Extended Data Fig. 2: Identification of ALK immunogenic peptides in mouse models.

(a, b) IFN-γ intracellular staining of CD4⁺-gated (a) and CD8⁺-gated (b) peripheral mononuclear cells (PBMCs) isolated from mice vaccinated with the indicated peptides and stimulated in vitro with the same peptide (%± SEM). Each dot represents a mouse (ALK_1058-1066, n = 3; remaining groups, N = 4 mice). (c) IFN-γ intracellular staining in CD4⁺ and CD8⁺ splenocytes isolated from a representative mouse (N = 3 independent mice) vaccinated with SLP7 and pulsed with 10 µg/mL of the same peptide. (d) Representative flow cytometric analysis of H-2K^d and H-2D^d expression on ASB-XIV and ASB-XIV^TAP2KO cells. Experiment performed three times. (e) H-2D^d and H-2K^d staining of ASB XIV^TAP2KO cells incubated with increasing concentrations of PGPGRVAKI peptide displayed as mean fluorescence intensity (MFI) ( ± SEM) (n = 3 independent experiments). (f) Schematic representation of the generation of the mEml4-Alk immortalized cell lines. (g) Sanger sequencing chromatogram showing the mElm4-Alk inversion. The mouse Eml4-Alk inversion involves exon 14 of Eml4 and exon 20 of mouse Alk. (h) Representative immunoblot analysis (N = 3 independent experiments with similar results) showing mEML4-ALK protein expression in two mEml4-Alk (mEml4-Alk¹ and mEml4-Alk²) immortalized cell lines. The K-Ras^G12D KP1233 cell line was used as a negative control; ALK SP8 antibody recognizes the murine EML4-ALK (arrow); ALK D5F3 antibody recognizes the human EML4-ALK in NCI-H3122. *Indicates a non-specific band recognized by the SP8 antibody. (i) Dose response curves of mElm4-Alk¹ cells to different ALK TKIs (crizotinib, N = 2 independent experiments; alectinib, N = 1; ceritinib, N = 1; brigatinib, N = 2 independent experiments; and lorlatinib, N = 2 independent experiments). (j) Representative immunoblot for the indicated proteins in mEml4-Alk¹ cells treated with crizotinib and lorlatinib at the indicated concentrations for 6 h (N = 2 independent experiments with similar results). (k) Subcutaneous tumor growth (mm³ ± SEM) of mEml4-Alk^-1 and mEml4-Alk^-2 immortalized cell lines in NSG and syngeneic BALB/c mice (N = 4 mice per group). (l) Quantification of volume changes compared with baseline tumor volume (change from baseline, % ± SEM) in syngeneic BALB/c mice injected subcutaneously with a mEml4-Alk cell line and treated as indicated. Red arrow indicates the end of treatment (N = 5 mice per group).

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