WO2022074098A1 - Méthode d'identification de néoantigènes du cancer - Google Patents

Méthode d'identification de néoantigènes du cancer Download PDF

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WO2022074098A1
WO2022074098A1 PCT/EP2021/077649 EP2021077649W WO2022074098A1 WO 2022074098 A1 WO2022074098 A1 WO 2022074098A1 EP 2021077649 W EP2021077649 W EP 2021077649W WO 2022074098 A1 WO2022074098 A1 WO 2022074098A1
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tumor
cells
peptide
peptides
hla
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Alena Gros Vidal
Andrea GARCÍA-GARIJO
María LOZANO-RABELLA
Anna YUSTE ESTEVANEZ
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Fundació Privada Institut D'investigació Oncològica De Vall Hebron
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response

Definitions

  • the invention relates to the field of immunotherapy and, more in particular, to a method for the identification of cancer cell-derived peptides which can be used in the immunotherapy of cancer.
  • IL-2 interleukin 2
  • adoptive cell transfer of ex vivo expanded tumorinfiltrating lymphocytes and monoclonal antibodies that “release the brakes” of T cells by blocking the inhibitory signals that lymphocytes receive in the tumor microenvironment are a few examples of immunotherapies that have shown encouraging results in patients with metastatic melanoma.
  • T lymphocytes which play an important role in the antitumor activity of cancer immunotherapies, mediate specific tumor recognition through the interaction of the T-cell receptor (TCR, expressed by T cells) with a peptide derived from tumor antigen presented by the major histocompatibility complex (MHC, expressed on the surface of target cells; referred to as human leucocyte antigen, HLA, in human).
  • TCR T-cell receptor
  • MHC major histocompatibility complex
  • HLA human leucocyte antigen
  • the TCR recognizes peptides presented in the context of HLA-II and, upon recognition, CD4+ T cells can acquire T helper functions that contribute to orchestrate the immune response.
  • HLA-II expression is typically restricted to professional antigen presenting cells such as dendritic cells, monocytes or B cells
  • HLA-II expression can be upregulated in epithelial cells, cancer cells and other cell types under inflammatory conditions, and can also lead to direct target recognition and lysis by CD4+ T cells.
  • CD8+ T cells can recognize peptides presented on HLA-L
  • HLA-II expression is ubiquitous and the recognition of specific peptides on HLA- I by CD8+ T cells leads to the acquisition of cytolytic activity and target cell lysis.
  • Much research has been devoted to identifying what peptides are presented on HLA and trying to exploit these therapeutically.
  • Accumulating evidence supports that lymphocytes targeting neo, non-self, peptides arising from tumor-specific genetic alterations play an important role in the antitumor efficacy of cancer immunotherapies.
  • TILs derived from NSCLC and melanoma are frequently enriched in mutation-specific lymphocytes.
  • Retrospective studies have shown that patients that exhibited complete tumor regressions following tumorinfiltrating lymphocyte (TIL) therapy have a higher tumor mutation burden and TILs from responders frequently contain neoantigen-specific lymphocytes.
  • TIL tumorinfiltrating lymphocyte
  • Active immunization strategies employed to treat patients rely on the identification of the non- synonymous mutations (NSM) by tumor whole exome sequencing (WES), in silico peptide HLA binding affinity prediction and prioritization of a variable number of candidate neoantigens, to manufacture RNA, synthetic short or long peptides or dendritic cell-based vaccines of unique compositions.
  • NSM non- synonymous mutations
  • WES tumor whole exome sequencing
  • tumor biopsies or tumor cell lines can be used as a source to identify HLA-I or HLA-II peptides via immunopeptidomics which relies on the study of the tumor HLA ligandome.
  • neoantigen identification from solid cancers is restricted to peptides presented on HLA-I, but not HLA-IL Moreover, this technique is limited by the need to generate an autologous tumor cell line or to obtain a tumor biopsy, which is often not possible.
  • immunopeptidomics based identification of neoantigens from the tumor biopsy or tumor cell lines can successfully identify all the mutated peptides presented on HLA-I or HLA-II is unclear.
  • T-cell therapies targeting neoantigens require the identification of T cells or T-cell receptors capable of recognizing neoantigens that can be used to treat patients with cancer.
  • T cells from a patient or donor need to be screened for neoantigen recognition in order to identify T cells and/or TCRs that recognize a specific antigen.
  • autologous T cells isolated from the blood or tumor cancer patients can be screened for recognition of peptides prioritized based on in silico HLA prediction binding algorithms or peptides identified on tumor cell surface HLA via immunopeptidomics by staining T cells using HLA multimers or measuring T-cell effector function (typically IFN-gamma release) following coculture with autologous antigen presenting cells (APCs) pulsed with the minimal mutated peptides.
  • APCs autologous antigen presenting cells
  • some in silico HLA binding prediction algorithms can inaccurately predict neoantigens and this frequently leads to the generation of highly complex and costly peptide or HLA multimer libraries.
  • this may miss clinically relevant neoantigen-specific lymphocytes.
  • T cell populations can be screened for neoantigen recognition using a personalized screening approach.
  • This latter strategy consists in screening T cells for recognition of all the NSM mutations identified, which can be encoded by minigene DNA constructs or synthesized in the form of long peptides and transfected into or pulsed onto autologous APCs, encoding for all the HLAI and HLAII molecules.
  • This approach is cumbersome and time-consuming and requires screening of one or more T cell populations of interest from each of the patients screened for neoantigen identification.
  • the authors of the present invention have developed an ex vivo immunopeptidomics-based method that allows to empirically identify the neoepitopes that can be naturally processed and presented on cell surface HLA-I and/or HLA-II molecules in a personalized fashion.
  • WES tumor and normal whole exome sequencing
  • NSM tumor-specific non-synonymous mutations
  • Enough tandem minigenes (TMGs; mutated minigenes stringed together) or peptide pools (PPs) are designed and synthesized in order to interrogate all the candidate neoantigens identified by WES.
  • the in vitro transcribed RNA generated using the TMGs as a template is then transfected or peptides are pulsed onto autologous APCs, such as B cells or immature dendritic cells of the patient of interest, which express the autologous HLA-I and HLA-II molecules. These cells are finally used as a source to elute ligands bound to HLA-I and/or HLA-II through immunopeptidomics.
  • This enables the empirical identification of the mutated peptides that are naturally processed and presented, leading to the direct identification of vaccine and T- cell therapy targets that could be used to develop personalized cancer immunotherapies.
  • Such an ex vivo personalized immunopeptidomics based approach is advantageous in that it overcomes the need to obtain a relatively large amount of tumor to perform HLA-I and HLA- II peptide elution, the dependence on in silico prediction, which often leads to the identification of peptides that are not really presented on the cell surface and are not accurate for HLA-II peptide prediction, and the need to screen T cells to validate the presentation of neoantigens on cell surface HLA.
  • by forcing the expression of all candidate NSM from the RNA encoding for mutated minigenes or peptides it could be more sensitive than immunopeptidomics based assays starting from fresh tumor biopsies or tumor cell lines.
  • the invention relates to a method for the isolation of a neoantigen peptide suitable for the generation of an immune response against a tumor found in a subject comprising the steps of:
  • step (iii) Recovering the peptides which are bound to the HLA complexes isolated in step (ii), said peptides being neoantigen peptides suitable for the generation of an immune response against a tumor.
  • the invention relates to a method for the preparation of an immunogenic or a vaccine composition for the generation of an immune response against a tumor containing a mutation comprising:
  • the invention relates to an immunogenic or vaccine composition which has been obtained by the method as defined in the previous paragraph.
  • the invention in another aspect, relates to a T-cell product comprising T-cells which specifically recognize the neoantigen peptide isolated by the method of the invention or a peptide comprising the sequence of said neoantigen.
  • the invention relates to an immunogenic or vaccine composition according to the invention or the T-cell product according to the invention for use in medicine or for use in a method of preventing or treating a disease in a subject which requires the generation of an immune response against the neoantigen in the subject.
  • the invention relates to method for obtaining a T-cell product comprising T-cells which specifically recognize the neoantigen isolated by the method of the invention or a peptide containing the sequence of said neoantigen comprising (i) Isolating a neoantigen peptide by the method according to the invention, wherein said neoantigen peptide comprises the mutation found in the tumor,
  • step (ii) Contacting the neoantigen peptide obtained in step (i) with a T cell population and
  • FIG. 1 Schematic representation of ex vivo immunopeptidomics-based assay for direct identification of candidate neoantigens for the development of vaccines and T-cell therapies.
  • a peripheral blood sample and a tumor biopsy are obtained from a patient. Tumor DNA is extracted from the tumor biopsy and whole-exome sequencing (WES) is performed to identify non-synonymous somatic mutations (NSMs) compared to normal DNA extracted from PBMCs.
  • WES whole-exome sequencing
  • NSMs non-synonymous somatic mutations
  • TMG Tandem minigene constructs encoding 25 minigenes, each representing one NSMs identified (mutant 25-mers), were used as templates to generate in vitro transcribed (IVT) RNA.
  • IVTT in vitro transcribed
  • autologous APCs are isolated and sometimes expanded ex vivo to obtain large numbers.
  • the mutated epitopes identified can be used to develop neoantigen-based vaccines or could be tested for recognition using autologous T cell populations or candidate TCRs that could be further expanded or used to generate an infusion bag enriched in neoantigen- reactive T cells for ACT (7).
  • Figure 2 Characterization of peptides eluted from HLA-I of VHIO-029 B cells transfected with TMG RNA encoding for the non-synonymous mutations identified by WES. a) Number of total unique peptides identified by LC- MS/MS of B cells transfected with individual TMGs 1-12. b) Length distribution of the unique peptides after pooling data from all TMG- electroporated B cells, c) Percentage of unique peptides predicted to bind to VHIO-029 HLA molecules according to NetMHCpan4.1 . Only peptides >7 AA were included. Peptides predicted to bind to any of VHIO-029 HLAs with EL ⁇ 0,5%-tile rank were considered strong binders, >0,5 to ⁇ 2 weak binders, and >2%-tile rank non-binders.
  • Figure 3 Number and category of peptides eluted from TMG-transfected B cells. Peptides eluted from TMG1-12-transfected B cells mapping to their corresponding TMG AA sequence are plotted. For each TMG the eluted peptides were classified as mutated, wild type or artificial junction peptides.
  • FIG. 4 Mutated peptides derived from NSM identified in VHIO-29 tumor predicted to bind to the patient’s HLA molecules. Dot plot shows the lowest %-tile rank (y-axis) of each mutated peptide derived from the mutations encoded in TMG1-12 (x-axis). Only peptides considered binders are displayed. Eluted peptides that were uniquely identified in electroporated B-cells are highlighted in black, peptides identified in both the autologous tumor cells (TCL) and electroporated B-cells are displayed in red and peptides predicted to bind but not eluted are shown in grey. NetMHCPan 4.1 was employed for prediction, all peptide lengths (8-14mers) were allowed. Peptides predicted to bind to any of VHIO-029 HLAs with ELL ⁇ 2%-tile rank were considered binders.
  • Figure 5 Characterization of peptides eluted from HLA-I from VHIO-029 tumor cell line, a) Number of total unique peptides identified by LC-MS/MS from VHIO-029 tumor cell line (TCL). b) Length distribution of peptides, c) Percentage of eluted peptides predicted to bind to VHIO-029 HLA molecules. Only peptides >7 AA were included. Peptides predicted to bind to any of VHIO-029 HLAs with ELL ⁇ 0.5%-tile rank were considered strong binders, >0,5 to ⁇ 2 weak binders, and >2%-tile rank non-binders.
  • FIG. 6 Recognition of GEMIN5 P .SI36OL and ETV1 P .E45SK by TILs and identification of HLA restriction element responsible fortheir presentation.
  • COS7 cells were transiently transfected with plasmids encoding for the individual HLA alleles of VHIO-029 and pulsed with the mutated peptide specified.
  • Anti-CD3 non-specific stimulation and B cells pulsed with the wt or mutated minimal peptide were used as controls. Recognition of the mutant peptide was assessed by measuring 4-1 BB upregulation on live CD8+ lymphocytes.
  • Figure 7 Number and category of peptides eluted from VHIO-029 PP-pulsed B cells. Peptides mapping to each individual PP1-PP6 are shown. For each PP, the eluted peptides were classified as mutated or wild type.
  • FIG. 8 Identification of neoantigens by screening VHIO-029 TILs for recognition of B cells transfected with TMGs.
  • TILs were expanded from 12 independent tumor fragments for 2-4 weeks in IL-2 and screened for recognition of B cells transfected with TMG1-12 RNA, encoding for all NSM identified by WES.
  • Media and anti-CD3 (OKT3) were used as negative and positive controls, respectively.
  • TIL were considered reactive to a specific TMG when they displayed >40 spots and > than twice the average background compared to the remaining TMGs.
  • TIL-3 was enriched for TMG1 recognition by selecting and expanding 4-1 BB+ TILs following co-culture with B cells transfected with TMG1 .
  • the resulting TIL-34-1 BB+ vs. TMG1 population and TIL-7 were co-cultured with B cells pulsed overnight with the individual 25- mers encoded by TMG1 .
  • Reactivity of TILs to B cells transfected with TMG1 RNA was used as a positive control.
  • the results of the IFN-y ELISPOT assay are shown. Experiments were performed without technical duplicates.
  • Figure 8 Characterization of peptides eluted from HLA-I of VHIO-0008 B cells transfected with TMG RNA encoding for the non-synonymous mutations identified by WES. a) Number of total unique peptides identified by LC- MS/MS of B cells transfected with individual TMGs 1-12. b) Length distribution of the unique peptides after pooling data from all TMG- electroporated B cells, c) Percentage of unique peptides predicted to bind to VHIO-008 HLA molecules. Only peptides >7 AA were included. Peptides predicted to bind to any of VHIO- 008 HLAs with EL ⁇ 0.5%-tile rank were considered strong binders, >0,5 to ⁇ 2 weak binders, and >2%-tile rank not binders.
  • Figure 9 Number and category of peptides eluted from TMG-transfected B cells. Peptides eluted from TMG1-12-transfected B cells mapping to their corresponding TMG AA sequence are plotted. For each TMG the eluted peptides were classified as mutated, wild type or artificial junction peptides
  • Figure 11 Characterization of peptides eluted from HLA-I from VHIO-008 tumor cell line, a) Number of total unique peptides identified by LC-MS/MS from VHIO-008 tumor cell line (TCL). b) Length distribution of peptides, c) Percentage of eluted peptides predicted to bind to VHIO-008 HLA molecules. Only peptides >7 AA were included. Peptides predicted to bind to any of VHIO-008 HLAs with EL ⁇ 0.5%-tile rank were considered strong binders, >0,5 to ⁇ 2 weak binders, and >2%-tile rank non-binders.
  • FIG. 14 Mutated peptides derived from selected NSM identified in VHIO-008 tumor predicted to bind to the patient’s HLA molecules. Dot plot showing the lowest %-tile rank (EL method; y-axis)) of each of the mutant peptides derived from TMG1-12 (x-axis) of VHIO-008 patient predicted to bind to the patient specific HLA molecules according to NetMHCPan 4.1. The peptides uniquely identified in electroporated B-cells are highlighted in black and the ones eluted and identified both in the transfected B cells and in the TCL are displayed in red. NetMHCPan 4.1 was employed for prediction, all peptide lengths (8-14mers) were allowed. Peptides predicted to bind to any of VHIO-008 HLAs with ELL ⁇ 2%-tile rank were considered binders.
  • EL method y-axis
  • TMGs encoding for mutations recognized by neoantigenspecific T cells were transfected independently in 3 different cell types (B cells, DCs and T cells) and were used as a source to elute HLA-I peptides to further identify their amino acid sequence by LC-MS/MS.
  • B cells, DCs and T cells were transfected independently in 3 different cell types (B cells, DCs and T cells) and were used as a source to elute HLA-I peptides to further identify their amino acid sequence by LC-MS/MS.
  • neoantigen-specific T cells were co-cultured against TMG-expressing APCs to ensure that the immunogenic peptides were being expressed, processed and presented.
  • the unelectroporated autologous tumor cell line was used as a reference to assess the HLA-I peptides presented by the tumor that can be detected by direct HLA-I elution or by the tumor-reactive T cells, a) Peptides eluted from TMG1-3-5 mapping to their corresponding TMG AA sequence are plotted for each transfected APC.
  • the eluted peptides (SEQ ID NOs: 104-121 ) were classified as mutated (black), wild type (wild) or artificial junction peptides (grey).
  • the mutated protein and AA the eluted peptides are derived from are specified for mutation and wt.
  • the immunogenic variants are highlighted, b) Recognition of the specific target was assessed by measuring 4- 1 BB upregulation after 20h of co-culture of IVT RNA-electroporated B cells electroporated with the indicated TMG and the effector populations shown.
  • the autologous tumor cell line was tested with and without pre-incubation with IFNg for 24 hours.
  • Anti- CD3 non-specific stimulation and electroporation with irrelevant TMGs were used as positive and negative controls, respectively.
  • Top panel displays the reactivity for MAGEA6_E168K- specific T cells (encoded in TMG1 ), the middle panel for PDS5A_Y1000F specific T cells and bottom MED13_P1691S specific T cells.
  • Two technical MS measurements of purified peptides were performed by LC-MS/MS for each sample. The results from 2 independent experiments are pooled, the conditions were performed twice when available (DCs and TCL only included in one experiment).
  • the invention relates to a method for the isolation of a neoantigen peptide suitable for the generation of an immune response against a tumor found in a subject comprising the steps of
  • step (ii) Recovering HLA complexes from the population of antigen-presenting cells and (iii) Recovering the peptides which are bound to the HLA complexes isolated in step (ii), said peptides being neoantigen peptide suitable for the generation of an immune response against a tumor.
  • nucleic acid peptide refers to peptides that are specific from cancer, tumor, or cell thereof and that are not encoded in a normal, non-mutated host genome.
  • the term “neoantigen” does not necessarily imply that the peptide that is isolated according to the method of the invention is capable of triggering an immune response, although it is likely that it will do so based on the properties used for its isolation when the method of the invention is carried out.
  • the “neoantigen peptide” can also be referred to as “putative neoantigen peptide”. Typically, neoantigens arise as a consequence of somatic mutations.
  • Neoantigens can also arise from the disruption of cellular mechanisms through the activity of viral proteins or as a result of the expression and presentation of foreign viral peptides.
  • Another example can be an exposure of a carcinogenic compound, which in some cases can lead to a somatic mutation as a consequence of aberrant DNA replication, aberrant RNA splicing and/or aberrant repair in the cancer, tumor, or cell thereof.
  • Neoantigens appearing in cancer, tumor, or cell thereof include (1 ) tumor antigens that arise from a tumor-specific mutation(s) which alters the amino acid sequence of genome encoded proteins; (2) tumor antigens having tumor specific expression that arises from retained introns, alternative open reading frames (ORFs) within coding genes, antisense transcripts, defective ribosomal products (DRiPs), "non-coding" regions of the genome, 5’ and 3’ untranslated regions (UTRs), overlapping yet out-of-frame alternative ORFs in annotated protein-coding genes, long non-coding RNAs (IncRNAs), pseudogenes and other transcripts currently annotated as non-protein coding; or (3) novel unannotated open reading frames (nuORFs) that arise from a tumor-specific mutation(s) in unannotated open reading frames.
  • ORFs alternative open reading frames
  • ORFs alternative open reading frames
  • DDRiPs defective ribosomal products
  • UTRs untranslated regions
  • tumor refers to, and is interchangeably used with one or more cancer cells, cancer tissues (including metastases), malignant tumor cells, or malignant tumor tissue, that can be placed or found in one or more anatomical locations in a human body.
  • patient includes both individuals that are diagnosed with a condition (e.g., cancer) as well as individuals undergoing examination and/or testing for the purpose of detecting or identifying a condition.
  • a patient having a tumor refers to both individuals that are diagnosed with a cancer as well as individuals that are suspected to have a cancer.
  • the cancer can be selected from the group consisting of skin cancer, lung cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, head and neck cancer, gastric cancer, intestinal cancer, pancreatic cancer, breast cancer, gynecological cancer and a cancer caused by a mismatch repair deficiency.
  • the skin cancer can be selected from the group consisting of basal cell carcinoma, squamous cell carcinoma, Merkel cell carcinoma, and melanoma.
  • the cancer can be a melanoma.
  • the cancer is a hypopharyngeal carcinoma.
  • the cancer is a primary cancer.
  • the cancer is a metastatic cancer.
  • immune response when used herein, has its ordinary meaning in the art, and refers to the action of a cell of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, neutrophils, etc.) and soluble macromolecules produced by any of these cells or the liver (e.g., antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from a subject of invading pathogens, cells or tissues infected with pathogens, or cancerous cells or other abnormal/diseased-associated cells.
  • a cell of the immune system e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, neutrophils, etc.
  • soluble macromolecules produced by any of these cells or the liver e.g., antibodies
  • Step (i) of the method of the invention effecting the presentation of tumor-specific peptide on HLA-expressing cells
  • the method of the present invention involves effecting the presentation of the tumor-specific peptides on HLA in the HLA-expressing cell isolated from the subject.
  • This step can be carried out by contacting a population of HLA-expressing cells isolated from the subject with at least one tumor-specific peptide or with a nucleic acid encoding said tumorspecific peptide under conditions adequate for the formation of a complex between said tumor-specific peptide and HLA molecules in said cells or between the tumor-specific peptide encoded by the nucleic acid and HLA molecules in said cells.
  • tumor-specific peptide refers to a peptide that contains one or more mutations with respect to the wild-type peptide, wherein the mutations are point mutations, substitutions and insertions or deletions.
  • the tumor-specific peptide results from a RNA splicing in the tumor cell which does not occur in the wild-type cell.
  • Tumor-specific peptides that can be used in step (i) in the present invention include, without limitation, (1 ) tumor antigens that arise from a tumor-specific mutation(s) which alters the amino acid sequence of genome encoded proteins; (2) tumor antigens having tumor specific expression that arises from retained introns, alternative open reading frames (ORFs) within coding genes, antisense transcripts, defective ribosomal products (DRiPs), "noncoding" regions of the genome, 5’ and 3’ untranslated regions (UTRs), overlapping yet out- of-frame alternative ORFs in annotated protein-coding genes, long non-coding RNAs (IncRNAs), pseudogenes and other transcripts currently annotated as non-protein coding; or (3) novel unannotated open reading frames (nuORFs) that arise from a tumor-specific mutation(s) in unannotated open reading frames.
  • ORFs alternative open reading frames
  • DDRiPs defective ribosomal products
  • UTRs untran
  • Tumor-specific peptides suitable for use in step (i) of the method of the invention can be identified by any method that allows the identification of peptides which contain mutations with respect to the wild-type peptide.
  • the omics data may cover the whole genome, exome, transcriptome, or portions thereof.
  • the identification of tumor-specific peptides is carried out by exome sequencing of the tumor cell derived from the patient and a non-tumor matched cell or tissue sample.
  • the identification of tumor-specific peptides is carried out by RNA sequencing.
  • DNA data may also be provided from an already established sequence record (e.g., SAM, BAM, FASTA, FASTQ, or VCF file) from a prior sequence determination-
  • the data sets are reflective of a tumor and a matched normal sample of the same patient to so obtain patient and tumor specific information.
  • genetic germ line alterations not exclusively present in the tumor e.g., silent mutation, SNP, etc.
  • the tumor sample may be from an initial tumor, from the tumor upon start of treatment, from a recurrent tumor or metastatic site, etc.
  • the matched normal sample of the patient may be blood, or non-diseased tissue from the same tissue type as the tumor.
  • the tumor-specific polypeptides to be used in step (i) of the method of the invention can be pre-filtered based on different criteria before introducing them or nucleic acids encoding them into an APC. Such filtering will increase the likelihood that the tumor-specific peptide, if selected in the subsequent steps of the method according to the present invention, acts as a neoantigen capable of therapeutically desirable response.
  • downstream analysis need not take into account silent mutations for the purpose of the methods presented herein.
  • preferred mutation analyses will provide in addition to the type of mutation (e.g., deletion, insertion, transversion, transition, translocation) also information of the impact of the mutation (e.g., non-sense, missense, etc.) and may as such serve as a first content filter through which silent mutations are eliminated.
  • neoantigens can be selected for further consideration where the mutation is a frame-shift, non-sense, and/or missense mutation.
  • tumor-specific peptides may also be subject to detailed analysis for sub-cellular location parameters. For example, tumor-specific peptides may be selected for further consideration if the tumor-specific peptides are identified as having a membrane associated location (e.g., are located at the outside of a cell membrane of a cell) and/or if an in silico structural calculation confirms that the tumor-specific peptide is likely to be solvent exposed, or presents a structurally stable epitope
  • tumorspecific peptides are especially suitable for use herein where omics (or other) analysis reveals that the tumor-specific peptides are actually expressed.
  • Identification of expression and expression level of a tumor-specific peptides can be performed in all manners known in the art and preferred methods include quantitative RNA (hnRNA or mRNA) analysis and/or quantitative proteomics analysis.
  • the threshold level for inclusion of tumorspecific peptides will be an expression level of at least 20 percent, at least 30 percent, at least 40 percent, or at least 50 percent of expression level of the corresponding matched normal sequence, thus ensuring that the tumor-specific peptides is at least potentially ‘visible’ to the immune system. Consequently, it is generally preferred that the omics analysis also includes an analysis of gene expression (transcriptomic analysis) to so help identify the level of expression for the gene with a mutation.
  • RNA sequence information may be obtained from reverse transcribed polyA+-RNA, which is in turn obtained from a tumor sample and a matched normal (healthy) sample of the same patient.
  • polyA+-RNA is typically preferred as a representation of the transcriptome
  • other forms of RNA hn-RNA, non-polyadenylated RNA, siRNA, miRNA, etc.
  • RNA quantification and sequencing is performed using RNA-seq, qPCR and/or rtPCR based methods, although various alternative methods (e.g., solid phase hybridization- based methods) are also deemed suitable.
  • transcriptomic analysis may be suitable (alone or in combination with genomic analysis) to identify and quantify genes having a cancer- and patient-specific mutation.
  • proteomics analysis can be performed in numerous manners to ascertain actual translation of the RNA of the tumor-specific peptides, and all known manners of proteomics analysis are contemplated herein.
  • particularly preferred proteomics methods include antibody-based methods and mass spectroscopic methods.
  • the proteomics analysis may not only provide qualitative or quantitative information about the protein per se, but may also include protein activity data where the protein has catalytic or other functional activity.
  • Further suitable methods of identification and even quantification of protein expression include various mass spectroscopic analyses (e.g., selective reaction monitoring (SRM), multiple reaction monitoring (MRM), and consecutive reaction monitoring (CRM)).
  • SRM selective reaction monitoring
  • MRM multiple reaction monitoring
  • CCM consecutive reaction monitoring
  • tumor-specific peptides will be visible to the immune system as the tumor-specific peptides also need to be presented on the MHC complex of the patient. Indeed, only a fraction of the tumor-specific peptides will have sufficient affinity for presentation, and the large diversity of MHC complexes will preclude use of most, if not all, common tumor-specific peptides. Consequently, in the context of present invention it should thus be readily apparent that tumor-specific peptides will be more likely effective where the tumor-specific peptides are bound to and presented by the MHC complexes on the cell surface.
  • the tumor-specific peptide used in step (i) of the method of the invention is preselected in vitro using isolated HLA molecules and/or in silico for its capacity for binding to HLA prior to its incorporation in the HLA complexes of the HLA- expressing cells used in step (i).
  • the preselection step requires determining the particular HLA-type of a patient followed by prediction of the binding of the tumor-specific peptide to the HLA.
  • the HLA- type determination includes at least three MHC-I sub-types (e.g., HLA-A, HLA-B, HLA-C) and at least three MHC-II sub-types (e.g., HLA-DP, HLA-DQ, HLA-DR), preferably with each subtype being determined to at least 4-digit depth. However, greater depth (e.g., 6 digit, 8 digit) is also contemplated herein.
  • a structural solution for the HLA-type is calculated or obtained from a database, which is then used in a docking model in silico to determine binding affinity of the (typically filtered) tumor-specific peptides to the HLA structural solution.
  • suitable systems for determination of binding affinities include the NetMHC platform (see e.g., Nucleic Acids Res. 2008 Jul 1 ; 36(Web Server issue): W509-W512.).
  • Tumor-specific peptides with high affinity e.g., having a KD of less than 200 nM, less than 100 nM, less than 75 nM, less than 50 nM
  • a previously determined HLA-type are then selected for therapy creation, along with the knowledge of the MHC-I/II subtype.
  • HLA determination can be performed using various methods in wet-chemistry that are well known in the art, and all of these methods are deemed suitable for use herein.
  • the HLA-type can also be predicted from omics data in silico using a reference sequence containing most or all of the known and/or common HLA-types as is shown in more detail below. Given the substantial sequence similarity within the HLA haplotypes and exceptionally high variability of the loci, some established approaches therefore rely on specific HLA enrichment and sequencing techniques.
  • algorithms are also frequently used to accurately predict HLA genotyping based from NGS. Some of these algorithms include OptiType, PHLAt, HLA-arcas or HLA-LA.
  • contemplated methods further employ one or more reference sequences that include a plurality of sequences of known and distinct HLA alleles.
  • a typical reference sequence may be a synthetic (without corresponding human or other mammalian counterpart) sequence that includes sequence segments of at least one HLA-type with multiple HLA-alleles of that HLA-type.
  • suitable reference sequences include a collection of known genomic sequences for at least 50 different alleles of HLA-A.
  • the reference sequence may also include a collection of known RNA sequences for at least 50 different alleles of HLA- A.
  • the reference sequence is not limited to 50 alleles of HLA-A, but may have alternative composition with respect to HLA-type and number/composition of alleles.
  • the reference sequence will be in a computer readable format and will be provided from a database or other data storage device.
  • suitable reference sequence formats include FASTA, FASTQ, EMBL, GCG, or GenBank format, and may be directly obtained or built from data of a public data repository (e.g., IMGT, the International ImMunoGeneTics information system, or The Allele Frequency Net Database, EUROSTAM, URL: www.allelefrequencies.net).
  • the reference sequence may also be built from individual known HLA-alleles based on one or more predetermined criteria such as allele frequency, ethnic allele distribution, common or rare allele types, etc.
  • tumor-specific peptides and HLA-type are identified, further computational analysis can be performed by docking tumor-specific peptides to the HLA and determining best binders (e.g., lowest KD, for example, less than 500nM, or less than 250nM, or less than 150nM, or less than 50nM), for example, using NetMHC. It should be appreciated that such approach will not only identify specific tumor-specific peptides that are genuine to the patient and tumor, but also those tumor-specific peptides that are most likely to be presented on a cell and as such most likely to elicit an immune response with therapeutic effect.
  • best binders e.g., lowest KD, for example, less than 500nM, or less than 250nM, or less than 150nM, or less than 50nM
  • HLA-matched tumor-specific peptides can be biochemically validated in vitro prior to inclusion in the nucleic acid encoding the epitope as payload into the HLA-expressing cells as is further discussed below.
  • matching of the patient’s HLA-type to the patient- and tumor-specific peptides can be done using systems other than NetMHC, and suitable systems include NetMHC II, NetMHCpan, IEDB Analysis Resource (URL immuneepitope.org), RankPep, PREDEP, SVMHC, Epipredict, HLABinding, and others (see e.g., J Immunol Methods 2011 ;374:1 4).
  • the collection of tumor-specific peptides sequences in which the position of the altered amino acid is moved can be used.
  • modifications to the tumor-specific peptides may be implemented by adding N- and/or C-terminal modifications to further increase binding of the expressed neoantigen to the patient’s HLA-type.
  • tumor-specific peptides may be native as identified or further modified to better match a particular HLA-type.
  • binding of corresponding wildtype sequences can be calculated to ensure high differential affinities. For example, especially preferred high differential affinities in MHC binding between the neoantigen and its corresponding wildtype sequence are at least 2-fold, at least 5 -fold, at least 10-fold, at least 100-fold, at least 500- fold, at least 1000-fold, etc.
  • the at least one tumor-specific peptide can bind in silico to an HLA class I molecule with a stability>2 h. In some configurations, the at least one tumor-specific peptide can bind in silico to an HLA class I molecule an affinity of ⁇ 500 nM. In some embodiments, the at least one tumor-specific peptide can bind in silico to an HLA class I molecule with an affinity of ⁇ 250 nM.
  • the at least one tumor-specific peptide can bind in silico to an HLA Class I molecule with an affinity of ⁇ 550 nM, ⁇ 500 nM, ⁇ 450 nM, ⁇ 400 nM, ⁇ 350 nM, ⁇ 300 nM, ⁇ 250 nM, or ⁇ 200 nM.
  • the at least one tumor-specific peptide can bind in vitro to an HLA class I molecule with an affinity of ⁇ 4.7 log (IC50, nM), ⁇ 4.6 log (IC50, nM), ⁇ 4.5 log (IC50, nM), ⁇ 4.4 log (IC50, nM), ⁇ 4.3 log (IC50, nM), ⁇ 4.2 log (IC50, nM), ⁇ 4.1 log (IC50, nM), ⁇ 4.0 log nM), ⁇ 3.9 log (IC50, nM), ⁇ 3.8 log (IC50, nM), or ⁇ 3.7 log (IC50, nM).
  • the at least one tumor-specific peptide can bind in vitro to an HLA class I molecule with an affinity of ⁇ 4.7 log (IC50, nM). In some embodiments, the at least one tumor-specific peptide can bind in vitro to an HLA class I molecule with an affinity of ⁇ 3.8 log (IC50, nM). In some embodiments, the at least one tumor-specific peptide can bind in vitro to an HLA class I molecule with an affinity of ⁇ 3.7 log (IC50, nM). In some embodiments, the at least one tumor-specific peptide can bind in vitro to an HLA class I molecule with an affinity of ⁇ 3.2 log (IC50, nM).
  • the at least one tumor-specific peptide is selected if it belongs to the subset of peptides within the top 2-quantile (i.e. above the median), in the top 3-quantile, in the top 4-quantile, in the top 5-quantile, in the top 6-quantile, in the top 7-quantile, in the top 8-quantile, in the top 9-quantile, in the top 10-quantile, in the top 50-quantille, in the top 100- quantile (percentile), in the top 1000-quantile or higher quantiles in terms of their in silico binding affinity to HLA class I, within the top quantile in terms of their in silico binding affinity to HLA class I.
  • the at least one tumor-specific peptide is selected if it belongs to the top 50, top 40, top 30, top 20, top 10, top 5, top 4, top 3, top 2, top 1 , top 0,5, top 0,4, top 0,3, top 0,2, top 0,1 percentile rank.
  • the tumor-specific peptides may be compared against a database that contains known human sequences (e.g., of the patient or a collection of patients) to so avoid use of a human-identical sequence.
  • filtering may also include removal of tumor-specific peptides that are due to SNPs in the patient where the SNPs are present in both the tumor and the matched normal sequence.
  • dbSNP The Single Nucleotide Polymorphism Database
  • NCBI National Center for Biotechnology Information
  • NHGRI National Human Genome Research Institute
  • SNPs single nucleotide polymorphisms
  • STRs microsatellite markers or short tandem repeats
  • MNPs multinucleotide polymorphisms
  • heterozygous sequences and (6) named variants.
  • the dbSNP accepts apparently neutral polymorphisms, polymorphisms corresponding to known phenotypes, and regions of no variation.
  • the tumor-specific peptides are preselected prior to their exposure to the HLA-expressing cells by identifying the presence of MHC-binding motif.
  • the MHC-binding motif means a structural feature that is common in peptides binding to particular MHC molecules (allelic polymorphism) and is necessary for forming stable complexes with the MHC molecules.
  • the peptide length varies from 12 to 18 amino acids, and even longer peptides can bind because both ends of the peptide-binding groove are open.
  • Most of MHC II molecules can accommodate up to 4 residues (called “anchor residues”) related to the binding, at relative positions P1 , P4, P6 and P9 contained in a nonameric core region. However, this core region may vary in distance from the N terminus of the peptide. In many cases, 2 to 4 N-terminal residues precede the core region.
  • the P1 anchor residue is located at position 3, 4, or 5 in many peptides that can form with complexes with the MHC II molecules.
  • Peptides eluted from, for example, HLA-DR molecules can share a hydrophobic P1 anchor such as tyrosine, phenylalanine, tryptophan, methionine, leucine, isoleucine, or valine.
  • the positions and types of the anchor residues can be estimated from the peptide-binding motifs of frequently occurring MHC molecules.
  • a computer algorithm that permits motif validation in peptide sequences can be obtained from, for example, “Tepitope” (www.vaccinome.com, J. Hammer, Nutley, USA).
  • the tumor-specific peptides are preselected prior to their exposure to the HLA-expressing cells by measuring in vitro its capacity to bind to MHC.
  • the ability to bind to MHC may be tested by a method generally known to those skilled in the art using the detected peptide itself (e.g., a synthetic peptide may be utilized) and a desired MHC molecule (Kropshofer H et al., J. Exp. Med. 1992, 175, 1799-1803; Vogt A B et al., J. Immunol. 1994, 153, 1665-1673; and Sloan V S et al., Nature 1995, 375, 802-806).
  • cellular binding assay using MHC molecule-expressing cell lines and biotinylated peptides may be used to test the ability to bind to MHC (Arndt S 0 et al., EMBO J., 2000, 19, 1241-1251 ).
  • the relative ability of each peptide to bind to MHC may be determined by measuring a concentration necessary to reduce the binding of a labelled reporter peptide to 50% (IC50).
  • each identified peptide may be used as such a peptide, or a peptide having a sequence common in identified peptides (core sequence) may be used.
  • the peptide to be detected is considered to depend on the types of MHC molecule allotypes, the strength of binding affinity for a MHC molecule, etc.
  • the patient and tumorspecific peptides may be filtered to remove those known sequences, yielding a sequence set with a plurality of tumor-specific peptides having substantially reduced false positives.
  • the tumor -specific peptide used in step (i) of the method of the invention has not been preselected in vitro using isolated HLA molecules and/or in silico for its capacity for binding to HLA prior to its incorporation in the HLA complexes of the HLA-expressing cells used in step (i).
  • the tumor-specific peptides can be at least 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more amino acid residues in length.
  • the tumor-specific peptides can be at most 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500 or less amino acid residues in length.
  • the tumor-specific peptides has a total length of at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, or at least 500 amino acids.
  • the tumor-specific peptides have a total length of at most 8, at most 9, at most 10, at most 11 , at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21 , at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, at most 30, at most 40, at most 50, at most 60, at most 70, at most 80, at most 90, at most 100, at most 150, at most 200, at most 250, at most 300, at most 350, at most 400, at most 450, or at most 500 amino acids.
  • the tumor-specific peptides can have a pl value of about 0.5 and about 12, about 2 and about 10, or about 4 and about 8. In some embodiments, the tumor-specific peptides can have a pi value of at least 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or more. In some embodiments, the tumor-specific peptides can have a pi value of at most 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or less.
  • the tumor specific peptide is encoded by a region of the tumor DNA which contains a mutation with respect to a reference DNA.
  • the mutation is a misssense mutation.
  • the amino acid which carries the mutation with respect to the wildtype peptide appears in the peptide in the vicinity of a wild-type N-terminal region, of a wildtype C-terminal region or is flanked by N-terminal and a C-terminal regions.
  • the length of the N- and C-terminal flanking regions is not particularly limitative as long as the resulting peptide can be presented in the HLA molecule.
  • the N-terminal flanking region is of at least 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33,
  • the N-terminal flanking region is of at most 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9,
  • the C-terminal flanking region is of at least 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33,
  • the C-terminal flanking region is of at most 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9,
  • the position in the tumor-specific peptide containing the missense mutation is centrally located with respect to the N-terminal and C-terminal regions.
  • the N-terminal and/or the C-terminal flaking regions consist each of at least 7 amino acids.
  • the N-terminal and the C-terminal regions consist each of 12 amino acids.
  • the tumor-specific peptide further comprises modifications which increase in vivo half-life, cellular targeting, antigen uptake, antigen processing, MHC affinity, MHC stability, or antigen presentation.
  • the modification is conjugation to a carrier protein, conjugation to a ligand, conjugation to an antibody, PEGylation, polysialylation HESylation, recombinant PEG mimetics, Fc fusion, albumin fusion, nanoparticle attachment, nanoparticulate encapsulation, cholesterol fusion, iron fusion, acylation, amidation, glycosylation, side chain oxidation, phosphorylation, biotinylation, the addition of a surface active material, the addition of amino acid mimetics, or the addition of unnatural amino acids.
  • the HLA-expressing cells isolated from the same subjects are contacted with the tumor-specific peptide, with a plurality of tumorspecific peptides, with a nucleic acid encoding the peptide, with a nucleic acid encoding a plurality of tumor-specific peptides or with a plurality of nucleic acids encoding one or more tumor-specific peptides.
  • HLA-expressing cell refers to cells that contain the gene loci encoding the human MHC class I or class II proteins and that, accordingly, are capable of displaying antigens complexed with major histocompatibility complexes (MHCs) on their surfaces presenting them there to specific cells, in particular cytotoxic T-lymphocytes or T- helper cells.
  • MHCs major histocompatibility complexes
  • Suitable HLA-expressing cells that can be used in the present invention include professional antigen-presenting cells (APC) as well as other cells or cell lines which present antigens originating inside the cell or antigens taken up from outside the cell to cytotoxic T cells.
  • APC professional antigen-presenting cells
  • HEK293 cells or COS7 cells could be used.
  • the cells that can be used in the method of the present invention can be genetically modified so that they express HLAs specific from the subject.
  • the cells are transfected with the polynucleotides encoding the HLAs from the subjects.
  • the transfection can be transient or stable.
  • the endogenous HLA genes of the cells are deleted or mutated (e.g.
  • HLA-expressing cells for use according to the present invention include professional antigen-presenting cells, such as macrophages, B cells, dendritic cells.
  • Dendritic cells are leukocyte populations that present intracellular antigens as well as antigens captured in peripheral tissues to T cells via both MHC class II and I antigen presentation pathways. It is well known that dendritic cells are potent inducers of immune responses and the activation of these cells is a critical step for the induction of antitumoral immunity. Dendritic cells are conveniently categorized as "immature” and “mature” cells, which can be used as a simple way to discriminate between two well characterized phenotypes.
  • Immature dendritic cells are characterized as antigen presenting cells with a high capacity for antigen uptake and processing, which correlates with the high expression of Fey receptor and mannose receptor.
  • the mature phenotype is typically characterized by a lower expression of these markers, but a high expression of cell surface molecules responsible for T cell activation such as class I and class II MHC, adhesion molecules (e. g. CD54 and CD11 ) and costimulatory molecules (e. g., CD40, CD80, CD86 and 4-1 BBL).
  • DCs are obtained from any tissue where they reside, including nonlymphoid tissues such as the epidermis of the skin (Langerhans cells) and lymphoid tissues such as the spleen, bone marrow, lymph nodes and thymus as well as the circulatory system including blood and peripheral blood. Because DCs occur in low numbers in any tissues in which they reside, DCs may be enriched or isolated for use. Any of a number of procedures entailing repetitive density gradient separation, positive selection, negative selection, differential adherence or a combination thereof may be used to obtain enriched populations of DCs. Once the DCs are obtained, they may be cultured in appropriate culture medium to expand the cell population and/or maintain the DCs in a state for optimal antigen uptake, processing and presentation.
  • nonlymphoid tissues such as the epidermis of the skin (Langerhans cells) and lymphoid tissues such as the spleen, bone marrow, lymph nodes and thymus as well as the circulatory system including blood and peripheral blood. Because DC
  • HLA-expressing cells for use according to the present invention include a non-professional antigen presenting cells such as T cells, PBMC and CD14+ monocytes.
  • the HLA-expressing cell is not a cell obtained from the tumor of the subject in which the tumor is found or a cell from a cell line derived from the tumor of the subject in which the tumor is found.
  • the HLA-expressing cell is a cell obtained from the tumor of the subject in which the tumor is found or a cell from a cell line derived from the tumor of subject in which the tumor is found. In some embodiments, the HLA-expressing cell is a cell obtained from a tumor or from a cell line derived from a tumor which is of the same or different type as the tumor of the subject in which the tumor is found. In this case, the cells are treated so as to remove the endogenous HLA and then modified to express the HLA from the subject in which the tumor is found.
  • the HLA-expressing cell is not a cell obtained from a tumor or from a cell line derived from a tumor which is of the same or different type as the tumor of the subject in which the tumor is found.
  • the HLA-expressing cells express in their surface MHC of either class I or class II.
  • Proteins of MHC class I are present on the surface of almost all cells of the body, including most tumor cells.
  • MHC class I proteins are loaded with antigens that usually originate from endogenous proteins or from pathogens present inside cells, and are then presented to naive or cytotoxic T-lymphocytes (CTLs).
  • CTLs cytotoxic T-lymphocytes
  • MHC class II proteins are present on dendritic cells, B- lymphocytes, macrophages and other antigen-presenting cells. They mainly present peptides, which are processed from external antigen sources, i.e. outside of the cells, to T-helper (Th) cells.
  • MHC molecules of class I are heterodimers that consist of two polypeptide chains, a and P2- microglobulin (B2M). The two chains are linked non-covalently via interaction of B2M and the a3 domain. These molecules and are capable of binding a peptide of about 8 to 12 amino acids, but usually 9 or 10 amino acids, if this peptide has suitable binding motifs, and presenting it to cytotoxic T-lymphocytes.
  • the peptide bound by the MHC molecules of class I originates from an endogenous protein antigen.
  • the heavy chain of the MHC molecules of class I is preferably an classical HLA such as HLA-A, HLA-B or HLA-C monomer or a non- classical (MHC-lb), such as HLA-E, HLA-F, HLA-G and HLA-H molecule.
  • HLA-A HLA-A
  • HLA-B HLA-C monomer
  • MHC-lb non- classical
  • MHC molecules of class II consist of an a-chain and a p-chain and are capable of binding a peptide of about 10 to 24 amino acids if this peptide has suitable binding motifs, and presenting it to T-helper cells.
  • the peptide bound by the MHC molecules of class II usually originates from exogenous protein antigen, proteins present in intracellular vesicles or membrane bound proteins.
  • the a-chain and the P-chain are in particular HLA-DR, HLA-DQ, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB and HLA-DP monomers.
  • HLA- A HLA- A
  • HLA-B HLA-C
  • all of these genes have a varying number of alleles. Closely related alleles are combined in subgroups of a certain allele.
  • IMGT/HLA http://www.ebi.ac.uk/ipd/imgt/hla/).
  • HLA-A gene locus
  • HLA-A*02 allele family serological antigen
  • allele subtypes assigned in numbers and in the order in which DNA sequences have been determined e.g. HLA- A*02:01
  • Alleles that differ only by synonymous nucleotide substitutions (also called silent or non-coding substitutions) within the coding sequence are distinguished by the use of the third set of digits (e.g. HLA-A*02:01 :01 ).
  • the HLA class I molecules can be selected from the group consisting of HLA- A*01 :01 , HLA-B*07:02, HLA A*02:01 , HLA-B*07:03, HLA-A*02:02, HLA-B*08:01 , HLA-
  • HLA-B*15:01 A*02:03, HLA-B*15:01 , HLA-A*02:05, HLA-B*15:02, HLA-A*02:06, HLA-B*15:03, HLA-
  • the present invention contemplates any method that allows the presentation of the tumorspecific peptides on the cell surface as complexes with HLA, including pulsing the cells with the peptides so that the peptides can interact with HLA and transfecting the cell with a nucleic acid encoding the peptide so that the peptides are expressed inside the cell from the nucleic acid by the transcriptional and translational machinery of the cell, processed by the cell and bound to the HLA and displayed on the cell surface.
  • Peptide pulsing is accomplished in vitro/ex vivo by exposing HLA-expressing cells to a solution containing the tumor-specific peptides and optionally subsequently removing the antigenic peptide and washing away the unbound excess peptides from the mixture.
  • the HLA-expressing cells can be combined with a plurality of tumor antigen peptides for a few seconds, minutes, or hours, for example about 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 5 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, or more.
  • the concentration of each tumor specific peptide used in the contacting step may be any concentration of about 0.1 , 0.5, 1 , 2, 3, 5, or 10 pg/mL.
  • the concentration of the tumor antigen peptide is about 0.1 to 200 pg/mL, such as about 0.1 to 0.5, 0.5 to 1 , 1 to 10, 10 to 50, 50 to Any concentration of 100, 100 to 150, or 150 to 200 pg/mL.
  • the skilled artisan will know the incubation, temperature and time period sufficient to allow effective binding, processing and loading. Incubation steps are typically from between about 1 to 2 to 4 to 20 hours or longer, at temperatures of between about 25 degrees to 37 degrees centigrade (or higher) and/or may be overnight at about 4 degrees centigrade and the like.
  • the HLA-expressing cells are loaded with the tumor-specific peptides by the method known as "painting".
  • GPI glycosyl- phosphotidylinositol
  • This method requires the expression of the P2 10 microglobulin and the HLA-allele in Schneider S2 Drosophila melanogaster cells, known to support GPI modification.
  • the proteins are incubated together with a purified tumor-specifc peptide which results in a trimolecular complex capable of efficiently inserting itself into the membranes of autologous cells. In essence, these protein mixtures were used to "paint" the APC surface. Using this method, cell coating occurs rapidly and is protein concentration dependent.
  • the peptide library when the cells are pulsed with a peptide library containing a plurality of different peptide, can be designed so that it contains peptides showing the highest likelihood of presentation on HLA using any of the algorithms mentioned above.
  • the algorithm takes into account the HLA binding prediction based on NetMHCpan 4.0, the number of minimal epitopes with ⁇ 2 percentile rank predicted to bind to HLA from each mutated 25-mer, and the variant allele frequency, among other factors.
  • HLA-cells bypass the need for gene transfer into the APC and permits control of antigenic peptide densities at the cell surfaces.
  • HLA-expressing cells can be transfected with nucleic acids encoding the tumorspecific peptides according to the invention.
  • the polynucleotide may be, e.g., DNA, cDNA, PNA, CNA, RNA, either single-and/or double-stranded, or native or stabilized forms of polynucleotides, such as e.g. polynucleotides with a phosphorothioate backbone, or combinations thereof and it may or may not contain introns so long as it codes for the peptide.
  • additional elements can be incorporated into the polynucleotide or TMG design.
  • Suitable modifications that could be used for increasing processing and presentation include the use of the 3’-UTR of the beta globin gene, a signal sequence or the DC LAMP trafficking sequence.
  • in vitro translation is used to produce the peptide.
  • Many exemplary systems exist that one skilled in the art could utilize e.g., Retie Lysate IVT Kit, Life Technologies, Waltham, MA).
  • the nucleic acid is introduced in the cell by any gene delivery method known in the art.
  • Transfection methods include a variety of techniques such as electroporation, protein-based, lipid-based and cationic ion based nucleic acid delivery complexes, viral vectors, “gene gun” delivery and various other techniques known to those of skill in the art.
  • the introduced polynucleotide can be stably maintained in the host cell or may be transiently expressed.
  • an mRNA is introduced into a DC and is transiently expressed.
  • Stable maintenance typically requires that the introduced polynucleotide either contains an origin of replication compatible with the host cell or integrates into a replicon of the host cell such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome.
  • a number of vectors are capable of mediating transfer of genes to mammalian cells, as is known in the art and described herein.
  • the nucleic acid encoding the tumor-specific peptides is included into a viral vector.
  • viral vectors include retroviral vectors, adenovirus vectors, adeno- associated virus vectors, alphavirus vectors and the like.
  • Alphavirus vectors such as Semliki Forest virus-based vectors and Sindbis virus-based vectors.
  • the viral vectors contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are known in the art.
  • Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.).
  • sources such as Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.).
  • consensus ribosome binding sites can be inserted immediately 5' of the start codon to enhance expression.
  • the tumor whole exome sequencing used to identify tumor-specific peptide results in the identification of a set of tumor-specific peptides.
  • the method of the invention can be carried out using a plurality of peptides, either by pulsing the cell with a peptide library or, alternatively, by introducing into the cell a nucleic acid library encoding the different peptides or a nucleic acid encoding a plurality of peptides can be provided.
  • the present invention contemplates pulsing the cell with a peptide library comprising different tumor-specific peptides or transfecting with one or more nucleic acids encoding the plurality of tumor-specific peptides.
  • the cells are pulsed with a composition comprising a plurality of tumorspecific peptides.
  • the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 tumor-specific peptides.
  • the cell is contacted with a plurality of nucleic acids, each of them encoding one or more tumor-specific peptides.
  • the composition comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 of the isolated polynucleotides.
  • the cell is contacted with at least one polynucleotide which encodes a plurality of tumor-specific peptides.
  • These nucleic acids are referred to in the present invention as tandem minigenes (TMG).
  • TMG tandem minigenes
  • the tandem minigene comprises nucleotide sequences encoding at least 1 , at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 tumor specific peptides, which may the identical or different.
  • the cells may be pulsed with the peptides as such or with a peptide library or transfected with a nucleic acid encoding the peptide, with a nucleic acid encoding a plurality of peptides (the TMG) or with a plurality of nucleic acids encoding different tumor specific peptides.
  • the TMG is designed so that its sequence encodes for peptides showing the highest likelihood of presentation on HLA using any of the algorithms mentioned above.
  • the algorithm takes into account the HLA binding prediction based on NetMHCpan 4.0, the number of minimal epitopes with ⁇ 2 percentile rank predicted to bind to HLA from each tumor-specific peptide, and the variant allele frequency, among other factors.
  • the library when the cells are transfected with a nucleic acid library comprising a plurality of nucleic acids, each of them encoding a tumor-specific peptide, the library can be designed so that it is formed by nucleic acid sequences encoding for peptides showing the highest likelihood of presentation on HLA using any of the algorithms mentioned above.
  • the algorithm takes into account the HLA binding prediction based on NetMHCpan 4.0, the number of minimal epitopes with ⁇ 2 percentile rank predicted to bind to HLA from each mutated 25-mer, and the variant allele frequency, among other factors.
  • the cells are transfected with a nucleic acid encoding the tumor-specific peptide, with a TMG, i.e. a nucleic acid encoding a plurality of tumor-specific peptides, or with a plurality of TMG encoding different tumor specific peptides.
  • the HLA that is going to be analyzed is an HLAs corresponding to MHC class II ((DP, DM, DO, DQ, and DR)
  • the cells are pulsed with a tumor-specific peptide or with a composition comprising a plurality of tumor-specific peptides.
  • dendritic cells are activated after having been contacted with the tumor-specific peptide or peptides or with nucleic acid encoding the tumor-specific peptide or peptides.
  • Activation of dendritic cells initiates the process that converts immature DCs, which are phenotypically similar to skin Langerhans cells, to mature, antigen presenting cells that can migrate to the lymph nodes. This process results in the gradual and progressive loss of the powerful antigen uptake capacity that characterizes the immature dendritic cell, and in the up-regulation of expression of co-stimulatory cell surface molecules and various cytokines.
  • Various stimuli can initiate the maturation of DCs.
  • maturation induces several chemokine receptors, including CCR7, which direct the cells to the T cell regions of draining lymph nodes, where the mature DCs activate T cells against the antigens presented on the DC surface in the context of class I and class II MHC molecules.
  • CCR7 chemokine receptors
  • activation and “maturation”, and “activated” and “mature” describe the process of inducing and completing the transition from an immature DC (partially characterized by the ability to take up antigen) to a mature DC (partially characterized by the ability to effectively stimulate de novo T cell responses).
  • the terms typically are used interchangeably in the art.
  • MCM monocyte conditioned media
  • DCs In vitro maturation of DCs can therefore be designed to induce the immune system to favor one type of immune response over another, i.e., to polarize the immune response.
  • Directional maturation of DCs describes the notion that the outcome of the maturation process dictates the type of ensuing immune response that results from treatment with the matured DCs.
  • directional maturation results in a DC population that produces cytokines that direct a T cell response polarized to either a Th 1 -type or Th2-type response.
  • DCs express up to nine different Toll-like receptors (TLR1 through TLR9), each of which can be used to trigger maturation.
  • TLR1 through TLR9 Toll-like receptors
  • interferon gamma IFN-y
  • IFN-y interferon gamma
  • Factors that can be used in the directional maturation of activated DCs can therefore include for example, Interleukin 1 beta (IL-b), Interleukin 6 (IL-6), and tumor necrosis factor alpha (TNFa).
  • Other maturation factors include prostaglandin E2 (PGE2), poly- dldC, vasointestinal peptide (VIP), bacterial lipopolysaccharide (LPS), as well as mycobacteria or components of mycobacteria, such as specific cell wall constituents.
  • Additional maturation factors include for example, an imidazoquinoline compound, e.g., R848 (WO 00/47719, incorporated herein by reference in its entirety), a synthetic double stranded polyribonucleotide, agonists of a Tolllike receptor (TLR), such as TLR3, TLR4, TLR7 and/or TLR9, a sequence of nucleic acids containing unmethylated CpG motifs known to induce the maturation of DC, and the like. Further, a combination of any of the above agents can be used in inducing the maturation of dendritic precursor cells. Fully mature dendritic cells differ qualitatively and quantitatively from immature DCs.
  • TLR Tolllike receptor
  • DCs express higher levels of MHC class I and class II antigens, and higher levels of T cell costimulatory molecules, i.e., CD80 and CD86.
  • TLRs Suitable ligands of TLRs that can be used to induce maturation of the HLA-expressing cells and, more in particular, of dendritic cells have been explained in detailed in Kaisho et al. (Biochimica et Biophysica Acta 1589 (2002) 1-13), the entire contents of which are hereby incorporated by reference.
  • maturation of the dendritic cells is achieved by the incubation of the cells with a one or more of the following:
  • TLR ligand mix comprising R848, Polyl:C and IFN-y
  • a cytokine mix comprising PGE2, IL-1 b, IL-6 and TNFa and
  • the HLA-expressing cells are expanded ex vivo prior to being contacted with the tumor specific peptides or the nucleic acids encoding the tumor specific peptides.
  • Suitable methods for inducing the expansion of HLA- expressing cells include, without limitation, treatment of the T cells with IL-2 or stimulated with anti-CD3 in presence of co-stimulation (4-1 BB, anti-CD28) or irradiated feeders, as well as treatment of B cells with IL-4 and co-stimulation through ligation of CD40, transformation with EBV or through the induction of pro-oncogenic signaling.
  • Step (ii) of the method of the invention recovery of HLA complexes from the HLA-expressing cells
  • Step (ii) of the method according to the present invention comprise the recovery of HLA complexes from the HLA-expressing cells once they have been contacted with the tumorspecific peptides or with the nucleic acid encoding said tumor-specific peptide under conditions adequate for the formation of a complex between said tumor-specific peptide and HLA molecules in said cells or between the tumor-specific peptide encoded by the nucleic acid and HLA molecules in said cells.
  • step (ii) of the method according to the present invention comprises the recovery of HLA-I complexes (the classical HLA-A, HLA-B or HLA-C monomer or a non- classical (MHC-lb), such as HLA-E, HLA-F, HLA-G and HLA-H).
  • step (ii) of the method according to the present invention comprises the recovery of HLA-II complexes (complexes containing the HLA-DR, HLA-DQ, HLA-DP, HLA-DM, HLA-DOA, HLA-DOB and HLA-DP monomers).
  • the cell membrane of the cell may be lysed. This lysis may be carried out by a method generally known to those skilled in the art, for example, freezing-thawing, use of a surfactant, or a combination thereof.
  • a surfactant for example, Triton X-100 (TX100), Nonidet P-40 (NP- 40), Tween 20, Tween 80, n-octylglucoside, ZWITTERGENT, Lubrol, sodium deoxycholate, IGEPAL or CHAPS may be used as the surfactant.
  • Protease inhibitors are added to prevent proteolysis.
  • phosphatase inhibitors can be added to prevent dephosphorylation of phosphopeptides.
  • iodacetamide is added as well to alkylate cysteines thereby inhibiting disulphide bond formation. Cell debris and nucleus are removed by centrifugation from the cell lysate containing the solubilized MHC molecule-peptide complexes.
  • the cell lysate containing the solubilized MHC molecule-peptide complexes may be subjected to immunoprecipitation or immunoaffinity chromatography to purify the MHC molecule-peptide complexes.
  • An antibody that is specific for each MHC molecule and is suitable for immunoprecipitation or immunoaffinity chromatography (anti-MHC I molecule antibody, for example, anti-HLA-A antibody, anti-HLA-B antibody, anti-HLA-C antibody, or anti-HLA-ABC antibody, etc.; or anti-MHC II molecule antibody, preferably anti-HLA-DR antibody, anti-HLA-DQ antibody, or anti-HLA-DP antibody) may be used for these methods.
  • the specific antibody is preferably a monoclonal antibody and may be conjugated with beads (e.g., Sepharose beads or agarose beads) through a covalent bond or a noncovalent bond, for example, via protein A.
  • beads e.g., Sepharose beads or agarose beads
  • the amino group of the antibody may be covalently bonded to CNBr-activated Sepharose so that the antibody is immobilized thereon.
  • a commercially available product may be purchased as the monoclonal antibody, or the monoclonal antibody may be purified from the supernatant of each corresponding hybridoma cell using protein A- or protein G-affin ity chromatography.
  • the MHC molecules may be immunoisolated, for example, by performing incubation while rotating the antibody-beads together with the cell lysate for a few hours. Also, the antibodybeads bound with the MHC molecule-peptide complexes may be washed in an Eppendorf tube. The results of immunoprecipitation may be analyzed by SDS-PAGE and Western blotting using an antibody that recognizes a denatured MHC molecule. This procedure can also be performed in a high-throughput format using 96-well plates as described by Marino et al. (Methods Mol Biol., 2019;1913:67-79). Step (Hi) of the method of the invention (recovery and characterization of the peptides which are bound to the HLA complexes)
  • Step (iii) of the method according to the present invention comprises recovering the peptides which are bound to the HLA complexes isolated in step (ii), said peptides being neoantigen peptides suitable for the generation of an immune response against a tumor.
  • the peptides which are bound to the HLA complexes isolated in step (ii) are recovered by eluting the peptides from the complexes formed with the MHC molecules.
  • the peptides may be eluted by a method generally known to those skilled in the art, for example, by use of a diluted acid, for example, diluted acetonitrile (Jardetzky T S et al., Nature 1991 353, 326- 329), diluted acetic acid and heating (Rudensky A Y et al., Nature 1991 , 353, 622-626; and Chicz R M et aL, Nature 1992, 358, 764-768), or diluted trifluoroacetic acid (Kropshofer H et al., J Exp Med 1992, 175, 1799-1803).
  • the peptides are preferably eluted with diluted trifluoroacetic acid, for example, at room temperature.
  • these complexes may be washed with water or a low-salt buffer solution in order to remove surfactant residues.
  • a Tris buffer solution, a phosphate buffer solution, or an acetate buffer solution having a concentration of 0.5 to 10 mM may be used as the low-salt buffer solution.
  • the MHC molecule-peptide complexes may be washed with ultrapure water for HPLC. After the elution of the antibody-bound pMHC complexes with acidic solutions, the eluted peptides can be separated from the HLA chains by ultrafiltration.
  • the ultrafiltration may be performed in an ultrafiltration tube having, for example, a cutoff value of 30 kD, 20 kD, 10 kD, or 5 kD and a tube volume of 0.5 to 1.0 ml.
  • the inside of the ultrafiltration tube may be washed, for example, 4 to 12 times, with a washing solution.
  • the eluted peptides may be dried by use of freeze drying or a centrifugal evaporator.
  • the eluted peptide mixture is preferably concentrated, desalted and/or fractionated, for example, using reverse-phase chromatography and anion-exchange chromatography or cation-exchange chromatography in combination or reverse-phase chromatography alone using, for example, via C18 and/or strong cation exchange (SCX) prefractionation.
  • SCX strong cation exchange
  • the peptide mixture thus eluted and purified may be fractionated and subjected to sequence analysis by use of liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to identify the amino acid sequence of each peptide.
  • the amino acid sequence of each peptide in the peptide mixture can be determined by sequence analysis according to any method known in the art. Typically, methods which show a sensitivity sufficient for determining the sequences of peptides in fmol amounts are preferred.
  • the identification reveals the specific sequence of the neoantigen peptides presented on HLA molecules.
  • the fractionation may be performed on a HPLC mode using a fused-silica micro-capillary column connected either to nanoflow electrospray ion source in a mass spectrometer or to a microfractionation apparatus that spots fractions on a plate for MALDI analysis.
  • Electrospray ionization tandem mass spectrometry (ESI-MS) or MALDI-post source decay (PSD) MS may be used as a mass spectrometry technique, and ESI-MS is preferred.
  • Data- dependent acquisition mode (DDA) is preferred over data-independent acquisition (DIA), although DIA methods such as SWATH can also be considered.
  • sequence analysis the amino acid sequence of each peptide may be determined by various approaches generally known to those skilled in the art.
  • the sequence analysis may be performed by the computer analysis of a peptide fragment spectrum using, for example, MASCOT, or SEQUEST or Andromeda or PEAKS softwares. These algorithms preferably employ protein and nucleotide sequence databases, for conducting the cross-correlation analysis of experimentally and theoretically prepared tandem mass spectra. This permits automatic high-throughput sequence analysis.
  • PEAKS DB protein identification integrates database search with de novo sequencing for peptide identification. Softwares used for peptide identification need to be statistically validated to avoid false positives.
  • the most accepted resUlt for validatibn is the determihation of the false discovery rate (FbR).
  • FbR false discovery rate
  • the software searches the protein database including the reference prbteome and the tumor-specific peptides selected for (i) to find a peptide that maximizes the peptide-spectrum matching score.
  • the software also compares the MS/MS spectrum in the data to a decoy (control) database with the same size. Since all the decoy identifications are false, this data can be used to calculate the FDR. By adjusting the FDR thresholds, the accuracy of the data can be traded with the sensitivity (humber of reported identifications).
  • an FDR of 1-5% is used, which means that 1 % to 5% of the peptides identified are false positives.
  • the FDR can be increased if desired.
  • Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry may be performed for the qualitative analysis of all of the peptides obtained by elution.
  • the MALDI-TOF analysis can provide a rough outline regarding the complexity of the peptide mixture and the presence of a primary peptide.
  • substances that have passed through a microcapillary column may be analyzed using a UV detector at a detection wavelength of 214 nm.
  • the peak area of the peptide to be analyzed may be compared with the peak areas of serial amounts of standard peptides (control) to estimate the amount of the peptide.
  • This step allows the identification of neaontigens from one or more tumor-specific peptides that are suitable for the generation of an immune response against the tumor.
  • a set of HLA-expressing cells exposed to the tumor-specific peptide or to the nucleic acid encoding the tumor-specific peptide as well as a set of HLA-expressing cells unexposed to the tumor-specific peptide or to the nucleic acid as a negative control should be prepared and analyzed by comparison.
  • a neoantigen peptide that can be detected in the antigen-presenting cells exposed to a tumorspecific peptide, but not in the antigen-presenting cells unexposed to this tumor-specific peptide represents a more accurate and reliable peptide identification that is suitable for immunization.
  • the method according to the present invention further comprises the characterization of the neoantigens identified by said method. It will be understood that the neoantigens identified by the method according to the present invention may be characterized in the form they appear after step (iii) or that longer peptides comprising the sequence of the neoantigen can be used for further characterization.
  • the neoantigens or the peptides containing the neoantigen sequence are characterized in terms of the capacity to form a complex with an HLA multimer which is capable of binding to T cells as explained in Bentzen and Hadrup (Cancer Immunol Immunother. 2017, 66(5):657-666).
  • the neoantigens or the fragments thereof are characterized in terms of their capacity to activate tumor-infiltrating lymphocytes isolated from the tumor.
  • the neoantigens or the peptides containing the neoantigen sequence are additionally or alternatively characterized by their capacity to activate or stimulate a T- cell population of interest.
  • the T cell population of interest is a population of tumor-infiltrating lymphocytes.
  • the T-cell population is provided as a preparation of peripheral blood lymphocytes or subpopulations thereof, wherein the activation assay can be carried out with or without in vitro sensitization of the PBMC.
  • the method according to the present invention further comprises the determination of the capacity of the neoantigens or of the peptides containing the neoantigen sequence to activate tumor-infiltrating lymphocytes isolated from the tumor.
  • the TILs are isolated from the same tumor from which the sequences of the tumor-specific peptides used in step (i) have been obtained.
  • the TILs can be expanded prior to their contacting with the neoantigens or with the peptides containing the neoantigen sequence using any method known in the art.
  • the TILs are expanded over the course of a few weeks with a high dose of IL-2.
  • TIL can also be further expanded in a "rapid expansion protocol" (REP), which uses anti-CD3 antibodies or a combination of anti- CD3 and/or anti-CD28 antibodies activation in presence or absence of irradiated feeders for a typical period of two weeks.
  • REP rapid expansion protocol
  • the expanded TILs can then be contacted with the neoantigen or with the peptides containing the neoantigen sequence presented in a HLA-expressing cell and the activation of the TIL can be detected by measuring activation of the cytotoxic (CD8) and helper (CD4) lymphocytes present in the TIL population.
  • CD8 cytotoxic
  • CD4 helper
  • Activation of the CD8 T cells can be detected by measuring the upregulation of T cell costimulatory molecules such as 0X40 or 4-1 BB,CD25, CD69 or cytokines such as, but not limited to IFNy, TNF-a, IL-2, IL-10, IL-4, IL-5, IL-17 or other secreted effector molecules such as Granzyme, perforin, or measuring T cell degranulation.
  • T cell costimulatory molecules such as 0X40 or 4-1 BB,CD25, CD69 or cytokines such as, but not limited to IFNy, TNF-a, IL-2, IL-10, IL-4, IL-5, IL-17 or other secreted effector molecules such as Granzyme, perforin, or measuring T cell degranulation.
  • T cell costimulatory molecules such as 0X40 or 4-1 BB,CD25, CD69 or cytokines such as, but not limited to IFNy, TNF-a, IL-2, IL-10
  • the method according to the present invention further comprises the determination of the capacity of the neoantigens or of the peptides containing the neoantigen sequence to activate peripheral blood lymphocytes (PBMC), with or without in vitro sensitization.
  • PBMC peripheral blood lymphocytes
  • helper T cells The cellular response of helper T cells may be measured by various in vitro methods generally known to those skilled in the art.
  • HLA molecule-expressing cells e.g., monocytes, macrophages, or dendritic cells
  • helper T cells are cultured together with helper T cells in the presence of the peptide to be evaluated.
  • the uptake of radioactive material-labeled thymidine (T) during DNA replication may be measured with helper T cell proliferation as an index.
  • 5-bromo-2'-deoxyuridine (Brdll) may be used instead of thymidine.
  • helper T cells that have taken up Brdll during DNA replication are treated with a monoclonal antibody against BrdU.
  • the amount of BrdU taken up may be measured using an enzymatically or fluorescently labeled secondary antibody (e.g., 5-Bromo-2'- deoxyuridine Labeling & Detection Kit III, Roche-Biochem, Cat No. 1 444 611 ).
  • an enzymatically or fluorescently labeled secondary antibody e.g., 5-Bromo-2'- deoxyuridine Labeling & Detection Kit III, Roche-Biochem, Cat No. 1 444 611 .
  • Naive Primary T cell Assay (Proimmune Ltd.) which employs flow cytometry using the dilution of a fluorescent dye label 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) by the proliferation of helper T cells as an index may be used in the measurement.
  • the cellular response of helper T cells may be evaluated by measuring various cytokines produced from the helper T cells, instead of measuring the cell proliferation.
  • cytokines examples include IL-2, IL-4, IL-6, IL-10, IL-12, TNF-a IFN-y, and transforming growth factor-p (TGF- ).
  • TGF- transforming growth factor-p
  • methods for measuring the cytokines include various methods generally known to those skilled in the art, for example, ELISA and ELISPOT.
  • the peptides which are isolated in step (iii) of the method of the invention can be further tested for one or more properties, including:
  • the peptides are selected if they have been isolated by the method of the invention simultaneously as a peptide which is presented by a cell after being pulsed with the tumor-specific peptides in step (i) of the method of the invention and as a peptide presented by a cell which has been transfected with a TMG as defined in step (i) of the method of the invention.
  • Such peptides which have been identified using the two different alternatives provided in step (i) of the method of the invention would be potentially interesting for their subsequent use in therapy as such or forming part of longer peptides as they would represent peptides that result from the processing of endogenous antigens and which are typically presented by the tumor cells and that result from exogenous presentation by APCs, which could further potentiate the antitumor T cell response. and vaccine the or
  • the invention relates to method for the preparation of an immunogenic or a vaccine composition for the generation of an immune response against a tumor containing a mutation comprising:
  • the invention relates to an immunogenic or vaccine composition which has been obtained by the method as defined in the invention.
  • an "immunogenic composition” is a composition that comprises an antigen, such as a neoantigen or a peptide containing the neoantigen sequence, where administration of the composition to a subject results in the development in the subject of a humoral and/or a cellular immune response to the antigen.
  • an antigen such as a neoantigen or a peptide containing the neoantigen sequence
  • a “vaccine composition” refers to compositions that affect the course of the disease by causing an effect on cells of the adaptive immune response, namely, B cells and/or T cells.
  • the effect of vaccines can include, for example, induction of cell-mediated immunity or alteration of the response of the T cell to its antigen.
  • Vaccines can be used for therapeutic administration or prophylactic administration.
  • the immunogenic and vaccine compositions according to the invention comprise an immunogenic active principle, which may be a neoantigen peptide identified by the method according to the present invention, a peptide containing the neoantigen sequence, a plurality of neoantigen peptides identified by the method according to the present invention, a plurality of peptides containing neoantigen sequences identified by the method according to the present invention, a nucleic acid encoding a neoantigen peptide identified by the method according to the present invention, a nucleic acid encoding a peptide containing the neoantigen sequence identified by the method according to the present invention, a plurality of nucleic acids encoding one or more neoantigen peptides or one or more peptides containing the neoantigen sequences identified by the method according to the present invention or a nucleic acid encoding a plurality of neoantigen peptides
  • the term “peptide comprising the sequence of the neoantigen” refers to a peptide which results from the extension of the neoantigen peptide in at least 1 amino acid, wherein the extension occurs at the N-terminus, at the C-terminus or at both ends.
  • the peptide comprising the sequence of the neoantigen result from the addition of 1 , 2, 3, 4, 5, 6, 7 or more amino acids to the N-terminus of the neoantigen peptide.
  • the peptide comprising the sequence of the neoantigen result from the addition of 1 , 2, 3, 4, 5, 6, 7 or more amino acids to the C-terminus of the neoantigen peptide.
  • the peptide comprising the sequence of the neoantigen results from the addition of 1 , 2, 3, 4, 5, 6, 7 or more amino acids to the N- and to the C-terminus of the neoantigen peptide.
  • a person skilled in the art will be able to select preferred peptides, polypeptide or combination thereof by testing, for example, the generation of T-cells in vitro as well as their efficiency and overall presence, the proliferation, affinity and expansion of certain T-cells specific for certain peptides, and the functionality of the T-cells, e.g. by analyzing the IFN-y production or tumor killing by T-cells. Usually, the most efficient peptides are then combined as a vaccine.
  • the preferred peptides can be further tested using in silico prediction of the HLA- binding affinity and/or immunogenicity for example by using one or more of the algorithms mentioned above suitable for the pre-selection of tumor-specific peptides in step (i) of the method of the invention, or by testing the RNA expression level of the peptide in the tumor.
  • the immunogenic or vaccine composition according to the invention comprise a neoantigen peptide isolated by a method according to the present invention or a peptide comprising the sequence of the neoantigen.
  • the immunogenic or vaccine composition according to the invention comprise a plurality of distinct neoantigen peptides isolated by the method according to the present invention, a plurality of peptides comprising the sequences of neoantigen peptides isolated by the method according to the present invention.
  • the immunogenic or vaccine composition comprises a plurality of peptides comprising the sequences of the neoantigen peptides isolated by the method according to the invention
  • the immunogenic or vaccine composition may comprise a plurality of peptides comprising the sequence of the same neoantigen peptide or a plurality of peptides comprising the sequences from different neoantigens.
  • plural of neoantigen peptides or “plurality of peptides comprising the sequence of different neoantigens” is meant that the peptides forming part of the immunogenic or vaccine composition can vary by length, amino acid sequence or both.
  • the at least two distinct peptides are derived from the same polypeptide.
  • a suitable vaccine will preferably contain between 1 and 20 neoantigen peptides or peptides comprising the sequence of the neoantigens, more preferably at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 different neoantigen peptides or peptides comprising the sequence of the neoantigen or neoantigens, further preferred 6, 7, 8, 9, 10 11 , 12, 13, or 14 different neoantigen peptides comprising the sequence of the neoantigen or neoantigens, and most preferably 12, 13 or 14 different neoantigen peptides comprising the sequence of the neoantigen or neoantigens.
  • the peptide or peptides forming part of the immune and vaccine compositions according to the present invention can be a variety of lengths, either in their neutral (uncharged) forms or in forms which are salts, and either free of modifications such as glycosylation, side chain oxidation, or phosphorylation or containing these modifications, subject to the condition that the modification does not destroy the biological activity of the polypeptides as herein described.
  • the size of the neoantigen peptide or the peptides comprising the sequences of the neoantigens forming part of the immune and vaccine compositions according to the present invention may comprise, but is not limited to, about 5, about 6, about 7, about 8, about 9, about 10, about 11 , about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21 , about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31 , about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41 , about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or greater amino molecule residues, and any range derivable therein.
  • the neoantigen peptide molecules are equal to or less than 50 amino acids.
  • the particular neoantigen peptide or peptides or the peptide or peptides comprising the sequence of the neoantigen or neoantigens forming part of the immune and vaccine compositions according to the present invention are: for HLA Class I 7-13 residues or less in length and usually consist of between about 8 and about 12 residues, particularly 9 or 10 residues; for MHC Class II, 10-24 residues.
  • a longer peptide may be designed in several ways.
  • a longer peptide could consist of either: (1 ) individual binding peptides with extensions of 2-5 or more amino acids toward the N- and C-terminus of each corresponding gene product; (2) a concatenation of some or all of the binding peptides with extended sequences for each.
  • sequencing reveals a long (>10 residues) neoepitope sequence present in the tumor (e.g.
  • a longer peptide would consist of: (3) the entire stretch of novel tumor-specific amino acids -- thus bypassing the need for computational prediction or in vitro testing of peptide binding to HLA proteins.
  • use of a longer peptide allows endogenous processing by patient cells and may lead to more effective antigen presentation and induction of T cell responses.
  • neoantigen peptide or peptides or the peptide or peptides comprising the sequence of the neoantigen peptide or peptides forming part of the immune and vaccine compositions according to the present invention bind an HLA protein.
  • the neoantigen peptide or peptide binds an HLA protein with greater affinity than a wild-type peptide.
  • the neoantigen peptide, the peptides, the peptide or peptides comprising the sequence of the neoantigen peptide or peptides or fragments thereof have an IC50 of at least less than 5000 nM, at least less than 500 nM, at least less then 250 nM, at least less than 200 nM, at least less than 150 nM, at least less than 100 nM, at least less than 50 nM or less.
  • neoantigen peptide or peptides or the peptide or peptides comprising the sequence of the neoantigen peptide or peptides forming part of the immune and vaccine compositions according to the present invention do not induce an autoimmune response and/or invoke immunological tolerance when administered to a subject.
  • neoantigen peptide or peptides or the peptide or peptides comprising the sequence of the neoantigen peptide or peptides having the desired activity may be modified as necessary to provide certain desired attributes, e.g. improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide to bind the desired MHC molecule and activate the appropriate T cell.
  • the neoantigenic peptide or the peptide comprising the sequence of the neoantigens may be subject to various changes, such as substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use, such as improved MHC binding.
  • substitutions By conservative substitutions is meant replacing an amino acid residue with another which is biologically and/or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another.
  • the substitutions include combinations such as Gly, Ala; Vai, lie, Leu, Met; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • the effect of single amino acid substitutions may also be probed using D-amino acids.
  • modifications may be made using well known peptide synthesis procedures, as described in e.g., Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (N.Y., Academic Press), pp. 1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, HL, Pierce), 2d Ed. (1984).
  • neoantigen peptide or peptides or the peptide or peptides comprising the sequence of the neoantigen or neoantigens can also be modified by extending or decreasing the compound’s amino acid sequence, e.g., by the addition or deletion of amino acids.
  • the peptides, polypeptides or analogs can also be modified by altering the order or composition of certain residues, it being readily appreciated that certain amino acid residues essential for biological activity, e.g., those at critical contact sites or conserved residues, may generally not be altered without an adverse effect on biological activity.
  • non-critical amino acids need not be limited to those naturally occurring in proteins, such as L-a-amino acids, or their D-isomers, but may include non-natural amino acids as well, such as p-y-6-amino acids, as well as many derivatives of L-a-amino acids.
  • a series of peptides with single amino acid substitutions are employed to determine the effect of electrostatic charge, hydrophobicity, etc. on binding. For instance, a series of positively charged (e.g., Lys or Arg) or negatively charged (e.g., Glu) amino acid substitutions are made along the length of the peptide revealing different patterns of sensitivity towards various MHC molecules and T cell receptors.
  • a series of positively charged (e.g., Lys or Arg) or negatively charged (e.g., Glu) amino acid substitutions are made along the length of the peptide revealing different patterns of sensitivity towards various MHC molecules and T cell receptors.
  • multiple substitutions using small, relatively neutral moieties such as Ala, Gly, Pro, or similar residues may be employed.
  • the substitutions may be homo-oligomers or hetero-oligomers.
  • residues which are substituted or added depend on the spacing necessary between essential contact points and certain functional attributes which are sought (e.g., hydrophobicity versus hydrophilicity). Increased binding affinity for an MHC molecule or T cell receptor may also be achieved by such substitutions, compared to the affinity of the parent peptide. In any event, such substitutions should employ amino acid residues or other molecular fragments chosen to avoid, for example, steric and charge interference which might disrupt binding.
  • Amino acid substitutions are typically of single residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final peptide. Substitutional variants are those in which at least one residue of a peptide has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Table when it is desired to finely modulate the characteristics of the peptide.
  • Substantial changes in function are made by selecting substitutions that are less conservative than those in above Table, i.e. , selecting residues that differ more significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain.
  • substitutions which in general are expected to produce the greatest changes in peptide properties will be those in which (a) hydrophilic residue, e.g. seryl, is substituted for (or by) a hydrophobic residue, e.g.
  • leucyl isoleucyl, phenylalanyl, valyl or alanyl
  • a residue having an electropositive side chain e.g., lysl, arginyl, or histidyl
  • an electronegative residue e.g. glutamyl or aspartyl
  • a residue having a bulky side chain e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine.
  • neoantigen peptide or peptides or the peptide or peptides comprising the sequence or sequences of the neoantigen or neoantigens forming part of the immune and vaccine compositions according to the present invention may also comprise isosteres of two or more residues in the neoantigenic peptide or in the peptides comprising the sequence of the neoantigens.
  • An isostere as defined here is a sequence of two or more residues that can be substituted for a second sequence because the steric conformation of the first sequence fits a binding site specific for the second sequence.
  • peptide backbone modifications well known to those skilled in the art. Such modifications include modifications of the amide nitrogen, the a-carbon, amide carbonyl, complete replacement of the amide bond, extensions, deletions or backbone crosslinks.
  • Modifications of peptides and polypeptides with various amino acid mimetics or unnatural amino acids are particularly useful in increasing the stability of the peptide and polypeptide in vivo, Stability can be assayed in a number of ways.
  • peptidases and various biological media such as human plasma and serum, have been used to test stability.
  • Half life of the peptides of the present disclosure is conveniently determined using a 25% human serum (v/v) assay.
  • the protocol is generally as follows. Pooled human serum (Type AB, nonheat inactivated) is delipidated by centrifugation before use. The serum is then diluted to 25% with RPMI tissue culture media and used to test peptide stability.
  • reaction solution is removed and added to either 6% aqueous trichloracetic acid or ethanol.
  • cloudy reaction sample is cooled (4°C) for 15 minutes and then spun to pellet the precipitated serum proteins.
  • the presence of the peptides is then determined by reversed-phase HPLC using stability-specific chromatography conditions.
  • the vaccine composition comprises mutant peptides and mutant polypeptides corresponding to tumor specific neoantigens identified by the methods described herein or peptides comprising the sequences of the neoantigens.
  • the different peptides and/or polypeptides are selected so that one vaccine composition comprises peptides and/or polypeptides capable of associating with different MHC molecules, such as different MHC class I molecules.
  • one vaccine composition comprises peptides and/or polypeptides capable of associating with the most frequently occurring MHC class I molecules.
  • vaccine compositions according to the disclosure comprises different fragments capable of associating with at least 2 preferred, more preferably at least 3 preferred, even more preferably at least 4 preferred MHC class I molecules.
  • the immunogenic or vaccine compositions comprise a nucleic acid encoding a neoantigen peptide identified by the method according to the present invention, a plurality nucleic acids encoding each of them one or more neoantigen peptides identified by the method according to the present invention, a nucleic acid encoding a plurality of neoantigen peptides identified by the method according to the present invention, a nucleic acid encoding a peptide comprising the sequence of a neoantigen peptide identified by the method according to the present invention, a plurality of nucleic acids encoding one or more peptides comprising the sequence of the neoantigen peptides identified by the method according to the present invention or a nucleic acid encoding a plurality of peptides comprising each of them the sequence of one or more neoantigen peptides identified by the method according to the present invention.
  • the immunogenic or vaccine compositions comprise a tandem minigene (TMG), i.e. or a nucleic acid encoding a plurality of neoantigen peptides identified by the method according to the present invention or a nucleic acid encoding a plurality of peptides comprising the sequence of the neoantigen peptides identified by the method according to the present invention.
  • TMG tandem minigene
  • the structure of the TMGs has been explained above in the context of the TMGs encoding a plurality of tumor-specific peptides and applies equally to the TMGs forming part of the vaccine compositions according to the present invention.
  • the tandem minigene comprises nucleotide sequences encoding at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 neoantigens or peptides comprising the sequence of the neoantigen peptides, which may be identical or different. In one embodiment, all neoantigens encoded in the TMG derive from the same protein.
  • the neoantigens encoded in the TMG derive from at least two different proteins. In one embodiment, all fragments encoded in the TMG derive from the same neoantigen. In another embodiment, the peptides containing the sequence of the neoantigen encoded in the TMG derive from the at least two different neoantigens.
  • the vaccine composition is capable of raising a specific cytotoxic T-cells response and/or a specific helper T cell response.
  • the vaccine composition can further comprise an adjuvant and/or a carrier.
  • an adjuvant and/or a carrier examples of useful adjuvants and carriers are given herein below.
  • the peptides and/or polypeptides in the composition can be associated with a carrier such as e.g. a protein or an antigen- presenting cell such as e.g. a dendritic cell (DC) capable of presenting the peptide to a T- cell.
  • Adjuvants are any substance whose admixture into the vaccine composition increases or otherwise modifies the immune response to the mutant peptide.
  • Carriers are scaffold structures, for example a polypeptide or a polysaccharide, to which the neoantigenic peptides, is capable of being associated.
  • adjuvants are conjugated covalently or non-covalently to the peptides or polypeptides of the disclosure.
  • an adjuvant to increase the immune response to an antigen is typically manifested by a significant increase in immune-mediated reaction, or reduction in disease symptoms.
  • an increase in humoral immunity is typically manifested by a significant increase in the titer of antibodies raised to the antigen
  • an increase in T-cell activity is typically manifested in increased cell proliferation, or cellular cytotoxicity, or cytokine secretion.
  • An adjuvant may also alter an immune response, for example, by changing a primarily humoral or Th response into a primarily cellular, or Th response.
  • Suitable adjuvants include, but are not limited to 1018 ISS, aluminium salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31 , Imiquimod, ImuFact IMP321 , IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51 , OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTeLRTM.
  • CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in a vaccine setting.
  • CpG oligonucleotides act by activating the innate (non-adaptive) immune system via Toll-like receptors (TLR), mainly TLR9.
  • TLR Toll-like receptors
  • CpG triggered TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cellular vaccines and polysaccharide conjugates in both prophylactic and therapeutic vaccines.
  • TH1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund’s adjuvant (IFA) that normally promote a TH2 bias.
  • IFA incomplete Freund’s adjuvant
  • CpG oligonucleotides show even greater adjuvant activity when formulated or co-administered with other adjuvants or in formulations such as microparticles, nano particles, lipid emulsions or similar formulations, which are especially necessary for inducing a strong response when the antigen is relatively weak. They also accelerate the immune response and enabled the antigen doses to be reduced by approximately two orders of magnitude, with comparable antibody responses to the full-dose vaccine without CpG in some experiments
  • useful adjuvants include, but are not limited to, chemically modified CpGs (e.g. CpR, Idera), Poly(l:C)(e.g. polyi:CI2U), non-CpG bacterial DNA or RNA as well as immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP- 547632, pazopanib, ZD2171 , AZD2171 , ipilimumab, tremelimumab, and SC58175, which may act therapeutically and/or as an adjuvant.
  • CpGs e.g. CpR, Idera
  • non-CpG bacterial DNA or RNA as well as immunoactive small
  • adjuvants and additives useful in the context of the present disclosure can readily be determined by the skilled artisan without undue experimentation.
  • Additional adjuvants include colonystimulating factors, such as Granulocyte Macrophage Colony Stimulating Factor (GM-CSF, sargramostim).
  • GM-CSF Granulocyte Macrophage Colony Stimulating Factor
  • a vaccine composition according to the present disclosure may comprise more than one different adjuvant.
  • the disclosure encompasses a therapeutic composition comprising any adjuvant substance including any of the above or combinations thereof. It is also contemplated that the peptide, polypeptide or nucleic acid, and the adjuvant can be administered separately in any appropriate sequence.
  • a carrier may be present independently of an adjuvant.
  • the function of a carrier can for example be to increase the molecular weight of in particular mutant in order to increase their activity or immunogenicity, to confer stability, to increase the biological activity, or to increase serum half-life.
  • a carrier may aid presenting peptides to TEP cells.
  • the carrier may be any suitable carrier known to the person skilled in the art, for example a protein or an antigen presenting cell.
  • a carrier protein could be but is not limited to keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid.
  • the carrier For immunization of humans, the carrier must be a physiologically acceptable carrier acceptable to humans and safe. However, tetanus toxoid and/or diptheria toxoid are suitable carriers in one instance of the disclosure. Alternatively, the carrier may be dextrans for example Sepharose.
  • Cytotoxic T-cells recognize an antigen in the form of a peptide bound to an MHC molecule rather than the intact foreign antigen itself.
  • the peptide-MHC complex itself is located at the cell surface of an antigen presenting cell.
  • an activation of CTLs is only possible if a trimeric complex of peptide antigen, MHC molecule, and APC is present.
  • it may enhance the immune response if not only the peptide is used for activation of CTLs, but if additionally APCs expressing the respective peptide-MHC complex are added. Therefore, in some instances the vaccine composition according to the present disclosure additionally contains at least one antigen presenting cell.
  • the antigen-presenting cell typically has MHC class I or II molecule on its surface, and in one instance is substantially incapable of itself loading the MHC class I or II molecule with the selected antigen. As is described in more detail below, the MHC class I or II molecule may readily be loaded with the selected antigen in vitro.
  • the antigen presenting cells are dendritic cells.
  • the dendritic cells are autologous dendritic cells that are pulsed with the neoantigenic peptide.
  • the peptide may be any suitable peptide that gives rise to an appropriate T-cell response as identified by the method of the present invention. Immunization using autologous dendritic cells pulsed with peptides from a tumor associated antigen is disclosed in Murphy et al. (1996) The Prostate 29, 371-380 and Tjua et al. (1997) The Prostate 32, 272-278.
  • the vaccine composition containing at least one antigen presenting cell is pulsed or loaded with one or more peptides of the present disclosure.
  • peripheral blood mononuclear cells PBMCs isolated from a patient may be loaded with peptides ex vivo and injected back into the patient.
  • the antigen presenting cell comprises an expression construct encoding a peptide of the present disclosure.
  • the polynucleotide may be any suitable polynucleotide and it is preferred that it is capable of transiently or stably transducing the dendritic cell, thus resulting in the presentation of a peptide and induction of immunity.
  • T cells may be activated and expanded in cell culture by incubation with the neoantigen identified according to the present invention and antigen presenting cells. Once activated, T cells undergo a complex cascade of cell signaling which leads to the transcription and expression of many gene products.
  • the invention described herein takes advantage of gene products specific of activated T cells for the identification and isolation of T cells with desired antigen specificity.
  • neoantigen isolated by the method according to the present invention or for a peptide comprising the sequence of said neoantigen.
  • the T cell which is specific for the neoantigen isolated by the method according to the present invention or for a peptide comprising the sequence of said neoantigen isolated by a method that comprises contacting a sample comprising T cells with the neoantigen or the peptide comprising the sequence of the neoantigen, which causes the activation of a T cell specific for the neoantigen.
  • the sample may be incubated with the antigen for 1 to 7 days.
  • the sample may also be incubated with the antigen for less than 1 day.
  • the sample may also be incubated with the antigen for less than 16 hours.
  • the sample may also be incubated with the antigen for less than 12 hours.
  • the sample may also be incubated with the antigen for less than 8 hours.
  • the sample may also be incubated with the antigen for less than 4 hours.
  • the sample may also be incubated with the antigen for less than 2 hours.
  • a library comprising at least two neoantigens can be incubated with T cells.
  • T cells can be prepared using standard methods that start from a tissue such as blood, a lymph node, or a tumor.
  • a T cell specific for the neoantigen or the peptide comprising the neoantigen may then be isolated by selecting for T cells that express gene products of T cells activated as described above.
  • Subsets of activated T cells may be isolated by selecting for T cells with subsetspecific gene products or cell surface markers (e.g., CD4 vs. CD8).
  • the T cells may be present in any sample comprising mononuclear cells.
  • the sample may be isolated from the peripheral blood or cerebral spinal fluid of a cancer patient
  • Mononuclear cells may be enriched in the sample by using centrifugation techniques known to those in the art including, but not limited to, Ficoll(R) gradients.
  • T cells may also be enriched in the sample by using positive selection, negative selection, or a combination thereof for expression of gene products of activated T cells.
  • the gene product for identifying or negatively selecting for activated T cells may be a cell surface marker or cytokine, or a combination thereof.
  • Cell surface markers for identifying activated T cells include, but are not limited to, CD69, CD4, CD8, CD25, HLA-DR, CD28, and CD134.
  • CD69 is an early activation marker found on B and T lymphocytes, NK cells and granulocytes.
  • CD25 is an IL-2 receptor and is a marker for activated T cells and B cells.
  • CD4 is a TCR coreceptor and is marker for thymocytes, TH1- and TH2-type cells, monocytes, and macrophages.
  • CD8 is also a TCR coreceptor and is marker for cytotoxic T cells.
  • CD134 is expressed only in activated CD4+ T cells.
  • Cell surface markers for negatively selecting for activated T cells include, but are not limited to, CD36, CD40, and CD44.
  • CD28 acts as a stimulatory T-cell activation pathway independent of the T-cell receptor pathway and is expressed on CD4+ and CD8+ cells.
  • CD36 is a membrane glycoprotein and is a marker for platelets, monocytes and endothelial cells.
  • CD40 is a marker for B cells, macrophages and dendritic cells.
  • CD44 is a marker for macrophages and other phagocytic cells.
  • Subsets of T cells may be isolated by using positive selection, negative selection, or a combination thereof for expression of cell surface gene products of helper T cells or cytotoxic T cells (e.g., CD4 vs. CD8).
  • Cytokines for identifying activated cells of the present invention include, but are not limited to cytokines produced by TH1-type T cells (cell-mediated response) and TH2-type T cells (antibody response). Cytokines for identifying activated TH 1 -type T cells include, but are not limited to, IL-2, gamma interferon (gammalFN) and tissue necrosis factor alpha (TNFa). Cytokines for identifying activated TH2-type T cells include, but not limited to, IL-4, IL-5, IL- 10 and IL-13. Subsets of T cells may also be isolated by using positive selection, negative selection, or a combination thereof for expression of cytokine gene products of helper T cells or cytotoxic T cells (e.g., gammalFN vs. IL4).
  • cytotoxic T cells e.g., gammalFN vs. IL4
  • An activated TH 1 -type T cell specific for an antigen of interest may be isolated by identifying cells that express CD69, CD4, CD25, IL-2, IFNgamma, TNFa, or a combination thereof.
  • An activated TH 1 -type T cell specific for an antigen of interest may also be isolated by identifying cells that express CD69 and CD4 together with IFNgamma or TNFa.
  • An activated TH2-type T cell specific for an antigen of interest may be isolated by identifying cells that express CD69, CD4, IL-4, IL-5, IL-10, IL-13, or a combination thereof.
  • a combination of an activated TH1- type T cell and a TH2-type T cell specific for an antigen of interest may be isolated by identifying cells that express CD69, CD4, CD25, IL-2, IFNgamma, TNFa, or a combination thereof and cells that express CD69, CD4, IL-4, IL-5, IL-10, IL-13, or a combination thereof.
  • the gene products used for positive or negative selection of the activated T cells of the present invention may be identified by immunoselection techniques known to those in the art which utilize antibodies including, but not limited to, fluorescence activated cell sorting (FACS), magnetic cell sorting, panning, and chromatography.
  • Immunoselection of two or more markers on activated T cells may be performed in one or more steps, wherein each step positively or negatively selects for one or more markers.
  • FACS fluorescence activated cell sorting
  • the two or more different antibodies may be labeled with different fluorophores.
  • Magnetic cell sorting may be performed using super-paramagnetic microbeads composed of iron oxide and a polysaccharide coat.
  • the microbeads may be approximately 50 nanometers in diameter, and have a volume about one-millionth that of a typical mammalian cell.
  • the microbeads are preferably small enough to remain in colloidal suspension, which permits rapid, efficient binding to cell surface antigens.
  • the microbeads preferably do not interfere with flow cytometry, are biodegradable, and have negligible effects on cellular functions.
  • the antibody coupling to the microbeads may be direct or indirect, via a second antibody to a ligand such as fluorescein.
  • the antibody may be of classes IgG, IgM, IgA, IgD, and IgE, or fragments or derivatives thereof, including Fab, F(ab')2, Fd, and single chain antibodies, diabodies, bispecific antibodies, bifunctional antibodies and derivatives thereof.
  • the antibody may be a monoclonal antibody, polyclonal antibody, affinity purified antibody, or mixtures thereof which exhibits sufficient binding specificity to an epitope of a gene product specific for activated T cells, or a sequence derived therefrom.
  • the antibody may also be a chimeric antibody.
  • the antibody may directly bind to the gene product and may be used for cell selection.
  • magnetofluorescent liposomes Schottamine-like molecules
  • conventional fluorescently labeled antibodies may not be sensitive enough to detect the presence of the cell surface expressed gene product.
  • Fluorophore-containing liposomes may be conjugated to antibodies with the specificity of interest, thereby allowing detection of the cell surface expressed gene product.
  • intracellular gene products such as cytokines, the antibody may be used after permeabilizing the cells.
  • the intracellular gene product if it is ultimately secreted from the cell may be detected as it is secreted through the cell membrane using a "catch" antibody on the cell surface.
  • the catch antibody may be a double antibody that is specific for two different antigens: (i) the secreted gene product of interest and (ii) a cell surface protein.
  • the cell surface protein may be any surface marker present on T cells, in particular, or lymphocytes, in general, (e.g., CD45).
  • the catch antibody may first bind to the cell surface protein and then bind to the intracellular gene product of interest as it is secreted through the membrane, thereby retaining the gene product on the cell surface.
  • a labeled antibody specific for the captured gene product may then be used to bind to the captured gene product, which allows the selection of the activated T cell (Manz, et al. Proc. Natl. Acad. Sci. USA 92:1921-1925, 1995, incorporated herein by reference).
  • the T cells isolated by the present invention may be enriched by at least 90 percent from whole blood.
  • the T cells may also be enriched by at least 95 percent from whole blood.
  • the T cells may also be enriched by at least 98 percent from whole blood.
  • the T cells may also be enriched by at least 90 percent from whole blood.
  • the invention in another aspect, relates to a T-cell product which comprises the T-cells specific for the neoantigen identified by the method of the invention or for a peptide containing the sequence of the neoantigen.
  • the invention relates to method for obtaining a T-cell product comprising T-cells which specific for the neoantigen identified by the method of the invention or a peptide containing the sequence of said neoantigen comprising
  • step (ii) Contacting the neoantigen peptide obtained in step (i) with a T cell population and
  • the T cells targeting neoantigens identified through this invention can be used as a source to sequence the T-cell receptor conferring specificity of the T cells to the specific neoantigen. This sequence, further modified by codon optimization and used to genetically engineer PBMCs of the patient studied to express and redirect the T cells to said neoantigen. These T cells can be expanded ex vivo for patient infusion.
  • the invention relates to an immunogenic or vaccine composition according to the invention for use in medicine.
  • the invention relates to an immunogenic or vaccine composition according to the invention for use in a method of preventing or treating a disease in a subject which requires the generation of an immune response against the neoantigen or a peptide comprising the sequence of the neoantigen in the subject.
  • the invention relates to an immunogenic or vaccine composition according to the invention for use in a method of preventing or treating a disease in a subject which requires the generation of an immune response against the neoantigen or a peptide comprising the sequence of the neoantigen in the subject and wherein the neoantigen peptide of the immunogenic or vaccine composition has been isolated by the method of the invention.
  • the invention relates to a method for preventing or treating a disease in a subject which requires the generation of an immune response against the neoantigen or a peptide comprising the sequence of the neoantigen in the subject, which comprises a) Isolating a neoantigen peptide by the method according to the invention, wherein said neoantigen peptide comprises the mutation found in the tumor, b) Formulating the neoantigen peptide, a peptide comprising the sequence of the neoantigen peptide, a nucleic acid encoding said neoantigen peptide or encoding the peptide comprising the sequence of the neoantigen peptide peptide thereof as an immunogenic composition, and c) administering the immunogenic composition to the subject in need thereof.
  • the disease which requires the generation of an immune response against the neoantigen or a peptide comprising the sequence of the neoantigen is a cancer in which the cancer cells express the neoantigen.
  • the cancer to be treated is the cancer which appear in the subject from which the tumor-specific peptides used in step (i) are identified.
  • the invention relates to a T-cell product according to the invention for use in medicine.
  • the invention relates to a T-cell product according to the invention for use in a method of preventing or treating a disease in a subject which requires the generation of an immune response against the neoantigen or a peptide comprising the sequence of the neoantigen in the subject.
  • the disease which requires the generation of an immune response against the neoantigen or a peptide comprising the sequence of the neoantigen is a cancer in which the cancer cells express the neoantigen.
  • the cancer to be treated is the cancer which appear in the subject from which the tumor-specific peptides used in step (i) are identified.
  • the type of cancer that can be treated by the method according to the present invention is nor particularly limitative of the scope of the invention provided that at least one tumor-specific peptide can be isolated by the method according to the present invention. Accordingly, the present invention encompasses methods for the treatment of any of the cancer types mentioned in the context of the method of the invention for the isolation of tumor-specific peptides, i.e. skin cancer, lung cancer, bladder cancer, colorectal cancer, gastrointestinal cancer, esophageal cancer, gastric cancer, intestinal cancer, breast cancer, and a cancer caused by a mismatch repair deficiency.
  • the skin cancer can be selected from the group consisting of basal cell carcinoma, squamous cell carcinoma, merkel cell carcinoma, and melanoma.
  • the disclosure further provides a method of inducing a tumor specific immune response in a subject, vaccinating against a tumor, treating and or alleviating a symptom of cancer in a subject by administering the subject an immunogenic or vaccine composition of the disclosure.
  • the immunogenic or vaccine composition is administered in an amount sufficient to induce a CTL response.
  • the induction of the immune response immunizes the subject against the development of cancer.
  • a primary immune response is induced, while in other aspects, a secondary immune response is induced.
  • the disclosure also provides methods of inducing resistance to growth of a cancer in a subject, comprising administering to the subject a therapeutically effective amount of the neoantigens and/or the immunogenic and vaccine compositions disclosed herein.
  • the subject to be treated may have been newly diagnosed with cancer.
  • the subject may have been diagnosed with a cancer that has recurred after being previously treated with standard-of-care therapies.
  • the subject is one who has not been previously treated with any therapeutic approaches that are immunosuppressive.
  • the neoantigens and/or the immunogenic and vaccine compositions comprising neoantigens or peptides comprising the sequence of the neoantigens disclosed herein as well as the T-cell products which specifically recognize the neoantigens disclosed herein are administered pre-operatively; for example prior to surgery to reduce tumor burden.
  • the neoantigens, the peptides comprising the sequence of the neoantigens and/or the immunogenic compositions may be administered up to 24 hours, up to 36 hours, up to 48 hours or up to 72 hours before surgery.
  • the immunogenic composition may be administered about 48 hours to about 72 hours before surgery.
  • the administration is by intravenous bolus.
  • the neoantigens, peptides comprising the sequence of the neoantigens and/or the immunogenic an vaccine compositions comprising neoantigens or peptides comprising the sequence of the neoantigens disclosed herein are administered post-operatively.
  • the neoantigens, peptides comprising the sequence of the neoantigens and/or the immunogenic compositions may be administered up to 24 hours, up to 36 hours, up to 48 hours or up to 72 hours after surgery.
  • the immunogenic composition may be administered about 48 hours to about 72 hours after surgery.
  • the peptide or its variant may be prepared for intravenous (i.v.) injection, subcutaneous (s.c,) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.
  • Preferred methods of peptide injection include s.c., i.d., i.p., i.m., and i.v.
  • Preferred methods of DNA injection include i.d., i.m., s.c., i.p. and i.v.
  • doses of between 1 and 500 mg 50 mg and 1.5 mg, preferably 125 mg to 500 mg, of peptide or DNA may be given and will depend from the respective peptide or DNA. Doses of this range were successfully used in previous trials (Brunsvig P F, et al., Cancer Immunol Immunother. 2006; 55(12):1553-1564; M. Staehler, et al., ASCO meeting 2007; Abstract No 3017).
  • Other methods of administration of the immunogenic or vaccine composition are known to those skilled in the art.
  • the immunogenic and vaccine compositions may be compiled so that the selection, number and/or amount of peptides present in the composition is/are tissue, cancer, and/or patientspecific. For instance, the exact selection of peptides can be guided by expression patterns of the parent proteins in a given tissue to avoid side effects. The selection may be dependent on the specific type of cancer, the status of the disease, earlier treatment regimens, the immune status of the patient, and, of course, the HLA-haplotype of the patient. Furthermore, the immunogenic and vaccine compositions according to the disclosure can contain individualized components, according to personal needs of the particular patient. Examples include varying the amounts of peptides according to the expression of the related neoantigen in the particular patient, unwanted side effects due to personal allergies or other treatments, and adjustments for secondary treatments following a first round or scheme of treatment.
  • the respective pharmaceutical composition for treatment of this cancer may be present in high amounts and/or more than one peptide specific for this particularly protein or pathway of this protein may be included.
  • compositions comprising the neoantigen peptide or peptides or the peptide comprising the sequence of the neoantigen of the peptides comprising the sequences of one or more neoantigens of the invention may be administered to an individual already suffering from cancer.
  • compositions are administered to a patient in an amount sufficient to elicit an effective CTL response to the tumor antigen and to cure or at least partially arrest symptoms and/or complications.
  • Amounts effective for this use will depend on, e.g., the neoantigen peptide composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician, but generally range for the initial immunization (that is for therapeutic or prophylactic administration) from about 1 .0 mg to about 50,000 mg of peptide for a 70 kg patient, followed by boosting dosages or from about 1.0 mg to about 10,000 mg of peptide pursuant to a boosting regimen over weeks to months depending upon the patient’s response and condition by measuring specific CTL activity in the patient’s blood.
  • the peptide and compositions of the present disclosure may generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations, especially when the cancer has metastasized. In such cases, in view of the minimization of extraneous substances and the relative nontoxic nature of the peptide, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these peptide compositions.
  • administration should begin at the detection or surgical removal of tumors. This is followed by boosting doses until at least symptoms are substantially abated and for a period thereafter.
  • compositions for therapeutic treatment are intended for parenteral, topical, nasal, oral or local administration.
  • the pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly.
  • the compositions may be administered at the site of surgical excision to induce a local immune response to the tumor.
  • compositions for parenteral administration which comprise a solution of the peptides and vaccine compositions are dissolved or suspended in an acceptable carrier, preferably an aqueous carrier.
  • an acceptable carrier preferably an aqueous carrier.
  • aqueous carriers may be used, e.g..water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like.
  • compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered.
  • the resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • the immunogenic and vaccine compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • concentration of neoantigen peptides in the immunogenic and vaccine compositions can vary widely, i.e., from less than about 0.1 %, usually at or at least about 2% to as much as 20% to 50% or more by weight, and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of administration selected.
  • neoantigen peptides isolated by the method according to the present invention may also be administered via liposomes, which target the peptides to a particular cells tissue, such as lymphoid tissue.
  • liposomes are also useful in increasing the half-life of the peptides. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
  • the peptide to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, e.g., a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions.
  • a molecule which binds to e.g., a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions.
  • liposomes filled with a desired peptide of the disclosure can be directed to the site of lymphoid cells, where the liposomes then deliver the selected therapeutic/immunogenic peptide compositions.
  • Liposomes for use in the disclosure are formed from standard vesicle forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol.
  • lipids are generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream.
  • a variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9;467 (1980), USAU.S. Patent Nos. 4,235,871 ,4501728USA 4,501 ,728, 4,837,028, and 5,019,369.
  • a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells.
  • a liposome suspension containing a peptide may be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.
  • nontoxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.
  • a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient, that is, one or more peptides of the disclosure, and more preferably at a concentration of 25%-75%.
  • the immunogenic and vaccine compositions are preferably supplied in finely divided form along with a surfactant and propellant.
  • Typical percentages of peptides are 0.01 %-20% by weight, preferably 1 %-10%.
  • the surfactant must, of course, be nontoxic, and preferably soluble in the propellant.
  • Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride.
  • nucleic acids encoding one or more neoantigen peptides or peptides comprising the sequence of the neoantigens or a plurality of distinct nucleic acids encoding different neoantigen peptides or peptides comprising the sequence of the neoantigens can also be administered to the patient.
  • nucleic acids can be delivered directly, as "naked DNA". This approach is described, for instance, in Wolff et al., Science 247: 1465-1468 (1990) as well as U.S. Patent Nos. 5,580,859 and 5,589,406.
  • the nucleic acids can also be administered using ballistic delivery as described, for instance, in U.S. Patent No. 5,204,253. Particles comprised solely of DNA can be administered. Alternatively, DNA can be adhered to particles, such as gold particles.
  • the nucleic acids encoding one or more neoantigen peptides or peptides comprising the sequence of the neoantigens or a plurality of distinct nucleic acids encoding different neoantigen peptides or peptides comprising the sequence of the neoantigens are administered in the form of RNA.
  • the nucleic acids can also be delivered complexed to cationic compounds, such as cationic lipids.
  • the nucleic acids encoding the neoantigen peptides or peptides comprising the sequence of the neoantigens can also be delivered by attenuated viral hosts, such as vaccinia or fowl pox.
  • This approach involves the use of vaccinia virus as a vector to express nucleotide sequences that encode the neoantigen peptides.
  • the recombinant vaccinia virus Upon introduction into an acutely or chronically infected host or into a non-infected host, the recombinant vaccinia virus expresses the neoantigen peptide, and thereby elicits a host CTL response.
  • the therapy is carried out by a prime-boost strategy.
  • priming is carried out using an adenoviral vector, preferably a chimpanzee adenoviral vector (ChAdV) and boosting is carried out by a self-amplifying or replicating, synthetic viral
  • vectors useful for therapeutic administration or for immunization with the neoantigenic peptides or peptides comprising the sequence of the neoantigens using the nucleic acids encoding the peptides or peptides comprising the sequence of the neoantigens e.g., Salmonella typhi vectors and the like, will be apparent to those skilled in the art from the description herein.
  • a preferred means of administering nucleic acids encoding the neoantigen peptide or peptides comprising the sequence of the neoantigens uses TMGs, i.e. tandem minigene constructs encoding multiple neoantigen peptides.
  • the TMG used in the therapeutic methods of the invention consist of constructs that encode multiple neoantigen peptides identified by the method according to the present invention, and thus, differ from the TMGs used in the first step of the method of the invention to express the tumor-specific peptide in the HLA- expressing cells.
  • the amino acid sequences of the epitopes are reverse translated.
  • a human codon usage table is used to guide the codon choice for each amino acid.
  • the exact nucleotide sequence encoding for the neoantigen obtained as a result of tumor DNA and/or RNA sequencing can be used.
  • MHC presentation of the epitopes may be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL epitopes.
  • the TMG is converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene.
  • Overlapping oligonucleotides (30-100 bases long) are synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides are joined using T4 DNA ligase.
  • This synthetic minigene, encoding the CTL epitope polypeptide can then be cloned into a desired expression vector.
  • additional flanking promoter and 5’ and 3’ sequences can be added to enable the expression of the polypeptide from a DNA fragment instead of a vector or plasmid.
  • the DNA can be used as such or, alternatively, converted in vitro to RNA and then used the RNA for therapeutic purposes.
  • Methods for the generation of RNA from a DNA of interest are well known in the art.
  • the DNA is introduced into a vector wherein it is flanked by a promoter and by a polyA sequence as well as by other regulatory sequences needed for the in vitro generation of RNA.
  • Standard regulatory sequences well known to those of skill in the art are included in the vector to ensure expression in the target cells.
  • Several vector elements are required: a promoter with a down-stream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g. ampicillin or kanamycin resistance).
  • E. coli origin of replication e.g. ampicillin or kanamycin resistance
  • Numerous promoters can be used for this purpose, e.g., the human cytomegalovirus (hCMV) promoter. See, U.S. Patent Nos, 5,580,859 and 5,589,466 for other suitable promoter sequences.
  • introns are required for efficient gene expression, and one or more synthetic or naturally-occurring introns could be incorporated into the transcribed region of the minigene.
  • mRNA stabilization sequences can also be considered for increasing minigene expression.
  • immunostimulatory sequences ISSs or CpGs
  • a bicistronic expression vector to allow production of the minigene- encoded epitopes and a second protein included to enhance or decrease immunogenicity
  • proteins or polypeptides that could beneficially enhance the immune response if co-expressed include cytokines (e.g., IL2, IL12, GM-CSF), cytokineinducing molecules (e.g. LelF) or costimulatory molecules.
  • Helper (HTL) epitopes could be joined to intracellular targeting signals and expressed separately from the CTL epitopes. This would allow direction of the HTL epitopes to a cell compartment different than the CTL epitopes.
  • immunosuppressive molecules e.g. TGF-P
  • TGF-P immunosuppressive molecules
  • the TMG is cloned into the polylinker region downstream of the promoter.
  • This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements included in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as a master cell bank and a working cell bank.
  • Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffer saline (PBS). A variety of methods have been described, and new techniques may become available. As noted above, nucleic acids are conveniently formulated with cationic lipids. In addition, glycolipids, fusogenic liposomes, peptides and compounds referred to collectively as protective, interactive, non-condensing (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.
  • PINC protective, interactive, non-condensing
  • the nucleic acids encoding the neoantigen peptide or peptides comprising the sequence of the neoantigens are administered in the form of RNA.
  • the RNA is obtained in vitro by transcription of the corresponding DNA, in which case the DNA is inserted into a vector that contains the adequate signals for allowing in vitro transcription and generation of functional RNA, such as a promoter and 3’ polyadenylation sequences.
  • Electroporation can be used for "naked" DNA or for RNA, whereas cationic lipids allow direct in vitro transfection of DNA or RNA.
  • a plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow enrichment of transfected cells using fluorescence activated cell sorting (FACS). These cells are then chromium-51 labeled and used as target cells for epitope-specific CTL lines. Cytolysis, detected by 51 Cr release, indicates production of MHC presentation of mini gene-encoded CTL epitopes.
  • GFP green fluorescent protein
  • In vivo immunogenicity is a second approach for functional testing of minigene DNA formulations or the corresponding RNA.
  • Transgenic mice expressing appropriate human MHC molecules are immunized with the DNA product.
  • the dose and route of administration are formulation dependent (e.g. IM for DNA in PBS, IP for lipid-complexed DNA).
  • Twenty- one days after immunization splenocytes are harvested and restimulated for 1 week in the presence of peptides encoding each epitope being tested.
  • These effector cells (CTLs) are assayed for cytolysis of peptide-loaded, chromium-51 labeled target cells using standard techniques. Lysis of target cells sensitized by MHC loading of peptides corresponding to minigene encoded epitopes demonstrates DNA vaccine function for in vivo induction of CTLs.
  • Neoantigen peptides may be used to elicit CTL ex vivo, as well.
  • the resulting CTL can be used to treat chronic tumors in patients that do not respond to other conventional forms of therapy, or will not respond to a peptide vaccine approach of therapy.
  • Ex vivo CTL responses to a particular tumor antigen are induced by incubating in tissue culture the patient’s CTL precursor cells (CTLp) together with a source of antigen-presenting cells (HLA-expressing cells) and the appropriate neoantigen peptide or expressing a polynucleotide encoding for the neoantigen peptide or comprising the neoantigen peptide or peptides.
  • CTLp CTL precursor cells
  • HLA-expressing cells source of antigen-presenting cells
  • the cells After an appropriate incubation time (typically 1-4 weeks), in which the CTLp are activated and mature and expand into effector CTL, the cells are infused back into the patient, where they will destroy their specific target cell (i.e., a tumor cell).
  • the culture of stimulator cells is maintained in an appropriate serum-free medium.
  • an amount of neoantigen peptide or peptides comprising the sequence of the neoantigens is added to the stimulator cell culture, of sufficient quantity to become loaded onto the human Class I molecules to be expressed on the surface of the stimulator cells.
  • a sufficient amount of peptide is an amount that will allow about 200, and preferably 200 or more, human Class I MHC molecules loaded with peptide to be expressed on the surface of each stimulator cell.
  • the stimulator cells are incubated with >0.1 ug/ml peptide.
  • the stimulator cells are incubated with > 3, 4, 5, 10, 15, or more g/ml peptide.
  • Resting or precursor CD8+ cells are then incubated in culture with the appropriate stimulator cells for a time period sufficient to activate the CD8+ cells.
  • the CD8+ cells are activated in an antigen-specific manner.
  • the ratio of resting or precursor CD8+ (effector) cells to stimulator cells may vary from individual to individual and may further depend upon variables such as the amenability of an individual’s lymphocytes to culturing conditions and the nature and severity of the disease condition or other condition for which the within- described treatment modality is used.
  • the lymphocyte:stimulator cell ratio is in the range of about 0.1 :1 to 300:1 .
  • the effector/stimulator culture may be maintained for as long a time as is necessary to stimulate a therapeutically useable or effective number of CD8+ cells.
  • CTL CTL precursor
  • Peptide loading of empty major histocompatibility complex molecules on cells allows the induction of primary cytotoxic T lymphocyte responses.
  • Peptide loading of empty major histocompatibility complex molecules on cells enables the induction of primary cytotoxic T lymphocyte responses.
  • mutant cell lines do not exist for every human MHC allele, it is advantageous to use a technique to remove endogenous MHC-associated peptides from the surface of APC, followed by loading the resulting empty MHC molecules with the immunogenic peptides of interest.
  • the use of non-transformed (non-tumorigenic), non-infected cells, and preferably, autologous cells of patients as APC is desirable for the design of CTL induction protocols directed towards development of ex vivo CTL therapies.
  • This application discloses methods for stripping the endogenous MHC-associated peptides from the surface of APC followed by the loading of desired peptides.
  • a stable MHC class I molecule is a trimeric complex formed of the following elements: 1 ) a peptide usually of 8 - 12 residues, 2) a transmembrane heavy polymorphic protein chain which bears the peptide-binding site in its a1 and a2 domains, and 3) a non-covalently associated non-polymorphic light chain, P2microglobuiin. Removing the bound peptides and/or dissociating the P2microglobulin from the complex renders the MHC class I molecules nonfunctional and unstable, resulting in rapid degradation. All MHC class I molecules isolated from PBMCs have endogenous peptides bound to them. Therefore, the first step is to remove all endogenous peptides bound to MHC class I molecules on the APC without causing their degradation before exogenous peptides can be added to them.
  • Two possible ways to free up MHC class I molecules of bound peptides include lowering the culture temperature from 37°C to 26°C overnight to destablize P2-microglobulin and stripping the endogenous peptides from the cell using a mild acid treatment.
  • the methods release previously bound peptides into the extracellular environment allowing new exogenous peptides to bind to the empty class I molecules.
  • the cold-temperature incubation method enables exogenous peptides to bind efficiently to the MHC complex, but requires an overnight incubation at 26°C which may slow the cell’s metabolic rate. It is also likely that cells not actively synthesizing MHC molecules (e.g., resting PBMC) would not produce high amounts of empty surface MHC molecules by the cold temperature procedure.
  • Harsh acid stripping involves extraction of the peptides with trifluoroacetic acid, pH 2, or acid denaturation of the immunoaffinity purified class l-peptide complexes. These methods are not feasible for CTL induction, since it is important to remove the endogenous peptides while preserving APC viability and an optimal metabolic state which is critical for antigen presentation.
  • Mild acid solutions of pH 3 such as glycine or citrate-phosphate buffers have been used to identify endogenous peptides and to identify tumor associated T cell epitopes, The treatment is especially effective, in that only the MHC class I molecules are destabilized (and associated peptides released), while other surface antigens remain intact, including MHC class II molecules.
  • Activated and or neoantigen-specific CD8+ and or CD4+ cells may be effectively separated from the stimulator cells using one of a variety of known methods.
  • monoclonal antibodies specific for the stimulator cells, for the peptides loaded onto the stimulator cells, or for the CD8+ cells (or a segment thereof) may be utilized to bind their appropriate complementary ligand.
  • Antibody-tagged molecules may then be extracted from the stimulator-effector cell admixture via appropriate means, e.g., via well-known immunoprecipitation or immunoassay methods.
  • Effective, cytotoxic amounts of the activated T cells can vary between in vitro and in vivo uses, as well as with the amount and type of cells that are the ultimate target of these killer cells. The amount will also vary depending on the condition of the patient and should be determined via consideration of all appropriate factors by the practitioner. Preferably, however, about 1 X 10 6 to about 1 X 10 12 , more preferably about 1 X 10 8 to about 1 X 10 11 , and even more preferably, about 1 X 10 9 to about 1 X 10 1 ° activated CD8+ cells are utilized for adult humans, compared to about 5 X 10 6 - 5 X 10 7 cells used in mice.
  • the activated or neoantigen specific T cells are harvested from the cell culture prior to administration to the individual being treated. It is important to note, however, that unlike other present and proposed treatment modalities, the present method uses a cell culture system that is not tumorigenic. Therefore, if complete separation of stimulator cells and activated CD8+ cells is not achieved, there is no inherent danger known to be associated with the administration of a small number of stimulator cells, whereas administration of mammalian tumor promoting cells may be extremely hazardous.
  • Methods of re-introducing cellular components are known in the art and include procedures such as those exemplified in U.S. Patent No. 4,844,893 to Honsik, et al. and U.S. Patent No. 4,690,915 to Rosenberg.
  • administration of activated CD8+ and or CD4+ cells via intravenous infusion is appropriate.
  • the modified T cell is adoptively transferred to the patient.
  • Adoptive cell transfer is an effective form of immunotherapy and involves the transfer of immune cells with antitumor activity into cancer patients.
  • Lymphocytes used for adoptive transfer can be derived from the blood, the stroma of resected tumors and genetically modified peripheral blood lymphocytes although other sources of such cells are known in the art.
  • the lymphocytes employed in ACT can be administered in a single dose. Such administration can be by injection, e.g., intravenous injection.
  • the lymphocytes can be administered in multiple doses. Dosing may be once, twice, three times, four times, five times, six times, or more than six times per year.
  • Dosing may be once a month, once every two weeks, once a week, or once every other day. Administration of cytotoxic lymphocytes can continue as long as necessary.
  • the methods described herein can be used to determine the immunorepertoire of a subject.
  • the immunorepertoire is analyzed: before a treatment, during a treatment, and/or after a treatment.
  • the treatment is a cancer treatment.
  • the immunotherapy comprises an adoptive cell transfer of T cells.
  • the T cells comprise a recombinant TCR or a chimeric antigen receptor.
  • the immunorepertoire provides information to provide a targeted therapy.
  • TIL tumor cell line
  • tumor fragments were cultured in individual wells of a 24-well plate in T cell media containing IL-2 at 37°C and 5% CO2 and expanded independently for 15-30 days and cryopreserved. Fresh media containing IL-2 was added at day 5 and media was changed or TILs were split when confluent every other day thereafter.
  • PBMCs were obtained using a Ficol-Hypaque gradient and cryopreserved for DNA extraction for WES and to expand B cells ex vivo.
  • genomic DNA and total RNA were purified from a cell pellet of VHIO-029 tumor cell line (TCL) and VHIO-008 TCL at early passage. A small aliquot of PBMCs was pelleted to extract normal DNA. WES libraries were generated by exome capture of approximately 20,000 coding genes using SureSelect human All exon V6 kit (Agilent Technologies) and paired-end sequencing was performed on a HiSeq sequencer (Illumina) at Macrogen. The average sequencing depth ranged from 100-150 for each of the individual libraries generated. Alignments of WES to the reference human genome build hg19 were performed using novoalign MPI from novocraft.
  • NSM for generation of TMGs were selected based on the presence the tumor exome. Mutations were manually vetted using IGV. TMG1-TMG12 encoded for minigenes arranged based on their predicted binding affinity to HLA from highest to lowest as well as other variables such as higher variant allele frequency and others.
  • RNA sequencing library was also prepared from tumor biopsies using Illumina TruSeq RNA library prep kit. RNA alignment was performed using STAR. Duplicates and marked using Picard’s Mark Duplicate tools. Fragments per million mapped reads (FPKM) values were calculated using cufflinks. The levels of specific transcripts encoding for putative NSM variants was calculated as FPKM and used to assess expression of candidate mutations.
  • TMGs were constructed as previously described (Lu et al., Clin. Cancer Res. 2014;20:3401- 10). For each nonsynonymous variant identified by WES, we constructed a minigene, consisting of the mutant amino acid flanked by 12 amino acids of the WT protein sequence. Up to 24 minigenes were concatenated to generate a TMG construct. TMG plasmids were synthesized by Gene Script. One microgram of linearized (Notl; New England Biolabs) plasmid DNA was used as a template to generate in vitro-transcribed TMG RNA using the HiScribe T7 ARCA mRNA kit (New England Biolabs), as instructed by the manufacturer.
  • HLA was determined from the WES data using the PHLAT algorithm. Eluted ligand likelihood (ELL) percentile rank scores for binding to the patient’s HLA molecules were obtained for all unique peptides >8 AA in length eluted from electroporated B-cells or from the cell line using NetMHCpan 4.0. The threshold for binding was set to ⁇ 2%-tile rank. Crude or HPLC purified minimal or 25-mer peptides were purchased from JPT.
  • B cells were isolated from PBMCs by positive selection using CD19 + microbeads (Miltenyi Biotec). B cells were expanded through CD40-CD40L stimulation by culturing cells for 4-5 days with irradiated NIH3T3 feeder cells constitutively expressing CD40L at 37°C in 5% CO2 in B cell medium (Iscove’s IMDM media + 10% human AB serum Biowest + 100U/ml Penicillin and 100 ug/ml streptomycin + 2mM L-Glutamine, supplemented with 200u/ml IL-4 (Peprotech). Up to three rounds of stimulation and expansion were performed consecutively. B cells were used fresh or cryopreserved at day 5 to 6. When used after cryopreservation, cells were thawed into B cell medium + DNAse (1 :1000) one day prior to electroporation.
  • CD4+ T cells were isolated from PBMCs by positive selection using CD4 + microbeads (Miltenyi Biotech) and subsequently expanded through a rapid expansion protocol for 14 days. Irradiated allogeneic PBMCs (50 Gy) pooled from three donors were used as feeder cells in T cell medium supplemented with 30 ng/ml anti-CD3 (OKT3, Biolegend) and 3,000 IU of interleukin (IL)-2 (Proleukin). After day 6, half of the medium was replaced with fresh T cell medium containing IL-2 every other day.
  • IL interleukin
  • Immature dendritic cells were generated from PBMC using the plastic-adherence method.
  • Cells were cultured in DC medium (RPMI supplemented with 5% human serum, 100 U/ml penicillin, 100 pg/ml streptomycin, 2 mM l-glutamine (Life Technologies), 800 lU/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 200 U/ml IL-4 (Peprotech).
  • DC medium RPMI supplemented with 5% human serum, 100 U/ml penicillin, 100 pg/ml streptomycin, 2 mM l-glutamine (Life Technologies), 800 lU/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 200 U/ml IL-4 (Peprotech).
  • DC medium RPMI supplemented with 5% human serum, 100 U/ml penicillin, 100 pg/ml streptomycin,
  • a total of 4-5x10 6 immature DCs or 1-2 * 10 7 B cells or T cells were resuspended in 10Oul of Opti-MEM media and transferred into a sterile 0.2-cm cuvette (VWR electroporation cuvettes). 12-18 ug of the RNA derived from a specific TMG from the patient or from an irrelevant TMG (as a control) were added. As a positive control for electroporation, 2.5 x 10 6 B cells were electroporated with 8 ug of GFP-RNA. Cells were electroporated at 400V, 0.5 ms and 1 pulse or at 150 V, 10-20 ms and one pulse using a ECM 830 BTX- Electroporator.
  • peptide pulsing For peptide pulsing, the individual 25-mers were synthesized by JPT and were grouped into peptide pools (PPs), each containing up to 26 peptides. Each peptide pool consisted of a mix of all the mutant 25-mers encoded by one TMG construct. Twelve PPs containing the same mutant peptides as VHIO-029 TMG1-12 were generated. A total of 1 x 10 7 B cells were resuspended in 700 pl of B-cell medium supplemented with 200 U/ml of IL-4, transferred to a sterile 15 ml Falcon and pulsed for 20 h with 5 pg/ml each of the PP mix (PP1 to PP6) at 37 °C and 5% CO2. Cells were then washed three times with PBS and dry cell pellets were snap frozen until further analysis.
  • PPs peptide pools
  • Purified anti-HLA-l clone W6/32 was cross-linked to protein-A Sepharose 4B conjugate beads (Invitrogen) at a concentration of 10 mg of antibodies per 1 ml volume of beads. To this end, antibodies were incubated with beads for 1 h at RT in the orbital shaker. Cross-linking was performed by addition of Dimethyl pimelimidate dihydrochloride (Sigma- Aldrich) in 0.2 M Sodium Borate buffer pH 9 (Applichem) at a final concentration of 20 mM for 30 min. Finally, beads with linked antibodies were incubated with 0.2 M ethanolamine pH 8 (Sigma) for 2 h. Anti-HLAI Sepharose4B beads were stored at 4°C until use.
  • the HLA-I peptide affinity chromatography was performed using a 96-well single-use microplate with 3 pm glass fiber and 10 pm polypropylene membranes (Agilent). Sep-Pak tC18 100 mg Sorbent 96-well plates (Waters) were used for peptide purification and concentration as previously described (Marino F. et al., Methods Mol Biol. 2019;1913:67-79). A vacuum manifold was used for washing steps.
  • Tumor cells used as a source for pHLA purification and peptide elution were expanded up to 5.5 x 10 7 cells for VHIO-029 and up to 3e8 for VHIO-008 TCL, harvested by trypsinization washed with PBS, snap frozen and stored as dry cell pellet at -80 °C until use.
  • B cells 20h after electroporation cells were harvested, washed with PBS and snap-frozen and stored as dry cell pellets at -80 C. Snap-frozen pellets were re-suspended in PBS containing 0.6% CHAPS and Protease inhibitor Cocktail Complete (Roche) by gentle shaking (250 rpm) for 1 h at 4°C.
  • the cell lysates were sonicated using Misonix 3000 for 4 cycles (1 .20 min in total) at an output level 3, 20 sec on, 20 sec off. Lysates were cleared by centrifugation at 15.000 rpm at 4 °C for 1 h (Eppendorf Centrifuge) to obtain the soluble fraction containing the pHLA complexes.
  • HLA complexes Fresh lysates were loaded on the corresponding wells and were allowed to flow by gravity into the collection plate at 4 °C. Each well was washed as follows: 4 x 2 ml of 150 mM sodium chloride (NaCI) (Panreac Applichem) in 20 mM Tris- HCI pH 8, 4 x 2 ml of 400 mM NaCI in 20 mM Tris-HCI pH 8 and again with 4 x 2 ml of 150 mM NaCI in 20 mM Tris-HCI pH 8. Finally, wells were washed twice with 2 ml of 20 mM Tris- HCI pH 8.
  • NaCI sodium chloride
  • HLA-I peptides were eluted with 500 pl of 32,5% ACN in 0.1 % TFA.
  • Eluted peptides were transferred into Eppendorf tubes that were immediately snap-freeze with liquid N2 and stored at -80°C. HLA class I heavy chains and the P2m molecules were also recovered from the C18 plates using 300 pl of 80% ACN in 0.1 % TFA.
  • HLA-I peptides were completely lyophilized (LyoQuest, Telstar), and then cleaned and desalted with TopTips (PolyLC Inc.). Briefly after lyophilization, samples were resuspended in 20 pL of 20%ACN + 0.1 % FA, sonicated for 2min in a water bath and then loaded on TopTips that were previously conditioned with 20%ACN + 0.1 % FA buffer. After three washes with 20%+0.1 %FA buffer, peptides were eluted with 5% NH4OH in 30% MetOH. Peptides were finally dried using vacuum centrifugation (SpeedVac, Savant, Thermo Scientific).
  • Peptides were diluted in 3% ACN, 1 % FA. Samples were loaded to a 300 pm * 5 cm Acclaim Pep-Map nanoViper, C18 (Thermo Scientific) at a flow rate of 15 pl/min using a Thermo Scientific Dionex Ultimate 3000 chromatographic system (Thermo Scientific).
  • the column outlet was directly connected to an Advion TriVersa NanoMate (Advion) fitted on an Orbitrap Fusion LumosTM Tribrid (Thermo Scientific).
  • the mass spectrometer was operated in a data-dependent acquisition (DDA) mode.
  • Survey MS scans were acquired in the orbitrap with the resolution (defined at 200 m/z) set to 120,000.
  • the lock mass was user-defined at 445.12 m/z in each Orbitrap scan.
  • the top speed (most intense) ions per scan were fragmented in the linear ion trap (CID) and detected in the Orbitrap with the resolution set to 30,000.
  • Quadrupole isolation was employed to selectively isolate peptides of 300-650 m/z.
  • the predictive automatic gain control (pAGC) target was set to 1.5e5.
  • the maximum injection time was set to 50ms for MS1 and 150ms for MS2 scan. Included charged states were 2 and 3.
  • Target ions already selected for MS/MS were dynamically excluded for 10 s.
  • the mass tolerance of this dynamic exclusion was set to ⁇ 2.5 ppm from the calculated monoisotopic mass.
  • Spray voltage in the NanoMate source was set to 1 .6 kV.
  • RF Lens were tuned to 30%.
  • Minimal signal required to trigger MS to MS/MS switch was set to 5000 and activation Q was 0.250.
  • the spectrometer was working in positive polarity mode and singly charge state precursors were rejected for fragmentation.
  • PEAKS-X Pro studio was used to identify the peptide sequences using a personalized database for each patient containing the human proteome (Swiss-Prot ), the most frequently observed contaminants in proteomics plus the AA sequences of all NSM identified by tumor WES.
  • the database included the exact AA sequence of all the TMGs encoding for the concatenated mutated minigenes for VHIO-029 or VHIO-008.
  • FDR False discovery rate
  • TIL In order to enrich for peptide-reactive TIL, in some instances TIL were co-incubated with peptide-pulsed or TMG RNA-electroporated B cells and 20 h later 4-1 BB+ cells were sorted using a BD FACSAria and expanded ex vivo for 14 days in presence of anti-CD3, irradiated allogeneic feeders and 3000 lU/ml of IL-2.
  • T cell populations of interest including ex vivo expanded TIL or enriched populations of tumor or TMG-reactive lymphocytes were co-incubated overnight with either peptide pulsed or TMG RNA electroporated B cells.
  • Minimal peptides were pulsed for 1 h, while 25- residue peptides were incubated with APCs overnight prior to the co-culture.
  • T cell reactivity was measured 20-24 h later using an IFN-y ELISPOT assay and detection of 4-1BB expression on the CD8+ T cells by flow cytometry.
  • T cells were thawed into T cell medium supplemented with 3000 IU IL-2 and DNAse two to three days before coincubation with target cells.
  • T cells were washed and replaced with cytokine- free T cell medium.
  • cytokine- free T cell medium typically, equal volumes (100 pL) of T cells and APCs or tumor cells were mixed together in a 96-well plate. 2 x 10 4 to 5 x 10 4 T cells were coincubated with 1 x 10 5 or 3 x 10 5 peptide-pulsed or TMG-electroporated DCs or B cells, respectively. All cocultures were performed in the absence of exogenously added cytokines. For all the assays, plate-bound OKT3 (1 pg/mL; Miltenyi Biotec) was used as a positive control. Media, DMSO (Sigma-Aldrich), and/or irrelevant TMGs or peptides were used as negative controls. Analysis and Statistics
  • Fig. 1 shows a scheme of the ex vivo immunopeptidomics-based method according to the present invention.
  • the method aims to empirically identify the neoepitopes that can be naturally processed and presented on cell surface HLA-I and/or HLA-II molecules in a personalized fashion.
  • Fig. 1 Here we tested the ability of the method depicted in Fig. 1 to identify clinically relevant candidate neoantigens that could be used to vaccinate or to generate personalized T-cell therapies for patient VHIO-029. Briefly, tumor and normal DNA was used to identify all the tumor-specific NSM through WES and this information was used to design and construct 12 TMGs, each encoding for up to 24 NSM and covering all 283 NSM mutations identified.
  • Minigenes encoded by each TMG were designed based on their likelihood of presentation on HLA using an algorithm that takes into account the HLA binding prediction based on NetMHCpan 4.0, the number of minimal epitopes with ⁇ 2 percentile rank predicted to bind to HLA from each mutated 25-mer, and the variant allele frequency, among other factors.
  • TMG12 encoded for those that had lowest average predicted binding affinity ranging 4.3749 to up to greater than 6 %- tile rank).
  • ex vivo stimulated B-cells from VHIO-029 patient were electroporated with each individual TMG RNA, cells were rested 20h in the incubator and harvested, pelleted and snap-frozen.
  • cell pellets were lysed and centrifuged to clear the supernatant containing the pHLA-l complexes.
  • immunoaffinity chromatography Protein- A Sepharose beads were cross-linked with anti HLA-I antibody and added into 12 independent wells from a 96-well plate. The lysates were passed by gravity flow first through the plate with anti-HLA-l specific beads.
  • Peptides predicted to bind to at least one HLA-I allele of VHIO- 029 with a minimal percentile rank of ⁇ 2 were considered binders. Approximately 86% of the peptides were predicted to bind ( ⁇ 2%-tile rank) to at least one of the HLA-I molecules of VHIO-029, and 80% of the peptides were considered strong binders (>0.5%-tile rank) based on this analysis (Fig. 2c). Altogether, this supports that the peptides identified were presented by HLA-I on the cell surface of electroporated B cells.
  • the peptides mapping to TMGs were categorized into: 1 ) mutated, when containing a mutant residue identified by tumor WES, 2) wild type, if derived from part of a minigene that does not contain a mutant residue, and 3) artificial junction peptides, if the peptide resulted from the concatenation of two consecutive minigenes.
  • TMG1 Six mutated peptides were eluted only from B cells expressing TMG1 , being the TMG with the highest number of candidate neoantigens identified. Despite no mutated peptides were identified from B cells electroporated with TMG5-TMG12, artificial junction peptides and wild-type peptides could be identified in TMG6, 7, 10, 11 and 12, supporting that the TMGs were being expressed processed and some peptides were being presented.
  • mutated and artificial junction peptides identified in cells electroporated with a specific TMG uniquely mapped to the AA sequence of that specific minigene and not the rest of the TMG sequences included in the personalized database, supporting that the peptides identified were eluted as a result of the expression and processing of the specific TMG electroporated.
  • the AA sequences of the 11 peptides identified using this approach and their predicted binding affinity to HLA is shown in Table 2.
  • aName of the protein and the AA position and specific change bMutated AA is shown in bold c Binding prediction NetMHCpan4.1.
  • SB strong binder (%-tile rank ⁇ 0.5); WB, weak binder (%-tile rank >0.5 and ⁇ 2); NB, non-binder (%-tile rank >2) dVariant allele frequency represents the percent of mutated reads of the total reads elevel of gene expression relative to all genes detected by RNAseq f++, >7% VAF by WES and >10% VAF by RNA sequencing and >20 percentile gene expression;
  • HLA-I peptide elution from VHIO-029 tumor cell line resulted in the identification of three mutated peptides derived from GEMIN5 P .SI36OL, ETV1 P .E45SK and CD74 P .EI64K.
  • EX vivo expanded TILs from VHIO-029 tumor biopsy recognized two of the three neoantigens identified derived from GEMIN5 p .si36oi_and ETV1 P .E455K, while they displayed less recognition of the corresponding wt counterparts (Fig. 6).
  • the neoantigens recognized by TILs were HLA- A*11 :01 and HLA-B*35:01 restricted, respectively, as predicted by NetMHCPan4.1 (Fig. 6).
  • all three peptides, including the two that were found to be immunogenic were detected using autologous B cells expressing either TMG1 or TMG2 as a source for HLA-I immunopeptidomics.
  • the genomic variants encoding for 3 of the mutated peptides (SEQ ID NO: 9- 11 ) exclusively detected in TMG electroporated B cells were not detected by RNA sequencing in VHIO-029 cell line and the genes encoding for these variants were detected at relatively low level.
  • the lack of detection of these mutated peptides in HLA-I of VHIO-029 cell line was consistent with the lack of detection by RNA sequencing.
  • these mutated peptides can be synthesized and pulsed either individually or grouped into peptide pools (PPs) onto antigen presenting cells (APCs).
  • PPs peptide pools
  • APCs antigen presenting cells
  • B cells were pulsed overnight individually with VHIO-029 PP1-PP6, as a representative example, cells were washed thoroughly to remove excess peptides and cells were snap frozen and used to isolate pHLA-l complexes, elute and purify peptides as previously described. Mass spectrometry LC-MS/MS was carried out using an Orbitrap Lumos. Of the 3161 unique peptides identified by pHLA-l elution from peptide-pulsed B cells, 22 were mutated and 17 were wild type peptides that mapped to the AA sequences from PP1-PP6 ( Figure 7). Using this approach, we identified the immunogenic mutated peptide derived from GEMIN5p.si36oi.
  • aName of the protein and the AA position and specific change bMutated AA is shown in bold c Binding prediction NetMHCpan4.1 .
  • SB strong binder (%-tile rank ⁇ 0.5); WB, weak binder (%-tile rank >0.5 and ⁇ 2); NB, non-binder (%-tile rank >2) dVariant allele frequency represents the percent of mutated reads of the total reads elevel of gene expression relative to all genes detected by RNAseq f ++ >7% VAF by WES and >10% VAF by RNA sequencing and >20 percentile gene expression;
  • mutated peptides were identified via immunopeptidomics of both peptide-pulsed and TMG-transfected B cells. These 5 peptides included two out of the three mutated peptides that were detected following pHLA-l elution of the VHIO-029 tumor cell line, including GEMIN5 P .SI36OL a neoantigen recognized by TIL. Interestingly, 6 mutated peptides were uniquely presented by HLA-I of TMG1-6 transfected B cells, but not in PP1-PP6 pulsed B cells. In the other hand, 17 mutated peptides derived from 12 unique NSM were exclusively detected in peptide-pulsed B cells, but not TMG-transfected cells.
  • TMG-electroporated or peptide-pulsed B cells have previously been used to screen T cells for neoantigen recognition through a co-culture with autologous lymphocyte populations isolated from the tumor or blood of a patient.
  • a neoantigen can only be identified if a T cell capable of recognizing it is interrogated.
  • certain neoantigens might be overseen, despite they are processed and presented, due to the limited repertoire of TCRs and limited availability of T cell populations.
  • NeoPepi-HLAScan was capable of identifying additional candidate neoantigens that were not detected by screening T cells using a personalized screening approach.
  • TILs screened for recognition of B cells electroporated with TMG1-12 recognized TMG1 (Fig. 8a).
  • TIL displayed recognition of PP1 (not shown).
  • Further testing for recognition of the individual mutated 25-mer peptides encoded by TMG1 and included in PP1 revealed the recognition of GEMIN5 P .SI36OL and ETV1 P .E45SK (Fig. 8b). These two neoantigens were detected via immunopeptidomics of VHIO-029 cell line, as well as from TMG1 electroporated B cells.
  • HLA-I peptides from TMG1-TMG12 transfected B cells and PP1-PP6-pulsed B cells detected 9 and 21 additional mutated peptides, respectively.
  • Those expressed by the cell line at RNA level have the potential of being presented on HLA-I by the tumor cell line or APCs, and could be recognized by T cells.
  • B cells transfected with TMG RNA encoding for mutated minigenes or B cells pulsed with mutated 25-mers identified three and two, respectively, candidate neoantigens that were detected in the VHIO-29 tumor cell line, two of which were immunogenic.
  • candidate neoantigens that were detected in the VHIO-29 tumor cell line, two of which were immunogenic.
  • this method can be used to identify candidate neoantigens for vaccines and T-cell therapies, overcoming the need to obtain relatively large amount of tumor material or to generate a cell line to perform immunopeptidomics, and also overcoming the need of performing cumbersome and time-consuming immunological screening assays.
  • this method allows the selection of empirically validated peptides presented on HLA in an autologous setting, overcoming the need to prioritize peptides based on prediction algorithms that typically give rise to hundreds of candidates. This is particularly important, given that only a limited number of candidates can be included in each personalized vaccine.
  • NeoPepi-HLAScan using TMG or PPs identified 2 or 10 additional mutated peptides (from 2 and 9 different variants identified by WES, respectively), that were not detected in the tumor cell line, despite they were expressed based on RNA transcript levels.
  • PRM targeted sequencing analysis at high resolution to detect low amounts of these peptides in the cell line is in progress, it is possible that by forcing the expression of the mutated minigenes encoded by TMGs or by pulsing B cells with high amounts of peptides, this technique appears to be more sensitive at detecting mutated peptides.
  • NeoPepi- HLAScan is an attractive personalized high-throughput approach to identify vaccine and T-cell therapy targets that could be used to develop personalized cancer immunotherapies.
  • NeoPepi-HLAScan was selected.
  • patient VHIO-008 with hypopharyngeal carcinoma was selected.
  • TMGs TMGs
  • the selected 266 minigenes encoded by each TMG were grouped based on their likelihood of presentation on HLA using an algorithm that takes into account the HLA binding prediction based on NetMHCpan 4.0, the number of minimal epitopes with ⁇ 2 percentile rank predicted to bind to HLA from each mutated 25-mer, and the variant allele frequency, among other factors, but without taking into account RNA expression level.
  • 12 TMG constructs were designed, each encoding from 14 to up to 26 NSM.
  • ex vivo stimulated B cells from VHIO-008 patient were electroporated with each individual TMG RNA.
  • Cells were rested 20h in the incubator and harvested, pelleted and snap-frozen in order to elute the pHLA-l complexes.
  • immunoaffinity chromatography peptides were dissociated from the HLA complex using acidic elution, concentrated, desalted and two technical replicate MS measurements of purified peptides were performed by LC-MS/MS using an Orbitrap Fusion Lumos mass spectrometer.
  • PEAKS-X Pro software was employed to search the experimental spectra against a personalized database including all proteins annotated in Swiss-Prot as well as the AA sequences of all the TMGs constructed for VHIO-008.
  • TILs from VHIO-008 tumor biopsy recognized one of the three neoantigens identified, RPL14 P .H2OY, but did not recognize the corresponding wildtype counterpart.
  • the neoantigen recognized by TILs was HLA-B*07:02 restricted (Fig. 12), as predicted by NetMHCPan 4.1.
  • Two of the three neoantigens (RPL14 P .H2OY, PSMD12 P .H2OM) were detected by eluting HLA-I peptides from VHIO-008 B cells electroporated with TMG3 and TMG5 RNAs, including one that was immunogenic, demonstrating that this method can detect a large proportion of the neoantigens presented by the tumor cell line.
  • aName of the protein and the AA position and specific change bMutated AA is shown in bold c Binding prediction NetMHCpan4.1.
  • SB strong binder (%-tile rank ⁇ 0.5); WB, weak binder (%-tile rank >0.5 and ⁇ 2); NB, non-binder (%-tile rank >2) dVariant allele frequency represents the percent of mutated reads of the total reads elevel of gene expression relative to all genes detected by RNAseq f ++ >7% VAF by WES and >10% VAF by RNA sequencing and >20 percentile gene expression;
  • NeoPepi-HLAScan was able to capture another neoantigen, MAGEB2 P .EI6?Q that was recognized by two TIL populations expanded from VHIO-008 tumor biopsy despite this specific neoantigen was not detected when eluting peptides from HLA-I of VHIO-008 tumor cell line.
  • TIL populations were able to selectively recognize the mutated peptide but not the wildtype counterpart when presented on the HLA allele HLA- A*03:01 (Fig. 13).
  • This, together with the lack of identification in the tumor of these 41 additional mutated peptides identified by NeoPepi-HLAScan further support that the elution of peptides presented on the HLA class-l context from TMG-electroporated APCs can be more sensitive at detecting putative neoantigens than the elution of HLA class-l complexes from the autologous tumor cells.
  • mutated peptides that were exclusively detected in TMG-electroporated B cells (SEQ ID NO: 30, 44, 51 , 55, 65, 66, 69, 75, 76, 78, 81 , 87, 88, 89, 94, 102) derived from variants present at a relatively high variant allele frequency by WES of the tumor cell line, but were not detected by RNA sequencing, which would indicate that these mutated peptides are not efficiently transcribed and are not presented on the surface of tumor cells.
  • the elution of peptides from HLA class-1 molecules of B cells transfected with TMG RNA encoding for selected mutated minigenes identified by WES in VHIO-008 hypopharyngeal carcinoma resulted in the detection of the immunogenic neoantigen that was detected by immunopeptidomics in VHIO-008 tumor cell line.
  • 73 additional mutated peptides were identified in TMG-electroporated B cells but not detected in the autologous tumor, including MAGEB2p.E167Q, an additional immunogenic neoantigen.
  • NeoPepi-HLAScan detects a greater number of mutated peptides compared to HLA-I peptide elution of tumor cells, including a greater number of immunogenic neoantigens that could be clinically relevant for cancer treatment, providing an alternative strategy for their identification.
  • the proposed method can also be used to empirically prioritize the selection of mutant peptides included in personalized therapeutic vaccines.
  • NeoPepi-HLAScan empirically identified 75 mutated peptides, including two that are targeted by T-cells (Fig. 14). This prioritization is especially important given that vaccines typically include a limited number of candidate neoantigens for vaccine generation. Thus, selecting mutant peptides among those eluted from TMG transfected B-cells will ensure that they can be effectively processed and presented naturally by a cell and will reduce the number of compounds that are not relevant for patient treatment.
  • NeoPepi-HLAScan can also identify targetable neoantigens for cancer immunotherapy not only in melanoma, but potentially in any tumor where non- synonymous mutations as well as other candidate neoantigens can be identified.
  • NeoPepi-HLAScan to identify immunogenic neoantigens by using other types of antigen-presenting cells (APCs).
  • APCs antigen-presenting cells
  • TMG1 -enriched T cells specifically recognize a peptide derived from a mutation in MAGEA6 P .EI68K
  • TMG3-enriched T cells recognized a mutated peptide derived from PDS5A p .YioooF
  • TMG5-enriched T cells recognized a neoantigen derived from mutated MED13 p .Pi69is (published data, not shown). All the enriched neoantigen- reactive cells recognized the autologous tumor cell line 3998mel to different extent.
  • NeoPepi- HLAScan could capture these immunogenic peptides in TMG-electroporated B cells and compare the use of other types of TMG-electroporated APCs.
  • immature DC from peripheral blood, which are known as the masters of the immune system in antigen presentation, T cells, which can also act as APC in some biological contexts, and autologous B cells.
  • T cells which can also act as APC in some biological contexts
  • autologous B cells The autologous tumor cell line was also expanded and used as a reference.
  • TMGs TMG1 , 3, 5
  • FACS FACS
  • peptides were dissociated from the HLA complex using acidic elution, concentrated, desalted and mass spectrometry LC-MS/MS was carried out using an Orbitrap Lumos. Two technical replicates MS measurements of purified peptides were performed for each sample.
  • PEAKS-X Pro software was employed to search the experimental spectra against a personalized database including all proteins annotated in Swiss-Prot as well as the AA sequences of the TMGs encoding for to the NSM identified by WES.
  • NeoPepi-HLAScan we were able to identify the immunogenic mutated peptide MAGEA6 P .EI68K in all types of TMG 1 -electroporated APCs as well as in the autologous tumor cell line.
  • T cells co-cultured with TMG 1 -electroporated APCs and with the autologous tumor cell line were capable of recognizing the corresponding target as observed by 4-1 BB expression, corroborating that the mutated peptide MAGEA6 P .EI68K was being processed and presented on the surface in all the cells studied.
  • the mutated peptide MED13 p .Pi69is encoded by TMG5 was also identified in all types of TMG5- electroporated APCs, which again, was corroborated by the upregulation of 4-1 BB on T cells upon co-culture with TMG5-electroporated APCs. In contrast, this mutation was not detected by directly eluting the HLA-I peptides from the autologous TCL although neoantigen-specific T cells were capable of recognizing the tumor (as shown in fig 15).
  • NeoPepi-HLAScan using alternative APCs could be of interest in order to detect immunogenic variants potentially induced by inflammation.
  • NeoPepi- HLAScan was able to detect 3 mutated peptides derived from 2 NSM detected by tumor WES, that were not previously detected through the elution of HLA-I peptides from the cell line.
  • NeoPepi-HLAScan consistently identifies immunogenic mutated peptides in an additional melanoma patient. Moreover, these results indicate that NeoPepi-HLAScan can be more sensitive at detecting immunogenic neoantigens than the direct elution of peptides from TCL. And last, we showed that NeoPepi-HLAScan is a flexible tool that can exploit alternative APC sources such as DC or T cells, and thus, patients where B cells are not available could also benefit from this method.

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Abstract

L'invention concerne des méthodes immunopeptidomiques ex vivo pour l'identification et l'isolement de néoantigènes dérivés du cancer qui peuvent être naturellement traités et présentés sur des molécules HLA-I et/ou HLA-II de surface cellulaire d'une manière personnalisée. L'invention concerne également l'utilisation des néoantigènes pour la préparation de vaccins et de compositions immunogènes pour le traitement du cancer.
PCT/EP2021/077649 2020-10-08 2021-10-07 Méthode d'identification de néoantigènes du cancer WO2022074098A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113272419A (zh) * 2018-12-04 2021-08-17 南托米克斯有限责任公司 制备治疗性t淋巴细胞的方法
CN117174166A (zh) * 2023-10-26 2023-12-05 北京基石京准诊断科技有限公司 基于三代测序数据的肿瘤新抗原预测方法及系统

Citations (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4235871A (en) 1978-02-24 1980-11-25 Papahadjopoulos Demetrios P Method of encapsulating biologically active materials in lipid vesicles
US4501728A (en) 1983-01-06 1985-02-26 Technology Unlimited, Inc. Masking of liposomes from RES recognition
US4690915A (en) 1985-08-08 1987-09-01 The United States Of America As Represented By The Department Of Health And Human Services Adoptive immunotherapy as a treatment modality in humans
US4837028A (en) 1986-12-24 1989-06-06 Liposome Technology, Inc. Liposomes with enhanced circulation time
US4844893A (en) 1986-10-07 1989-07-04 Scripps Clinic And Research Foundation EX vivo effector cell activation for target cell killing
US5019369A (en) 1984-10-22 1991-05-28 Vestar, Inc. Method of targeting tumors in humans
US5204253A (en) 1990-05-29 1993-04-20 E. I. Du Pont De Nemours And Company Method and apparatus for introducing biological substances into living cells
US5580859A (en) 1989-03-21 1996-12-03 Vical Incorporated Delivery of exogenous DNA sequences in a mammal
US5589406A (en) 1993-07-30 1996-12-31 Ag Technology Co., Ltd. Method of making TFT display
WO2000047719A2 (fr) 1999-02-11 2000-08-17 3M Innovative Properties Company Maturation de cellules dendritiques a l'aide de composes modifiant la reponse immunitaire
WO2019105485A1 (fr) * 2017-12-01 2019-06-06 上海桀蒙生物技术有限公司 Procédé de préparation de vaccin personnalisé contre le cancer

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4235871A (en) 1978-02-24 1980-11-25 Papahadjopoulos Demetrios P Method of encapsulating biologically active materials in lipid vesicles
US4501728A (en) 1983-01-06 1985-02-26 Technology Unlimited, Inc. Masking of liposomes from RES recognition
US5019369A (en) 1984-10-22 1991-05-28 Vestar, Inc. Method of targeting tumors in humans
US4690915A (en) 1985-08-08 1987-09-01 The United States Of America As Represented By The Department Of Health And Human Services Adoptive immunotherapy as a treatment modality in humans
US4844893A (en) 1986-10-07 1989-07-04 Scripps Clinic And Research Foundation EX vivo effector cell activation for target cell killing
US4837028A (en) 1986-12-24 1989-06-06 Liposome Technology, Inc. Liposomes with enhanced circulation time
US5589466A (en) 1989-03-21 1996-12-31 Vical Incorporated Induction of a protective immune response in a mammal by injecting a DNA sequence
US5580859A (en) 1989-03-21 1996-12-03 Vical Incorporated Delivery of exogenous DNA sequences in a mammal
US5204253A (en) 1990-05-29 1993-04-20 E. I. Du Pont De Nemours And Company Method and apparatus for introducing biological substances into living cells
US5589406A (en) 1993-07-30 1996-12-31 Ag Technology Co., Ltd. Method of making TFT display
WO2000047719A2 (fr) 1999-02-11 2000-08-17 3M Innovative Properties Company Maturation de cellules dendritiques a l'aide de composes modifiant la reponse immunitaire
WO2019105485A1 (fr) * 2017-12-01 2019-06-06 上海桀蒙生物技术有限公司 Procédé de préparation de vaccin personnalisé contre le cancer
US20200368336A1 (en) * 2017-12-01 2020-11-26 Shanghai Jenomed Biotech Co., Ltd. Method for preparing personalized cancer vaccine

Non-Patent Citations (30)

* Cited by examiner, † Cited by third party
Title
ALLISON A C, DEV BIOL STAND, vol. 92, 1998, pages 3 - 11
ANDREA GARCIA-GARIJO ET AL: "Determinants for Neoantigen Identification", FRONTIERS IN IMMUNOLOGY, vol. 10, no. 1392, 24 June 2019 (2019-06-24), XP055630095, DOI: 10.3389/fimmu.2019.01392 *
ARNDT S 0 ET AL., EMBO J., vol. 19, 2000, pages 1241 - 1251
BARANYMERRIFIELD: "The Peptides", 1979, ACADEMIC PRESS, pages: 1 - 284
BENTZENHADRUP, CANCER IMMUNOL IMMUNOTHER, vol. 66, no. 5, 2017, pages 657 - 666
BRÄUNLEIN EVA ET AL: "Identification and Characterization of Neoantigens As Well As Respective Immune Responses in Cancer Patients", FRONTIERS IN IMMUNOLOGY, vol. 8, 30 November 2017 (2017-11-30), XP055791665, DOI: 10.3389/fimmu.2017.01702 *
BRUNSVIG P F ET AL., CANCER IMMUNOL IMMUNOTHER, vol. 55, no. 12, 2006, pages 1553 - 1564
CASTIELLO ET AL., CANCER IMMUNOL IMMUNOTHER, vol. 60, no. 4, April 2011 (2011-04-01), pages 457 - 466
CHICZ R M ET AL., NATURE, vol. 358, 1992, pages 764 - 768
DUPUIS M ET AL., CELL IMMUNOL, vol. 186, no. 1, 1998, pages 18 - 27
FANGJUN CHEN ET AL: "Neoantigen identification strategies enable personalized immunotherapy in refractory solid tumors", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 129, no. 5, 5 March 2019 (2019-03-05), GB, pages 2056 - 2070, XP055717584, ISSN: 0021-9738, DOI: 10.1172/JCI99538 *
GROS A ET AL., NAT MED, vol. 22, no. 4, April 2016 (2016-04-01), pages 433 - 8
J IMMUNOL METHODS, vol. 374, 2011, pages 1 4
JARDETZKY T S ET AL., NATURE, vol. 353, 1991, pages 622 - 626
KAISHO, BIOCHIMICA ET BIOPHYSICA ACTA, vol. 1589, 2002, pages 1 - 13
KROPSHOFER H ET AL., J EXP MED, vol. 175, 1992, pages 1799 - 1803
KROPSHOFER H ET AL., J. EXP. MED., vol. 175, 1992, pages 1799 - 1803
LU ET AL., CLIN. CANCER RES., vol. 20, 2014, pages 3401 - 10
MANZ ET AL., PROC. NATL. ACAD. SCI. USA, vol. 92, 1995, pages 1921 - 1925
MARINO F. ET AL., METHODS MOL BIOL, vol. 1913, 2019, pages 67 - 79
MERRIFIELD, SCIENCE, vol. 232, 1986, pages 341 - 347
MURPHY ET AL., THE PROSTATE, vol. 29, 1996, pages 371 - 380
NUCLEIC ACIDS RES., vol. 36, 1 July 2008 (2008-07-01), pages W509 - W512
SCHEFFOLD ET AL., NATURE MED, vol. 6, 2000, pages 107 - 110
SHELLY KALAORA ET AL: "Use of HLA peptidomics and whole exome sequencing to identify human immunogenic neo-antigens", ONCOTARGET, vol. 7, no. 5, 2 February 2016 (2016-02-02), XP055602237, DOI: 10.18632/oncotarget.6960 *
SLOAN V S ET AL., NATURE, vol. 375, 1995, pages 802 - 806
SZOKA ET AL., ANN.REV. BIOPHYS. BIOENG., vol. 9, 1980, pages 467
TJUA ET AL., THE PROSTATE, vol. 32, 1997, pages 272 - 278
VOGT A B ET AL., J. IMMUNOL., vol. 153, 1994, pages 1665 - 1673
WOLFF ET AL., SCIENCE, vol. 247, 1990, pages 1465 - 1468

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113272419A (zh) * 2018-12-04 2021-08-17 南托米克斯有限责任公司 制备治疗性t淋巴细胞的方法
CN117174166A (zh) * 2023-10-26 2023-12-05 北京基石京准诊断科技有限公司 基于三代测序数据的肿瘤新抗原预测方法及系统
CN117174166B (zh) * 2023-10-26 2024-03-26 北京基石生命科技有限公司 基于三代测序数据的肿瘤新抗原预测方法及系统

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