IMMUNOTHERAPY TARGETING TUMOR NEOANTIGENIC PEPTIDES

Abstract

The present disclosure relates to a method for selecting a tumor neoantigenic peptide wherein said method comprises: a step of identifying, among mRNA sequences from cancer cells of a subject, a fusion transcript sequence comprising a transposable element (TE) sequence and an exonic sequence, and including an open reading frame (ORF), and a step of selecting a tumor neoantigenic peptide of at least 8 amino acids, encoded by a part of said ORF of the fusion transcript sequence, wherein said ORF overlaps the junction between the TE and the exonic sequence, is pure TE and/or is non-canonical, and wherein said tumor neoantigenic peptide binds to at least one Major Histocompatibility Complex (MHC) molecule of said subject. The present disclosure also relates to tumor neoantigenic peptide obtained according to the present method, vaccine or immunogenic composition, antibodies and immune cells derived thereof and their use in therapy of cancer.

Claims

1. A method for selecting a tumor neoantigenic peptide wherein said method comprises a step of identifying, among mRNA sequences from cancer cells of a subject, a fusion transcript sequence comprising a transposable element (TE) sequence and an exonic sequence, and including an open reading frame (ORF), and a step of selecting a tumor neoantigenic peptide of at least 8 amino acids, encoded by a part of said ORF of the fusion transcript sequence, wherein said ORF overlaps the junction between the TE and the exonic sequence, is pure TE and/or is non-canonical, and wherein said tumor neoantigenic peptide binds to at least one Major Histocompatibility Complex (MHC) molecule of said subject.

2. A method according to claim 1, wherein said tumor neoantigenic peptide is 8 to 11 amino acids long, and binds to at least one MHC class I molecule of said subject, or wherein said tumor neoantigenic peptide is from 13 to 25 amino acids long, and binds to at least one MHC class II molecule of said subject.

3. A tumor neoantigenic peptide comprising at least 8 amino acids, wherein said neoantigenic peptide is encoded by a part of an open reading frame (ORF) from a fusion transcript comprising a transposable element (TE) sequence and an exonic sequence, and wherein said ORF overlaps the junction between the TE and the exonic sequence, is pure TE and/or is non-canonical.

4. A tumor neoantigenic peptide, according to claim 3, wherein the fusion transcript is selected from SEQ ID NO:118-17492.

5. The tumor neoantigenic peptide of claim 3 comprising at least 8 amino acids of any of SEQ ID NO: 1-48, 50-117, and 17493-17496.

6. The isolated tumor neoantigenic peptide according to claim 3, wherein said neoantigenic peptide is expressed at higher levels in tumor cells compared to normal healthy cells; is expressed in at least 1% of subjects from a population of subjects suffering from cancer; and/or binds MHC class I or class II with a Kd binding affinity of less than about 10.sup.?5 M.

7. A population of autologous dendritic cells or antigen presenting cells that have been pulsed with one or more of the peptides of claim 3 or transfected with a polynucleotide encoding one or more of the peptides of claim 3.

8. A vaccine or immunogenic composition capable of raising a specific T-cell response comprising a. one or more neoantigenic peptides as defined in claim 3, optionally with a physiologically acceptable buffer, carrier, or excipient, and/or optionally with an adjuvant or immunostimulant; b. one or more polynucleotides encoding a neoantigenic peptide as defined in claim 3, optionally linked to a heterologous regulatory control nucleotide sequence; and/or c. a population of antigen presenting cells that have been pulsed with one or more of the peptides or transfected with a polynucleotide encoding one or more of the peptides of claim 3.

9. An antibody, or an antigen-binding fragment thereof, a T cell receptor (TCR), or a chimeric antigen receptor (CAR) that specifically binds a neoantigenic peptide as defined in claim 3, optionally in association with an MHC molecule, with a Kd affinity of about 10.sup.?6 M or less.

10. A method of producing an antibody, TCR or CAR that specifically binds a neoantigenic peptide as defined in claim 3, comprising the step of selecting an antibody, TCR or CAR that binds to a tumor neoantigen peptide of claim 3, optionally in association with an MHC or HLA molecule, with a Kd binding affinity of about 10.sup.?6 M or less.

11. An antibody, TCR or CAR produced by the method of claim 10, wherein said antibody is a multispecific antibody that further targets at least an immune cell antigen, optionally wherein the immune cell is a T cell, a NK cell or a dendritic cell, optionally wherein the targeted antigen is CD3, CD16, CD30 or a TCR, and/or wherein said TCR is made soluble and fused to an antibody fragment directed to a T cell antigen, optionally wherein the targeted antigen is CD3 or CD16.

12. A T cell receptor according to claim 9, wherein said T cell receptor is made soluble and fused to an antibody fragment directed to a T cell antigen, optionally wherein the targeted antigen is CD3 or CD16.

13. An antibody according to claim 9, wherein said antibody is a multispecific antibody that further targets at least an immune cell antigen, optionally wherein the immune cell is a T cell, a NK cell or a dendritic cell, optionally wherein the targeted antigen is CD3, CD16, CD30 or a TCR.

14. A polynucleotide encoding a neoantigenic peptide as defined in claim 3, or an antibody, a CAR or a TCR that specifically binds a neoantigenic peptide as defined in claim 3, optionally linked to a heterologous regulatory control sequence.

15. A vector comprising the polynucleotide of claim 14.

16. An immune cell that specifically binds to one or more neoantigenic peptides as defined in claim 3.

17. The immune cell of claim 16, which is an allogenic or autologous cell selected from the group consisting of: T cell, NK cell, CD4+/CD8+, TILs/tumor derived CD8 T cells, central memory CD8+ T cells, Treg, MAIT, and Y? T cell.

18. The immune cell according to claim 17, which is a T-cell comprising a T cell receptor or CAR that specifically binds one or more neoantigenic peptides of claim 3.

19. A method of using the neoantigenic peptide of claim 3, or a nucleic acid encoding the peptide of claim 3, for inhibiting cancer cell proliferation, or for cancer vaccination therapy of a subject, or for treating cancer in a subject.

20. A method of using an antibody, TCR or CAR that binds the neoantigenic peptide of claim 3 for inhibiting cancer cell proliferation, or for treatment of cancer in a subject in need thereof.

21. A method of using the immune cells of claim 16 for cell therapy of cancer.

22. The method of claim 19 comprising administering at least one further therapeutic agent.

23. The method of claim 19 wherein said at least one further therapeutic agent is a chemotherapeutic agent, or an immunotherapeutic agent, optionally a checkpoint inhibitor.

24. The method of claim 19 wherein the subject is suffering from NSCLC or is at risk of suffering from NSCLC.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0313] FIG. 1: Tumor neoantigenic peptides (or TE-derived epitopes) having a predicted affinity for MHC alleles of less than 500 nM, identified by the in silico method according to the disclosure in the tumor mouse lines B16F10-OVA cells (A) and in MCA101-OVA cells (B) and identified both in the two lines (C).

[0314] FIG. 2: (A) RT-PCR gels of amplification of the fusion transcript sequence encoding the neoantigenic peptide N25, in cDNA of tumor mouse lines B16F10-OVA and MCA101-OVA. (B) RT-PCR gels of amplification of the fusion transcript sequence encoding the neoantigenic peptide N26, in cDNA of tumor mouse lines B16F10, B16F10-OVA and MCA101-OVA.

[0315] FIG. 3: (A) Detection of peptide-reactive IFNg-secreting cells by ELISPOT in inguinal lymph nodes from immunized animals with DMSO (negative control), OVA (ovalbumine) (positive control), peptide N25 or peptide N26. (B) IFNg spots for 10?cells for immunized animals with DMSO (negative control), SIINFEKL (positive control), N25 or N26 peptide.

[0316] FIG. 4: (A) Evolution of the tumor volume (mm3) in mice beforehand immunized with DMSO, OVA or N25L peptide, following the days after the injection of tumor cells B16F10-OVA into said immunized mice. (B) Evolution of the tumor volume (mm3) in mice beforehand immunized with DMSO, OVA or N26L peptide, following the days after the injection of tumor cells B16F10-OVA into said immunized mice.

[0317] FIG. 5: TCGA data sets for 784 luminal, 100 HER2+, 197 TNBC, 112 normal breast tissue, 516 primary lung adenocarcinomas (primary tumor) and 59 normal lung tissue (solid tissue normal), were analyzed by the method for identifying fusion transcript sequence encoded tumor neoantigenic peptide described. (A) Number of fusion transcript sequence (TE-exon fusions) in different subtypes of breast cancer (HER2+, TNBC, normal breast tissue and luminal). (B) Number of fusion transcript sequence (TE-exon fusions) in different subtypes of lung cancer (primary lung adenocarcinomas, normal lung tissue).

[0318] FIG. 6: 8-9 amino acid-long peptides predicted from TE-gene fusion products from each sample were tested in silico for binding to the predicted HLA alleles expressed in the same sample. Shown are peptides with predicted affinity below 500 nM for at least one HLA-A, -B, or -C allele from each sample. (A) Samples of different subtypes of breast cancer (HER2+, TNBC, normal breast tissue and luminal). (B) Samples of different subtypes of lung cancer (non-small cell lung cancer, normal lung tissue).

[0319] FIG. 7: Distribution of tumor-specific peptides per patient across breast tumor subtypes. (A) Numbers of tumor-specific HLA-binding peptides per subtypes of breast cancer patient are shown. (B) Numbers of predicted tumor neoantigenic peptides shared across luminal subtypes samples (n=784) (abscissa). (C) Numbers of predicted tumor neoantigenic peptides shared across HER2+ subtypes samples (n=100) (abscissa). (D) Numbers of predicted tumor neoantigenic peptides shared across TNBC subtypes samples (n=197) (abscissa).

[0320] FIG. 8: (A) Numbers of tumor-specific HLA-binding peptides per primary lung adenocarcinomas (LUAD) sample (lung cancer). (B) Distribution of tumor-specific peptides per patient across lung adenocarcinomas. Numbers of predicted tumor neoantigenic peptides shared across primary tumor subtypes samples (n=516) (abscissa).

[0321] FIG. 9: Reconstruction of the fusion nucleotide sequence when the donor is the exon (A) and when the donor is the TE (B).

[0322] FIG. 10: Binding of chimeric transcripts-derived peptides to HLA-A2. Binding to HLA-A2 allele of predicted peptides from the most frequent chimeric fusions were validated by flow cytometry using tetramer formation assay. The results are shown as percentage of binding relative to positive control. Dotted line indicates the threshold considered to confirm the binding to this allele.

[0323] FIG. 11. Immunogenicity of fusion transcripts-derived peptides and reactive CD8+ T cells generation. (A) Percentage of tetramer positive CD8 T cells for the indicated fusion transcript-derived peptides induced by autologous moDCs in immunogenicity assays for the 6 healthy donors analyzed. (B) Cytokine secretion of CTL-clones after stimulation with different concentration of specific peptide. On the right is listed the CTL-clones generated and their peptide specificity. (C) Killing assay for CTL-clone 9 in co-culture with target cells loaded with 2 different peptide concentration in combination with anti-MHC-I antibodies or Isotype control (Left panel), or with un-loaded targets cells at different ratios (Right panel). (D) Killing assays for CTL-clone 9, 80 and 64 when co-cultured with peptide unloaded target cells in combination with anti-MHCI-I antibodies or isotype control. Effector:Target ratio is indicated in each individual plot. H1650 were used as target cells for each plot of this figure.

[0324] FIG. 12. Expression of TCR recognizing fusion-derived peptides. Transduced Jurkat-reporter cells with TCR sequence derived from CTL-clone 9 co-cultured with target cells alone, or loaded with 2 different peptide concentration. Plots show percentage of positive Jurkat cells for the 3 reporter genes evaluated by flow cytometry, using H1650 cell line as target cells (upper plots) or H1395 cell line as target cells (lower plots). Negative control: non-transduced Jurkat cells. No peptide: transduced Jurkat cells co-cultured with peptide unloaded target cells. Positive control: Transduced Jurkat cells stimulated with PMA/ionomycin.

[0325] FIG. 13. Tumor infiltrating lymphocytes recognizing fusion transcripts-derived peptides. Percentage of tetramer positive CD8 T cells for the indicated fusion transcript-derived peptides found in tumor infiltrating lymphocytes (TILs) expanded in the presence of fusion transcripts-derived peptide's mix+IL2 (A) or only with IL-2 (B).

[0326] FIG. 14. Phenotype of CD8+ T cells recognizing fusion transcripts-derived peptides in LUAD patient's derived samples. Percentage of tetramer positive CD8 T cells recognizing fusion transcripts-derived peptides present in tumor, juxta tumor, lymph nodes and blood samples derived from LUAD Patient 2 (A, upper panel) and Patient 3 (B, upper panel). In lower panel of figure (A) and (B) is shown the percentage of Na?ve (CCR7+CD45+), Central Memory (CM, CCR7+CD45?), Effector Memory (EM, CCR7?CD45?) and Terminal Effector (TE, CCR7?CD45+) cells of tetramer positive parental cell population.

[0327] FIG. 15. Immunopeptidomics analysis of lung tumor samples. Fusion transcript-derived peptide sequences were searched in public MHC-I immunopeptidomes datasets. Each column represents a different sample. Each row represents a different peptide sequence (specify on the right). Colored squares indicate in which sample is found each fusion transcript-derived peptide. Publications describing each sample data-sets are annotated on the top.

EXAMPLES

1. Example 1: Identification of Fusion Transcript Sequence Encoded Tumor Neoantigenic Peptide

1.1 Proof of Concept in Mice

[0328] To detect individual and shared tumor neoantigenic peptide issued from fusion transcripts sequences, a bioinformatics pipeline has been developed. This pipeline is designed to identify tumor-specific mRNA sequences composed in part of a TE sequence and in part of an exonic sequence. This pipeline implies determining the MHC alleles. For each human sample, the Class I and Class II MHC alleles can be determined using the seq2hla (v2.2) tool (bitbucket.org/sebastian_boegel/seq2hla). For mouse models, murine H-2 alleles are generally known. The bioinformatics method comprises the mapping of transcripts from RNA-sequencing against the reference genome. For the proof of concept analyses described here, mm10 was used for mouse and hg19 for human. Different versions of assembled genomes can be used for example hg19, hg38, mm9 or mm10. This mapping is carried out with STAR (v2.5.3a) (github.com/alexdobin/STAR), with the following setting: [0329] For allowing multi-hits mapping the parameter outFilterMultimapNmax which sets the maximum number of loci, the read is allowed to map to, is set at 1000, and [0330] For detecting the abnormal junction (fusion), the parameter chimSegmentMin which sets the minimum length of fusion segment, is set at 10, the parameter chimJunctionOverhangMin which sets the minimum overhang for a fusion junction is set at 10.

[0331] Normal (from SJ.out.tab output file) and abnormal (from Chimeric.out.junction output file) junctions are annotated using Ensembl and repeatmasker databases. Normal junctions define all the junctions that match the parameters used for the mapping (maximum intron length <=1 000 000 bp (set by alignIntronMax), same chromosome and well oriented) and abnormal ones are junctions that do not match with at least one of the previous criteria. This mean that a TE/Exon junction could be in both junction type but a Exon/Exon junction must be in normal file (SJ.out.tab). Transcript sequences comprising a junction between a TE sequence and an exonic sequence are extracted in silico. From the area of the transcript sequence which overlaps the junction, or downstream of the junction when out-of-frame (reading frame non-canonical), the software predicts, in all reading frames, all possible peptides of 8 or 9 mers. Then, the binding affinity of all these possible peptides for the MHC alleles previously defined for the matched sample is determined netMHCpan (v3.4) (cbs.dtu.dk/services/NetMHCpan/). There are currently more than a dozen various prediction algorithms for predicting the binding affinity of peptides, with NetMHC being the most widely used and validated algorithm for neoantigen prediction pipelines.

[0332] Peptides with either less than 500 nM or with a percentile rank less than 2% are considered as potential neo-antigens. Each splice site (donor or acceptor) is uniquely annotated as TE or as Exon. The part in the 5 end is qualified donor, and the part in the 3 is qualified acceptor.

[0333] Predicted HLA-binding peptides shared between cancer and normal tissues are excluded from further analyses.

[0334] This method has been applied to RNAseq data obtained from 7 well-characterized murine tumor cell lines (B16F10, B16F10-OVA, MCA101, MCA101-OVA, MC38, MC38-GFP, MC38-GFP-OVA). The cell lines with the extension-OVA corresponding to the same model but further expressing ovalbumin. In this study, this line is considered as the similar model, that is to say for example that an assay carried out on the cell line from B16F10-OVA is considered as a repeat of an assay carried out on the cell line from B16F10.

[0335] A list of candidate peptides has been obtained with these parameters (FIGS. 1A, 1B and 1C), some were specific to particular cell lines (FIGS. 1A and 1B), and some were shared between the two tumor cell lines (FIG. 1C).

[0336] For validation, we selected a range of peptides, expressed either in B16F10-OVA or MCA101-OVA, with predicted affinities less than 500 nM. Peptides were selected trying to optimize the ratio between number of reads and predicted affinity for MHC-I.

[0337] Four predicted tumor neoantigenic peptides were selected and characterized by identifying the TE and the exonic sequence (table 3).

TABLE-US-00002 TABLE 2 Characterization of 4 predicted tumor neoantigenic peptides selected by the method Peptide Cell line Donor Acceptor Predicted affinity N25 B16/B16-OVA ERV-MaLR Chmp3, exon2 H2-Db, 51.8937 (subfamily MTA) N26 MCA/MCA-OVA SINE-Alu(B1F) Angel2, exon2 H2-Kb, 392.0384 MC38-GFP/MC38-OVAGFP N90 MCA/MCA-OVA Predicted gene ERVL-MaLR H2-Kb, 403.8959 45873 (subfamily and 50.5416 ORR1A2-int) N94 MCA/MCA-OVA Rsrc1 ERV1 (subfamily H2-Kb, 431.0564 MC38-GFP/MC38-OVAGFP RLTR4_MM-int)

1.2 Validation by RT-PCR of the Fusion Transcript Sequence

[0338] First, a validation by regular RT-PCR has been performed, using primer pairs with one primer in the TE sequence, and the other one in the exonic sequence.

[0339] For the RNA extraction and reverse transcription, 3-5.10.sup.6 cells were lyzed in 500 ?L Trizol, and 100 ?L phenol-chloroform added to the lyzates prior centrifugation. Aqueous phase was collected, mixed in a 1:1 ratio with 100% EtOH and transferred to RNAeasy minikit columns. RNA was then collected following manufacturer's instructions (including on column DNAse treatment). After RNA elution, DNA contaminants were further removed by treatment with Turbo DNAse (Fisher scientific), according to manufacturer's instructions). RNA concentration was measured using a nanodrop, and 1 ?g of RNA used for reverse transcription. First strand synthesis was performed with Superscript III (Life technologies) using oligodT(15) as primers, according to manufacturer's instructions. Primers were ordered from Eurogentec. PCR reactions were performed using Taq polymerase. After identification of optimal conditions for each reaction, PCR products were extracted from agarose gels, and sequencing was performed using GATC lightrun. Sequence alignment was checked with APE software.

[0340] Using this approach, bands matching predicted size for N25, N26, N90 and N94 were detected, respectively in the cell lines identified in Table 1 (See FIG. 2A for N25). Interestingly, although N26 was detected only in MCA and MC38 cells in silico by RNAseq as previously described in the pipeline, using RT-PCR we detected a band corresponding to N26 in B16F10-OVA cells (FIG. 2B), indicating that this sequence is shared between three independent tumor cell lines (MCA, MC38 and B16F10). By re-analyzing the RNAseq data, we found that the N26 junction was present in B16F10-OVA cells, but below the detection threshold of the algorithm. Moreover, sequencing of the RT-PCR product showed exact match with sequences predicted by the algorithm.

1.3 In Vivo Immunization of Mice

[0341] To validate these candidates in vivo, short (9-mers) peptides corresponding to neoantigenic peptide which binds to the MHC class I sequences, were synthetized. For the in vivo assays, long (27-mers) peptides, which include the flanking regions to the predicted MHC-binding short peptides of 9 mers, were synthetized, because this length is better suited for in vivo immunization. B16F10 OVA and MCA101-OVA were maintained in RPMI, Glutamax, 10% FCS, 1% penicillin-streptomycin and passaged using TrypLE. Cells were kept in culture for a maximum of one month, and new vials were thawed for each in vivo experiment. C57BL6J recipient mice were immunized with 100 ?g long peptide (N25L or N26L), SIINFEKL peptide (short OVA peptide), OVA (Sigma) or DMSO, each with 50 ?g polyL:C, by subcutaneous injection into the flank. Immunizations were repeated 7 days after primary immunization. 3 days later (10 days after primary immunization), animals were sacrificed and numbers of peptide-specific IFNg-secreting CD8 T cells in inguinal lymph nodes were detected by ELISPOT (FIG. 3A). Short peptides (N25, N26, or SIINFEKL) or DMSO at 10 ?g.Math.mL.sup.?1 were used to restimulate T cells. Alternatively, 7 days after secondary immunization, animals were injected subcutaneously with 2.5.Math.10.sup.5 B16F10-OVA or 5.Math.10.sup.5 MCA-OVA cells in PBS. We found that N25, and to a lesser extent N26 were able to induce immune responses (FIG. 3B).

1.4 In Vivo Treatment of Mice with Tumor

[0342] To test whether these peptides were protective against tumor cells, we immunized C57BL6 mice with 100 mg peptides N25L or N26L, or OVA (control peptide) and 50 ?g polyL:C in PBS at d0 and d7, and at d14, we injected 2.5.10.sup.5 B16F10-OVA cells to mice immunized with OVA, N25L and N26L. B16F10 OVA and MCA101-OVA were maintained in RPMI, Glutamax, 10% FCS, 1% penicillin-streptomycin and passaged using TrypLE. Cells were kept in culture for a maximum of one month, and new vials were thawed for each in vivo experiment. C57BL6J recipient mice were immunized with 100 ?g long peptide (N25L or N26L), OVA (Sigma) or DMSO, each with 50 ?g polyL:C, by subcutaneous injection into the flank. Immunizations were repeated 7 days after primary immunization.

[0343] Short peptides (N25, N26, or SIINFEKL) or DMSO at 10 ?g.Math.mL.sup.?1 were used to restimulate T cells. Alternatively, 7 days after secondary immunization, animals were injected subcutaneously with 2.5.Math.10.sup.5 B16F10-OVA or 5.Math.10.sup.5 MCA-OVA cells in PBS. Tumor size was measured twice weekly using a manual caliper, and animal health status monitored throughout the experiment timeframe (FIGS. 4A and 4B). Animals were sacrificed when tumor volume reached 1 mm.sup.3. Strikingly, we observed that N25L significantly delayed the formation of B16OVA tumors, in a more efficient way than OVA. Moreover, we obtained a similar result upon N26L immunization.

2 Example 2: Identification of Human Lung Adenocarcinoma (LUAD) Neoantigenic Peptides Derived from Fusion Transcripts Composed of a TE Element and an Exonic Sequence

2.1 Material and Methods

[0344] RNA extraction. Tumour and juxtatumour samples were cut into pieces of #1 mm.sup.3 and resuspended in 700 ?l RTL lysis buffer (Quiagen) supplemented with 1% ?-mercaptoethanol and homogenized using Perecellys 24 Tissue Homogenizer (Bertin Technologies). Total RNA isolation was performed using RNeasy Micro Kit (Qiagen) following manufacturer instructions. Total RNA from tumour cell lines were extracted from 5.Math.10.sup.6 tumor cell lines using the same procedure.

[0345] PCR and Sequencing. Primers were designed using APE software. For each sample, 1 ?g of RNA was retrotranscribed into cDNA using SuperScript III Reverse transcriptase (ThermoFisher), as indicated by the provider. PCR reaction was performed using GoTaq G2 Hot Start Polymarase (Promega). All primers were used in a concentration of 0.5 ?M. Reactions were carried out in Veriti? 96-Well Thermal Cycler (ThermoFisher). PCR products were loaded in LabChip GX (Caliper LifeSciences) and analysed by LabChip GX Software (v4.2).

[0346] PCR reactions were repeated for those samples with an amplification product on the expected size. Then, the PCR products were run in a 2% agarose gel SYBR Free Dye (1/10000) (Invitrogen). The specific bands were cut and the DNA products were purified using QIAquick Gel Extraction Kit (Qiagen) following manufacturer instructions. Finally, these products were sequenced by EuroFins Scientific. The resulting sequences were compared to the expected one using Serial Cloner software.

[0347] Tetramer formation. HLA-A2 monomers were purchased from ImmunAware? and the formation of tetramers was evaluated with synthetic ER-derived peptides following manufacturer instructions. Briefly, synthetic HLA-A2 monomers were incubated with synthetic peptides during 48 h at 18? C. Tetramerization was done by further incubation of monomers with biotinylated-sepharose. Finally, tetramer formation was measured by flow cytometry using a PE-conjugated anti-?2-microglobulin antibody. As a positive control we used a peptide derived from CMV provided by the manufacturer.

[0348] In experiments addressed to evaluate the presence of specific CD8+ T cells, the tetramerization step was performed by incubating the monomers with different combinations of fluorescent streptavidin (PE, APC, PE-Cy5, PE-CF594, BV421, BV711 and FITC).

[0349] Priming of na?ve CTLs. PBMCs were obtained by Ficoll gradient separation from HLA-A2+ healthy blood donors. CD14+, CD4+ and CD8+ cells were purified by positive selection using magnetic beads (Miltenyi Biotec). While CD4+ and CD8+ T cells were cryopreserved until the experiment day, CD14+ fraction was cultured in the presence of IL-4 (50 ng/mL) and GM-CSF (10 ng/mL) at 106 cells/mL during 5 days to obtain moDCs. After this period of time, the moDCs were maturated with LPS and incubated with synthetic ER-derived peptides at a final concentration of 1 ?g/mL for 2 hours. Finally, peptide-loaded moDCs were co-cultured with autologous CD4+ and CD8+ T cells in culture medium supplemented with with IL-2 (10 U/ml) and IL-7 (100 ng/ml). The ER-derived peptide stimulation of specific CD8+ CTL populations was assessed by MHC-I tetramer staining by flow cytometry using a combination of two-color tetramer for each peptide.

[0350] Tetramer Staining. Cells were resuspended in PBS, stained with Live/Dead Aqua-405 nm (ThermoFisher) during 20 minutes at 4? C. and washed once. After that, cells were resuspended in PBS-1% BSA containing the mix of SA-coupled tetramers and incubated in the dark at room temperature during 20 minutes. Without further washing, surface antibodies were added in PBS-1% BSA and cells were incubated 20 minutes in the dark at 4? C. Surface antibodies were a combination of anti?CD3-BV650+ anti?CD8-PECy7 in combination with anti-CCR7-AF700+ anti?CD45RA-BUV395 when required. Finally, cells were washed twice and resuspended in FACS buffer for flow cytometry analysis.

[0351] CTL-clones generation. Tetramer positive cells were single-cell FACS sorted (ARIA-sorter, BD) in U bottom 96-well plates. Sorted cells were collected in 100 ?l of RPMI 10% human serum AB (Sigma-Aldrich) containing 150.000 feeders' cells. Finally, 100 ?l of AIM-medium containing IL-2 (3000 IU/ml) and anti?CD3 (100 ?g/ml, OKT3 clone from Miltenyi) were added and cells were cultured during 15-20 days maximum. When evident cell growth was observed in wells, we perform a second round of expansions with new feeders' cells for an additional period of 15 days maximum. Cells were feed and split as necessary during this period with the same culture media (AIM-RPMI 50/50+5% Human Serum) but only containing IL-2 at 500 IU/ml. Finally, expanded clones were checked for their specificity by FACs-tetramer staining and only clones with >85% of tetramer positive clones were used for further analysis.

[0352] Killing assays. To perform killing assays, xCELLigence RTCA S16 Real Time Cell Analyzer was used. H1650 cell-line were plated at 0.5?10.sup.6 cells/ml in pre-coated 16 well plates. One day after, cells were incubated or not during 1 h with different concentration of the correspondent synthetic peptides. After that, cells were washed twice with culture medium and incubated or not for additional 30 minutes with anti-MHC-I antibodies (clone W6/32, 50 ?g/well) or isotype control at the same concentration. Without additional wash, CTL-clones were added at the correspondent ratio. The complete assay was done in free-serum culture medium in a final volume of 200 at 37? C. connected to the xCELLigence system. Impedance variation (cell-index) was measured in real-time during 40 h. Each condition was performed by duplicates.

[0353] Cytokine secretion and Jurkat cells activation. 50.000 H1650 cells were plated in 96-well plate in culture medium supplemented with 5% of fetal bovine serum. The day after, cells were culture during 1-2 h with synthetic peptides at different final concentrations. After that, cells were washed twice, CTL-clones were added at 1:1 ratio and co-cultured during 18 h with peptide-loaded target cells. Culture supernatants were collected and cytokine concentration analyzed by cytokine beads arrays (CBA, BD Biosciences) following manufacturer's instructions.

[0354] The same experiment was performed using transduced Jurkat cells instead of CTL-clones and two different types of target cells: H1650 and H1395 cell lines. In this assay, after co-cultured with peptide-loaded target cells, Jurkat cells were assessed by flow cytometry analyzing the expression of reporter markers. PMA/Ionomycin was used as positive control to activate Jurkat cells.

[0355] Tissues and Blood samples. Lung tumor, juxta tumor and lymoh nodes samples were cut into small pieces and digested using a mix of collagenase-I (2 mg/ml), hyaluronidase (2 mg/ml) and DNasa (25 ?g/ml) in a final volume of 2 ml culture medium (CO.sub.2 independent medium+5) during 40 min at 37? C. After digestion single cell suspensions were collected through a cell Strainer and washed. Tumor and Juxta tumor suspensions were enriched on lymphocyte fractions by a ficoll gradient. After that cells were staining for tetramer analysis by FACs as described before.

[0356] Blood samples were seeded on a ficoll gradient and PBMCs were isolated. After that, PBMCs were enriched for CD8+ T cells using EasyStep Human CD8+ T cell Enrichment Kit (STEMCELL Technologies). Finally, enriched cells were stained for tetramer analysis as described before.

[0357] Tumor infiltrating lymphocytes (TILs) cultures. Tumor tissue was cut into small pieces (1-3 mm.sup.3 size, 6-12 pieces maximum). Each tumor fragment was transferred into individual wells from 24-well plates and cultured in a final volume of 2 ml RPMI 10% Human Serum+IL-2 6000 IU/ml. Cells were feed/split as necessary during 15-20 days and cryopreserve or analyzed for tetramer staining.

[0358] TCR cloning. Total RNA was extracted from CTL-clones and retrotranscribed into cDNA using SuperScript III (ThermoFisher). TCR? and ? were amplified by PCR as described in Li et al 2019. DNA products were run in 2% agarose gels and sequenced after gel band extraction (Qiagen). TCR V regions (? and ?) were concatenated with murine TCR constant chain and cloned into a PEW-pEF1A-inactEGFP vector and amplified in transformed bacteria.

[0359] Jurkat transduction. Lentivirus particles were produced by HEK-293FT cell line transfected with TCR-expression plasmids together with envelope (pVSVG) and packaging (psPAX2) plasmids. After 64 h, supernatant was collected and lentivirus particles were concentrated using 100 kDa centrifugal filter (Sigma-Aldrich). Lentivirus suspension was transferred by spinoculation into TCR-negative Jurkat cells expressing reporter genes (NFAT-GPF, NF-KB-CFP and AP-1-mCherry). After 5 days, transduction efficiency was evaluated by FACS using anti-murine TCR-? antibody (Clone H57-597). This Jurkat cells were described in Rosskopf S. et al. 2018.

[0360] Mass spectrometry data analysis. Public immunopeptidomics raw data derived from MHC-eluted peptides were analysed using ProteomeDiscoverer 1.4 (ThermoFisher) with the following parameters: no-enzyme, peptide length 8-15 aa, precursor mass tolerance 20 ppm and fragment mass tolerance 0.02 Da. Methionine was enabled as variable modification and a false discovery rate (FDR) of 1% was applied. MS/MS spectra were searched against the human proteome from Uniprot/SwissProt (updated 6 Mar. 2020) concatenated with the list of all fusion transcripts-derived proteins from lung TCGA projects. Finally, peptides matching with Uniprot database or with translated fusion transcripts present in lung normal samples were discarded.

2.2 Results: Identification of Fusion Transcript Sequences Encoding Tumor Neoantigenic Peptide in Human Subject

2.2.1 Characterization of Neoantigens

[0361] First the TE-Exon fusion transcript landscape was characterized in normal samples from TCGA public database. A total of 8876 unique fusions were identified in 679 normal samples from 19 different tissues (bile duct, bladder, brain, breast, cervical, colon, head and neck, kidneys, liver, pancreas, PCPG, prostate, rectum, sarcoma, skin, thymus, thyroid, uterine). Specific fusions to each tissue type were found with a very small portion of pan-tissue fusion transcripts. These results suggest that a dedicated tissue specific regulatory mechanism is associated with these fusion transcripts.

[0362] Then the number of identified fusions in 514 LUAD samples from TCGA has been compared to their 59 normal associated pulmonary samples present in TCGA. On average, 235 fusions were identified in NSCLC samples, compared with 200 in healthy lung samples (Wilcoxon pvalue=9?10..sup.?10). 8269 total unique fusions were identified in NSCLC tumors.

[0363] A first category of fusions called TSF (tumor specific fusion) was obtained as those found in at least 1% of tumor samples and in none of the normal samples. 210 fusions were thus defined as TSF.

[0364] Some high-frequency fusion transcripts in tumors and low frequency in normal cells may also be good candidates for neo-antigens. Thus, a second category called TAF (tumor associated fusion) was notably defined as fusions present in less than 4% of normal tissues, notably less than 2%, and more than 10% of the tumors and that is over expressed in tumors compared to normal tissue samples.

[0365] Tables 3 and 4 (see below) describe the fusion according to whether the Exon or the TE is the donor. The first column indicates the frequency of the fusion in the NSCLC cohort. The columns Donor and Acceptor introduce the type of each element. All the columns starting with Donor (respectively Acceptor) are information relative to the donor (resp. the acceptor). The sequence of the fusion can be retrieved as follow: [0366] Donor sequence: on chromosome Donor_Chromosome_X starting from Donor_start_X to Donor_Breakpoint_X on strand Donor_strand_X [0367] Acceptor sequence: on chromosome Acceptor_Chromosome_X starting from Acceptor_Breakpoint_X to Acceptor_end_X on strand Donor_strand_X
Care should be taken to take the reverse complement of the sequence if the fusion is present on the minus strand.

[0368] Fusion Sequence: [0369] In order to reconstruct the fusion nucleotide sequence, the sequence of the donor on chromosome Donor_Chromosome_X from Donor_start_X to Donor_Breakpoint_X on strand Donor_strand_X and the acceptor sequence on the chromosome Acceptor_Chromosome_X starting from Acceptor_Breakpoint_X to Acceptor_end_X on the strand Acceptor_strand_X have been extracted from the Ensembl HG19 human assembly database. It is to be noted that the use of the Ensembl HG19 human database is not limitative and that any other adapted database may be used such as NCBI reference Sequence Database (RefSeq). [0370] Care should be taken to take the reverse complement of the sequence if the fusion is present on the minus strand. [0371] The fusion sequence consists of the donor sequence followed by the acceptor sequence.

[0372] Nucleotide Sequence of the Fusion Transcript:

[0373] On the basis of the known canonical transcripts in which the exon is involved, all the fusion transcripts were reconstructed.

[0374] When the donor is the exon (see FIG. 9A) [0375] it starts with the beginning of the canonical transcript to the donor exon and replace the complete canonical exon sequence with the fusion sequence. In this case, the fusion transcript stops after the TE sequence of the acceptor.

[0376] When the donor is the TE (FIG. 9 B) [0377] The sequence begins at the canonical position of the acceptor exon in the transcript and forget all exons upstream. The canonical sequence of the acceptor exon was replaced with the fusion sequence and the transcript was reconstructed until the end.

[0378] Each nucleotide sequence of size k (i.e. from 24 to 75 nucleotides) of the fusion transcript (translation of the first k-mer starts at the first nucleotide of the fusion transcript, translation of the second k-mer starts at the second nucleotide of the fusion transcript, etc.) was then translated into a peptide sequence.

[0379] The obtained peptides are then further analyzed with NetMHCpan for MHC binding prediction. Affinity for binding to at least one of the known human alleles was thus predicted, (see also example 1 for further illustration) for each k-mer present in the sequence.

TABLE-US-00003 TABLE 3 Coordinates of the fusion sequences for which the donor is the exon. The names of the columns are the following: 1. Frequency in LUAD cohort 2. Donor Chromosome Exon 3. Donor start Exon 4. Donor Breakpoint Exon 5. Donor strand Exon 6. Donor transcript (i.e. Donor_tx_name_Exon) 7. Acceptor Chromosome TE 8. Acceptor Breakpoint TE 9. Acceptor end TE 10. Acceptor strand TE 11. Fusion type The fusion transcript sequence of table 3 correspond in the same order to SEQ ID NO: 118- 431 (typically line 1 is SEQ ID NO:118, line 2 is SEQ ID NO: 119, line 3 corresponds to SEQ ID NO: 120 and 121, because of the 2 donor transcripts (ENST00000296474 and ENST00000344206 respectively as indicated in col. 6), etc.). 1 2 3 4 5 6 7 8 9 10 11 31% chr19 42265157 42265435 + ENST00000199764 chr19 42274707 42275202 + TSA 17% chr12 71509630 71509630 ? ENST00000549357 chr12 71504233 71504376 ? TSA 15% chr3 49927357 49927357 ? ENST00000296474; chr3 49927248 49927342 ? TSA ENST00000344206 12% chr5 82554349 82554496 + ENST00000282268; chr5 82606608 82606935 + TSA ENST00000338635; ENST00000396027; ENST00000511817 10% chr3 98600384 98600384 ? ENST00000449482; chr3 98586282 98586295 ? TSF ENST00000326840; ENST00000326857 8% chrX 100169327 100169610 ? ENST00000328526; chrX 100143801 100143850 ? TSF ENST00000372956 7% chrX 100177782 100177782 ? ENST00000328526; chrX 100143801 100143850 ? TSF ENST00000372956 6% chr1 225156461 225156576 + ENST00000430092; chr1 225157336 225158402 + TSF ENST00000400952; ENST00000366849; ENST00000439375 6% chr8 63502273 63502353 + ENST00000523211; chr8 63546747 63547118 + TSF ENST00000328472 4% chr4 57319769 57319927 + ENST00000514888; chr4 57321183 57321327 + TSF ENST00000264221; ENST00000505164; ENST00000399688; ENST00000512576 4% chr12 113623819 113623826 + ENST00000552495 chr12 113623998 113624117 + TSF 4% chr3 32280465 32280611 + ENST00000458535; chr3 32324083 32324625 + TSF ENST00000307526 4% chr19 3868963 3868963 ? ENST00000586578; chr19 3855694 3856396 ? TSF ENST00000262961; ENST00000438164; ENST00000587212; ENST00000439086 4% chr4 56230241 56230438 + ENST00000264228 chr4 56252510 56252750 + TSF 3% chr10 126205749 126205840 + ENST00000368842 chr10 126251911 126252288 + TSF 3% chr1 241803184 241803184 ? ENST00000366554; chr1 241771682 241771840 ? TSF ENST00000331838 3% chr12 56742313 56742313 ? ENST00000314128 chr12 56740986 56741274 ? TSF ENST00000557235 3% chr1 234546191 234546191 ? ENST00000040877 chr1 234545319 234545408 ? TSF 3% chr7 81964451 81964451 ? ENST00000356860; chr7 81929467 81929664 ? TSF ENST00000356253; ENST00000423588 3% chr2 89160398 89160398 ? ENST00000390239 chr2 89129384 89129429 ? TSF 3% chr5 54993674 54993674 ? ENST00000396865; chr5 54993040 54993109 ? TSF ENST00000539768; ENST00000318672; ENST00000508124; ENST00000511233; ENST00000503891; ENST00000513993; ENST00000505563; ENST00000506624; ENST00000507109 3% chrX 119708406 119708406 ? ENST00000404115 chrX 119705855 119706010 ? TSF 3% chr2 135223685 135223685 ? ENST00000281924 chr2 135216236 135216307 ? TSF 3% chr11 20981978 20982106 + ENST00000298925; chr11 21041136 21041271 + TSF ENST00000357134; ENST00000325319; ENST00000532434 2% chr13 53307354 53307354 ? ENST00000431550; chr13 53304269 53304818 ? TSF ENST00000448904; ENST00000377962 2% chr3 138289160 138289160 ? ENST00000264982; chr3 138261631 138262493 ? TSF ENST00000542237; ENST00000484888; ENST00000474781; ENST00000481834; ENST00000468900; ENST00000462419; ENST00000464035 2% chr1 1255836 1255836 ? ENST00000435064; chr1 1255085 1255253 ? TSF ENST00000540437; ENST00000450926; ENST00000545578; ENST00000528879; ENST00000434694; ENST00000526797; ENST00000527719; ENST00000530031; ENST00000534345; ENST00000498476 2% chr2 135470770 135470770 ? ENST00000281924 chr2 135443800 135443808 ? TSF 2% chr5 23976106 23976159 + ENST00000512559; chr5 24177946 24178380 + TSF ENST00000507936 2% chr9 125054028 125054119 + ENST00000297908; chr9 125068067 125068171 + TSF ENST00000344641; ENST00000373723; ENST00000373729; ENST00000394315 2% chrX 117900807 117900939 + ENST00000371666 chrX 117902549 117902902 + TSF 2% chr5 31493314 31493314 ? ENST00000511367; chr5 31489188 31489272 ? TSF ENST00000344624; ENST00000442743; ENST00000513349 2% chr2 89416833 89416936 ? ENST00000490686 chr2 89370042 89370075 ? TSF 2% chr20 29632611 29632721 + ENST00000278882; chr20 29652086 29652324 + TSF ENST00000358464 2% chr14 66096210; 66096324 + ENST00000360689; chr14 66099743 66101298 + TSF 66096217 ENST00000394586; ENST00000342677; ENST00000394585; ENST00000358307; ENST00000557164 2% chr9 130678687 130678687 ? ENST00000335791 chr9 130678527 130678564 ? TSF 2% chr1 180283827 180283827 ? ENST00000367595 chr1 180281457 180281879 ? TSF 2% chr20 44333136 44333136 ? ENST00000335769 chr20 44322980 44322991 ? TSF 2% chr2 89512908 89512947 ? ENST00000498435 chr2 89389297 89389324 ? TSF 2% chr8 62546242 62546242 ? ENST00000541428; chr8 62544521 62544571 ? TSF ENST00000379454; ENST00000522919; ENST00000356457; ENST00000519234; ENST00000518068; ENST00000517903; ENST00000445642; ENST00000517847; ENST00000522835 2% chr4 1102131 1102131 ? ENST00000382968; chr4 1101132 1101138 ? TSF ENST00000433731; ENST00000511620; ENST00000510715; ENST00000333673 2% chr6 24551662 24551662 ? ENST00000430948; chr6 24548828 24548834 ? TSF ENST00000535378; ENST00000378214; ENST00000543707 2% chr1 11115838 11115838 ? ENST00000490101; chr1 11115464 11115465 ? TSF ENST00000376957 2% chr7 55270210 55270401 + ENST00000455089 chr7 55272949 55272949 + TSF 2% chr8 42924698; 42924802 + ENST00000534420; chr8 42925245 42925476 + TSF 42924750 ENST00000302279; ENST00000533998; ENST00000342116; ENST00000531266; ENST00000533336; ENST00000525699; ENST00000529687 2% chr2 176860281; 176860286 ? ENST00000392540; chr2 176859008 176859011 ? TSF 176860286 ENST00000272748; ENST00000544803; ENST00000445472 2% chr1 63955754 63955754 ? ENST00000371092; chr1 63952444 63952998 ? TSF ENST00000271002; ENST00000489099; ENST00000283568 2% chr16 2825452 2825452 ? ENST00000262306; chr16 2823822 2823948 ? TSF ENST00000409906; ENST00000409477; ENST00000494946 2% chr7 16900083; 16900124 ? ENST00000402239; chr7 16894536 16894625 ? TSF 16900124 ENST00000310398; ENST00000414935 2% chr17 79532346; 79532531 ? ENST00000374747; chr17 79527702 79527706 ? TSF 79532531 ENST00000539314; ENST00000572760; ENST00000573876; ENST00000331134; ENST00000573519; ENST00000571714; ENST00000572824; ENST00000573212 2% chr19 55178145; 55178200 + ENST00000391733 chr19 55178294 55178458 + TSF 55178148 ENST00000391736; ENST00000270452; ENST00000430952; ENST00000391734; ENST00000434286 2% chr3 48715987; 48715997 ? ENST00000413374; chr3 48702393 48702506 ? TSF 48715997 ENST00000341520; ENST00000416649; ENST00000294129 2% chr3 137906397; 137906441 + ENST00000469044; chr3 137907243 137907252 + TSF 137906427 ENST00000461600; ENST00000461822; ENST00000470821; ENST00000471709; ENST00000538260; ENST00000463485; ENST00000393058 2% chr12 93873164 93873248 + ENST00000549982 chr12 93876129 93876286 + TSF ENST00000361630; ENST00000552217; ENST00000393128; ENST00000547098; ENST00000549561; ENST00000548545 2% chr1 53558226 53558226 ? ENST00000371494 chr1 53556655 53556736 ? TSF 2% chr2 143743517 143743590 + ENST00000264170; chr2 143745513 143745682 + TSF ENST00000375773; ENST00000409512 1% chr12 117537030 117537030 ? ENST00000470612; chr12 117513606 117513652 ? TSF ENST00000335209; ENST00000541210; ENST00000462502; ENST00000392545 1% chr11 63365533 63365533 ? ENST00000323646; chr11 63360665 63361041 ? TSF ENST00000415826 1% chr16 14782022 14782022 ? ENST00000438167 chr16 14779702 14779829 ? TSF ENST00000567462 1% chr7 22532184 22532184 ? ENST00000406890 chr7 22512853 22512874 ? TSF ENST00000404369; ENST00000424363 1% chr5 1802435 1802488 + ENST00000274137; chr5 1811112 1811428 + TSF ENST00000469176 1% chr7 93516573 93516573 ? ENST00000451238; chr7 93492029 93492238 ? TSF ENST00000222543 1% chr14 106235574 106235574 ? ENST00000390551 chr14 106216701 106216951 ? TSF 1% chr22 47071365 47071449 + ENST00000406902; chr22 47078308 47078350 + TSF ENST00000361034; ENST00000408031 1% chr16 16381600 16381719 + ENST00000399336; chr16 16382422 16382605 + TSF ENST00000263012; ENST00000538468 1% chr7 22532184 22532184 ? ENST00000406890; chr7 22531483 22531483 ? TSF ENST00000404369; ENST00000424363 1% chr18 56807181 56807267 + ENST00000587834; chr18 56814218 56814267 + TSF ENST00000299714; ENST00000588875 1% chr22 48885405 48885516 + ENST00000402357; chr22 48915770 48916108 + TSF ENST00000336769 1% chr10 75555297 75555421 + ENST00000604729; chr10 75556079 75556138 + TSF ENST00000603114; ENST00000604524; ENST00000398706; ENST00000605216; ENST00000433366; ENST00000492395; ENST00000603187; ENST00000412198; ENST00000604754 1% chr16 4401233 4401233 ? ENST00000577031; chr16 4398819 4398944 ? TSF ENST00000318059; ENST00000571986; ENST00000576217; ENST00000571178 1% chr1 220240644 220240644 ? ENST00000322067; chr1 220237757 220238024 ? TSF ENST00000469520; ENST00000354807; ENST00000544404; ENST00000414869; ENST00000463953; ENST00000498791; ENST00000480959 1% chr14 106092109 106092109 ? ENST00000390543 chr14 105470421 105470731 ? TSF 1% chr1 53569018 53569018 ? ENST00000371494 chr1 53568657 53568729 ? TSF 1% chr9 5163913 5164179 ? ENST00000381641 chr9 4992433 4992489 ? TSF 1% chr7 8198157 8198157 ? ENST00000402384; chr7 8197727 8198053 ? TSF ENST00000406470; ENST00000265577; ENST00000396675; ENST00000339809; ENST00000401396; ENST00000422063; ENST00000407906; ENST00000317367 1% chr8 17872093 17872349 + ENST00000325083; chr8 17873210 17873221 + TSF ENST00000519253; ENST00000327578; ENST00000522275 1% chr2 192546672; 192546743 + ENST00000307849; chr2 192548016 192548103 + TSF 192546682 ENST00000451500; ENST00000425611; ENST00000435931; ENST00000307834; ENST00000410026; ENST00000409510 1% chr11 67786242 67786362 + ENST00000539229; chr11 67786535 67786588 + TSF ENST00000316367; ENST00000007633; ENST00000342456 1% chr8 62546242 62546242 ? ENST00000541428; chr8 62544404 62544571 ? TSF ENST00000379454; ENST00000522919; ENST00000356457; ENST00000519234; ENST00000518068; ENST00000517903; ENST00000445642; ENST00000517847; ENST00000522835 1% chr12 102547647 102547754 + ENST00000327680; chr12 102548605 102548938 + TSF ENST00000378128; ENST00000541394; ENST00000358383; ENST00000392911; ENST00000412715; ENST00000417507; ENST00000457614 1% chr19 18710375 18710375 ? ENST00000392386 chr19 18709961 18710215 ? TSF 1% chr10 79796952 79797062 + ENST00000435275; chr4 176584518 176584519 + TSF ENST00000440692; ENST00000372360; ENST00000360830 1% chrX 41598637 41598637 ? ENST00000421587; chrX 41557348 41557352 ? TSF ENST00000318588; ENST00000361962; ENST00000378163; ENST00000378158; ENST00000378166; ENST00000442742; ENST00000378154 1% chr10 5037511 5037511 ? ENST00000380753; chr10 5023140 5023482 ? TSF ENST00000421196; ENST00000407674 1% chr20 37384500 37384682 + ENST00000243903 chr20 37390296 37390409 + TSF 1% chr4 169086398 169086477 + ENST00000359299 chr4 169090666 169090754 + TSF 1% chr22 23165476 23165642 + ENST00000390317 chr22 23175722 23175755 + TSF 1% chr4 25759156 25759156 ? ENST00000399878; chr4 25723603 25724054 ? TSF ENST00000264868; ENST00000502949; ENST00000510448

TABLE-US-00004 TABLE 4 Coordinates of the fusion sequences for which the donor is the TE. The names of the columns are the following: 1. Frequency in LUAD cohort 2. Donor Chromosome TE 3. Donor start TE 4. Donor Breakpoint TE 5. Donor strand TE 6. Acceptor Chromosome exon 7. Acceptor Breakpoint exon 8. Acceptor end exon 9. Acceptor strand exon 10. Acceptor transcript 11. Fusion type The fusion transcript sequences of table 4 correspond in the same order to SEQ ID NO: 432-910 (with same reasoning as table 3 above). 1 2 3 4 5 6 7 8 9 10 11 51% chr12 122430912 122431655 + chr12 122437622 122437850 + ENST00000288912 TSA 50% chr12 122430912 122431578 + chr12 122437622 122437850 + ENST00000288912 TSA 42% chr8 104389530 104389551 + chr8 104390255 104390471 + ENST00000330295; TSA ENST00000520337 36% chr12 122430912 122431615 + chr12 122437622 122437850 + ENST00000288912 TSA 36% chr17 80440912 80441180 + chr17 80441592 80441655; + ENST00000390006; TSA 80441659 ENST00000309794; ENST00000345415; ENST00000457415; ENST00000577432; ENST00000584513; ENST00000412079 32% chr20 44420870 44421070 + chr20 44421316 44421386 + ENST00000372622; TSA ENST00000449078; ENST00000456939 32% chr6 80021194 80022085 ? chr6 79924739 79924739 ? ENST00000275036; TSF ENST00000344726 24% chr7 116364854 116364901 + chr7 116371722 116371913 + ENST00000397752; TSA ENST00000318493; ENST00000436117 23% chr17 70713482 70713885 ? chr17 70645407 70645407 ? ENST00000255559; TSF ENST00000542342; ENST00000582769 22% chr10 5077666 5077808 + chr10 5138602 5138769 + ENST00000602997; TSA ENST00000605149; ENST00000380554 19% chr10 5059958 50092 ? chr10 5043873 5043873 ? ENST00000380753; TSA ENST00000421196; ENST00000407674; ENST00000604507; ENST00000455190 18% chr11 423924 423942 ? chr11 421198 421198 ? ENST00000332826 TSA 17% chr5 66178759 66178848 + chr5 66195779 66195810 + ENST00000404260; TSA ENST00000403625; ENST00000406374; ENST00000406039; ENST00000452953; ENST00000432817; ENST00000434115; ENST00000411628; ENST00000490016; ENST00000403666; ENST00000450827 16% chr4 100015102 10002105 ? chr4 100006367 100006367 ? ENST00000296412; TSA ENST00000512659; ENST00000503130; ENST00000502590; ENST00000505652 16% chr11 93467948 93468129 ? chr11 93467826 93467826 ? ENST00000393259; TSA ENST00000527169 15% chr17 70670228 70670643 ? chr17 70645407 70645407 ? ENST00000255559; TSA ENST00000542342; ENST00000582769 14% chr5 822923 823504 ? chr5 822010 822010 ? ENST00000424784; TSA ENST00000283441 12% chr20 31764878 31764929 + chr20 31765953 31766034 + ENST00000253362; TSA ENST00000354932 10% chr5 1474878 1475076 ? chr5 1474800 1474800 ? ENST00000475622; TSF ENST00000283415 9% chr19 14129234 14129243 + chr19 14141522 14141549; + ENST00000585987; TSF 14141760 ENST00000431365 9% chr7 48039432 48039725 ? chr7 48035743 48035743 ? ENST00000453071; TSF ENST00000297325; ENST00000412371; ENST00000412142; ENST00000395572; ENST00000453192; ENST00000438771 8% chr10 33505378 33505616 ? chr10 33502645 33502645 ? ENST00000432372; TSF ENST00000374875; ENST00000265371; ENST00000374867; ENST00000395995; ENST00000374821; ENST00000374822; ENST00000374823; ENST00000374816 7% chrX 107293989 107294242 + chrX 107301268 107301431 + ENST00000415430; TSF ENST00000217957; ENST00000458383 5% chr7 7558677 7558760 ? chr7 7557468 7557468 ? ENST00000399429; TSF ENST00000444268 5% chr3 182832430 182833262 ? chr3 182812393 182812393 ? ENST00000265594; TSF ENST00000497830; ENST00000495767; ENST00000476176; ENST00000487634; ENST00000466650; ENST00000486226 5% chr3 98584565 98586135 ? chr3 98568442 98568442 ? ENST00000449482; TSF ENST00000326840; ENST00000326857 4% chr2 143794737 143794842 + chr2 143797997 143798227 + ENST00000264170; TSF ENST00000409512 4% chr10 5056485 5057095 ? chr10 5043873 5043873 ? ENST00000380753; TSF ENST00000421196; ENST00000407674; ENST00000604507; ENST00000455190 4% chr12 122430912 122432103 + chr12 122437622 122437850 + ENST00000288912 TSF 4% chr12 122430912 122431795 + chr12 122437622 122437850 + ENST00000288912 TSF 4% chr22 22899861 22899965 ? chr22 22893511 22893511 ? ENST00000406503; TSF ENST00000398743; ENST00000398741; ENST00000543184; ENST00000405655; ENST00000402697; ENST00000439106; ENST00000438888; ENST00000420709; ENST00000403441; ENST00000442481 4% chr12 117498474 117498567 ? chr12 117494691 117494691 ? ENST00000470612; TSF ENST00000335209; ENST00000392545; ENST00000462502 4% chr8 104389530 104389536 + chr8 104390255 104390471 + ENST00000330295; TSF ENST00000520337 4% chr2 38982396 38983253 ? chr2 38977336 38977336 ? ENST00000313117; TSF ENST00000425778; ENST00000425941; ENST00000446327; ENST00000409276; ENST00000431066; ENST00000443213 4% chr5 66178759 66179020 + chr5 66195779 66195810 + ENST00000404260; TSF ENST00000403625; ENST00000406374; ENST00000406039; ENST00000452953; ENST00000432817; ENST00000434115; ENST00000411628; ENST00000490016; ENST00000403666; ENST00000450827 4% chr20 45337040 45337192 + chr20 45353680 45354963 + ENST00000359271 TSF 4% chr4 57559029 57559845 ? chr4 57522178 57522178 ? ENST00000420433; TSF ENST00000554144; ENST00000508121; ENST00000557328 4% chr7 56018506 56018522 + chr7 56020872 56021011 + ENST00000426595 TSF 4% chr6 117763522 117763597 ? chr6 117739669 117739669 ? ENST00000368507; TSF ENST00000368508 3% chr19 1114639 1114676 ? chr19 1114421 1114421 ? ENST00000361757; TSF ENST00000587024; ENST00000438103 3% chr5 147244208 147245387 + chr5 147261009 147261211 + ENST00000296694 TSF 3% chr17 39974832 39974893 + chr17 39975462 39975651 + ENST00000321562 TSF 3% chr12 122430912 122432282 + chr12 122437622 122437850 + ENST00000288912 TSF 3% chr4 57559029 57559885 ? chr4 57522178 57522178 ? ENST00000420433; TSF ENST00000554144; ENST00000508121; ENST00000557328 3% chr4 872984 873014 ? chr4 871597 871597 ? ENST00000314167; TSF ENST00000511163 3% chr22 42509838 42511212 ? chr22 42483179 42483179 ? ENST00000602404; TSF ENST00000498737 3% chr22 29117270 29117506 ? chr22 29115473 29115473 ? ENST00000348295; TSF ENST00000382566; ENST00000382578; ENST00000404276; ENST00000328354; ENST00000405598; ENST00000382580; ENST00000433728; ENST00000402731; ENST00000403642; ENST00000439200; ENST00000448511 3% chr8 117816470 117816753 + chr8 117861127 117861256; + ENST00000517820; TSF 117861276 ENST00000520733 3% chr16 66461229 66461334 + chr16 66503505 66503768 + ENST00000536005 TSF 3% chr17 70713482 70713885 ? chr17 70709120 70709120 ? ENST00000581581 TSF 3% chr17 39976225 39976301 + chr17 39976521 39976713 + ENST00000321562; TSF ENST00000455106; ENST00000544340 3% chr1 223720296 223720355 ? chr1 223718212 223718212 ? ENST00000430824; TSF ENST00000366872 3% chrX 151404470 151410406 ? chrX 151393317 151393317 ? ENST00000370314; TSF ENST00000535043 3% chr16 74833176 74833256 ? chr16 74774013 74774013 ? ENST00000219368; TSF ENST00000567683; ENST00000569949 2% chr16 4708255 4708353 + chr16 4714710 4714776 + ENST00000262370; TSF ENST00000415496; ENST00000536343; ENST00000587747; ENST00000399577; ENST00000588994; ENST00000586183; ENST00000590790 2% chr1 1422590 1422685 + chr1 1423243 1423294 + ENST00000308647 TSF 2% chr7 44158104 44158351 ? chr7 44157663 44157663 ? ENST00000406581; TSF ENST00000223361; ENST00000452185; ENST00000433715; ENST00000456038; ENST00000418438 2% chr10 5059958 5060092 ? chr10 5043777 5043873 ? ENST00000380753; TSF ENST00000421196; ENST00000407674; ENST00000604507; ENST00000455190 2% chr8 121656402 121662337 ? chr8 121644891 121644891 ? ENST00000395601; TSF ENST00000517992 2% chr15 43467899 43467988 + chr15 43470805 43470909 + ENST00000260403 TSF 2% chr5 58596413 58596862 ? chr5 58511794 58511794 ? ENST00000340635; TSF ENST00000360047; ENST00000507116; ENST00000503258; ENST00000405755; ENST00000502484; ENST00000546160; ENST00000309641; ENST00000502575 2% chr14 106258470 106258725 ? chr14 106209234 106209408 ? ENST00000390548; TSF ENST00000390549; ENST00000390542 2% chrX 149996757 149996779 ? chrX 149963959 149963959 ? ENST00000370377; TSF ENST00000466436; ENST00000418547 2% chr14 106258470 106258725 ? chr14 106237569 106237742 ? ENST00000390551 TSF 2% chrX 123617772 123617816 ? chrX 123615814 123615814 ? ENST00000371130; TSF ENST00000422452 2% chrX 100654643 100654732 ? chrX 100653934 100653934 ? ENST00000218516 TSF 2% chr1 156716126 156716133 ? chr1 156715165 156715165 ? ENST00000357325; TSF ENST00000537739; ENST00000368209; ENST00000368206 2% chr18 70839859 70840073 ? chr18 70829208 70829208 ? ENST00000581011; TSF ENST00000581862 2% chr5 132737208 132737281 ? chr5 132736678 132736678 ? ENST00000265342; TSF ENST00000510685 2% chr2 89366766 89370031 ? chr2 89292018 89292223 ? ENST00000495489 TSF 2% chr6 32745845 32746483 ? chr6 32731247 32731247 ? ENST00000411527; TSF ENST00000435145; ENST00000437316 2% chr1 236485526 236485567 ? chr1 236433294 236433294 ? ENST00000354619; TSF ENST00000327333 2% chr1 224527533 224527861 + chr1 224553581 224553693 + ENST00000465271; TSF ENST00000366858; ENST00000366857; ENST00000366856 2% chr16 74834260 74834274 ? chr16 74774013 74774013 ? ENST00000219368; TSF ENST00000567683; ENST00000569949 2% chr18 71976895 71977013 ? chr18 71930712 71930712 ? ENST00000340533; TSF ENST00000494131; ENST00000397914 2% chr2 85823332 85823377 + chr2 85823642 85823772 + ENST00000441634; TSF ENST00000306368; ENST00000414390; ENST00000443647; ENST00000456023 2% chr14 92119780 9211 9864 ? chr14 92105594 92105594 ? ENST00000256343; TSF ENST00000557036 2% chr4 57559029 57559962 ? chr4 57522178 57522178 ? ENST00000420433; TSF ENST00000554144; ENST00000508121; ENST00000557328 2% chr8 144690895 144690934 ? chr8 144690296 144690296 ? ENST00000220966; TSF ENST00000433751 2% chr12 104681506 104681628 + chr12 104682709 104682818 + ENST00000378070; TSF ENST00000525566; ENST00000429002; ENST00000526691; ENST00000388854; ENST00000542918 2% chr5; 52897435; 52897704 +; chr5 52899282 52899360 + ENST00000502423; TSF chr5 52897700 + ENST00000296684; ENST00000506974; ENST00000506765 2% chr11 60694460 60694542 + chr11 60694676 60694890 + ENST00000453848; TSF ENST00000005286 2% chr1 23762004 23762216 ? chr1 23761111 23761111 ? ENST00000495646; TSF ENST00000336689; ENST00000437606 2% chr7 50763231 50763289 ? chr7 50742355 50742355 ? ENST00000439599; TSF ENST00000398812; ENST00000403097; ENST00000357271; ENST00000401949; ENST00000439044 2% chr15 34452835 34453416 ? chr15 34446885 34446885 ? ENST00000256544; TSF ENST00000557877; ENST00000560108; ENST00000559515 2% chr12 6458902 6458996 ? chr12 6458387 6458387 ? ENST00000360168; TSF ENST00000358945; ENST00000540037; ENST00000228916; ENST00000543768 2% chr7 95053024 95053104 ? chr7 95045609 95045609 ? ENST00000536183; TSF ENST00000455123; ENST00000433091; ENST00000222572 2% chr19 11140024 11140045 + chr19 11141406 11141569 + ENST00000358026; TSF ENST00000344626; ENST00000429416; ENST00000541122; ENST00000589677; ENST00000444061; ENST00000590574; ENST00000413806; ENST00000450717 2% chr12 58189709 58189746 + chr12 58189960 58189980; + ENST00000540550; TSF 58190044; ENST00000457189; 58190366 ENST00000454289; ENST00000323833; ENST00000350762 2% chr14 70466641 70466673 + chr14 70477471 70477663 + ENST00000361956; TSF ENST00000381280 2% chr10 3133726 3133857 + chr10 3141467 3141544 + ENST00000607886; TSF ENST00000381125; ENST00000381075; ENST00000407806 2% chr7 134212336 134212386 + chr7 134215479 134215562 + ENST00000359579 TSF 2% chr2 135250006 135250213 ? chr2 135223796 135223796 ? ENST00000281924 TSF 2% chr7 30468003 30468120 ? chr7 30465326 30465326 ? ENST00000222823 TSF 2% chr17 90088 90313 ? chr17 69527 69527 ? ENST00000331302; TSF ENST00000323434; ENST00000536489 1% chr7 6423751 6423803 + chr7 6426843 6426914 + ENST00000348035; TSF ENST00000356142 1% chr7 95053024 95053140 ? chr7 95045609 95045609 ? ENST00000536183; TSF ENST00000455123; ENST00000433091; ENST00000222572 1% chr10 5049827 5050220 ? chr10 5043873 5043873 ? ENST00000380753; TSF ENST00000421196; ENST00000407674; ENST00000604507; ENST00000455190 1% chr15 43431174 43431330 + chr15 43440953 43441077 + ENST00000564698; TSF ENST00000260403; ENST00000564494 1% chr19 50492001 50492051 ? chr19 50491749 50491749 ? ENST00000593919; TSF ENST00000316763; ENST00000377011; ENST00000599538; ENST00000443401; ENST00000601341; ENST00000594948; ENST00000601912; ENST00000594092; ENST00000593912 1% chr1 26595323 26595855 + chr1 26595951 26596105 + ENST00000451429; TSF ENST00000476272; ENST00000252992; ENST00000453146 1% chr20 43561150 43561175 + chr20 43561713 43561826 + ENST00000255136; TSF ENST00000217073 % chr10 5059958 5060092 ? chr10 5043783 5043873 ? ENST00000380753; TSF ENST00000421196; ENST00000407674; ENST00000604507; ENST00000455190 1% chr2 26947307 26947428 + chr2 26950535 26951436 + ENST00000302909 TSF 1% chr10 99600727 99601513 + chr10 99619215 99619340 + ENST00000370602 TSF 1% chr20 58402976 58403213 + chr20 58411560 58411615 + ENST00000359926; TSF ENST00000371015; ENST00000395639; ENST00000541461; ENST00000355648; ENST00000361300; ENST00000395636 1% chr14 105641713 105641815 ? chr14 105639598 105639598 ? ENST00000392568 TSF 1% chr5 34913998 34914032 ? chr5 34913683 34913683 ? ENST00000382038; TSF ENST00000341754 1% chr4 39516293 39516533 ? chr4 39515804 39515804 ENST00000316423; TSF ENST00000501493; ENST00000506179; ENST00000515021; ENST00000514106; ENST00000509391; ENST00000505698 1% chr8 140818301 140818376 ? chr8 140744445 140744445 ? ENST00000389327; TSF ENST00000389328; ENST00000520857; ENST00000438773 1% chr20 44442461 44442685 + chr20 44443023 44443109 + ENST00000356455; TSF ENST00000405520; ENST00000335046; ENST00000372568 1% chr17 66244121 66244199 + chr17 66244785 66244846 + ENST00000584837 TSF 1% chr17 17051276 17051346 + chr17 17053458 17053547 + ENST00000395811; TSF ENST00000444976; ENST00000395804; ENST00000341712; ENST00000584067 1% chr6 39267897 39268149 ? chr6 39267513 39267513 ? ENST00000373231 TSF 1% chr20 25840234 25840376 ? chr20 25755948 25755948; ? ENST00000376403; TSF 25755972 ENST00000584071 1% chr2 90168849 90168893 + chr2 90193334 90193424 + ENST00000390275 TSF 1% chr19 50174724 50174836 + chr19 50176955 50177005; + ENST00000441864; TSF 50177034 ENST00000246785; ENST00000598306; ENST00000600947 1% chr11 85339 945 85340247 + chr11 85342731 85342852 + ENST00000358867; TSF ENST00000534341 1% chr19 17421511 17421655 + chr19 17424832 17424912 + ENST00000593466; TSF ENST00000359866; ENST00000596582 1% chr3 32323981 32324151 + chr3 32398865 32399038 + ENST00000307526 TSF 1% chr4 57559029 57559925 ? chr4 57522178 57522178 ? ENST00000420433; TSF ENST00000554144; ENST00000508121; ENST00000557328 1% chrX 107265928 107266261 + chrX 107301268 107301431 + ENST00000415430; TSF ENST00000217957; ENST00000458383 1% chr2 143715736 143715823 + chr2 143718193 143718339 + ENST00000264170; TSF ENST00000375773; ENST00000409512 1% chr20 25841897 25842039 ? chr20 25755948 25755948; ? ENST00000376403; TSF 25755972 ENST00000584071 1% chr8 98817265 98817331 + chr8 98817581 98817692 + ENST00000445593; TSF ENST00000521545; ENST00000517924 1% chr17 45698288 45698367 + chr17 45699 34 45699286 + ENST00000530173; TSF ENST00000322157; ENST00000544660; ENST00000528565 1% chr19 46194383 46194670 ? chr19 46191824 46191824 ? ENST00000342669; TSF ENST00000588301; ENST00000590212 1% chr21 38272435 38272892 ? chr21 38269431 38269431 ? ENST00000336648; TSF ENST00000399120 1% chr4 162585892 162585968 ? chr4 162577646 162577646 ? ENST00000306100; TSF ENST00000379164; ENST00000536695; ENST00000427802 1% chr4 40352026 40352049 + chr4 40355996 40356537 + ENST00000310169 TSF 1% chr20 25843554 25843696 ? chr20 25755948 25755948; ? ENST00000376403; TSF 25755972 ENST00000584071 1% chrX 138072586 138072670 ? chrX 137939841 137939841 ? ENST00000370603; TSF ENST00000436198; ENST00000455663; ENST00000448673 1% chr2 97560868 97560977 ? chr2 97559788 97559788 ? ENST00000327896; TSF ENST00000417561; ENST00000490605 1% chr11 60933230 60933962 ? chr11 60901679 60901679 ? ENST00000301765; TSF ENST00000538036 1% chr9 130929707 130929818 ? chr9 130929443 130929443 ? ENST00000372954; TSF ENST00000393608; ENST00000541172; ENST00000325721; ENST00000357558; ENST00000538431; ENST00000277465; ENST00000372948; ENST00000372938; ENST00000415526 1% chr21 39513161 39513404 + chr21 39528398 39528496 + ENST00000357704; TSF ENST00000400477 1% chr8 63314966 63315263 + chr8 63492098 63492235 + ENST00000523211; TSF ENST00000524201; ENST00000328472 1% chr4 186460593 186460994 ? chr22 23243156 23243475 + ENST00000390323 TSF 1% chr12 8864869 8864879 + chr12 8866407 8866637 + ENST00000537189 TSF 1% chr10 5059958 5060040 ? chr10 5043873 5043873 ? ENST00000380753; TSF ENST00000421196; ENST00000407674; ENST00000604507; ENST00000455190 1% chr7 65418154 65418399 + chr7 65419061 65419287; + ENST00000360768; TSF 65419400 ENST00000434382 1% chr1 156305244 156305264 ? chr1 156304709 156304709 ENST00000295688; TSF ENST00000368258; ENST00000413555; ENST00000496684; ENST00000478640; ENST00000415548

[0380] The peptides were then further screened against a reference proteome, typically for human subject against all sequences present in Uniprot (representing all the sequences encoded in the human exome). Peptides were considered equal to those in Uniprot if they had the same amino acid sequence or if they only differed in the amino acid in the first or last position. All these equal sequences were then discarded from the candidate list. 117 peptide sequences derived from these 230 fusion transcripts where thus predicted to bind to HLA-A2: 01 (see table 5 below).

TABLE-US-00005 TABLE5 PeptidesLUAD SEQID Peptidesequence 1 RLLHLESFL 2 TLMNLVQVL 3 ILHSLVTGV 4 FMMEQVGLA 5 AMDGKELSL 6 TLAYGKYYI 7 GLIQLIWLA 8 GMVDGGSNI 9 YLWTTFFPL 10 ALWEAKMII 11 WLSSRVTQL 12 AILPKANTV 13 VLLFEVELV 14 GLDTGLQGM 15 SLLDGTQLF 16 GLPTGYLFV 17 LLDRFGYHV 18 SLLEETQAI 19 MLLVQPAEL 20 GLLNISHTA 21 HLYEPWFPV 22 YLQGLPLPL 23 KAVEGILAV 24 MIYEENNRL 25 YLPYFLKSL 26 GLYSLSSVV 27 LMISRTPEV 28 LLGGPSVFL 29 ILSGYGPCV 30 FLPDLDRPL 31 AMDGKELSL 32 RMDFEDLGL 33 TLIFNPTEI 34 LLPGLLLLL 35 LLLVHQHAV 36 FLDDAPPGT 37 VLIRYVWTL 38 YLCGHLHTL 39 VLSQLTILI 40 TLGGLMPVL 41 FLQGSITFI 42 MLLLYIWQV 43 YLKIMPVHL 44 HTLGGLMPV 45 YIMARVLFV 46 FILRTDHYI 47 IMSSAIAYL 48 FIIGILQLA 49 YLLQEIYGI 50 GVFPVVIQA 51 ALVHLPSQL 52 GLHPAKPQV 53 MLVTWELAL 54 VLLTNTIWL 55 ALVHLPSQL 56 CLIDEMPEA 57 ALMGGFMKT 58 LLLHLPLXL 59 TLQDKNLGL 60 ILANLPPAL 61 PLWDGMAGL 62 GLDHQTHPL 63 GMFLLPPQL 64 RLADHLSFC 65 RMRDQLPAL 66 GLLHAEVAL 67 SLQNCQVSV 68 VISAFPSEV 69 ALAIAALEL 70 VLDGLDVLL 71 ELFPPLFMA 72 FLIVAEILI 73 IVAEILISL 74 KAVEGILAV 75 YLPHLPQVL 76 MLLDPMGGI 77 RLLHLESFL 78 YLAYILYFV 79 LMTSSIMSV 80 MLMKTVWQA 81 SLQPEDMAL 82 KILTYFPMV 83 FLGTRVTRV 84 SLMQSGSPV 85 VLMWTMAHL 86 LLGETKVYV 87 KILTYFPMV 88 SLLERGLEA 89 VLSSLNVPL 90 FLERKSIRV 91 FVGSSTFYL 92 FLYTGDFFL 93 SVGPFALTV 94 NLALPLPKV 95 VLESGLYQV 96 MLVAITVLI 97 FMDDAKILF 98 ALVHLPSQL 99 ILTASITSI 100 AMDGKELSL 101 SLGWNISGV 102 MISAFPNEV 103 RLTHELPGI 104 LLFSDGEKV 105 RLNESTTFV 106 KLEELKSFV 107 SINEEIQTV 108 RLHDGPLRA 109 MISAFPNEV 110 ILHTSVPFL 111 YLENMVSGV 112 QLLGRLESL 113 RLLHLESFL 114 ALLRQMEGI 115 TLNKDFQEV 116 IMEQGDLSV 117 RLLHLESFL

2.2.2 Validation on HLA-A2 Associated Peptides

[0381] Given that HLA-A2 allele is expressed in almost 50% of the Caucasian population, together with the existence of different technical tools, validations were focused on HLA-A2-associated peptides.

[0382] In the following paragraphs TE-Exon derived-transcripts is used interchangeably with fusion transcripts and the term TE-derived peptides is used interchangeably with fusion transcripts-derived peptides.

[0383] Expression of TE-Exon Derived-Transcripts in Lung Adenocarcinoma Samples

[0384] To experimentally validate the predicted TE-Exon transcripts, the expression by PCR in LUAD tumor samples and tumor cell lines was validated firstly. Specific primers for each chimeric fusion were thus designed, in order to have one of them binding to the TE part and the other to the Exon part of the fusion. The results were further confirmed by sequencing of the PCR products.

[0385] In particular, specific primers were designed in such a way that the forward primer was binding in the donor sequence and the reverse primer was binding in the acceptor sequence of the reconstructed fusion sequence. PCR reactions were run on RNA derived from lung tumor samples and human tumor cell lines. Amplifications products were seeded on agarose gels and bands found on the expected size were cut and sequenced. Finally, sequenced PCR products were compared with the reconstructed fusion sequence.

[0386] Using this approach, it was possible to confirm the presence of predicted fusion transcripts both in LUAD tumor samples and tumor cell lines. Table 6 below summarizes the results found for 8 of the most frequent chimeric fusions with a predicted peptide associated to bind with high affinity to HLA-A2 allele.

TABLE-US-00006 Most frequent fusion transcript validation. The most frequent fusions peptides were validated by PCR in 15 LUAD tumor samples and 6 LUAD tumor cell lines. The status Yes or No in the table below indicates the presence or absence of the PCR product on the expected size. When the PCR product was further validated by sequencing, is denoted as Yes. TE-Exon fusion derived-peptides asociated to bind HLA-A2 Frequency 119 48 28 24 peptide sequence RLLHLESFL MLMKTVWQA FLGTRVTRV AILPKANTV LUAD H1975 Yes Yes No No tumor H1650 Yes No No No cell lines H1299 Yes No No No A549 Yes Yes No No H2052 Yes No No No HCC827 Yes Yes No No LUAD Tumor 1 Yes No No No tumor Tumor 2 Yes Yes No No samples Tumor 3 Yes No No No Tumor 4 Yes No No No Tumor 5 Yes No No No Tumor 6 Yes No No Yes Tumor 7 Yes No No No Tumor 8 Yes No No No Tumor 9 Yes No No No Tumor 10 Yes Yes Yes Yes Tumor 11 Yes Yes No No Tumor 12 Yes Yes Yes Yes Tumor 13 Yes Yes No Yes Tumor 14 Yes Yes Yes Yes Tumor 15 No No No No TE-Exon fusion derived-peptides asociated to bind HLA-A2 Frequency 23 19 18 16 peptide sequence YLPYFLKSL AMDGKELSL FLIVAEILI RLADHLSFC LUAD H1975 Yes No No No tumor H1650 No No No Yes cell lines H1299 No No No Yes A549 Yes No No No H2052 No No No No HCC827 Yes yes No No LUAD Tumor 1 Yes Yes No Yes tumor Tumor 2 Yes Yes No No samples Tumor 3 Yes No No Yes Tumor 4 Yes Yes No No Tumor 5 No No No No Tumor 6 Yes Yes Yes Yes Tumor 7 No Yes No No Tumor 8 Yes Yes No No Tumor 9 No Yes Yes No Tumor 10 Yes Yes Yes No Tumor 11 Yes Yes Yes No Tumor 12 Yes Yes No No Tumor 13 Yes No No Yes Tumor 14 Yes Yes No Yes Tumor 15 No Yes No No

[0387] Binding of ER-Derived Peptides to HLA-A2 Molecule

[0388] Once confirmed the expression of chimeric transcripts, the derived-peptides were synthetized and their binding to HLA-A2 was confirmed. Because monomer stabilization and tetramer formation are only possible in the presence of a high affinity binding peptide, the formation of HLA-A2 tetramers was estimated in the presence of synthetized peptides by flow cytometry. All predicted peptides were able to stabilize tetramer formation, showing a percentage of fluorescence higher than 50% relative to positive control. As positive control, a known high affinity binding peptide to HLA-A2 derived from Cytomegalovirus (CMV) was used. This result confirmed the predicted high affinity binding to HLA-A2 allele. FIG. 10 shows the result for 10 peptides derived from the most frequent fusions peptides.

[0389] Immunogenicity of ER-Derived Peptides

[0390] The following step after binding validation to HLA-A2 allele, was to test the immunogenicity of predicted peptides. Priming assays were thus performed to test the ability of identified peptides to expand specific cytotoxic T cells. PBMCs from HLA-A2+ healthy donors were used to generate monocyte derived-DCs (moDCs). After loading the moDCs with a mix of synthetic peptides, autologous co-culture was performed with CD4+ and CD8+ T cells. Finally, the expansion of specific CD8+ T cells was analysed by flow cytometry using two-colours tetramer staining. As a control of specific expansion, the co-culture was performed in the absence of peptides. By using this approach in one donor, it has been possible to identify and expand specific CD8+ T cells recognizing 6 of the most frequent chimeric fusion derived-peptides (RLLHLESFL, LLGETKVYV, AILPKANTV, RLADHLSFC, FLIVAEILI, YLWTTFFPL). This result is evidenced by an increase in at least one magnitude order of the percentage of tetramer positive cells compared to control test among total CD8+ T cells. The same experiment was performed in order to evaluate the response in additional 5 donors. FIG. 11A summarizes the results obtained for the total of 6 donors analyzed in which we found specific CD8+T expansions for 21 of the most frequent fusions transcripts-derived peptides (YLWTTFFPL, FLGTRVTRV, RLADHLSFC, LLGETKVYV, MLVTWELAL, MLMKTVWQA, SLMQSGSPV, AILPKANTV, AMDGKELSL, LLDRFGYHV, GLLNISHTA, ILTASITSI, ILSGYGPCV, RQAPGFHHA, GLPSHVELA, ILHSLVTGV, LLHLESFLV, VLLTNTIWL, LLTSWHLYL). These experiments show that these peptides are able to induce an immune response and confirms the immunogenicity of ER-derived peptides.

[0391] Generation of Cytotoxic T Lymphocytes Clones Recognizing ER-Derived Peptides

[0392] Expanded CD8+ tetramer positive T-cells from immunogenicity assays (FIG. 11A) were single cell FACS-sorted in order to generate cytotoxic T lymphocytes (CTLs) clones. We generated 10 clones recognizing 5 different ER-derived peptides: YLWTTFFPL, LLGETKVYV, MLVTWELAL, MLMKTVWQA, RLADHLSF. These peptides are listed in Table 5 as peptide 9, 86, 53, 80 and 64 respectively. We will refer to these numbers to indicate the specificity of each generated CTL-clone. Example, CTL-clone 9 recognize ER-derived peptide 9.

[0393] In order to evaluate the cytotoxic capacity of generated CTL-clones, two different functional assays were conducted using the H1650 cell line as target cells. This is a LUAD-derived tumor cell line expressing HLA-A2 allele.

[0394] First, we measure the ability of CTL-clones to secret cytokines after exposure to ER-derived peptides. After co-cultured CTL-clones with target cells loaded with the specific ER-derived peptides during 18 h, secretion of INF-?, TNF and Granzyme-B (Gr-B) was measured in culture supernatants. All CTL-clones were activated after exposure specific ER-derived peptides, secreting cytokines in a dose-dependent manner (FIG. 11B).

[0395] In a second set of experiments, CTL clones killing capacity was assessed. CTL-clones were co-cultured in different conditions with target cells loaded or not with ER-derived peptides. Using xCELLigence system we measure the real-time impedance variation in a target cells monolayer. In these assays, a decrease in cell-index is related with a decrease in the number of cells in the monolayer reflecting cell viability.

[0396] When CTL-clone 9 was co-culture in 1:1 ratio with target cells loaded with ER-derived peptide 9, we saw a decrease in cell-index over time compared to the control cells (target cells alone). This decrease in the cell index was inhibited when co-culture is performed in presence of blocking anti-MHC-I antibody (+ anti-MHC-I). Performing the co-culture using the same concentration of isotype control (+ isotype) did not inhibit the decrease in cell-index. Moreover, these decrease increases when target cells were loaded with higher concentration of peptide (1 ?M compared to 1 uM) (FIG. 11C, left panel). This result show that a cytotoxic T cell that recognizes a peptide identified by the methods disclosed herein, CTL clone 9, is killing target tumor cells.

[0397] We reasoned that if ER-derived peptides are naturally expressed and presented by target cells, we should be able to kill them by co-culturing with CTL-clones without external addition of peptides. To this aim, we performed co-culture of CTL-clone 9 with H1650 target cells at different ratios to find the one in which effectors are sufficient to kill target cells. In the right panel of FIG. 11C, we saw that CTL-9 was able to kill target cells at a ratio effector-target 4:1 compared with the control cells (target cells alone). Moreover, killing is increased at bigger ratios (8:1). No killing of target cells was evidenced at lower ratios (2:1).

[0398] Finally, similar experiments were performed with CTL-clone 9, CTL-clone 64, and CTL-clone 80 showing a specific killing of target cells that could be also inhibited when the co-culture is performed in the presence of anti-MCH-I antibodies (FIG. 11D).

[0399] All together, these results confirm that cytotoxic T cells that recognizes several different peptides identified by the methods disclosed herein are able to recognize and kill tumor cells expressing specific fusion transcripts-derived peptides and that this effect is due to the specific recognition of peptides in the context of MHC-I molecules. Moreover, the fact that CTL-clones are able to kill target cells without addition of external peptides, indicates that fusion transcripts-derived peptides 9, 64 and 80 are naturally expressed and presented by H1650 LUAD tumor cell line.

[0400] Generation of Engineered T-Cells Recognizing Fusion-Derived Peptides

[0401] Jurkat cells transduced with lentiviral vector encoding for CTL-9 TCR sequence were co-cultured with two different target cells, H1650 and H1395. Both are LUAD-derived cell lines expressing HLA-A2 allele. TCR-mediated activation of Jurkat cells was evaluated by flow cytometry analyzing an increase in the fluorescence of reporter genes (NFAT-GPF, NF-KB-CFP and AP-1-mCherry). Preliminary results show that Jurkat cells are activated when co-cultured with both target cells compared to negative control (non-transduced Jurkat cells). Furthermore, this activation increased in a dose-dependent manner when the co-culture was performed with target cells loaded with specific peptides. PMA/ionomycin was used as positive control (FIG. 12). These results are in line with the results shown in FIGS. 11 C and D, suggesting that LUAD-derived tumor cells express TE-derived peptides. Furthermore, we demonstrated the potential use of CTL-clones TCR sequences in the development of engineered T cells.

[0402] Presence of CD8+ Cells Recognizing Fusion-Derived Peptides in LUAD Patients

[0403] We aimed to identify presence of CTL cells recognizing fusion-derived peptides in LUAD tumor samples.

[0404] In a first set of experiments tumor infiltrating lymphocytes (TILs) expanded with a mix of TE-derived peptides and 11-2, or only with 11-2, were analyzed by tetramer staining. As is shown in FIGS. 13 A and B, we found CD8+ T-cells cells recognizing fusion-derived peptides in TILs derived from LUAD patients.

[0405] Then, we analyzed whether if we detect tetramer positive cells and their phenotype in non-expanded CD8+ T cells derived from fresh tumor samples. Using this strategy, we analyzed CD8+ T cells present in Tumor, juxta-tumor, invaded lymph-nodes and blood derived from LUAD patient samples. Phenotype was determined considering the expression of surface markers CCR7 and CD45RA as Na?ve (CCR7+CD45+), Central Memory (CM, CCR7+CD45RA?) Effector Memory (EM, CCR7?CD45?) and Terminal Effectors (TE, CCR7?CD45+). Interestingly, tetramer positive cells found in tumor tissues shared preferentially a memory phenotype whereas na?ve cells (CCR7+CD45+) are found mostly cells derived from lymph nodes (FIGS. 14 A and B). Patient 2 and 3 are the same in FIG. 13 and FIG. 14.

[0406] All samples tested derived from HLA-A2+ patients.

[0407] Presence of tetramer positive cells with a memory phenotype in tumor tissues, together with the presence of tetramer positive cells in TILs, are consistent with an immune response generated against TE-derived peptides in these patients. Moreover, the existence of na?ve tetramer positive cells in lymph nodes suggest the potential capacity to generate an immune response against these particularly TE-derived peptides.

[0408] Peptide Identification by Mass Spectrometry in LUAD Biopsies.

[0409] Presentation by MHC class I molecules on the tumour cell surface is required for ER-derived peptides in order to be recognized by cytotoxic T cells. In order to confirm that predicted peptides are express on MHC class I molecules, public data from MHC I immunopeptidome derived from 3 LUAD biopsies (Laumont C M et al., Noncoding regions are the main source of targetable tumor-specific antigens Sci Transl Med. 2018 10(470)) were used. OpenMS Software was used to analyse the raw data uploaded to PRIDE database from MHC-I immunopurification of 3 LUAD tumours (PXD009752, PXD009754 and PXD009755). Having in mind that data-dependent acquisition in proteomics only allows the identification of those sequences contained in a target database (generally the whole human proteome); the peptides as per the present application had not been previously identified because they derive from non-coding sequences. The MS/MS identifications incorporating the sequences of the herein predicted peptides in the target database has been re-analyzed. Five peptides among the 3 samples biopsies (peptides ID: 3304, 269, 757, 1810, 3953) were found. To perform this analysis, all predicted peptides derived from chimeric fusions present in at least 5 samples in the TCGA binding to any MHC I allele were considered. This result confirms the expression of chimeric fusion-derived peptides on MHC class I molecules in LUAD tumors.

[0410] Later, we extended our analysis to new lung immunopeptidomics datasets (Bulik-Sullivan et al. Nat. Biotec 2018, Chong et al. Nat. Comm. 2020 and Javitt et al. Front Immunol 2019). Of note, all datasets were generated with fresh lung tumor samples with the exception of Javitt et al. Front Immunol 2019 containing LUAD tumor cell line. For this second analysis, ProteomeDiscoverer 1.4 Software was used to identify the ER-derived peptides. Considering the 4 datasets, 23 unique ER-derived peptides were present in at least one of the total 19 immunopeptidomic samples. In FIG. 15, ER-derived peptides (rows) identified in each MHC sample (column) are indicated with a grey square. On the right, the peptide sequence found is indicated. Interestingly, some of them were observed in more than 1 MHC sample indicating that they are shared across samples. These results confirm that fusion transcripts-derived peptides are processed and presented by HLA-I molecules on tumor cells surface.

[0411] Peptide RLADHLSFC derived from a fusion transcript where the gene part of the fusion is a tumor suppressor gene (Fusion ID: chr22:29117506:->chr22:29115473:-/gene involved: CHEK2) and peptide GLPSHVELA derived from a fusion transcript where the gene part is an oncogene (Fusion ID: chr6:117763597:->chr6:117739669:-/gene involved: ROS1). Interestingly, both peptides were found to be immunogenic (FIG. 11 A) and particularly for peptide RLADHLSFC, results show in FIG. 11 D indicate that could be express by H1650 cell line. Furthermore, we found TILs recognizing peptide GLPSHVELA (FIG. 12A), which indicates that this fusion transcript-derived peptide could be express in LUAD tumor samples.

3 Example 3: Identification Neoantigenic Peptides Derived from Fusion Transcripts Composed of a TE Element and an Exonic Sequence from Various Cancer Samples

[0412] 9184 samples from 32 different cancer types (Acute Myeloid Leukemia, Adrenocortical Carcinoma, Bladder Urothelial Carcinoma, Breast Ductal Carcinoma, Breast Lobular Carcinoma, Cervical Carcinoma, Cholangiocarcinoma, Colorectal Adenocarcinoma, Esophageal Carcinoma, Gastric Adenocarcinoma, Glioblastoma Multiforme, Head and Neck Squamous Cell Carcinoma, Hepatocellular Carcinoma, Kidney Chromophobe Carcinoma, Kidney Clear Cell Carcinoma, Kidney Papillary Cell Carcinoma, Lower Grade Glioma, Lung Adenocarcinoma, Lung Squamous Cell Carcinoma, Mesothelioma, Ovarian Serous Adenocarcinoma, Pancreatic Ductal Adenocarcinoma, Paraganglioma & Pheochromocytoma, Prostate Adenocarcinoma, Sarcoma, Skin Cutaneous Melanoma, Testicular Germ Cell Cancer, Thymoma, Thyroid Papillary Carcinoma, Uterine Carcinosarcoma, Uterine Corpus Endometrioid Carcinoma and Uveal Melanoma) were analyzed according to the method as previously described.

[0413] Fusion transcripts of SEQ ID NO: 911-17492 were identified.

[0414] In the following tables, columns will be referenced as follow: [0415] 1. Frequency in the cohort [0416] 2. Donor Chromosome Exon/2 Donor Chromosome Exon [0417] 3. Donor start Exon/3 Donor start TE [0418] 4. Donor Breakpoint Exon/4Donor Breakpoint TE [0419] 5. Donor strand Exon/5 Donor strand TE [0420] 6. Donor transcript (i.e. Donor_tx_name_Exon)/6 Acceptor chromosome exon [0421] 7. Acceptor Chromosome TE/7 acceptor breakpoint exon [0422] 8. Acceptor Breakpoint TE/8 acceptor end exon [0423] 9. Acceptor end TE/9 acceptor strand exon [0424] 10. Acceptor strand TE/10 acceptor transcript (i.e. Acceptor_tx_name_Exon) [0425] 11. Fusion type/11 fusion type

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DESCRIPTION OF THE SEQUENCES

[0426]

TABLE-US-00072 SEQ ID NO Description 1-117 LUAD peptide sequences from P1 application 118-431 Table 3 from P1 application Fusion sequences with exon donor 432-910 Table 4 from P1 application Fusion sequence with TE donor 911-972 New sequences-TCGA-ACC Fusion sequences with exon donor 973-1237 New sequences-TCGA-BLCA Fusion sequences with exon donor 1238-1466 New sequences-TCGA-BRCA Fusion sequences with exon donor 1467-1632 New sequences-TCGA-CESC Fusion sequences with exon donor 1633-1696 New sequences-TCGA-CHOL Fusion sequences with exon donor 1697-1801 New sequences-TCGA-COAD Fusion sequences with exon donor 1802-1925 New sequences-TCGA-DLBC Fusion sequences with exon donor 1926-2205 New sequences-TCGA-ESCA Fusion sequences with exon donor 2206-2393 New sequences-TCGA-GBM Fusion sequences with exon donor 2394-2608 New sequences-TCGA-HNSC Fusion sequences with exon donor 2609-2721 New sequences-TCGA-KICH Fusion sequences with exon donor 2722-2890 New sequences -TCGA-KIRC Fusion sequences with exon donor 2891-2986 New sequences-TCGA-KIRP Fusion sequences with exon donor 2987-3277 New sequences-TCGA-LGG Fusion sequences with exon donor 3278-3435 New sequences-TCGA-LIHC Fusion sequences with exon donor 3436-3624 New sequences-TCGA-LUAD Fusion sequences with exon donor 3625-3946 New sequences-TCGA-LUSC Fusion sequences with exon donor 3947-4001 New sequences-TCGA-MESO Fusion sequences with exon donor 4002-4597 New sequences-TCGA-OV Fusion sequences with exon donor 4598-4660 New sequences-TCGA-PAAD Fusion sequences with exon donor 4661-4811 New sequences-TCGA-PCPG Fusion sequences with exon donor 4812-4970 New sequences-TCGA-PRAD Fusion sequences with exon donor 4971-5030 New sequences-TCGA-READ Fusion sequences with exon donor 5031-5150 New sequences-TCGA-SARC Fusion sequences with exon donor 5151-5266 New sequences-TCGA-SKCM Fusion sequences with exon donor 5267-5628 New sequences-TCGA-STAD Fusion sequences with exon donor 5629-5876 New sequences-TCGA-TGCT Fusion sequences with exon donor 5877-5979 New sequences-TCGA-THCA Fusion sequences with exon donor 5980-6119 New sequences-TCGA-THYM Fusion sequences with exon donor 6120-6190 New sequences-TCGA-UCEC Fusion sequences with exon donor 6191-6247 New sequences-TCGA-UCS Fusion sequences with exon donor 6248-6315 New sequences-TCGA-UVM Fusion sequences with exon donor 6316-6441 New sequences-TCGA-ACC Fusion sequences with TE donor 6442-6810 New sequences-TCGA-BLCA Fusion sequences with TE donor 6811-7275 New sequences-TCGA-BRCA Fusion sequences with TE donor 7276-7515 New sequences-TCGA-CESC Fusion sequences with TE donor 7516-7638 New sequences-TCGA-CHOL Fusion sequences with TE donor 7639-7817 New sequences-TCGA-COAD Fusion sequences with TE donor 7818-8055 New sequences-TCGA-DLBC Fusion sequences with TE donor 8056-8703 New sequences-TCGA-ESCA Fusion sequences with TE donor 8704-9163 New sequences-TCGA-GBM Fusion sequences with TE donor 9164-9653 New sequences-TCGA-HNSC Fusion sequences with TE donor 9654-9883 New sequences-TCGA-KICH Fusion sequences with TE donor 9884-10277 New sequences-TCGA-KIRC Fusion sequences with TE donor 10278-10454 New sequences-TCGA-KIRP Fusion sequences with TE donor 10455-11125 New sequences-TCGA-LGG Fusion sequences with TE donor 11126-11547 New sequences-TCGA-LIHC Fusion sequences with TE donor 11548-11890 New sequences-TCGA-LUAD Fusion sequences with TE donor 11891-12574 New sequences-TCGA-LUSC Fusion sequences with TE donor 12575-12698 New sequences-TCGA-MESO Fusion sequences with TE donor 12699-13750 New sequences-TCGA-OV Fusion sequences with TE donor 13751-13828 New sequences-TCGA-PAAD Fusion sequences with TE donor 13829-14029 New sequences-TCGA-PCPG Fusion sequences with TE donor 14030-14419 New sequences-TCGA-PRAD Fusion sequences with TE donor 14420-14524 New sequences-TCGA-READ Fusion sequences with TE donor 14525-14776 New sequences-TCGA-SARC Fusion sequences with TE donor 14777-15051 New sequences-TCGA-SKCM Fusion sequences with TE donor 15052-15902 New sequences-TCGA-STAD Fusion sequences with TE donor 15903-16545 New sequences-TCGA-TGCT Fusion sequences with TE donor 16546-16688 New sequences-TCGA-THCA Fusion sequences with TE donor 16689-16951 New sequences-TCGA-THYM Fusion sequences with TE donor 16952-17101 New sequences-TCGA-UCEC Fusion sequences with TE donor 17102-17261 New sequences-TCGA-UCS Fusion sequences with TE donor 17262-17492 New sequences-TCGA-UVM Fusion sequences with TE donor

TABLE-US-LTS-00001 LENGTHY TABLES The patent application contains a lengthy table section. A copy of the table is available in electronic form from the USPTO web site (). An electronic copy of the table will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3).