib co-structure was compared to those of crizotinib, ceritinib, and alectinib. Structural superposition was carried out in PyMOL (distributed by Schrodinger Inc.) using the entire kinase domain protein molecules. The PDB codes used are 2XP2 for crizotinib, 4MKC for ceritinib and 3AOX for alectinib. Figures were prepared using the PyMOL software. Supplementary results: Activity of brigatinib against native ROS1 and clinically relevant resistance mutants: To explore the cellular activity of brigatinib against ROS1, the ability of brigatinib to inhibit viability of Ba/F3 cells dependent on expression of 4 different ROS1 fusion genes was assessed (Supplementary Figure 2A). The potent ROS1 inhibitor crizotinib (1) was also examined for comparison. Brigatinib potently inhibited viability of Ba/F3 cells expressing FIG-, CD74-, SDC4-, or EZR-ROS1 fusions (IC 50 s from 16 nm to 41 nm) with potency similar to that of crizotinib (IC 50 s 17 to 52 nm); similar results were seen in HCC78 cells, a NSCLC cell line containing an SLC34A2-ROS1 fusion (brigatinib and crizotinib GI 50 s 95 and 132 nm, respectively). In NSCLC and engineered Ba/F3 cells, inhibition of viability was associated with suppression of ROS1-dependent signaling (Supplementary Figure 3A). Introduction of a mutation at the gatekeeper residue (L2026M) of a CD74-ROS1 fusion had no effect on the potency of brigatinib, while crizotinib potency was reduced 5-fold (Supplementary Figure 2A). However, neither brigatinib nor crizotinib maintained substantial activity against CD74-ROS1 with a G2032R mutation shown to confer clinical resistance to crizotinib (2) (IC 50 s 1100 and 1600 nm, respectively). 3

4 The in vivo activity of brigatinib against ROS1 was assessed in Ba/F3 cells dependent on CD74-ROS1 expression, grown as subcutaneous tumors (Supplementary Figure 3B). Daily oral administration of brigatinib (10, 25, and 50 mg/kg) led to a dose-dependent inhibition of tumor growth, with a dose level of 25 mg/kg inducing tumor stasis and 50 mg/kg inducing tumor regression by >90%. In comparison, a 50 mg/kg daily dose of crizotinib induced tumor stasis, and 200 mg/kg induced tumor regression to the degree observed with 50 mg/kg brigatinib. Overall, in cellular and in vivo assays, brigatinib inhibits ROS1 with potency similar to that observed against ALK (Table 1; Figure 2; Supplementary Figure 2A; Supplementary Figure 3B). However, while brigatinib maintains substantial activity against L2026M mutant ROS1, it does not maintain activity against G2032R mutant ROS1. Activity of brigatinib against native FLT3 and clinically relevant resistance mutants: To explore the cellular activity of brigatinib against FLT3, the ability of brigatinib to inhibit viability of Ba/F3 cells dependent on activated FLT3 (FLT3-ITD), or 3 variants containing secondary mutations associated with clinical resistance to FLT3 inhibitors (3), was assessed (Supplementary Figure 2A). The potent FLT3 inhibitor ponatinib was also examined for comparison. Consistent with previous results (4), ponatinib potently inhibited both FLT3-ITD and a variant with a F691I mutation at the gatekeeper residue (IC 50 s <0.04 nm), but demonstrated somewhat reduced activity against an F691L gatekeeper mutant (IC nm) and greatly reduced activity against a D835Y activation-loop mutant (IC nm). Brigatinib inhibited FLT3-ITD with potency (IC nm) ~11-fold reduced compared with ALK. Interestingly, in contrast to ponatinib, F691L and D835Y mutations only led to a modest further reduction of brigatinib potency (IC 50 s nm), while an F691I mutation led to a strong reduction in potency (IC nm). Activity of brigatinib against native EGFR and clinically relevant mutants: 4

5 The cellular activity of brigatinib against native EGFR was examined by measuring its effect on ligand-stimulated phosphorylation of EGFR, compared to that of erlotinib (Supplementary Figure 2A). In H358 cells, a NSCLC cell line, brigatinib had no effect on EGFR phosphorylation (IC 50 >3000 nm) while erlotinib potently inhibited phosphorylation (IC nm). To explore the cellular activity of brigatinib against mutant variants of EGFR, the ability of brigatinib to inhibit viability of Ba/F3 cells dependent on their activity was assessed and compared to that of erlotinib (Supplementary Figure 2A). Cell lines examined contained EGFR with the most common activating mutations (dele746-a750 [Del] or L858R), or EGFR with both a common activating mutation and a T790M resistance mutation (Del/T790M or L858R/T790M). As expected, erlotinib potently inhibited EGFR Del and L858R (IC 50 s nm) but not variants containing T790M (IC 50 s 6688 to >10000 nm). Brigatinib inhibited EGFR Del and L858R with potencies (IC 50 s nm) 7-28-fold reduced compared to ALK (IC nm). The potencies of brigatinib against T790M containing variants (IC 50 s nm) were reduced somewhat further (by fold compared to ALK). Interestingly, there was an even greater disparity between the potency required to inhibit EGFR T790M variants compared to ALK based on analysis of IC 90 values, though this was not the case for EGFR Del and L858R (Supplementary Figure 2A). While brigatinib inhibited EGFR Del and L858R with potencies (IC 90 s nm) that were 8-32-fold reduced compared to ALK (IC nm), T790M containing variants (IC 90 s nm) were inhibited with potencies fold reduced compared to ALK. The different curve slopes are shown in Supplementary Figure 2B. Overall, in cellular assays, brigatinib maintains activity against EGFR Del, with potency only ~7-fold reduced compared to ALK, while exhibiting substantially reduced activity against EGFR L858R and variants with T790M (19-78-fold reduced potency compared to ALK depending on the assessment). Brigatinib does not inhibit native EGFR. 5

6 Activity of brigatinib against IGF-1R and INSR: The cellular activity of brigatinib against IGF-1R and INSR was examined by determining its effects on ligand-stimulated phosphorylation of these receptors in cellular assays. NVP- TAE684, an ALK inhibitor with structural similarity to brigatinib that also inhibits IGF-1R and INSR (5) (Supplementary Figure 4), was examined for comparison. In HepG2 cells, a human hepatocarcinoma cell line, brigatinib inhibited IGF-1-stimulated phosphorylation of IGF-1R (IC nm) with 5-fold reduced potency compared to NVP-TAE684 (IC nm). In H-4-II-E cells, a rat hepatoma cell line, brigatinib inhibited insulin-stimulated phosphorylation of INSR (IC nm) with 32-fold reduced potency compare to NVP-TAE684 (IC 50 of 293 nm). Overall, in cellular assays brigatinib inhibits IGF-1R with ~11-fold reduced potency compared to ALK and does not inhibit INSR (Table 1, Supplementary Figure 4). 6

7 Supplementary Table 1. Brigatinib in vitro activity (IC 50 s) in a kinase panel (N=289) Kinase a 10 nm Kinase a 250 nm Kinase a >250 nm brigatinib b IC 50 IC 50 IC 50 Additional kinases that were inhibited by <90% at 1 µm ALK (C1156Y) 0.6 RPS6kA2 13 PRKD2 285 ABL2 GRK4 PAK5 ALK 0.6 LTK 14 CSK 329 ADRBK1 GRK5 PAK6 FER 1.3 YES1 19 BRSK1 338 ADRBK2 GRK6 PASK ALK (F1174L) 1.4 RET (V804M) 22 FGFR3 358 AKT1 GRK7 PBK/TOPK FLT3 (D835Y) 1.5 CLK1 23 FMS 358 AKT2 GSK3a PDGFRa EGFR (L858R) 1.5 PTK2B 24 SIK2 466 AKT3 GSK3b PDGFRa (D842V) ALK (L1196M) 1.7 RPS6KA3 26 FGR 492 ARAF HIPK1 PDGFRa (T674I) ROS1 1.9 ERBB4/HER4 27 TAOK1 493 AXL HIPK2 PDGFRa (V561D) FLT3 2.1 RET (V804L) 27 ABL1 500 BMX HIPK3 PDGFRb FES 3.5 CAMK2D 29 LCK 512 BRAF HIPK4 PDK1 FAK/PTK2 3.9 EGFR (L858R,T790M) 29 PLK1 611 BRAF(V600E) IKBKB PIM1 PTK6 4.1 CHEK1 30 BTK 674 CAMK1D IRAK1 PIM2 TSSK1 4.4 RPS6KA1 30 VEGFR2 816 CAMK2A IRAK4 PKA ALK (G1202R) 4.9 FGFR1 (V561M) 41 MELK 895 CAMK4 ITK PKC epsilon CHEK2 (I157T) 5.6 ERBB2 42 PRKG1 >1000 CAMKIa JAK1 PKCbII CHEK2 6.5 RPS6KA6 42 STK4 >1000 CAMKIIb JAK3 PKCeta ALK (R1275Q) 6.6 INSRR 45 TEK >1000 CAMKK1 JNK1a1 PKCG NUAK1 47 NEK9 >1000 CDK1 (+Cyclin B) JNK2a2 PKCtheta CAMK2G 48 AURKB >1000 CDK2 (+Cyclin A) LIMK1 PKG1b LRRK2 51 AURKC >1000 CDK2 (+Cyclin E) LYNB PKN2 FRK 52 MERTK >1000 CDK3 (+Cyclin E) MAP2K1 PLK2 FLT4 58 EPHA1 >1000 CDK4 (+Cyclin D1) MAP2K2 PLK3 RET 65 EPHA7 >1000 CDK5 (+p25) MAP3K14 PRKCA EGFR 67 EPHB1 >1000 CDK5 (+p35) MAP3K5 PRKCB IGF-1R 73 NTRK2 >1000 CDK6 (+Cyclin D1) MAP3K7 PRKCD CAMKK2 82 CDK6 (+Cyclin D3) MAP3K8 PRKCI KIT (D816V) 83 CDK7 (+Cyclin H) MAP4K4 PRKCZ MNK1 T385D 88 CDK9 (+Cyclin K) MAPK1 PRKG2 MARK2 93 CDK9 (+Cyclin T1) MAPK10 PRKX PRKD3 95 CHUK MAPK11 RAF1 PHKg2 108 CK1d MAPK13 RIPK2 KIT (D816H) 111 CK1epsilon MAPK14 ROCK1 STK CK2a MAPK3 ROCK2 BRSK2 125 CK2a2 MAPKAPK2 RPS6KA4 MARK3 127 CLK3 MAPKAPK3 RPS6KA5 MARK1 127 CLK4 MAPKAPK5 RPS6KB2 FGFR1 128 CSK MATK SGK1 (d1-59, S422D) BLK 136 CSNK1A1 MET SGK2 TSSK2 138 CSNK1G1 MINK1 SGK3 AURKA 146 CSNK1G2 MNK2 SRPK1 JAK2 154 CSNK1G3 MRC alpha SRPK2 INSR 160 DAPK1 MRC beta SRPK3 CSK (T341M) 165 DAPK2 MST1R STK11 ABL1 (Q252H) 171 DAPK3 MTOR STK17A FGFR4 181 DCLK2 MUSK STK24 KIT (V560G) 195 DDR2 MYLK STK26 PRKD1 197 DMPK MYLK2 STK3 FYN 198 DYRK1A NEK1 STK33 HCK 198 DYRK1B NEK11 SYK FGFR2 (N549H) 203 DYRK2 NEK2 TAOK2 MAP3K9 218 DYRK3 NEK3 TAOK3 FGFR2 228 DYRK4 NEK4 TBK1 CLK2 240 EEF2K NEK6 TEC LYN 241 EPHA2 NEK7 TGFBR2 ABL1 (T315I) 242 EPHA3 NLK TTK EPHA4 NTRK1 TXK EPHA5 NTRK3 TYK2 EPHA8 P38a (T106M) TYRO3 EPHB2 P38g VRK1 EPHB3 P70S6K WEE1 EPHB4 PAK1 WNK2 ERBB2 PAK2 WNK3 FLT1 PAK3 ZAK/MLTK GCK/MAP4K2 PAK4 ZAP70 a The list of kinases and their mutants are arranged based on ascending brigatinib IC 50 values. b These kinases are listed alphabetically. 7

8 Supplementary Figure 1. Chemical structures of brigatinib, crizotinib, ceritinib, and alectinib. 8

9 A Dependent genetic IC 50 ± SD (nm) IC 90 ± SD (nm) Kinase alteration a Brigatinib Comparator Brigatinib ALK EML4-ALK v1 14 ± ± 11 Crizotinib 38 ± 10 FIG-ROS1 31±15 45 ± 21 SDC4-ROS1 16 ± 2 17 ± 1 ROS1 EZR-ROS1 41 ± 1 52 ± 4 CD74-ROS1 18 ± 3 25 ± 12 Crizotinib ND CD74-ROS1 (L2026M) 17 ± ± 44 CD74-ROS1 (G2032R) 1100 ± ± 339 FLT3-ITD 158 ± 42 <0.04 FLT3 FLT3-ITD (F691I) 1155 ± 190 <0.04 FLT3-ITD (F691L) 263 ± ± 14 Ponatinib ND FLT3-ITD (D835Y) 211 ± ± 160 EGFR ( Del) 95 ± ± ± 128 EGFR (L858R) 397 ± ± ± 182 EGFR EGFR ( Del/T790M) 272 ± ± 2461 Erlotinib 2461 ± 724 EGFR (L858R/T790M) 489 ± 138 > ± 635 EGFR b > c ND Fusion negative Parental line 3214 ± 725 NA ND a Potency assessed by viability assays using Ba/F3 cells whose survival was dependent on activity of the indicated fusion protein, except native EGFR. b Native EGFR inhibition was assessed by evaluation of p-egfr inhibition of H358 NSCLC cells. c N = 1; NA, not applicable; ND, not determined B Supplementary Figure 2. Brigatinib-mediated inhibition of native or mutant ALK-, ROS1-, FLT3-, and EGFR-driven tumor activity in vitro. (A) Brigatinib- or comparator-mediated inhibition of viability of cells harboring ALK, ROS, FLT3, or EGFR or relevant mutants. (B) In vitro viability of Ba/F3 cells harboring EML4-ALK, EGFR ( Del [D]), EGFR Del/T790M cells treated with increasing concentrations of brigatinib for 72 h. Brigatinib IC 50 (red dotted lines) and IC 90 (blue dotted lines) values corresponding to the three cell lines are depicted. 9

10 A Brigatinib HCC78 (SLC34A2-ROS1) (nm) pros1 ROS1 pakt perk1/2 Brigatinib 0 Ba/F3 (CD74-ROS1) pros1 ROS1 pakt perk1/2 Crizotinib pros1 ROS1 pakt perk1/2 Crizotinib pros1 ROS1 pakt perk1/2 Tumor Volume (mm 3 ) B Brigatinib Vehicle 10 mg/kg 25 mg/kg 50 mg/kg Days Post Treatment Initiation Tumor Volume (mm 3 ) Crizotinib Vehicle 50 mg/kg 100 mg/kg 200 mg/kg Days Post Treatment Initiation Supplementary Figure 3. Cellular and in vivo activity of brigatinib in ROS1 harboring models, compared with crizotinib. (A) Inhibition of ROS1 phosphorylation and downstream signaling in ROS1+ cellular models by brigatinib or crizotinib. SLC34A2-ROS1+ HCC78 cells and CD74 ROS1+ Ba/F3 cells were exposed to increasing concentrations (0-10,000 nm) of crizotinib or brigatinib for 1 hour, and cell lysates were immunoblotted to detect the indicated proteins. (B) TKI efficacy in CB-17/SCID mice bearing Ba/F3 cells expressing CD74-ROS1. Tumors were established by subcutaneous injection of CD74-ROS1 Ba/F3 tumor cells into the right flank of ~8-week old CB-17/SCID female mice. Brigatinib or crizotinib was administered orally as indicated for 14 days and mean tumor volume was assessed as described in the Methods; mean tumor volume ± standard error (SE) is shown for each group. 10

11 Supplementary Figure 4. Comparison of IGF-1R and INSR inhibitory activity of brigatinib with NVP-TAE684. TKI-mediated inhibition of IGF-1R and INSR phosphorylation in vitro. HepG2 cells (expressing IGF-1R) and H-4-II-E cells (expressing INSR) were serum-starved for 18 h and then treated with increasing concentrations of either brigatinib or NVP-TAE684 for 30 min, followed by stimulation for 10 minutes with 10 nm IGF-1 or 100 nm insulin, respectively. Cells lysates were immunoblotted to detect the indicated proteins. 11

12 Karpas-299 (ALCL) H3122 (NSCLC) Supplementary Figure 5. Inhibition of ALK phosphorylation in ALK+ cellular models by brigatinib, compared with crizotinib. In vitro cellular signaling activity was assessed in ALK+ cell lines (Karpas-299 [ALCL] and H3122 [NSCLC]) by treating with the indicated concentrations of each inhibitor (brigatinib or crizotinib) for 1 h, and analyzing levels of p-alk (Tyr1604), total ALK, and p-erk in lysates via immunoblotting. 12

13 13

14 Supplementary Figure 6. Inhibition of ALK phosphorylation and downstream signaling, and tolerability of brigatinib in vivo, compared with crizotinib. (A) Levels of phosphorylated ALK (p-alk) (mean ± SD) evaluated by ELISA in Karpas-299 tumors from SCID Beige female mice treated with the indicated doses of brigatinib or crizotinib, relative to tumors from mice treated with vehicle (n=3 mice/group). Plasma TKI levels are also shown (blue data points; mean ± SD). Mice bearing Karpas-299 tumors were administered TKI or vehicle once daily for 4 days. Tumors and plasma were collected 2, 10, and 24 h after the last dose of TKI; for the vehicle-treated animals, tumors were collected at 6 h post last dose. SD, standard deviation. (B) Inhibition of ALK phosphorylation and downstream signaling (at 6 and 24 h) in tumors from mice subcutaneously implanted with Karpas-299 cells and treated with a single dose of brigatinib (10, 25, or 50 mg/kg) or vehicle. Tumors were collected and lysed at indicated times and immunoblotted for target and downstream signaling proteins. (C) Comparison of drug exposure levels in CB-17/SCID female mice and in humans following treatment with brigatinib or crizotinib. (D) Change in bodyweight (mean ± SE) of mice implanted subcutaneously with Karpas-299 and treated with brigatinib or crizotinib at the indicated doses (see Figure 2C). 14

15 A B ALK mutant Crizotinib IC 50 ± SD (nm) TKI activity, IC 50 TKI activity, IC 90 Brigatinib IC 50 ± SD (nm) Ceritinib IC 50 ± SD (nm) Alectinib IC 50 ± SD (nm) Crizotinib IC 90 ± SD (nm) Brigatinib IC 90 ± SD (nm) Ceritinib IC 90 ± SD (nm) Alectinib IC 90 ± SD (nm) Native 107 ± ± 1 37 ± 8 25 ± ± ± ± ± 5 T1151Tins 1109 ± 453^ 114 ± ± ± ± 413^ 416 ± ± ± 130 L1152R 844 ± 100^ 11 ± ± ± ± 206^ 34 ± ± ± 59 L1152P 721 ± ± ± ± ± ± ± ± 14 C1156Y 529 ± 152^ 45 ± ± ± ± 266^ 129 ± ± ± 57 I1171N 532 ± 122^ 124 ± ± ± 130^ 1058 ± 258^ 278 ± ± ± 681^ F1174C 238 ± ± ± 70^ 31 ± ± ± ± 159^ 116 ± 55 F1174L 253 ± 90^ 55 ± ± ± ± 124^ 137 ± ± ± 8 F1174V 257 ± 101^ 64 ± ± 55^ 46 ± ± 282^ 195 ± ± 110^ 206 ± 164 V1180L 170 ± ± 3 16 ± ± ± ± ± ± 700 L1196M 589 ± 97^ 41 ± ± ± ± 138^ 118 ± ± ± 105 L1198F 17 ± 6 82 ± ± ± ± ± ± ± 85 G1202R 617 ± 76^ 184 ± ± 73^ 695 ± 260^ 1638 ± 236^ 762 ± 249 # 1042 ± 214^ 1886 ± 651^ D1203N 459 ± 176^ 79 ± ± ± ± 426^ 276 ± ± ± 114 S1206F 199 ± 61^ 43 ± ± ± ± 155^ 120 ± ± ± 86 S1206Y 179 ± 62^ 36 ± ± ± ± 127^ 109 ± ± ± 36 E1210K 240 ± ± ± ± ± ± ± ± 85 G1269A 509 ± 146^ 9 ± 5 29 ± ± ± 306^ 22 ± ± ± 66 Parental* 1237 ± ± ± ± ± ± ± ± 3033 *Untransformed Ba/F3 cells grown in the presence of IL-3. Red shading indicates that the IC 50 or IC 90 value exceeds the total steady-state plasma level in patients for that TKI (C ave ; see Figure 4A) ^Denotes ALK mutations that have been previously associated with clinical resistance (see Figure 4A). # The IC 90 value for G1202R exceeds the C ave in patients dosed with 90 mg, but not 180 mg, brigatinib (see Figure 4A). ALK Mutant IC 50 Fold Change from Parental Ba/F Native T1151Tins L1152R L1152P C1156Y I1171N 100 F1174C F1174L F1174V V1180L L1196M 10 L1198F G1202R D1203N S1206F S1206Y 1 E1210K G1269A Crizotinib Brigatinib Ceritinib Alectinib Supplementary Figure 7. TKI activity against parental, and native and mutant EML4-ALK driven Ba/F3 cells (A) IC 50 and IC 90 values (nm) of crizotinib, brigatinib, ceritinib, and alectinib 15

16 in Ba/F3 cells harboring native-eml4-alk or 17 mutant variants, and in ALK- (parental) cells presented in Figure 4B. Data for each cell line are derived from at least 4 independent experiments; error bars=sd. (B) Ratio of IC 50 values between ALK-negative (parental) and native or mutant ALK cell lines for each TKI. 16

17 Supplementary Figure 8. TKI antitumor activity against native and mutant EML4-ALK tumors, TKI plasma levels, and tolerability, in vivo. (A) Quantification of TKI-mediated antitumor activity in in vivo Ba/F3 tumors expressing L1196M-mutant, native, or G1202R-mutant ALK (see Figure 5A), and evaluation of levels of TKI in mouse plasma. Data shown for the native EML4-ALK efficacy study were derived from 2 separate studies: crizotinib and brigatinib were analyzed side-by-side in one study and ceritinib and alectinib in a second study. To allow comparison of the results, brigatinib (vehicle and 50 mg/kg qd) was also included in the second study. In this second study, the growth of tumors in mice treated with brigatinib vehicle was similar to that in mice treated with ceritinib and alectinib vehicles and the antitumor activity of brigatinib (89% tumor regression) was similar to that observed in the first study (95% tumor regression). (B) Change in bodyweight (mean ± SE) of CB-17/SCID mice implanted subcutaneously with G1202R-mutant ALK+ Ba/F3 cells and treated with the indicated TKIs at the specified doses (see Figure 5A). 17

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