NVL-655 Is a Selective and Brain-Penetrant Inhibitor of Diverse ALK-Mutant Oncoproteins, Including Lorlatinib-Resistant Compound Mutations

Abstract

Three generations of tyrosine kinase inhibitors (TKI) have been approved for anaplastic lymphoma kinase (ALK) fusion-positive non-small cell lung cancer. However, none address the combined need for broad resistance coverage, brain activity, and avoidance of clinically dose-limiting TRK inhibition. NVL-655 is a rationally designed TKI with >50-fold selectivity for ALK over 96% of the kinome tested. In vitro, NVL-655 inhibits diverse ALK fusions, activating alterations, and resistance mutations, showing ≥100-fold improved potency against ALKG1202R single and compound mutations over approved ALK TKIs. In vivo, it induces regression across 12 tumor models, including intracranial and patient-derived xenografts. NVL-655 inhibits ALK over TRK with 22-fold to >874-fold selectivity. These preclinical findings are supported by three case studies from an ongoing first-in-human phase I/II trial of NVL-655 which demonstrate preliminary proof-of-concept clinical activity in heavily pretreated patients with ALK fusion-positive non-small cell lung cancer, including in patients with brain metastases and single or compound ALK resistance mutations. Significance: By combining broad activity against single and compound ALK resistance mutations, brain penetrance, and selectivity, NVL-655 addresses key limitations of currently approved ALK inhibitors and has the potential to represent a distinct advancement as a fourth-generation inhibitor for patients with ALK-driven cancers.

Figure 1.

Design and biochemical activity of NVL-655. A, Chemical structure of NVL-655. B, X-ray structure of NVL-655 (cyan) in the binding pocket of ALK^{G1202R/L1196M}, with key residues highlighted in yellow and hydrogen bonds in red. C, Classification of ALK TKIs into 1G, 2G, 3G, or next generation. D, Positioning of NVL-655 with respect to Leu1198 in the crystal structure of ALK^{G1202R/L1196M} (left, yellow; PDB: 9GBE) or Tyr619 in TRKB (right, orange; PDB: 4AT3) based on α-carbon superposition. Red disc indicates a steric clash based on van der Waals overlap. E, Activity of eight TKIs against ALK (gray) and ALK^{G1202R/L1196M} (blue) in biochemical assays. Geometric mean and SD are plotted, with numerical values of the means shown. “IC₅₀ >” is treated as “IC₅₀ =” for plotting. F, Biochemical activity of NVL-655 (blue) and lorlatinib (orange) against ALK with single amino acid substitution or insertion. Geometric mean and SD are plotted. Graph indicates relative potency to NVL-655. G, Kinome selectivity tree for NVL-655. Twelve kinases inhibited with IC₅₀ within 50-fold of ALK are indicated.

Figure 2.

NVL-655 inhibits the viability of ALK-driven cancer cell lines. A, Heat map showing the activity in cell viability assays against ALK fusion with the WT kinase domain. Karpas299 harbors the NPM1–ALK fusion, not EML4–ALK. B, Same as A but for ALK fusion with single amino acid mutation (substitution, insertion, or deletion). C, Plot showing the IC₅₀ for ALK WT (gray) vs. G1202R (blue) in Ba/F3 EML4–ALK v1, along with the associated fold change in IC₅₀. Red indicates an increase in IC₅₀, whereas green indicates a decrease in IC₅₀. D, Same as A but for ALK fusion with compound mutations. E, Same with C but for G1202R/L1196M. F, Same as A but for ALK nonfusion mutations. Potency values reflect geometric mean, and “IC₅₀ <” and “IC₅₀ >” are treated as “IC₅₀ =” for plotting and heat map coloring. Errors, repeats, and maximal effects (E_max) are provided in Supplementary Figs. S5 and S6. Grayed-out entries indicate data not determined (ND). Average potencies (blue) reflect the geometric mean across the cell lines within each category. Amp, amplification.

Figure 3.

Selectivity for ALK vs. TRK. A, Activity against TRKA, TRKB, and/or TRKC in three assay modalities (biochemical, cell viability, and cellular phosphorylation assays). Geometric mean and SD are plotted. “IC₅₀ >” and “IC₅₀ <” are treated as “IC₅₀ =” for plotting. B, Heat map showing the selectivity window calculated from cellular TRKB phosphorylation IC₅₀ (Supplementary Fig. S10A) divided by Ba/F3 EML4–ALK v1 cell viability IC₅₀ (Supplementary Fig. S4). “Selectivity <” and “selectivity >” are treated as “selectivity =” for heat map coloring. C, Graphical representation of the heat map in B. “Selectivity <” and “selectivity >” are treated as “selectivity =” for plotting. N.C., noncalculable.

Figure 4.

NVL-655 inhibits subcutaneous ALK-driven tumor xenografts in mice. A–D, Change in tumor volume over time plotted as mean ± SEM for models harboring ALK fusion with a WT kinase domain (A), G1202R single mutation (B), I1171N single mutation (C), or G1202R compound mutations (D). Horizontal gray lines denote mean starting tumor volume of the vehicle group. All treatments were administered orally twice daily, except alectinib 50 mg/kg and lorlatinib 10 mg/kg, which were administered orally once daily. Each group contained 4–9 mice.

Figure 5.

NVL-655 inhibits intracranial ALK-driven tumor xenografts in mice. A–D, NVL-655 inhibited tumor growth in the YU-1077 intracranial model. A, Superimposed MRI images from individual mice with brain tumor masses highlighted in cyan. B, Brain tumor volume over time based on MRI reconstruction, plotted as mean ± SEM. Horizontal gray line indicates initial tumor volume of the vehicle group. * indicates that six mice remained at the final treatment timepoint in the alectinib treatment group. C, Survival analysis. D–G, NVL-655 inhibited the Ba/F3 EML4–ALK v1 G1202R/L1196M luciferase intracranial model. D, Bioluminescence images indicating a change in tumor burden over 10 days of treatment. E, Brain bioluminescence over time plotted as mean ± SEM. Plotting for each treatment group stopped when the first animal was lost. Horizontal gray line indicates initial bioluminescence of the vehicle group. F, Body weight over time plotted as mean ± SEM. Horizontal gray line indicates initial body weight. G, Survival analysis. All treatment was administered orally twice daily, except alectinib 10 mg/kg, which was administered orally once daily.

Figure 6.

Preliminary clinical activity of NVL-655. A, Left, Representative CT images demonstrating a confirmed PR to NVL-655 in a patient with EML4–ALK v3 fusion–positive lung adenocarcinoma with ALK^{G1202R/F1174C} compound resistance mutation after prior alectinib, lorlatinib, and chemotherapy. The patient received NVL-655 at 150 mg once daily. Yellow circles indicate liver metastases (top) and mesenteric and retroperitoneal lymph nodes (bottom) that decreased in size over the course of treatment. Right, Analysis of ctDNA indicated clearance of ALK fusion and mutation alleles. Not detected is imputed as 0% for graphing. B, Left, Representative CT images demonstrating a confirmed PR to NVL-655 in a patient with EML4–ALK v3 fusion–positive lung adenocarcinoma with ALK^G1202R and ALK^G1269A resistance mutations after prior alectinib, brigatinib, lorlatinib, and chemotherapy. The patient received NVL-655 at 150 mg once daily. Yellow arrows indicate lung metastases that became barely appreciable. Right, Analysis of ctDNA indicated clearance of ALK fusion and mutation alleles. Not detected is imputed as 0% for graphing. C, Representative images of MRI demonstrating a confirmed CNS CR to NVL-655 in a patient with EML4–ALK v1 fusion–positive lung adenocarcinoma without baseline detectable resistance mutation after prior crizotinib, ceritinib, lorlatinib, and chemotherapy. The patient received NVL-655 at 50 mg once daily. Yellow arrows indicate multiple brain metastases at baseline which completely disappeared after 6 weeks of therapy.