Immunogenic EGFR peptide compositions and their use in cancer treatment

Abstract

Compositions comprising EGFR mutant peptides that bind to HLA class I and / or HLA class II complexes, as well as compositions comprising a plurality of such peptides, are provided. Methods for treating EGFR mutant cancers using the peptides of the embodiments are also provided. Methods for expanding relevant populations of immune effector cells, such as T cells, are also provided.

Description

[Technical Field]

[0001] Reference to related applications This application is an international application under the Patent Cooperation Treaty, claiming priority to U.S. Provisional Patent Application No. 62 / 906,688, filed on 26 September 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] Reference to electronically submitted sequence lists In accordance with the EFS-Web legal framework and 37 CFR·1.821-825 (see MPEP·2442.03(a)), a sequence listing in the form of an ASCII-compliant text file (titled "Sequence_Listing_3000072-001977_ST25.txt", created on September 22, 2020, and measuring 243,949 bytes) was submitted concurrently with this application, and this file contains the entire contents of the Sequence Listing. [Background technology]

[0003] 1. Field of Invention This invention generally relates to the fields of immunology and medicine. More particularly, it relates to cancer-specific peptides that bind to HLA class I and HLA class II molecules.

[0004] 2. Explanation of related technologies T-cell-based therapies have shown significant potential as a method for treating many cancers; unfortunately, this approach is also hindered by a lack of immunogenic antigen targets for common cancers and potential toxicity to non-cancerous tissues. These T-cell-based therapies may include ACT (adoptive cell transfer) and vaccination approaches. ACT generally involves injecting a large number of self-activated tumor-specific T cells into a patient, for example, to treat cancer. Generally, three steps are usually required to elicit an effective anti-tumor T-cell response: priming and activation of antigen-specific T cells, migration of activated T cells to the tumor site, and recognition and killing of the tumor by antigen-specific T cells. The selection of target antigens is crucial for inducing effective antigen-specific T cells.

[0005] Neoantigen peptides derived from proteins encoding tumor mutations, presented on the tumor cell surface by human leukocyte antigen (HLA) molecules, may serve as promising antigens for generating effective immune responses. However, due to the diversity of HLA and the inherent nature of most tumor-associated mutations, few neoanthigens are shared among patients, and vaccine-induced clinical responses remain rare. Therefore, methods are needed to identify and evaluate novel epitopes for cancers that express neoanthigens. [Overview of the project]

[0006] In some embodiments, the present invention provides a method for treating a subject having EGFR-mutated cancer, comprising administering to the subject a first HLA-binding peptide from at least EGFR (Epidermal Growth Factor Receptor) and at least a first EGFR inhibitor, wherein the peptide comprises a mutated amino acid sequence related to wild-type human EGFR that matches the mutation in the subject's cancer. In some embodiments, the EGFR-derived HLA-binding peptide binds to an HLA class I molecule. In some embodiments, the HLA class I-binding peptide has a length of 8, 9, 10, 11, 12, or 13 amino acids. In further embodiments, the HLA class I-binding peptide has a length of 9, 10, or 11 amino acids. In some embodiments, the EGFR-derived HLA-binding peptide binds to an HLA class II molecule. In some embodiments, the HLA class II-binding peptide has a length of 13 to 30 amino acids. In further embodiments, the HLA class II-binding peptide has a length of 15 to 23 amino acids.

[0007] In some embodiments, the method comprises the step of administering at least first and second HLA-binding peptides derived from EGFR, wherein each peptide contains a mutant amino acid sequence associated with wild-type human EGFR that matches the mutation in the target cancer. In some embodiments, the first HLA-binding peptide from EGFR binds to an HLA class I molecule, and the second HLA-binding peptide from EGFR binds to an HLA class II molecule. In some embodiments, the method comprises the step of administering multiple HLA-binding peptides derived from EGFR, wherein each peptide contains a mutant amino acid sequence associated with wild-type human EGFR that matches the mutation in the target cancer. In some embodiments, the multiple HLA-binding peptides include peptides that bind to both HLA class I and HLA class II molecules. In some embodiments, the method comprises administering 2 to 30 different HLA-binding peptides to a target. In further embodiments, the method comprises the step of administering 5 to 30 different HLA-binding peptides to a target.

[0008] In some embodiments, the target HLA genotype has been determined. In some embodiments, the EGFR-derived HLA-binding peptide is predicted to bind to the HLA type (also called the type) supported by the target. In some embodiments, the EGFR-derived HLA-binding peptide is predicted to bind to the HLA type supported by the target using an automated analysis program. In some embodiments, EGFR-mutated cancers express EGFR polypeptides with amino acid substitutions, deletions, or insertions that are related to wild-type EGFR. In some ways, EGFR-mutated cancers include E709K, E709A, E709H, G719A, G719S, G719C, S768I, H773L, T790M, L858R, L861Q, 709_710del>D, 745_749del, 745_750del, 746_750del, 746_751del, 746_751del>A, 74 6_752del>V,747_750del,747_751del,747_751del>P,747_753del,747_753del>S,747_753de l>Q,747_750del>P,747_752del,751_759del>N,752_759del,744_745ins>KIPVAI,745_746ins >IPVAIK,745_746ins>VPVAIK,745_746ins>TPVAIK,763_764ins>FQEA,764_765ins>HH,766_7 67ins>AI,768_770dupSVD,769_770ins>ASV,770_771ins>G,770_771ins>NPG,770_771ins>SVD This group includes mutations selected from the following: 771_772ins>N, 771_772ins>H, 772_773ins>NP, 772_773ins>PR, 773_774ins>H, 773_774ins>PH, 773_774ins>NPH, 773_774ins>AH, 774_775ins>HV, and 775_776ins>YVMA. In some aspects, EGFR-mutated cancers include G719A, G719S, G719C, S768I, H773L, T790M, L858R, L861Q, 709_710del>D, 746_750del, 746_752del>V, 747_751del, 747_751del>P, 747_753del>S, 747_750del>P, 747_752del, and 744_745in. This group includes mutations selected from the group consisting of s>KIPVAI,745_746ins>IPVAIK,763_764ins>FQEA,768_770dupSVD,769_770ins>ASV,770_771ins>SVD,772_773ins>PR,773_774ins>H,773_774ins>PH,773_774ins>NPH, and773_774ins>AH. In some embodiments, EGFR-mutated cancers include mutations selected from the group consisting of L858R, H773L, T790M, E709V, 747_751Del, V774MandS768I.

[0009] In some embodiments, the EGFR inhibitor is administered before the HLA-binding peptide from EGFR. In other embodiments, the EGFR inhibitor is administered after, or essentially simultaneously with, the HLA-binding peptide derived from EGFR. In some embodiments, the HLA-binding peptide derived from mutant EGFR is administered together with a TLR ligand. In some embodiments, the TLR ligand is a TLR2, TLR3, TLR4, TLR7, TLR8, or TLR9 agonist. In some embodiments, the TLR ligand is a TLR7 agonist. In some embodiments, the TLR7 agonist is CL075, CL097, CL264, CL307, GS-9620, Poly(dT), imiquimod, gardiquimod, regiquimod (R848), loxoribine, or ssRNA oligonucleotide. In some embodiments, the TLR7 agonist is imiquimod. In some embodiments, the EGFR inhibitor is a tyrosine kinase inhibitor. In some embodiments, the EGFR inhibitor is an EGFR-binding antibody. In some embodiments, the EGFR inhibitor is osimertinib, erlotinib, gefitinib, cetuximab, matsuzumab, panitumumab, AEE788, CI-1033, HKI-272, HKI-357, or EKB-569.

[0010] In some aspects, the cancer is lung cancer. In some aspects, the lung cancer is non-small cell lung cancer. In some aspects, the lung cancer is metastatic lung cancer. In some aspects, the lung cancer is lung adenocarcinoma. In some aspects, the cancer is EGFR inhibitor resistant.

[0011] In another embodiment, the present invention provides an immunogenic composition comprising at least first and second HLA-binding peptides derived from EGFR, wherein the first and second peptides comprise mutant amino acid sequences related to wild-type human EGFR, consistent with mutations in human EGFR-mutated cancer, the first HLA-binding peptide derived from EGFR binds to HLA class I molecules, and the second HLA-binding peptide derived from EGFR binds to HLA class II molecules. In some embodiments, the HLA-binding peptides are formulated in a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is an aqueous carrier. In some embodiments, the pharmaceutically acceptable carrier is a salt solution. In some embodiments, the pharmaceutically acceptable carrier is a saline solution, preferably an isotonic saline solution. In some embodiments, the HLA class I-binding peptide is an amino acid with a length of 8, 9, 10, 11, 12, or 13. In further embodiments, the HLA class I-binding peptide is an amino acid with a length of 9, 10, or 11. In some embodiments, the HLA class II-binding peptide is 13 to 30 amino acids long. In a further embodiment, the HLA class II binding peptide is 15 to 23 amino acids long. In some embodiments, the composition comprises multiple HLA binding peptides derived from EGFR, each of which contains a mutated amino acid sequence related to wild-type human EGFR, corresponding to an EGFR mutation in human EGFR-mutated cancer. In some embodiments, the composition comprises 2 to 30 different HLA binding peptides for a subject. In some embodiments, the composition comprises at least two HLA class I binding peptides and at least one HLA class II binding peptide. In some embodiments, the composition comprises at least one HLA class I binding peptide and at least two HLA class II binding peptides. In some embodiments, the composition comprises at least two HLA class I binding peptides and at least two HLA class II binding peptides. In some embodiments, the composition comprises at least three HLA class I binding peptides and at least three HLA class II binding peptides.

[0012] In some embodiments, the EGFR-derived HLA-binding peptide includes amino acid substitutions, deletions, or insertions related to wild-type EGFR. In some aspects, HLA-binding peptides are related to wild-type human EGFR, including E709K, E709A, E709H, G719A, G719S, G719C, S768I, H773L, T790M, L858R, L861Q, 709_710del>D, 745_749del, 745_750del, 746_750del, 746_751del, and 74 6_751del>A,746_752del>V,747_750del,747_751del,747_751del>P,747_753del,747_753del>S ,747_753del>Q,747_750del>P,747_752del,751_759del>N,752_759del,744_745ins>KIPVAI,74 5_746ins>IPVAIK,745_746ins>VPVAIK,745_746ins>TPVAIK,763_764ins>FQEA,764_765ins>HH ,766_767ins>AI,768_770dupSVD,769_770ins>ASV,770_771ins>G,770_771ins>NPG,770_771ins This group includes mutations selected from the following: >SVD, 771_772ins>N, 771_772ins>H, 772_773ins>NP, 772_773ins>PR, 773_774ins>H, 773_774ins>PH, 773_774ins>NPH, 773_774ins>AH, 774_775ins>HV, and 775_776ins>YVMA. In some aspects, HLA-binding peptides are related to wild-type human EGFR, including G719A, G719S, G719C, S768I, H773L, T790M, L858R, L861Q, 709_710del>D, 746_750del, 746_752del>V, 747_751del, 747_751del>P, 747_753del>S, 747_750del>P, 747_752del, This group includes mutations selected from the group consisting of 744_745ins>KIPVAI, 745_746ins>IPVAIK, 763_764ins>FQEA, 768_770dupSVD, 769_770ins>ASV, 770_771ins>SVD, 772_773ins>PR, 773_774ins>H, 773_774ins>PH, 773_774ins>NPH, and 773_774ins>AH. In some embodiments, the HLA-binding peptide contains mutations selected from the group consisting of L858R, H773L, T790M, E709V, 747_751Del, V774M, and S768I, which are related to wild-type human EGFR.

[0013] In some embodiments, the composition further comprises an EGFR inhibitor. In some embodiments, the composition further comprises a TLR ligand. In some embodiments, the TLR ligand is a TLR2, TLR3, TLR4, TLR7, TLR8, or TLR9 agonist. In some embodiments, the composition further comprises a TLR7 agonist. In some embodiments, the TLR7 agonist is CL075, CL097, CL264, CL307, GS-9620, Poly(dT), imiquimod, gardiquimod, regiquimod (R848), loxoribine, or ssRNA oligonucleotide. In further embodiments, the TLR7 agonist is imiquimod. In some embodiments, the HLA-binding peptide is complexed with an HLA class I and / or HLA class II molecule. In some embodiments, the composition further comprises antigen-presenting cells. In some embodiments, the antigen-presenting cells include dendritic cells. In some embodiments, the HLA-binding peptide is contained within liposomes, lipid-containing nanoparticles, or a lipid-based carrier. In some embodiments, the composition further comprises an adjuvant component.

[0014] In yet another embodiment, the present invention provides a method for treating a subject comprising administering to the subject an effective amount of the composition of the present invention (for example, the composition of the present invention containing an effective amount), wherein the subject has an EGFR-mutated cancer, and the EGFR-derived HLA-binding peptide in the composition contains a mutation consistent with that of the EGFR-mutated cancer in the subject. In some embodiments, the method further comprises the step of sequencing the EGFR gene in the subject's cancer. In some embodiments, the subject's HLA genotype has been determined. In some embodiments, the EGFR-derived HLA-binding peptide is predicted to bind to an HLA class I or HLA class II type present in the subject. In a further embodiment, the EGFR mutant peptides included in the composition are selected using an algorithm that predicts HLA binding to the HLA type expressed in the subject. In some embodiments, the peptides included in the composition are selected based on predicted HLA affinity and / or predicted HLA ranking. In a further embodiment, the peptides included in the composition are selected based on predicted HLA affinity and predicted HLA ranking. In a further embodiment, the algorithm used to select the HLA-binding peptide is selected from NetMHC4.0, NetMHCpan3.0, NetMHCpan4.0, NetMHCII2.2 and / or NetMHCII2.3 [see, for example, Andreatta et al. (2015) and Jensen et al. (2018), each of which is incorporated herein by reference].

[0015] In some embodiments, the method further includes administering an EGFR inhibitor to the target. In some embodiments, the EGFR inhibitor is administered before the HLA-binding peptide from mutant EGFR. In some embodiments, the EGFR inhibitor is administered after, or essentially simultaneously with, the HLA-binding peptide derived from mutant EGFR. In some embodiments, the EGFR inhibitor is an EGFR-binding antibody. In some embodiments, the EGFR inhibitor is osimertinib, erlotinib, gefitinib, cetuximab, matuzumab, panitumumab, AEE788, CI-1033, HKI-272, HKI-357, or EKB-569. In some embodiments, the EGFR inhibitor is a tyrosine kinase inhibitor. In some embodiments, the HLA-binding peptide derived from EGFR is administered together with a TLR ligand. In some embodiments, the TRL ligand is a TLR2, TLR3, TLR4, TLR7, TLR8, or TLR9 agonist, preferably a TLR7 agonist. In some embodiments, the TLR7 agonist is CL075, CL097, CL264, CL307, GS-9620, Poly(dT), imiquimod, gardiquimod, regiquimod (R848), loxoribine, or ssRNA oligonucleotide. In a further embodiment, the TLR7 agonist is imiquimod.

[0016] In some aspects, the cancer is lung cancer. In some aspects, the lung cancer is non-small cell lung cancer. In some aspects, the lung cancer is metastatic lung cancer. In some aspects, the lung cancer is lung adenocarcinoma. In some aspects, the cancer is EGFR inhibitor resistant. In some aspects, the composition is administered by parenteral administration, intravenous injection, intramuscular injection, inhalation, or subcutaneous injection. In some aspects, the composition is formulated in an aqueous carrier. In some aspects, the aqueous carrier is a salt solution. In some aspects, the aqueous carrier is isotonic saline. In some aspects, the composition is administered at least two, three, four, or five times. In some aspects, there are two, three, four, five, six, or seven days between administrations. In some aspects, the method further comprises a step of administering further anticancer therapy. In some aspects, the further anticancer therapy is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, or surgery. In some aspects, the immunotherapy includes at least one immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-PD1 or anti-CTLA-4 monoclonal antibody. In some embodiments, the immunotherapy is a combination of immune checkpoint inhibitors.

[0017] In yet another embodiment, the present invention provides a method for producing EGFR-mutated cancer-specific immune effector cells, comprising “obtaining” a starting population of immune effector cells and contacting the starting population of immune effector cells with a composition of the present invention to generate EGFR-mutated cancer-specific immune effector cells. In some embodiments, contact is further defined as co-culturing a starting population of immune effector cells with antigen-presenting cells (APCs), where the APCs present the HLA-binding peptide of the present invention on their surface. In some embodiments, the APCs are dendritic cells. In some embodiments, the immune effector cells are T cells, peripheral blood lymphocytes, NK cells, invariant NK cells, or NKT cells. In some embodiments, the immune effector cells are differentiated from mesenchymal stem cells (MSCs) or induced pluripotent stem (iPS) cells. In some embodiments, the T cells are CD8 + T cells, CD4 + These are T cells, or γδ T cells. In some embodiments, the starting population of T cells is CD8 + T cells or CD4 + These are T cells. In certain contexts, T cells are cytotoxic T lymphocytes (CTLs). In some embodiments, “obtaining” includes isolating a starting population of immunoeffector cells from peripheral blood mononuclear cells (PBMCs).

[0018] In yet another embodiment, the present invention provides EGFR-mutated cancer-specific T cells produced by the method of the present invention.

[0019] In yet another embodiment, the present invention provides a pharmaceutical composition comprising EGFR-mutated cancer-specific T cells produced by the method of the present invention.

[0020] In yet another aspect, the present invention provides a method for treating cancer in a subject, comprising administering an effective amount of the EGFR-mutated cancer-specific T cells of the present invention to the subject.

[0021] In yet another embodiment, the present invention provides a composition comprising an effective amount of the present invention's EGFR-mutated cancer-specific T cells for the treatment of cancer in a subject.

[0022] As used herein, "essentially free" means, with respect to a particular ingredient, that ingredient is intentionally not included in the composition and / or is present only as a contaminant or in trace amounts.

[0023] As used herein, "a" or "an" may mean one or more.

[0024] In this specification, the term “or” means “and / or” unless it is explicitly indicated that it refers only to the options or that the options are mutually exclusive, but this disclosure supports the definitions of options only and “and / or.” As used herein, the term “another” means at least two or more.

[0025] Throughout this specification, the term “about” is used to indicate that a value includes inherent variations in the variability present between the device, the method used to determine the value, or the subject of study.

[0026] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, the detailed description, specific examples, and preferred examples of the present invention are illustrative, and it should be understood that various changes and modifications that will become apparent to those skilled in the art through this detailed description fall within the spirit and scope of the present invention. [Brief explanation of the drawing]

[0027] The following drawings form part of this specification and are included to further illustrate specific aspects of the invention. The invention will be better understood by referring to one or more of these drawings in combination with a detailed description of the specific examples presented herein.

[0028] [Figure 1-1]Figures 1A-1H show the personalized peptide vaccine (PPV) trial design and patient outcomes. (Figure 1A) Neoantigen peptide vaccine production pipeline leading to immunization in patients with advanced non-small cell lung cancer (NSCLC). DNA derived from lung tumor biopsies was sequenced using a panel of 508 tumor-related genes, while high-resolution HLA typing was performed on patients' peripheral blood. Neoantigen vaccine peptides were selected primarily based on HLA class I and class II binding prediction (see Methods). Each patient was divided into two cocktails and immunized weekly with a saline-based mixture of short and long neoantigen peptides, administered to the opposite limb for 12 weeks. Arrows indicate the week of vaccination. Blood was collected at weeks 0, 4, 8, and 12, as indicated by syringe symbols. [Figure 1-2] (Figure 1B) Clinical event schedule for 24 NSCLC patients who received PPV. Dark bars (labeled neoantigen vaccine), PPV immunization period. Light bars (labeled EGFR inhibitor), EGFR inhibitor treatment period. [Figure 1-3] (Figure 1C) Fourteen PPV patients were divided into three groups based on their EGFR mutation status and the use of EGFR inhibitors during vaccination. [Figure 1-4] (Figure 1D-1F) Number of vaccine peptides, number of targeted mutations, and predicted vaccine peptide binding affinity, stratified by patient cohort, clinical response, and progression-free survival. Black dots represent non-EGFR neoantigen peptides, and gray dots represent EGFR neoantigen peptides; the proportion of the latter is listed below. [Figure 1-5](Figure 1G) Survival analysis of the three patient groups showed that patients in group 3 experienced significantly longer overall survival compared to the other groups (group 2 vs. group 3, P=0.038). (Figure 1H) Comparison of progression-free survival among patients who experienced objective clinical response (CR / PR, n=7), stable disease (SD, n=9), or disease progression (PD, n=8). Survival analysis was performed using the Log-rank (Mantel-Cox) test. Two-sided unpaired t-tests or Mann-Whitney U tests were used to analyze statistically significant differences between groups. P<0.05 was considered statistically significant.

[0029] [Figure 2] Figure 2 shows the time course of immunization in 24 PPV patients. Each square represents one week. Dark squares and arrows indicate weeks in which vaccination was received, while white squares indicate weeks in which vaccination was not administered. According to the trial design, patients were to receive weekly vaccinations for a minimum of 12 weeks. With the exception of 9, 19, and 20 patients, all patients completed the 12-week vaccination period and expired due to progressive disease during the initial trial period. Patients were given the opportunity to continue immunization beyond 12 weeks if they wished.

[0030] [Figure 3] Figure 3 shows the treatment and clinical outcomes of 24 PPV patients divided into groups.

[0031] Figures 4A and 4B show a summary of clinical baseline characteristics for each group. [Figure 4-1] (Figure 4A) This figure shows the clinical and demographic characteristics of the PPV group at baseline, and provides a statistical comparison of the PPV-1 group (EGFR-WT, PPV only), PPV-2 group (EGFR mutant, PPV only), and PPV-3 group (EGFR mutant, PPV + EGFRi). SQ: Squamous cell carcinoma, AD: Adenocarcinoma. Continuous data are shown as mean + standard deviation (SD). [Figure 4-2](Figure 4B) EGFR inhibitor treatment history of PPV-2 and PPV-3 patients. TKI: Tyrosine kinase inhibitor. Statistical significance between groups was analyzed using a two-tailed unpaired t-test or a chi-square test. P<0.05 was considered statistically significant.

[0032] [Figure 5-1] Figures 5A-5C show the clinical response of PPV patients by group. (Figure 5A) Summary of immunized patient responses by group, showing progression-free survival, overall survival, and clinical response assessed using RECIST 1.1 criteria 12–18 weeks after the start of PPV. [Figure 5-2] (Figure 5B) Measured total tumor volume (sum of all target lesions) of PPV patients during the course of treatment. The treatment outcomes for each patient were as follows: Pt.1 PD; Pt.4 PD; Pt.6 SD; Pt.7 SD; Pt.9 SD; Pt.10 PD; Pt.19 PD; Pt.11 CR or PR; Pt.14 CR or PR; Pt.16 SD; Pt.17 CR or PR; Pt.18 PD; Pt.20 PD; Pt.24 PD; Pt.3 SD; Pt.5 CR or PR; Pt.8 CR or PR; Pt.12 CR or PR; Pt.13 SD; Pt.15 SD; Pt.21 SD; Pt.22 CR or PR; Pt.23 PD. * Patient 1 developed pleural effusion at week 12. ** A follow-up CT scan was taken for patient 11 at week 24. Tumor measurements for patient 2 are not shown due to the absence of measurable tumors. Follow-up CT scans were not available for patients 13 and 15, but other clinical follow-up information regarding response and survival was available. PPV, Personalized Peptide Vaccine; PPV-1, PPV Group 1; PPV-2, PPV Group 2; PPV-3, PPV Group 3. MUT, Variant; CR, Complete Response; PR, Partial Response; PD, Disease Progression; SD, Disease Stable. [Figure 5-3] (Figure 5C) List of treatment-related adverse events.

[0033] [Figure 6]Figures 6A–6C show representative CT scans of selected PPV patients, including trial EGFR inhibitor failures prior to vaccination. (Figure 6A) Serial CT scans showing the disappearance of pleural effusion in patient 2 (group 1) 10 weeks after PPV treatment. (Figure 6B) CT scans of patient 23 showing progression of liver metastases during PPV treatment. Patient 24 similarly showed lung tumor progression 11 weeks after the start of immunotherapy. (Figure 6C) Serial CT scans showing trial EGFR inhibitor failures prior to PPV vaccination, followed by objective clinical responses after the start of PPV treatment in patients 17, 5, 8, and 12.

[0034] [Figure 7-1] Figures 7A-7F show the clinical response of patients after individualized neoantigen peptide vaccination. (Figure 7A) CT scan showing regression of two lung lesions from patient 17 with a complete response. [Figure 7-2] (Figure 7B) Tissue biopsy confirmed that the remaining lung CT signal consisted only of fibrous tissue without viable tumor cells. (Figure 7C) Bone metastases in 17 patients, evaluated by T2-weighted magnetic resonance imaging (MRI), disappeared 18 weeks after the start of neoantigen vaccination. These bone metastases were considered non-targeted lesions according to RECIST (version 1, bone lesion measurability). [Figure 7-3] (Figure 7D) Two additional patients in Group 2 showed an objective clinical response to PPV. Patient 11 experienced regression of the lung tumor in addition to the disappearance of obstructive atelectasis 24 weeks after the start of PPV (white arrow, right), but the pneumothorax did not change (gray arrow). Patient 14's cervical metastases showed significant regression 12 weeks after the start of PPV treatment (right). [Figure 7-4] (Figure 7E) CT scans showing lung tumor regression in patients 5, 8, 12, and 22. All showed a partial clinical response after PPV treatment. [Figure 7-5](Figure 7F) Change in total tumor volume 3-4 months after PPV administration compared to baseline after preliminary treatment in PPV trial patients. *Patient 1 developed pleural effusion at week 12. CR: complete remission; PR: partial remission.

[0035] [Figure 8-1] Figures 8A-8E show survival analyses for various patient groups. (Figure 8A) Analysis of progression-free survival (PFS) for PPV patients by group. (Figure 8B) Analysis of overall survival (OS) for PPV patients by group. [Figure 8-2] (Figure 8C) Comparison of PFS between patients with disease control (CR + PR + SD; n=16) or PD (n = 8 ted EGFR patients, Group 2 patients, or Group 3 patients). And, comparison of overall survival between patients who experienced objective clinical response (CR / PR, n=7), disease stability (SD, n=9), or disease progression (PD, n=8). (Figure 8D) Comparison of PFS between patients who used the third-generation TKI osimertinib and those who did not. All 16 cases are shown, either in Group 2 or Group 3. [Figure 8-3] (Figure 8E) Comparison of overall survival between patients who received osimertinib and those who did not among 16 patients with variant EGFR. Survival analysis was performed using the log-rank (Mantel-Cox) test, with P<0.05 considered statistically significant (bold). The 95% confidence interval (C1) is for the hazard ratio.

[0036] [Figure 9-1] Figures 9A–9C show vaccine peptide analysis, clinical response, and progression-free survival rates for each group. (Figure 9A) The number of different HLA class I and class I molecules involved with the vaccine peptide, as predicted by HLA peptide binding affinity. Each dot represents one PPV patient. (Figure 9B) The number of administered vaccine peptides was limited to HLA class I (short) or HLA class II (long). Each dot or circle represents one PPV patient. [Figure 9-2](Figure 9C) Peptide delta scores of vaccine peptides. The delta score is calculated by subtracting the predicted binding affinity of the mutant neoantigen peptide from the binding affinity of the corresponding wild-type peptide. Each point represents one vaccine peptide. Gray: EGFR neoantigen peptide; Black: Non-EGFR neoantigen peptide. No significant differences were found between groups using a two-sided unpaired t-test, and a P<0.05 was considered significant. MUT: Mutant; WT: Wild-type; PD: Disease progression; SD: Disease stability; PR: Partial response; CR: Complete response.

[0037] [Figure 10-1] (Figure 10A) Factors associated with survival in patients who received personalized peptide vaccines, as determined by univariate analysis. [Figure 10-2] (Figure 10B) Kaplan-Meier analysis showing that pleural effusion and tumor volume are two risk factors affecting overall survival in PPV trial patients. Survival analysis was performed using the Log-rank (Mantel-Cox) test, with P<0.05 considered significant (bold). SD: Standard deviation; EGFRi; EGFR inhibitor; ECOG: Eastern Cooperative Oncology Group; PS: Performance status; *SQ: Squamous cell carcinoma; AD: Adenocarcinoma. EGFRi failure category 1: Failed 1st generation EGFRi; 2 / 3: Failed 2nd or 3rd generation EGFRi.

[0038] [Figure 11-1] Figures 11A–11I demonstrate that EGFR neoantigen peptides are immunogenic, shared, and exhibit specific HLA class I binding preference. (Figure 11A) Interferon-γ (IFN-γ) ELISA assays performed on peptide pool-stimulated patient PBMC supernatants showed vaccine peptide pool-specific responses, primarily in six PPV patients: 5, 8, 14, 17, 21, and 22. Five of the six patients experienced an objective clinical response after PPV (Pt. 11, Pt. 14, Pt. 17, Pt. 5, Pt. 8, Pt. 12, Pt. 22). [Figure 11-2] (Figure 11B) Deconvolution of individual vaccine peptide responsiveness by IFN-γ ELISA in patients 5, 8, 14, and 17 revealed a high immune response in patients 5, 8, and 14 to the HLA-A*1101-specific EGFR-L858R peptide KITDFGRAK (SEQ ID NO: 4). Similarly, patient 17, a complete responder, showed a high response to the HLA*C1502-specific EGFR-T790M peptide LTSTVQLIM (SEQ ID NO: 65). Individual peptide responsiveness in other PPV research patients is shown in Figure 12. [Figure 11-3] (Figures 11C and 11D) Summary of IFN-γ ELISPOT assay and HLA tetramer-based staining revealed that the frequency of PBMCs in HLA-A*1101 / KITDFGRAK (SEQ ID NO: 4)-specific CD8+ T cells in patients 5, 8, and 14, and in HLA-C*1502 / LTSTVQLIM (SEQ ID NO: 65)-specific CD8+ T cells in patient 17, significantly increased over the course of PPV. [Figure 11-4] (Figure 11E) The ELISPOT assay showed that post-PPV PBMCs from patients 5, 8, 14, and 17 specifically recognized the mutant EGFR neoantigen peptide, but not the corresponding wild-type (WT) EGFR peptide. [Figure 11-5](Figure 11F) Predicted HLA peptide binding affinity for EGFR protein sequencing and mutant EGFR-L858R (box region of larger peptide SEQ ID NO: 980) and T790M (box region of larger peptide SEQ ID NO: 982) peptides and their corresponding WT epitopes (box region of larger peptide SEQ ID NO: 979 and box region of larger peptide SEQ ID NO: 981, respectively). (Figures 11G & 11H) Neoantigens derived from the most common EGFR variants, L858R (light gray) and Exon 19 deletion (Ex19del, medium gray), showed significant binding selectivity for HLA class I allotypes within the A3 superfamily, while other less common EGFR point mutations (S768I, T790M, and L861Q, dark gray) showed binding selectivity to the A2 and C3 superfamilies. [Figure 11-6] (Figure 11I) Enlarged view showing individual HLA class I allotypes with the highest number (<500 nM affinity) of predictive binding EGFR neoantigens for the most common shared EGFR mutations in lung cancer. Black arrows indicate A*1101-limited KITDFGRAK peptide (SEQ ID NO: 4) and C*1502-limited LTSTVQLIM peptide (SEQ ID NO: 65). Two-tailed unpaired t-tests were used for statistical significance analysis between groups. P<0.05 was considered statistically significant.

[0039] [Figure 12-1] Figures 12A and 12B show ELISA-based immunomonitoring of PPV-induced immune responses. (Figure 12A) Results of IFN-γ ELISA assays measuring peripheral blood reactivity to individual vaccine peptides. Peptide counts correspond to those listed in Table 5. At the indicated time point, a twofold change is measured compared to the peptide-free control (No). IC, irrelevant peptide control. Clinical responses are indicated as either (CR) or (PR). [Figure 12-2] (Continued from Figure 12A) [Figure 12-3] (Continued from Figure 12A) [Figure 12-4] (Continued from Figure 12A) [Figure 12-5] (Continued from Figure 12A) [Figure 12-6] (Figure 12B) Summary diagram showing the total vaccine peptide-specific immunoreactivity (ComboScore, see Methods) for each patient, along with their associated group, histology, and clinical outcome.

[0040] [Figure 13-1] Figures 13A–13H show ex vivo peripheral blood immune monitoring of ELISPOT-based vaccine-induced responses. Results from the IFN-γ ELISPOT assay demonstrate PPV-induced immune responses to selected vaccine peptides (2.5 x 10⁵ cells per well) in eight immunized patients, including seven clinical responders (Figures 13A, 13B, and 13D). The EGFR(L858R) neoantigen (hereinafter also known as Neo Ag) peptide KITDFGRAK (SEQ ID NO: 4) induced high IFN-γ responses in three different response groups Pt. 5, 8, and 14 (Figures 13A, 13B, and 13D). [Figure 13-2] (Figure 13E) However, no immune response to this vaccine peptide was detected in SD Pt.16 (Figure 13E). [Figure 13-3](Figure 13F) Complete response Pt.17 showed a high immune response to the LTSTVQLIM (SEQ ID NO: 65) Neo Ag peptide containing a shared EGFR (T790M) mutation (Figure 13F). (Figure 13G) Two further PR patients showed a CD8+ T cell response to Neo Ag encoding private mutations: AQP12A (L28R) peptide KARLPVGAY (SEQ ID NO: 875) in Pt.11 (Figure 13C) and FGFR1 (R734W) peptide YMMMWDCWHAV (SEQ ID NO: 964) in Pt.22 (Figure 13G). Immune responses to long CD4+-limited EGFR Neo Ag peptides were also induced in three patients: EGFR(L858R)-containing peptide HVKITDFGRAKLLGAEE (SEQ ID NO: 826) in Pts. 8 and 16 (Figures 13B and 13E), and H773L / V774MNeoAg peptide MASVDNPLMCRLLGICL (SEQ ID NO: 958) in Pt. 22 (Figure 13G). A summary of the line graphs showing the number of IFN-γ spots (standardized) per 106 PBMCs for each vaccine peptide that induced a response is also provided. The peptide identification numbers (P1, P2, etc.) correspond to those listed in Table 5, including SEQ ID NO: 874 (RLPVGAYEV); SEQ ID NO: 880 (VMASVDNPL); SEQ ID NO: 1 (HVKITDFGR); SEQ ID NO: 16 (VKITDFGRAK); SEQ ID NO: 32 (AIKESPKANK); SEQ ID NO: 914 (KIPVAIKESPKANKEIL); SEQ ID NO: 687 (STVQLIMQL); SEQ ID NO: 956 (NPLMCRLLGI); SEQ ID NO: 15 (TDFGRAKLL); and SEQ ID NO: 884 (RLSISFENLDTAKKKLP). A two-tailed unpaired t-test was used for statistical significance analysis between groups. A P<0.05 value was considered statistically significant. *P<0.05, **P<0.01, ***P<0.001. [Figure 13-4] Same as above

[0041] [Figure 14-1]Figure 14A-14C shows HLA / peptide tetramer-based immunomonitoring of vaccine-induced CD8+ T cell responses. List of custom-synthesized EGFR neoantigen tetramers used in the immunomonitoring analysis: KITDFGRAK (SEQ ID NO: 4); VKITDFGRAK (SEQ ID NO: 16); HVKITDFGR (SEQ ID NO: 1); AIKESPKANK (SEQ ID NO: 32); AIKTSPKANK (SEQ ID NO: 20); LTSTVQLIM (SEQ ID NO: 65) [Figure 14-2] (Figures 14B and 14C) Tetramer staining results of ex vivo PBMCs collected before and after vaccination at the indicated time points. (Figure 14B) EGFR neoantigen-specific CD8+ T cell populations were observed for PPV Pt. 5, 8, 14, and 16 (L858R; SEQ ID NO: 4) and 17 (T790M; SEQ ID NO: 65). [Figure 14-3] (Figure 14C) shows an example of negative EGFR neoantigen tetramer staining of PBMCs collected from vaccinated Pts.3, 5, 8, 14, and 16.

[0042] [Figure 15-1] Figures 15A-15C show the HLA class I and class II superfamily peptide binding analysis of shared EGFR neoantigens. HLA binding prediction was performed for EGFR neoantigen peptides containing 10 of the most common EGFR mutations in lung cancer, including five shared point mutations (S768I, H773L, T790M, L858R, and L861Q) and five common Exon 19 deletions (legend). (Figure 15A) The number of 9, 10, or 11-mer EGFR neoantigen peptides was predicted to bind to 100 of the most common HLA class I allotypes worldwide with predicted binding affinities of 500 nM or less. The allotypes were divided into HLA superfamilies, revealing different superfamily binding selectivity for different shared EGFR mutations. [Figure 15-2] (Continued from Figure 15A) [Figure 15-3] (Figure 15B) Number of 9, 10, or 11-mer EGFR neoantigen peptides predicted to bind to HLA class I allotypes with a predicted binding affinity of 5000 nM or less. [Figure 15-4] (Continued from Figure 15B) [Figure 15-5] (Figure 15C) Number of 17-mer EGFR neoantigen peptides predicted to bind to HLA class II allotypes with a predicted binding affinity of 500 nM or less. Binding prediction was performed using NetMHCpan4.0 for HLA class I peptides and NetMHCII2.3 for HLA class II peptides. The grouping of HLA class I and II superfamilies was adapted from Sidney et al., 2008, Harjanto et al., 2014, and Jensen et al., 2018. BX: Unclassifiable HLA-B allotype.

[0043] [Figure 16-1] Figures 16A-16H show that neoantigen vaccination increased the frequency and number of EGFR-L858R neoantigen-specific T cell clones in peripheral blood and cancer. (Figures 16A and 16B) Percentage change in TCRVβ-CDR3 clonal score in patient PBMCs after PPV (stratified by patient group and progression-free survival). (Figure 16C) Flow sorting of CD8+ tetramer+ (Tet+) T cells from PPV PBMCs of patient 5 10 months after HLA-A*1101 / KITDFGRAK (SEQ ID NO: 4) tetramer staining. Sorted Tet+ cells were single-cell TCRα / β sequencing. (Figure 16D) TCRVβ-CDR3 sequencing was performed on PBMCs of patient 5 and tumor biopsies taken before or after PPV treatment. [Figure 16-2](Figure 16D) The Venn diagram and dot plot show the number and frequency of overlapping CDR3 clones between sample pairs. Fifty-two highly reliable Tet+Vβ-CDR3 clones selected after PPV showed a high degree of overlap with both PBMC and TILCDR3 clones (gray dots). PPV induced a significant increase in the frequency of neoantigen-specific Tet+ clones (small boxes), and 13 new Tet+ clones also appeared after PPV (light gray dots). [Figure 16-3] (Figure 16E) Immunization also induced other CDR3 clone populations in both PBMCs and TILs (black arrows) in PBMCs (Figure 16E) and induced TIL frequencies in the top 10 existing Tet+ CDR3 clones at pre- and post-PPV time points. (Figure 16F) PBMC and TIL frequencies of 12 vaccine-induced Tet+ CDR3 clones before and after PPV. [Figure 16-4] (Figure 16G) Single sequencing of selected Tet+ clones facilitated the cloning of Vα-N1 and Vβ-N1 using the HLA-A*1101 / KITDFGRAK (SEQ ID NO: 4) tetramer. A549 cells were also engineered to express minigenes encoding HLA-A*1101 and / or KITDFGRAK (SEQ ID NO: 4) peptides. [Figure 16-5] (Figure 16H) TCR-N1 modified T cells co-cultured with A11. Kit-transduced A549 cells produced significantly more IFN-γ than control T cells, as measured by IFN-γ ELISA (top) (P<0.001). TCR-N1 T cells did not recognize the control parental strains A549, A549-A11, and A549.KIT target cells, and showed neoantigen-specific responsiveness (bottom). Statistical comparisons were measured against the control. Two-tailed unpaired t-tests or Mann-Whitney U tests were used to analyze statistical significance between groups. P<0.05 was considered statistically significant. **, P<0.01; ***, P<0.001.

[0044] [Figure 17-1](FIG.17A) This figure shows the change in PMBC frequency during PPV treatment in patient 5 for 52 TCR-VβCDR3 clones identified by the single-cell TCR sequence of KITDFGRAK (SEQ ID NO: 4) tetramer-positive CD8+ (Tet+) T cells derived from 10 Mo PBMCs. The Y-axis shows the CDR3 frequency in selected Tet+ cells, and the X-axis shows the CDR3 frequency in PBMCs at the indicated time point. Correlation coefficients are shown. [Figure 17-2] (FIG. 17B) Maximum polyploidy of 52 Tet+CDR3 clones in peripheral blood during the course of PPV treatment. Induced clones were not detected in pre-treated PBMCs. [Figure 17-3] (FIG.17C) Excluding 52 Tet+ clones, the same analysis as in Figure 17A is performed and compared with the CDR3 frequency in tumor samples collected before PPV or after 12Mo. [Figure 17-4] (FIG.17D) Treatment of H1975 (EGFR-L858R / T790M) and H1299 (EGFR-WT) lung cancer cells with the EGFR inhibitor ocimatinib significantly reduced phosphorus-EGFR and phosphorus-ERK in H1975 cells, as shown in the Western blot. EGFRi concentrations are shown in μM. C1 and C2 are untreated control cells. [Figure 17-5] (FIG.17E) Heatmap showing RNA transcript changes in H1975 and H1299 cells after exposure to EGFRi (24h) or EGFR (24h) + IFN-γ (12h). [Figure 17-6] (FIG.17F) Gene signature changes in Jak-Stat, TNFα, and TRAIL signaling in H1975 cells (circles) and H1299 cells (squares) after EGFRi or EGFRi + IFNγ treatment. [Figure 17-7] (FIG.17G) RNA sequencing analysis of H1975 cells after EGFRi treatment.

[0045] [Figure 18-1]Figures 18A-18M: Immunomodulation by EGFR inhibitors promotes immune cell infiltration, tumor antigen presentation, and T cell activation. H1975 (EGFR-L858R / T790M) and H1299 (EGFR-WT) cell lines were treated with the EGFR inhibitor (EGFRi) osimertinib, and RNA sequencing analysis was performed at 0, 12, or 24 hours after treatment. RNA sequencing was also performed on 4 patient tumors, 2 on-EGFRi cases, and 2 off-EGFRi cases. (Figure 18A) In H1975 cells after EGFRi treatment, relative transcript expression levels of genes related to cell division, cell cycle, apoptosis, and cell survival decreased, and this trend was reflected in the on-EGFRi patient tumor samples. (Figure 18B) EGFRi upregulated the expression of immune-related genes associated with antigen presentation and immune cell transport in H1975 cells. [Figure 18-2] (Figure 18C) Changes in gene expression pathways in EGFRi-treated H1975 and H1299 cell lines. In H1975 cells, HLA expression, STAT signaling, TRAIL signaling, apoptosis, TNF-α signaling, and NF-κB signaling were upregulated, while EGFR signaling, MAPK signaling, PI3K signaling, cell cycle, MYC signaling, and EMT signaling were downregulated after 24 hours of EGFRi treatment. In H1299 cells, TRAIL signaling, apoptosis, STAT signaling, and EMT signaling were upregulated, while TNF-α signaling, NFkappaB signaling, HLA expression, EGFR signaling, MAPK signaling, PI3K signaling, cell cycle, and MYC signaling were downregulated after 24 hours of EGFRi treatment. [Figure 18-3](Figure 18D) EGFRi treatment of H1975 cells (circles) and H1299 cells (squares) upregulated genes related to TRAIL signaling while downregulating genes related to EGFR signaling and proliferation rate. (Figure 18E) Patients undergoing EGFRi treatment showed similar changes in EGFR signaling, proliferation rate, and TRAIL signaling as EGFRi-treated H1975 cells. [Figure 18-4] (Figures 18F and 18G) Luminex analysis of H1975 cell supernatant confirmed changes in 10 chemokines and cytokines at the protein level. Statistical comparisons were measured compared to controls. (Figure 18H) Migration assays showed that EGFRi treatment of H1975 cells increased the migration of PBMC monocytes and CD4+ T cells and activated CD8+ tumor-infiltrating lymphocytes (TILs) toward H1975 cell supernatant. [Figure 18-5] (Figure 18I) HLA class I surface expression increased in H1975 cells after EGFRi treatment, but not in H1299 cells. (Figure 18J) Tumor antigen-specific CD8+ T cells showed significantly increased IFN-γ secretion in response to recognition of the congener antigen on EGFRi-treated H1975 cells compared to untreated cells. [Figure 18-6] (Figures 18K and 18L) The immune cell content of patient tumor specimens was supplemented from RNA-seq data using a method that takes into account immune cell type-specific gene expression. [Figure 18-7] (Figure 18M) A proposed mechanistic model to explain how EGFRi therapy may synergistically interact with PPV to enhance immune cell transport and T cell activation at tumor sites. Two-sided unpaired t-tests or Mann-Whitney U tests were used to analyze statistical significance between groups. P<0.05 was considered statistically significant. *, P<0.05; **, P<0.01. [Modes for carrying out the invention]

[0046] I. The present invention In some embodiments, peptides derived from mutant EGFR neoantigens are provided that are recognized and bind to HLA class I and / or HLA class II molecules. In particular, peptides are identified that are predicted to bind to specific HLA molecular carriers by cancer patients with a given EGFR mutant cancer. These peptides, or cocktails of such peptides (e.g., including both HLA class I and HLA class II binding peptides), can be used to stimulate an effective immune response against EGFR mutant cancer. Thus, such peptides and peptide cocktails can be administered directly to subjects to simulate an in vivo immune response in subjects with EGFR mutant cancer (e.g., lung cancer). Alternatively, the peptides of the embodiments can be used in anti-cancer immunotherapy compositions to activate and expand immune effector cells (e.g., T cells) ex vivo.

[0047] II. Definition As used herein, “essentially not present” with respect to a particular component is used herein to mean that none of the particular component is intentionally included in the composition and / or is present only as a contaminant or in trace amounts. Therefore, the total amount of a particular component resulting from unintended contamination of the composition is far less than 0.01%. Most preferably, the composition is one in which the amount of the particular component cannot be detected by standard analytical methods.

[0048] As used herein, "a" or "an" may mean one or more. As used herein, when used with the word "including", the word "a" or "an" may mean one or more.

[0049] The use of the term “or” (sometimes written as “or”) in the claims is used to mean “and / or” unless it is explicitly indicated that it refers only to alternatives, or alternatives are mutually exclusive, but the disclosure supports definitions that refer only to alternatives and “and / or”.

[0050] Throughout this application, the term “about” is used to indicate that the values ​​include inherent variations in the apparatus, the methods used to determine the values, or the variations present among the subjects of study.

[0051] "Treatment" and "to treat" mean the administration or application of a therapeutic agent to a subject, or the implementation of a procedure or modality to a subject, with the aim of obtaining therapeutic benefits from a disease or health-related condition. For example, a procedure may include the administration of T-cell therapy.

[0052] The terms "subject" and "patient" are used interchangeably to refer to either humans or non-humans, such as primates, mammals, and vertebrates. In certain embodiments, "subject" refers to humans.

[0053] As used throughout this application, the terms “therapeutic benefit” or “therapeutic effectiveness” refer to any medical treatment of the condition that promotes or enhances the health of the subject. This includes, but is not limited to, a reduction in the frequency or severity of the signs or symptoms of the disease. For example, cancer treatment may include, for instance, a reduction in tumor size, a reduction in tumor invasiveness, a reduction in the rate of cancer growth, or a prevention of metastasis. Cancer treatment may also mean extending the survival time of a subject with cancer.

[0054] "Anticancer drugs" can, for example, negatively affect cancer cells / tumors in a subject, by promoting the death of cancer cells, inducing apoptosis in cancer cells, slowing the growth rate of cancer cells, reducing the incidence or number of metastases, shrinking tumor size, inhibiting tumor growth, reducing blood supply to tumors or cancer cells, promoting the immune response against cancer cells or tumors, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.

[0055] As used herein, the term “antibody” is used in its broadest sense and particularly includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, insofar as they exhibit the desired biological activity.

[0056] The phrase "medicinal or pharmacologically acceptable" refers to molecular entities and compositions that, where necessary, do not produce harmful, allergic, or other adverse reactions when administered to animals, such as humans. The preparation of pharmaceutical compositions containing antibodies or additional active ingredients is known to those skilled in the art in light of the disclosure of this invention. Furthermore, for administration to animals (e.g., humans), it is understood that preparations should meet sterility, pyrogenicity, general safety, and purity standards as required by the FDA's Bureau of Biological Standards.

[0057] As used herein, “pharmaceutically acceptable carrier” includes any and all aqueous solvents (e.g., water, alcohol / aqueous solution, physiological saline solution, parenteral vehicles (e.g., sodium chloride, ringer's dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and organic esters for injection (e.g., ethyl oleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobial or antifungal agents, chelating agents, and inert gases), isotonic agents, absorption retarders, salts, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, dyes, fluids, and nutritional supplements, as is known to those skilled in the art, including materials and combinations thereof. The pH and precise concentrations of the various components in the pharmaceutical composition are adjusted according to well-known parameters.

[0058] The term “unit dose” or “dosage” refers to a physically distinct unit appropriate for use in the subject, each unit comprising a predetermined amount of the therapeutic composition calculated to produce the desired response discussed above in relation to its administration, i.e., the appropriate route and treatment regimen. The amount administered depends on the desired effect, depending on both the number of treatments and the unit dose. The actual dosage of the composition of this embodiment administered to a patient or subject may be determined by physical and physiological factors such as the subject’s body weight, age, health, and sex, the type of disease being treated, the degree of disease penetration, previous or concurrent therapeutic interventions, the patient’s idiopathy, the route of administration, and the potency, stability, and toxicity of the particular therapeutic substance. For example, the dose may also include a range of approximately 1 μg / kg / body weight to approximately 1000 mg / kg / body weight per dose (including the intervening dose) and any achievable range within that range. In non-limiting examples of the derivable range from the numbers enumerated herein, doses can be administered in ranges such as approximately 5 μg / kg / body weight to approximately 100 mg / kg / body weight, or approximately 5 μg / kg / body weight to approximately 500 mg / kg / body weight. The physician responsible for administration will, in any case, determine the concentration of the active ingredient in the composition and the appropriate dose for each individual subject. In some embodiments, the dose of antigen-specific T cell infusion may contain approximately 100 million to approximately 30 billion cells, for example, 10, 15, or 20 billion cells.

[0059] The term "immune checkpoint" refers to molecules, such as proteins, in the immune system that signal components to maintain balance in the immune response. Known immune checkpoint proteins include CTLA-4, PD1 and its ligands PD-L1 and PD-L2, as well as LAG-3, BTLA, B7H3, B7H4, TIM3, and KIR. It is recognized in the art that pathways involving LAG3, BTLA, B7H3, B7H4, TIM3, and KIR constitute immune checkpoint pathways similar to the CTLA-4 and PD-1-dependent pathway (see, e.g., Pardoll, 2012; Mellman et al., 2011).

[0060] An "immune checkpoint inhibitor" refers to any compound that inhibits the function of an immune checkpoint protein. Inhibition includes reduced function and complete blockade. Specifically, immune checkpoint proteins are human immune checkpoint proteins. Therefore, an immune checkpoint protein inhibitor is specifically an inhibitor of human immune checkpoint proteins.

[0061] As used herein, “protective immune response” refers to the response of the mammalian host’s immune system to cancer. Protective immune responses may provide therapeutic effects for cancer, such as reducing tumor size or increasing survival.

[0062] As used herein, the term “antigen” refers to a molecule that can be bound by an antibody or a T cell receptor. Antigens may commonly be used to induce humoral and / or cellular immune responses that lead to the production of B and / or T lymphocytes.

[0063] The terms “tumor-associated antigen,” “tumor antigen,” and “cancer cell antigen” are used interchangeably herein. In each case, these terms refer to proteins, glycoproteins, or carbohydrates that are specifically or preferentially expressed by cancer cells.

[0064] The term “chimeric antigen receptor (CAR),” as used herein, may refer to, for example, an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immune receptor, and encompasses engineered receptors that impart artificial specificity to specific immune effector cells. CARs can be used to confer the specificity of monoclonal antibodies to T cells, thereby enabling the generation of a large number of specific T cells, for example, for use in adoptive cell therapy. In certain embodiments, CARs direct, for example, cell specificity to tumor-associated antigens. In some embodiments, CARs include an intracellular activation domain, a transmembrane domain, and an extracellular domain including a tumor-associated antigen binding region. In certain aspects, CARs include a fusion of single-chain variable fragments (scFv) derived from a monoclonal antibody, which are fused to a CD3 zeta, a transmembrane domain, and an endodomain. Specificity of other CAR designs may be derived from ligands of receptors (e.g., peptides) or from pattern recognition receptors (e.g., Dectins). In certain cases, the spacing of the antigen recognition domains may be modified to reduce activation-induced cell death. In certain cases, the CAR may include domains for further co-stimulatory signaling, such as CD3ζ, FcR, CD27, CD28, CD137, DAP10, and / or OX40. In some cases, molecules including co-stimulatory molecules, imaging reporter genes (e.g., for positron emission tomography), prodrugs, homing receptors, chemokines, chemokine receptors, cytokines, and gene products that conditionally remove T cells upon addition of cytokine receptors may be co-expressed with the CAR.

[0065] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) has a specific percentage (e.g., 80%, 85%, 90%, or 95%) of "sequence identity" or "homology" to another sequence, meaning that when aligned, the percentage of bases (or amino acids) is the same when comparing the two sequences. This alignment and the percentage of homology or sequence identity can be determined using a software program known in the art (e.g., as described in CURRENT PROTOCOLS IN MOLECULARBIOLOGY (FMAusubel et al., 1987) Supplement 30, Section 7.7.18, Table 7.7.1). Preferably, default parameters are used for alignment. A preferred alignment program is BLAST, which uses default parameters. In particular, preferred programs are BLASTN and BLASTP, which use the following default parameters: Geneticcode=standard;filter=none;strand=both;cutoff=60;expect=10;Matrix=BLOSUM62;Descriptions=50array;sort by=HIGH SCORE;Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. III. EGFR Mutation Peptides

[0066] The embodiments disclosed in this invention relate to tumor antigen-specific peptides, such as EGFR peptides, which include a mutant EGFR sequence (e.g., a peptide having an insertion, substitution, or deletion relative to the wild-type EGR sequence). In certain embodiments, the tumor antigen-specific peptide has the amino acid sequence of the EGFR mutant peptide. In some embodiments, the peptide is 50, 45, 40, 35, 30, 25, 20, or 15 amino acids or less in length. In certain aspects, for example, any of those in Tables 1-3 are fused to a polypeptide having a non-EGFR amino acid sequence (i.e., a heterologous polypeptide). In some embodiments, the tumor antigen-specific peptide may have an amino acid sequence having at least 80, 85, 90, 95, 96, 97, 98, 99, or 100 percent sequence identity with the peptide sequences in Tables 1-3, or a sequence that conforms to those in Tables 1-3 but includes 1, 2, or 3 amino acid substitutions or deletions compared to those in Tables 1-3.

[0067] As used herein, the term “peptide” encompasses an amino acid chain comprising 7 to 35 amino acid residues, preferably 8 to 35 amino acid residues, more preferably 8 to 25 amino acids, or an amino acid chain having a length of 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, 34, or 35 amino acids, or any range therein that can be derived. For example, the EGFR mutant peptides disclosed herein may, in some embodiments, comprise or consist of any one of the sequences in Tables 1 to 3. As used herein, “antigenic peptide” is a peptide that, when introduced into a vertebrate, can stimulate antibody production in the vertebrate, i.e., is antigenic, and where the antibody can selectively recognize and / or bind to the antigenic peptide. Antigenic peptides may include immunoreactive EGFR mutant peptides and may include additional sequences. Further sequences may be derived from native antigens and may be heterogeneous; such sequences may or may not be immunogenic. In some embodiments, the tumor antigen-specific peptide (e.g., EGFR mutant peptide) can selectively bind to specific HLA class I or HLA class II complexes. In certain embodiments, the EGFR mutant peptide is 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, or 35 amino acids in length, or any range within which this can be derived. Preferably, the tumor antigen-specific peptide (e.g., EGFR mutant peptide) is 8 to 35 amino acids long. In some embodiments, the tumor antigen-specific peptide (e.g., EGFR mutant peptide) is 8 to 10 amino acids long.

[0068] As those skilled in the art will understand, MHC molecules can bind to peptides of various sizes, but typically cannot bind to full-length proteins. While it has traditionally been described that MHC class I molecules bind to peptides 8–11 amino acids long, it has been shown that peptides up to 15 amino acids can also bind to MHC class I molecules by swelling in the middle of the binding site or by extending outward from the MHC class I binding groove (Guo et al., 1992; Burrows et al., 2006; Samino et al., 2006; Stryhn et al., 2000; Collins et al., 1994; Blanchard and Shastri, 2008). Furthermore, recent studies have also demonstrated that longer peptides can be more efficiently endocytized, processed, and presented by antigen-presenting cells (Zwaveling et al., 2002; Bijker et al., 2007; Melief and van der Burg, 2008; Quintarelli et al., 2011). As shown by Zwaveling et al. (2002), peptides up to 35 amino acids in length can selectively bind to and be effective against class II MHC. As will be readily apparent to those skilled in the art, naturally occurring full-length tumor antigens, such as mutant EGFR, would not be useful for selectively binding to class II MHC so that they are endocytized and generate T cell proliferation. In general, naturally occurring full-length tumor antigen proteins do not exhibit these properties and are therefore not useful for these immunotherapeutic purposes.

[0069] In certain embodiments, tumor antigen-specific peptides (e.g., EGFR mutant peptides) are immunogenic or antigenic. As shown in the following examples, various tumor antigen-specific peptides (e.g., EGFR mutant peptides) disclosed in this invention can promote T cell proliferation. Such peptides are expected to be used to induce some degree of protective immunity.

[0070] Tumor antigen-specific peptides (e.g., EGFR mutant peptides) may be recombinant peptides, synthetic peptides, purified peptides, immobilized peptides, detectably labeled peptides, encapsulated peptides, or vector-expressed peptides (e.g., peptides encoded by nucleic acids in a vector containing a heterologous promoter operably linked to the nucleic acid). In some embodiments, synthetic tumor antigen-specific peptides (e.g., EGFR mutant peptides) can be administered to subjects such as human patients to induce an immune response in the subject. Synthetic peptides may exhibit certain advantages compared to recombinantly expressed peptides, such as a reduced risk of bacterial contamination. Tumor antigen-specific peptides (e.g., EGFR mutant peptides) may also be included in pharmaceutical compositions, such as vaccine compositions, which are formulated for administration to mammalian or human subjects.

[0071] A. Examples of HLA class I and II library peptides for peptide selection procedures Table 1 below shows examples of final peptide compositions of EGFR mutant peptides predicted to bind to specific HLA class I compositions. The peptides are ranked based on their predicted binding properties. In this example, the patient has the L858R mutation and possesses two different specific HLA-A, HLA-B, and HLA-C genes.

[0072] An exemplary procedure for selecting HLA class I peptides (HLA class II peptides can be selected using the same procedure): 1. The patient's tumor is genetically analyzed to check for the presence of EGFR mutations. 2. The patient's HLA class I and HLA class II are typed to determine which HLA molecules are expressed. 3. If the patient harbors an EGFR mutation, HLA peptide binding prediction is performed to determine whether the peptide comprising the EGFR mutation can bind to either HLA class I or HLA class II molecules. For the top 20 most common EGFR mutations, these predicted peptides are listed in the included table. 4. Patient example in Table 1: As a result of EGFR sequencing, the patient has an L858R EGFR mutation, and HLA class I typing shows HLA-A * 0101, A * 0301, B * 0702, B * 4601, C * 0102, C * 0701. 5. From the specific peptide list (see Tables 2 and 3), obtain all peptides listed for these 6 HLA types, and combine them into one list. 6. Peptides are individually scored according to their predicted affinity (nM) and predicted percentile as follows: [Table A] 7. Sum the affinity score (max = 3) and the percentile score (max = 3) to obtain the final total score (maximum total score = 6) for each peptide. 8. Peptides are ranked according to their total scores. 9. Peptides with the highest total scores are prioritized for inclusion in the immunogenic composition (vaccine).

[0073] The HLA Class I Library contains the top 20 EGFR mutations, covering approximately 95% of EGFRmut patients (see Table 2 below). The library contains 364 total EGFR mutation peptides, including 342 mutation-specific peptides and 16 peptides shared by two mutations. The library includes 100 HLA class I allotypes (HLA-A, 30; HLA-B, 48; and HLA-C, 22).

[0074] The HLA class 2 library contains the top 20 EGFR mutations and includes 429 all EGFR mutant peptides (see Table 3 below). The top 20 EFGR mutations are A763_Y764insFQEA, D770_N771insSVD, E746_A750del, E746_S752del>V, G719A, G719C, G719S, H773_V774insH, I744_K745insKIPVAI, L747_A750del>P, L747_P753del>S, L747_S752del, L747_T751del, L747_T751del>P, L858R, L861Q, P772_H773insPR, S768I, T790M, and V769_D770insASV. Table 1: Exemplary HLA class I peptide libraries and related characteristics [Table 1] Table 2: Peptide HLA class I libraries and related characteristics [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] Table 3: Peptide HLA class II libraries and related characteristics [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13] [Table 3-14] [Table 3-15] [Table 3-16]

[0075] B. Cell-permeable peptides In some embodiments, immunotherapy can utilize tumor antigen-specific peptides (e.g., EGFR mutant peptides) disclosed herein, related to cell penetrators such as liposomes or cell-permeable peptides (CPPs). Antitumor immunity can be enhanced using antigen-presenting cells (e.g., dendritic cells) pulsed with the peptides (Celluzzi et al., 1996; Young et al., 1996). Liposomes and CPPs are described in further detail below. In some embodiments, immunotherapy can utilize nucleic acids encoding tumor antigen-specific peptides (e.g., EGFR mutant peptides) disclosed herein, where the nucleic acids are delivered, for example, in a viral or non-viral vector.

[0076] Tumor antigen-specific peptides (e.g., EGFR mutant peptides) can also associate with or covalently bind to cell-permeable peptides (CPPs). Cell-permeable peptides that can covalently bind to tumor antigen-specific peptides (e.g., EGFR mutant peptides) include, for example, HIV Tat, herpesvirus VP22, Drosophila Antennapedia homeobox gene products, signal sequences, fusion sequences, or protegrin I. Covalent binding of peptides to CPPs can extend peptide presentation by dendritic cells and thus enhance antitumor immunity (Wang and Wang, 2002). In some embodiments, tumor antigen-specific peptides disclosed herein (e.g., EGFR mutant peptides) (e.g., contained within a peptide or polyepitope string) can be covalently bound to CPPs (e.g., via peptide bonds) to generate fusion proteins. In another embodiment, a tumor antigen-specific peptide (e.g., an EGFR mutant peptide) or a nucleic acid encoding a tumor antigen-specific peptide can be encapsulated within a liposome (e.g., a multilayer, vesicle, or multivesicle liposome, exocytic vesicle, or exosome) or associated with a liposome.

[0077] As used herein, “association” means a physical association, a chemical association, or both. For example, associations may include covalent bonding, hydrophobic interactions, encapsulation, surface adsorption, and the like.

[0078] As used herein, “cell penetrator” means a composition or compound that enhances the intracellular delivery of peptide / polyepitope strings to antigen-presenting cells. For example, a cell penetrator may be a lipid that, when associated with a peptide, enhances its ability to cross the plasma membrane. Alternatively, a cell penetrator may be a peptide. Cell-penetrating peptides (CPPs) are known in the art and include, for example, the HIV Tat protein (Frankel and Pabo, 1988), the HSV VP22 protein (Elliott and O'Hare, 1997), and fibroblast growth factor (Lin et al., 1995).

[0079] Cell-permeable peptides (or "protein transduction domains") have been identified from the Drosophila Antennapedia homeobox gene (Antp), HIV Tat, and the third helix of herpesvirus VP22, all of which contain positively charged domains enriched with arginine and lysine residues (Schwarze et al., 2000; Schwarze et al., 1999). Hydrophobic peptides derived from signal sequences have also been identified as cell-permeable peptides (Rojas et al., 1996; Rojas et al., 1998; Du et al., 1998). Binding these peptides to marker proteins such as β-galactosidase has been shown to confer efficient internalization of marker proteins into cells, and chimeric lineframe fusion proteins containing these peptides have been used to deliver proteins to a wide range of cell types both in vitro and in vivo (Drin et al., 2002). The fusion of these cell-permeable peptides with tumor antigen-specific peptides (e.g., EGFR mutant peptides) as disclosed in this invention may enhance the cellular uptake of polypeptides.

[0080] In some embodiments, cellular uptake is facilitated by the attachment of lipids (e.g., stearates or myristylates) to polypeptides. Lipidization has been shown to enhance the passage of peptides into cells. Attachment of lipid moieties is another method, as disclosed in the present invention, that increases polypeptide uptake by cells. Cellular uptake is discussed further below.

[0081] The tumor antigen-specific peptides disclosed in this invention (e.g., EGFR mutant peptides) may be included in liposomal vaccine compositions. For example, the liposomal composition may be or may contain a proteoliposome composition. Methods for producing proteoliposome compositions that may be used in conjunction with the disclosure of this invention are described, for example, by Neelapu et al. (2007) and Popescu et al. (2007). In some embodiments, the proteoliposome composition may be used to treat melanoma.

[0082] By enhancing the uptake of tumor antigen-specific polypeptides, it may be possible to reduce the amount of protein or peptide required for treatment. This, in turn, could significantly reduce the cost of treatment and increase the supply of therapeutic agents. Lower dosages may also minimize the potential immunogenicity of the peptides and suppress toxic side effects.

[0083] In some embodiments, tumor antigen-specific peptides (e.g., EGFR mutant peptides) can be associated with nanoparticles to form nanoparticle-polypeptide complexes. In some embodiments, the nanoparticles are liposomes or other lipid-based nanoparticles such as lipid-based vesicles (e.g., DOTAP: cholesterol vesicles). In other embodiments, the nanoparticles are iron oxide-based superparamagnetic nanoparticles. Superparamagnetic nanoparticles in a diameter range of approximately 10–100 nm are small enough to avoid sequestration by the spleen but large enough to avoid clearance by the liver. Particles of this size can penetrate very small capillaries and be effectively distributed throughout body tissues. Superparamagnetic nanoparticle-polypeptide complexes can be used as MRI contrast agents to identify and track cells that take up tumor antigen-specific peptides (e.g., EGFR mutant peptides). In some embodiments, the nanoparticles are semiconductor nanocrystals or semiconductor quantum dots, both of which can be used for optical imaging. In further embodiments, the nanoparticles may be nanoshells containing a gold layer on a silica core. One advantage of nanoshells is that polypeptides can be bound to the gold layer using standard chemistry. In other embodiments, the nanoparticles may be fullerenes or nanotubes (Gupta et al., 2005).

[0084] Peptides are rapidly removed from circulation by the kidneys and are susceptible to degradation by serum proteases. By associating tumor antigen-specific peptides (e.g., EGFR mutant peptides) with nanoparticles, the nanoparticle-polypeptide complexes disclosed in this invention can be protected from degradation and / or reduced by renal clearance. This can increase the blood half-life of the polypeptide, thereby reducing the polypeptide dose required for effective treatment. Furthermore, this can reduce the cost of treatment and minimize immunological problems and therapeutic toxic reactions.

[0085] C. Polyepitope Strings In some embodiments, tumor antigen-specific peptides (e.g., EGFR mutant peptides) are included in or contain a polyepitope string. A polyepitope string is a peptide or polypeptide containing multiple antigenic epitopes derived from one or more antigens that are linked together. Polyepitope strings can be used to induce an immune response in subjects such as human subjects. Polyepitope strings have been previously used to target malaria and other pathogens (Baraldo et al., 2005; Moorthy et al., 2004; Baird et al., 2004). A polyepitope string can refer to a nucleic acid (e.g., a nucleic acid encoding multiple antigens including an EGFR mutant peptide) or a peptide or polypeptide (e.g., containing multiple antigens including an EGFR mutant peptide). Polyepitope strings can be included in cancer vaccine compositions.

[0086] D. Biological functional equivalent The tumor antigen-specific peptides disclosed in this invention (e.g., EGFR mutant peptides) are HLA class proteins (e.g., HLA-A *0101) Modifications may include amino acid substitutions, insertions, and / or deletions that do not alter the respective interactions with the binding region. Such biofunctional equivalents of tumor antigen-specific peptides (e.g., EGFR mutant peptides) may be molecules having similar or other desired features (e.g., binding to specific HLA class I or HLA class II complexes). As a non-limiting example, certain amino acids may be substituted with other amino acids in the tumor antigen-specific peptides (e.g., EGFR mutant peptides) disclosed herein without significant loss of interaction ability, as demonstrated by peptides that remain detectably unchanged in binding to HLA. In some embodiments, the tumor antigen-specific peptides have substitutional mutations at anchor residues, such as substitutional mutations at one, two, or all of the 1(P1), 2(P2), and / or 9(P9) positions. Thus, the tumor antigen-specific peptides (e.g., EGFR mutant peptides) (or nucleic acids encoding such peptides) disclosed herein are intended to be modified in sequence and / or structure, but without alteration of biological utility or activity, and within the scope of the compositions and methods disclosed herein.

[0087] It will also be well understood by those skilled in the art that a key aspect of the definition of a biologically functionally equivalent peptide is the concept that there is a limit to the number of changes that can be made within a defined portion of the molecule, while still maintaining an acceptable level of equivalent biological activity. A biologically functionally equivalent peptide is thus defined here as a peptide in which specific amino acids, rather than most or all, can be substituted. Of course, multiple distinct peptides with different substitutions can be readily prepared and used in accordance with the disclosures of this invention.

[0088] Those skilled in the art will also recognize that when certain residues, for example residues in a particular epitope, are shown to be particularly important to the biological or structural properties of a peptide, such residues are generally not convertible. This may be the case in the disclosure of the present invention, as mutations in tumor antigen-specific peptides (e.g., EGFR mutant peptides) disclosed herein may result in species-specific loss, and subsequently reduce the usefulness of the resulting peptide for use in the methods disclosed herein. Therefore, peptides that are antigenic (e.g., specifically bind to HLA class I or HLA class II complexes) and contain conserved amino acid substitutions are understood to be included in the disclosure of the present invention. Conservative substitutions are the least likely to dramatically alter the activity of a protein. “Conservative amino acid substitution” means substituting one amino acid with a chemically similar amino acid, i.e., substituting one nonpolar amino acid with another nonpolar amino acid; substituting one polar amino acid with another polar amino acid; substituting an acidic residue with another acidic amino acid, etc.

[0089] Amino acid substitutions that may be used when modifying tumor antigen-specific peptides (e.g., EGFR mutant peptides) disclosed herein are generally based on the relative similarity of amino acid side-chain substituents (e.g., their hydrophobicity, hydrophilicity, charge, size, etc.). Analysis of the size, shape, and type of amino acid side-chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine are all of similar size; and phenylalanine, tryptophan, and tyrosine all have substantially similar shapes. Based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine are defined herein as biofunctional equivalents. In some embodiments, the mutations may enhance TCR-pMHC interactions and / or peptide-MHC binding.

[0090] The disclosure of the present invention also intends to include isoforms of tumor antigen-specific peptides (e.g., EGFR mutant peptides) disclosed herein. While the isoforms contain the same number and types of amino acids as the peptides disclosed herein, they have different molecular structures. The isoforms intended by the disclosure of the present invention are isoforms having the same properties as the peptides disclosed herein as described herein.

[0091] Non-standard amino acids can be incorporated into proteins by chemical modification of existing amino acids or by de novo synthesis of peptides disclosed herein. Non-standard amino acids refer to amino acids that have a chemical structure different from the 20 standard amino acids encoded by the genetic code.

[0092] In selected embodiments, the disclosure of the present invention intends to be a chemical derivative of a tumor antigen-specific peptide (e.g., an EGFR mutant peptide) disclosed herein. A “chemical derivative” means a peptide having one or more residues that are chemically derivatized by the reaction of a functional side chain group and retain biological activity and utility. Examples of such derivatized peptides include those in which a free amino group is derivatized to form a specific salt, or by alkylation and / or acylation, a p-toluenesulfonyl group, a carbobenzoxy group, a t-butylosicarbonyl group, a chloroacetyl group, a formyl or acetyl group. A free carboxyl group can be derivatized to form an organic or inorganic salt, a methyl or ethyl ester, or another type of ester or hydrazide, preferably an amide (primary or secondary). Chemical derivatives may include peptides containing one or more naturally occurring amino acid derivatives of 20 standard amino acids. For example, 4-hydroxyproline may be substituted with serine, and ornithine may be substituted with lysine.

[0093] It should be noted that all amino acid residue sequences herein are represented by formulas in which the left and right orientations are in the conventional direction from the amino terminus to the carboxyl terminus. Furthermore, it should be noted that a dash at the start or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino acid residues. The amino acids described herein are preferably in "L" isomer form. However, residues in "D" isomer form may be substituted with any L-amino acid residue, as long as the desired functional properties described herein are maintained by the protein.

[0094] Preferred tumor antigen-specific peptides (e.g., EGFR mutant peptides) or their analogues preferably bind specifically or preferentially to specific HLA class I or HLA class II complexes. Whether, and to what extent, a particular tumor antigen-specific peptide or labeled peptide, or its analogue, can bind to a particular HLA class I or HLA class II complex can be determined using in vitro assays [e.g., enzyme-linked immunosorbent assay (ELISA), immunoblotting, immunoprecipitation, radioimmunoassay (RIA), immunostaining, latex agglutination, indirect hemagglutination assay (IHA), complement fixation, indirect immunofluorescence assay (FA), turbidimetry, flow cytometry, chemiluminescence assay, lateral flow immunoassay, u-capture assay, mass spectrometry, particle-based assay, inhibition assay, and / or avidity assay].

[0095] E. Nucleic acids encoding tumor antigen-specific peptides In one embodiment, the disclosure of the present invention provides nucleic acids encoding isolated antigen-specific peptides containing sequences having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with peptides selected from Tables 1-3, or the peptides may have one, two, three, or four point mutations (e.g., substitution mutations) compared to peptides selected from Tables 1-3. As described above, such tumor antigen-specific peptides may be, for example, 8-35 amino acid lengths, or any range within that range that can be induced.

[0096] Some embodiments disclosed herein provide recombinant tumor antigen-specific peptides (e.g., EGFR mutant peptides) that can specifically bind to specific HLA class I or HLA class II complexes. Thus, nucleic acids encoding tumor antigen-specific peptides can be operably ligated to expression vectors using methods well known in the field of molecular biology, and the peptides can be produced in a suitable expression system. The nucleic acids encoding tumor antigen-specific peptides disclosed herein can be incorporated into any expression vector that ensures good expression of the peptide. Possible expression vectors include, but are not limited to, cosmids, plasmids, or modified viruses (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses), as long as the vector is suitable for transforming host cells.

[0097] A recombinant expression vector "suitable for host cell transformation" means that the expression vector comprises a nucleic acid molecule disclosed in the present invention and a regulatory sequence operably linked to the nucleic acid molecule, selected based on the host cell used for expression. The terms "operatively linked" or "operably linked" are used interchangeably and are intended to mean that nucleic acids are linked to regulatory sequences in a manner that enables nucleic acid expression.

[0098] Accordingly, the disclosure of the present invention provides a recombinant expression vector comprising a nucleic acid encoding a tumor antigen-specific peptide, and regulatory sequences necessary for the transcription and translation of the inserted protein sequence. Suitable regulatory sequences may be derived from various sources, including bacterial, fungal, or viral genes (see, for example, the regulatory sequences described in Goeddel (1990)).

[0099] The selection of appropriate regulatory sequences generally depends on the selected host cell and can be easily achieved by those skilled in the art. Examples of such regulatory sequences include transcription promoters and enhancers or RNA polymerase binding sequences, and ribosome binding sequences containing translation initiation signals. Furthermore, depending on the selected host cell and the vector used, other sequences (e.g., origins of replication, further DNA limiting sites, enhancers, and sequences conferring transcriptional inducibility) may be incorporated into the expression vector. It will also be understood that the required regulatory sequences may be supplied by native proteins and / or their adjacent regions.

[0100] Recombinant expression vectors may also include selection marker genes to facilitate the selection of host cells transformed or transfected with recombinant tumor antigen-specific peptides (e.g., EGFR mutant peptides) disclosed herein. Examples of selection marker genes are genes encoding proteins such as G418 and higromycin that confer resistance to specific drugs, β-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. Transcription of selection marker genes is monitored by changes in the concentration of selection marker proteins such as β-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. If the selection marker gene encodes a protein that confers antibiotic resistance, such as neomycin-resistant transformed cells, it can be selected with G418. Cells incorporating the selection marker gene survive, while other cells die. This allows for visualization and assay of the expression of the recombinant expression vector, and in particular, for determining the effects of mutations on expression and phenotype. It is understood that selection markers may be introduced onto a vector other than the nucleic acid of interest.

[0101] Recombinant expression vectors can be introduced into host cells to produce transformed host cells. The term “transformed host cell” is intended to include prokaryotic and eukaryotic cells transformed or transfected with the recombinant expression vectors disclosed in this invention. The terms “transformed with,” “transfected with,” “transformation,” and “transfection” are intended to encompass the introduction of nucleic acids (e.g., vectors) into cells by one of many possible techniques known in the art. Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins disclosed in this invention may be expressed in bacterial cells (e.g., E. coli), insect cells (using baculoviruses), yeast cells, or mammalian cells.

[0102] The nucleic acid molecules disclosed in this invention can also be chemically synthesized using standard techniques. Various methods for chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis, which is fully automated in commercially available DNA synthesizers, as well as peptide synthesis (see, for example, U.S. Patents 4,598,049; 4,458,066; 4,401,796; and 4,373,071).

[0103] IV. Antigen-specific cell therapy Embodiments disclosed in the present invention involve antigen-specific cells [e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4)]. + T cells, CD8 + This relates to obtaining T cells (or gamma delta T cells), NK cells, invariant NK cells, NKT cells, mesenchymal stem cells (MSCs), or induced pluripotent stem (iPS) cells and administering them to cancer cells as targeted immunotherapy. In particular, these cells are antigen-specific T cells (e.g., mutant EGFR-specific T cells). Several basic approaches for inducing, activating, and expanding functional antitumor effector cells have been described over the past 20 years. These include autologous cells such as tumor-infiltrating lymphocytes (TILs); T cells activated ex vivo using autologous DCs, lymphocytes, artificial antigen-presenting cells (APCs), or T cell ligands and activating antibodies coated beads, or cells isolated by capturing target cell membranes; allogeneic cells that spontaneously express anti-host tumor T cell receptors (TCRs); and non-tumor-specific autologous or allogeneic cells that have been genetically reprogrammed or "directed" to express tumor-reactive TCR molecules or chimeric TCR molecules that exhibit antibody-like tumor recognition ability known as "T-bodies." These approaches have given rise to numerous protocols for T cell preparation and immunization that may be used in the methods described herein.

[0104] B. T cell preparation In some embodiments, T cells originate from blood, bone marrow, lymph, umbilical cord, or lymphoid organs. In some embodiments, the cells are human cells. The cells are typically primary cells, such as those isolated directly from the subject and / or isolated and frozen from the subject. In some embodiments, cells are defined by their function, activation state, maturity, differentiation potential, expansion, recirculation, localization, and / or persistence, antigen specificity, antigen receptor type, presence in a specific organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation, such as T cells or other cell types (e.g., entire T cell population, CD4). + cells, CD8 +The method comprises one or more subsets of cells (and their subpopulations). With respect to the subject being treated, the cells may be allogeneic and / or autologous. In some embodiments, such as ready-made technologies, the cells are pluripotent and / or multipotent, such as stem cells including induced pluripotent stem cells (iPSCs). In some embodiments, the method includes isolating cells from a subject, preparing them, processing them, culturing them, and / or manipulating them, and reintroducing them to the same patient before or after cryopreservation, as described herein.

[0105] T cell subtypes and subpopulations (e.g., CD4) + and / or CD8 + Among T cells, naive T cells (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes, for example, stem cell memory T (TSC) M ), Central Memory T (TC M ), Effector Memory T (T EM ), or terminal differentiated effector memory T cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, spontaneously generated and adaptive regulatory T (Treg) cells, helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, and δ / γ T cells.

[0106] In some embodiments, one or more T cell populations are enriched or depleted for cells that are positive for a specific marker, such as a surface marker, or negative for a specific marker. In some cases, such markers are absent or expressed at relatively low levels on a particular population of T cells (e.g., non-memory cells), but are present or expressed at relatively high levels on a particular other population of T cells (e.g., memory cells).

[0107] In some embodiments, T cells are isolated from PBMC samples by negative selection of markers expressed on non-T cells (e.g., B cells, monocytes, or other leukocytes (e.g., CD14)). In some embodiments, CD4 + or CD8 + The selection stage is CD4 + Helper T cells and CD8 + Used to isolate cytotoxic T cells. Such CD4 + and CD8 + The population can be further subdivided into subpopulations by positive or negative selection for markers expressed at relatively high levels on one or more naive T cell subpopulations, memory T cell subpopulations, and / or effector T cell subpopulations.

[0108] In some embodiments, CD8 + T cells are further enriched or depleted, for example, by positive or negative selection based on surface antigens associated with each subpopulation, for naive, central memory, effector memory, and / or central memory stem cells. In some embodiments, central memory T(T) CM Cell enrichment is performed to increase potency, for example, to improve long-term survival, growth, and / or engraftment after administration, which in some embodiments is particularly robust in such subpopulations. See Terakura et al., 2012; Wang et al., 2012.

[0109] In some embodiments, the T cells are autologous T cells. In this method, the tumor sample is obtained from the patient, and a single-cell suspension is obtained. The single-cell suspension is processed in any suitable manner, for example, mechanically (e.g., gentleMACS). (商標)Tumors can be deaggregated using a dissociator (Miltenyi Biotec, Auburn, CA) or enzymatically (e.g., collagenase or DNase). Single-cell suspensions of tumor enzyme digests are cultured in interleukin-2 (IL-2). Cells are then confluenced (e.g., approximately 2 × 10⁻⁶). 6 The cells are cultured until they reach the lymphocyte stage, for example, for about 5 to 21 days, preferably for about 10 to 14 days.

[0110] Cultured T cells can be pooled and rapidly proliferated. Rapid proliferation provides at least a 50-fold increase in the number of antigen-specific T cells over a period of about 10 to about 14 days (e.g., 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more). More preferably, rapid proliferation provides at least a 200-fold increase over a period of about 10 to about 14 days (e.g., 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, or more).

[0111] Proliferation can be achieved by any of the many methods known in the art. For example, T cells can be rapidly proliferated using nonspecific T cell receptor stimulation in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin-15 (IL-15), with IL-2 being preferred. Nonspecific T cell receptor stimulation is performed using approximately 30 ng / ml of OKT3, mouse monoclonal anti-CD3 antibody (Ortho-McNeil). (登録商標)This may include (available from Raritan, NJ). Alternatively, T cells can be rapidly proliferated by in vitro stimulation of PBMCs with one or more cancer antigens (including their antigenic portion such as an epitope, or cells) which can be selectively expressed from a vector such as a human leukocyte antigen A2 (HLA-A2) binding peptide, in the presence of a T cell growth factor such as 300 IU / ml of IL-2 or IL-15, with IL-2 preferred. In vitro-induced T cells are rapidly expanded by restirritation with the same(multiple) cancer antigens pulsed onto HLA-A2-expressing antigen-presenting cells. Alternatively, T cells can be restirritated with radiation, autologous lymphocytes, or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0112] Autologous T cells can be modified to express T cell growth factors that promote the proliferation and activation of autologous T cells. Suitable T cell growth factors include, for example, IL-2, IL-7, IL-15, and IL-12. Appropriate modification methods are known in the art. See, for example, Sambrook et al., 2001; and Ausubel et al., 1994. In particular, modified autologous T cells express T cell growth factors at high levels. T cell growth factor coding sequences (e.g., the IL-12 sequence) are readily available in the field, just as promoters are, and operable linking to these T cell growth factor coding sequences promotes high levels of expression.

[0113] C. Antigen-presenting cells Antigen-presenting cells (APCs), such as macrophages, B lymphocytes, and dendritic cells, are distinguished by the expression of specific MHC molecules. APCs take up antigens and re-express a portion of those antigens along with MHC molecules on their outer membrane. The major histocompatibility complex (MHC) is a large genetic complex with multiple loci. MHC loci encode two major classes of MHC membrane molecules called class I and class II MHC. Helper T lymphocytes generally recognize antigens bound to MHC class II molecules, while cytotoxic T lymphocytes recognize antigens bound to MHC class I molecules. In humans, MHC is called the HLA complex, and in mice, it is called the H-2 complex. In some embodiments, the EGFR mutant peptide of the embodiment is expressed in antigen-presenting cells. Such cells provide engineered APCs that can be used to specifically proliferate immune effector cells specific to the mutant EGFR antigen of interest.

[0114] In some cases, aAPC is useful in preparing the therapeutic compositions and cell therapy products of this embodiment. For general guidance on the preparation and use of antigen presentation systems, see, for example, the U.S. Patents: U.S. Patent Nos. 6,225,042, 6,355,479, 6,362,001 and 6,790,662; U.S. Patent Application Publication Nos. 2009 / 0017000 and 2009 / 0004142; and International Publication No. WO2007 / 103009.

[0115] The aAPC system may include at least one exogenous co-stimulatory molecule. Any appropriate number and combination of co-stimulatory molecules may be used. Co-stimulatory molecules may be selected from, for example, co-stimulatory molecules and adhesion molecules. Exemplary co-stimulatory molecules include CD86, CD64 (FcγRI), 41BB ligand, and IL-21. Adhesion molecules may include single-pass transmembrane immunoglobulin (Ig) superfamily proteins such as carbohydrate-binding glycoproteins like selectins, transmembrane glycoproteins like integrins, calcium-dependent proteins like cadherins, and intercellular adhesion molecules (ICAMs) that facilitate intercellular or cell-matrix contact. Exemplary adhesion molecules include LFA-3 and ICAM (e.g., ICAM-1). Techniques, methods, and reagents useful for the selection, cloning, preparation, and expression of exemplary co-stimulatory molecules, including co-stimulatory molecules and adhesion molecules, are exemplified, for example, in U.S. Patent Nos. 6,225,042, 6,355,479, and 6,362,001.

[0116] V. Treatment method Provided herein are methods for treating or delaying the progression of cancer in an individual, which include administering an effective dose to the individual in antigen-specific cell therapy (e.g., mutant EGFR-specific T cell therapy). Adaptive T cell therapy using genetically modified TCR-transduced T cells (in which the TCR is bound to another bioreactive protein (e.g., anti-CD3)) is also provided in the present invention. In further embodiments, methods for treating cancer are provided, comprising immunizing a target with a purified tumor antigen or an immunodominant tumor antigen-specific peptide.

[0117] The EGFR mutant peptides provided herein may be used to develop cancer vaccines or immunogens (e.g., a mixture of an adjuvant and a peptide or modified peptide that encodes a polynucleotide and corresponds to an expression product such as an inactivated virus or other microbial vaccine). These peptide-specific vaccines or immunogens may be used to directly immunize cancer patients in vivo to induce an antitumor immune response, or to proliferate antigen-specific T cells in vitro using peptide or polynucleotide-loaded APC stimulation. These numerous T cells can be adoptedly transferred into patients to induce tumor regression.

[0118] Examples of cancers planned for treatment include lung cancer, head and neck cancer, breast cancer, pancreatic cancer, prostate cancer, kidney cancer, bone cancer, testicular cancer, cervical cancer, gastrointestinal cancer, lymphoma, preneoplastic lesions in the lungs, colon cancer, melanoma, and bladder cancer.

[0119] In some embodiments, T cells are autologous. However, the cells can be allogeneic. In some embodiments, T cells are isolated from the patient themselves so that the cells are autologous. If the T cells are allogeneic, they can be pooled from several donors. The cells are administered to the target in an amount sufficient to control, reduce, or eliminate the symptoms and signs of the disease being treated.

[0120] In some embodiments, the subject may be subjected to non-myeloablative lymphodepleting chemotherapy prior to T-cell therapy. Non-myeloablative lymphodepleting chemotherapy may be any appropriate such treatment that can be administered by any appropriate route. Non-myeloablative lymphodepleting chemotherapy may include, for example, the administration of cyclophosphamide and fludarabine, particularly when the cancer is melanoma, which may be metastatic. An exemplary route for administering cyclophosphamide and fludarabine is intravenous. Similarly, any appropriate doses of cyclophosphamide and fludarabine can be administered. In particular, approximately 60 mg / kg of cyclophosphamide is administered for 2 days, followed by approximately 25 mg / m2 of fludarabine for 5 days.

[0121] In certain embodiments, T cell growth factors that promote the proliferation and activation of autologous T cells are administered simultaneously with or after the autologous T cells. The T cell growth factor can be any suitable growth factor that promotes the proliferation and activation of autologous T cells. Examples of suitable T cell growth factors include IL-2, IL-7, IL-15, and IL-12, which can be used alone or in various combinations such as IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, "IL-2, IL-7, and IL-15", IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL-2. IL-12 is a preferred T cell growth factor.

[0122] T cells can be administered intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, intraorbitally, implantably, by inhalation, intrathecally, intraventricularly, or intranasally. The appropriate dosage of T cell therapy may be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, medical history and response to treatment, and the discretion of the attending physician.

[0123] Intratumoral injection or injection into the tumor vascular system is particularly intended for individual, solid, and accessible tumors. Local, local, or systemic administration may also be appropriate. For tumors larger than 4 cm, the administered volume is approximately 4–10 ml (especially 10 ml), while for tumors smaller than 4 cm, a volume of approximately 1–3 ml (especially 3 ml) is used. Multiple injections administered as a single dose contain volumes of approximately 0.1 to 0.5 ml.

[0124] B. Pharmaceutical Compositions Pharmaceutical compositions and formulations comprising antigen-specific immune cells (e.g., T cells) or receptors (e.g., TCRs) and pharmaceutically acceptable carriers are also provided in the present invention. Vaccine compositions for pharmaceutically acceptable use in a subject may comprise tumor antigen peptide (e.g., EGFR mutation peptide) compositions and pharmaceutically acceptable carriers disclosed herein.

[0125] The pharmaceutical compositions and formulations described herein can be prepared by mixing an active ingredient (such as an antibody or polypeptide) of desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22nd edition, 2012) in the form of a lyophilized formulation or aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dose and concentration used and include buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzesonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); and low molecular weight (less than about 10 residues) polypropylene Examples of such substances include, but are not limited to, peptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as glycine, glutamine, asparagine, histidine, arginine, or lysine; other carbohydrates including monosaccharides, disaccharides, glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Examples of pharmaceutically acceptable carriers as described herein include soluble neutral-active hyaluronidase glycoproteins (sHASEGP), such as rHuPH20[HYLENEX]. (登録商標) The invention further includes intra-tissue drug dispersants such as human soluble PH-20 hyaluronidase glycoprotein, such as Baxter International, Inc. Specific exemplary sHASEGPs containing rHuPH20 and methods of use are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one embodiment, sHASEGP is conjugated with one or more additional glycosaminoglycans, such as chondroitinase.

[0126] C. Combination therapy In certain embodiments, the compositions and methods of this embodiment include an antigen-specific immune cell population or TCR in combination with at least one additional therapy. The additional therapy may be radiotherapy, surgery (e.g., tumor resection and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination thereof. This additional therapy may take the form of adjuvant therapy or neoadjuvant therapy.

[0127] In some embodiments, the additional treatment is the administration of a small molecule enzyme inhibitor or an anti-metastatic agent. In some embodiments, the additional treatment is the administration of an adverse reaction inhibitor (e.g., a drug intended to reduce the occurrence and / or severity of adverse reactions to the treatment, e.g., an anti-nausea agent). In some embodiments, the additional treatment is radiotherapy. In some embodiments, the additional treatment is surgery. In some embodiments, the additional treatment is a combination of radiotherapy and surgery. In some embodiments, the additional treatment is gamma irradiation. In some embodiments, the additional treatment is a treatment targeting the PBK / AKT / mTOR pathway, such as an HSP90 inhibitor, a tubulin inhibitor, an apoptosis inhibitor, and / or a chemopreventative agent. The additional treatment may be one or more chemopreventative agents known in the art.

[0128] Immunotherapy can be administered in various combinations in conjunction with additional cancer treatments such as immune checkpoint therapy. Doses may be spaced simultaneously or at intervals ranging from minutes to days to weeks. In embodiments where immunotherapy is provided to the patient separately from additive therapies, it is generally ensured that no significant time elapses between each delivery so that the two compounds can still exert a beneficial combined effect on the patient. In such cases, it is intended that the patient may be provided with antibody therapy and anticancer therapy within approximately 12–24 hours or 72 hours of each other, or more specifically, within approximately 6–12 hours of each other. In some situations, it may be desirable to significantly extend the treatment period, with several days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) between each delivery.

[0129] Various combinations can be used. In the following example, antigen-specific immunotherapy, peptides, or TCRs are indicated as "A," and anticancer therapy is indicated as "B": A / B / AB / A / BB / B / AA / A / BA / B / BB / A / AA / B / B / BB / A / B / B B / B / B / AB / B / A / BA / A / B / BA / B / A / BA / B / B / AB / B / A / A B / A / B / AB / A / A / BA / A / A / BB / A / A / AA / B / A / AA / A / B / A

[0130] The administration of any compound or treatment in this embodiment to a patient follows a general protocol for the administration of such compounds, taking into account the toxicity of the drug (if any). Therefore, in some embodiments, there is a step to monitor toxicity resulting from the combination therapy.

[0131] 2.Chemotherapy A wide variety of chemotherapeutic agents may be used in accordance with this embodiment. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to mean a compound or composition administered in the treatment of cancer. These drugs or agents are classified according to the mode of their activity within cells, for example, whether and at what stage they affect the cell cycle. Alternatively, drugs may be characterized based on their ability to directly crosslink DNA, insert into DNA, or induce chromosomal and mitotic abnormalities by affecting nucleic acid synthesis.

[0132] Examples of chemotherapeutic agents include: Alkylating agents such as thiotepa and cyclosphosphamide; Alkyl sulfonates such as busulfan, improsulfan, and piposulfan; Aziridines such as benzodopa, carboquone, meturedopa, and uredopa; Ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; Acetogenins (especially bullatacin and bullatacinone); The synthetic analogue topotecan, which includes a camptothecin; bryostatin; callystatin; CC-1065 (including adozelesin, carzelesin, and bizelesinsyntheticanalogues); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189, and CB1-TM1); eleutherobin; pancratistatin; asarcodictyin; spongistatin; Nitrogen mustards such as chlorambucil, chlornaphazine, chorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracilmustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; Antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammal I and calicheamicin omega I1); dynemicins, including dynemicin A; bisphosphonates like clodronate; an esperamicin; similar neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins(mitomycinC, mycophenolic acid, Examples include nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; Antimetabolites such as methotrexate and 5-fluorouracil (5-FU); denopterin, pteropterin, and folic acid analogues such as trimetrexate; Purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxfluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; Anti-adrenals such as mitotane and trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinicacid;eniluracil;amsacrine;bestrabucil;bisantrene;edatraxate;defofamine;demecolcine; diaziquone; elformithine;elliptinium acetate; an epothilone; etoglucid;galliumnitrate; hydroxyurea; Maytansine and maytansinoids such as ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, for example paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (for example CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine;Carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, and any pharmaceutically acceptable salts, acids, or derivatives thereof.

[0133] 3. Radiation therapy Other factors that cause DNA damage and have been widely used include what is commonly known as gamma rays, X-rays, and / or directed delivery of radioisotopes to tumor cells. Other forms of DNA damage factors such as microwaves, proton beam irradiation (US Patents 5,760,395 and 4,870,287), and UV irradiation are also intended. All of these factors are most likely to affect widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dose range for X-rays ranges from 50 to 200 roentgens per day over long periods (3 to 4 weeks) to single doses of 2,000 to 6,000 roentgens. The dose range for radioisotopes varies widely and depends on the half-life of the isotope, the intensity and type of radiation emitted, and uptake by nascent cells.

[0134] 4. Immunotherapy Those skilled in the art will understand that additional immunotherapy may be used in combination with or in conjunction with the methods of the embodiments. In relation to cancer treatment, immunotherapeutic agents generally rely on the use of immune effector cells and molecules to target and destroy cancer cells. Rituximab (RITUXAN) (登録商標)) is an example of such a thing. Immune effectors can be, for example, antibodies that are specific to certain markers on the surface of tumor cells. Antibodies may act alone as therapeutic effectors, or they may recruit other cells to actually influence cell killing. Antibodies can also bind to drugs or toxins (chemotherapeutic agents, radionuclides, lysine A chain, cholera toxin, pertussis toxin, etc.) and serve as targeting agents. Alternatively, effectors may be lymphocytes that carry surface molecules that interact directly or indirectly with tumor cell targets. Various effector cells include cytotoxic T cells and MK cells.

[0135] Antibody-drug conjugates (ADCs) are emerging as a groundbreaking approach to the development of cancer treatments. Cancer is one of the leading causes of death worldwide. Antibody-drug conjugates (ADCs) contain monoclonal antibodies (MAbs) covalently bound to cytotoxic drugs. This approach combines the high specificity of MAbs to their antigen targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver the payload (drug) to tumor cells enriched with the antigen. Targeted drug delivery also minimizes exposure to normal tissues, resulting in reduced toxicity and improved therapeutic index. (ADCETRIS, 2011) (登録商標) (Brentuximab vedotin), KADCYLA in 2013 (登録商標) The FDA's approval of two ADC therapies (trastuzumab emtansine or T-DM1) confirmed the validity of this approach. Currently, there are more than 30 ADC candidate therapies in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization become increasingly mature, the discovery and development of new ADCs increasingly depend on the identification and confirmation of new targets suitable for this approach, as well as the generation of target MAbs. Two criteria for selecting an ADC target are upregulation / high levels of expression in tumor cells and robust internalization.

[0136] In one embodiment of immunotherapy, tumor cells must be targetable, that is, they must carry certain markers that are not present in most other cells. Many tumor markers exist, and any of these may be suitable for targeting in the context of this embodiment. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sial Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. An alternative form of immunotherapy is to combine anticancer effects with immunostimulatory effects. Immunostimulatory molecules may also include cytokines such as IL-2, IL-4, IL-12, GM-CSF, and γ-IFN, chemokines such as MIP-1, MCP-1, and IL-8, and growth factors such as FLT3 ligand.

[0137] Examples of immunotherapies currently under development or in use include: Immunoadjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patents 5,801,005 and 5,739,169, Hui and Hashimoto, 1998, and Christodoulides et al., 1998); Cytokine therapy, for example, interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); Gene therapy, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; US Patents 5,830,880 and 5,846,945); and Monoclonal antibodies, such as anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; US Patent 5,824,311). It is intended that one or more anticancer therapies may be used in conjunction with the antibody therapies described herein.

[0138] In some embodiments, the immunotherapy may be an immune checkpoint inhibitor. Immune checkpoints either turn up or turn down signals (e.g., costimulatory molecules). Inhibitory immune checkpoints that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuator (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activator gene-3 (LAG3), programmed death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and T cell activation V domain Ig suppressor (VISTA). In particular, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0139] Immune checkpoint inhibitors may be drugs such as small molecules, ligands, or recombinant receptor forms, or in particular antibodies such as human antibodies (e.g., International Patent Publication WO2015-016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012; both incorporated herein by reference). Known inhibitors of immune checkpoint proteins or their analogues may be used, in particular chimeric, humanized, or human forms of antibodies. As those skilled in the art will know, alternative and / or equivalent names may be used for the particular antibodies referred to herein. Such alternative and / or equivalent names are interchangeable in the context of this disclosure. For example, lambrolizumab is known under the alternative and equivalent names MK-3475 and Pembrolizumab.

[0140] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partner. In a specific aspect, the PD-1 ligand binding partner is PDL1 and / or PDL2. In another embodiment, the PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In a specific aspect, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, the PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In a specific aspect, the PDL2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patent Nos. US8735553, US8354509, and US8008449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the methods provided herein are known in the art, as described in U.S. Patent Application Publication Nos. 2014 / 0294898, 2014 / 022021, and 2011 / 0008369 (all of which are incorporated herein by reference).

[0141] In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising the extracellular or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP224. Nivolumab is MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO (登録商標)also known as, and is the anti-PD-1 antibody described in WO2006 / 121168. Pembrolizumab is MK-3475, Merck 3475, lambrolizumab, KEYTRUDA (登録商標) , and also known as SCH-900475, and is the anti-PD-1 antibody described in WO2009 / 114335. The anti-PD-1 antibody described in WO2009 / 101611 is also known as CT-011, hBAT or hBAT-1. AMP-224, also known as B7-DCIg, is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342.

[0142] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and acts as an "off" switch when bound to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits an inhibitory signal to T cells. CTLA4 is similar to CD28, a T cell costimulatory protein, and both molecules bind to CD80 and CD86 (also referred to as B7-1 and B7-2, respectively) on antigen-presenting cells. CTLA4 transmits an inhibitory signal to T cells, while CD28 transmits a stimulatory signal. Intracellular CTLA4 is also found in regulatory T cells, and its function may be important. Activation of T cells via the T cell receptor and CD28 leads to increased expression of CTLA-4, an inhibitory receptor of B7 molecules.

[0143] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody), an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or an oligopeptide.

[0144] Anti-human CTLA-4 antibodies (or VH and / or VL domains derived therefrom) suitable for use in this method can be produced using methods well known in the art. Alternatively, anti-CTLA-4 antibodies recognized in the art can be used. For example, anti-CTLA-4 antibodies such as U.S. Patent Nos. 8,119,129, WO01 / 14424, WO98 / 42752; WO00 / 37504 (CP675,206, also known as tremelimumab, formerly ticilimumab), U.S. Patent Nos. 6,207,156; Hurwitz et al. (1998) Proc NatlAcadSciUSA95(17)10067-10071; Camacho et al. (2004) J ClinOncology 22(145): Abstract No. 2505 (Antibody CP-675,206); and Mokyr et al. (1998) CancerRes 58:5301-5304 may be used in the manner disclosed herein. The teachings of each of these publications are incorporated herein by reference. Regarding binding to CTLA-4, antibodies that compete with any of these antibodies recognized in the art may also be used. For example, humanized CTLA-4 antibodies are described in WO2001 / 014424, WO2000 / 037504, and U.S. Patent No. 8,017,114, all of which are incorporated herein by reference.

[0145] Exemplary anti-CTLA-4 antibodies include ipilimumab (10D1, MDX010, MDX101, and Yervoy) (登録商標)The antibody is also known as ipilimumab or its antigen-binding fragment and variants (see, for example, WO 01 / 14424). In other embodiments, the antibody comprises the heavy and light chain CDRs or VRs of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, and the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for binding to and / or binding to the same epitope on CTLA-4 as the antibody described above. In another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the antibody described above (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0146] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors, such as those described in US5844905, US5885796, and WO1995 / 001994 and WO1998 / 042752, all of which are incorporated herein by reference. Also included are immunoadhesion factors, such as those described in U.S. Patent No. US8329867, which are incorporated herein by reference.

[0147] 5.Surgery Approximately 60% of cancer patients undergo some form of surgery, including prophylactic, diagnostic, or staging, curative, and palliative surgery. Curative surgery involves the physical removal, excision, and / or destruction of all or part of the cancerous tissue, and may be used in conjunction with other therapies such as the procedures of this embodiment, chemotherapy, radiotherapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor excision means the physical removal of at least a portion of the tumor. Surgical treatments, in addition to tumor excision, include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs procedure).

[0148] When cancerous cells, tissue, or part or all of a tumor are removed, a cavity may form in the body. Treatment can be achieved by perfusion, direct injection, or local application of the area with additional anticancer treatment. Such treatment can be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or every 1, 2, 3, 4, and 5 weeks, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These treatments can also be of varying doses.

[0149] 6. Other medications Other agents may be used in combination with specific aspects of this embodiment to improve the therapeutic efficacy of the treatment. These additional agents include agents that affect the upregulation of cell surface receptors and GAP binding, cell growth inhibitors and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferating cells to apoptosis inducers, or other biological agents. Increased intercellular signaling by increasing the number of GAP bindings would likely increase the anti-hyperproliferative effect on adjacent hyperproliferating cell populations. In other embodiments, cell growth inhibitors or differentiation agents may be used in combination with specific aspects of this embodiment to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors may improve the efficacy of this embodiment. Examples of cell adhesion inhibitors include local adhesion kinase (FAK) inhibitors and lovastatin. Other agents that increase the sensitivity of hyperproliferating cells to apoptosis, such as the antibody c225, may be used in combination with specific aspects of this embodiment to improve therapeutic efficacy.

[0150] VI. Manufactured articles or kits The present invention also provides manufactured articles or kits containing antigen-specific immune cells, TCRs, or antigen peptides (e.g., EGFR peptides). The manufactured articles or kits may further include a package insert containing instructions for using the antigen-specific immune cells to treat or delay the progression of cancer in an individual, or to enhance the immune function of an individual with cancer. Any of the antigen-specific immune cells described herein may be included in the manufactured articles or kits. Suitable containers include, for example, bottles, vials, bags, and syringes. Containers can be formed from a variety of materials, such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloys (e.g., stainless steel or Hastelloy). In some embodiments, the container holds the formulation, and labels on or associated with the container may indicate instructions for use. The manufactured articles or kits may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and accompanying documentation with instructions for use. In some embodiments, the manufactured articles further include one or more other agents (e.g., chemotherapeutic agents and antitumor agents). Suitable containers for one or more drugs include, for example, bottles, vials, bags, and syringes.

[0151] VII. Examples The following embodiments are included to illustrate preferred specific examples of the present invention. Those skilled in the art will understand that the methods disclosed in the following embodiments represent the techniques found by the inventors and therefore function well in the practice of the present invention. However, those skilled in the art will understand that, considering the disclosures of the present invention, many modifications are possible in the specific embodiments disclosed and yielding similar results without departing from the spirit and scope of the invention. [Examples]

[0152] Results and Methods A. Results Patient characteristics, PPV administration, and efficacy evaluation. What has already been reported is that for the widely shared EGFR-L858R mutation, CD8 + This case study describes a stage IV NSCLC patient who experienced a significant regression of multiple lung cancers following PPV, which is associated with a T-cell response (Li et al., 2016). Based on this case study, a phase Ib clinical trial of PPV in stage III / IV NSCLC patients was initiated to assess the safety and feasibility of PPV. Secondary endpoints included evaluating the immunogenicity of individualized and shared NeoAg, as well as the potential clinical survival effect (Figure 1A). Twenty-four patients (18 with adenocarcinoma and 6 with squamous cell carcinoma) were successfully enrolled and underwent 12 weekly immunizations with PPV followed by CT scans to assess the response. Clinical response was assessed using RECIST criteria 3–4 months after the start of PPV. Patients were given the option to continue PPV beyond 12 weeks if desired; the immunization timeline for all 24 patients is shown in Figure 2. Three patients (Pts. 1, 6, and 24) received alternative salvage therapy after disease progression following 12 weeks of immunization and discontinued the study. Five additional patients did not complete CT-based staging: three patients (Pts. 9, 19, and 20) showed no disease progression during the 12 weeks following vaccination, and two patients (Pts. 13 and 15) were followed up at an external hospital as informed by the authors. A complete summary of patient treatment and clinical outcomes is shown in Figure 1B.

[0153] All 24 NSCLC patients had failed multiple lines of conventional therapy, including surgery, radiotherapy, and / or chemotherapy. Of the patients with tumors bearing EGFR gene mutations, 16 had previously progressed with EGFRi therapy, and 9 of these patients were unresponsive to two or more different EGFRi investigational drugs. These 16 patients were given the option to discontinue or continue EGFRi therapy simultaneously with PPV immunization; 9 chose to continue EGFRi administration, and 7 chose to discontinue EGFRi therapy. Apart from these 9 patients, none of the other 15 patients in the PPV trial received any other treatment during the follow-up period. Although patients were not initially randomized into separate cohorts, when the 24 patients were divided into the following three groups, clear response and survival profiles emerged: EGFR wild-type (WT) patients treated with PPV alone (Group 1), EGFR mutant patients treated with PPV alone (Group 2), and EGFR mutant patients treated with EGFRi concurrently (Group 3) (Figure 1C; Figure 3). There were no significant differences in the clinical characteristics of the three patient groups at baseline (Figure 4A). Detailed EGFR inhibitor history for patients in Groups 2 and 3 is shown in Figure 4B.

[0154] Designing individualized neoantigen vaccines. Large-scale whole-exome sequencing studies have demonstrated that NSCLC is one of the most mutagenic cancers of all, with individual patient tumors often expressing 100 or more nonsynonymous somatic mutations (Rizva et al, 2015; Lawrence et al, 2013). Based on such a profile, commercially available HLA class I peptide binding prediction algorithms typically generate hundreds of potential NeoAg peptide candidates, far more than can be incorporated into a single multiepitope peptide vaccine.

[0155] However, most of these mutations are thought to be "branch" mutations, expressed by tumor subclones but not by all tumor cells. These mutations are also unlikely to constitute driver mutations essential for tumor survival (Hao et al., 2016). To focus on targeting shared driver mutations, we chose to perform mutation profiling on a panel of 508 known cancer-associated genes using DNA from fresh tumor tissue obtained by needle biopsy (Table 4). Vaccine peptides were selected primarily based on the highest predicted binding affinity of NeoAg encoding mutations against each patient's individual HLA class I and class II allotypes (Table 5, Methods). Each patient was immunized with a unique, individualized mixture of short and long NeoAg peptides dissolved in saline, divided into two pools, and subcutaneously administered to the opposite limb. Imiquimod was applied topically after each immunization to provide costimulatory signals through Toll-like receptor (TLR) 7 (Figure 1A).

[0156] Table 4. Panel of 508 cancer-related genes. [Table 4-1] [Table 4-2]

[0157] Of the 24 enrolled patients, an average of 6.1 coding mutations per tumor were detected (range, 1 to 20). Of the 16 patients with EGFR mutations, 14 harbored common EGFR driver mutations (7 cases of L858R point mutation, 7 cases of Exon 19 deletion), of which 2 cases were accompanied by the T790M mutation known to confer resistance to first-generation EGFRi. The other 2 additional patients harbored the relatively rare H773L mutation. The number of administered vaccine peptides ranged from 5 to 14 per patient (average 9.4), which was mainly determined by the number of NeoAgs predicted to bind to the patient's HLA class I (average 6.5 peptides) or HLA class II (average 2.9 peptides). Patients in Group 2 and Group 3 were administered an average of 4 mutant EGFR peptides each (2.8 short peptides and 1.2 long peptides). The number of immunized peptides or the average predicted peptide binding affinity did not differ significantly between groups; however, patients in Group 2 and Group 3 overall received vaccines targeting fewer somatic mutations (Figures 1D to 1F). This was due to the intentional targeting of individual EGFR mutations using multiple NeoAg peptides, an approach that succeeded in inducing lung tumor regression in the first case trial patient of the present inventors (Li et al., 2016; Table 5).

[0158] Table 5: Individualized neoantigen peptides and predicted HLA binding affinities. * Peptide binding affinity predicted by NetMHC4.0, NetMHCpan4.0 (HLA class I) and NetMHCII2.3 (HLA class II) ** Delta score = WT peptide affinity minus mutant peptide affinity [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10]

[0159] PPV (Personalized Peptide Vaccine) induced clinical responses in multiple patients with EGFR-mutated tumors. The clinical outcomes of all 24 patients are shown in Table 6, and group outcomes are summarized in Figures 5A and 5B and Table 7. Aside from transient Grade 1 rash, fatigue, and / or fever experienced by three patients, no other treatment-related adverse events were observed (Figure 5C). Of the EGFR-WT patients in Group 1, four showed stable disease (SD) after 12 weeks of PPV, and four showed disease progression (PD). Patient 2 experienced pleural effusion clearance, but no tumor regression was observed (Figures 6A and 6B). In contrast, objective clinical responses were observed in 3 out of 7 patients in the two groups: 2 PRs (Pt. 11 and 14) and 1 CR (Pt. 17), in which no viable tumor cells were found on post-treatment biopsy (Figures 7A-7D). Furthermore, of the 9 patients in Group 3, 4 experienced partial response (PR), and 4 further patients were considered to have SD (Figures 7E and 7F; Figure 5A). Patients in Group 3 responded to EGFRi treatment despite having a history of disease progression (Figure 6C).

[0160] Table 6: Clinical outcomes of 24 patients receiving individualized neoantigen vaccines. F: Female, M: Male, SR: Surgical resection, RT: Radiotherapy, CM: Chemotherapy, EGFRi: EGFR inhibitor, PFS: Progression-free survival, CR: Complete response, PR: Partial response, PD: Disease progression, SD: Disease stable. [Table 6-1] [Table 6-2]

[0161] Table 7-1: EGFR neoantigen vaccine peptides associated with patient clinical response. * Peptide contained in vaccines of PR patients from Li et al., 2016. + Pt.17 is a complete responder. [Table 7-1-1] [Table 7-1-2]

[0162] Table 7-2: EGFR neoantigen vaccine peptides associated with patient clinical response. *Peptide contained in vaccines of PR patients from Li et al., 2016. ++ The two patients each expressed the listed HLA class II allotypes. [Table 7-2]

[0163] Table 7-3: EGFR neoantigen vaccine peptides associated with patient clinical response. * Peptide contained in vaccines of PR patients from Li et al., 2016. ** The prevalence of T790M is rare in untreated patients (less than 2%), but is acquired in ~50% of EGFRi-treated patients who develop resistance. [Table 7-3-1] [Table 7-3-2]

[0164] Table 7-4: EGFR neoantigen vaccine peptides associated with patient clinical response. *Peptide contained in vaccines of PR patients from Li et al., 2016. [Table 7-4]

[0165] Notably, none of the patients in Group 3 had undergone a drug-free or "drug-free period" from EGFRi treatment prior to the trial treatment, which supports the idea that NeoAg vaccination played a significant role in these clinical responses. In particular, patients in Group 3 experienced a longer median OS compared to patients in Group 2 (13.8 vs. 7.6 months, P=0.038, Figure 1G; Figures 8A and 8B). Patients who experienced clinical benefit (PR / CR or SD) also showed significantly extended OS and PFS (Figure 1H; Figure 8C). However, neither clinical response nor progression-free survival (PFS) was associated with the number of immunizing peptides, peptide length, predicted HLA binding affinity, number of engaged HLA molecules, peptide delta score, or EGFRi treatment history (Figures 1D-1F; 8D and 8D; 9A-9C). PR / CR patients received significantly fewer somatic mutation-targeting vaccine treatments compared to PD patients (P=0.014). This was a result of the seven responding patients receiving a significantly higher proportion of EGFR NeoAg peptides in their vaccine (P<0.001, Figure 1F), supporting the idea that EGFR NeoAg vaccination was associated with the observed clinical response. Univariate analysis showed that the presence of pleural effusion and increased tumor burden were two risk factors that adversely impacted patient survival outcomes (Figures 10A and 10B); both are known risk factors for NSCLC (Morgensztern et al., 2012a; Morgensztern et al., 2012b).

[0166] PPV (also known as personalized vaccine) induced a NeoAg-specific T cell response to shared EGFR mutations. To better understand the nature of the antitumor response, serial immunomonitoring was performed on patient peripheral blood mononuclear cell (PBMC) samples collected before or after PPV (see Methods). Vaccine-induced immune responses were first screened by stimulating individual patient PBMCs with their immunopeptide pools and measuring specific interferon-γ (IFN-γ) secretion by ELISA (Figure 11A). In this assay, peptide pool-specific responses were detected in 6 of the 20 patients evaluated, all from either Group 2 (2 out of 5) or Group 3 (4 out of 7). Peptide deconvolution revealed individual NeoAg peptide-specific IFN-γ responses in 7 additional PPV patients (Figure 12A; Table 5). Based on these results, we calculated an immune response combo score (IRC) that describes the breadth, intensity, and persistence of the PPV-specific IFN-γ response (see Methods; Figure 12B). Of the seven patients with the lowest combo score (IRC = zero), five were in Group 1, and only one (Pt. 12 in Group 3) achieved an objective clinical response. In contrast, all six patients with the highest IRC were in Group 2 or Group 3, and five of these patients experienced a clinical response with a PFS longer than the median PFS period (Figure 12B).

[0167] Immune monitoring based on ELISPOT revealed that in 5 out of 6 responding patients who showed a vaccine-induced response, EGFR mutations constituted the primary target of the NeoAg-specific T cell response. Pt.11 is HLA-A * A moderate IFN-γ response was produced to the mutant AQP12A(L28R)NeoAg peptide limited to 0301, but HLA-A * No detectable response was produced to the EGFR(H773L) NeoAg vaccine peptide limited to 0201 (Figure 13). In contrast, three different responses were observed in HLA-A. * 1101+ patients (Pts. 5, 8, and 14) contain highly shared EGFR-L858R mutations, A* 1101 Limited Edition Peptide KITDFG R It produced a dominant immunoreactivity against AK (SEQ ID NO: 4) (Figure 11B). Similarly, CR Pt.17 showed a dominant immunoreactivity against HLA-C * Limited edition 1502 T790M-containing peptide LTSTVQLI M A strong response was observed to (SEQ ID NO: 65). ELISPOT and HLA / peptide tetramer staining assays showed specific CD8 for both of these NeoAg peptides. + T cell responses were confirmed, and both assays showed an increase in T cell frequency during immunization, up to 3 months (Figures 11C and 11D; Figure 13; Figure 14). Importantly, vaccine-induced T cells from four patients were functionally distinguishable between wild-type EGFR peptide and mutant EGFR peptide (Figure 11E).

[0168] Patient 22 (PR) is A * In addition to generating a potent response to the 0201-limited FGFR1(R734W) NeoAg peptide, the compound contains the long DRB1 EGFR mutation H773L / V774M. * 0901 Limited Edition NeoAg Peptide (MASVDNP) LM Vaccine-induced response to CRLLGICL) (SEQ ID NO: 958) also occurred. Both Pts. 8 and 16 contain a long HLA class II limited EGFR NeoAg peptide (HVKITDFG) with an L858R mutation. R An immune response to AKLLGAEE) (SEQ ID NO: 826) was produced (Figure 13). However, three other patients (5, 12, 14) vaccinated with the same length L858R peptide did not produce a detectable antigen-specific immune response, despite sharing the relevant HLA class II assignment type with Pt. 8 and 16. KITDFG R Four individuals immunized with AK peptide (SEQ ID NO: 4) showed HLA-A * Of the 1101+ patients, Pt.16 had detectable CD8 for this NeoAg. +This patient was a loan patient who did not develop a T cell response and was the only one of four patients who did not experience a clinically stable disease (SD). In summary, these results suggest that multiple different EGFR mutations are associated with objective clinical response in NSCLC patients, and that NeoAg vaccine-specific CD4 + and CD8 + This provides evidence that it could be an immunogenic target for T cell responses.

[0169] Surprisingly, KITDFG R AK NeoAg peptide (SEQ ID NO: 4) has a lower affinity (163 nM) than the corresponding WT peptide (20 nM) for HLA-A * It is expected to bind to 1101, but still well within the typical range for HLA binders of moderate affinity (Figure 11F). In contrast, the EGFR-T790M mutation converts the peptide C-terminal anchor from a polar threonine residue to a hydrophobic methionine residue, thus NeoAg LTSTVQLI M HLA-C peptide (SEQ ID NO: 65) * Binding to 1502 is more favorable than that of the WT peptide (Figure 11F). A global analysis of HLA peptide binding selectivity revealed a significant distortion of EGFR NeoAg presentation at the HLA superfamily level: L858R and Exon 19 deletion mutants both contain >80% of all EGFR mutations, while HLA-A * The A3 superfamily members, including 1101, shared S768I, T790M, and L861Q mutants, primarily produce NeoAg with a high content of binding-favoring basic amino acids, and are therefore more hydrophobic, and thus HLA-C *It is oriented (favored) to bind members of the A2, B15, B27, and C3 superfamilies, including 1502 (Figure 11G-11I; Kobayashi et al., 2016). In contrast, HLA class I allotypes within the A1, A24, B8, and C7 superfamilies are not expected to bind to and present most covalent EGFR NeoAg (Figures 15A and 15B). HLA class II molecules are predicted to bind to peptides containing a wide range of EGFR mutations, and the class II superfamily is not expected to show skewed binding preference, except for the DP1 and DP3 allotypes (Figure 15C; Sidney et al., 2008; Harjanto et al., 2014; Jensen et al., 2018).

[0170] PPV promoted the proliferation and tumor invasion of EGFR NeoAg-specific T cells. TCR Vβ-CDR3 sequencing was performed on DNA isolated from PBMCs before and after vaccination collected from 17 PPV patients. Clonality scores were calculated at each time point. After PPV, an increase in T cell clonality was observed in 10 patients, a decrease in clonality in 5 patients, and no change in 2 patients (mean change +13.2%, Figure 16A). In the 5 patients with the longest PFS (>9 months), an increase in T cell clonality was observed after PPV, particularly pronounced in patients 5 and 8 (69.8% and 95.5%, respectively, Figure 16B). These results were consistent with the expansion of NeoAg-specific T cells detected by immune monitoring in these two patients after PPV (Figures 12; 13; 14).

[0171] Patient 5 experienced an objective clinical response after PPV, and no disease progression was observed even after more than 20 months, providing a unique opportunity to analyze the long-term dynamics of the NeoAg-specific immune response in this patient. * 1101 / KITDFG R Using the AK (SEQ ID NO: 4) tetramer, EGFR(L858R)-specific CD8 was extracted from PBMCs collected 12 months after the start of PPV.+ T cells were selected (Figure 16C; Figure 14). The selected NeoAg-specific cells then underwent single-cell TCRα / β sequencing, from which 52 highly reliable TCR clones were identified (Tet + (See Methods). In addition, TCR Vβ-CDR3 sequencing of tumor-infiltrating lymphocytes (TILs) was performed on tumor biopsies taken before treatment or 12 months after PPV, allowing for a detailed comparison of CDR3 frequencies in blood and tumor compartments before and after immunization (Table 7). As shown in Figure 16D, overlapping CDR3 clones in both PBMCs and TIL pre-PPV were NeoAg-specific Tetra. + Clones were present in the blood at a higher frequency (Figures 17A and 17B).

[0172] In contrast, while post-vaccination samples showed only half the number of overlapping CDR3 clones between blood and TIL, these clones were present at significantly higher frequencies within the TIL compartment. Importantly, NeoAg-specific Tet + Clones showed a significant frequency increase in both PBMC and TIL compartments after immunization, including 13 new T cell clones that were not detected in pre-treated samples (Figure 16D; Figure 17C). Comparison of PBMC CDR3 sequences before and after PPV showed a subset of T cells (Tet + (Including clones) increased after immunization, but the frequency decreased in most other T cell clones. In contrast, at the tumor site, almost all CDR3 clones were present in 40 T cells. + This included 35 clones, showing an increased frequency after immunization (Figure 16D).

[0173] One striking feature revealed by this analysis was that 52 Tet selected after PPV + The fact that 40 of the clones were already present at a high frequency in PBMC and TIL samples before PPV suggests that spontaneous priming of EGFR NeoAg-specific cells occurred before immunization. However, these Tet +The majority of CDR3 clones showed a significant increase in PBMC frequency over a 12-week PPV course associated with patient clinical response, consistent with IFN-γ immunomonitoring results (Figure 16E; Figure 7E; Figures 11A-11D). Interestingly, PPV also showed a significant increase in frequency several months later in both PBMCs and tumor sites, although it was not detected in pre-treatment PBMCs. + It appeared to stimulate clonal expansion (Figures 16D and 16F; Figure 17B). Such induction of Tet + One of the clones, Vβ-N1, showed a more than 500-fold increase in frequency at tumor sites.

[0174] This Tet + TCRα / β chains from the clone were subcloned into a lentiviral vector, and PBMC-derived T cells were transduced to express TCR-N1 (Figure 16G). EGFR NeoAg-specific recognition was achieved by transducing TCR-N1-transduced T cells to HLA-A * 1101 and / or KITDFG R This was confirmed by co-culturing A549 tumor target cells that had been engineered to express the AK(SEQ ID NO: 4) minigene (Figures 16G and 16H).

[0175] In summary, this data shows that Pt. 5 peptide vaccination is associated with NeoAg-specific CD8 in peripheral blood. + This stimulates a significant expansion of T cells, ultimately leading to the development of these T cells in the tumor site. + This provides evidence that it leads to an increase in the frequency of T cells. + It remains unclear whether the increased tumor infiltration by cells was caused by enhanced T cell transport to the tumor, T cell proliferation resulting from NeoAg recognition at the tumor site, or both.

[0176] EGFRi promotes immune cell infiltration, antigen presentation, and T cell activation. Patients in groups 2 and 3 showed similar objective clinical response rates after PPV, but patients in group 3 showed significantly longer OS and PFS (Figure 8). To better understand why patients in group 3 experienced better survival outcomes, we then evaluated possible mechanisms by which EGFRi treatment may synergistically interact with vaccination. Two lung cancer cell lines, H1975 (EGFR mutation: L858R) + / T790M + H1975 cells and H1299 (EGFR-WT) were treated with EGFRi or DMSO, and the cell supernatant and total RNA were collected at multiple time points after treatment. As expected, EGFRi-treated H1975 cells showed reduced EGFR signaling, as confirmed by both RNA-seq and Western blot analysis, in addition to reduced expression of genes related to MYC signaling, proliferation, cell cycle, and apoptosis and survival (Figures 18A, 18C, and 18D; Figure 17D).

[0177] Examination of immune-related genes revealed that EGFRi treatment increased transcription of genes related to TRAIL signaling and HLA class I and II antigen presentation, along with a co-decreasing of checkpoint genes (Figures 18B and 18D; Figures 17E and 17F). Transcripts encoding several chemokines and cytokines increased or decreased after EGFRi treatment, and Luminex analysis confirmed their changes at the protein level in the cell supernatant (Figures 18B, 17E, and 17F). Since EGFRi treatment upregulated CXCL1, CXCL2, and CCL2, chemokines known to promote immune cell migration, we then tested how peripheral blood leukocytes migrated in response to EGFRi or DMSO-treated H1975 cell supernatant (Figures 17E and 17F). CD4 from ex vivo PBMCs + Both T cells and CD14+ monocytes showed increased migration toward EGFRi-treated cell supernatant.

[0178] As expected, they required activation before they could upregulate their migratory capabilities, but CD8 +T cells also showed significantly increased migration in response to the same cell supernatant (Figure 18H). Surface HLA class I surface expression also increased in H1975 cells after EGFRi treatment, but not in H1299 cells, resulting in antigen-specific CD8 production of more IFN-γ after recognition of EGFRi-treated H1975 tumor cells. + T cells were generated (Figures 18I and 18J; Figure 17G). These results support the idea that EGFRi can promote immune cell infiltration and antigen presentation at tumor sites, and therefore enhance the anti-tumor immune response (Wantanabe et al., 2019; Im et al., 2016).

[0179] To determine whether tumor samples from Group 3 patients exhibited similar genetic signatures, RNA-seq analysis was performed on tumor biopsies from a small subset of PPV patients. The analyzed tumor specimens included two from patients not treated with EGFRi (Group 2 Pts. 16 and 24) and two from patients treated with EGFRi (Group 3 Pts. 12 and 23). Biopsies were taken during PPV immunization, with the exception of Pt. 24, which was obtained before PPV. Gene expression signatures related to cell cycle, cell division, and cell survival in EGFRi-treated Pts. 12 and 23 correlated well with those of EGFRi-treated H1975 cells, as did gene signatures related to EGFR signaling and proliferation rate (Figures 18A, 18D, and 18E).

[0180] Immune-related gene signatures from PPV patient tumors showed partial overlap with the H1975 signature, including some agreement with upregulated antigen presentation, which was most pronounced in PR patient 12. With the exception of CCL2 and IL1RN, most chemokine and cytokine features were largely unmatched and likely result from tumor specimens containing RNA derived from infiltrating normal immune cells and stromal cells. To individually complement the immune cell content of tumor specimens, tumor RNA-seq data were analyzed with reference to immune cell type-specific genes. As shown in Figure 18K, tumors from two EGFRi-treated patients included elevated levels of M2 macrophages and B cells, as well as decreased neutrophil infiltration, compared to two patients who did not receive EGFRi.

[0181] The on-EGFRi tumor biopsy from Pt. 23 was the only sample showing elevated levels of dendritic cells and CD4 + It was also associated with increased T cell and NK cell infiltration (Figure 18L). Notably, tumor-infiltrating CD8 + T cells were observed at elevated levels in all three patients on PPV compared to pre-PPV tumor samples from Pt.24 (Figure 18K). While the number of patient samples analyzed is too small to draw definitive conclusions, these results, taken together, suggest that EGFRi has the ability to alter gene expression within the tumor microenvironment to promote immune cell infiltration and antigen presentation.

[0182] In summary, our research supports the following model for explaining the therapeutic synergy between peptide vaccination and EGFRi therapy (Figure 18M): PPV administration stimulates the proliferation of circulating NeoAg-specific T cells, while EGFRi promotes enhanced antigen presentation and chemokine secretion at the tumor site. The increase in chemokines then increases the transport of immune cells, including activated T cells, to the tumor, where T cell recognition of congenital tumor antigens stimulates tumor cell destruction and IFN-γ production (Venugopalan et al., 2016).

[0183] Since IFN-γ is known to strongly upregulate antigen presentation and chemokine production (Schoebom et al., 2007; Schroder et al., 2004; Figures 17E and 17F), the combination of PPV and EGFRi may stimulate an initial antitumor T cell response, which subsequently initiates a "feed-forward" loop at the tumor site to maintain an antitumor immune response (Figure 18M). Such a sustained immune response may explain the prolonged PFS observed in PPV patients in Group 3, in addition to the long-term expansion of induced NeoAg-specific T cell clones observed in Patient 5.

[0184] Consideration. This is considered the first report demonstrating that peptide vaccination against shared NeoAg can induce objective clinical regression in multiple cancer patients. Due to the overwhelming prevalence of HLA diversity and individual mutations, no previous vaccine studies have reported immunizing one or more patients with potentially shared NeoAg peptides. However, in Asian NSCLC patients, HLA-A * The relatively high prevalence of both 1101 and EGFR(L858R) mutations predicted that -15% of our EGFR mutation patients would share this combined phenotype (Kobayashi et al., 2016; Gonzalez-Galarza et al., 2015).

[0185] This is A * 1101 Limited KITDFG R A vaccine commonly containing EGFR NeoAg peptides, including AK peptide (SEQ ID NO: 4) (Li et al., 2016), provided a unique opportunity to immunize five such patients (four in this study). Notably, four of the five patients experienced tumor regression within 12 weeks of NeoAg vaccination, and all four patients showed a significant increase or dominance of KITDFG during the time to clinical response. R AK (SEQ ID NO: 4) specific CD8 + T cell reactivity was demonstrated (Li et al., 2016). Multiple A *By demonstrating that patients with 1101 / EGFR(L858R) clinically responded to immunization with shared NeoAg peptides, this study provides a crucial first empirical concept in cancer vaccine research.

[0186] Our study also revealed that at least two other EGFR mutations can serve as immune sources in patients with NSCLC. Two additional clinical responses were associated with dominant vaccine-induced co4+ or cos+ T-cell responses to the H773L / V774M or T790M mutations, respectively (Figure 12A). T790M has a low prevalence in primary NSCLC but frequently manifests as a resistance mechanism to first-line EGFRi therapy (Kobayashi et al., 2005; Xu et al., 2017; El Nadi et al., 2018). T790M-containing LTSTVQLI M The peptide (SEQ ID NO: 65) was found in the only complete responder (Pt.17) in our study of CD8 + It is a dominant NeoAg target of T cells, and therefore HLA-C * This constitutes another promising potential shared target for approximately 7% of patients worldwide who express 1502.

[0187] In patients who responded, a non-EGFR NeoAg specific immune response was also detected, specifically CD8 against AQP12A (L28R) in Pt. 11 and FGFR1 (R734) in Pt. 22. + The T cell response was most pronounced. Although our study was not designed to directly compare the immunogenicity of EGFR NeoAg with that of other mutant genes, we observed that the dominance of immunoreactivity was focused on the mutant EGFR target, as evidenced by the discrepancies in IRC scores between patients in Group 1 and Groups 2 and 3 (Figure 12B). Since there is no reason to suspect that EGFR-derived NeoAg is inherently more immunogenic than those derived from other mutant proteins, this finding may be related to the use of EGFRi by patients in Groups 2 and 3, as will be further discussed below.

[0188] In addition, several non-responding patients developed PPV-induced T cell reactivity, including against mutant NeoAgs derived from 1DH2, MAP2K4, PIK3CA, TP53, and EGFR (746_750del). The present inventors hypothesize that the lack of clinical response in these patients may reflect a lack of NeoAg presentation by the patient's tumors for any number of potential reasons, including dysfunctional antigen processing, immune editing, HLA loss, or neoantigen promoter hypermethylation (McGranahan et al., 2017; Bedognetti et al., 2019; Rosenthal et al., 2019).

[0189] Since peptide-based cancer vaccine studies rarely report clinical responses after immunization, it is worth discussing the unique features of the authors' vaccination approach. CD4 + and CD8 + To activate both T cell-mediated immunities, we chose to immunize patients with a mixture of long and short NeoAg peptides, which often contain multiple peptides (up to 6) against the same target NeoAg (Table 5). The peptides were solubilized and administered in saline to avoid any inhibitory long-term antigen depot effect; vaccination was administered weekly to compensate for the typically short half-life of saline-solubilized peptides (Li et al., 2016; Overwijk et al., 2015).

[0190] Focusing on mutations called in a panel of 508 cancer-related genes, potential driver genes greatly simplified PPV design but also limited the number of NeoAg targets that could be identified per patient. This is a significant limitation of our study. However, this more focused approach made it possible to identify EGFR mutations as shared NeoAgs, which had promising therapeutic potential. As a vaccine adjuvant, topically applied Imiquimod Cream, a TLR7 agonist known to be moderately effective in activating local antigen-presenting cells, was used.

[0191] Based on the observed width, magnitude, and timing of the NeoAg-specific T cell response, our immunization approach appears to be most effective in boosting previously spontaneously primed T cell responses in patients, while its effectiveness in priming novel T cell responses is limited. Incorporating more potent and promising vaccine adjuvants (e.g., poly(I:C), anti-CD40, or STING agonists) into future peptide vaccine formulations may help address the lack of novel T cell priming. Nevertheless, PPV-mediated activation of existing immune responses was effective in inducing multiple clinical responses in our study. These findings strongly support NeoAg immunoreactivity prescreening, which will guide future vaccine designs for NSCLC patients (Malekzadeh et al., 2019).

[0192] EGFRi therapy did not affect the clinical response rate to PPV or the magnitude of the PPV-induced immune response, but it had a remarkably positive impact on patients' OS and PFS, despite previous failures as a monotherapy (Figure 1G; Figure 5B; Figure 6C; Figures 8A and 8B). Our mechanistic study suggests that the immunomodulatory effects of EGFRi may not only enhance the efficacy of cancer vaccines but also improve other T-cell-based immunotherapies such as checkpoint blockade or genetically engineered T-cell therapy.

[0193] However, these concepts need to be rigorously tested in future studies to confirm their effectiveness and usefulness. One of the more noteworthy findings from our study was that all seven PPV-responsive patients had tumors containing EGFR mutations, whereas none of the eight patients with EGFR-WT tumors responded to PPV. In light of the existing immune responses described above, we hypothesize that first-line EGFRi treatment may initially induce "immunogenic" tumor cell death, leading to spontaneous cross-priming of NeoAg-specific T cells (Pol et al., 2015; Goodridge et al., 2013), which is then followed by additional immunization with PPV immunization.

[0194] It remains unclear whether EGFR NeoAg-specific T cell priming is preferred over other tumor-associated NeoAgs. However, EGFRi drugs are known to irreversibly bind to mutated EGFR target proteins and may influence processing and subsequent NeoAg presentation by both APCs and tumor cells (Yamaoka et al., 2017). Further research is needed to clarify the precise role of EGFRi therapy in priming NeoAg-specific immune responses. It is important to note that some responding patients also developed specific immune responses to private NeoAgs that may have contributed to the observed clinical response (Figure 12).

[0195] While many significant challenges remain to be addressed in the identification of personalized NeoAg, these results provide the first evidence that shared NeoAg can constitute therapeutic vaccine targets capable of inducing immune responses and clinical outcomes in multiple cancer patients. HLA-A * Based on the prevalence of 1101 and L858R mutations, KITDFG R The AK NeoAg peptide (SEQ ID NO: 4) is estimated to be presented by up to 8.4% of Asians and up to 1.2% of North American NSCLC patients, making it one of the most widely shared NeoAgs in cancer (Kobayashi et al., 2016; Gonzalez-Galarza et al., 2015; Wirth and Kuhnel, 2017; Lu and Robbins, 2016).

[0196] The findings demonstrate that multiple other EGFR mutations may also exhibit immunogenicity in patients expressing specific HLA haplotypes, and that there is a natural antigen-presentation "synergy" between the most common EGFR mutations and the class I allotype HLA-A3 superfamily. These findings have important practical implications for future vaccine development and patient selection. Furthermore, since both tumor volume and pleural effusion were associated with worsened overall survival in PPV patients (Figures 10A and 10B), it is important to note that immunization of early NSCLC patients may be associated with better clinical outcomes, even before the development of EGFRi resistance. While the results of this phase 1b trial are promising, they need to be replicated and validated in relation to large-scale randomized vaccine trials. However, the presented data provide compelling rationale for initiating these trials.

[0197] B. Method Data report. Statistical methods were not used to predetermine the number of patient samples. This study was not randomized, and some researchers were not blinded during the experiment and outcome assessment.

[0198] Clinical trial design and procedures. A single-arm trial was conducted at Tianjin Beishen Hospital in China between November 2016 and December 2018. The trial protocol adhered to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the internationally recognized ChiCTR and the Tianjin Beishen Hospital Ethics Committee (Chinese Clinical Trial Registry (ChiCTR) No.: ChiCTR-IIR-16009867). The primary endpoints of this trial were safety and tolerability, and feasibility of an individualized approach. Secondary endpoints included antitumor immunoresponsiveness, clinical response, and potential survival effects. All patients submitted written informed consent prior to enrollment in this trial.

[0199] Patient eligibility. Twenty-four patients with stage III-IV NSCLC (18 with adenocarcinoma and 6 with squamous cell carcinoma) were enrolled in this clinical trial of a personalized neoantigen peptide vaccine (PPV), and immunization was successful. The 24 patients were selected according to the following selection criteria: Adult patients aged 18 years or older; all patients with stage III / IV NSCLC were classified according to the NCCN Clinical Oncology Guidelines, 3.2016 edition, non-small cell lung cancer, and 8th edition lung cancer staging classification. NSCLC diagnosis was confirmed by biopsy and pathological evaluation. Patients who had relapsed after failure of conventional treatment including surgery, chemotherapy, radiotherapy, and / or EGFR inhibitor (EGFRi) therapy, and who had not received aggressive treatment, showed a good or moderate Eastern Cooperative Oncology Group (ECOG) performance status (PS ≤ 3). Patients were not receiving other concurrent immunotherapy. Pre-treatment biopsy samples were available and showed at least one gene mutation. Patients had a life expectancy of more than 3 months.

[0200] The following patients were excluded: pregnant or lactating; patients with known or suspected autoimmune diseases or other immune system disorders; patients who had received systemic cytotoxic chemotherapy or investigational drugs for the treatment of metastatic NSCLC within 4 weeks prior to the administration of the personalized vaccine (excluding EGFRi); patients who had participated in a clinical trial involving other investigational drugs within 4 weeks prior to the first dose of the personalized vaccine; patients with hepatic or renal impairment, severe heart disease, coagulation disorders or hematopoietic disorders; patients with active infections requiring systemic treatment; and patients with other malignancies that are ongoing or have been treated within the past 5 years. Pre-administration tumor biopsy was required for the study, post-administration biopsy was optional, and additional consent was required for additional patients. The clinical characteristics of the study patients are shown in Figure 4A, and the EGFRi treatment history of 16 EGFR mutation patients is shown in Figure 4B.

[0201] Development of personalized neoantigen vaccines. Somatic Mutation Analysis: Tumor specimens from patients were obtained by fine-needle biopsy of tumor sites in the lungs or lymph nodes (Table 7). Tumor biopsies from individual patients were sequenced using a 508-gene panel in conjunction with standard clinical and pathological examination procedures at Tianjin Beijing Hospital (Tianjin, China). This genotyping panel was designed to detect shared, tumor-associated driver mutations (gene mutations associated with carcinogenesis and malignancy) within 508 cancer-related genes (Table 4). Tumor DNA was extracted from biopsy specimens according to the instructions for the TIANamp Genomic DNA Kit (TIANGEN, China) and profiled using hybridization-based exon capture followed by next-generation DNA sequencing and detected using Hiseq X-10 (Illumina, USA) (HengJia Medical Laboratory, Tianjin, China).

[0202] For somatic mutation calling, next-generation sequencing data from patient-derived tumors and corresponding PBMCs (as a source of normal germline DNA) were analyzed to identify specific coding sequence mutations, including single amino acid missense mutations and single, di, or trinucleotide mutations leading to small insertions / deletions (indels). Output from Illumina software was processed by Broad Picard Pipeline to obtain BAM files containing aligned reds with well-calibrated quality scores [bwa version 0.7.8, aligned to NCBI Human Reference Genome Build hg19].

[0203] Somatic single nucleotide variants (sSNVs), somatic small insertions, and deletions were all detected using Varscan2 (version 2.4.3). All indels (insertions and deletions) were manually reviewed using Integrative Genomics Viewer (version 2.4.1). All somatic mutations, insertions, and deletions were annotated using Annovar (version 2013-07-2811:32:41). Neoantigen peptides were selected based on nonsynonymous somatic mutations detected with a mutated variant allele frequency of 0.04 or higher.

[0204] HLA Typing: Peripheral blood samples were collected at enrollment for high-resolution HLA typing. Human leukocyte antigen (HLA) loci were typed using polymerase chain reaction-sequence-based typing (PCR-SBT) with a DNA amplification step (CapitalBio, China). In short, DNA was extracted from patients' peripheral blood according to the instructions for the Magic Beads DNA Extraction Kit (TANBead, China). Exons 2 and 3 of HLA class I genes (HLA-A, B, and C) and exon 2 of HLA class II α and β genes (HLA-DQ and DR) were amplified and purified, and the PCR products were sequenced using an ABI 3730XL DNA Analyzer (Applied Biosystems, USA). Sequence chromatograms were analyzed using ATF1.5 software (Conexio Genomics, Australia).

[0205] Vaccine peptide selection: Due to the large number of somatic mutations typically found in lung cancer, and the fact that the majority constitute individual “passenger” mutations, we chose to target somatic mutations detected from a focused panel of 508 tumor-associated genes. The rationale for this approach was a combination of two factors: (1) it would enable the targeting of mutations that are likely essential to the tumor phenotype and therefore less likely to be lost by immunoediting; and (2) it would increase the opportunity to identify common neoantigen targets that could potentially be beneficial to multiple NSCLC patients.

[0206] Non-synonymous coding mutations detected by a 508-gene panel were translated in silico, and the resulting neoantigen sequences were evaluated for their predicted binding affinity to patient HLA class I and class II molecules according to the HLA-peptide prediction algorithms NetMHC4.0, NetMHCpan3.0, NetMHCpan4.0, NetMHCII2.2, and NetMHCII2.3 (Andretta et al., 2015; Jensen et al., 2018). Immune-treated neoantigen peptides were selected primarily based on their highest predicted binding affinity to patient HLA class I and class II molecules. However, the vaccines were also designed to maximize the number of different HLA molecules involved and, where possible, minimize intra-HLA peptide competition.

[0207] Certain biochemical properties that could negatively affect the synthesizability or solubility of immunizing peptides (e.g., high hydrophobicity or the presence of multiple cysteines) were also considered. Furthermore, the aim was to design individual patient vaccines containing short / long vaccine peptides in a ratio of approximately 2:1, or as close as that to somatic mutation profiling and HLA / peptide bond prediction. For each patient, up to 14 peptides ranging from 9 to 17 amino acids in length, resulting from up to 12 independent mutations, were selected and prioritized. An average of 9.4 neoantigen peptides per patient were selected for peptide synthesis, including an average of 6.5 short HLA class I-limited peptides and 2.9 length HLA class II-limited peptides (Table 5).

[0208] Patient immunization: Immunopeptides were synthesized using standard solid-phase peptide chemistry, purified to >98% using reverse-phase high-performance liquid chromatography, and tested for sterility and endotoxin presence to ensure safety and tolerability using methodologies consistent with Good Manufacturing Practice [HengJia Neoantigen Biotechnology (Tianjin) Co., Ltd.]. As shown in Table 5, 5 to 14 peptides were synthesized per patient, individually solubilized in sterile phosphate-buffered saline (PBS), and mixed into two separate peptide cocktails, each containing 2 to 7 short peptides and 1 to 3 long peptides in a total volume of 1 ml.

[0209] Peptides that bind to the same HLA allotype were separated into different cocktails to reduce potential antigen competition. Patients received 200 μg of each peptide per immunization, administered subcutaneously to both limbs once a week for a minimum of 12 weeks. To provide simultaneous Toll-like receptor (TLR)-7 stimulation of pAPC, Aldara cream containing 5% imiquimod was applied topically to the vaccine site as a vaccine adjuvant immediately after administration of the peptide cocktail. Patients were permitted to continue immunization beyond 12 weeks if desired, in accordance with the patient's best interests. As shown in Figure 2 and Table 5, 11 of the 24 patients continued vaccination beyond 12 weeks.

[0210] Sample collection: Continuous peripheral blood mononuclear cell (PBMC) samples were collected before treatment and at 4, 8, and 12 weeks after vaccination. For patients with advanced cancer, a maximum of 15 ml of blood was collected per month, according to the regulations of Tianjin Beichen Hospital. Additional blood samples beyond the 12-week trial period were optional and required additional patient consent. Additional blood samples were collected from patients 5, 8, and 17 who all experienced an objective clinical response.

[0211] Live PBMCs were collected and stored at -80°C. While tumor biopsy after vaccination was optional, it was not required in this study due to the invasive nature of the procedure, the uncertainty of its feasibility, and the general reluctance of most patients. Tumor tissue obtained for further RNA sequencing and / or bulk T cell receptor VB CDR3 sequencing analysis was collected from PPV study patients 5, 12, 16, 23, and 24 after providing written informed consent.

[0212] Criteria for evaluating tumor reduction effectiveness: Objective evaluation of tumor reduction was performed according to the Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) guidelines. Computed tomography (CT) and / or magnetic resonance imaging (MRI) scans were used to measure selected target lesions. Patients were required to undergo at least one pre-treatment scan for baseline measurement and another scan 3–4 months after vaccination for response evaluation. Where possible, additional patient scans were performed monthly for the first 12 weeks after vaccination.

[0213] Target lesions with a minimum size of 10 mm (15 mm for malignant lymph nodes) were measured by three different radiologists at their longest diameter, and the average of three independent measurements was used for clinical evaluation. A maximum of two target lesions were measured per organ, and the two largest lesions were selected, for a total of up to five lesions. Tumor volume was calculated as the sum of the diameters of all target lesions (Figure 5B).

[0214] Clinical efficacy was evaluated as follows: CR: complete disappearance of all target lesions; PR: partial response defined as a 30% reduction in the total diameter of target lesions; PD: progressive disease defined as a minimum 20% increase in the total diameter of target lesions or the appearance of new lesions; SD: stable disease defined as a change in tumor volume insufficient to qualify for PR or PD, or PD. Clinical efficacy was evaluated 3–4 months after the initial immunization date.

[0215] Statistical design for clinical trials: Statistical analysis was primarily descriptive, listing the number of patients who experienced adverse events. All statistical tests were two-tailed, with an alpha level of 0.05. Confidence intervals were constructed at a significance level of 0.05. Further exploratory analyses of the data were performed where deemed appropriate.

[0216] Analysis of primary outcome measures: Treatment-related adverse events were classified and graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events (version 4.0) and analyzed based on these classifications. Safety assessments were performed from each vaccination day for the first 12 weeks of vaccine treatment and for 48 hours thereafter for continued vaccination beyond 12 weeks. For patients with extended follow-up periods, safety assessments were performed every two months. Disease-related symptoms observed at baseline (before treatment) were not reported unless they worsened after vaccination.

[0217] Analysis of secondary outcome measures: Secondary endpoints, including progression-free survival (PFS) and overall survival (OS), were aggregated using the Kaplan-Meier method. Immune response measurements via ELISA, ELISPOT, and tetramer analysis were descriptively summarized before the initial vaccination and every four weeks after each vaccination. PFS and OS for enrolled subjects were calculated from the registration date to disease progression and / or death, or to December 31, 2018, respectively. Subgroup analysis: Based on differing response and survival profiles, enrolled patients were analyzed in subgroups based on wild-type (WT) or mutant (Mut) EGFR mutation status. The analysis then considered whether EGFR inhibitor use was continued or discontinued before immunization initiation. These groups were defined as EGFR WT-PPV only (Group 1), EGFR Mut-PPV only (Group 2), and EGFR Mut-PPV + EGFRi (Group 3).

[0218] Enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunosorbent assay (ELISPOT): Peripheral blood mononuclear cells (PBMCs) collected at different time points before the initiation of immunization and after PPV were isolated by Ficoll density gradient centrifugation and counted in the presence of trypan blue dye to assess viability before cryopreservation. For ELISA analysis, 5 × 10⁶ cells in RPMI-1640 containing 10% FBS were used. 5 PBMCs were added to each well of a 96-well plate in a total volume of 250 μl. The PBMCs were incubated with 300 IU / ml interleukin (IL-2) in the presence of a vaccine peptide pool, individual vaccine peptides, or an unrelated control peptide (7.5 μg / ml) for 5 days in a humidified incubator at 37°C with 5% CO2.

[0219] 24 hours after peptide restimulation, IFN-γ concentrations in the cell supernatant were measured using a human IFN-γ ELISA kit (Dakewe, China) according to the manufacturer's instructions. IFN-γ secretion levels greater than 1.5 times the background signal of the non-peptide control were considered positive immune responses. Immune Response ComboScores (IRCs), considering the breadth, intensity, and duration of the vaccine-induced immune response, were calculated for each patient as follows: ComboScore = Sum of multiplier changes at all time points (pre-treatment, 4 weeks, 8 weeks, 12 weeks) > 1.5 for all vaccine peptides.

[0220] For the ELISpot assay, 2.5 × 10 5 PBMCs were prepared in RPMI-1640 containing 0.5% FBS, with a total volume of 150 μL per well in a 96-well plate. Ex vivo PBMCs were stimulated in triplicate with individual vaccine peptides at a final concentration of 10 μg / ml, and the plates were incubated in a humidified incubator containing 5% CO2 at 37°C for 36 hours. Spot detection was performed using the Human IFN-γ ELISpot PRO kit (MABTECH Inc., USA). 6 The data was normalized to the number of IFN-γ spots detected per PBMC.

[0221] Tetramer staining and flow cytometry statistical analysis: Selected custom phycoerythrin (PE) or APC-conjugated HLA peptide tetramers (Baylor College of Medicine, USA; MBL, Japan) were successfully generated (Figure 13A). PBMCs were thawed and resuspended in RPMI-1640 containing 0.5% fetal bovine serum (FBS). 100 μL of fluorophore-conjugated HLA / peptide tetramer-containing PBS-1% BSA (1:50 dilution) was added to 5×10 5 PBMCs, and the mixture was incubated for 20 minutes at room temperature in the dark. Cells were washed with PBS-1% BSA, stained with FITC or PE-conjugated anti-CD8 mAb (Biolegend, USA, 1:200 dilution), and incubated for 15 minutes. The cells were then washed and resuspended in 400 μL of PBS-1% BSA for flow cytometry analysis (LSRFortessa X-20 Analyzer).

[0222] Tumor RNA sequencing and bulk T cell receptor Vβ CDR3 sequencing analysis: After obtaining patient consent, post-vaccination tumor samples were collected from patients 12, 16, 23 and 24. RNA was isolated from the patient's tumor tissue using an RNA extraction kit (Qiagen, USA). The library was prepared using NEBNext (登録商標) Ultra TM RNA Library Prep Kit (Illumina, USA) according to the manufacturer's instructions. The library was purified with the AMPure XP system (Beckman Coulter, Germany), and the samples were sequenced using the Illumina NovaSeq platform (Illumina, USA).

[0223] For T-cell receptor (TCR) diversity analysis, DNA samples were extracted from patient PBMCs or pre- and post-treatment tumor biopsies from Patient 5 using a DNA extraction kit (Qiagen, USA), followed by library construction with two rounds of PCR-based amplification. CDR3 fragments were first amplified using specific primers for each V and J gene, and then the target fragments of the multiplexed PCR products were purified using magnetic beads (A63882, Beckman, Germany).

[0224] Next, PCR was performed using universal primers, and target fragments of 200-350 bp were searched for and purified using the QIAquick Gel Purification Kit (Qiagen, USA). The PCR products were then sequenced using the Illumina X10 platform. After removing the single-read CDR3 sequence, the remaining sequences were analyzed to evaluate the TCRVβ IMGT clonality of patients before and after treatment, as previously described (Tumeh et al., 2014).

[0225] Sequencing of single-cell T cell receptors: HLA-A derived from PBMC after PPV in patient 5 * 1101 / KITDFG R AK (Sequence No. 4) Tetramer + CD8 + T cells were sorted using a flow-based cell sorter (BD FACSAria III, USA) and imaged using a confocal microscope (Leica SP8, Germany). + Cells are divided into 1 × 10 cells per 1 ml of PBS. 6 Cells were prepared, loaded into a Chromium Single Cell Controller (10X Genomics, USA), and single-cell gel beads (GEMs) were generated in emulsion using the Single Cell 5' Library and Gel Bead Kit (10X Genomics, USA).

[0226] Captured cells were lysed, and the released RNA was barcoded by reverse transcription to generate barcode-containing cDNA in individual GEMs according to the manufacturer's instructions. V(D)J sequences were enriched by nested PCR amplification using specific primers targeting conserved TCR sequences. Sequence determination was performed on an Illumina NovaSeq platform with 150 bp (PE150) paired ends. V(D)J recombination, T cell diversity, and appropriate TCRα and β chain sequence pairing for each individual T cell were analyzed using Cellranger VDJ. + Single-cell sequencing of the cells identified 639 different TCRα / β pairs.

[0227] However, since tetramer-based cell sorting can result in contamination by non-antigen-specific T cells, we selected TCR clones that detected a minimum of 10 Vβ-CDR3 reads, resulting in 51 unique TCR clones. Four clones with Vβ-CDR3 sequences that showed little to no presence in PBMC or TIL samples were excluded from the analysis. Five further TCR clones with 7 or 8 CDR3 reads were then included based on their increasing frequency in PBMCs and / or TILs during PPV, as expected for neoantigen-specific T cell clones.

[0228] TCR cloning and validation: Variable region sequences of TCR Vα-N1 and Vβ-N1 chains, obtained by single-cell sequencing, were fused to a manipulated human constant region to enhance α- and β-chain pairing (Cohen et al., 2007). These modified Vα and Vβ chain sequences were synthesized and inserted into the lentiviral expression vector pCDH based on the EF1a promoter to create the lentivirus lenti-EF1a-TCR-N1. Healthy donor PBMCs were prepared using lymphocyte isolation medium (Stem Cell, Canada).

[0229] Dynabeads (登録商標)T cells were isolated using the Human T-Expander CD3 / CD28 kit (Thermofisher, USA), mixed with 3 ml of X-Vivo15 serum-free medium (Lonza, Switzerland), and cultured at 37°C in 5% CO2 for 48 hours. The cell density was reduced to 1 × 10⁶. 6 The packaged lentivirus lenti-EF1a-TCR-N1 was co-cultured with the solution adjusted to / mL at 37°C in 5% CO2 for 4 days. TCR-N1 expression and antigen specificity were compared with HLA-A * 1101 / KITDFG R This was confirmed by staining with the AK (sequence number 4) tetramer.

[0230] HLA-A via IRES sequence * A minigene encoding the KITDFGRAK peptide (SEQ ID NO: 4) linked to 1101 cDNA was synthesized and inserted into a lentiviral vector pCDH containing the EF1A promoter to construct the lentivirus lenti-EF1a-KIT-A11. The control lentiviral construct was KITDFG R AK (Sequence ID 4) minigene or HLA-A * The vectors contained vectors expressing one of the 1101 genes individually. Lentiviral transducer A549 lung cancer cells that stably express the target gene were selected through prinomycin-based selection.

[0231] HLA-A * Surface expression of 1101, A * The cells were stained with 1101-specific mAbs for confirmation, followed by flow cytometry analysis. Manipulated TCR-N1-T cells were co-cultured with parental A549, A549-A11, A549-KIT, or A549-A11.KIT cells (20,000 target cells per well) at effector-to-target ratios of 1:1, 2.5:1, 5:1, and 10:1 at 37°C in 5% CO2. Non-transduced, proliferating T cells from the same PBMC donor were used as controls. The supernatant was collected 24 hours after co-culture, and the level of IFN-γ secretion was evaluated by ELISA.

[0232] Immunoprecipitation and Western blot: Lung cancer cell lines H1975 (EGFR-L85R / T790M) and H1299 (EGFR-WT) (ATCC, USA) were treated with EGFRi osimertinib (LC Laboratories, USA) at different concentrations (0.1, 1, 2, and 5 μM) for different durations to optimize EGFRi treatment conditions. Cells were washed with cold PBS and lysed using lysis buffer (1% TritonX-100, Sigma, USA) and Halt Protease Inhibitor Cocktail 100X and 0.5 μM EDTA 100X (Thermo Scientific, USA).

[0233] The lysates were collected and centrifuged at high speed for 30 minutes at 4°C before measuring protein concentration with the Bradford assay kit (BioRad, USA). Preclearing was performed using 10 μL of Pierce Protein A / G Ultra Link resin (Thermo Scientific) per sample, and the mixture was incubated at 4°C for 2 hours. Immunoprecipitation was performed by using equal amounts of total protein and incubating the cell lysates with the following antibodies at a 500:1 dilution for 18 hours at 4°C: anti-phospho-EGFR (EMD Millipore), anti-EGFR, anti-p44 / 42 MAPK ERK1 / 2, anti-phospho-p44 / 42 MAPK ERK1 / 2, and anti-GAPDH (Cell Signaling Technology).

[0234] Protein A / G crosslinked beads were added and incubated for 4 hours before washing with cold PBS. The sample was flowed using an SDS-PAGE gradient (8-16%) gel (Invitrogen, USA). The protein was transferred to a PVDF membrane (Thermo Scientific, USA), the blot was blocked with 5% milk, and then incubated overnight with a specific antibody at a concentration of 1:1000. The blots were washed and incubated with peroxidase-conjugated anti-rabbit secondary antibody (1:1000) (Jackson Immuno Research, USA). The blots were developed using Super Signal West Pico PLUS Chemiluminescent enhanced horseradish peroxidase substrate (Thermo Scientific, USA) and visualized on X-ray film.

[0235] RNA sequencing of lung tumor cell lines: H1975 and H1299 lung cancer cells were treated with 1 μM EGFRi Osimertinib (LC Laboratories, USA) for 0, 12, or 24 hours; or 24 hours, followed by 1 μM EGFRi Osimertinib (LC Laboratories, USA) + 500 U / mL recombinant human IFNγ (R&D Systems, USA) for the last 12 hours of culture. The cells were lysed, and total RNA was prepared using the RNeasy Mini Kit (Qiagen, USA) according to the manufacturer's protocol. RNA sequencing was performed by the Avera Institute for Human Genetics (South Dakota, USA) as follows: Total RNA was evaluated for degradation on an RNA 6000 Nano chip run on a 2100 Bioanalyzer (Agilent, USA), with a mean RNA integrity score of 9.7 for the sample set.

[0236] The sequencing library was prepared using the TruSeq Stranded Total RNA Library Prep Kit (Illumina, Inc., USA) following a low sample procedure. Briefly, ribosomal RNA (rRNA) was depleted from the total RNA, the remaining RNA was purified, appropriately fragmented, and primed for cDNA synthesis. Blunt-end cDNAs were generated after the synthesis of the first and second strands. Adapter ligation was performed before enriching the cDNA fragments, following adenylation of the 3' blunt end. Quality control of the final library was performed by evaluating fragment size on a DNA1000 chip operated with a 2100 BioAnalyzer (Agilent, USA).

[0237] The average library yielded an insert bp size of 326. The concentration of each library was determined prior to sequencing by quantitative PCR (qPCR) using the KAPA Library Quantification Kit for Next-Generation Sequencing (KAPA Biosystems, USA). The libraries were normalized to 2 nM in 10 mM Tris-Cl (pH 8.5) containing 0.1% Tween 20 and then homogeneously pooled. The pooled libraries were denatured with 0.05 M NaOH and diluted to 20 pM. Cluster generation of the denatured libraries was performed using the HiSeq PE Cluster Kit v2 chemistry and flow cell according to the manufacturer's specifications (Illumina, Inc, USA).

[0238] The libraries were appropriately clustered with 1% PhiX spike-in. Sequencing-by-synthesis (SBS) was performed on a HiSeq2500 using v2 chemistry, reading paired 101bp ends, resulting in an average of 52.4 million paired end reads per sample. The sequence reading data was processed and converted to FASTQ format for downstream analysis using Illumina BaseSpace analysis software, FASTQ Generation v1.0.0.

[0239] RNA sequence data analysis: Quality control of patient and cell line RNAseq data was performed using FastQC v0.11.5, FastQ Screen v0.11.1, RSeQC v3.0.0, MultiQC v1.6, and a proprietary algorithm of the BostonGene platform (Wingett et al., 2018). Four patient samples with acceptable quality (good phred score, good sequencing content per base along read length, code read >50 M, adapter content <15%, <5% microbial / mouse-specific genome contamination) were selected for further analysis. RNAseq reads were pseudo-aligned to GENCODE v23 transcripts using Kalisto v0.42.4 (Bray et al., 2016). Transcripts with the transcription type [protein_coding,IG_C_gene, IG_D_gene, IG_J_gene, IG_V_gene, TR_C_gene, TR_V_gene, TR_D_gene, TR_J_gene] were selected, and then non-coding RNA transcripts and histone and mitochondrial transcripts were removed to obtain 20,062 protein-coding genes.

[0240] Gene expression was quantified as transcripts per million cells (TPM) and transformed log2 (Conesa et al., 2016). Changes in gene expression in EGFRi-treated cell lines were shown as relative (log) expression normalized to untreated control cells. Patient tumor gene expression was centered around the median value within the four patient samples, and gene expression relative to the median value of the control shown on the heatmap was observed. Seven pathway activity scores were calculated using PROGENy v1.4.1 (EGFR, MAPK, PI3K, TRAIL, TNFa, NFkB, JAK-STAT; Schubert et al., 2018). Other pathway signature scores were calculated using ssGSEA with our own Python implementation.

[0241] Pathway activity was represented as gene signatures downloaded from mSIGdB v6.2 (Subramanian et al., 2005) unless otherwise specified: "Cell cycle signature" - HALLMARK_G2M_CHECKPOINT, "Apoptosis signature" - HALLMARK_APOPTOSIS, MYCHALLMARK_MYC_TARGETS_V2, "EMT signature" - 788 - HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION, "IFN-0 signature" - HALLMARK_INTERFERON_GAMMA_RESPONSE, "HLA expression" - gene set (HLA-A, HLA-B, HLA-C).

[0242] The difference in pathway scores compared to the control was normalized separately for each pathway against its maximum value and displayed on a line plot. For a schematic representation, the maximum absolute deviation of the change in pathway activity score from the control point (0 hours) to 24 hours was calculated within each pathway. The pathway colorants on the schema correspond to the percentage of the maximum absolute deviation. Cell deconvolution was performed from RNAseq data using the quanTIseq approach (Finotello et al., 2018). Heatmaps, point plots, line plots, and bar plots were created using the pandas v0.23.4, matplotlib v2.1.1, and seaborn v0.9.0 Python packages (Liang et al., 2016).

[0243] Luminex assay: Duplicate samples of supernatant from untreated and 1 μM EGFRi-treated H1975 and H1299 cell lines were analyzed for the presence of CCL2, CXCL1, CXCL2, CXCL8, IP10, IL1RA, IL6, VEGFA, CSF2, and CSF3 proteins using a custom Luminex kit according to the manufacturer's instructions (R&D Systems, USA). 50 microliters of test supernatant and an appropriate microparticle cocktail were added to each well (1:10 dilution) and incubated in a microplate shaker at room temperature for 2 hours. The plates were washed three times, 50 μl of biotin-antibody cocktail (1:10 dilution) was added, incubated for 1 hour, and then washed three times.

[0244] After incubation with 50 μl of streptavidin (1:25 dilution) for 30 minutes, the plate was washed three times, the microparticles were resuspended in buffer, and the plate was read using a Luminex plate reader. EGFRi-induced changes were expressed as a multiplier increase or decrease compared to the measured baseline (0 hour) concentration. If the measured concentration fell below the detection level, the manufacturer's minimum detection threshold was used.

[0245] T cell function assay: T cell migration: H1975 cells were treated with 1 μM EGFRi Osimertinib (LC Laboratories, USA) for 24 hours. DMSO containing only the culture medium was used as a control. The cells were then washed and incubated in ImmunoCulut-XF T cell proliferation medium (Stem Cell Technology, Canada) for 24 hours. The cell supernatant was then collected and filtered using a Millex-GS filter. Healthy donor PBMCs or proliferating melanoma CD8 cells were used. + Tumor-infiltrating lymphocytes (TIL 3311 and TIL 3329) were thawed approximately 16 hours before performing the migration assay.

[0246] Place 650 μL of H1975 cell supernatant at the bottom of a Transwell plate (Corning, USA), and add 3 × 10⁶ cells to the upper well.5 Each healthy donor PBMC was incubated with the cells for 6 hours. Migratory cells were collected from the bottom of the well, stained with CD4, CD8, or CD14 (Biolegend) at 4°C for 30 minutes, washed with PBS, and fixed with 4% PFA. 50 μl of counting beads was added to each sample to obtain accurate cell counts. Samples were flow-through a Canto II flow cytometer and analyzed using a FlowJo V10.

[0247] HLA class I quantification and T cell antigen recognition: H1299 and H1975 cells were treated with 1 μM EGFRi Osimertinib (LC Laboratories) or DMSO control for 6 or 20 hours. Cells were harvested, stained for all class I (W6 / 32-APC, Thermo-Fisher, USA), washed, and fixed in 4% PFA. Samples were flow-through a Canto II flow cytometer and analyzed using FlowJo V10. H1975 cells were seeded at 50,000 cells per well in a 96-well plate and treated with EGFRi at concentrations of 0, 0.1, and 0.3 μM per well for 24 hours.

[0248] Next, H1975 cells were treated with 0, 10, or 100 nM of cognate HLA-A 100 nM one hour before washing. * Pulsation was performed with the 0101 limited VGLL1 peptide LSELETPGKY (SEQ ID NO: 978). Subsequently, VGL1 peptide antigen-specific CD8 + T cells were added in a 1:1 effector / target cell ratio and co-cultured overnight. IFN-γ in the cell supernatant after 24 hours was analyzed using a human IFN-γ ELISA kit (Invitrogen, USA), and the plates were scanned using SpectraMax. (登録商標) Read using an M5 / M5e multimode plate reader.

[0249] Statistical analysis: All statistical analyses were performed using GraphPad Prism 5 (GraphPad Software Inc., LaJolla, CA). Survival curves and survival rates were calculated using the Log-rank (Mantel-Cox) test, and overall survival rates were measured from the registration date to December 31, 2018, or until death. Two-tailed student's t-tests or Mann-Whitney U tests were used to analyze statistical significance between groups. A p-value of 0.05 or less was used as the threshold for determining statistical significance.

[0250] Data availability: Raw data of bulk RNA sequences, single-cell TCR vs. αβ chain sequences, and TCR VβCDR3 sequences of patients' tumors and cell lines, generated and analyzed during the current study, are available through the NCBI Sequence Read Archive (SRA) (code SRP188005). DNA sequencing data that may identify patients (for mutation detection) are not included in the informed consent of enrolled patients, and data that does not identify information can be shared upon reasonable request. All other data are available from the corresponding authors upon reasonable request.

[0251] All methods disclosed and claimed herein can be prepared and performed in light of this disclosure without excessive experimentation. While the compositions and methods of the present invention have been described in relation to preferred embodiments, it will be apparent to those skilled in the art that modifications can be applied to the methods and steps or order of steps of the methods herein without departing from the concept, spirit and scope of the invention.

[0252] More specifically, it will be understood that the same or similar results may be achieved by substituting the agents described herein with certain chemically and physiologically relevant agents. All such similar substitutions and modifications that are obvious to those skilled in the art are considered to fall within the spirit, scope and concept of the invention as defined by the appended claims.

[0253] References The following references are inserted herein by reference, to the extent that they supplement the content disclosed herein in an illustrative or otherwise detailed manner. Andreatta and Nielsen, Bioinformatics, 32:511-517, 2015. Bedognetti et al., J. Immunother.Cancer, 7:131, 2019. Bradley et al., Cancer. Immunol. Res., 3:602-609, 2015. Bray et al., Nat. Biotechnol., 34:525-527, 2016. Cafri et al., Nat. Commun., 10:449, 2019. Carreno et al., Science, 348:803-808, 2015. Chen et al., J. Clin. Invest., 129(5):2056-2070, 2019. Cohen et al., Cancer. Res., 67:3898-3903, 2007. Conesa et al., Genome. Biol., 17:13-31, 2016. El Kadi et al., Cancer Res., 78:6728-6735, 2018. Finn and Rammensee, Cold Spring Harb.Perspect. Biol., 10:a028829, 2018. Finotello and Trajanoski, Cancer. Immunol. Immun., 67:1031-1040, 2018. Frederick et al., Clin. Cancer. Res., 19:1225-1231, 2013. Galon and Bruni, Nat. Rev. Drug. Discov.,18:197-218, 2019. Gonzalez-Galarza et al., Nucleic Acids Res., 43:D784-788, 2015. Goodridge et al., J. Immunol., 191:1567-1577, 2013. Griffin et al., Oncotarget, 8:78174-78192, 2017. Hailemichael et al., Nat. Med., 19:465-472, 2013. Harjanto et al., PLoS One,9:e86655, 2014. Hao et al., Nat. Genet., 48:1500-1507, 2016. Hilf et al., Nature, 565:240-245, 2019. Hu et al., Nat. Rev. Immunol., 18:168-182, 2018. Iiizumi et al., Cancers, 11:266-279, 2019. Im et al., PLoS One,11:e0160004, 2016. Jemal et al., CA. Cancer J. Clin., 61:69-90, 2011. Jensen et al., Immunology, 154:394-406, 2018. Jia et al., Int. J. Cancer. 145(5):1432-1444, 2019. Keenan et al., Nat. Med., 25:389-402, 2019. Keskin et al., Nature, 565, 234-239, 2019. Khalili et al., Clin. Cancer. Res., 1:5329-5340, 2012. Knight et al., J. Clin. Invest., 123:1371-1381, 2013. Kobayashi et al., Cancer Sci., 107:1179-1186, 2016. Kobayashi et al., N. Engl. J. Med., 352:786-792, 2005. Kreiter et al., Nature, 520:692-696, 2015. Lawrence et al., Nature, 499:214-218, 2013. Levine et al., Cancer Cell, 35:10-15, 2019. Li and Gillanders, Ann. Oncol., 28:xii11-xii17, 2017. Li et al., Oncoimmunology, 5:e1238539, 2016. Liang et al., Ieee. Acm. T.Comput. Bi., 13:549-556, 2016. Liu and Mardis, Cell, 168:600-612, 2017. Lu and Robbins, Semin. Immunol., 28:22-27, 2016. Malekzadeh et al., J. Clin. Invest., 129:1109-1114,2019. McGranahan et al., Cell, 171:1259-1271, 2017. Morgensztern et al., J. Thorac. Oncol., 7:1485-1489, 2012a. Morgensztern et al., J. Thorac. Oncol., 7:1479-1484, 2012b. Munn and Bronte, Curr. Opin.Immunol., 39:1-6, 2016. Ott et al., Nature, 13:217-221, 2017. Overwijk, Nat. Med., 21:12-14, 2015. Parker et al., Adv. Cancer. Res., 128:95-139, 2015. Pol et al., Oncoimmunology, 4:e974411, 2015. Rizvi et al., Science, 348:124-128, 2015. Robinson et al., Nucleic. Acids Res., 43:D423-D431, 2014. Rosenthal et al., Nature, 567:479-485, 2019. Sahin et al., Nature, 547:222-226, 2017. Schoenborn and Wilson, Adv. Immunol., 96:41-101, 2007. Schroder et al., J. Leukoc. Biol., 75:163-189, 2004. Schubert et al., Nat. Commun., 9:20-30, 2018. Schumacher and Schreiber, Science, 348:69-74, 2015. Sidney et al., BMC Immunol., 22(9):12, 2008. Siegel et al., CA: Cancer J. Clin., 67:7-30, 2016. Subramanian et al., Proc. Natl. Acad. Sci., 102:15545-15550, 2005. Sumitro et al., PLoS One,27:e86655, 2014. Tran et al., Science, 344:641–645; Tumeh et al., Nature, 515:568–571, ​​2014. Venugopalan et al., Oncotarget,23:54137-54156. Wang et al., Mol. Cancer, 17:168, 2018. Watanabe et al., Cancer. Sci., 110:52–60. Wilmott et al., Clin. Cancer. Res., 18:1386–1394. Wingett and Andrews, F1000Res., 7:1338–1350, 2018. Wirth and Kuhnel, Front. Immunol., 8:1848; Xu et al., Oncotarget, 8:90557–90578, 2017. Yamaoka et al., Int. J. Mol. Sci., 18:E2420, Yossef et al., JCI Insight, 3:122647, 2018.

Claims

1. An immunogenic composition comprising at least first and second HLA-binding peptides derived from EGFR, wherein each of the first and second HLA-binding peptides comprises a mutant amino acid sequence corresponding to a mutation in human EGFR mutant cancer, relative to wild-type human EGFR. The first HLA-binding peptide derived from EGFR binds to HLA class I molecules, The second HLA-binding peptide derived from EGFR binds to HLA class II molecules, The first HLA-binding peptide derived from EGFR comprises at least one HLA-binding peptide containing the L858R mutation, The second HLA-binding peptide derived from EGFR comprises at least one HLA-binding peptide containing the L858R mutation, At least one HLA-binding peptide containing the L858R mutation of the first HLA-binding peptide derived from EGFR comprises an amino acid sequence having 100% identity with any one of SEQ ID NOs: 1 to 18. At least one HLA-binding peptide containing the L858R mutation of the second HLA-binding peptide derived from EGFR contains an amino acid sequence that is 100% identical to any one of SEQ ID NOs. 665 to 685. Immunogenic composition.

2. The composition according to claim 1, wherein the HLA-binding peptide is formulated in a pharmaceutically acceptable carrier.

3. The composition according to claim 2, wherein the pharmaceutically acceptable carrier is an aqueous carrier, a salt solution, physiological saline, and / or isotonic physiological saline.

4. The composition according to any one of claims 1 to 3, wherein the HLA class I bound peptide has an amino acid length of 8, 9, 10, 11, 12, or 13, or an amino acid length of 9, 10, or 11.

5. The composition according to any one of claims 1 to 4, wherein the HLA class II bound peptide is 13 to 30 amino acids long or 15 to 23 amino acids long.

6. The composition according to any one of claims 1 to 5, comprising a plurality of HLA-binding peptides derived from EGFR, wherein each HLA-binding peptide contains a mutant amino acid sequence relative to wild-type human EGFR that matches an EGFR mutation in human EGFR-mutated cancer.

7. The composition according to claim 6, comprising 2 to 30 different HLA-binding peptides for the target.

8. The composition according to claim 6 or 7, comprising at least two HLA class I binding peptides and at least one HLA class II binding peptide.

9. The composition according to claim 6 or 7, comprising at least one HLA class I binding peptide and at least two HLA class II binding peptides.

10. The composition according to any one of claims 7 to 9, comprising at least two HLA class I binding peptides and at least two HLA class II binding peptides, or comprising at least three HLA class I binding peptides and at least three HLA class II binding peptides.

11. The composition according to any one of claims 1 to 10, further comprising an EGFR inhibitor.

12. The composition according to any one of claims 1 to 11, further comprising a TLR ligand and / or a TLR-7 agonist.

13. The composition according to claim 12, wherein the TLR7 agonist is CL075, CL097, CL264, CL307, GS-9620, poly(dT), imiquimod, gardiquimod, regiquimod (R848), loxoribine, or an ssRNA oligonucleotide, or imiquimod.

14. The composition according to any one of claims 1 to 13, wherein the HLA-binding peptide is in complex with an HLA class I and / or HLA class II molecule.

15. The composition according to any one of claims 1 to 14, further comprising antigen-presenting cells.

16. The composition according to claim 15, wherein the antigen-presenting cells include dendritic cells.

17. The composition according to any one of claims 1 to 16, wherein the HLA-binding peptide is contained in liposomes, lipid-containing nanoparticles, or a lipid-based carrier.

18. The composition according to any one of claims 1 to 17, further comprising an adjuvant component.

19. A drug for treating a subject, comprising administering an effective amount of the composition described in any one of claims 1 to 18 to the subject, wherein the subject has EGFR-mutated cancer, and each EGFR-derived HLA-binding peptide in the composition contains a mutation that matches that of the EGFR-mutated cancer in the subject.

20. The agent according to claim 19, further comprising sequencing the EGFR gene in the target cancer.

21. The agent according to claim 19 or 20, wherein the target HLA genotype has been determined.

22. The agent according to claim 21, wherein the EGFR-derived HLA-binding peptide is expected to bind to HLA class I or HLA class II supported by the target.

23. The agent according to claim 21 or 22, wherein the HLA-binding peptide contained in the composition is selected using an algorithm that predicts HLA binding to the HLA type expressed in the target.

24. The agent according to any one of claims 21 to 23, wherein the HLA-binding peptide contained in the composition is selected based on predicted HLA affinity and / or predicted HLA ranking, or the peptide contained in the composition is selected based on predicted HLA affinity and predicted HLA ranking.

25. The agent according to claim 23, wherein the algorithm is selected from NetMHC4.0;NetMHCpan3.0, NetMHCpan4.0, NetMHCII2.2 and / or NetMHCII2.

3.

26. The agent according to any one of claims 19 to 25, further comprising administering it to an EGFR inhibitor.

27. The agent according to claim 26, wherein the EGFR inhibitor is administered before the HLA-binding peptide derived from mutant EGFR.

28. The agent according to claim 26, wherein the EGFR inhibitor is administered after or essentially simultaneously with the HLA-binding peptide derived from mutant EGFR.

29. The agent according to any one of claims 26 to 28, wherein the EGFR inhibitor is an EGFR-binding antibody and / or a tyrosine kinase inhibitor.

30. The agent according to any one of claims 26 to 29, wherein the EGFR inhibitor is osimertinib, erlotinib, gefitinib, cetuximab, matsuzumab, panitumumab, AEE788; CI-1033, HKI-272, HKI-357, or EKB-569.

31. The agent according to any one of claims 19 to 30, wherein an EGFR-derived HLA-binding peptide is administered together with a TLR ligand.

32. The agent according to claim 31, wherein the TLR ligand is a TLR7 agonist selected from CL075, CL097, CL264, CL307, GS-9620, poly(dT), imiquimod, gardiquimod, regiquimod (R848), loxoribine, or ssRNA oligonucleotides.

33. The agent according to claim 32, wherein the TLR7 agonist is imiquimod.

34. The drug according to any one of claims 19 to 33, wherein the cancer is lung cancer.

35. The agent according to claim 34, wherein the lung cancer is selected from non-small cell lung cancer, metastatic lung cancer, and / or lung adenocarcinoma.

36. The agent according to any one of claims 19 to 35, wherein the cancer is EGFR inhibitor resistant.

37. The agent according to any one of claims 19 to 36, wherein the composition is administered by parenteral administration, intravenous injection, intramuscular injection, inhalation, or subcutaneous injection.

38. The agent according to any one of claims 19 to 37, wherein the composition is administered at least twice.

39. The agent according to any one of claims 19 to 38, further comprising administering it for further anti-cancer treatment.

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