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WO2021071468A1 - Vaccin à néo-épitope et combinaisons et procédés de stimulation immunitaire - Google Patents

Vaccin à néo-épitope et combinaisons et procédés de stimulation immunitaire Download PDF

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Publication number
WO2021071468A1
WO2021071468A1 PCT/US2019/055054 US2019055054W WO2021071468A1 WO 2021071468 A1 WO2021071468 A1 WO 2021071468A1 US 2019055054 W US2019055054 W US 2019055054W WO 2021071468 A1 WO2021071468 A1 WO 2021071468A1
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administration
immune
tumor
regimen
vaccine
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Shahrooz Rabizadeh
Patrick Soon-Shiong
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Immunitybio Inc
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NantCell Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/208—IL-12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86

Definitions

  • the field of the invention is compositions and methods of cancer therapy, especially as it relates to protocols and compositions for cancer immunotherapy that utilize vaccine and immune modulators in a coordinated manner.
  • the immune system has been described as playing a dual role in cancer as it can protect against cancer development by detecting and eliminating tumor cells, but also in that it can promote cancer progression by selection of tumor cells that escape immune destruction.
  • This paradoxical role of the immune system in cancer is also known as cancer immunoediting (see e.g., Science. 2011;331:1565-70).
  • Cancer cells frequently employ various mechanisms to evade recognition and destruction by immune cells. For example, cancer cells can modulate the tumor microenvironment (TME) through recruitment of regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and immunosuppressive macrophages (M2 macrophages).
  • TME tumor microenvironment
  • Regs regulatory T cells
  • MDSCs myeloid-derived suppressor cells
  • M2 macrophages immunosuppressive macrophages
  • Cancer cells can also evade the immune system by down-regulating expression of certain MHC (major histocompatibility complex) molecules, which are deemed typically essential for T cells to recognize tumor-associated antigens (TAAs). Still further, cancer cells can secrete immune suppressive cytokines (e.g., TGF-b, IL-8) to establish a more immune suppressive environment.
  • immune suppressive cytokines e.g., TGF-b, IL-8
  • immune stimulants can be administered to increase an immune response to specific tumor antigens.
  • cytokines and derivatives are often used and include IL-2, IL-12, or IL-15, and chimeric forms thereof such as ALT-803 (see e.g., Altor BioScience Corporation, 2810 North Commerce Parkway, Miramar, FL 33025; ALT-803 FACT SHEET) or NHS-IL-12 (see e.g., Oncotarget 2014, Vol.5, No.7, 1869-1884).
  • ALT-803 see e.g., Altor BioScience Corporation, 2810 North Commerce Parkway, Miramar, FL 33025; ALT-803 FACT SHEET
  • NHS-IL-12 see e.g., Oncotarget 2014, Vol.5, No.7, 1869-1884.
  • checkpoint inhibitors have been used in the treatment of cancer, and in some cases yielded remarkable results.
  • inventive subject matter is directed to various compositions and methods of cancer immune therapy, in which various pharmaceutical compositions are administered to the patient to so elicit a therapeutically effective response.
  • such methods include prime/boost administration of a cancer vaccine with concurrent and overlapping immune stimulation and checkpoint inhibition regimes. It is further preferred that the so generated immune response is further modified/enhanced by administration of a pharmaceutical that triggers a Thl bias of the immune response.
  • the inventors contemplate a method of stimulating an immune response in an individual against a (typically solid) tumor.
  • the method includes a step of subjecting the individual to a timed treatment combination that includes a prime-boost vaccination regime, an immune stimulation regimen, a checkpoint inhibition regimen, and a Thl stimulation.
  • the immune stimulation regimen commences after the prime vaccination
  • the checkpoint inhibition regimen commences after the immune stimulation regimen
  • the Thl stimulation commences after the boost vaccination or subsequent administration of the vaccine composition.
  • the prime- boost vaccination regime comprises administration of a vaccine composition that elicits an immune response towards at least one of a tumor associated antigen and a tumor specific antigen, and it is generally preferred that the vaccine composition comprises a bacterial, a yeast, or a viral vaccine composition.
  • the prime-boost vaccination regime comprises administration of the vaccine composition in weekly intervals.
  • the immune stimulation regimen comprises administration of at least one of IL-2, IL-12, IL-15, IL-21 or a derivative thereof (which may include a targeting antibody, or an antibody fragment), and an especially preferred derivative is ALT-803.
  • the immune stimulation regimen may comprise administration of at least one of IL-2, IL-12, IL-15, IL-21 or a derivative thereof in 3-5 day intervals.
  • the checkpoint inhibition regimen comprises administration of an anti PD1 antibody, an anti-PD-Ll antibody, and/or an anti- CTLA4 antibody, which is preferably performed in 1-3 day intervals.
  • Contemplated Thl stimulations typically comprise administration of a cytokine or small molecule drug that biases an immune response towards a Thl response. Therefore, especially preferred cytokines include IL-12 or a derivative thereof (e.g., comprising an antibody portion that binds to a tumor cell or a necrotic component of a tumor cell).
  • the immune stimulation regimen and the checkpoint inhibition will regimen overlap, and/or the immune stimulation regimen, the checkpoint inhibition regimen, and the Thl stimulation are at least initiated during the prime -boost vaccination regime.
  • the inventors contemplate a method of treating a tumor in an individual that includes the following steps: (a) subjecting the individual to a vaccination regimen with a vaccine composition to elicit an immune response towards at least one of a tumor associated antigen and a tumor specific antigen, wherein the vaccination regimen comprises at least a first and a second administration of a vaccine composition; (b) subjecting the individual to an immune stimulation regimen after the first administration of the vaccine composition, wherein the immune stimulation regimen comprises at least a first and a second administration of an immune stimulatory composition; (c) subjecting the individual to a checkpoint inhibition regimen after the first administration of the immune stimulatory composition, wherein the checkpoint inhibition regimen comprises at least a first and a second administration of a checkpoint inhibitor; and (d) subjecting the individual to Thl stimulation after the second or subsequent administration of the vaccine composition, wherein the Thl stimulation comprises administration of a compound that biases an immune response towards a Thl response.
  • the first and the second administration of the vaccine composition, and optionally the second administration and a subsequent administration of the vaccine composition are between 5 and 10 days apart, and/or the first administration of the immune stimulatory composition is between the first and second administration of the vaccine composition. It is further contemplated that the first administration of the checkpoint inhibitor is between the first and second administration of the immune stimulatory composition, or concurrent with or after administration of the second administration of the vaccine composition, and/or that the immune stimulation regimen and the checkpoint inhibition regimen are performed between the first and a last administration of the vaccine composition. Therefore, the immune stimulation regimen and the checkpoint inhibition regimen may overlap.
  • the first and the second administration of the immune stimulatory composition are between 3-5 days apart, and/or the first and the second administration of the checkpoint inhibitor are between 1-3 days apart.
  • the compound that biases the immune response towards the Thl response further comprises an antibody or antibody fragment that binds to a tumor cell or a component of a necrotic cell.
  • the inventors also contemplate a coordinated use of a vaccine composition, an immune stimulatory composition, a checkpoint inhibitor, and a compound that biases an immune response towards a Thl response.
  • the immune stimulatory composition is administered after administration of a first dose of the vaccine composition
  • the checkpoint inhibitor is administered after a first dose of the immune stimulatory composition
  • the compound that biases the immune response towards the Thl response is administered after a second or subsequent administration of the vaccine composition.
  • the vaccine composition elicits an immune response towards at least one of a tumor associated antigen and a tumor specific antigen, and/or comprises a bacterial vaccine, a yeast vaccine, or a viral vaccine.
  • the immune stimulatory composition comprises a cytokine or a cytokine derivative (e.g., at least one of IL-2, IL-12, IL-15, IL-21 or a derivative thereof where the derivative thereof comprises a targeting antibody, or an antibody fragment, and/or ALT-803).
  • the checkpoint inhibitor comprises an antibody binding to PD1, PD-L1, or CTLA4.
  • the compound that biases the immune response towards the Thl response comprises IL-12, optionally coupled to an antibody or fragment thereof that binds to a tumor cell or a component of a necrotic tumor cell (e.g., NHS IL-12).
  • the vaccine composition is administered using a weekly schedule
  • the immune stimulatory composition is administered using a 3-5 day schedule
  • the checkpoint inhibitor is administered using a 1-3 day schedule.
  • Fig. 1 is a schematic illustration of a personalized vaccine preparation used in the inventive subject matter.
  • Fig. 2 is a graph depicting exemplary results for patient neoepitope filtering using expression and calculated MHC binding.
  • FIG. 3 is an exemplary schematic of various control experiments compared with a vaccination scheme according to the inventive subject matter.
  • Fig. 4 depicts photomicrographs of CD8 T cell infiltration of tumors in animals of the schemes or Fig. 3.
  • Fig. 5 is an exemplary graph of tumor volumes in animals treated using a scheme according to the inventive subject matter.
  • Figs. 6A-H show the procedures for identification of immunogenic neoepitopes.
  • Fig. 6A shows workflow for neoepitope discovery in subcutaneously implanted MC38 tumors.
  • Fig. 6B shows in-vitro binding score of MC38 neoepitopes.
  • Fig. 6C shows IENg ELIS POT analysis of naive mice vaccinated twice with pools of 9-mer neoepitopes. Splenocytes were harvested 1 week following the second vaccination. Shaded bars represent peptides that induced robust immune responses in repeated experiments.
  • Fig. 6D shows treatment schedule. Figs.
  • 6E-F shows day 18 IFNy ELIS POT analysis of mice vaccinated with (6E) four 9-mer or (6F) two 25-mer neoepitopes, alone or in combination with N-803 and/or anti- PD-L1.
  • Fig. 6G shows survival curves of tumor-bearing mice treated with N-803, anti-PD-Ll and four 9-mer neoepitope peptides (black line), N-803, anti-PD-Ll and PBS (dashed line), or no treatment (gray line).
  • Fig. 6H shows survival curves of mice treated with 9-mer neoepitope vaccine, N-803 and anti-PD-Ll stratified by antigen- specific IFNy-secreting cells per 106 cells in the peripheral blood on day 13, *P ⁇ 0.05.
  • Figs. 7A-H show combination therapy using a 9-mer neoepitope vaccine, N-803, anti-PD-Ll and NHS- IL12.
  • Fig. 7A shows treatment schedule.
  • Fig. 7B shows IENg ELI SPOT analysis on days 11, 18 and 25 of tumor growth against peptides contained within the vaccine (top) or MC38 neoepitopes not contained within the vaccine or P15e (bottom). Each column represents one mouse.
  • Fig. 7C shows tumor growth curves.
  • Fig. 7A-H show combination therapy using a 9-mer neoepitope vaccine, N-803, anti-PD-Ll and NHS- IL12.
  • Fig. 7A shows treatment schedule.
  • Fig. 7B shows IENg ELI SPOT analysis on days 11, 18 and 25 of tumor growth against peptides contained within the vaccine (top) or MC38 neoepitopes not contained within the vaccine or P15e (bottom). Each column
  • FIG. 7D shows tumor growth in mice treated with 9-mer neoepitope vaccine, N-803, anti-PD-Ll and NHS-IL12 stratified by antigen-specific IFNy-secreting cells per 106 cells in the peripheral blood on day 13.
  • Fig. 7E shows survival curves after re-challenge of naive animals or those with a previously regressed MC38 tumor following indicated treatment. Re-challenged animals were implanted with MC38 tumors on day 0 of survival curve and received no subsequent therapies. Arrow indicates depletion of CD8+ cells. Figs.
  • FIG. 7F-G show analysis of splenocytes from mice treated with 9- mer neoepitope vaccine, N-803, anti-PD-Ll and NHS- IL12, harvested on day 25 and stimulated overnight with either vaccine components or cascade antigens.
  • Fig. 7F shows ELISPOT analysis of the ratio of antigen- dependent TNFcxdFN secreting splenocytes.
  • Fig. 7G shows flow cytometric analysis of percent of CD8+ cells that were single IFNy (orange), single TNFoc (pink), or double (purple) producers.
  • Fig. 7H shows Tumor growth in mice treated with 9-mer neoepitope vaccine and NHS-IL12 according to the schedule in Fig. 7A.
  • Figs. 8A-B show tumor progression in mice treated with combination therapy using single-peptide vaccines or immune cell depletions.
  • Fig. 8A shows tumor growth in mice treated with N-803, anti-PD-Ll, NHS-IL12 and a neoepitope vaccine consisting of a single 9-mer neoepitope or a pool of four 9- mer neoepitopes. Mice were treated according to the schedule in Fig. 7A.
  • Fig. 8B shows timeline (left) and tumor growth (right) of depletion studies. Tumor-bearing mice were depleted of NK, CD4 or CD8 cells starting 3 days prior to the first vaccine (early depletion) or NHS-IL12 administration (late depletion).
  • Figs. 9A-D show tumor progression and tumor-infiltrating immune cells in mice treated with single, triple, or quadruple combinations of N-803, anti-PD-Ll, NHS-IL12 and 9-mer neoepitope vaccine. Mice were treated as previously described, and tumors were harvested on day 22 post- tumor implantation. Tumors were analyzed via (9A+9B) immunofluorescent analysis or (9C+9D) flow cytometry.
  • FIG. 9B shows Percent CD8+ cells (of total DAPI+ cells) in immunofluorescent sections.
  • Fig. 9C shows intratumoral Ml macrophages (top, CDllb+F4/80+CD38-i-) and M2 macrophages (CDllb+F4/80+CD206+).
  • Fig. 9D shows CD8+ TIL maturation (CD44/CD62L/CD127). *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001, **** P ⁇ 0.0001.
  • Figs. 10A-D show Gene expression and clonality of tumor-infiltrating immune cells after treatment with a 9-mer neoepitope vaccine, N-803, anti-PD-Ll and NHS-IL12.
  • Fig. 10A shows gene expression analysis of tumor-infiltrating leukocytes.
  • Fig. 10B shows clonality of TCRfl chains detected in tumor infiltrates **P ⁇ 0.001.
  • Fig. IOC shows number of TCR clones that comprise the top 25% of detected sequences.
  • Fig. 10D shows frequency of the top 100 TCR sequences detected in each sample. Each column represents one mouse, and each row represents a unique clone.
  • Figs. 11A-K show combination therapy utilizing adenoviral vectors.
  • Fig. 11A shows Visual representation of admixed and multi-epitope adenoviral vaccines targeting neoepitopes.
  • Fig. 11B shows treatment schedule.
  • Fig. 11C shows IFN ELIS POT analysis on day 25 of tumor growth against neoepitopes contained within the vaccine. Each row represents one mouse.
  • Fig. 11D shows immune responses generated in animals vaccinated with multi-epitope adenovirus relative to paired neoepitopes from animals treated with admixed adenovirus. Each row represents one mouse.
  • Fig. HE shows tumor growth curves.
  • Fig. 11A shows Visual representation of admixed and multi-epitope adenoviral vaccines targeting neoepitopes.
  • Fig. 11B shows treatment schedule.
  • Fig. 11C shows IFN E
  • Fig. 11G shows ratio of CD8:CD4 TIL in immunofluorescent sections. Fig.
  • FIG. 11H shows % FoxP3+ cells within immunofluorescent sections.
  • Fig. Ill shows tumor growth in mice treated as indicated in Fig. 11B utilizing an adenovirus targeting TWIST1.
  • Fig. 11 J shows IFN ELI SPOT analysis on day 25 of tumor growth against neoepitopes identified in the MC38 cell line. Each column represents one mouse.
  • Fig. 11K shows relative immunity of the immune responses generated in animals against neoepitopes in the adeno multi-epitope vaccinated animals as compared to animals vaccinated with adeno- TWIST1 vaccine. Each column represents one mouse.
  • cancer immune therapy can be administered using a coordinated orchestration of a combination of certain immunotherapeutic agents to so elicit an effective anti-tumor response in a mammal.
  • orchestrated treatments are based on a prime/boost vaccination regimen that includes an immune stimulation phase using one or more immune stimulatory cytokines and checkpoint inhibition phase using one or more checkpoint inhibitors, before the so elicited immune response is further maintained and/or modified towards a Thl response.
  • immune therapy using a prime/boost regiment with cancer vaccine can be significantly improved by stimulating the immune system after the prime vaccination with immune stimulatory cytokines, and by further modulating suppressive signals with checkpoint inhibitors, preferably at or during the boost phase of the vaccination. Stimulation and checkpoint inhibition are continued during the boost phase, and the so elicited immune response is then supported and/or further modulated towards a Thl response by administration of a second immunostimulatory cytokine, preferably in a tumor targeted manner.
  • contemplated compositions and methods will be suitable for a variety of tumors, including solid tumors, soft tissue tumors, and lymphatic tumors.
  • the term “tumor” refers to, and is interchangeably used with one or more cancer cells, cancer tissues, malignant tumor cells, or malignant tumor tissue, that can be placed or found in one or more anatomical locations in a human body.
  • the term “bind” refers to, and can be interchangeably used with a term “recognize” and/or “detect”, an interaction between two molecules with a high affinity with a K D of equal or less than 10 6 M, or equal or less than 10 7 M.
  • the term “provide” or “providing” refers to and includes any acts of manufacturing, generating, placing, enabling to use, or making ready to use.
  • the type and content of the vaccine composition to elicit an immune response towards at least one of a tumor associated antigen and a tumor specific antigen may vary considerably. Consequently, all manners of providing an immunogen to an individual are deemed suitable and include DNA vaccines, peptide vaccines, and especially recombinant vaccines.
  • the content of the vaccines may vary considerably and may include a tumor associated antigen or fragment thereof (e.g., MUC1, brachyury, CEA, etc.), a tumor specific antigen or fragment thereof (e.g., PSA, PSMA, HER2, etc.), and particularly tumor and patient specific neoantigens (i.e., neoepitopes).
  • the tumor-associated antigen refers any antigen that can be presented on the surface of the tumor cells, which includes an inflammation-associated peptide antigen, a tumor associated peptide antigen, a tumor specific peptide antigen, and a cancer neoepitope.
  • the tumor associated antigen is a tumor-specific, patient- specific neoepitope. It is contemplated that patient- and tumor-specific neoantigens or neoepitopes can be identified via analyzing and comparing omics data from diseased tissue and healthy tissue of a patient, (e.g., via whole genome sequencing and/or exome sequencing, etc.) as is described in US20120059670 and US20120066001. For example, the tumor associated antigens and neoepitopes (which are typically patient-specific and tumor-specific) can be identified from the omics data obtained from the cancer tissue of the patient or normal tissue (of the patient or a healthy individual), respectively.
  • Omics data of tumor and/or normal cells preferably comprise a genomic data set that includes genomic sequence information.
  • the genomic sequence information comprises DNA sequence information that is obtained from the patient (e.g., via tumor biopsy), most preferably from the tumor tissue (diseased tissue) and matched healthy tissue of the patient or a healthy individual.
  • the DNA sequence information can be obtained from a pancreatic cancer cell in the patient’s pancreas (and/or nearby areas for metastasized cells), and a normal pancreatic cells (non-cancerous cells) of the patient or a normal pancreatic cells from a healthy individual other than the patient.
  • DNA analysis is performed by whole genome sequencing and/or exome sequencing (typically at a coverage depth of at least lOx, more typically at least 20x) of both tumor and matched normal sample.
  • DNA data may also be provided from an already established sequence record (e.g., SAM, BAM, FASTA, FASTQ, or VCF file) from a prior sequence determination ⁇ Therefore, data sets may include unprocessed or processed data sets, and exemplary data sets include those having BAM format, SAM format, FASTQ format, or FASTA format.
  • the data sets are provided in BAM format or as BAMBAM diff objects (see e.g., US2012/0059670A1 and US2012/0066001A1).
  • the data sets are reflective of a tumor and a matched normal sample of the same patient to so obtain patient and tumor specific information.
  • genetic germ line alterations not giving rise to the tumor e.g., silent mutation, SNP, etc.
  • the tumor sample may be from an initial tumor, from the tumor upon start of treatment, from a recurrent tumor or metastatic site, etc.
  • the matched normal sample of the patient may be blood, or non-diseased tissue from the same tissue type as the tumor.
  • neoepitopes may then be subject to further detailed analysis and filtering using predefined structural and expression parameters, and sub-cellular location parameters.
  • predefined expression threshold e.g., at least 20%, 30%, 40%, 50%, or higher expression as compared to normal
  • membrane associated location e.g., are located at the outside of a cell membrane of a cell.
  • Further contemplated analyses will include structural calculations that delineate whether or not a neoepitope or a tumor associated antigen, or a self-lipid is likely to be solvent exposed, presents a structurally stable epitope, etc. Further details on identification of patient-specific neoantigens and/or cancer-specific, patient-specific neoantigens are described in detail in the international patent application No. PCT/US 16/56550.
  • the tumor-related antigen is a high- affinity binder to at least one MHC Class I sub-type or at least one MHC Class II sub-type of an HLA-type of the patient, which may be determined in silico using a de Bruijn graph approach as, for example, described in WO 2017/035392, or using conventional methods (e.g., antibody-based) known in the art.
  • the binding affinity of the human disease-related antigen is tested in silico to the determined HLA-type.
  • the preferred binding affinity can be measured by lowest KD, for example, less than 500nM, or less than 250nM, or less than 150nM, or less than 50nM, for example, using NetMHC.
  • the HLA-type determination includes at least three MHC-I sub-types (e.g., HLA-A, HLA-B, HLA-C, etc.) and at least three MHC-II sub-types (e.g., HLA-DP, HLA-DQ, HLA-DR, etc.), preferably with each subtype being determined to at least 4-digit depth.
  • MHC-I sub-types e.g., HLA-A, HLA-B, HLA-C, etc.
  • MHC-II sub-types e.g., HLA-DP, HLA-DQ, HLA-DR, etc.
  • matching of the patient’s HLA-type to the patient- and cancer-specific neoantigen can be done using systems other than NetMHC, and suitable systems include NetMHC II, NetMHCpan, IEDB Analysis Resource (URL immuneepitope.org), RankPep, PREDEP, SVMHC, Epipredict, HLABinding, and others (see e.g., J Immunol Methods 2011;374:1-4).
  • the collection of neoantigen sequences in which the position of the altered amino acid is moved can be used.
  • modifications to the neoantigens may be implemented by adding N- and/or C-terminal modifications to further increase binding of the expressed neoantigen to the patient’s HLA-type.
  • neoantigens may be native as identified or further modified to better match a particular HLA-type.
  • binding of corresponding wild type sequences can be calculated to ensure high differential affinities.
  • especially preferred high differential affinities in MHC binding between the neoantigen and its corresponding wild type sequence are at least 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, at least 500-fold, at least 1000-fold, etc.
  • the omics information (especially where the omics information comprises whole genome sequencing or exome sequencing, RNA sequence and transcription data, and (preferably quantitative) proteomics information) can also be used to determine the status of various cell signaling pathways.
  • Such pathway information and especially in conjunction with mutational information, may reveal further druggable targets within a cell that are independent from anatomical features of the tumor (e.g., presence of HER2 signaling in a non-breast cancer).
  • Particularly preferred pathway analyses that are based on omics information include those described in WO 2011/139345, WO 2013/062505, WO 2014/193982, WO 2014/059036, WO 2014/210611, WO 2015/184439, and WO 2016/118527.
  • omics data in contemplated treatments and uses will be employed to both, inform generation of immune therapeutic compositions as well as inform selection of chemotherapeutic drugs based on pathway information rather than tumor type and location. Therefore, suitable omics data include whole genome sequencing data, exome sequencing data, RNA sequence and transcription data, and proteomics data (e.g., quantitative proteomics data from mass spectroscopic analyses).
  • suitable omics data include whole genome sequencing data, exome sequencing data, RNA sequence and transcription data, and proteomics data (e.g., quantitative proteomics data from mass spectroscopic analyses).
  • the tumor associated antigen or neoepitopes can be a polytope.
  • a polytope refers a tandem array of two or more antigens (or neoepitopes) expressed as a single polypeptide.
  • two or more human disease-related antigens are separated by a linker or spacer peptides.
  • Any suitable length and order of peptide sequence for the linker or the spacer can be used.
  • the length of the linker peptide is between 3-30 amino acids, preferably between 5-20 amino acids, more preferably between 5-15 amino acids.
  • glycine-rich sequences e.g., gly-gly-ser-gly-gly, etc. are preferred to provide flexibility of the polytope between two antigens.
  • Fig. 1 The notable reduction in potentially useful neoepitopes as described above is exemplarily shown in Fig. 1 where a patients tumor tissue is compared using the same patient’ s normal tissue using omics analysis and neoepitope discovery.
  • an adenovirus is then genetically engineered to include an expression cassette that includes nucleic acid segments that encode one or more neoepitopes of the patient.
  • Such modified vims can then be used as a recombinant viral vaccine.
  • Fig. 2 exemplarily illustrates a selection process in which all neoepitopes identified by omics analysis (left bar) are first filtered by expression level.
  • neoepitopes are eliminated, resulting in a substantial reduction in potential neoepitopes (middle bar).
  • These filtered neoepitopes are further filtered against the patients MHC type, and as can be seen from the Figure (right bar), the number of neoepitopes that can be presented by the patient’s antigen presenting cells is once more dramatically reduced.
  • the neoepitopes can then be used in therapy for example in a recombinant vims/recombinant expression system.
  • a nucleic acid sequence encoding one or more tumor-associated antigen(s) and/or neoepitopes can be placed in an expression vector.
  • the recombinant nucleic acid is then inserted into an expression vector such that the nucleic acid can be delivered to an antigen presenting cell (e.g., dendritic cells, etc.) of the patient, or to transcribe the nucleic acid sequence in bacteria or yeast so that the recombinant protein encoded by the nucleic acid sequence can be, as a whole, or as fragments, delivered to the antigen presenting cell.
  • an antigen presenting cell e.g., dendritic cells, etc.
  • Any suitable expression vectors that can be used to express protein are contemplated.
  • a preferred expression vector may include those that can carry a cassette size of at least lk, preferably 2k, more preferably 5k base pairs.
  • a preferred expression vector includes a viral vector (e.g., non-replicating recombinant adenovirus genome, optionally with a deleted or non-functional El and/or E2b gene).
  • the expression vector is viral vector (e.g., an adenovirus, and especially AdV with El and E2b deleted)
  • the recombinant viruses including the recombinant nucleic acid may then be individually or in combination used as a therapeutic vaccine in a pharmaceutical composition, typically formulated as a sterile injectable composition with a vims titer of between 10 6 -10 13 vims particles, and more typically between 10 9 -10 12 vims particles per dosage unit.
  • the vims vaccine can be formulated with a virus titer at least 10 6 vims particles/day, or at least 10 8 vims particles/day, or at least 10 10 vims particles/day, or at least 10 11 vims particles/day.
  • virus may be employed to infect patient (or other HLA matched) cells ex vivo and the so infected cells are then transfused to the patient.
  • the expression vector can also be a bacterial vector that can be expressed in a genetically engineered bacterium, which expresses endotoxins at a level low enough not to cause an endotoxic response in human cells and/or insufficient to induce a CD- 14 mediated sepsis when introduced to the human body.
  • a bacteria strain with modified lipopolysaccharides includes ClearColi® BL21 (DE3) electrocompetent cells.
  • This bacteria strain is BL21 with a genotype F- ompT hsdSB (rB- mB-) gal dcm Ion /.(DE3 I tad lacUV5-T7 gene 1 indl sam7 nin5 ]) msbA148 AgutQAkdsD
  • AlpxLAlpxMApagPAlpxPAeptA encodes the modification of LPS to Lipid IVA, while one additional compensating mutation (msbA148) enables the cells to maintain viability in the presence of the LPS precursor lipid IVA.
  • mutations result in the deletion of the oligosaccharide chain from the LPS. More specifically, two of the six acyl chains are deleted.
  • the six acyl chains of the LPS are the trigger which is recognized by the Toll-like receptor 4 (TLR4) in complex with myeloid differentiation factor 2 (MD-2), causing activation of NF-kB and production of proinflammatory cytokines.
  • TLR4 Toll-like receptor 4
  • MD-2 myeloid differentiation factor 2
  • Lipid IVA which contains only four acyl chains, is not recognized by TLR4 and thus does not trigger the endotoxic response.
  • electrocompetent BL21 bacteria is provided as an example, the inventors contemplates that the genetically modified bacteria can be also chemically competent bacteria.
  • the expression vector can also be a yeast vector that can be expressed in yeast, preferably, in Saccharomyces cerevisiae (e.g., GI-400 series recombinant immunotherapeutic yeast strains, etc.) ⁇
  • yeast vector that can be expressed in yeast
  • Saccharomyces cerevisiae e.g., GI-400 series recombinant immunotherapeutic yeast strains, etc.
  • recombinant nucleic acids contemplated herein need not be limited to viral, yeast, or bacterial expression vectors, but may also include DNA vaccine vectors, linearized DNA, and mRNA, all of which can be transfected into suitable cells following protocols well known in the art.
  • the vaccine composition may change over the course of the vaccination regimen, in terms of formulation and in terms of antigen composition.
  • a prime vaccination may be performed using a recombinant bacterial or yeast expression system as described above, which is then followed in the boost phase using with an adenoviral vaccination.
  • the vaccination regimen may also use different routes of administration, including subcutaneous or intramuscular administration of a bacterial or yeast vaccine composition, and an intratumoral or intravenous administration of an adenoviral vaccine composition.
  • the prime vaccine composition may include both tumor associated antigens and neoepitopes, while the boost vaccination may include or encode only neoepitopes as the immunogenic agent.
  • the vaccine regimen may use various timelines for administration. However, it is generally contemplated that the timing of prime/boost will follow an anticipated timing of immune response with respect to antigen processing and presentation and T cell stimulation and proliferation. Therefore, the vaccine compositions will at least initially be administered at least 2 days apart, more typically within a window of 3-5 days, or 5-7 days, or 7-10 days, or 10-14 days, and in some cases even longer. For subsequent administrations in the boost phase (e.g., fourth, fifth, etc. administration), the interval may be longer, including several weeks, months, or even years. In some embodiments, the dose of the vims formulation can be gradually increased during the schedule, or gradually decreased during the schedule. In still other embodiments, several series of administration of virus formulation can be separated by an interval (e.g., one administration each for 3 consecutive days and one administration each for another 3 consecutive days with an interval of 7 days, etc.).
  • an interval e.g., one administration each for 3 consecutive days and one administration each for another 3 consecutive days with an interval of 7 days, etc.
  • such regimen comprises at least a first and a second administration of one or more immune stimulatory compositions, and it is generally preferred that the immune stimulatory composition comprises an immune stimulatory cytokine.
  • immune stimulatory cytokine especially preferred cytokines include various interleukins, and particularly IL-2, IL-12, IL-15, IL-17, IL-21, as well as other cytokines including Thl cytokines (e.g., IFN-g, TNFa, etc.).
  • Suitable dosages for the immune stimulation will typically be within the range of clinically acceptable dosages, which art well known in the art.
  • the immune stimulatory compositions may also include modified forms of the above noted proteins, and especially modified forms include those that increase serum half life time and/or reduce toxicity.
  • contemplated immune stimulating compositions may include truncated IL-2 that lacks the PEP portion, or ALT-803, which is an IL-15 based chimeric protein complex with IL-15 superagonist function.
  • Still further contemplated immune stimulatory compounds may also be target specific, for example, by coupling to an antibody or portion thereof, wherein the antibody typically binds a tumor epitope, a neoepitope, or a component of a necrotic cell (e.g., dsDNA, histones, etc.).
  • suitable dosages for the immune stimulation using such compounds will typically be within the range of clinically acceptable dosages, which art well known in the art.
  • immune stimulatory compounds include various TLR ligands and/or NOD ligands that enhance an immune response.
  • suitable TLR ligands include TLR2, TLR3, TLR4, TLR7, or TLR8, and especially include mRNA, dsDNA, ssRNA, CpG, glycolipids, etc.
  • immune stimulatory compounds may also include an 0X40 agonistic antibody. Thus, numerous immune stimulatory compounds are deemed suitable, and all reasonable combinations thereof.
  • the immune stimulatory compounds may change over the course of the treatment regimen, both in terms of type and the dosage of the immune stimulatory compounds.
  • initial stimulation maybe may be performed using more acute effect compounds (e.g., IL-2, IL-15), while subsequent administrations may employ immune stimulatory compounds with longer lasting effects (e.g., ALT- 803).
  • immune stimulatory compounds may also be administered in a target specific manner as noted above to reduce systemic (toxic) effects.
  • the immune stimulatory regimen may use different routes of administration, including subcutaneous or intramuscular administration, however, intratumoral or intravenous administration are particularly preferred.
  • the immune stimulatory regimen may use various timelines for administration.
  • the first administration will be after the prime administration of the vaccine as already noted above.
  • Subsequent administrations will generally be in relatively rapid succession to elicit a substantially continuous stimulatory effect. Therefore, the immune stimulatory compounds will be administered about 1 day apart, more typically within a window of 1-2 days, or 2-3 days, or 1-3 days, or 2-4 days, and in some cases even longer intervals.
  • the interval may be longer, including several weeks, months, or even years.
  • the immune stimulatory compounds are at least administered within the prime/boost vaccine regimen, and that the immune stimulatory regimen overlaps to at least some degree with a checkpoint inhibition regimen as described in more detail below. While not limiting to the inventive subject matter, it is believed that upon administration of the prime vaccination, an immune stimulatory phase that supports immune cell activation and a regimen that reduces checkpoint inhibition will modulate the immune system to provide an optimum response to the prime and contemporaneous/subsequent boost phase.
  • the checkpoint inhibition regimen comprises at least a first and a second administration of a checkpoint inhibitor, and all known checkpoint inhibitors are deemed suitable for use herein.
  • suitable checkpoint inhibitors include those that interfere with signaling through PD-1 (e.g., pembrozilumab, nivolumab), PD-L1 (e.g., atezolizumab, avelumab, durvalumab), and/or CTLA4 (e.g., ipilimumab).
  • PD-1 e.g., pembrozilumab, nivolumab
  • PD-L1 e.g., atezolizumab, avelumab, durvalumab
  • CTLA4 e.g., ipilimumab
  • Suitable dosages for the immune stimulation will typically be within the range of clinically acceptable dosages, which art well known in the art.
  • a tumor secrets soluble ligands to checkpoint receptors
  • antibodies scavenging such soluble factors may be used to reduce adverse effects of such soluble forms.
  • the checkpoint inhibition regimen may employ various timelines for administration.
  • the first administration will be after the first administration of the vaccine and also after the first administration of the immune stimulatory compound.
  • the immune stimulation and checkpoint inhibition will at least partially overlap. Subsequent administrations will generally be in relatively rapid succession to elicit a substantially continuous stimulatory effect.
  • the checkpoint inhibitors will be administered about 1 day apart, more typically within a window of 1-2 days, or 2-3 days, or 1-3 days, or 2-4 days, and in some cases even longer intervals.
  • the interval may be longer, including several weeks, months, or even years.
  • the compound is a cytokine, and especially IL-12.
  • the IL-12 is conjugated to an antibody or portion thereof, wherein the antibody binds to a tumor antigen, a tumor neoepitope, or a component of a necrotic cell.
  • especially preferred compounds include NHS IL-12 and derivatives thereof.
  • plant based compositions e.g., PLoS Negl Trop Dis.
  • deoxunucleotides e.g., Molecular Therapy, Vol.23, Issue 2, 222 - 223
  • ribavirin e.g., PLoS One. 2012; 7(7): e42094.
  • IL-4 and IL-10 IL-4 and IL-10.
  • Thl stimulation/immune support may employ various timelines for administration.
  • the first administration will be after the second or subsequent administration of the vaccine and also after the administrations of the immune stimulatory compound and checkpoint inhibitor.
  • the Thl stimulation will typically coincide with the boost phase of vaccination, and most typically provide a support function that maintains the immune stimulation. Subsequent administrations will generally be in relatively slow succession to maintain a continuous stimulatory effect.
  • the compounds that stimulate Thl will be administered, where desired, about 3 day apart, more typically within a window of 2-5 days, or 5-7 days, or 7-14, and in some cases even longer intervals.
  • the interval may be longer, including several weeks, months, or even years.
  • contemplated treatments may further include other therapeutic compositions that complement or otherwise support the methods described herein.
  • contemplated methods may include administration of autologous or heterologous NK cells to a patient, and particularly NK cells that are genetically modified to exhibit less inhibition.
  • the genetically modified NK cell may be a NK-92 derivative that is modified to have a reduced or abolished expression of at least one killer cell immunoglobulin-like receptor (KIR), which will render such cells constitutively activated.
  • KIR killer cell immunoglobulin-like receptor
  • KIRs may be deleted or that their expression may be suppressed (e.g., via miRNA, siRNA, etc.), including KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR2DS1, KIR2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DL1, KIR3DL2, KIR3DL3, and KIR3DS1.
  • modified cells may be prepared using protocols well known in the art. Alternatively, such cells may also be commercially obtained from NantKwest as aNK cells (‘activated natural killer cells). Such cells may then be further modified to express the co-stimulatory molecules as further discussed below.
  • contemplated NK cells suitable for use herein also include those that have abolished or silenced expression of NKG2A, which is an activating signal to Tregs and MDSCs.
  • the genetically engineered NK cell may also be an NK-92 derivative that is modified to express a high-affinity Fey receptor (CD16-158V).
  • a high-affinity Fey receptor CD16-158V.
  • Sequences for high- affinity variants of the Fey receptor are well known in the art, and all manners of generating and expression are deemed suitable for use herein. Expression of such receptor is believed to allow specific targeting of tumor cells using antibodies produced by the patient in response to the treatment contemplated herein, or supplied as therapeutic antibodies, where those antibodies are specific to a patient’s tumor cells (e.g., neoepitopes), a particular tumor type (e.g., her2neu, PSA, PSMA, etc.), or antigens associated with cancer (e.g., CEA-CAM).
  • tumor cells e.g., neoepitopes
  • a particular tumor type e.g., her2neu, PSA, PSMA, etc.
  • such cells may be commercially obtained from NantKwest as haNK cells (‘high- affinity natural killer cells) and may then be further modified (e.g., to express co stimulatory molecules).
  • genetically engineered NK cells may also be genetically engineered to express a chimeric T cell receptor.
  • the chimeric T cell receptor will have an scFv portion or other ectodomain with binding specificity against a tumor associated antigen, a tumor specific antigen, and/or a neoepitope of the patient as determined by the omics analysis.
  • such cells may be commercially obtained from NantKwest as taNK cells (‘target-activated natural killer cells’) and further modified as desired.
  • tumor associated antigens include CEA, MUC-1, CYPB1, PSA, Her-2, PSA, brachyury, etc.
  • the methods and uses contemplated herein also include cell based treatments with cells other than (or in addition to) NK cells.
  • suitable cell based treatments include T cell based treatments.
  • one or more features associated with T cells e.g., CD4+ T cells, CD8+ T cells, etc. can be detected.
  • contemplated omics analysis can identify specific neoepitopes (e.g., 8-mers to 12-mers for MHC I, 12-mers to 25-mers for MHC II, etc.) that can be used for the identification of neoepitope reactive T cells bearing a specific T cell receptor against the neoepitopes/MHC protein complexes.
  • the method can include harvesting the neoepitope reactive T cells.
  • the harvested T cells can be grown or expanded (or reactivated where exhausted) ex vivo in preparation for reintroduction to the patient.
  • the T cell receptor genes in the harvested T cells can be isolated and transferred into viruses, or other adoptive cell therapies systems (e.g., CAR-T, CAR-TANK, etc.).
  • the omics analyses can also provide one or more tumor associated antigens (TAAs). Therefore, one can also harvest T cells that have receptors that are sensitive to the TAAs identified from these analyses. These cells can be grown or cultured ex vivo and used in a similar therapeutic manner as discussed above.
  • the T cells can be identified by producing synthetic versions of the peptides and bind them with commercially produced MHC or MHC-like proteins, then using these ex vivo complexes to bind to the target T cells.
  • the harvested T cells can included T cells that have been activated by the patient’s immune response to the disease, exhausted T cells, or other T cells that are responsive to the discussed features. Examples
  • Example 1 As can be readily seen from Fig. 3, the timed sequence of prime/boost vaccination with immune stimulation and checkpoint inhibition and Thl stimulation had dramatic effect (tumor volumes were significantly reduced after 40 days of combination treatment) as compared to any treatment regimen that lacked one of the components. This remarkable picture is also reflected in the histopathological analysis shown in Fig. 4. Here, photomicrographs are shown with CD8+ tumor infiltrating cells stained. Once more, the timed sequence of prime/boost vaccination with immune stimulation, checkpoint inhibition, and Thl stimulation resulted in a significant increase in T cells infiltrating the tumor. Fig. 5 shows the results for a follow-up study indicating days after NHS IL-12 administration. Again, the effect of the timed sequence of prime/boost vaccination with immune stimulation and checkpoint inhibition and Thl stimulation is readily apparent and resulted in all but two cured animals.
  • Cell culture MC38 and RMA-S cells were grown in RPMI-1640 with L-glutamine (Corning) supplemented with 10% (v/v) fetal bovine serum (Atlanta Biologicals) and 1% (v/v) antibiotic/antimitotic solution (Corning). All cells were cultured at 37 °C, 5% C02.
  • Neoepitopes were ranked by RNA expression as well as allele frequency of the observed coding variant to offset issues arising from tumor heterogeneity.
  • NetMHC 3.4 http://www.cbs.dtu.dk/services/NetMHC-3.4/) (27,28) was used to predict neoepitope binding to a specific MHC H-2 allele.
  • Neoepitopes with predicted binding affinities ⁇ 500 nM were retained for further analysis.
  • Peptide synthesis Peptides were synthesized by Bio-Synthesis or GenScript to >85% purity.
  • PBMCs Peripheral blood mononuclear cells
  • Flow cytometric assays All antibodies used for flow cytometric analysis are fully described in Supplementary Table SI. Peptide binding assays were performed using RMA-S cells incubated with individual peptides at 50 pg/mL overnight. Following incubation, cells were stained with anti-MHC antibodies. Data were acquired using a FACS Calibur (BD Biosciences), and reported as an in vitro binding score, which is the percentage of RMA-S cells expressing MHC on their surface. Tumor-infiltrating leukocytes were stained for immune cell subsets and data were collected using an Attune NxT flow cytometer (ThermoFisher) or BD LSRFortessa (BD Biosciences).
  • CD8+ T cells live/CD45+/CD3+/CD8-i-; macrophages: live/CD45+/CD3- /CDllb+F4/80+.
  • splenocytes were cultured with indicated peptides for 4 hours, at which time GolgiPlug/GolgiStop (BD Biosciences) were added. Cultures were incubated an additional 20 hours, and fixed, permeabilized and stained for TNF and IFNy production. Data were collected using an Attune NxT flow cytometer.
  • Vaccination and treatment with immune-modulators Animals were vaccinated with pools of 9-mer or 25-mer neoepitope peptides (100 pg each peptide), emulsified in Montanide ISA 51 VG (Seppic), administered subcutaneously. Adenoviral vectors encoding TWIST 1 or neoepitopes were kindly produced and provided by the Etubics/NantCell Corporation. 1010 viral particles encoding the multi-epitope vims were administered subcutaneously. The admixed vims was administered by injecting animals with 1010 viral particles each of which is a single-neoepitope encoding vimses subcutaneously.
  • N-803 was kindly provided by Nantworks, and lpg was administered subcutaneously into animals.
  • Anti- PD-L1 (10F.9G2, BioXCell, 200 pg) was administered intraperitoneally.
  • Murine NHS-IL12 was administered at a dose of 50 pg.
  • NanoString and T cell receptor (TCR) sequencing Isolated tumor-infiltrating leukocytes were enriched using a CD45+ or CD4+/CD8+ murine TIL microbead kit
  • Nanostring data was normalized using the nSolver Analysis Software 4.0.
  • TCR diversity was assessed using genomic DNA purified from tumor-infiltrating CD4+ and CD8+ cells using the QIAamp DNA mini kit (Qiagen).
  • TCRfl chain sequencing was performed by Adaptive Biotechnologies and analyzed using the Immunoseq analyzer. The top 100 TCR sequences were analyzed.
  • the inventors also utilized a second metric, which ranked neoepitopes solely by their predicted MHC binding affinity. Thirteen peptides, representing the top 10 peptides from both metrics were synthesized. Seven of these 13 neoepitopes were capable of binding MHC in-vitro (Fig. 6B), and the immunogenicity of each peptide was assessed by vaccinating non-tumor bearing mice with pools of neoepitope peptides emulsified in Montanide ISA 51 VG adjuvant (Montanide).
  • neoepitopes were found to induce immune responses in repeated experiments using non-tumor bearing animals (Fig. 6C, reactive peptides highlighted with shaded bars). Based upon these observations, we chose to utilize a vaccination strategy incorporating a pool of four 9-mer neoepitope peptides (Jakl, 01fr99, Ptgfr and Trp53) emulsified in Montanide. When administered as a single agent, the neoepitope vaccine induced a low level of immunity, but failed to provide any survival benefit as compared to control animals.
  • the inventors combined it with the IL15 superagonist fusion protein N- 803 and anti-PD-Ll MAb.
  • N-803 and anti- PD-L1 synergized to increase the immunogenicity of the 9-mer, but not 25-mer neoepitope vaccines in tumor bearing animals (Figs. 6E and 6F).
  • This enhancement in immunogenicity seen when combining our 9-mer neoepitope vaccine with N-803 and anti-PD-Ll correlated with only a slight, but significant increase in the survival of treated, as compared to control, animals (Fig. 6G).
  • This survival benefit was seen only in animals mounting a robust immune response against vaccine components, as assessed by performing an ELISPOT using peripheral blood collected from animals on day 13 of tumor growth (Fig. 6H).
  • Fig. 7A Using the treatment regimen depicted in Fig. 7A, the inventors assayed the immune response in vaccinated animals on days 11, 18 and 25 of tumor growth, and observed that the magnitude of the immunity generated against vaccine components decreased over time. Interestingly, the inventors observed that the neoepitope vaccine also resulted in the in-situ expansion of T cells reactive against neoepitopes not contained in the vaccine. However, the magnitude of these de-novo immune responses also diminished over time despite continued vaccination (Fig. 7B). In an effort to promote the maintenance of neoepitope-reactive cells within the tumor microenvironment, the inventors incorporated a single injection of NHS- IL12 to the vaccine regimen on day 18 of tumor growth.
  • neoepitope vaccine Animals treated with the combination of anti-PD-Ll, NHS-IL12, N-803 and neoepitope vaccine were able to maintain a robust immune response against neoepitopes Ptgfr and Trp53, both of which are components of the neoepitope vaccine, along with additional neoepitopes expressed by MC38 tumors, but not included in the vaccine (Fig. 7B).
  • mice with N- 803, anti-PD-Ll and NHS-IL12 did not have T cells specific for a peptide (P15e) derived from GP70, an endogenous retro vims protein expressed by MC38 cells, but not to any of the neoepitopes assayed (Fig. 7B).
  • a vaccine consisting of a single neoepitope was capable of mediating tumor regression
  • tumor-bearing animals were treated with either a single 9-mer or a pool of all four neoepitope peptides in combination with N-803, anti-PD-Ll and NHS-IL12.
  • the pool of neoepitopes was more efficient at inducing the regression of MC38 tumors than any of the single peptide vaccinations (Fig. 8A).
  • animals were depleted of NK1.1+, CD4+ or CD8+ T cells either beginning on day 1 of tumor growth (early depletion) or day 15 of tumor growth (late depletion).
  • NK1.1+ cells Animals depleted of NK1.1+ cells early during tumor growth were able to resolve MC38 tumors with kinetics similar to those seen in non-depleted animals. Early depletion of CD4+ cells in treated animals was associated with a more rapid regression of tumors as compared to non-depleted animals. Late depletion of NK1.1+ cells was associated with only 2/10 animals controlling tumor growth. Late depletion of CD4+ cells was associated with a rate of tumor resolution similar to that seen in non-depleted treated animals. As expected, both early and late depletion of CD8+ cells were associated with a lack of response to treatment, resulting in progressive tumor growth (Fig. 8B).
  • Fig. 9A and 9B The quadruple treatment regimen was shown to maximally enhance the infiltration of CD8+ T cells into the tumor.
  • the addition of NHS- IL12 to the treatment regimen had a profound impact on both the innate and adaptive immune cells within the tumor microenvironment. It promoted the expansion of Ml macrophages with a coordinate contraction of M2 macrophages (Fig. 9C); also observed was a trend in the reduction of effector and central memory CD8+ T cells, and an expansion of CD8+ effector memory cells (Fig. 9D).
  • TCR T cell receptor
  • mice treated with the neoepitope vaccine, N-803 and anti-PD-Ll the top 25% of productive rearrangements was composed of 21, 57 and 126 clones.
  • the number decreased to 1, 2 and 6 in animals treated with N-803, anti-PD-Ll and NHS-IL12.
  • the number of clones were 3, 7 and 22.
  • NHS-IL12 drives the expansion of a limited number of clones, while the neoepitope vaccine broadens the repertoire, which associates with tumor clearance (Fig. IOC).
  • An analysis of the top 100 TCR sequences detected in each sample revealed that each animal, regardless of treatment, had a unique T cell repertoire (Fig. 10D).
  • Recombinant adenoviral vectors were produced that encoded either single neoepitopes or four neoepitopes in a single viral vector (Fig. 11 A). Following the treatment schedule outlined in Fig. 1 IB, tumor-bearing animals were treated using an admix of four vectors with each single neoepitope vector administered at spatially separated injection sites, or a single viral vector encoding four neoepitopes (multi-epitope). Both the admix and multi-epitope vectors resulted in comparable immunity generated against the four neoepitopes comprising the vaccine (Fig.
  • the inventors sought to determine if the degree of epitope spread detected on day 25 of tumor growth associated with a change in the rate of tumor growth in animals treated with the combination of neoepitope vaccine, N-803, anti-PD-Ll and NHS-IL12.
  • the inventors observed a positive association between a decreased rate of tumor growth and the generation of epitope spread to numerous neoepitopes.
  • mice were vaccinated with an adenoviral vector encoding the ‘self’ tumor-associated antigen TWIST1, which is expressed by the MC38 cell line (32), and has been used in prior anti-cancer vaccine studies (33,34). Tumor regression was observed in only 2/10 animals treated with a regimen incorporating the TWIST1 -targeted vaccine (Fig. 111). Interestingly, TWIST1 vaccination was associated with the expansion of T cells reactive against tumor neoepitopes (Fig. 11J); however, the neoepitope vaccine is more efficient at mediating epitope spread than a vaccine targeting the tumor-associated antigen (Fig. 11K).
  • the numbers expressing quantities of ingredients, properties such as concentration, reaction conditions, and so forth, used to describe and claim certain embodiments of the invention are to be understood as being modified in some instances by the term “about.” Accordingly, in some embodiments, the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment. In some embodiments, the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some embodiments of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as practicable.
  • the term “treat” , “treating” or “treatment” of any disease or disorder refers, in one embodiment, to the administration of one or more compounds or compositions for the purpose of ameliorating the disease or disorder (e.g., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof) .
  • “treat”, “treating”, or “treatment” refers to the administration of one or more compounds or compositions for the purpose of alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient.
  • “treat”, “treating”, or “treatment” refers to the administration of one or more compounds or compositions for the purpose of modulating the disease or disorder, either symptomatically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., breaking the escape phase of cancer immunoediting, induction of an elimination phase of cancer immunoediting, reinstatement of equilibrium phase of cancer immunoediting), or both.
  • “treat”, “treating”, or “treatment” refers to the administration of one or more compounds or compositions for the purpose of preventing or delaying the onset or development or progression of the disease or disorder.
  • treat may result, for example in the case of cancer in the stabilization of the disease, partial, or complete response. However, and especially where the cancer is treatment resistant, the terms “treat”, “treating”, and “treatment” do not imply a cure or even partial cure.
  • patient refers to a human (including adults and children) or other mammal that is diagnosed or suspected to have a disease, and especially cancer.

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Abstract

L'invention concerne le traitement d'un cancer par l'intermédiaire d'un régime thérapeutique coordonné qui utilise divers composés et compositions qui emploient une vaccination de primo-immunisation en combinaison avec un traitement immunomodulateur et la polarisation d'une réponse immunitaire vers un profil Th1.
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US12121551B2 (en) 2020-06-10 2024-10-22 Prokarium Limited Cancer therapy
US12214001B2 (en) 2020-06-10 2025-02-04 Prokarium Limited Cancer therapy

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