WO2022056491A1 - Treatment methods - Google Patents

Treatment methods Download PDF

Info

Publication number
WO2022056491A1
WO2022056491A1 PCT/US2021/050332 US2021050332W WO2022056491A1 WO 2022056491 A1 WO2022056491 A1 WO 2022056491A1 US 2021050332 W US2021050332 W US 2021050332W WO 2022056491 A1 WO2022056491 A1 WO 2022056491A1
Authority
WO
WIPO (PCT)
Prior art keywords
tumor antigens
subject
antigens
tumor
level
Prior art date
Application number
PCT/US2021/050332
Other languages
French (fr)
Inventor
Victoria L. DEVAULT
Hubert LAM
Hanna S. STAROBINETS
Jessica Baker Flechtner
Original Assignee
Genocea Biosciences, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genocea Biosciences, Inc. filed Critical Genocea Biosciences, Inc.
Publication of WO2022056491A1 publication Critical patent/WO2022056491A1/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B30/00—Methods of screening libraries
    • C40B30/06—Methods of screening libraries by measuring effects on living organisms, tissues or cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5032—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on intercellular interactions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G01N33/505—Cells of the immune system involving T-cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6866—Interferon
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/521—Chemokines
    • G01N2333/522—Alpha-chemokines, e.g. NAP-2, ENA-78, GRO-alpha/MGSA/NAP-3, GRO-beta/MIP-2alpha, GRO-gamma/MIP-2beta, IP-10, GCP-2, MIG, PBSF, PF-4 or KC
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/521—Chemokines
    • G01N2333/523—Beta-chemokines, e.g. RANTES, I-309/TCA-3, MIP-1alpha, MIP-1beta/ACT-2/LD78/SCIF, MCP-1/MCAF, MCP-2, MCP-3, LDCF-1or LDCF-2
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/525—Tumor necrosis factor [TNF]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/555—Interferons [IFN]
    • G01N2333/56—IFN-alpha
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/555—Interferons [IFN]
    • G01N2333/57—IFN-gamma
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70514—CD4
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)
    • G01N2333/96436—Granzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • Cancer is characterized by proliferation of abnormal cells. Many treatments include costly and painful surgeries and chemotherapies. Although there is a growing interest in cancer therapies that target cancerous cells using a patient’s own immune system, such therapies have had limited success.
  • the present invention features, inter alia, methods of identifying and/or selecting antigens that improve, increase and/or stimulate immune control of a tumor or cancer and methods of administering the same. Additionally, methods described herein include obtaining a subject response profile and selecting a subject for a cancer therapy based on a subject response profile.
  • the disclosure features a method of selecting a subject for cancer therapy.
  • the method comprises: a) obtaining a first response profile from the subject, the first response profile comprising a representation of (i) a first number of identified stimulatory tumor antigens and (ii) a first number of identified inhibitory tumor antigens, at a first time; b) obtaining a second response profile from the subject, the second response profile comprising a representation of (i) a second number of identified stimulatory tumor antigens and (ii) a second number of identified inhibitory tumor antigens, at a second time; c) comparing the first response profile to the second response profile; and if (i) the first number of stimulatory tumor antigens is different from the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is different from the second number of inhibitory tumor antigens, then selecting the subject as a candidate subject for initiation, continuation, modification, discontinuation or
  • the first time is prior to initiation of a cancer therapy.
  • the second time is after initiation of a cancer therapy.
  • the first time and second time are after initiation of a cancer therapy.
  • stimulatory antigens and/or inhibitory antigens are identified by: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more
  • the cancer therapy comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor (CPI) therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
  • CPI checkpoint inhibitor
  • chemotherapy iii
  • cell-based therapy iv
  • immunotherapy cytokine therapy
  • kinase inhibitors kinase inhibitors
  • the method comprises selecting the subject as a candidate subject for modification or discontinuation of the cancer therapy. In some embodiments, the method further comprises modifying or discontinuing the cancer therapy for the subject.
  • the method comprises selecting the subject as a candidate subject for continuation of the cancer therapy. In some embodiments, the method further comprises continuing the cancer therapy for the subject.
  • Another aspect of the disclosure features a method of selecting a subject for cancer therapy, where the method comprises: a) obtaining a first response profile from the subject, the first response profile comprising a representation of (i) a first number of identified stimulatory tumor antigens and (ii) a first number of identified inhibitory tumor antigens, at a first time; b) obtaining a second response profile from the subject, the second response profile comprising a representation of (i) a second number of identified stimulatory tumor antigens and (ii) a second number of identified inhibitory tumor antigens, at a second time; c) comparing the first response profile to the second response profile; and if (i) the first number of stimulatory tumor antigens is greater than the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is less than the second number of inhibitory tumor antigens, then selecting the subject as a candidate subject for modification or discontinuation of a cancer therapy.
  • the first time is prior to initiation of a cancer therapy.
  • the second time is after initiation of a cancer therapy.
  • the first time and second time are after initiation of a cancer therapy.
  • stimulatory antigens and/or inhibitory antigens are identified by: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more
  • the cancer therapy comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
  • Another aspect of the disclosure provides a method of selecting a subject for cancer therapy, where the method comprises: a) obtaining a first response profile from the subject, the first response profile comprising a representation of (i) a first number of identified stimulatory tumor antigens and (ii) a first number of identified inhibitory tumor antigens, at a first time; b) obtaining a second response profile from the subject, the second response profile comprising a representation of (i) a second number of identified stimulatory tumor antigens and (ii) a second number of identified inhibitory tumor antigens, at a second time; c) comparing the first response profile to the second response profile; and if (i) the first number of stimulatory tumor antigens is less than the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is greater than the second number of inhibitory tumor antigens, then selecting the subject as a candidate subject for continuation of a cancer therapy.
  • the method further comprises continuing the cancer therapy for the subject.
  • the first time is prior to initiation of a cancer therapy.
  • the second time is after initiation of a cancer therapy.
  • the first time and second time are after initiation of a cancer therapy.
  • stimulatory antigens and/or inhibitory antigens are identified by: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more
  • the cancer therapy comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
  • the disclosure provides a method of selecting a subject for cancer therapy, comprising: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) obtained from a subject at a first time, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes obtained from the subject at the first time, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control)
  • APCs antigen
  • the first time is prior to initiation of a cancer therapy.
  • the second time is after initiation of a cancer therapy.
  • the first time and second time are after initiation of a cancer therapy.
  • the cancer therapy comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cellbased therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
  • the method comprises selecting the subject as a candidate subject for modification or discontinuation of the cancer therapy.
  • the method further comprises modifying or discontinuing the cancer therapy for the subject. In some embodiments, if (i) the first number of stimulatory tumor antigens is less than the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is greater than the second number of inhibitory tumor antigens, then the method comprises selecting the subject as a candidate subject for continuation of the cancer therapy. In some embodiments, the method further comprises continuing the cancer therapy for the subject.
  • the present disclosure provides a method of selecting a subject for cancer therapy, comprising: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) obtained from a subject at a first time, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes obtained from the subject at the first time, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control
  • the method further comprises modifying or discontinuing the cancer therapy for the subject.
  • the first time is prior to initiation of a cancer therapy.
  • the second time is after initiation of a cancer therapy.
  • the first time and second time are after initiation of a cancer therapy.
  • the cancer therapy comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
  • the present disclosure describes a method of selecting a subject for cancer therapy, comprising: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) obtained from a subject at a first time, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes obtained from the subject at the first time, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control
  • the method further comprises continuing the cancer therapy for the subject.
  • the first time is prior to initiation of a cancer therapy.
  • the second time is after initiation of a cancer therapy.
  • the first time and second time are after initiation of a cancer therapy.
  • the cancer therapy comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
  • the present disclosure describes a method of selecting a cancer therapy for a subject, the method comprising: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secreti
  • APCs antigen
  • the cancer therapy or sequence of cancer therapies comprises
  • checkpoint inhibitor therapy (ii) chemotherapy, (iii) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
  • the checkpoint inhibitor therapy comprises one or more agents directed to PD-1, PD-L1, CTLA4, Lag-3, Tim-3, TIGIT, BTLA, CBLB, CISH, VISTA, B7-H3, CSF-1R, or GITR.
  • the checkpoint inhibitor therapy comprises two or more agents directed to PD-1, PD-L1, CTLA4, Lag-3, Tim-3, TIGIT, BTLA, CBLB, CISH, VISTA, B7-H3, CSF-1R, or GITR.
  • the checkpoint inhibitor therapy comprises one or more of pembrolizumab, nivolumab.
  • the method further comprises administering the selected cancer therapy or sequence of cancer therapies to the subject.
  • the cancer therapy or sequence of cancer therapies comprises
  • checkpoint inhibitor therapy (ii) chemotherapy, (iii) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
  • Another aspect of the disclosure features a method of treating cancer in a subject, the method comprising: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more
  • the cancer therapy or sequence of cancer therapies comprises
  • checkpoint inhibitor therapy (ii) chemotherapy, (iii) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
  • the checkpoint inhibitor therapy comprises one or more agents directed to PD-1, PD-L1, CTLA4, Lag-3, Tim-3, TIGIT, BTLA, CBLB, CISH, VISTA, B7-H3, CSF-1R, or GITR.
  • the checkpoint inhibitor therapy comprises two or more agents directed to PD-1, PD-L1, CTLA4, Lag-3, Tim-3, TIGIT, BTLA, CBLB, CISH, VISTA, B7-H3, CSF-1R, or GITR.
  • the checkpoint inhibitor therapy comprises one or more of pembrolizumab, nivolumab.
  • the one or more immune mediators are selected from the group consisting of cytokines, soluble mediators, and cell surface markers expressed by the lymphocytes. In some embodiments, the one or more immune mediators are cytokines.
  • the one or more cytokines are selected from the group consisting of TRAIL, IFN- gamma, IL-12p70, IL-2, TNF-alpha, MIP1 -alpha, MIPl-beta, CXCL9, CXCL10, MCP1, RANTES, IL-1 beta, IL-4, IL-6, IL-8, IL-9, IL-10, IL-13, IL-15, CXCL11, IL-3, IL-5, IL-17, IL- 18, IL-21, IL-22, IL-23 A, IL-24, IL-27, IL-31, IL-32, TGF-beta, CSF, GM-CSF, TRANCE (also known as RANK L), MIP3 -alpha, fractalkine, aldesleukin, IFN-alpha-2a, IFN-alpha-2b, and pegIFN-alpha-2b.
  • the one or more immune mediators are soluble mediators.
  • the one or more soluble mediators are selected from the group consisting of granzyme A, granzyme B, sFas, sFasL, perforin, and granulysin.
  • the one or more immune mediators are cell surface markers.
  • the one or more cell surface markers are selected from the group consisting of CD107a, CD107b, CD25, CD69, CD45RA, CD45RO, CD137 (4-1BB), CD44, CD62L, CD27, CCR7, CD154 (CD40L), KLRG-1, CD71, HLA-DR, CD122 (IL-2RB), CD28, IL7Ra (CD127), CD38, CD26, CD134 (OX-40), CTLA-4 (CD 152), LAG-3, TIM-3 (CD366), CD39, PD-1 (CD279), FoxP3, TIGIT, CD 160, BTLA, 2B4 (CD244), and KLRG1.
  • the lymphocytes comprise CD4 + T cells. In some embodiments, the lymphocytes comprise CD8 + T cells. In some embodiments, lymphocyte inhibition and/or suppression is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, or 200% higher or lower than a control level.
  • lymphocyte inhibition and/or suppression is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 1, 2, or 3 standard deviations greater or lower than the mean of a control level. In some embodiments, lymphocyte inhibition and/or suppression is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) greater or lower than a median response level to a control.
  • MADs median absolute deviations
  • lymphocyte stimulation is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, or 200% higher or lower than a control level. In some embodiments, lymphocyte stimulation is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 1, 2, or 3 standard deviations higher or lower than the mean of a control level. In some embodiments, lymphocyte stimulation is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) greater or lower than a median response level to a control.
  • MADs median absolute deviations
  • Figure 1 shows exemplary elements of GEN-009-101, a first-in-human Phase l/2a study testing personalized vaccines designed using the ATLAS platform for feasibility, safety, immunogenicity and clinical activity in selected solid tumors.
  • Panel A shows Schedule 1, an exemplary dosing regimen.
  • Panel B shows a schematic of the overall design of the clinical study.
  • Figure 2 shows representative results of in vitro stimulated FluoroSpot assays on CD4+ and CD8+ T cells enriched from PBMCs collected at baseline (prior to vaccination) and at Day 50 from each of 5 patients (patients A, B, C, E, and F).
  • Figure 3 shows representative results of ex vivo FluoroSpot assays and in vitro stimulated FluoroSpot assays on CD4+ and CD8+ T cells enriched from PBMCs collected at baseline (prior to vaccination) and at Day 50 from a representative patient (patient E).
  • Panels A and B ex vivo FluoroSpot assays.
  • Panel C in vitro stimulated FluoroSpot assays.
  • Figure 4 shows representative summary results of ex vivo FluoroSpot assays and in vitro stimulated FluoroSpot assays on total PBMC or PBMCs depleted of CD4+ or CD8+ T cells collected at baseline (prior to vaccination) and at Day 50 from patients A-H and K. Data are reported as the proportion of peptides positive by the DFReq test. Circles represent baseline, squares represent D50 time point. Panel A shows ex vivo FluoroSpot assays for patients A-H, and K. Panel B shows in vitro stimulated FluoroSpot assays for patients A-H, and K. Panel C shows the proportion of SLPs scored positive by any assay for patients A-H, and K.
  • Figure 5 shows data for and the status of each patient and includes, for each patient, the tumor type, stage of cancer at diagnosis, period of time from diagnosis, prior therapies the patient received, the patient’s calculated tumor mutational burden (TMB), the number of stimulatory and inhibitory neoantigens identified for each patient, and the number of peptides in the example vaccine administered.
  • the graph indicates the status of each patient at different time points within the example vaccination regimen. The timing of example vaccination is indicated by the vertical arrows.
  • the color of the horizontal bars indicates the stage of cancer at diagnosis.
  • a blue horizontal arrow indicates that the patient has not yet completed the vaccination regimen (i.e., is within the dosing period).
  • a black horizontal arrow indicates that the patient has completed the vaccination regimen (i.e., is past the treatment period or post vaccination schedule).
  • a black circle indicates a status of “NED” or no evidence of disease.
  • Figure 6 shows representative results of ex vivo dual-analyte FluoroSpot assays on CD4+ and CD8+ T cells enriched from PBMCs of three representative patients (patients A and E; low response patient H).
  • Bulk PBMCs were isolated from the patients at baseline (prior to vaccination) and at the indicated timepoints over the course of their treatment.
  • the secretion of IFN-y and granzyme B (GrB) was quantified via ex vivo dual-analyte FluoroSpot after stimulation with overlapping peptide pools (OLPs) spanning the patient-specific SLPs used for immunization.
  • OTPs overlapping peptide pools
  • Panel A data are expressed as mean ( ⁇ SEM) spot forming cells (SFC) per million PBMCs to each of the four pools.
  • Figure 7 shows a spider plot for an idealized patient, illustrating the method of assessing GEN 009 vaccine-specific contribution to clinical responses in Part B of the study.
  • Each point on the plot represents assessment of the idealized patient’s target lesions.
  • the X axis shows time and treatment.
  • the left side of the graph shows the period of the idealized patient’s initial assessment and standard-of-care (SOC) treatment.
  • the right side of the graph shows the period of standard-of-care treatment combined with GEN 009 vaccination (SOC & Vaccine).
  • the Y axis shows percent change in the sum, or diameter, of the idealized patient’s target lesions.
  • Baseline is defined as the patient’s lesions at initial assessment.
  • PD Progressive disease
  • PR partial response
  • CR complete response
  • Figure 8 shows results for patient -058, enrolled in the Main Part B Study.
  • Panel A Spider plot, as described for Figure 7.
  • Panel B Results of in vitro stimulated FluoroSpot assays performed at baseline and at Day 50 post-initial vaccination. The height of each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN- y and TNF-a, for each SLP included in the patient’s vaccine, as indicated. CD4+ and CD8+ T cell responses are presented in the left and right panels, respectively.
  • Panel C Aggregate results of in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination.
  • PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ⁇ SD per 20,000 T cells (IVS). The dark bars indicate total IFNy responses, the lighter bars indicate total TNF ⁇ responses.
  • Panel D Radiographic imaging of mediastinal tumor, showing tumor mass at 3 timepoints (Baseline, Pre-vaccine, Postvaccine).
  • Panel E Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 10 6 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
  • Figure 9 shows results for patient -105, enrolled in the Main Part B Study.
  • Panel A Spider plot, as described for Figure 7.
  • Panel B, top Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed with PBMCs collected at baseline and at the indicated days after initial vaccination with GEN 009. The height of each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine.
  • SFU mean cytokine spot forming units
  • Panel B bottom: Results of in vitro stimulated FluoroSpot assays performed on CD4+ and CD8+ T cells enriched from PBMC at baseline and at Day 50 after initial vaccination.
  • the height of each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN-y (left axis) and TNF-a (right axis), for each SLP included in the patient’s vaccine, as indicated.
  • SFC cytokine spot forming cells
  • IFN-y left axis
  • TNF-a right axis
  • CD4+ and CD8+ T cell responses are presented in the left and right panels, respectively.
  • Panel C Summary results of ATLAS screens performed at baseline and at Day 50 after initial vaccination with GEN 009.
  • Each pie chart shows percentages of the patient’s tumor mutations that elicit stimulatory T cell responses (stimulatory antigens) or inhibitory T cell responses (inhibigens).
  • Panel D Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ⁇ SD per 10 6 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNF ⁇ responses.
  • Panel E Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 10 6 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN- 009 dosing, with black triangles representing days of vaccination.
  • Figure 10 shows results for patient -001, enrolled in the Main Part B Study.
  • Panel A Spider plot, as described for Figure 7.
  • Panel B, top Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed on PBMCs collected at baseline and at the indicated days after initial vaccination with GEN 009. The height of each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine.
  • SFU mean cytokine spot forming units
  • Panel B bottom: Results of in vitro stimulated FluoroSpot assays performed on CD4+ and CD8+ T cell enriched from PBMC at baseline and at Day 50 after initial vaccination.
  • the height of each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN-y (left axis) and TNF-a (right axis), for each SLP included in the patient’s vaccine, as indicated.
  • SFC cytokine spot forming cells
  • IFN-y left axis
  • TNF-a right axis
  • CD4+ and CD8+ T cell responses are presented in the left and right panels, respectively.
  • Panel C Summary results of ATLAS screens performed at baseline and at Day 50 after initial vaccination with GEN 009.
  • Each pie chart shows percentages of the patient’s expressed tumor mutations that elicit stimulatory T cell responses (stimulatory antigens) or inhibitory T cell responses (inhibigens).
  • Panel D Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown.
  • Panel E Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 10 6 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
  • Figure 11 shows results for patient -057, enrolled in the Main Part B Study.
  • Panel A Spider plot, as described for Figure 7.
  • Panel B, top Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed on PBMCs collected at baseline and at the indicated days after initial vaccination with GEN 009.
  • the height of each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine.
  • SFU cytokine spot forming units
  • Panel B bottom: Results of in vitro stimulated FluoroSpot assays performed on CD4+ and CD8+ T cells enriched from PBMC at baseline and at Day 50 after initial vaccination.
  • the height of each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN-y (left axis) and TNF-a (right axis), for each SLP included in the patient’s vaccine, as indicated.
  • SFC cytokine spot forming cells
  • IFN-y left axis
  • TNF-a right axis
  • CD4+ and CD8+ T cell responses are presented in the left and right panels, respectively.
  • Panel C Summary results of ATLAS screens performed at baseline and at Day 50 after initial vaccination with GEN 009.
  • Each pie chart shows percentages of the patient’s tumor mutations that elicit stimulatory T cell responses (stimulatory antigens) or inhibitory T cell responses (inhibigens).
  • Panel D Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ⁇ SD per 10 6 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNF ⁇ responses.
  • Panel E Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 10 6 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN- 009 dosing, with black triangles representing days of vaccination.
  • Figure 12 shows results for patient -031, enrolled in the Main Part B Study.
  • Panel A Spider plot, as described for Figure 7.
  • Panel B, top Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed on PBMCs collected at baseline and at the indicated days after initial vaccination with GEN 009. The height of each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine.
  • SFU mean cytokine spot forming units
  • Panel B Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ⁇ SD per 10 6 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNFa responses.
  • Panel C Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume.
  • Values for IFNy are reported on the right axis as mean background subtracted SFC per 10 6 cells.
  • the corresponding patient tumor scans are indicated on the left axis (open triangles).
  • the dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%).
  • the grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
  • FIG. 13 shows results of all ex vivo dual-color FluoroSpot assays plotted over time.
  • Peripheral blood mononuclear cells were collected from patients participating in the Main Part B Study at the indicated time points (day 1 is the day of the first GEN-009 vaccination) and viably frozen.
  • Thawed PBMCs were stimulated with pools containing the vaccine antigens (or controls).
  • Results are shown as the mean cytokine spot forming cells (SFC) per million PBMC (+/- SEM) for each subject contributing data at each time point. Not all patients contributed data for each time point: the N at each time point is indicated.
  • SFC mean cytokine spot forming cells
  • Figure 14 shows summary results for all patients enrolled in the Main Part B Study.
  • Panel A is a composite of all spider plots, as described for Figure 7, showing the percent target tumor lesion change for each patient, from the time of consent and initial assessment.
  • Panel B is a composite of all spider plots showing the percent target tumor lesion change for each patient from the time the patient received GEN-009 dose 1 (i.e., baseline is defined at day 0 of the study).
  • Panel C is a swimmers’ plot showing timelines of PD-1 standard-of-care therapy, GEN-009 dosing, and outcomes for each patient.
  • Panel D shows aggregate results for all patients of dual-analyte immunogenicity assays performed at screening and at Day 50 post-initial vaccination.
  • PBMC, CD4+ T cell and CD8+ T cell responses are shown. Values presented are the means of background subtracted SFCs ⁇ SEM per 20,000 T cells. The dark bars indicate total IFNy responses, the lighter bars indicate total TNF ⁇ responses.
  • Figure 15 shows results for patient -106, enrolled in the Part B Refractory Study.
  • Panel A Spider plot, as described for Figure 7.
  • Panel B, top Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed on PBMCs collected at baseline and at the indicated days after initial vaccination with GEN 009. The height of each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine.
  • SFU mean cytokine spot forming units
  • the DI time point represents post-checkpoint inhibitor therapy and post-chemotherapy, pre-vaccine responses; therefore, increases in frequency of PBMC responses at subsequent time points can be interpreted as attributable to GEN 009 vaccination.
  • Panel B bottom: Results of in vitro stimulated FluoroSpot assays on CD4+ and CD8+ T cells enriched from PBMCs performed at baseline and at Day 50 after initial vaccination.
  • the height of each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN-y (left axis) and TNF-a (right axis), for each SLP included in the patient’s vaccine, as indicated.
  • SFC cytokine spot forming cells
  • IFN-y left axis
  • TNF-a right axis
  • CD4+ and CD8+ T cell responses are presented in the left and right panels, respectively.
  • Panel C Summary results of ATLAS screens performed at baseline and at Day 50 after initial vaccination with GEN 009.
  • Each pie chart shows percentages of the patient’s expressed tumor mutations that elicit stimulatory T cell responses (stimulatory antigens) or inhibitory T cell responses (inhibigens).
  • Panel D Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ⁇ SD per 10 6 T cells (ex vivo, top panel) or 20,000 T cells (in vitro stimulated, bottom panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNF ⁇ responses.
  • Panel E Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 10 6 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
  • Figure 16 shows results for patient -109, enrolled in the Part B Refractory Study.
  • Panel A Spider plot, as described for Figure 7.
  • Panel B Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 postinitial vaccination.
  • PBMC, CD4+ and CD8+ responses are shown.
  • Values presented are the means of background subtracted SFCs ⁇ SD per 10 6 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNF ⁇ responses.
  • Figure 17 shows results for patient -027, enrolled in the Part B Refractory Study.
  • Panel A Spider plot, as described for Figure 7.
  • Panel B Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 10 6 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN- 009 dosing, with black triangles representing days of vaccination.
  • Figure 18 shows results for patient -110, enrolled in the Part B Refractory Study.
  • Panel A Spider plot, as described for Figure 7.
  • Panel B Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 postinitial vaccination.
  • PBMC, CD4+ and CD8+ responses are shown.
  • Values presented are the means of background subtracted SFCs ⁇ SD per 10 6 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNF ⁇ responses.
  • Figure 19 shows results for patient -060, enrolled in the Part B Refractory Study.
  • Panel A Spider plot, as described for Figure 7.
  • Figure 20 shows summary results for all patients enrolled in the Main Part B Refractory Study.
  • Panel A is a composite of all spider plots, as described for Figure 7, showing the percent target tumor lesion change for each patient, from the time of consent and initial assessment.
  • Panel B is a composite of all spider plots showing the percent target tumor lesion change for each patient from the time the patient received GEN-009 dose 1 (i.e., baseline is defined at day 0 of the study).
  • Panel C is a swimmers’ plot showing timelines of PD-1 standard- of-care therapy, GEN-009 dosing, and outcomes for each patient.
  • Panel D shows aggregate results for all patients of dual-analyte immunogenicity assays performed at screening and at Day 50 post-initial vaccination.
  • PBMC, CD4+ T cell and CD8+ T cell responses are shown. Values presented are the means of background subtracted SFCs ⁇ SEM per 20,000 T cells. The dark bars indicate total IFNy responses, the lighter bars indicate total TNF ⁇
  • Figure 21 shows representative summary results for CD4+ and CD8+ T cell responsiveness, prior to and after vaccination with GEN 009.
  • the bars labeled “ATLAS” indicate the overall percentages of antigens that elicited robust CD4+ or CD8+ T cell responses in initial ATLAS screening of the selected Part B patients and were consequently included in the GEN 009 vaccine compositions administered to these patients.
  • the bars labeled “Post GEN-009 Treatment” indicate the overall percentages of antigens that elicited CD4+ or CD8+ T cell responses in ATLAS screening of the selected Part B patients following their vaccination with GEN 009. The comparison shows changes in the breadth of T cell responses.
  • Panel B is a graph showing aggregate IFNy responses (ex vivo FluoroSpot assays) as SFCs ⁇ SEM per million PBMCs over several timepoints, from screening to 6 months after patients received GEN 009 dose 3.
  • Panel C shows the average number of neoantigens that elicited positive CD4+ and CD8+ T cell responses, as determined by ATLAS, at the time of screening and at Day 50 post-initial vaccination, broken out for Main Part B (Controlled) and Part B Refractory (Refractory) patients.
  • Panel D shows the total number of non-synonymous mutations (tumor mutational burden) for each cohort at the time of screening.
  • Figure 22 shows overall characterization of stimulatory antigens (neoantigens), inhibigens, and non-responsive antigens identified in 37 patients screened for inclusion in the Part A Study, the Main Part B Study, and the Part B Refractory Study of GEN-009.
  • Panel B Total proportion of stimulatory antigens (neoantigens), inhibigens, and non-responsive antigens.
  • Figure 23 shows the overall proportion of CD4 + and CD8 + stimulatory antigens (neoantigens), inhibigens, and non-responsive antigens identified in 37 patients screened for inclusion in the Part A Study, the Main Part B Study, and the Part B Refractory Study of GEN- 009. Results are expressed as a percentage of total CD4 + and CD8 + responses.
  • Figure 24 shows overall characterization, against total mutations screened, of somatic mutations, stimulatory antigens (neoantigens), and inhibigens identified in 37 patients screened for inclusion in the Part A Study, the Main Part B Study, and the Part B Refractory Study of GEN-009.
  • Panel A Number of mutations screened ( ⁇ SEM) by tumor type.
  • Panel B Number of inhibigens detected, by tumor type, as a percent of total mutations screened.
  • Figure 25 shows the proportion of stimulatory antigens and inhibigens identified by ATLAS screens for each patient at initial screening at D50 post-initiation of vaccinations, broken out by T cell subset, tumor type and checkpoint inhibition responsiveness vs. nonresponsiveness. For each set of bars, the top bar shows results obtained at initial screening, and the bottom bar shows results obtained at Day 50.
  • Middle rows individual patient responses, with patient ID indicated to the left of each set.
  • Bottom row pooled responses by tumor type.
  • Figure 26 shows the results of 16-plex patient-specific somatic assays for detection of circulating tumor DNA (ctDNA). Assays were performed on plasma samples taken at initial screening (Baseline) and at D50 post initiation of vaccination (Post-Tx). Two patients enrolled in the Main Part B Study are represented, one in each graph. Results are expressed as the percent frequency of variant alleles in plasma, and show disappearance of ctDNA postvaccination.
  • Figure 27 shows a representative workflow and representative results for mouse ATLAS (m ATLAS) screening, preliminary to Bl 6F 10 melanoma challenge and vaccination studies.
  • Panel A m ATLAS workflow.
  • Panel B Representative results of mATLAS screening for IFNy only.
  • Panel A shows results of IFNy ELISPOT performed on splenocytes from vaccinated mice of the first study (top graph) and of the third study (bottom graph) after in vitro stimulation with overlapping peptides spanning vaccine components. Statistical analysis was by multiple T-tests (top graph) and one-way ANOVA (bottom graph; p ⁇ 0.0001).
  • Panel B Mean tumor growth kinetics following vaccination with adjuvant only, protective vaccine, or protective vaccine plus inhibigen (first study).
  • Panel C Mean tumor growth kinetics following vaccination with adjuvant only, protective vaccine, or protective vaccine plus inhibigen (second study).
  • Panel D Mean tumor growth kinetics following vaccination with adjuvant only, or inhibigen plus adjuvant (third study).
  • Figure 29 shows immunohistochemically stained sections and quantitation of tumors from vaccinated mice.
  • Panel A shows representative tumor sections stained with antibodies specific for mouse CD4 or CD8a (T cells), FoxP3 (Tregs), F4/80 (macrophages), or CD11c (dendritic cells), in pink/red, and counterstained for nuclei with hematoxylin (blue), for assessment of infiltrating immune cell populations.
  • Mice were vaccinated with adjuvant alone (Adjuvant, top row), protective vaccine (Protective, middle row), or protective vaccine plus inhibigen (MMP9FS) (+ Inhibigen, bottom row).
  • Panel B shows quantification of Panel A images.
  • Panel C shows representative tumor sections stained for the endothelial marker CD31, for assessment of tumor vasculature. Mice were vaccinated with protective vaccine (Protective, top row), or protective vaccine plus inhibigen MMP9FS (+ Inhibigen, bottom row).
  • Panel D shows tumor sections stained for metabolic markers LDHA, ARG1, and CD73, for assessment of metabolic status. Mice were vaccinated with protective vaccine plus inhibigen MMP9FS.
  • Panel E shows quantification of Panel D images, together with quantification of images (not shown) of tumor sections from mice vaccinated with adjuvant alone, or with protective vaccine.
  • Panel F shows quantification of representative tumor sections immuno-histochemically stained for CD8 T cell markers, from mice vaccinated with adjuvant only, protective vaccine, or MMP9FS only (top graph), or mice vaccinated with adjuvant only, protective vaccine, or protective vaccine plus MMP9FS (bottom graph).
  • tumors were resected on day 11 of the study (early-stage tumors).
  • Image quantification in Panels B, E, and F was determined by Imaged software thresholding on positive stain (pink) divided by tissue area (total image area - white non-tissue). Statistical analysis was performed by one-way ANOVA with multiple comparisons.
  • Figure 30 is a schematic showing workflow for MHC competition experiments.
  • FIG 31 shows results of MHC competition experiments. Splenocytes collected from mice vaccinated with adjuvant only, protective vaccine, or the protective vaccine plus inhibigen MMP9FS were incubated with pulsed APCs (Together or Separate). After incubation, an IFNy ELISPOT was performed. Panel A: Mean IFNy Spot Forming Cells (SFC) per IxlO 5 APCs and 5xl0 5 splenocytes ( ⁇ SEM) pooled from three mice per vaccination group. Panel B: Representative wells.
  • SFC Mean IFNy Spot Forming Cells
  • ⁇ SEM splenocytes
  • FIG 32 shows results of flow cytometry experiments. Splenocytes collected from mice vaccinated with adjuvant only, protective vaccine, or the protective vaccine plus inhibigen MMP9FS and incubated with the pulsed APCs (Together) were further restimulated with respective antigen peptides, stained for intracellular cytokines, and gated by flow cytometry on live, CD45 + , CD19 nes /NKl.l nes , CD3 + , CD8 + T cells. Splenocytes were pooled from four mice from each vaccination group. IFNy expressing T cells are shown graphed as a percentage of CD3 + T cells.
  • Figure 33 is a schematic showing workflow for B 16F10 melanoma challenge and vaccination studies in conjunction with checkpoint inhibitor.
  • Figure 34 shows results of IFNy ELISPOT performed on splenocytes from vaccinated mice, with and without checkpoint inhibition, after in vitro stimulation with overlapping peptides spanning vaccine components.
  • the experimental groups received protective vaccine or protective vaccine plus inhibigen MMP9FS, in each case combined with anti-PD-1, anti-CTLA-4, or no checkpoint inhibitor.
  • Figure 35 shows mean tumor growth kinetics following vaccination in conjunction with checkpoint inhibitors.
  • Panel A Tumor growth for groups vaccinated with protective vaccine, or with protective vaccine plus inhibigen, and treated with anti-PD-1 checkpoint inhibitor (first study).
  • Panel B Tumor growth for groups vaccinated with protective vaccine or protective vaccine plus inhibigen, and treated with anti-CTLA-4 checkpoint inhibitor (first study).
  • Panel C Tumor growth for groups vaccinated with protective vaccine, or with inhibigen plus adjuvant, and treated with anti-PD-1, anti-PD-Ll, or anti-CTLA-4 checkpoint inhibitor (second study).
  • Panel D Tumor growth for groups vaccinated with protective vaccine, or with inhibigen plus adjuvant, and treated with a combination of anti-PD-1 plus anti-CTLA-4 checkpoint inhibitors (second study). Statistical analyses were performed by one-way ANOVA with multiple comparisons.
  • Figure 36 shows representative immunohistochemically stained tumor sections from mice vaccinated with adjuvant alone (Adjuvant, top row), the protective vaccine (Protective, middle row), or the protective vaccine plus inhibigen (+ Inhibigen, bottom row), with and without anti-PD-1, anti-CTLA-4, or anti-PD-1 plus anti-CTLA-4 checkpoint therapy.
  • the tumor sections were stained with an anti-mouse CD8a antibody (pink) or CD4 antibody (not pictured) and counterstained with hematoxylin (blue). Endogenous melanin deposits in tumors are visible (brown).
  • Figure 37 shows image quantification of tumor sections by ImageJ software thresholding on positive stain (pink) divided by tissue area (total image area - white non-tissue).
  • the top panel shows results for CD8 + tumor infiltrating lymphocytes (TILs); the bottom panel shows results for CD4 + TILs.
  • TILs tumor infiltrating lymphocytes
  • Each symbol in the graph represents data from one mouse, the horizontal line indicates the mean ⁇ SD.
  • Statistical analyses were performed by one-way ANOVA with multiple comparisons.
  • Figure 38 shows immunohistochemically stained sections and quantitation of tumors from vaccinated mice. Tumor sections were stained with antibodies specific for checkpoint markers CTLA-4 and PD-L1. Panel A shows representative tumor sections stained for CTLA-4 (top) and PD-L1 (bottom). Panel B shows image quantification of tumor sections stained for CTLA-4 and PD-L1, from mice vaccinated with adjuvant alone (None), protective vaccine (Protective), or protective vaccine plus inhibigen MMP9FS (+ Inhibigen), with and without anti-PD-1 and/or anti-CTLA-4 checkpoint therapy. Quantification of all immunohistochemical images was performed using ImageJ assessment of pink staining area. Graphs depict mean and standard deviation; statistical analysis is ANOVA with multiple comparisons, * p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001, ****p ⁇ 0.0001.
  • FIG 39 Panel A shows results of IFNy ELISPOT performed on splenocytes from vaccinated mice after in vitro stimulation with overlapping peptides spanning vaccine components, at day 21 of the study.
  • Panel B shows a time course of IFNy ELISPOT at days 4, 8, and 21 of the study.
  • Statistical analysis was by multiple T-tests.
  • Panel C shows flow-cytometry results for IFNy-expressing cells (left), and CD44- or CD69-expressing cells (right) from splenocytes of vaccinated mice of the first study, after non-specific stimulation with PMA/ionomycin or anti-CD3 antibody and treatment with Golgi stop and Golgi plug.
  • Panel D shows flow-cytometry results for IFNv- expressing cells from splenocytes of vaccinated mice harvested at day 4 of the second study. Cells were re-stimulated with PMA/ionomycin.
  • Panel E shows flow-cytometry results for cells expressing markers CD69, PD-1, 4- IBB (CD 137), or CD62L, from splenocytes harvested at day 4 of the second study.
  • Results are shown as the proportion of viable CD3 + T cells from a pool of 4 mice per group, for mice vaccinated with adjuvant alone (Adjuvant), protective vaccine (Protective vaccine), or protective vaccine plus inhibigen MMP9FS (Protective vaccine + Inhibigen). Responses were recalled with specific peptide stimulation from the vaccination.
  • Panel F shows flow-cytometry results for myeloid populations (Ml and M2 macrophages, and myeloid-derived suppressor cells [MDSC’s]) from splenocytes of vaccinated mice.
  • Figure 40 shows the effect of inhibigens on T cell proliferation in splenocytes of vaccinated mice from Figure 39.
  • Panel A shows total CD3 + T cell proliferation in the presence of IL-2 and anti-CD3 antibody.
  • Panel B shows specific proliferation of CD8 + T cells in response to anti-CD3 antibodies by quantitation of cell trace violet.
  • Figure 41 shows the effect of inhibigens on TCR signal strength in splenocytes of vaccinated mice from Figure 39.
  • Splenocytes were non-specifically stimulated with excess anti- CD3, at a concentration 5-fold higher than what is normally used, stained for activation markers CD69 and 4-1BB (CD137), and analyzed by flow cytometry. Results are expressed as the percentage of CD3 + T cells expressing the indicated marker, and as Mean Fluoresence Intensity (MFI) on CD3 + T cells.
  • MFI Mean Fluoresence Intensity
  • Figure 42 shows the effect of inhibigens on activation-induced cell death (AICD) in splenocytes of vaccinated mice from Figure 39.
  • Splenocytes were non-specifically stimulated with excess aCD3, at a concentration 5-fold higher than what is normally used, stained for cleaved Caspase-3 in conjunction with surface markers, and analyzed by flow cytometry.
  • PMA/ionomycin at 5-fold greater concentrations was used as a positive AICD control.
  • Results are expressed as the percentage of CD3 + T cells expressing cleaved Caspase-3.
  • Figure 43 shows gene expression of selected markers CD44, CTLA-4 and LDHA (Panel A) and PD-1, NK1 and Tox2 (Panel B) in draining lymph nodes collected from vaccinated mice. Gene expression was assessed by RNAseq. Results are expressed as RNA counts per million reads. Mice were vaccinated with adjuvant only (Adjuvant), protective vaccine plus inhibigen (Inhibitory or MMP9FS), or protective vaccine (Protective), as indicated on the x-axis. Panel C shows volcano plots, comparing representative groups of differentially expressed genes; significantly upregulated genes are shown in red, downregulated genes are shown in blue.
  • Figure 44 shows viability of cells incubated for 24 hrs in vitro with peptide corresponding to inhibigen MMP9FS.
  • Panel A shows results for BMDCs from B16F10 tumorbearing mice.
  • Panel B shows results for CD4 + T cells from B16F10 tumor-bearing mice and OT- II mice.
  • Panel C shows results for CD8 + T cells from B16F10 tumor-bearing mice and OT-I mice.
  • Figure 45 shows functional expression of the indicated markers, characterized by flow cytometry.
  • OT-I and OT-II T cells were incubated ⁇ bone marrow dendritic cells (BMDC’s), with PMA/ionomycin ⁇ inhibigen (MMP9FS). T cell activation was assessed 5 days later by flow cytometry.
  • BMDC bone marrow dendritic cells
  • MMP9FS PMA/ionomycin ⁇ inhibigen
  • Figure 46 shows representative results for adoptive transfer of T cells into a mouse B16F10 melanoma model.
  • Panel A is a schematic representation of the experiment.
  • Panel B shows mean tumor growth kinetics following adoptive transfer of T cells.
  • Figure 47 shows mean tumor growth kinetics following vaccination in conjunction with a combination of checkpoint inhibitors. Groups were vaccinated with protective vaccine, protective vaccine plus inhibigen, or inhibigen plus adjuvant, or adjuvant only (control), and treated with a combination of anti -PD-1 and CTLA-4 checkpoint inhibitors. Statistical analyses were performed by one-way ANOVA with multiple comparisons. [0086] Figure 48 shows results of IFNy ELISPOT performed on splenocytes from vaccinated mice, with and without checkpoint inhibition, after in vitro stimulation with overlapping peptides spanning vaccine components.
  • M30 and Trp2 peptides correspond to stimulatory antigens of the same name
  • S10 peptides correspond to stimulatory antigen Gal3Stl
  • In21 peptides correspond to inhibigen MMP9FS.
  • Panel A shows representative results for mice receiving protective vaccine and the checkpoint inhibitors indicated on the x axis.
  • Panel B shows representative results for mice receiving protective vaccine plus inhibigen and the checkpoint inhibitors indicated on the x axis.
  • a peptide presented by an antigen presenting cell “activates” a lymphocyte if lymphocyte activity is detectably modulated after exposure to the peptide presented by the APC under conditions that permit antigen-specific recognition to occur.
  • Any indicator of lymphocyte activity can be evaluated to determine whether a lymphocyte is activated, e.g., T cell proliferation, phosphorylation or dephosphorylation of a receptor, calcium flux, cytoskeletal rearrangement, increased or decreased expression and/or secretion of immune mediators such as cytokines or soluble mediators, increased or decreased expression of one or more cell surface markers.
  • administration typically refers to the administration of a composition to a subject or system.
  • routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human.
  • administration may be systemic or local.
  • administration may be enteral or parenteral.
  • administration may be by injection (e.g., intramuscular, intravenous, or subcutaneous injection).
  • injection may involve bolus injection, drip, perfusion, or infusion.
  • administration may be topical.
  • administration may involve electro-osmosis, hemodialysis, infiltration, iontophoresis, irrigation, and/or occlusive dressing.
  • administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing.
  • administration may involve continuous dosing.
  • Antigen refers to a molecule (e.g., a polypeptide) that elicits a specific immune response.
  • Antigen-specific immunological responses also known as adaptive immune responses, are mediated by lymphocytes (e.g., T cells, B cells, NK cells) that express antigen receptors (e.g., T cell receptors, B cell receptors).
  • lymphocytes e.g., T cells, B cells, NK cells
  • an antigen is a T cell antigen, and elicits a cellular immune response.
  • an antigen is a B cell antigen, and elicits a humoral (/. ⁇ ., antibody) response.
  • an antigen is both a T cell antigen and a B cell antigen.
  • the term “antigen” encompasses both a full-length polypeptide as well as a portion or immunogenic fragment of the polypeptide, and a peptide epitope within the polypeptides (e.g., a peptide epitope bound by a Major Histocompatibility Complex (MHC) molecule (e.g., MHC class I, or MHC class II)).
  • MHC Major Histocompatibility Complex
  • Antigen presenting cell refers to a cell that presents peptides on MHC class I and/or MHC class II molecules for recognition by T cells.
  • APC include both professional APC (e.g., dendritic cells, macrophages, B cells), which have the ability to stimulate naive lymphocytes, and non-professional APC (e.g., fibroblasts, epithelial cells, endothelial cells, glial cells).
  • APC are able to internalize (e.g., endocytose) members of a library (e.g., cells of a library of bacterial cells) that express heterologous polypeptides as candidate antigens.
  • Autolysin polypeptide is a polypeptide that facilitates or mediates autolysis of a cell (e.g., a bacterial cell) that has been internalized by a eukaryotic cell.
  • an autolysin polypeptide is a bacterial autolysin polypeptide.
  • Autolysin polypeptides include, and are not limited to, polypeptides whose sequences are disclosed in GenBank® under Acc. Nos. NP_388823.1, NP_266427.1, and P0AGC3.1.
  • cancer refers to a disease, disorder, or condition in which cells exhibit relatively abnormal, uncontrolled, and/or autonomous growth, so that they display an abnormally elevated proliferation rate and/or aberrant growth phenotype characterized by a significant loss of control of cell proliferation.
  • a cancer may be characterized by one or more tumors.
  • adrenocortical carcinoma astrocytoma, basal cell carcinoma, carcinoid, cardiac, cholangiocarcinoma, chordoma, chronic myeloproliferative neoplasms, craniopharyngioma, ductal carcinoma in situ, ependymoma, intraocular melanoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, glioma, histiocytosis, leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia, myelogenous leukemia, myeloid leukemia), lymphoma (e.g., Burkitt lymphoma [n ALL), acute myeloid leukemia (AML), chronic lymphocytic leuk
  • Cytolysin polypeptide' is a polypeptide that has the ability to form pores in a membrane of a eukaryotic cell.
  • a cytolysin polypeptide when expressed in host cell (e.g., a bacterial cell) that has been internalized by a eukaryotic cell, facilitates release of host cell components (e.g., host cell macromolecules, such as host cell polypeptides) into the cytosol of the internalizing cell.
  • a cytolysin polypeptide is bacterial cytolysin polypeptide.
  • a cytolysin polypeptide is a cytoplasmic cytolysin polypeptide.
  • Cytolysin polypeptides include, and are not limited to, polypeptides whose sequences are disclosed in U.S. Pat. No. 6,004,815, and in GenBank® under Acc. Nos. NP_463733.1, NP_979614, NP_834769, YP_084586, YP_895748, YP_694620, YP_012823, NP_346351, YP_597752, BAB41212.2, NP_561079.1, YP 001198769, and NP_359331.1.
  • Cytoplasmic cytolysin polypeptide is a cytolysin polypeptide that has the ability to form pores in a membrane of a eukaryotic cell, and that is expressed as a cytoplasmic polypeptide in a bacterial cell.
  • a cytoplasmic cytolysin polypeptide is not significantly secreted by a bacterial cell.
  • Cytoplasmic cytolysin polypeptides can be provided by a variety of means. In some embodiments, a cytoplasmic cytolysin polypeptide is provided as a nucleic acid encoding the cytoplasmic ccytolysin polypeptide.
  • a cytoplasmic cytolysin polypeptide is provided attached to a bead.
  • a cytoplasmic cytolysin polypeptide has a sequence that is altered relative to the sequence of a secreted cytolysin polypeptide (e.g., altered by deletion or alteration of a signal sequence to render it nonfunctional).
  • a cytoplasmic cytolysin polypeptide is cytoplasmic because it is expressed in a secretion-incompetent cell.
  • a cytoplasmic cytolysin polypeptide is cytoplasmic because it is expressed in a cell that does not recognize and mediate secretion of a signal sequence linked to the cytolysin polypeptide.
  • a cytoplasmic cytolysin polypeptide is a bacterial cytolysin polypeptide.
  • heterologous refers to genes or polypeptides, refers to a gene or polypeptide that does not naturally occur in the organism in which it is present and/or being expressed, and/or that has been introduced into the organism by the hand of man.
  • a heterologous polypeptide is a tumor antigen described herein.
  • Immune mediator refers to any molecule that affects the cells and processes involved in immune responses. Immune mediators include cytokines, chemokines, soluble proteins, and cell surface markers.
  • an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent.
  • the effect of a particular agent or treatment may be direct or indirect.
  • an appropriate reference measurement may be or may comprise a measurement in a comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment.
  • a peptide presented by an antigen presenting cell “stimulates” or is “stimulatory” to a lymphocyte if the lymphocyte is activated to a phenotype associated with beneficial responses, after exposure to the peptide presented by the APC under conditions that permit antigen-specific recognition to occur, as observed by, e.g., T cell proliferation, phosphorylation or dephosphorylation of a receptor, calcium flux, cytoskeletal rearrangement, increased or decreased expression and/or secretion of immune mediators such as cytokines or soluble mediators, increased or decreased expression of one or more cell surface markers, relative to a control.
  • a peptide presented by an antigen presenting cell “suppresses”, “inhibits” or is “inhibitory” to a lymphocyte if the lymphocyte is activated to a phenotype associated with deleterious or non-beneficial responses, after exposure to the peptide presented by the APC under conditions that permit antigen-specific recognition to occur, as observed by, e.g., phosphorylation or dephosphorylation of a receptor, calcium flux, cytoskeletal rearrangement, increased or decreased expression and/or secretion of immune mediators such as cytokines or soluble mediators, increased or decreased expression of one or more cell surface markers, relative to a control.
  • an “inhibitory antigen” is an antigen that inhibits, suppresses, impairs and/or reduces immune control of a tumor or cancer.
  • an inhibitory antigen promotes tumor growth, enables tumor growth, increases and/or enables tumor metastasis, and/or accelerates tumor growth.
  • an inhibitory antigen stimulates one or more lymphocyte responses that are deleterious or non-beneficial to a subject; and/or inhibits and/or suppresses one or more lymphocyte responses that are beneficial to a subject.
  • an inhibitory antigen is the target of one or more lymphocyte responses that are deleterious or non-beneficial to a subject; and/or inhibits and/or suppresses one or more lymphocyte responses that are beneficial to a subject.
  • an “invasin polypeptide” is a polypeptide that facilitates or mediates uptake of a cell (e.g., a bacterial cell) by a eukaryotic cell. Expression of an invasin polypeptide in a noninvasive bacterial cell confers on the cell the ability to enter a eukaryotic cell.
  • an invasin polypeptide is a bacterial invasin polypeptide.
  • an invasin polypeptide is a Yersinia invasin polypeptide (e.g., a Yersinia invasin polypeptide comprising a sequence disclosed in GenBank® under Acc. No. YP 070195.1).
  • Listeriolysin O (LLO)'.
  • the terms “listeriolysin O” or “LLO” refer to a listeriolysin O polypeptide of Listeria monocytogenes and truncated forms thereof that retain pore-forming ability (e.g., cytoplasmic forms of LLO, including truncated forms lacking a signal sequence).
  • an LLO is a cytoplasmic LLO. Exemplary LLO sequences are shown in Table 1, below.
  • polypeptide generally has its art- recognized meaning of a polymer of at least three amino acids. Those of ordinary skill in the art will appreciate, however, that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having the complete sequence recited herein (or in a reference or database specifically mentioned herein), but also to encompass polypeptides that represent functional fragments (/. ⁇ ., fragments retaining at least one activity) and immunogenic fragments of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity.
  • Primary cells refers to cells from an organism that have not been immortalized in vitro.
  • primary cells are cells taken directly from a subject (e.g., a human).
  • primary cells are progeny of cells taken from a subject (e.g., cells that have been passaged in vitro).
  • Primary cells include cells that have been stimulated to proliferate in culture.
  • response refers to an alteration in a subject’s condition that occurs as a result of, or correlates with, treatment.
  • a response is a beneficial response.
  • a beneficial response can include stabilization of a subject’s condition (e.g., prevention or delay of deterioration expected or typically observed to occur absent the treatment), amelioration (e.g., reduction in frequency and/or intensity) of one or more symptoms of the condition, and/or improvement in the prospects for cure of the condition, etc.
  • a beneficial response can include: the subject has a positive clinical response to cancer therapy or a combination of therapies; the subject has a spontaneous response to a cancer; the subject is in partial or complete remission from cancer; the subject has cleared a cancer; the subject has not had a relapse, recurrence or metastasis of a cancer; the subject has a positive cancer prognosis; the subject has not experienced toxic responses or side effects to a cancer therapy or combination of therapies.
  • the beneficial responses occurred in the past, or are ongoing.
  • a response is a deleterious or non-beneficial response.
  • a deleterious or non-beneficial response can include deterioration of a subject’s condition, lack of amelioration (e.g., no reduction in frequency and/or intensity) of one or more symptoms of the condition, and/or degradation in the prospects for cure of the condition, etc.
  • a deleterious or non-beneficial response can include: the subject has a negative clinical response to cancer therapy or a combination of therapies; the subject is not in remission from cancer; the subject has not cleared a cancer; the subject has had a relapse, recurrence or metastasis of a cancer; the subject has a negative cancer prognosis; the subject has experienced toxic responses or side effects to a cancer therapy or combination of therapies.
  • the deleterious or non-beneficial responses occurred in the past, or are ongoing.
  • a beneficial response in the context of a cell, organ, tissue, or cell component, e.g., a lymphocyte, “response”, “responsive”, or “responsiveness” refers to an alteration in cellular activity that occurs as a result of, or correlates with, administration of or exposure to an agent, e.g. a tumor antigen.
  • a beneficial response can include increased expression and/or secretion of immune mediators associated with positive clinical responses or outcomes in a subject.
  • a beneficial response can include decreased expression and/or secretion of immune mediators associated with negative clinical response or outcomes in a subject.
  • a deleterious or non-beneficial response can include increased expression and/or secretion of immune mediators associated with negative clinical responses or outcomes in a subject.
  • a deleterious or non- beneficial response can include decreased expression and/or secretion of immune mediators associated with positive clinical responses or outcomes in a subject.
  • a response is a clinical response.
  • a response is a cellular response.
  • a response is a direct response.
  • a response is an indirect response.
  • “non-response”, “non-responsive”, or “nonresponsiveness” mean minimal response or no detectable response.
  • a “minimal response” includes no detectable response.
  • presence, extent, and/or nature of response can be measured and/or characterized according to particular criteria.
  • criteria can include clinical criteria and/or objective criteria.
  • techniques for assessing response can include, but are not limited to, clinical examination, positron emission tomography, chest X-ray, CT scan, MRI, ultrasound, endoscopy, laparoscopy, presence or level of a particular marker in a sample, cytology, and/or histology.
  • a response of interest is a response of a tumor to a therapy
  • a response of interest is a response of a tumor to a therapy
  • methods and guidelines for assessing response to treatment are discussed in Therasse et al., J. Natl. Cancer Inst., 2000, 92(3):205-216; and Seymour et al., Lancet Oncol., 2017, 18:el43-52.
  • the exact response criteria can be selected in any appropriate manner, provided that when comparing groups of tumors, patients or experimental organism, and/or cells, organs, tissues, or cell components, the groups to be compared are assessed based on the same or comparable criteria for determining response rate.
  • One of ordinary skill in the art will be able to select appropriate criteria.
  • a “stimulatory antigen” is an antigen that improves, increases and/or stimulates immune control of a tumor or cancer.
  • a stimulatory antigen is the target of an immune response that reduces, kills, shrinks, resorbs, and/or eradicates tumor growth; does not enable tumor growth; decreases tumor metastasis, and/or decelerates tumor growth.
  • a stimulatory antigen inhibits and/or suppresses one or more lymphocyte responses that are deleterious or non-beneficial to a subject; and/or stimulates one or more lymphocyte responses that are beneficial to a subject.
  • Tumor refers to an abnormal growth of cells or tissue.
  • a tumor may comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic.
  • a tumor is associated with, or is a manifestation of, a cancer.
  • a tumor may be a disperse tumor or a liquid tumor.
  • a tumor may be a solid tumor.
  • Neoantigens potential cancer rejection antigens that are entirely absent from the normal human genome, are postulated to be relevant to tumor control; however, attempts to define them and their role in tumor clearance has been hindered by the paucity of available tools to define them in a biologically relevant and unbiased way (Schumacher and Schreiber, 2015 Science 348:69-74, Gilchuk et al., 2015 Curr Opin Immunol 34:43-51)
  • NSCLC non-small cell lung carcinoma
  • T cells that have been educated in the context of the tumor microenvironment sometimes are sub-optimally activated, have low avidity, and ultimately fail to recognize the tumor cells that express antigen.
  • tumors are complex and comprise numerous cell types with varying degrees of expression of mutated genes, making it difficult to generate polyclonal T cell responses that are adequate to control tumor growth.
  • researchers in the field have proposed that it is important in cancer subjects to identify the mutations that are “potential tumor antigens” in addition to those that are confirmed in the cancer subject to be recognized by their T cells.
  • Cancer immune therapies boost immune responses, mainly T cell responses, to kill cancer cells while sparing normal cells.
  • the success of checkpoint blockade immunotherapies in producing durable remission in a significant subset of cancer patients has reinforced that immunotherapeutic interventions can result in tumor control. Additionally, they have demonstrated the importance of tumor reactive T cells in antitumor efficacy.
  • checkpoint inhibitor therapy is effective in only 20%-30% of treated patients. Therefore, there remains a large unmet need for safe and effective immune therapies that might be applicable to a broader range of tumor types.
  • Neoantigens may induce reactive T cells that can mediate the killing of cancer cells by the host immune system (1, 2).
  • Neoantigens represent dominant targets in tumor-infiltrating lymphocyte populations in patients benefiting from adoptive T cell therapy, and a neoantigen specific T cell population was sufficient to induce tumor regression in mouse and man (3, 4).
  • a neoantigen-specific CD4 T cell product caused regression of a metastatic cholangiocarcinoma (12).
  • neoantigens Because of the tumor exclusivity of neoantigens, they can serve as tumor-specific targets for T cell-mediated recognition and destruction of tumor cells. Cancer vaccines targeting neoantigens are expected to be effective in activating T cells that can recognize and kill tumors. Several clinical trials have been initiated to directly test neoantigen vaccines. Most solid tumors harbor over 100 non-synonymous mutations; however, not all mutated proteins are processed and presented to T cells by pHLA. So far, most neoantigen vaccines depend on algorithms to identify which mutations detected in tumors are the appropriate neoantigens for inclusion in the vaccine.
  • tumor antigens e.g., tumor specific antigens (TSAs, or neoantigens), tumor associated antigens (TAAs), or cancer/testis antigens (CTAs)
  • TSAs tumor specific antigens
  • TAAs tumor associated antigens
  • CTAs cancer/testis antigens
  • tumor antigens includes both tumor antigens and potential tumor antigens.
  • methods of the present disclosure identified stimulatory tumor antigens that were not identified by known algorithms. Further, methods of the present disclosure identified suppressive and/or inhibitory tumor antigens that are not identifiable by known algorithms.
  • Methods of the present disclosure also identified polypeptides that are potential tumor antigens, z.e., polypeptides that activate T cells of non-cancerous subjects, but not T cells of subjects suffering from cancer.
  • the present disclosure also provides methods of selecting tumor antigens and potential tumor antigens, methods of using the selected tumor antigens and potential tumor antigens, immunogenic compositions comprising the selected tumor antigens and potential tumor antigens, and methods of manufacturing immunogenic compositions.
  • the present disclosure further provides methods for identifying stimulatory and/or inhibitory antigens in a particular subject suffering from cancer.
  • potential tumor antigens may be identified from a tumor sample from the subject; a library of bacterial cells or beads comprising a plurality of tumor antigens may be generated, where each bacterial cell or bead of the library comprises a different tumor antigen; APCs from the patient can then be contacted with and internatize the bacterial cells or beads.
  • the subject’s T cells are then exposed to APCs expressing the potential antigens.
  • Stimulatory and inhibitory antigens may then be identified based on the measuring T cell response to the different antigens.
  • the present disclosure also provides methods for monitoring disease progression, and effectiveness or suitability of therapies in a particular subject suffering from cancer, based on comparison of stimulatory and/or inhibitory antigen profiles obtained at different times. Also provided are methods for selecting a cancer therapy for a subject, and methods of treating cancer in a subject.
  • a library is a collection of members (e.g., cells or non-cellular particles, such as virus particles, liposomes, or beads (e.g., beads coated with polypeptides, such as in vitro translated polypeptides, e.g., affinity beads, e.g., antibody coated beads, or NTA-Ni beads bound to polypeptides of interest).
  • members of a library include (e.g., internally express or carry) polypeptides of interest described herein.
  • members of a library are cells that internally express polypeptides of interest described herein.
  • members of a library which are particles carry, and/or are bound to, polypeptides of interest.
  • a library is designed to be internalized by human antigen presenting cells so that peptides from library members, including peptides from internally expressed polypeptides of interest, are presented on MHC molecules of the antigen presenting cells for recognition by T cells.
  • Libraries can be used in assays that detect peptides presented by human MHC class I and MHC class II molecules.
  • Polypeptides expressed by the internalized library members are digested in intracellular endocytic compartments (e.g., phagosomes, endosomes, lysosomes) of the human cells and presented on MHC class II molecules, which are recognized by human CD4 + T cells.
  • library members include a cytolysin polypeptide, in addition to a polypeptide of interest.
  • library members include an invasin polypeptide, in addition to the polypeptide of interest.
  • library members include an autolysin polypeptide, in addition to the polypeptide of interest.
  • library members are provided with cells that express a cytolysin polypeptide (i.e., the cytolysin and polypeptide of interest are not expressed in the same cell, and an antigen presenting cell is exposed to members that include the cytolysin and members that include the polypeptide of interest, such that the antigen presenting cell internalizes both, and such that the cytolysin facilitates delivery of polypeptides of interest to the MHC class I pathway of the antigen presenting cell).
  • a cytolysin polypeptide can be constitutively expressed in a cell, or it can be under the control of an inducible expression system (e.g., an inducible promoter).
  • a cytolysin is expressed under the control of an inducible promoter to minimize cytotoxicity to the cell that expresses the cytolysin.
  • a cytolysin polypeptide perforates intracellular compartments in the human cell, allowing polypeptides expressed by the library members to gain access to the cytosol of the human cell. Polypeptides released into the cytosol are presented on MHC class I molecules, which are recognized by CD8 + T cells.
  • a library can include any type of cell or particle that can be internalized by and deliver a polypeptide of interest (and a cytolysin polypeptide, in applications where a cytolysin polypeptide is desirable) to, antigen presenting cells for use in methods described herein.
  • a polypeptide of interest and a cytolysin polypeptide, in applications where a cytolysin polypeptide is desirable
  • antigen presenting cells for use in methods described herein.
  • the term “cell” is used throughout the present specification to refer to a library member, it is understood that, in some embodiments, the library member is a non-cellular particle, such as a virus particle, liposome, or bead.
  • members of the library include polynucleotides that encode the polypeptide of interest (and cytolysin polypeptide), and can be induced to express the polypeptide of interest (and cytolysin polypeptide) prior to, and/or during internalization by antigen presenting cells.
  • the cytolysin polypeptide is heterologous to the library cell in which it is expressed, and facilitates delivery of polypeptides expressed by the library cell into the cytosol of a human cell that has internalized the library cell.
  • Cytolysin polypeptides include bacterial cytolysin polypeptides, such as listeriolysin O (LLO), streptolysin O (SLO), and perfringolysin O (PFO). Additional cytolysin polypeptides are described in U.S. Pat. 6,004,815.
  • library members express LLO.
  • a cytolysin polypeptide is not significantly secreted by the library cell (e.g., less than 20%, 10%, 5%, or 1% of the cytolysin polypeptide produced by the cell is secreted).
  • the cytolysin polypeptide is a cytoplasmic cytolysin polypeptide, such as a cytoplasmic LLO polypeptide (e.g., a form of LLO which lacks the N-terminal signal sequence, as described in Higgins et al., Mol. Microbiol. 31(6): 1631-1641, 1999).
  • Exemplary cytolysin polypeptide sequences are shown in Table 1.
  • the listeriolysin O (A3-25) sequence shown in the second row of Table 1 has a deletion of residues 3-25, relative to the LLO sequence in shown in the first row of Table 1, and is a cytoplasmic LLO polypeptide.
  • a cytolysin is expressed constitutively in a library host cell.
  • a cytolysin is expressed under the control of an inducible promoter. Cytolysin polypeptides can be expressed from the same vector, or from a different vector, as the polypeptide of interest in a library cell.
  • a library member (e.g., a library member which is a bacterial cell) includes an invasin that facilitates uptake by the antigen presenting cell.
  • a library member includes an autolysin that facilitates autolysis of the library member within the antigen presenting cell.
  • a library member includes both an invasin and an autolysin.
  • a library member which is an E. coll cell includes an invasin and/or an autolysin.
  • library cells that express an invasin and/or autolysin are used in methods that also employ non-professional antigen presenting cells or antigen presenting cells that are from cell lines. Isberg et al.
  • an autolysin has a feature that permits delayed lysis, e.g., the autolysin is temperature-sensitive or time-sensitive (see, e.g., Chang et al., 1995, J.
  • cytolysins also include addiction (poison/antidote) autolysins, (see, e.g., Magnuson R, et al., 1996, J. Biol. Chem. 271(31), 18705- 18710; Smith A S, et al., 991, Mol. Microbiol. 26(5), 961-970).
  • members of the library include bacterial cells.
  • the library includes non-pathogenic, non-virulent bacterial cells.
  • bacteria for use as library members include E. coli, mycobacteria, Listeria monocytogenes, Shigella flexneri, Bacillus subtilis, or Salmonella.
  • members of the library include eukaryotic cells (e.g., yeast cells).
  • members of the library include viruses (e.g., bacteriophages).
  • members of the library include liposomes. Methods for preparing liposomes that include a cytolysin and other agents are described in Kyung-Dall et al., U.S. Pat. No. 5,643,599.
  • members of the library include beads. Methods for preparing libraries comprised of beads are described, e.g., in Lam et al., Nature 354: 82-84, 1991, U.S. Pat. Nos. 5,510,240 and 7,262,269, and references cited therein.
  • a library is constructed by cloning polynucleotides encoding polypeptides of interest, or portions thereof, into vectors that express the polypeptides of interest in cells of the library.
  • the polynucleotides can be synthetically synthesized.
  • the polynucleotides can be cloned by designing primers that amplify the polynucleotides.
  • Primers can be designed using available software, such as Primer3Plus (available the following URL: bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi; see Rozen and Skaletsky, In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp. 365-386, 2000). Other methods for designing primers are known to those of skill in the art. In some embodiments, primers are constructed so as to produce polypeptides that are truncated, and/or lack hydrophobic regions (e.g., signal sequences or transmembrane regions) to promote efficient expression.
  • hydrophobic regions e.g., signal sequences or transmembrane regions
  • the location of predicted signal sequences and predicted signal sequence cleavage sites in a given open reading frame (ORF) sequence can be determined using available software, see, e.g., Dyrlov et al, J. Mol. Biol., 340:783-795, 2004, and the following URL: cbs.dtu.dk/services/SignalP/).
  • ORF open reading frame
  • Primers can also be designed to include sequences that facilitate subsequent cloning steps.
  • ORFs can be amplified directly from genomic DNA (e.g., genomic DNA of a tumor cell), or from polynucleotides produced by reverse transcription (RT-PCR) of mRNAs expressed by the tumor cell. RT-PCR of mRNA is useful, e.g., when the genomic sequence of interest contains intronic regions. PCR-amplified ORFs are cloned into an appropriate vector, and size, sequence, and expression of ORFs can be verified prior to use in immunological assays.
  • a polynucleotide encoding a polypeptide of interest is linked to a sequence encoding a tag (e.g., an N-terminal or C-terminal epitope tag) or a reporter protein (e.g., a fluorescent protein).
  • a tag e.g., an N-terminal or C-terminal epitope tag
  • a reporter protein e.g., a fluorescent protein.
  • Useful epitope tags include, for example, a polyhistidine (His) tag, a V5 epitope tag from the P and V protein of paramyxovirus, a hemagglutinin (HA) tag, a myc tag, and others.
  • His polyhistidine
  • HA hemagglutinin
  • a polynucleotide encoding a polypeptide of interest is fused to a sequence encoding a tag which is a known antigenic epitope (e.g., an MHC class I- and/or MHC class Il-restricted T cell epitope of a model antigen such as an ovalbumin), and which can be used to verify that a polypeptide of interest is expressed and that the polypeptide-tag fusion protein is processed and presented in antigen presentation assays.
  • a tag includes a T cell epitope of a murine T cell (e.g., a murine T cell line).
  • a polynucleotide encoding a polypeptide of interest is linked to a tag that facilitates purification and a tag that is a known antigenic epitope.
  • Useful reporter proteins include naturally occurring fluorescent proteins and their derivatives, for example, Green Fluorescent Protein (Aequorea Victoria) and Neon Green (Branchiostoma lanceolatum). Panels of synthetically derived fluorescent and chromogenic proteins are also available from commercial sources. [0128] Polynucleotides encoding a polypeptide of interest are cloned into an expression vector for introduction into library host cells.
  • expression vectors include elements that drive production of polypeptides of interest encoded by a polynucleotide in library host cells (e.g., promoter and other regulatory elements).
  • polypeptide expression is controlled by an inducible element (e.g., an inducible promoter, e.g., an IPTG- or arabinose- inducible promoter, or an IPTG-inducible phage T7 RNA polymerase system, a lactose (lac) promoter, a tryptophan (trp) promoter, a tac promoter, a trc promoter, a phage lambda promoter, an alkaline phosphatase (phoA) promoter, to give just a few examples; see Cantrell, Meth, in Mol. Biol., 23525 -2 6, Humana Press, Casali and Preston, Eds.).
  • an inducible element e.g., an inducible promoter, e.g., an IPTG- or arabinose- inducible promoter, or an IPTG-inducible phage T7 RNA polymerase system
  • lactose (lac) promoter e.g.,
  • polypeptides are expressed as cytoplasmic polypeptides.
  • the vector used for polypeptide expression is a vector that has a high copy number in a library host cell. In some embodiments, the vector used for expression has a copy number that is more than 25, 50, 75, 100, 150, 200, or 250 copies per cell. In some embodiments, the vector used for expression has a ColEl origin of replication.
  • Useful vectors for polypeptide expression in bacteria include pET vectors (Novagen), Gateway® pDEST vectors (Invitrogen), pGEX vectors (Amersham Biosciences), pPRO vectors (BD Biosciences), pB AD vectors (Invitrogen), pLEX vectors (Invitrogen), pMALTM vectors (New England BioLabs), pGEMEX vectors (Promega), and pQE vectors (Qiagen).
  • Vector systems for producing phage libraries are known and include Novagen T7Select® vectors, and New England Biolabs Ph D.TM Peptide Display Cloning System.
  • library host cells express (either constitutively, or when induced, depending on the selected expression system) a polypeptide of interest to at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the total cellular protein.
  • the level a polypeptide available in or on a library member e.g., cell, virus particle, liposome, bead
  • a library member e.g., cell, virus particle, liposome, bead
  • site-specific recombinases or rare-cutting restriction enzymes can be used to transfer polynucleotides between expression vectors in the proper orientation and reading frame (Walhout et al., Meth. EnzymoL 328:575-592, 2000; Marsischky et al., Genome Res. 14:2020-202, 2004; Blommel et al., Protein Expr. Purif 47:562-570, 2006).
  • expressed polypeptides e.g., purified or partially purified polypeptides
  • liposomal membranes e.g., as described in Wassef et al., U.S. Pat. No. 4,863,874; Wheatley et al., U.S. Pat. No. 4,921,757; Huang et al., U.S. Pat. No. 4,925,661; or Martin et al., U.S. Pat. No. 5,225,212.
  • a library can be designed to include full length polypeptides and/or portions of polypeptides. Expression of full length polypeptides maximizes epitopes available for presentation by a human antigen presenting cell, thereby increasing the likelihood of identifying an antigen. However, in some embodiments, it is useful to express portions of polypeptides, or polypeptides that are otherwise altered, to achieve efficient expression.
  • polynucleotides encoding polypeptides that are large (e.g., greater than 1,000 amino acids), that have extended hydrophobic regions, signal peptides, transmembrane domains, or domains that cause cellular toxicity are modified (e.g., by C-terminal truncation, N-terminal truncation, or internal deletion) to reduce cytotoxicity and permit efficient expression a library cell, which in turn facilitates presentation of the encoded polypeptides on human cells.
  • Other types of modifications such as point mutations or codon optimization, may also be used to enhance expression.
  • a library can be designed to express polypeptides from at least 5%, 10%, 15%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of ORFs in a target cell (e.g., tumor cell).
  • a target cell e.g., tumor cell
  • a library expresses at least 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2500, 5000, 10,000, or more different polypeptides of interest, each of which may represent a polypeptide encoded by a single full length polynucleotide or portion thereof.
  • assays may focus on identifying antigens that are secreted polypeptides, cell surface-expressed polypeptides, or virulence determinants, e.g., to identify antigens that are likely to be targets of both humoral and cell mediated immune responses.
  • libraries can include tags or reporter proteins that allow one to easily purify, analyze, or evaluate MHC presentation, of the polypeptide of interest.
  • polypeptides expressed by a library include C- terminal tags that include both an MHC class I and an MHC class Il-restricted T cell epitope from a model antigen, such as chicken ovalbumin (OVA).
  • OVA chicken ovalbumin
  • Library protein expression and MHC presentation is validated using these epitopes.
  • the epitopes are OVA247- 265 and OVA258-265 respectfully, corresponding to positions in the amino acid sequence found in GenBank® under Acc. No. NP 990483.
  • T cell hybridomas e.g., B3Z T hybridoma cells, which are H2-K b restricted, and KZO T hybridoma cells, which are H2-A k restricted
  • T cell hybridomas e.g., B3Z T hybridoma cells, which are H2-K b restricted, and KZO T hybridoma cells, which are H2-A k restricted
  • Sets of library members e.g., bacterial cells
  • an array e.g., on a solid support, such as a 96-well plate
  • members in each location express a different polypeptide of interest, or a different set of polypeptides of interest.
  • library members also have utility in assays to identify B cell antigens.
  • lysate prepared from library members that include polypeptides of interest can be used to screen a sample comprising antibodies (e.g, a serum sample) from a subject (e.g, a subject who has been exposed to an infectious agent of interest, a subject who has cancer, and/or a control subject), to determine whether antibodies present in the subject react with the polypeptide of interest.
  • Suitable methods for evaluating antibody reactivity are known and include, e.g., ELISA assays.
  • methods and compositions described herein can be used to identify and/or detect immune responses to a polypeptide of interest.
  • a polypeptide of interest is encoded by an ORF from a target tumor cell, and members of a library include (e.g., internally express or carry) ORFs from a target tumor cell.
  • members of a library include (e.g., internally express or carry) ORFs from a target tumor cell.
  • a library can be used in methods described herein to assess immune responses to one or more polypeptides of interest encoded by one or more ORFs.
  • methods of the disclosure identify one or more polypeptides of interest as stimulatory antigens (e.g., that stimulate an immune response, e.g., a T cell response, e.g., expression and/or secretion of one or more immune mediators).
  • methods of the disclosure identify one or more polypeptides of interest as antigens or potential antigens that have minimal or no effect on an immune response (e.g., expression and/or secretion of one or more immune mediators).
  • methods of the disclosure identify one or more polypeptides of interest as inhibitory and/or suppressive antigens (e.g., that inhibit, suppress, down-regulate, impair, and/or prevent an immune response, e.g., a T cell response, e.g., expression and/or secretion of one or more immune mediators).
  • methods of the disclosure identify one or more polypeptides of interest as tumor antigens or potential tumor antigens, e.g., tumor specific antigens (TSAs, or neoantigens), tumor associated antigens (TAAs), or cancer/testis antigens (CTAs).
  • TSAs tumor specific antigens
  • TAAs tumor associated antigens
  • CTAs cancer/testis antigens
  • a polypeptide of interest is a putative tumor antigen
  • methods and compositions described herein can be used to identify and/or detect immune responses to one or more putative tumor antigens.
  • members of a library include (e.g., internally express or carry) putative tumor antigens (e.g., a polypeptide previously identified (e.g., by a third party) as a tumor antigen, e.g., identified as a tumor antigen using a method other than a method of the present disclosure).
  • a putative tumor antigen is a tumor antigen described herein.
  • such libraries can be used to assess whether and/or the extent to which such putative tumor antigen mediates an immune response.
  • methods of the disclosure identify one or more putative tumor antigens as stimulatory antigens.
  • methods of the disclosure identify one or more putative tumor antigens as antigens that have minimal or no effect on an immune response.
  • methods of the disclosure identify one or more putative tumor antigens as inhibitory and/or suppressive antigens.
  • a polypeptide of interest is a pre-selected tumor antigen
  • methods and compositions described herein can be used to identify and/or detect immune responses to one or more pre-selected tumor antigens.
  • members of a library include (e.g., internally express or carry) one or more polypeptides identified as tumor antigens using a method of the present disclosure and/or using a method other than a method of the present disclosure.
  • such libraries can be used to assess whether and/or the extent to which such tumor antigens mediate an immune response by an immune cell from one or more subjects (e.g., a subject who has cancer and/or a control subject) to obtain one or more response profiles described herein.
  • methods of the disclosure identify one or more pre-selected tumor antigens as stimulatory antigens for one or more subjects.
  • methods of the disclosure identify one or more pre-selected tumor antigens as antigens that have minimal or no effect on an immune response for one or more subjects.
  • methods of the disclosure identify one or more pre-selected tumor antigens as inhibitory and/or suppressive antigens for one or more subjects.
  • a polypeptide of interest is a known tumor antigen
  • methods and compositions described herein can be used to identify and/or detect immune responses to one or more known tumor antigens.
  • members of a library include (e.g., internally express or carry) one or more polypeptides identified as a tumor antigen using a method of the present disclosure and/or using a method other than a method of the present disclosure.
  • such libraries can be used to assess whether and/or the extent to which such tumor antigens mediate an immune response by an immune cell from one or more subjects (e.g., a subject who has cancer and/or a control subject) to obtain one or more response profiles described herein.
  • methods of the disclosure identify one or more known tumor antigens as stimulatory antigens for one or more subjects. In some embodiments, methods of the disclosure identify one or more known tumor antigens as antigens that have minimal or no effect on an immune response for one or more subjects. In some embodiments, methods of the disclosure identify one or more known tumor antigens as inhibitory and/or suppressive antigens for one or more subjects.
  • a polypeptide of interest is a potential tumor antigen
  • methods and compositions described herein can be used to identify and/or detect immune responses to one or more potential tumor antigens.
  • members of a library include (e.g., internally express or carry) one or more polypeptides identified as being of interest, e.g., encoding mutations associated with a tumor, using a method of the present disclosure and/or using a method other than a method of the present disclosure.
  • such libraries can be used to assess whether and/or the extent to which such polypeptides mediate an immune response by an immune cell from one or more subjects (e.g., a subject who has cancer and/or a control subject) to obtain one or more response profiles described herein.
  • methods of the disclosure identify one or more polypeptides as stimulatory antigens for one or more subjects.
  • methods of the disclosure identify one or more polypeptides as antigens that have minimal or no effect on an immune response for one or more subjects.
  • methods of the disclosure identify one or more polypeptides as inhibitory and/or suppressive antigens for one or more subjects.
  • Polypeptides of interest used in methods and systems described herein include tumor antigens and potential tumor antigens, e.g., tumor specific antigens (TSAs, or neoantigens), tumor associated antigens (TAAs), and/or cancer/testis antigens (CTAs).
  • TSAs tumor specific antigens
  • TAAs tumor associated antigens
  • CTAs cancer/testis antigens
  • Exemplary tumor antigens include, e.g., MART-l/MelanA (MART -I or MLANA), gplOO (Pmel 17 or SILV), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3 (also known as HIP8), BAGE, GAGE-1, GAGE-2, pl 5, Calcitonin, Calretinin, Carcinoembryonic antigen (CEA), Chromogranin, Cytokeratin, Desmin, Epithelial membrane protein (EMA), Factor VIII, Glial fibrillary acidic protein (GFAP), Gross cystic disease fluid protein (GCDFP-15), HMB-45, Human chorionic gonadotropin (hCG), inhibin, lymphocyte marker, MART-1 (Melan-A), Myo DI, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase (PLAP), prostate-specific anti
  • TSAs Tumor specific antigens
  • TSAs are tumor antigens that are not encoded in normal host genome (see, e.g., Yarchoan et al., Nat. Rev. Cancer. 2017 Feb 24. doi: 10.1038/nrc.2016.154; Gubin et al., J. Clin. Invest. 125:3413-3421 (2015)).
  • TSAs arise from somatic mutations and/or other genetic alterations.
  • TSAs arise from missense or in-frame mutations.
  • TSAs arise from frame-shift mutations or loss-of-stop-codon mutations.
  • TSAs arise from insertion or deletion mutations.
  • TSAs arise from duplication or repeat expansion mutations. In some embodiments, TSAs arise from splice variants or improper splicing. In some embodiments, TSAs arise from gene fusions. In some embodiments, TSAs arise from translocations. In some embodiments, TSAs include oncogenic viral proteins. For example, as with Merkel cell carcinoma (MCC) associated with the Merkel cell polyomavirus (MCPy V) and cancers of the cervix, oropharynx and other sites associated with the human papillomavirus (HPV), TSAs include proteins encoded by viral open reading frames.
  • MCC Merkel cell carcinoma
  • MCPy V Merkel cell polyomavirus
  • HPV human papillomavirus
  • TSAs are specific (personal) to a subject.
  • TSAs are shared by more than one subject, e.g., less than 1%, 1-3%, 1-5%, 1-10% , or more of subjects suffering from a cancer.
  • TSAs shared by more than one subject may be known or pre-selected.
  • a TSA is encoded by an open reading frame from a virus.
  • a library can be designed to express polypeptides from one of the following viruses: an immunodeficiency virus (e.g., a human immunodeficiency virus (HIV), e.g., HIV-1, HIV-2), a hepatitis virus (e.g., hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis A virus, non-A and non-B hepatitis virus), a herpes virus (e.g., herpes simplex virus type I (HSV- 1), HSV-2, Varicella-zoster virus, Epstein Barr virus, human cytomegalovirus, human herpesvirus 6 (HHV-6), HHV-7, HHV-8), a poxvirus (e.g., variola, vaccinia, monkeypox, Molluscum contagiosum virus), an influenza virus,
  • an immunodeficiency virus
  • Tumor specific antigens are known in the art, any of which can be used in methods described herein.
  • gene sequences encoding polypeptides that are potential or putative neoantigens are determined by sequencing the genome and/or exome of tumor tissue and healthy tissue from a subject having cancer using next generation sequencing technologies.
  • genes that are selected based on their frequency of mutation and ability to encode a potential or putative neoantigen are sequenced using next- generation sequencing technology.
  • Next-generation sequencing applies to genome sequencing, genome resequencing, transcriptome profiling (RNA-Seq), DNA-protein interactions (ChlP- sequencing), and epigenome characterization (de Magalhaes et al.
  • Nextgeneration sequencing can be used to rapidly reveal the presence of discrete mutations such as coding mutations in individual tumors, e.g., single amino acid changes (e.g., missense mutations, in-frame mutations) or novel stretches of amino acids generated by frame-shift insertions, deletions, gene fusions, read-through mutations in stop codons, duplication or repeat expansion mutations, and translation of splice variants or improperly spliced introns, and translocations (e.g., “neoORFs”).
  • single amino acid changes e.g., missense mutations, in-frame mutations
  • novel stretches of amino acids generated by frame-shift insertions, deletions, gene fusions, read-through mutations in stop codons, duplication or repeat expansion mutations e.g., “neoORFs”.
  • Another method for identifying potential or putative neoantigens is direct protein sequencing. Protein sequencing of enzymatic digests using multidimensional MS techniques (MSn) including tandem mass spectrometry (MS/MS)) can also be used to identify neoantigens. Such proteomic approaches can be used for rapid, highly automated analysis (see, e.g., Gevaert et al., Electrophoresis 21 : 1145-1154 (2000)). High-throughput methods for de novo sequencing of unknown proteins can also be used to analyze the proteome of a subject’s tumor to identify expressed potential or putative neoantigens. For example, meta shotgun protein sequencing may be used to identify expressed potential or putative neoantigens (see e.g., Guthals et al. (2012) Molecular and Cellular Proteomics 11(10): 1084-96).
  • Potential or putative neoantigens may also be identified using MHC multimers to identify neoantigen-specific T cell responses.
  • MHC tetramerbased screening techniques see e.g., Hombrink et al. (2011) PLoS One; 6(8): e22523; Hadrup et al. (2009) Nature Methods, 6(7):520-26; van Rooij et al. (2013) Journal of Clinical Oncology, 31 : 1-4; and Heemskerk et al. (2013) EMBO Journal, 32(2): 194-203).
  • one or more known or pre-selected tumor specific antigens, or one or more potential or putative tumor specific antigens identified using one of these methods can be included in a library described herein.
  • Tumor associated antigens include proteins encoded in a normal genome (see, e.g., Ward et al., Adv. Immunol. 130:25-74 (2016)).
  • TAAs are either normal differentiation antigens or aberrantly expressed normal proteins.
  • Overexpressed normal proteins that possess growth/ survival -promoting functions such as Wilms tumor 1 (WT1) (Ohminami et al., Blood 95:286-293 (2000)) or Her2/neu (Kawashima et al., Cancer Res. 59:431-435 (1999)), are TAAs that directly participate in the oncogenic process.
  • WT1 Wilms tumor 1
  • Her2/neu Kawashima et al., Cancer Res. 59:431-435 (1999)
  • TAAs Post- translational modifications, such as phosphorylation, of proteins may also lead to formation of TAAs (Doyle, J. Biol. Chem. 281 :32676-32683 (2006); Cobbold, Sci. Transl. Med. 5:203ral25 (2013)).
  • TAAs are generally shared by more than one subject, e.g., less than 1%, 1-3%, 1-5%, 1- 10%, 1-20%, or more of subjects suffering from a cancer.
  • TAAs are known or pre-selected tumor antigens.
  • TAAs are potential or putative tumor antigens.
  • CTAs Cancer/testis antigens
  • reproductive tissues for example, testes, fetal ovaries and trophoblasts
  • MHC class I molecules see, e.g., Coulie et al., Nat. Rev. Cancer 14: 135-146 (2014); Simpson et al., Nat. Rev. Cancer 5:615-625 (2005); Scanlan et al., Immunol. Rev. 188:22-32 (2002)).
  • Methods of the present disclosure utilize human antigen presenting cells.
  • Human antigen presenting cells express ligands for antigen receptors and other immune activation molecules on human lymphocytes. Given differences in MHC peptide binding specificities and antigen processing enzymes between species, antigens processed and presented by human cells are more likely to be physiologically relevant human antigens in vivo than antigens identified in non-human systems. Accordingly, methods of identifying these antigens employ human cells to present candidate tumor antigen polypeptides. Any human cell that internalizes library members and presents polypeptides expressed by the library members on MHC molecules can be used as an antigen presenting cell according to the present disclosure. In some embodiments, human cells used for antigen presentation are primary human cells.
  • the cells can include peripheral blood mononuclear cells (PBMC) of a human.
  • peripheral blood cells are separated into subsets (e.g., subsets comprising dendritic cells, macrophages, monocytes, B cells, or combinations thereof) prior to use in an antigen presentation assay.
  • a subset of cells that expresses MHC class II is selected from peripheral blood.
  • a cell population including dendritic cells is isolated from peripheral blood.
  • a subset of dendritic cells is isolated (e.g., plasmacytoid, myeloid, or a subset thereof).
  • Human dendritic cell markers include CDlc, CDla, CD303, CD304, CD141, and CD209. Cells can be selected based on expression of one or more of these markers (e.g., cells that express CD303, CDlc, and CD141).
  • Dendritic cells can be isolated by positive selection from peripheral blood using commercially available kits (e.g., from Miltenyi Biotec Inc.). In some embodiments, the dendritic cells are expanded ex vivo prior to use in an assay. Dendritic cells can also be produced by culturing peripheral blood cells under conditions that promote differentiation of monocyte precursors into dendritic cells in vitro. These conditions typically include culturing the cells in the presence of cytokines such as GM-CSF and IL-4 (see, e.g., Inaba et al., Isolation of dendritic cells, Curr. Protoc. Immunol. May; Chapter 3: Unit 3.7, 2001).
  • cytokines such as GM-CSF and IL-4
  • CD34 + hematopoietic stem and progenitor cells are isolated from peripheral blood or bone marrow and expanded in vitro in culture conditions that include one or more of Flt3-L, IL-1, IL-3, and c-kit ligand.
  • immortalized cells that express human MHC molecules are used for antigen presentation.
  • assays can employ COS cells transfected with human MHC molecules or HeLa cells.
  • both the antigen presenting cells and immune cells used in the method are derived from the same subject (e.g., autologous T cells and APC are used).
  • DC dendritic cells
  • DC are used with T- and DC-depleted cells in an assay, at a ratio of 1 :2, 1 :3, 1 :4, or 1 :5.
  • the antigen presenting cells and immune cells used in the method are derived from different subjects (e.g., heterologous T cells and APC are used).
  • Antigen presenting cells can be isolated from sources other than peripheral blood.
  • antigen presenting cells can be taken from a mucosal tissue (e.g., nose, mouth, bronchial tissue, tracheal tissue, the gastrointestinal tract, the genital tract (e.g., vaginal tissue), or associated lymphoid tissue), peritoneal cavity, lymph nodes, spleen, bone marrow, thymus, lung, liver, kidney, neuronal tissue, endocrine tissue, or other tissue, for use in screening assays.
  • cells are taken from a tissue that is the site of an active immune response (e.g, an ulcer, sore, or abscess). Cells may be isolated from tissue removed surgically, via lavage, or other means.
  • Antigen presenting cells useful in methods described herein are not limited to “professional” antigen presenting cells.
  • non-professional antigen presenting cells can be utilized effectively in the practice of methods of the present disclosure.
  • Non-professional antigen presenting cells include fibroblasts, epithelial cells, endothelial cells, neuronal/glial cells, lymphoid or myeloid cells that are not professional antigen presenting cells (e.g, T cells, neutrophils), muscle cells, liver cells, and other types of cells.
  • Antigen presenting cells are cultured with library members that express a polypeptide of interest (and, if desired, a cytolysin polypeptide) under conditions in which the antigen presenting cells internalize, process and present polypeptides expressed by the library members on MHC molecules.
  • library members are killed or inactivated prior to culture with the antigen presenting cells.
  • Cells or viruses can be inactivated by any appropriate agent (e.g., fixation with organic solvents, irradiation, freezing).
  • the library members are cells that express ORFs linked to a tag (e.g., a tag which comprises one or more known T cell epitopes) or reporter protein, expression of which has been verified prior to the culturing.
  • antigen presenting cells are incubated with library members at 37°C for between 30 minutes and 5 hours (e.g., for 45 min. to 1.5 hours). After the incubation, the antigen presenting cells can be washed to remove library members that have not been internalized. In certain embodiments, the antigen presenting cells are non-adherent, and washing requires centrifugation of the cells. The washed antigen presenting cells can be incubated at 37°C for an additional period of time (e.g., 30 min. to 2 hours) prior to exposure to lymphocytes, to allow antigen processing. In some embodiments, it is desirable to fix and kill the antigen presenting cells prior to exposure to lymphocytes (e.g., by treating the cells with 1% paraformaldehyde).
  • antigen presenting cell and library member numbers can be varied, so long as the library members provide quantities of polypeptides of interest sufficient for presentation on MHC molecules.
  • antigen presenting cells are provided in an array, and are contacted with sets of library cells, each set expressing a different polypeptide of interest.
  • each location in the array includes 1 x 10 3 - 1 x 10 6 antigen presenting cells, and the cells are contacted with 1 x 10 3 - 1 x 10 8 library cells which are bacterial cells.
  • antigen presenting cells can be freshly isolated, maintained in culture, and/or thawed from frozen storage prior to incubation with library cells, or after incubation with library cells.
  • Human Lymphocytes are tested for antigenspecific reactivity to antigen presenting cells, e.g., antigen presenting cells that have been incubated with libraries expressing polypeptides of interest as described above.
  • the methods of the present disclosure permit rapid identification of human antigens using pools of lymphocytes isolated from an individual, or progeny of the cells. The detection of antigen-specific responses does not rely on laborious procedures to isolate individual T cell clones.
  • the human lymphocytes are primary lymphocytes.
  • human lymphocytes are NKT cells, gamma-delta T cells, or NK cells.
  • a population of lymphocytes having a specific marker or other feature can be used.
  • a population of T lymphocytes is isolated.
  • a population of CD4 + T cells is isolated.
  • a population of CD8 + T cells is isolated.
  • CD8 + T cells recognize peptide antigens presented in the context of MHC class I molecules.
  • the CD8 + T cells are used with antigen presenting cells that have been exposed to library host cells that co-express a cytolysin polypeptide, in addition to a polypeptide of interest.
  • T cell subsets that express other cell surface markers may also be isolated, e.g., to provide cells having a particular phenotype. These include CLA (for skin-homing T cells), CD25, CD30, CD69, CD 154 (for activated T cells), CD45RO (for memory T cells), CD294 (for Th2 cells), y/5 TCR- expressing cells, CD3 and CD56 (for NK T cells). Other subsets can also be selected.
  • Lymphocytes can be isolated, and separated, by any means known in the art (e.g., using antibody -based methods such as those that employ magnetic bead separation, panning, or flow cytometry). Reagents to identify and isolate human lymphocytes and subsets thereof are well known and commercially available.
  • Lymphocytes for use in methods described herein can be isolated from peripheral blood mononuclear cells, or from other tissues in a human.
  • lymphocytes are taken from tumors, lymph nodes, a mucosal tissue (e.g., nose, mouth, bronchial tissue, tracheal tissue, the gastrointestinal tract, the genital tract (e.g., vaginal tissue), or associated lymphoid tissue), peritoneal cavity, spleen, thymus, lung, liver, kidney, neuronal tissue, endocrine tissue, peritoneal cavity, bone marrow, or other tissues.
  • cells are taken from a tissue that is the site of an active immune response (e.g., an ulcer, sore, or abscess). Cells may be isolated from tissue removed surgically, via lavage, or other means.
  • Lymphocytes taken from an individual can be maintained in culture or frozen until use in antigen presentation assays.
  • freshly isolated lymphocytes can be stimulated in vitro by antigen presenting cells exposed to library cells as described above.
  • these lymphocytes exhibit detectable stimulation without the need for prior non-antigen specific expansion.
  • primary lymphocytes also elicit detectable antigenspecific responses when first stimulated non-specifically in vitro.
  • lymphocytes are stimulated to proliferate in vitro in a non-antigen specific manner, prior to use in an antigen presentation assay.
  • Lymphocytes can also be stimulated in an antigen-specific manner prior to use in an antigen presentation assay.
  • cells are stimulated to proliferate by a library (e.g., prior to use in an antigen presentation assay that employs the library). Expanding cells in vitro provides greater numbers of cells for use in assays.
  • Primary T cells can be stimulated to expand, e.g., by exposure to a polyclonal T cell mitogen, such as phytohemagglutinin or concanavalin, by treatment with antibodies that stimulate proliferation, or by treatment with particles coated with the antibodies.
  • T cells are expanded by treatment with anti-CD2, anti-CD3, and anti-CD28 antibodies.
  • T cells are expanded by treatment with interleukin-2.
  • lymphocytes are thawed from frozen storage and expanded (e.g., stimulated to proliferate, e.g., in a non-antigen specific manner or in an antigen-specific manner) prior to contacting with antigen presenting cells.
  • lymphocytes are thawed from frozen storage and are not expanded prior to contacting with antigen presenting cells.
  • lymphocytes are freshly isolated and expanded (e.g., stimulated to proliferate, e.g., in a non-antigen specific manner or in an antigen-specific manner) prior to contacting with antigen presenting cells.
  • T cells are cultured with antigen presenting cells prepared according to the methods described above, under conditions that permit T cell recognition of peptides presented by MHC molecules on the antigen presenting cells.
  • T cells are incubated with antigen presenting cells at 37°C for between 12-48 hours (e.g., for 24 hours).
  • T cells are incubated with antigen presenting cells at 37°C for 3, 4, 5, 6, 7, or 8 days. Numbers of antigen presenting cells and T cells can be varied.
  • the ratio of T cells to antigen presenting cells in a given assay is 1 : 10, 1 :5, 1 :2, 1 : 1, 2: 1, 5: 1, 10: 1, 20: 1, 25: 1, 30: 1, 32: 1, 35:1 or 40: 1.
  • antigen presenting cells are provided in an array (e.g., in a 96-well plate), wherein cells in each location of the array have been contacted with sets of library cells, each set including a different polypeptide of interest.
  • each location in the array includes 1 x 10 3 - 1 x 10 6 antigen presenting cells, and the cells are contacted with 1 X 10 3 - 1 X 10 6 T cells.
  • lymphocyte activation can be detected by any means known in the art, e.g., T cell proliferation, phosphorylation or dephosphorylation of a receptor, calcium flux, cytoskeletal rearrangement, increased or decreased expression and/or secretion of immune mediators such as cytokines or soluble mediators, increased or decreased expression of one or more cell surface markers.
  • culture supernatants are harvested and assayed for increased and/or decreased expression and/or secretion of one or more polypeptides associated with activation, e.g., a cytokine, soluble mediator, cell surface marker, or other immune mediator.
  • the one or more cytokines are selected from TRAIL, IFN-gamma, IL-12p70, IL-2, TNF-alpha, MIP1 -alpha, MIPl-beta, CXCL9, CXCL10, MCP1, RANTES, IL-1 beta, IL-4, IL-6, IL-8, IL-9, IL-10, IL-13, IL-15, CXCL11, IL-3, IL-5, IL-17, IL- 18, IL-21, IL-22, IL-23 A, IL-24, IL-27, IL-31, IL-32, TGF-beta, CSF, GM-CSF, TRANCE (also known as RANK L), MIP3 -alpha, and fractalkine.
  • the one or more soluble mediators are selected from granzyme A, granzyme B, sFas, sFasL, perforin, and granulysin.
  • the one or more cell surface markers are selected from CD107a, CD107b, CD25, CD69, CD45RA, CD45RO, CD137 (4-1BB), CD44, CD62L, CD27, CCR7, CD 154 (CD40L), KLRG-1, CD71, HLA-DR, CD 122 (IL-2RB), CD28, IL7Ra (CD 127), CD38, CD26, CD134 (OX-40), CTLA-4 (CD152), LAG-3, TIM-3 (CD366), CD39, PD-1 (CD279), FoxP3, TIGIT, CD160, BTLA, 2B4 (CD244), and KLRG1.
  • Cytokine secretion in culture supernatants can be detected, e.g., by ELISA, bead array, e.g., with a Luminex® analyzer. Cytokine production can also be assayed by RT-PCR of mRNA isolated from the T cells, or by ELISPOT analysis of cytokines released by the T cells.
  • proliferation of T cells in the cultures is determined (e.g., by detecting 3 H thymidine incorporation).
  • target cell lysis is determined (e.g., by detecting T cell dependent lysis of antigen presenting cells labeled with Na2 51 CrO4). Target cell lysis assays are typically performed with CD8 + T cells.
  • Protocols for these detection methods are known. See, e.g., Current Protocols In Immunology, John E. Coligan et al. (eds), Wiley and Sons, New York, N.Y., 2007.
  • appropriate controls are used in these detection methods, e.g, to adjust for non-antigen specific background activation, to confirm the presenting capacity of antigen presenting cells, and to confirm the viability of lymphocytes.
  • antigen presenting cells and lymphocytes used in the method are from the same individual. In some embodiments, antigen presenting cells and lymphocytes used in the method are from different individuals.
  • antigen presentation assays are repeated using lymphocytes from the same individual that have undergone one or more previous rounds of exposure to antigen presenting cells, e.g, to enhance detection of responses, or to enhance weak initial responses. In some embodiments, antigen presentation assays are repeated using antigen presenting cells from the same individual that have undergone one or more previous rounds of exposure to a library, e.g, to enhance detection of responses, or to enhance weak initial responses.
  • antigen presentation assays are repeated using lymphocytes from the same individual that have undergone one or more previous rounds of exposure to antigen presenting cells, and antigen presenting cells from the same individual that have undergone one or more previous rounds of exposure to a library, e.g., to enhance detection of responses, or to enhance weak initial responses.
  • antigen presentation assays are repeated using antigen presenting cells and lymphocytes from different individuals, e.g., to identify antigens recognized by multiple individuals, or compare reactivities that differ between individuals.
  • One advantage of methods described herein is their ability to identify clinically relevant human antigens.
  • Humans that have cancer may have lymphocytes that specifically recognize tumor antigens, which are the product of an adaptive immune response arising from prior exposure.
  • these cells are present at a higher frequency than cells from an individual who does not have cancer, and/or the cells are readily reactivated when reexposed to the proper antigenic stimulus (e.g., the cells are “memory” cells).
  • the cells are “memory” cells.
  • the individual has been recently diagnosed with cancer (e.g., the individual was diagnosed less than one year, three months, two months, one month, or two weeks, prior to isolation of lymphocytes and/or antigen presenting cells from the individual). In some embodiments, the individual was first diagnosed with cancer more than three months, six months, or one year prior to isolation of lymphocytes and/or antigen presenting cells.
  • lymphocytes are screened against antigen presenting cells that have been contacted with a library of cells whose members express or carry polypeptides of interest, and the lymphocytes are from an individual who has not been diagnosed with cancer.
  • lymphocytes are used to determine background (i.e., non-antigen- specific) reactivities.
  • lymphocytes are used to identify antigens, reactivity to which exists in non-cancer individuals.
  • Cells from multiple donors can be collected and assayed in methods described herein.
  • cells from multiple donors are assayed in order to determine if a given tumor antigen is reactive in a broad portion of the population, or to identify multiple tumor antigens that can be later combined to produce an immunogenic composition that will be effective in a broad portion of the population.
  • Antigen presentation assays are useful in the context of both infectious and non- infectious diseases.
  • the methods described herein are applicable to any context in which a rapid evaluation of human cellular immunity is beneficial.
  • antigenic reactivity to polypeptides that are differentially expressed by neoplastic cells e.g., tumor cells
  • sets of nucleic acids differentially expressed by neoplastic cells have been identified using established techniques such as subtractive hybridization.
  • Methods described herein can be used to identify antigens that were functional in a subject in which an anti -tumor immune response occurred.
  • methods are used to evaluate whether a subject has lymphocytes that react to a tumor antigen or set of tumor antigens.
  • antigen presentation assays are used to examine reactivity to autoantigens in cells of an individual, e.g., an individual predisposed to, or suffering from, an autoimmune condition. Such methods can be used to provide diagnostic or prognostic indicators of the individual’s disease state, or to identify autoantigens.
  • libraries that include an array of human polypeptides are prepared.
  • libraries that include polypeptides from infectious agents which are suspected of eliciting cross-reactive responses to autoantigens are prepared.
  • antigens from infectious agents thought to elicit cross-reactive autoimmune responses see Barzilai et al.. Curr Opin Rheumatol. , 19(6):636-43, 2007; Ayada et al... Ann N Y Acad Sci., 1108:594-602, 2007;
  • the present disclosure includes methods in which polypeptides of interest are included in a library (e.g., expressed in library cells or carried in or on particles or beads). After members of the library are internalized by antigen presenting cells, the polypeptides of interest are proteolytically processed within the antigen presenting cells, and peptide fragments of the polypeptides are presented on MHC molecules expressed in the antigen presenting cells.
  • the identity of the polypeptide that stimulates a human lymphocyte in an assay described herein can be determined from examination of the set of library cells that were provided to the antigen presenting cells that produced the stimulation. In some embodiments, it is useful to map the epitope within the polypeptide that is bound by MHC molecules to produce the observed stimulation. This epitope, or the longer polypeptide from which it is derived (both of which are referred to as an “antigen” herein) can form the basis for an immunogenic composition, or for an antigenic stimulus in future antigen presentation assays.
  • epitopes are identified by generating deletion mutants of the polypeptide of interest and testing these for the ability to stimulate lymphocytes. Deletions that lose the ability to stimulate lymphocytes, when processed and presented by antigen presenting cells, have lost the peptide epitope. In some embodiments, epitopes are identified by synthesizing peptides corresponding to portions of the polypeptide of interest and testing the peptides for the ability to stimulate lymphocytes (e.g., in antigen presentation assays in which antigen presenting cells are pulsed with the peptides).
  • MHC bound peptides involve lysis of the antigen presenting cells that include the antigenic peptide, affinity purification of the MHC molecules from cell lysates, and subsequent elution and analysis of peptides from the MHC (Falk, K. et al. Nature 351 :290, 1991, and U.S. Pat. No. 5,989,565).
  • T cell receptors that have been expanded in response to the antigen.
  • Clonal T cell receptors are identified by DNA sequencing of the T cell receptor repertoire (Howie et al, 2015 Sci Trans Med 7:301). By identifying TCR specificity and function, TCRs can be transfected into other cell types and used in functional studies or for novel immunotherapies.
  • the disclosure provides methods of identifying one or more immune responses of a subject.
  • one or more immune responses of a subject are determined by a) providing a library described herein that includes a panel of tumor antigens (e.g., known tumor antigens, tumor antigens described herein, or tumor antigens, potential tumor antigens, and/or other polypeptides of interest identified using a method described herein); b) contacting the library with antigen presenting cells from the subject; c) contacting the antigen presenting cells with lymphocytes from the subject; and d) determining whether one or more lymphocytes are stimulated by, inhibited and/or suppressed by, activated by, or non-responsive to one or more tumor antigens presented by one or more antigen presenting cells.
  • the library includes about 1, 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more tumor antigens.
  • a subject is (i) a cancer subject who has not received a cancer therapy; (ii) a cancer subject who has not responded and/or is not responding and/or has responded negatively, clinically to a cancer therapy; or (iii) a subject who has not been diagnosed with a cancer.
  • lymphocyte stimulation, non-stimulation, inhibition and/or suppression, activation, and/or non-responsiveness is determined by assessing levels of one or more expressed or secreted cytokines or other immune mediators described herein.
  • levels of one or more expressed or secreted cytokines that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, higher than a control level indicates lymphocyte stimulation.
  • a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 standard deviations greater than the mean of a control level indicates lymphocyte stimulation.
  • a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) greater than a median response level to a control indicates lymphocyte stimulation.
  • a control is a negative control, for example, a clone expressing Neon Green (NG).
  • NG Neon Green
  • a level of one or more expressed or secreted cytokines that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, lower than a control level indicates lymphocyte inhibition and/or suppression.
  • a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 standard deviations lower than the mean of a control level indicates lymphocyte inhibition and/or suppression.
  • a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) lower than a median response level to a control indicates lymphocyte inhibition and/or suppression.
  • a control is a negative control, for example, a clone expressing Neon Green (NG).
  • levels of one or more expressed or secreted cytokines that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, higher or lower than a control level indicates lymphocyte activation.
  • a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 standard deviations greater or lower than the mean of a control level indicates lymphocyte activation.
  • a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) greater or lower than a median response level to a control indicates lymphocyte activation.
  • MADs median absolute deviations
  • a control is a negative control, for example, a clone expressing Neon Green (NG).
  • NG Neon Green
  • a level of one or more expressed or secreted cytokines that is within about 20%, 15%, 10%, 5%, or less, of a control level indicates lymphocyte non-responsiveness or nonstimulation.
  • a level of one or more expressed or secreted cytokines that is less than 1 or 2 standard deviations higher or lower than the mean of a control level indicates lymphocyte non-responsiveness or non-stimulation.
  • a level of one or more expressed or secreted cytokines that is less than 1 or 2 median absolute deviations (MADs) higher or lower than a median response level to a control indicates lymphocyte non- responsiveness or non-stimulation.
  • MADs median absolute deviations
  • a subject response profile can include a quantification, identification, and/or representation of a panel of different cytokines (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more cytokines) and of the total number of tumor antigens (e.g., of all or a portion of different tumor antigens from the library) that stimulate, do not stimulate, inhibit and/or suppress, activate, or have no or minimal effect on production, expression or secretion of each member of the panel of cytokines.
  • cytokines e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more cytokines
  • tumor antigens e.g., of all or a portion of different tumor antigens from the library
  • the disclosure provides methods for obtaining a subject response profile from a test subject (a “subject response profile”).
  • the subject response profile of a test subject is obtained by a) providing a library described herein that includes a panel of tumor antigens (e.g., known tumor antigens, tumor antigens described herein, or tumor antigens, potential tumor antigens, and/or other polypeptides of interest identified using a method described herein); b) contacting the library with antigen presenting cells from the test subject; c) contacting the antigen presenting cells with lymphocytes from the test subject; and d) determining whether one or more lymphocytes are stimulated by, inhibited and/or suppressed by, activated by, or non-responsive to one or more tumor antigens presented by one or more antigen presenting cells, to obtain the subject response profile.
  • the library includes about 1, 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 1000, or more tumor antigens.
  • the subject response profile can include a quantification, identification, and/or representation of all or a portion of the panel of tumor antigens, identified by the methods of the disclosure, that stimulate lymphocytes, that do not stimulate lymphocytes, that inhibit and/or suppress lymphocytes, that activate lymphocytes, or to which lymphocytes are non-responsive.
  • the subject response profile further includes a quantification, identification, and/or representation of the level of expression or secretion of one or more immune mediators, e.g., one or more cytokines.
  • the subject response profile includes a quantification, identification, and/or representation of all or a portion of the panel of tumor antigens, identified by the methods of the disclosure, that stimulate expression or secretion of one or more immune mediators, that inhibit and/or suppress expression or secretion of one or more immune mediators, and/or which do not, or minimally, affect expression or secretion of immune mediators.
  • the subject response profile further includes a quantification, identification, and/or representation of the level of expression or secretion of one or more immune mediators, e.g., one or more cytokines.
  • the subject response profile includes a quantification, identification, and/or representation of all or a portion of the panel of stimulatory and/or inhibitory antigens, identified by methods of the disclosure.
  • a subject response profile is obtained for a subject at one or more times before initiation of treatment a cancer therapy, such as (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, or (iv) any combination of the foregoing.
  • a subject response profile is a representation of (i) a number of identified stimulatory tumor antigens and (ii) a number of identified inhibitory tumor antigens, at a given time, for the subject.
  • a subject response profile is obtained for a subject at one or more times after initiation of treatment a cancer therapy, such as (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, or (iv) any combination of the foregoing.
  • a subject response profile is a representation of (i) a number of identified stimulatory tumor antigens and (ii) a number of identified inhibitory tumor antigens, at a given time, for the subject.
  • a subject response profile can change over a period of time, e.g., over a course of cancer therapy.
  • a change in subject response profile can be used to predict responsiveness to a cancer therapy and/or determine suitability or efficacy of a cancer therapy.
  • change in subject response profile can be used to determine a course of treatment for a subject (e.g., initiation, continuation, modification, discontinuation or non-initiation of a cancer therapy, or of a sequence of cancer therapies).
  • a first subject response profile is obtained before or after a subject is treated with a cancer therapy described herein. In some embodiments, a first subject response profile is obtained after a subject is treated with a cancer therapy described herein. In some embodiments, a second subject response profile is obtained after a subject is treated with a cancer therapy described herein. In some embodiments, a second subject response profile is obtained after a subject has ceased treatment with a cancer therapy described herein.
  • a second subject response profile is obtained from the subject about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or about 7 days, about 1 week, 2 week, 3 week, 4 week, 5 week, 6 weeks, 7 weeks, or about 8 weeks, about 1 month, 2 months, 3 months, 4 months, 5 months, or about 6 months, about 1 year, 2 years, 3 years, 4 years, or about 5 years, after the first subject response profile is obtained.
  • subject response profile is obtained from a subject at least once per week during treatment with a cancer therapy. In some embodiments, subject response profile is obtained from a subject at least once per month during treatment with a cancer therapy.
  • a first subject response profile is compared to a second subject response profile.
  • a change between a first subject response profile and a second subject response profile is determined by a change in number of stimulatory antigens identified in a subject.
  • a change between a first subject response profile and a second subject response profile is determined by a change in number of inhibitory antigens identified in a subject.
  • a first subject response profile does not differ from a second subject response profile if the identified tumor antigens that stimulate lymphocytes in the first subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that stimulate lymphocytes in the second subject response profile; if the identified tumor antigens that do not stimulate lymphocytes in the first subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that do not stimulate lymphocytes in the second subject response profile; if the identified tumor antigens that inhibit and/or suppress lymphocytes in the first subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that inhibit and/or suppress lymphocytes in the second subject response profile; if the identified tumor antigens that activate lymphocytes in the first subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that activate lymphocytes in the second subjectresponse profile; and/
  • a first subject response profile differs from a second subject response profile if the identified tumor antigens that stimulate lymphocytes in the first subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that stimulate lymphocytes in the second subject response profile; if the identified tumor antigens that do not stimulate lymphocytes in the first subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that do not stimulate lymphocytes in the second subject response profile; if the identified tumor antigens that inhibit and/or suppress lymphocytes in the first subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that inhibit and/or suppress lymphocytes in the second subject response profile; if the identified tumor antigens that activate lymphocytes in the first subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that activate lymphocytes in the second subjectresponse profile; and/or if the identified tumor antigens that activate lymphocyte
  • a first subject response profile does not differ from a second subject response profile if the identified tumor antigens that stimulate lymphocytes in the first subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that stimulate lymphocytes in the second subject response profile; if the identified tumor antigens that do not stimulate lymphocytes in the first subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that do not stimulate lymphocytes in the second subject response profile; if the identified tumor antigens that inhibit and/or suppress lymphocytes in the first subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that inhibit and/or suppress lymphocytes in the second subject response profile; if the identified tumor antigens that activate lymphocytes in the first subject response profile differ
  • a first subject response profile differs from a second subject response profile if the identified tumor antigens that stimulate lymphocytes in the first subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%, or more, from the identified tumor antigens that stimulate lymphocytes in the second subject response profile if the identified tumor antigens that do not stimulate lymphocytes in the first subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20% ,or more, from the identified tumor antigens that do not stimulate lymphocytes in the second subject response profile; and/or if the identified tumor antigens that inhibit and/or suppress lymphocytes in the first subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%, or more, from the identified tumor antigens that inhibit and/or suppress lymphocytes in the second subject response profile; if the identified tumor antigens that activate lymphocytes in the first subject response profile differ by more than 5%, 6%, 7%,
  • a second subject response profile can include a quantification, identification, and/or representation of one or more cytokines and the total number of tumor antigens (e.g., of the same tumor antigens included in a first subject response profile) that stimulate, do not stimulate, inhibit and/or suppress, or have no or minimal effect on cytokine production, expression and/or secretion.
  • the second subject response profile can include a quantification, identification, and/or representation of a panel of different cytokines (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more (e.g., all) of the cytokines included in the first subject response profile) and the total number of tumor antigens (e.g., of the same tumor antigens included in thefirst subject response profile) that stimulate, do not stimulate, inhibit and/or suppress, or have no or minimal effect on production, expression and/or secretion of the panel of cytokines.
  • cytokines e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more (e.g., all) of the cytokines included in the first subject response profile
  • tumor antigens e.g., of the same tumor antigens included in thefirst subject response profile
  • a first subject response profile is not different from a second subject response profile if the total number of antigens that stimulate expression and/or secretion of one or more cytokines included in the first subject response profile differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the total number of antigens that stimulate the same one or more cytokines included in second subject response profile; if the total number of antigens that do not stimulate expression and/or secretion of one or more cytokines included in the first subject response profile differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the total number of antigens that do not stimulate the same one or more cytokines included in the second subject response profile; if the total number of antigens that inhibit and/or suppress one or more cytokines included in the first subject response profile differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the total number of antigens that inhibit and/or suppress expression and/or secretion of the same one or more cytok
  • a first subject response profile differs from a second response profile if the total number of antigens that stimulate expression and/or secretion of one or more cytokines included in the first subject response profile differs by more than 5, 6, 7, 8, 9, 10, 20, or more, from the total number of antigens that stimulate the same one or more cytokines included in the second subject response profile; if the total number of antigens that do not stimulate expression and/or secretion of one or more cytokines included in the first subject response profile differs by more than 5, 6, 7, 8, 9, 10, 20, or more, from the total number of antigens that do not stimulate the same one or more cytokines included in the second subject response profile ; if the total number of antigens that inhibit and/or suppress expression and/or secretion of one or more cytokines included in the first subject response profile differs by more than 5, 6, 7, 8, 9, 10, 20, or more, from the total number of antigens that inhibit and/or suppress the same one or more cytokines included in
  • determining a course of therapy includes initiation, continuation, modification, and/or discontinuation or in some cases non-initiation of a cancer therapy (e.g., a cancer therapy described herein), or of a course of cancer therapies.
  • methods include, e.g., comparing at least two subject response profiles obtained from a cancer subject who has not received a cancer therapy, who has not responded and/or is not responding and/or has responded negatively, clinically to a cancer therapy.
  • a first subject response profile is obtained at a first time (e.g., before initiation of a cancer therapy or after initiation of a cancer therapy) and a second subject response profile is obtained at a second time (e.g., after initiation of a cancer therapy), as described herein.
  • a change in subject response profile comprises a change in number of stimulatory antigens and/or inhibitory antigens detected in a subject.
  • a change in subject response profile is measured as a change in a subject’s lymphocytes level of expression or secretion of one or more immune mediators, e.g., one or more cytokines to a particular tumor antigen.
  • a change in subject response profile is identified when a lymphocyte would no longer be activated (i.e., activated to secrete/express a marker associated with a deleterious or beneficial immune response).
  • a change in subject response profile is identified when a lymphocyte initially is not activated (i.e., activated to secrete/express a marker associated with a deleterious or beneficial immune response) by a particular tumor antigen developes a recognition/response to that particular tumor antigen.
  • an increase in number of stimulatory antigens in a second subject response profile relative to a first subject response profile and/or (ii) a decrease in number of inhibitory antigens in a second subject response profile relative to a first subject response profile indicates that the subject should continue a course of cancer therapy.
  • a decrease in number of stimulatory antigens in a second subject response profile, relative to a first subject response profile, and/or (ii) an increase in number of inhibitory in a second subject response profile, relative to a first subject response profile indicates that the subject should modify the course of cancer therapy or should initiate a new course of cancer therapy (e.g., combine with one or more other modalities).
  • determining the course of therapy and/or sequence of cancer therapies is based on the number and/or characteristics of the identified stimulatory and inhibitory antigens in a subject response profile.
  • a cancer therapy and/or sequence of cancer therapies is selected based on identification of one or more inhibitory antigens that reduce and/or inhibit an immune response by, e.g., at least 50%, 60%, 70%, 80%, 90%, or 100%, relative to control.
  • a cancer therapy e.g., an immunogenic composition comprising one or more of the identified stimulatory antigens, checkpoint inhibitor therapy, chemotherapy, cell-based therapy, immunotherapy, cytokine therapy, and/or kinase inhibitors
  • a cancer therapy and/or sequence of cancer therapies is selected based on identification of one or more stimulatory antigens that increase an immune response by, e.g., at least 50%, 60%, 70%, 80%, 90%, or 100%, relative to control.
  • a cancer therapy e.g., an immunogenic composition comprising one or more of the identified stimulatory antigens, checkpoint inhibitor therapy, chemotherapy, cell-based therapy, immunotherapy, cytokine therapy, and/or kinase inhibitors
  • a cancer therapy and/or sequence of cancer therapies is selected based on the presence or absence of identified dominant stimulatory antigens in a subject response profile. In some embodiments, a cancer therapy and/or sequence of cancer therapies is based on the presence or absence of identified dominant inhibitory antigens in a subject response profile.
  • a dominant inhibitory antigen is one that abrogates the effects of a protective cancer vaccine and results in, e.g., hyperprogresssion of a tumor.
  • a sub-dominant inhibitory antigen is one that partially abrogates the effects of a protective cancer vaccine.
  • a cancer therapy and/or sequence of cancer therapies is selected based on the number of identified stimulatory and/or inhibitory antigens in a subject response profile. In some embodiments, a cancer therapy and/or sequence of cancer therapies is based on the ratio of identified stimulatory and/or inhibitory antigens in a subject response profile.
  • immune responses can be usefully defined in terms of their integrated, functional end-effects.
  • Dhabar et al. (2014) have proposed that immune responses can be categorized as being immunoprotective, immunopathological, and immunoregulatory/inhibitory. While these categories provide useful constructs with which to organize ideas, an overall in vivo immune response is likely to consist of several types of responses with varying amounts of dominance from each category.
  • Immunoprotective or beneficial responses are defined as responses that promote efficient wound healing, eliminate infections and cancer, and mediate vaccine-induced immunological memory. These responses are associated with cytokines and mediators such as IFN-gamma, IL-12, IL-2, granzyme B, CD107, etc.
  • Immunopathological or deleterious responses are defined as those that are directed against self (autoimmune disease like multiple sclerosis, arthritis, lupus) or innocuous antigens (asthma, allergies) and responses involving chronic, non-resolving inflammation. These responses can also be associated with molecules that are implicated in immunoprotective responses, but also include immune mediators such as TNF-alpha, IL-10, IL-13, IL-17, IL-4, IgE, histamine, etc. Immunoregulatory responses are defined as those that involve immune cells and factors that regulate (mostly down- regulate) the function of other immune cells. Recent studies suggest that there is an arm of the immune system that functions to inhibit immune responses.
  • regulatory CD4 + CD25+FoxP3 + T cells, IL-10, and TGF-beta have been shown to have immunoregulatory/inhibitory functions.
  • the physiological function of these factors is to keep pro-inflammatory, allergic, and autoimmune responses in check, but they may also suppress antitumor immunity and be indicative of negative prognosis for cancer.
  • the expression of co-stimulatory molecules often decreases, and the expression of co-inhibitory ligands increases.
  • MHC molecules are often down-regulated on tumor cells, favoring their escape.
  • the tumor micro-environment including stromal cells, tumor associated immune cells, and other cell types, produce many inhibitory factors, such as, IL-10, TGF-P, and IDO.
  • Inhibitory immune cells including T regs, Tri cells, immature DCs (iDCs), pDCs, and MDSC can be found in the tumor microenvironment. (Y Li UT GSBS Thesis 2016). Examples of mediators and their immune effects are shown in Table 2.
  • a stimulatory antigen is a tumor antigen (e.g., a tumor antigen described herein) that stimulates one or more lymphocyte responses that are beneficial to the subject.
  • a stimulatory antigen is a tumor antigen (e.g., a tumor antigen described herein) that inhibits and/or suppresses one or more lymphocyte responses that are deleterious or non-beneficial to the subject.
  • immune responses that may lead to beneficial anti -tumor responses (e.g., that may enhance immune control of a tumor) include but are not limited to 1) cytotoxic CD8 + T cells which can effectively kill cancer cells and release the mediators perforin and/or granzymes to drive tumor cell death; and 2) CD4 + Thl T cells which play an important role in host defense and can secrete IL-2, IFN-gamma and TNF-alpha. These are induced by IL- 12, IL-2, and IFN gamma among other cytokines.
  • an inhibitory antigen is a tumor antigen (e.g., a tumor antigen described herein) that stimulates one or more lymphocyte responses that are deleterious or non-beneficial to the subject.
  • an inhibitory antigen is a tumor antigen (e.g., a tumor antigen described herein) that inhibits and/or suppresses one or more lymphocyte responses that are beneficial to the subject.
  • immune responses that may lead to deleterious or non-beneficial anti -tumor responses (e.g., that may impair or reduce control of a tumor) include but are not limited to 1) T regulatory cells which are a population of T cells that can suppress an immune response and secrete immunosuppressive cytokines such as TGF-beta and IL-10 and express the molecules CD25 and FoxP3; and 2) Th2 cells which target responses against allergens but are not productive against cancer. These are induced by increased IL-4 and IL- 10 and can secrete IL-4, IL-5, IL-6, IL-9 and IL-13.
  • T regulatory cells which are a population of T cells that can suppress an immune response and secrete immunosuppressive cytokines such as TGF-beta and IL-10 and express the molecules CD25 and FoxP3
  • Th2 cells which target responses against allergens but are not productive against cancer.
  • the disclosure provides methods and systems for identifying and selecting tumor antigens, e.g., stimulatory and/or inhibitory antigens.
  • one or more selected antigen is a stimulatory antigen.
  • a stimulatory antigen may be selected based on the measured immune response to the antigen using a method of the disclosure.
  • a stimulatory antigen may be selected if the antigen produces an immune response that stimulates the expression and/or release of one or more of any cytokine associated with a beneficial response, as shown for example, in Table 2.
  • the cytokine comprises one or more of IL-2, IFN-gamma and TNF-alpha.
  • a stimulatory antigen may be selected if the antigen produces an immune response that inhibits the expression and/or release of one or more of any of the cytokines associated with a deleterious response, as shown for example, in Table 2.
  • the cytokine comprises one or more of TGF-beta and IL-10.
  • a composition comprising the one or more selected tumor antigens is administered to a cancer subject before, during, and/or after administration of a cancer therapy.
  • the disclosure provides methods for selecting tumor antigens identified by the methods herein based on comparison of a subject response profile to a subsequently obtained subject response profile from the same subject.
  • the disclosure also provides methods for selecting (or de-selecting) tumor antigens identified by the methods herein, based on association with desirable or beneficial responses.
  • the disclosure also provides methods for selecting (or deselecting) tumor antigens identified by the methods herein, based on association with undesirable, deleterious or non-beneficial responses.
  • the methods for selecting tumor antigens are combined. The methods may be combined in any order, e.g.
  • selection may be carried out by comparison of a subject response profile to a subsequently obtained subject response profile, followed by selection based on association with a desirable (or undesirable) response; or, selection may be carried out based on association with a desirable (or undesirable) response, followed by comparison of the subject response profile to a subsequently obtained subject response profile.
  • Methods for identifying tumor antigens and potential tumor antigens are provided herein.
  • Methods for generating or obtaining a subject response profile are provided herein.
  • Methods for generating or obtaining as subsequent subject response profile e.g. a subject response profile obtained before and during treatment, are provided herein.
  • Methods for comparison of a subject response profile to a subsequently obtained subject response profile are provided herein.
  • Methods for determining whether a subject response profile has changed over time are provided herein.
  • a first subject response profile and a second subject response profile are generated or obtained using the same plurality of polypeptides of interest. In some embodiments, a first subject response profile and a second subject response profile are generated or obtained using the same plurality of tumor antigens.
  • the subject response profile includes a quantification, identification, and/or representation of one or more tumor antigens that stimulate lymphocytes, that do not stimulate lymphocytes, that inhibit and/or suppress lymphocytes, that activate lymphocytes, and/or to which lymphocytes are non-responsive.
  • one or more tumor antigens are identified as inhibiting and/or suppressing lymphocytes in the subject (e.g., identified from a first subject response profile), and the same one or more tumor antigens are identified as stimulating lymphocytes in the subject at a later timepoint (e.g., identified from a second subject response profile).
  • one or more tumor antigens are identified as stimulating lymphocytes in the subject (e.g., identified from a first subject response profile) and the same one or more tumor antigens are identified as inhibiting and/or suppressing lymphocytes in the subject at a later point in time (e.g., identified in a second subject response profile).
  • one or more tumor antigens or potential tumor antigens are identified as eliciting minimal or no response from lymphocytes in the subject (e.g., identified from a first subject response profile), and the same one or more tumor antigens are identified as stimulating, or inhibiting and/or suppressing lymphocytes in the subject at a later point in time (e.g., identified from a second subject response profile).
  • one or more tumor antigens are identified as stimulating, or inhibiting and/or suppressing, lymphocytes in the subject (e.g., identified from a first subject response profile), and the same one or more tumor antigens are identified as eliciting minimal or no response from lymphocytes in the subject at a later point in time (e.g., identified from a second subject response profile).
  • Tumor antigens may be identified and/or selected (or de-selected) based on association with desirable or beneficial responses. Tumor antigens may be identified and/or selected (or de-selected) based on association with undesirable, deleterious or non-beneficial responses. Tumor antigens may be identified and/or selected (or de-selected) based on a combination of the preceding methods, applied in any order.
  • a stimulatory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a beneficial response is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, higher than a control level indicates lymphocyte stimulation. In some embodiments, a stimulatory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a beneficial response is at least 1, 2, 3, 4 or 5 standard deviations greater than the mean of a control level indicates lymphocyte stimulation.
  • a stimulatory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a beneficial response is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) greater than a median response level to a control indicates lymphocyte stimulation.
  • a control is a negative control, for example, a clone expressing Neon Green (NG).
  • a stimulatory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a deleterious response is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, lower than a control level indicates lymphocyte inhibition and/or suppression. In some embodiments, a stimulatory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a deleterious response is at least 1, 2, 3, 4 or 5 standard deviations lower than the mean of a control level indicates lymphocyte inhibition and/or suppression.
  • a stimulatory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a deleterious response is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) lower than a median response level to a control that indicates lymphocyte inhibition and/or suppression.
  • a control is a negative control, for example, a clone expressing Neon Green (NG).
  • one or more selected antigen is an inhibitory antigen.
  • An inhibitory antigen may be de-selected based on a measured immune response to the antigen using a method of the disclosure.
  • An inhibitory antigen may be selected if the antigen produces an immune response that stimulates the expression and/or release of one or more cytokines associated with a deleterious response, as shown for example, in Table 2.
  • the cytokine comprises one or more of TGF-beta and IL-10.
  • An inhibitory antigen may be selected if the antigen produces an immune response that inhibits the expression and/or release of one or more of any cytokine associated with a beneficial response, as shown for example, in Table 2.
  • the cytokine comprises one or more of IL-2, IFN-gamma and TNF-alpha.
  • an inhibitory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a deleterious response is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, higher than a control level indicates lymphocyte stimulation. In some embodiments, an inhibitory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a deleterious response is at least 1, 2, 3, 4 or 5 standard deviations greater than the mean of a control level indicates lymphocyte stimulation.
  • an inhibitory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a deleterious response is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) greater than a median response level to a control that indicates lymphocyte stimulation.
  • a control is a negative control, for example, a clone expressing Neon Green (NG).
  • an inhibitory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a beneficial response is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, lower than a control level that indicates lymphocyte inhibition and/or suppression. In some embodiments, an inhibitory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a beneficial response is at least 1, 2, 3, 4 or 5 standard deviations lower than the mean of a control level that indicates lymphocyte inhibition and/or suppression.
  • an inhibitory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a beneficial response is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) lower than a median response level to a control that indicates lymphocyte inhibition and/or suppression.
  • a control is a negative control, for example, a clone expressing Neon Green (NG).
  • a tumor antigen suitable for use in any method or composition of the disclosure may be produced by any available means, such as recombinantly or synthetically (see, e.g., Jaradat Amino Acids 50:39-68 (2016); Behrendt et al., J. Pept. Sci. 22:4-27 (2016)).
  • a tumor antigen may be recombinantly produced by utilizing a host cell system engineered to express a tumor antigen-encoding nucleic acid.
  • a tumor antigen may be produced by activating endogenous genes.
  • a tumor antigen may be partially or fully prepared by chemical synthesis.
  • any expression system can be used.
  • known expression systems include, for example, E. coll. egg, baculovirus, plant, yeast, or mammalian cells.
  • recombinant tumor antigen suitable for the present invention are produced in mammalian cells.
  • Non-limiting examples of mammalian cells include BALB/c mouse myeloma line (NSO/1, ECACC No: 85110503); human retinoblasts (PER.C6, CruCell, Leiden, The Netherlands); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (HEK293 or 293 cells subcloned for growth in suspension culture, Graham et al., J.
  • human fibrosarcoma cell line e.g., HT1080
  • baby hamster kidney cells BHK21, ATCC CCL 10
  • Chinese hamster ovary cells +/-DHFR CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980
  • mouse sertoli cells TM4, Mather, Biol.
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • the present disclosure provides recombinant tumor antigen produced from human cells. In some embodiments, the present disclosure provides recombinant tumor antigen produced from CHO cells or HT1080 cells.
  • cells that are engineered to express a recombinant tumor antigen may comprise a transgene that encodes a recombinant tumor antigen described herein.
  • the nucleic acids encoding recombinant tumor antigen may contain regulatory sequences, gene control sequences, promoters, non-coding sequences and/or other appropriate sequences for expressing the recombinant tumor antigen.
  • the coding region is operably linked with one or more of these nucleic acid components.
  • the coding region of a transgene may include one or more silent mutations to optimize codon usage for a particular cell type.
  • the codons of a tumor antigen transgene may be optimized for expression in a vertebrate cell.
  • the codons of a tumor antigen transgene may be optimized for expression in a mammalian cell.
  • the codons of a tumor antigen transgene may be optimized for expression in a human cell.
  • compositions that include a tumor antigen or tumor antigens identified or selected by methods described herein, nucleic acids encoding the tumor antigens, and methods of using the compositions.
  • a composition includes tumor antigens that are peptides 8-40 amino acids, 8- 60 amino acids, 8-100, 8-150, or 8-200 amino acids in length (e.g., MHC binding peptides, e.g., peptides 23-29, 24-28, 25-27, 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24, 8-23, 8-22, 8-21, 8-20, 8- 15, 8-12 amino acids in length).
  • a composition includes one or more tumor antigens that are about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the length of the full-length polypeptides.
  • a composition includes one or more tumor antigens that are truncated by about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids, relative to the full-length polypeptides.
  • the compositions can include tumor antigens that are, or that comprise, MHC class I-binding peptides, MHC class Il-binding peptides, or both MHC class I and MHC class Il-binding peptides.
  • Compositions can include a single tumor antigen, or multiple tumor antigens.
  • a composition includes a set of two, three, four, five, six, seven, eight, nine, ten, or more tumor antigens.
  • a composition includes ten, fifteen, twenty, twenty -five, thirty, or more tumor antigens.
  • the tumor antigens or peptides are provided as one or more fusion proteins.
  • a composition comprises nucleic acids encoding the tumor antigens or peptides.
  • the nucleic acids encoding the tumor antigens or peptides are provided as one or more fusion constructs.
  • an immunogenic composition includes a tumor antigen linked to a carrier protein.
  • carrier proteins include, e.g., toxins and toxoids (chemical or genetic), which may or may not be mutant, such as anthrax toxin, PA and DNI (PharmAthene, Inc.), diphtheria toxoid (Massachusetts State Biological Labs; Serum Institute of India, Ltd.) or CRM 197, tetanus toxin, tetanus toxoid (Massachusetts State Biological Labs; Serum Institute of India, Ltd.), tetanus toxin fragment Z, exotoxin A or mutants of exotoxin A of Pseudomonas aeruginosa, bacterial flagellin, pneumolysin, an outer membrane protein of Neisseria meningitidis (strain available from the ATCC (American Type Culture Collection, Manassas, Va.)), Pse
  • coli heat labile enterotoxin shiga-like toxin
  • human LTB protein a protein extract from whole bacterial cells, and any other protein that can be cross-linked by a linker.
  • Other useful carrier proteins include high density lipoprotein (HDL), bovine serum albumin (BSA), P40, and chicken riboflavin. Many carrier proteins are commercially available (e.g., from Sigma Aldrich.).
  • the disclosure also provides nucleic acids encoding the tumor antigens.
  • the nucleic acids can be used to produce expression vectors, e.g., for recombinant production of the tumor antigens, or for nucleic acid-based administration in vivo (e.g., DNA vaccination).
  • an immunogenic composition may be suitable for administration to a human patient, and vaccine preparation may conform to USFDA guidelines.
  • an immunogenic composition is suitable for administration to a nonhuman animal.
  • an immunogenic composition is substantially free of either endotoxins or exotoxins.
  • Endotoxins include pyrogens, such as lipopolysaccharide (LPS) molecules.
  • An immunogenic composition may also be substantially free of inactive protein fragments.
  • an immunogenic composition has lower levels of pyrogens than industrial water, tap water, or distilled water. Other components of the immunogenic composition may be purified using methods known in the art, such as ion-exchange chromatography, ultrafiltration, or distillation.
  • the pyrogens may be inactivated or destroyed prior to administration to a patient.
  • Raw materials for immunogenic compositions such as water, buffers, salts and other chemicals may also be screened and depyrogenated. All materials in a immunogenic composition may be sterile, and each lot of the composition may be tested for sterility.
  • the endotoxin levels in the immunogenic composition fall below the levels set by the USFDA, for example 0.2 endotoxin (EU)/kg of product for an intrathecal injectable composition; 5 EU/kg of product for a non- intrathecal injectable composition, and 0.25-0.5 EU/ml for sterile water.
  • EU endotoxin
  • an immunogenic composition (e.g., a vaccine and/or a vaccine formulation) comprising a polypeptide contains less than 5%, 2%, 1%, 0.5%, 0.2%, 0.1% of other, undesired unpolypeptides, relative to the amount of desired polypeptides. In some embodiments, an immunogenic composition contains less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% DNA and/or RNA.
  • Immunogenic compositions can be prepared as formulations suitable for route of administration.
  • Formulations suitable for parenteral administration such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, intranasal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials.
  • Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
  • Immunogenic compositions described herein may include an adjuvant.
  • Adjuvants can be used as vaccine delivery systems and/or for their immunostimulatory properties.
  • Vaccine delivery systems are often particulate formulations, e.g., emulsions, microparticles, immune- stimulating complexes (ISCOMs), which may be, for example, particles and/or matrices, and liposomes.
  • Immunostimulatory adjuvants include ISCOMS or may be derived from pathogens and can represent pathogen associated molecular patterns (PAMP), e.g., lipopolysaccharides (LPS), monophosphoryl lipid (MPL), or CpG-containing DNA, which activate cells of the innate immune system.
  • PAMP pathogen associated molecular patterns
  • LPS lipopolysaccharides
  • MPL monophosphoryl lipid
  • CpG-containing DNA which activate cells of the innate immune system.
  • Adjuvants may also be classified as organic and inorganic.
  • Inorganic adjuvants include aluminum salts such as aluminum phosphate, amorphous aluminum hydroxyphosphate sulfate, and aluminum hydroxide, which are commonly used in human vaccines.
  • Organic adjuvants comprise organic molecules including macromolecules.
  • An example of an organic adjuvant is cholera toxin.
  • Adjuvants may also be classified by the response they induce, and adjuvants can activate more than one type of immunostimulatory response.
  • the adjuvant induces the activation of CD4+ T cells.
  • the adjuvant may induce activation of TH1 cells and/or activation of TH17 cells and/or activation of TH2 cells. Alternately, the adjuvant may induce activation of TH1 cells and/or TH17 cells but not activation of TH2 cells, or vice versa.
  • the adjuvant induces activation of CD8+ T cells.
  • the adjuvant may induce activation of Natural Killer T (NKT) cells.
  • NKT Natural Killer T
  • the adjuvant induces the activation of TH1 cells or TH17 cells or TH2 cells. In other embodiments, the adjuvant induces the activation of B cells. In yet other embodiments, the adjuvant induces the activation of APCs. These categories are not mutually exclusive; in some cases, an adjuvant activates more than one type of cell.
  • an adjuvant stimulates an immune response by increasing the numbers or activity of APCs such as dendritic cells.
  • an adjuvant promotes the maturation of APCs such as dendritic cells.
  • the adjuvant is or comprises a saponin.
  • a saponin adjuvant is immunostimulatory.
  • a saponin is a triterpene glycoside, such as those isolated from the bark of the Quillaja saponaria tree.
  • a saponin extract from a biological source can be further fractionated (e.g., by chromatography) to isolate the portions of the extract with the best adjuvant activity and with acceptable toxicity.
  • fractions A and C Typical fractions of extract from Quillaja saponaria tree used as adjuvants are known as fractions A and C.
  • An exemplary saponin adjuvant is QS-21 (fraction C), which is available from Antigenics.
  • QS-21 is an oligosaccharide-conjugated small molecule.
  • QS-21 may be admixed with a lipid such as 3D-MPL or cholesterol.
  • a particular form of saponins that may be used in vaccine formulations described herein is immunostimulating complexes (ISCOMs).
  • ISCOMs are an art-recognized class of adjuvants, that generally comprise Quillaja saponin fractions and lipids (e.g., cholesterol and phospholipids such as phosphatidyl choline).
  • an ISCOM is assembled together with a polypeptide or nucleic acid of interest.
  • different saponin fractions may be used in different ratios.
  • the different saponin fractions may either exist together in the same particles or have substantially only one fraction per particle (such that the indicated ratio of fractions A and C are generated by mixing together particles with the different fractions).
  • substantially refers to less than 20%, 15%, 10%, 5%, 4%, 3%, 2% or even 1%.
  • Such adjuvants may comprise fraction A and fraction C mixed into a ratio of 70-95 A: 30-5 C, such as 70 A : 30 C to 75 A : 25 C; 75 A : 25 C to 80 A : 20 C; 80 A : 20 C to 85 A : 15 C; 85 A : 15 C to 90 A : 10 C; 90 A : 10 C to 95 A : 5 C; or 95 A : 5 C to 99 A : 1 C.
  • ISCOMatrix produced by CSL, and AblSCO 100 and 300, produced by Isconova, are ISCOM matrices comprising saponin, cholesterol and phospholipid (lipids from cell membranes), which form cage-like structures typically 40-50 nm in diameter.
  • Posintro produced by Nordic Vaccines, is an ISCOM matrix where the immunogen is bound to the particle by a multitude of different mechanisms, e.g., electrostatic interaction by charge modification, incorporation of chelating groups, or direct binding.
  • the adjuvant is Matrix-M2 (MM2).
  • the Matrix-M2 adjuvant comprises saponin fractions purified from Quillaja saponaria (soapbark tree) bark, phosphatidylcholine and cholesterol.
  • the adjuvant is diluted in normal saline, for example 0.9% saline.
  • the adjuvant is a TLR ligand.
  • TLRs are proteins that may be found on leukocyte membranes, and recognize foreign antigens (including microbial antigens).
  • An exemplary TLR ligand is IC-31, which is available from Intercell.
  • IC-31 comprises an anti -microbial peptide, KLK, and an immunostimulatory oligodeoxynucleotide, ODNla.
  • IC- 31 has TLR9 agonist activity.
  • Another example is CpG-containing DNA.
  • Vaxlmmune is CpG 7909 (a (CpG)- containing oligodeoxy-nucleotide)
  • Actilon is CpG 10101 (a (CpG)-containing oligodeoxynucleotide).
  • the adjuvant is a nanoemulsion.
  • One exemplary nanoemulsion adjuvant is Nanostat Vaccine, produced by Nanobio. This nanoemulsion is a high- energy, oil-in-water emulsion. This nanoemulsion typically has a size of 150-400 nanometers, and includes surfactants to provide stability. More information about Nanostat can be found in US Patents 6,015,832, 6,506,803, 6,559,189, 6,635,676, and 7,314,624.
  • an adjuvant includes a cytokine.
  • the cytokine is an interleukin such as IL-1, IL-6, IL-12, IL-17 and IL-23.
  • the cytokine is granulocyte-macrophage colony-stimulating factor (GM-CSF).
  • the adjuvant may include cytokine as a purified polypeptide.
  • the adjuvant may include nucleic acids encoding the cytokine.
  • Adjuvants may be covalently bound to antigens (e.g., the polypeptides described above).
  • the adjuvant may be a protein which induces inflammatory responses through activation of APCs.
  • one or more of these proteins can be recombinantly fused with an antigen of choice, such that the resultant fusion molecule promotes dendritic cell maturation, activates dendritic cells to produce cytokines and chemokines, and ultimately, enhances presentation of the antigen to T cells and initiation of T cell responses (see Wu et al., Cancer Res 2005; 65(11), pp 4947-4954).
  • Other exemplary adjuvants that may be covalently bound to antigens comprise polysaccharides, synthetic peptides, lipopeptides, and nucleic acids.
  • the adjuvant can be used alone or in combination of two or more kinds.
  • Adjuvants may be directly conjugated to antigens.
  • Adjuvants may be administered in therapeutically effective amounts, for example, an amount that produces the desired effect (e.g., immunostimulatory effect) for which it is administered.
  • Adjuvants may also be combined to increase the magnitude of the immune response to the antigen.
  • the same adjuvant or mixture of adjuvants is present in each dose of an immunogenic composition (e.g., a vaccine and/or a vaccine formulation).
  • an adjuvant may be administered with a first dose of an immunogenic composition and not with subsequent doses (e.g., additional dose(s) or maintenance dose(s)).
  • a strong adjuvant may be administered with the first dose of an immunogenic composition and a weaker adjuvant or lower dose of the strong adjuvant may be administered with subsequent doses.
  • the adjuvant can be administered before the administration of the antigen, concurrent with the administration of the antigen or after the administration of the antigen to a subject (sometimes within 1, 2, 6, or 12 hours, and sometimes within 1, 2, or 5 days). Certain adjuvants are appropriate for human patients, non-human animals, or both.
  • an immunogenic composition e.g., a vaccine, a vaccine formulation and/or a pharmaceutical composition, may include one or more additional components.
  • an immunogenic composition may include one or more stabilizers such as sugars (such as sucrose, glucose, or fructose), phosphate (such as sodium phosphate dibasic, potassium phosphate monobasic, dibasic potassium phosphate, or monosodium phosphate), glutamate (such as monosodium L-glutamate), gelatin (such as processed gelatin, hydrolyzed gelatin, or porcine gelatin), amino acids (such as arginine, asparagine, histidine, L-histidine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and the alkyl esters thereof), inosine, or sodium borate.
  • stabilizers such as sugars (such as sucrose, glucose, or fructose), phosphate (such as sodium phosphate dibasic, potassium phosphate monobasic, dibasic potassium phosphate, or monosodium
  • an immunogenic composition includes one or more buffers such as a mixture of sodium bicarbonate and ascorbic acid.
  • an immunogenic composition may be administered in saline (e.g., 0.9% saline), such as phosphate buffered saline (PBS), or distilled water.
  • saline e.g. 0.9% saline
  • PBS phosphate buffered saline
  • an immunogenic composition includes one or more surfactants such as polysorbate 20 (Tween 20), polysorbate 80 (Tween 80), Polyethylene glycol tert-octylphenyl ether t-Octylphenoxypolyethoxyethanol 4-(l,l,3,3-Tetramethylbutyl)phenyl- polyethylene glycol (TRITON X-100); Polyoxy ethylenesorbitan monolaurate Polyethylene glycol sorbitan monolaurate (TWEEN 20); and 4-(l,l,3,3-Tetramethylbutyl)phenol polymer with formaldehyde and oxirane (TYLOXAPOL).
  • a surfactant can be ionic or nonionic.
  • an immunogenic composition includes one or more salts such as sodium chloride, ammonium chloride, calcium chloride, or potassium chloride.
  • a preservative is included in an immunogenic composition. In other embodiments, no preservative is used. A preservative is most often used in multi-dose vaccine vials, and is less often needed in single-dose vaccine vials. In certain embodiments, the preservative is 2-phenoxyethanol, methyl and propyl parabens, benzyl alcohol, and/or sorbic acid.
  • an immunogenic composition is a controlled-release formulation.
  • an immunogenic composition is administered to a subject according to a dosing regimen or dosing schedule.
  • the amount of antigen in each immunogenic composition dose is selected to be a therapeutically effective amount, which induces a prophylactic or therapeutic response, as described above, in either a single dose or over multiple doses.
  • a dose is without significant adverse side effects in typical immunogenic compositions. Such amount will vary depending upon which specific antigen is employed.
  • a single dose will comprise about 100 to about 1500 pg total peptide.
  • a total volume of a single dose is 0.5 mL to 1.0 mL.
  • a single dose will comprise more than one antigen, for example, 2, 3, 4, 5 or more.
  • a dosing regimen comprises an initial dose of an immunogenic composition and at least one additional dose of the immunogenic composition.
  • an additional dose is administered about 3 weeks following the initial dose.
  • an additional dose is administered about 6 weeks following the initial dose.
  • an additional dose is administered about 12 weeks following the initial dose.
  • an additional dose is administered about 24 weeks following the initial dose.
  • the dosing regimen comprises administration of different immunogenic compositions, e.g., 2, 3, 4, 5, 6, 7, 8, or more different immunogenic compositions comprising antigens.
  • each dose comprises administering different immunogenic compositions, e.g., in succession.
  • each dose comprises administering the same set of different immunogenic compostions.
  • a dosing regimen can include an initial dose of 2, 3, 4, 5, 6, 7, 8, or more different immunogenic compositions, and at least one additional dose of the 2, 3, 4, 5, 6, 7, 8, or more different immunogenic compositions.
  • an immunogenic composition comprises one antigen.
  • an immunogenic composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.
  • a dosing regimen can include an initial dose of 2, 3, 4, 5, 6, 7, 8, or more different immunogenic compositions (e.g., where each immunogenic composition can separately include 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens), and at least one additional dose of the 2, 3, 4, 5, 6, 7, 8, or more different immunogenic compositions.
  • a dosing regimen can include an initial dose of 4 different immunogenic compositions, where each immunogenic composition comprises 1, 2, 3, 4 or 5 different antigens.
  • such dosing regimen further includes at least 1 (e.g., at least 2, 3, 4, 5, 6, or more) additional doses of the 4 different immunogenic compositions.
  • a second dose is administered about 1, 2, 3, 4, or 5 weeks after the initial dose; a third dose is administered about 1, 2, 3, 4, or 5 weeks after the second dose; a fourth dose is administered about 2, 4, 6, 8, 10, 12, 14, 16, or 18 weeks after the third dose; and a fifth dose is administered about 2, 4, 6, 8, 10, 12, 14, 16, or 18 weeks after the fourth dose.
  • tumor antigens are used in diagnostic assays.
  • compositions including the tumor antigens can be provided in kits, e.g., for detecting antibody reactivity, or cellular reactivity, in a sample from an individual.
  • tumor antigen compositions are used to induce an immune response in a subject.
  • the subject is a human.
  • the subject is a non-human animal.
  • the tumor antigen compositions can be used to raise antibodies (e.g., in a non-human animal, such as a mouse, rat, hamster, or goat), e.g., for use in diagnostic assays, and for therapeutic applications.
  • a tumor antigen discovered by a method described herein may be a potent T cell and/or B cell antigen.
  • Preparations of antibodies may be produced by immunizing a subject with the tumor antigen and isolating antiserum from the subject. Methods for eliciting high titers of high affinity, antigenspecific antibodies, and for isolating the tumor antigen-specific antibodies from antisera, are known in the art.
  • the tumor antigen compositions are used to raise monoclonal antibodies, e.g., human monoclonal antibodies.
  • a tumor antigen composition is used to induce an immune response in a human subject to provide a therapeutic response.
  • a tumor antigen composition is used to induce an immune response in a human subject that redirects an undesirable immune response.
  • a tumor antigen composition elicits an immune response that causes the subject to have a positive clinical response described herein, e.g., as compared to a subject who has not been administered the tumor antigen composition.
  • a tumor antigen composition elicits an immune response that causes the subject to have an improved clinical response, e.g., as compared to a subject who has not been administered the tumor antigen composition.
  • a tumor antigen composition is used to induce an immune response in a human subject for palliative effect.
  • the response can be complete or partial therapy.
  • a tumor antigen composition is used to induce an immune response in a human subject to provide a prophylactic response.
  • the response can be complete or partial protection.
  • immunogenicity of a tumor antigen is evaluated in vivo.
  • humoral responses to a tumor antigen are evaluated (e.g., by detecting antibody titers to the administered tumor antigen).
  • cellular immune responses to a tumor antigen are evaluated, e.g., by detecting the frequency of antigen-specific cells in a sample from the subject (e.g., by staining T cells from the subject with MHC/peptide tetramers containing the antigenic peptide, to detect antigen-specific T cells, or by detecting antigen-specific cells using an antigen presentation assay such as an assay described herein).
  • the ability of a tumor antigen or antigens to elicit protective or therapeutic immunity is evaluated in an animal model. In some embodiments, the ability of a tumor antigen or antigens to stimulate or to suppress and/or inhibit immunity is evaluated in an animal model.
  • a tumor is or comprises a hematologic malignancy, including but not limited to, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, AIDS-related lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, Langerhans cell histiocytosis, multiple myeloma, or myeloproliferative neoplasms.
  • a tumor is or comprises a solid tumor, including but not limited to breast carcinoma, a squamous cell carcinoma, a colon cancer, a head and neck cancer, ovarian cancer, a lung cancer, mesothelioma, a genitourinary cancer, a rectal cancer, a gastric cancer, or an esophageal cancer.
  • a tumor is or comprises an advanced tumor, and/or a refractory tumor.
  • a tumor is characterized as advanced when certain pathologies are observed in a tumor (e.g., in a tissue sample, such as a biopsy sample, obtained from a tumor) and/or when cancer patients with such tumors are typically considered not to be candidates for conventional chemotherapy.
  • pathologies characterizing tumors as advanced can include tumor size, altered expression of genetic markers, invasion of adjacent organs and/ or lymph nodes by tumor cells.
  • a tumor is characterized as refractory when patients having such a tumor are resistant to one or more known therapeutic modalities (e.g., one or more conventional chemotherapy regimens) and/or when a particular patient has demonstrated resistance (e.g., lack of responsiveness) to one or more such known therapeutic modalities.
  • one or more known therapeutic modalities e.g., one or more conventional chemotherapy regimens
  • resistance e.g., lack of responsiveness
  • the present disclosure provides methods and systems related to cancer therapy.
  • the present disclosure is not limited to any specific cancer therapy, and any known or developed cancer therapy is encompassed by the present disclosure.
  • Known cancer therapies include, e.g., administration of chemotherapeutic agents, radiation therapy, surgical excision, chemotherapy following surgical excision of tumor, adjuvant therapy, localized hypothermia or hyperthermia, anti-tumor antibodies, and anti-angiogenic agents.
  • cancer and/or adjuvant therapy includes a TLR agonist (e.g., CpG, Poly I:C, etc., see, e.g., Wittig et al., Crit. Rev. Oncol. Hematol.
  • the cancer therapy is or comprises oncolytic virus therapy, e.g., talimogene leherparepvec. (see, e.g., Fukuhara et al., Cancer Sci. 107: 1373-1379 (2016)).
  • the cancer therapy is or comprises bi-specific antibody therapy (e.g., Choi et al., 2011 Expert Opin Biol Ther, Huehls et al., 2015, Immunol and Cell Biol).
  • the cancer therapy is or comprises cellular therapy such as chimeric antigen receptor T (CAR-T) cells, TCR-transduced T cells, dendritic cells, tumor infiltrating lymphocytes (TIL), or natural killer (NK) cells (e.g., as reviewed in Sharpe and Mount, 2015, Dis Model Meeh 8:337-50).
  • CAR-T chimeric antigen receptor T
  • TIL tumor infiltrating lymphocytes
  • NK natural killer cells
  • a cancer therapy is or comprises an immunotherapy.
  • an immunotherapy is or comprises immune checkpoint blockade therapy (see, e.g., Martin-Liberal et al., Cancer Treat. Rev. 54:74-86 (2017); Menon et al., Cancers (Basel) 8: 106 (2016)), or immune suppression blockade therapy.
  • Certain cancer cells thrive by taking advantage of immune checkpoint pathways as a major mechanism of immune resistance, particularly with respect to T cells that are specific for tumor antigens. For example, certain cancer cells may overexpress one or more immune checkpoint proteins responsible for inhibiting a cytotoxic T cell response.
  • immune checkpoint blockade therapy may be administered to overcome the inhibitory signals and permit and/or augment an immune attack against cancer cells.
  • Immune checkpoint blockade therapy may facilitate immune cell responses against cancer cells by decreasing, inhibiting, or abrogating signaling by negative immune response regulators (e.g., CTLA-4).
  • a cancer therapy or may stimulate or enhance signaling of positive regulators of immune response (e.g., CD28).
  • immune checkpoint blockade and immune suppression blockade therapy include agents targeting one or more of A2AR, B7-H4, BTLA, CTLA-4, CD28, CD40, CD137, GITR, IDO, KIR, LAG-3, PD-1, PD-L1, 0X40, TIM-3, TIGIT, CBLB, CISH, B7-H3, CSF-1R, VISTA, .
  • immune checkpoint blockade agents include the following monoclonal antibodies: ipilimumab (targets CTLA-4); tremelimumab (targets CTLA- 4); atezolizumab (targets PD-L1); pembrolizumab (targets PD-1); nivolumab (targets PD-1); avelumab; durvalumab; spartalizumab; camrelizumab; cabiralizumab; cemiplimab; dostarlimab; sintilmab; tislelizumab; and toripalirnab.
  • an immunotherapy is or comprises administration of one or more of PD-1 or PD-L1 blockade therapies. In some embodiments, an immunotherapy is or comprises administration of one or more of CTLA-4 blockade therapies.
  • an immunotherapy can include any of ipilumimab and tremelimumab which target CTLA-4; pembrolizumab, nivolumab, avelumab, durvalumab, and atezoluzumab, which target PD-1; or combinations thereof.
  • immunotherapy involves administration of an agent that acts as a blockade of cytotoxic T-lymphocyte- associated protein 4 (CTLA-4).
  • CTLA-4 cytotoxic T-lymphocyte-associated protein 4
  • immunotherapy involves treatment with an agent that interferes with an interaction involving CTLA-4 (e.g., with CD80 or CD86).
  • immunotherapy involves administration of one or more of tremelimumab and/or ipilimumab.
  • immunotherapy e.g. immune checkpoint blockade therapy
  • an agent e.g. antibody agent
  • PD-1 programmed cell death 1
  • immunotherapy involves treatment with an agent that interferes with an interaction involving PD-1 (e.g., with PD-L1).
  • immunotherapy involves administration of an agent (e.g. antibody agent) that specifically interacts with PD-1 or with PD-L1.
  • agent e.g. antibody agent
  • immunotherapy e.g. immune checkpoint blockade therapy
  • immune suppression blockade agents include: Vista (B7- H5, v-domain Ig suppressor of T cell activation) inhibitors; Lag-3 (lymphocyte-activation gene 3, CD223) inhibitors; IDO (indolemamine-pyrrole-2,3,-dioxygenase-l,2) inhibitors; KIR receptor family (killer cell immunoglobulin-like receptor) inhibitors; CD47 inhibitors; and TIGIT (T cell immunoreceptor with Ig and ITIM domain) inhibitors.
  • PD-1 is expressed on T cells, B cells, and certain myeloid cells; however, its role is best characterized in T cells. PD-1 expression on T cells is induced by antigen stimulation. Unlike CTLA-4, which limits early T-cell activation, PD-1 mainly exerts its inhibitory effect on T cells in the periphery where T cells encounter PD-1 ligands. Two ligands of PD-1 have been identified so far, PD-L1 and PD-L2, which are expressed by a large range of cell types, including tumor cells, monocyte-derived myeloid dendritic cells, epithelial cells, T cells, and B cells.
  • tumor cells and myeloid cells are thought to be main cell types mediating T-cell suppression through PD-1 ligation. It is still unclear whether effects of PD-L1 and PD-L2 on PD-1 downstream signaling are dependent on cell type that expresses a given ligand. Moreover, there are differences between PD-L1- versus PD-L2-induced effects, which remain to be fully elucidated.
  • PD-1 ligation inhibits T cell activation only upon T cell receptor engagement.
  • PD-1 has an intracellular “immunoreceptor tyrosine-based inhibition motif’ or (ITIM) and an immunoreceptor tyrosine-based switch motif. It has been shown that PD-1 ligation leads to recruitment of phosphatases called “src homology 2 domain-containing tyrosine phosphatases,” or SHP-1 and SHP-2, to immunoreceptor tyrosine-based switch motif.
  • ITIM immunoreceptor tyrosine-based inhibition motif
  • SHP-1 and SHP-2 immunoreceptor tyrosine-based switch motif.
  • PD-1 ligation has been shown to interfere with signaling molecules, such as phosphatidylinositol-4,5-bisphosphate 3-kinase and Ras, which are important for T-cell proliferation, cytokine secretion, and metabolism.
  • signaling molecules such as phosphatidylinositol-4,5-bisphosphate 3-kinase and Ras
  • HlV human immunodeficiency virus
  • Ligation of PD-1 has also been shown to induce metabolic alterations in T cells. Metabolic reprogramming of T cells from glycolysis to lipolysis is a consequence of PD-1 -mediated impairment of T-cell effector function.
  • PD-l-induced defects in mitochondrial respiration and glycolysis leads to impaired T-cell effector function that could be reversed by a mammalian target of rapamycin inhibition. Since most of the identified mechanisms of PD-1 -mediated T-cell suppression are based on in vitro or ex vivo experiments, it remains to be demonstrated that these same mechanisms are responsible for T-cell exhaustion in vivo.
  • PD-1 is a type I membrane protein of 288 amino acids and is a member of the extended CD28/CTLA-4 family of T cell regulators.
  • PD-1 protein structure includes an extracellular IgV domain followed by a transmembrane region and an intracellular tail, which contains two phosphorylation sites located in an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif, which suggests that PD-1 negatively regulates T cell receptor signals. This is consistent with binding of SHP-1 and SHP-2 phosphatases to PD-1 cytoplasmic tail upon ligand binding.
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • PD-1 ligation up- regulates E3 -ubiquitin ligases CBL-b and c-CBL that trigger T cell receptor down-modulation.
  • PD-1 is expressed on the surface of activated T cells, B cells, and macrophages, suggesting that compared to CTLA-4, PD-1 more broadly negatively regulates immune responses.
  • CTLA-4 is a member of the immunoglobulin superfamily that is expressed by activated T cells and transmits an inhibitory signal to T cells.
  • CTLA-4 is structurally similar to T cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86 on antigen- presenting cells.
  • CTLA-4 binds CD80 and CD86 with greater affinity than CD28, thus enabling it to outcompete CD28 for its ligands.
  • CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. T cell activation through T cell receptor and CD28 leads to increased expression of CTLA-4.
  • CTLA-4 acts in T cells remains somewhat elusive. Biochemical evidence suggests that CTLA-4 recruits a phosphatase to a T cell receptor, thus attenuating the signal. It has also been suggested that CTLA-4 may function in vivo by capturing and removing CD80 and CD86 from membranes of antigen-presenting cells, thus making these antigens unavailable for triggering of CD28.
  • CTLA-4 protein contains an extracellular V domain, a transmembrane domain, and a cytoplasmic tail.
  • CTLA-4 has an intracellular domain that is similar to that of CD28, in that it has no intrinsic catalytic activity and contains one YVKM motif able to bind PI3K, PP2A and SHP-2, as well as one proline-rich motif able to bind SH3 -containing proteins.
  • One role of CTLA-4 in inhibiting T cell responses seems to directly involve SHP-2 and PP2A dephosphorylation of T cell receptor-proximal signaling proteins, such as CD3 and LAT.
  • CTLA- 4 can also affect signaling indirectly, via competition with CD28 for CD80 and/or CD86 binding.
  • ipilimumab is a human IgGl antibody with specificity for CTLA-4.
  • another CTLA-4 blockade therapy, tremelimumab is a human IgG2 antibody.
  • an immunotherapyis or comprises immune activation therapy.
  • immune activators include: CD40 agonists; GITR (glucocorticoid-induced TNF-R-related protein, CD357) agonists; 0X40 (CD134) agonists; 4- 1BB (CD137) agonists; ICOS (inducible T cell stimulator); CD278 agonists; IL-2 (interleukin 2) agonists; and interferon agonists.
  • an immunotherapy is or comprises a combination of one or more immune checkpoint blockade agents, immune suppression blockade agents, and/or immune activators, or a combination of one or more immune checkpoint blockade agents, immune suppression blockade agents, and/or immune activators, and other cancer therapies.
  • an immune checkpoint blockade therapy is and/or has been administered to an individual.
  • Examples 4 and 5 present certain FDA-approved dosing information for PD-1 and CTLA-4 blockade regimens, respectively.
  • an immune checkpoint blockade therapy is administered in accordance with the present invention according to such an approved protocol.
  • an immune checkpoint blockade therapy is administered in a pharmaceutical composition that also comprises a physiologically acceptable carrier or excipient.
  • a pharmaceutical composition is sterile.
  • a pharmaceutical composition is formulated for a particular mode of administration.
  • suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, sugars such as mannitol, sucrose, or others, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, etc., as well as combinations thereof.
  • salt solutions e.g., NaCl
  • saline e.g., buffered saline
  • alcohols e.glycerol
  • ethanol e.glycerol
  • gum arabic e.glycerol
  • vegetable oils e.glycerol
  • benzyl alcohols polyethylene glycols
  • a pharmaceutical preparation can, if desired, comprise one or more auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like) which do not deleteriously react with the active compounds or interference with their activity.
  • auxiliary agents e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like
  • a water-soluble carrier suitable for intravenous administration is used.
  • a pharmaceutical composition or medicament can contain an amount (typically a minor amount) of wetting or emulsifying agents, and/or of pH buffering agents.
  • a pharmaceutical composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.
  • a pharmaceutical composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharine, cellulose, magnesium carbonate, etc.
  • a pharmaceutical composition can be formulated in accordance with routine procedures as a pharmaceutical composition adapted for administration to human beings.
  • a composition for intravenous administration typically is a solution in sterile isotonic aqueous buffer.
  • a composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection.
  • ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachet indicating the quantity of active agent.
  • a composition where a composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water.
  • an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • an immune checkpoint blockade therapy can be formulated in a neutral form; in some embodiments it may be formulated in a salt form.
  • pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
  • compositions for use in accordance with the present invention may be administered by any appropriate route.
  • a pharmaceutical composition is administered intravenously.
  • a pharmaceutical composition is administered subcutaneously.
  • a pharmaceutical composition is administered by direct administration to a target tissue, such as heart or muscle (e.g., intramuscular), or nervous system (e.g., direct injection into the brain; intraventricularly; intrathecally).
  • a pharmaceutical composition is administered parenterally, transdermally, or transmucosally (e.g., orally or nasally). More than one route can be used concurrently, if desired.
  • an immune checkpoint blockade therapy can be administered alone, or in conjunction with other immunomodulatory agents.
  • the term, “in conjunction with,” indicates that a first an immune checkpoint blockade therapy is administered prior to, at about the same time as, or following another an immune checkpoint blockade therapy.
  • a first an immune checkpoint blockade therapy can be mixed into a composition containing one or more different an immune checkpoint blockade therapies, and thereby administered contemporaneously; alternatively, in some embodiments, the agent can be administered contemporaneously, without mixing (e.g., by “piggybacking” delivery of the agent on the intravenous line by which the an immune checkpoint blockade therapy is also administered, or vice versa).
  • an immune checkpoint blockade therapy can be administered separately (e.g., not admixed), but within a short time frame (e.g., within 24 hours) of administration of another an immune checkpoint blockade therapy.
  • subjects treated with an immune checkpoint blockade therapy are administered one or more immunosuppressants.
  • one or more immunosuppressants are administered to decrease, inhibit, or prevent an undesired autoimmune response (e.g., enterocolitis, hepatitis, dermatitis (including toxic epidermal necrolysis), neuropathy, and/or endocrinopathy), for example, hypothyroidism.
  • exemplary immunosuppressants include steroids, antibodies, immunoglobulin fusion proteins, and the like.
  • an immunosuppressant inhibits B cell activity (e.g. rituximab).
  • an immunosuppressant is a decoy polypeptide antigen.
  • an immune checkpoint blockade therapy is administered in a therapeutically effective amount (e.g., a dosage amount and/or according to a dosage regimen that has been shown, when administered to a relevant population, to be sufficient to treat cancer, such as by ameliorating symptoms associated with the cancer, preventing or delaying the onset of the cancer, and/or also lessening the severity or frequency of symptoms of cancer).
  • a therapeutically effective amount e.g., a dosage amount and/or according to a dosage regimen that has been shown, when administered to a relevant population, to be sufficient to treat cancer, such as by ameliorating symptoms associated with the cancer, preventing or delaying the onset of the cancer, and/or also lessening the severity or frequency of symptoms of cancer.
  • long term clinical benefit is observed after treatment with an immune checkpoint blockade therapy, including, for example, PD-1 blockade such as pembrolizumab, CTLA-4 blockade such as ipilimumab, and/or other agents.
  • a dose which will be therapeutically effective for the treatment of cancer in a given patient may depend, at least to some extent, on the nature and extent of cancer, and can be determined by standard clinical techniques.
  • one or more in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges.
  • a particular dose to be employed in the treatment of a given individual may depend on the route of administration, the extent of cancer, and/or one or more other factors deemed relevant in the judgment of a practitioner in light of patient’s circumstances.
  • effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems (e.g., as described by the U.S. Department of Health and Human Services, Food and Drug Administration, and Center for Drug Evaluation and Research in “Guidance for Industry: Estimating Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers”, Pharmacology and Toxicology, July 2005).
  • a therapeutically effective amount of an immune checkpoint blockade therapy can be, for example, more than about 0.01 mg/kg, more than about 0.05 mg/kg, more than about 0.1 mg/kg, more than about 0.5 mg/kg, more than about 1.0 mg/kg, more than about 1.5 mg/kg, more than about 2.0 mg/kg, more than about 2.5 mg/kg, more than about 5.0 mg/kg, more than about 7.5 mg/kg, more than about 10 mg/kg, more than about 12.5 mg/kg, more than about 15 mg/kg, more than about 17.5 mg/kg, more than about 20 mg/kg, more than about 22.5 mg/kg, or more than about 25 mg/kg body weight.
  • a therapeutically effective amount can be about 0.01-25 mg/kg, about 0.01-20 mg/kg, about 0. QI- 15 mg/kg, about 0.01-10 mg/kg, about 0.01-7.5 mg/kg, about 0.01-5 mg/kg, about 0.01-4 mg/kg, about 0.01-3 mg/kg, about 0.01-2 mg/kg, about 0.01-1.5 mg/kg, about 0.01-1.0 mg/kg, about 0.01-0.5 mg/kg, about 0.01-0.1 mg/kg, about 1-20 mg/kg, about 4-20 mg/kg, about 5-15 mg/kg, about 5-10 mg/kg body weight.
  • a therapeutically effective amount is about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1.0 mg/kg, about 1.1 mg/kg, about 1.2 mg/kg, about 1.3 mg/kg about 1.4 mg/kg, about 1.5 mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, about 1.9 mg/kg, about 2.0 mg/kg, about 2.5 mg/kg, about 3.0 mg/kg, about 4.0 mg/kg, about 5.0 mg/kg, about 6.0 mg/kg, about 7.0 mg/kg, about 8.0 mg/kg, about 9.0 mg/kg, about 10.0 mg/kg, about 11.0 mg/kg, about 12.0 mg/kg, about 13.0 mg/kg, about 14.0 mg/kg, about 1
  • the therapeutically effective amount is no greater than about 30 mg/kg, no greater than about 20 mg/kg, no greater than about 15 mg/kg, no greater than about 10 mg/kg, no greater than about 7.5 mg/kg, no greater than about 5 mg/kg, no greater than about 4 mg/kg, no greater than about 3 mg/kg, no greater than about 2 mg/kg, or no greater than about 1 mg/kg body weight or less.
  • the administered dose for a particular individual is varied (e.g., increased or decreased) over time, depending on the needs of the individual.
  • a loading dose (e.g., an initial higher dose) of a therapeutic composition may be given at the beginning of a course of treatment, followed by administration of a decreased maintenance dose (e.g., a subsequent lower dose) of the therapeutic composition.
  • a loading dose may clear out an initial and, in some cases massive, accumulation of undesirable materials (e.g., fatty materials and/or tumor cells, etc) in tissues (e.g., in the liver), and maintenance dosing may delay, reduce, or prevent buildup of fatty materials after initial clearance.
  • a loading dose and maintenance dose amounts, intervals, and duration of treatment may be determined by any available method, such as those exemplified herein and those known in the art.
  • a loading dose amount is about 0.01-1 mg/kg, about 0.01-5 mg/kg, about 0.01-10 mg/kg, about 0.1-10 mg/kg, about 0.1-20 mg/kg, about 0.1-25 mg/kg, about 0.1-30 mg/kg, about 0.1-5 mg/kg, about 0.1-2 mg/kg, about 0.1-1 mg/kg, or about 0.1-0.5 mg/kg body weight.
  • a maintenance dose amount is about 0-10 mg/kg, about 0-5 mg/kg, about 0-2 mg/kg, about 0-1 mg/kg, about 0-0.5 mg/kg, about 0-0.4 mg/kg, about 0-0.3 mg/kg, about 0-0.2 mg/kg, about 0- 0.1 mg/kg body weight.
  • a loading dose is administered to an individual at regular intervals for a given period of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months) and/or a given number of doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more doses), followed by maintenance dosing.
  • a maintenance dose ranges from 0 - 2 mg/kg, about 0-1.5 mg/kg, about 0-1.0 mg/kg, about 0-0.75 mg/kg, about 0-0.5 mg/kg, about 0-0.4 mg/kg, about 0-0.3 mg/kg, about 0-0.2 mg/kg, or about 0-0.1 mg/kg body weight.
  • a maintenance dose is about 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, or 2.0 mg/kg body weight.
  • maintenance dosing is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months. In some embodiments, maintenance dosing is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years. In some embodiments, maintenance dosing is administered indefinitely (e.g., for life time).
  • a therapeutically effective amount of an immune checkpoint blockade therapy may be administered as a one-time dose or administered at intervals, depending on the nature and extent of the cancer, and on an ongoing basis.
  • Administration at an “interval,” as used herein indicates that the therapeutically effective amount is administered periodically (as distinguished from a one-time dose).
  • an interval can be determined by standard clinical techniques.
  • an immunomodulatory agent e.g. immune checkpoint modulator
  • the administration interval for a single individual need not be a fixed interval, but can be varied over time, depending on the needs and rate of recovery of the individual.
  • the term “bimonthly” has its art understood meaning, referring to administration once per two months (i.e., once every two months); the term “monthly” means administration once per month; the term “triweekly” means administration once per three weeks (i.e., once every three weeks); the term “biweekly” means administration once per two weeks (i.e., once every two weeks); the term “weekly” means administration once per week; and the term “daily” means administration once per day.
  • the invention additionally pertains to a pharmaceutical composition comprising an immune checkpoint blockade therapy, as described herein; in some embodiments in a container (e.g., a vial, bottle, bag for intravenous administration, syringe, etc.) with a label containing instructions for administration of the composition for treatment of cancer.
  • a container e.g., a vial, bottle, bag for intravenous administration, syringe, etc.
  • the present disclosure provides methods and systems related to subjects who do not respond and/or have not responded; or respond and/or have responded (e.g., clinically responsive, e.g., clinically positively responsive or clinically negatively responsive) to a cancer therapy.
  • subjects respond and/or have responded positively clinically to a cancer therapy.
  • subjects respond and/or have responded negatively clinically to a cancer therapy.
  • subjects do not respond and/or have not responded (e.g., clinically non-responsive or refractory) to a cancer therapy.
  • Whether a subject responds positively, responds negatively, and/or fails to respond to a cancer therapy can be measured and/or characterized according to particular criteria.
  • criteria can include clinical criteria and/or objective criteria.
  • techniques for assessing response can include, but are not limited to, clinical examination, positron emission tomography, chest X-ray, CT scan, MRI, ultrasound, endoscopy, laparoscopy, presence or level of a particular marker in a sample, cytology, and/or histology.
  • a positive response, a negative response, and/or no response, of a tumor to a therapy can be assessed by ones skilled in the art using a variety of established techniques for assessing such response, including, for example, for determining one or more of tumor burden, tumor size, tumor stage, etc. Methods and guidelines for assessing response to treatment are discussed in Therasse et al., J. Natl. Cancer Inst., 2000, 92(3):205-216; and Seymour et al., Lancet Oncol., 2017, 18:el43-52.
  • a responsive subject exhibits a decrease in tumor burden, tumor size, and/or tumor stage upon administration of a cancer therapy.
  • a non-responsive or refractory subject does not exhibit a decrease in tumor burden, tumor size, or tumor stage upon administration of a cancer therapy.
  • a non-responsive or refractory subject exhibits an increase in tumor burden, tumor size, or tumor stage upon administration of a cancer therapy.
  • a cancer subject is identified and/or selected for administration of a cancer therapy as described herein. In some embodiments, the cancer therapy is administered to the subject.
  • the subject upon administration of the cancer therapy, the subject exhibits a positive clinical response to the cancer therapy, e.g., exhibits an improvement based on one or more clinical and/or objective criteria (e.g., exhibits a decrease in tumor burden, tumor size, and/or tumor stage).
  • the clinical response is more positive than a clinical response to the cancer therapy administered to a cancer subject who is identified (using a method described herein) as a cancer subject who should not initiate, and/or should modify (e.g., reduce and/or combine with one or more other modalities), and/or should discontinue the cancer therapy, and/or should initiate an alternative cancer therapy.
  • Methods described herein can include preparing and/or providing a report, such as in electronic, web-based, or paper form.
  • the report can include one or more outputs from a method described herein, e.g., a set of stimulatory and/or inhibitory antigens described herein.
  • a report is generated, such as in paper or electronic form, which identifies the presence or absence of one or more tumor antigens (e.g., one or more stimulatory and/or inhibitory and/or suppressive tumor antigens, or tumor antigens to which lymphocytes are not responsive, described herein) for a cancer patient, and optionally, a recommended course of cancer therapy.
  • the report includes an identifier for the cancer patient.
  • the report is in web-based form.
  • a report includes information on prognosis, resistance, or potential or suggested therapeutic options.
  • the report can include information on the likely effectiveness of a therapeutic option, the acceptability of a therapeutic option, or the advisability of applying the therapeutic option to a cancer patient, e.g., identified in the report.
  • the report can include information, or a recommendation, on the administration of a cancer therapy, e.g., the administration of a pre-selected dosage or in a preselected treatment regimen, e.g., in combination with one or more alternative cancer therapies, to the patient.
  • the report can be delivered, e.g., to an entity described herein, within 7, 14, 21, 30, or 45 days from performing a method described herein.
  • the report is a personalized cancer treatment report.
  • a report is generated to memorialize each time a cancer subject is tested using a method described herein.
  • the cancer subject can be reevaluated at intervals, such as every month, every two months, every six months or every year, or more or less frequently, to monitor the subject for responsiveness to a cancer therapy and/or for an improvement in one or more cancer symptoms, e.g., described herein.
  • the report can record at least the treatment history of the cancer subject.
  • the method further includes providing a report to another party.
  • the other party can be, for example, the cancer subject, a caregiver, a physician, an oncologist, a hospital, clinic, third-party payor, insurance company or a government office.
  • an immunogenic composition described herein e.g., an immunogenic composition comprising one or more stimulatory antigens described herein
  • Combination therapy refers to those situations in which a subject or population of subjects is simultaneously exposed to two or more therapeutic agents (e.g., an immunogenic composition and a cancer therapy).
  • the two or more therapies may be administered simultaneously (e.g., concurrently).
  • such therapies may be administered sequentially (e.g., all “doses” of a first therapeutic agent are administered prior to administration of any doses of a second therapeutic agent).
  • “administration” of combination therapy may involve administration of one or more agents or modalities to a subject receiving the other agents or modalities in the combination.
  • combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).
  • an immunogenic composition described herein e.g., an immunogenic composition comprising one or more stimulatory antigens described herein
  • one or more cancer therapies such as an immunotherapy.
  • an immunogenic composition described herein e.g., an immunogenic composition comprising one or more stimulatory antigens described herein
  • an immunogenic composition described herein is administered to a patient that has received or is recieving an immunotherapy.
  • immunotherapy involves administration of an agent that acts as a blockade of cytotoxic T-lymphocyte- associated protein 4 (CTLA-4).
  • CTLA-4 cytotoxic T-lymphocyte-associated protein 4
  • immunotherapy involves treatment with an agent that interferes with an interaction involving CTLA-4 (e.g., with CD80 or CD86).
  • immunotherapy involves administration of one or more of tremelimumab and/or ipilimumab.
  • immunotherapy e.g. immune checkpoint blockade therapy
  • an agent e.g. antibody agent
  • PD-1 programmed cell death 1
  • immunotherapy involves treatment with an agent that interferes with an interaction involving PD-1 (e.g., with PD-L1).
  • immunotherapy involves administration of an agent (e.g. antibody agent) that specifically interacts with PD-1 or with PD-L1.
  • agent e.g. antibody agent
  • immunotherapy e.g. immune checkpoint blockade therapy
  • a patient is receiving or has received an immunotherapy for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 12 weeks, or longer prior to receiving an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein).
  • a patient is receiving or has received an immunotherapy for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 12 months, or longer prior to receiving an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein).
  • the immunotherapy treatment is continued which the subject receives the immunogenic composition.
  • the immunotherapy treatment and immunogenic composition is administered concurrently to the subject.
  • the subject exhibits no cancer progression after 12 months after treatment with the immunogenic composition.
  • the efficacy of the immunogenic composition and/or the immunotherapy is improved in the subject over a specified period of time related to the subject receiving only the immunogenic composition or the immunotherapy alone.
  • the efficacy of the immunogenic composition and/or the immunotherapy is measured or indicated by a decrease in target lesion sum or diameter based on a cancer patient’s tumor size.
  • the decrease in target lesion sum or diameter comprises at least a 10%, 20%, 30%, 40%, or 50% or more decrease in target lesion sum or diameter compared to the control level.
  • the efficacy of the immunogenic composition and/or the immunotherapy is measured or indicated by a decrease in cancer progression. In some embodiments, the efficacy of the immunogenic composition is measured or indicated by an increase in humoral response and/or an increase in cellular response.
  • an immunogenic composition described herein e.g., an immunogenic composition comprising one or more stimulatory antigens described herein
  • a patient that has received or is recieving one or more additional therapeutic agents, such as a cancer therapeutic agent, e.g., a chemotherapeutic agent or a biological agent.
  • an immunogenic composition described herein e.g., an immunogenic composition comprising one or more stimulatory antigens described herein
  • is administered to a subject who has received, is receiving, and/or will receive therapy with another therapeutic agent or modality e.g., with a chemotherapeutic agent, surgery, radiation, or a combination thereof.
  • Some embodiments of combination therapy modalities provided by the present disclosure provide, for example, administration of an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein) and additional agent(s) in a single pharmaceutical formulation. Some embodiments provide administration of an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein) and administration of an additional therapeutic agent in separate pharmaceutical formulations.
  • chemotherapeutic agents that can be used in combination with an immunomodulatory agent described herein include platinum compounds (e.g., cisplatin, carboplatin, and oxaliplatin), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, nitrogen mustard, thiotepa, melphalan, busulfan, procarbazine, streptozocin, temozolomide, dacarbazine, and bendamustine), antitumor antibiotics (e.g., daunorubicin, doxorubicin, idarubicin, epirubicin, mitoxantrone, bleomycin, mytomycin C, plicamycin, and dactinomycin), taxanes (e.g., paclitaxel and docetaxel), antimetabolites (e.g., 5-fluorouracil, cytarabine, premetrexed
  • Examples of biological agents that can be used in the compositions and methods described herein include monoclonal antibodies (e.g., rituximab, cetuximab, panetumumab, tositumomab, trastuzumab, alemtuzumab, gemtuzumab ozogamicin, bevacizumab, catumaxomab, denosumab, obinutuzumab, ofatumumab, ramucirumab, pertuzumab, ipilimumab, nivolumab, nimotuzumab, lambrolizumab, pidilizumab, siltuximab, BMS-936559, RG7446/MPDL3280A, MEDI4736, tremelimumab, dostarlimab, siniilmab, tisleliumab, or toripalimab, trastuzum
  • an immunogenic composition described herein e.g., an immunogenic composition comprising one or more stimulatory antigens described herein
  • another agent for the treatment of cancer either in the same or in different pharmaceutical compositions.
  • the additional agent is an anticancer agent.
  • the additional agent affects (e.g, inhibits) histone modifications, such as histone acetylation or histone methylation.
  • an additional anticancer agent is selected from the group consisting of chemotherapeutics (such as 2CdA, 5-FU, 6-Mercaptopurine, 6-TG, AbraxaneTM, Accutane®, Actinomycin-D, Adriamycin®, Alimta®, all-trans retinoic acid, amethopterin, Ara-C, Azacitadine, BCNU, Blenoxane®, Camptosar®, CeeNU®, Clofarabine, ClolarTM, Cytoxan®, daunorubicin hydrochloride, DaunoXome®, Dacogen®, DIC, Doxil®, Ellence®, Eloxatin®, Emcyt®, etoposide phosphate, Fludara®, FUDR®, Gemzar®, Gleevec®, hexamethylmelamine, Hycamtin®, Hydrea®, Idamycin®, Ifex®, ixabepilone, Ixem
  • an immunogenic composition described herein e.g., an immunogenic composition comprising one or more stimulatory antigens described herein
  • additional agents that can be used in combination with an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein) as set forth above are for illustrative purposes and not intended to be limiting.
  • the combinations embraced by this disclosure include, without limitation, an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein) and at least one additional agent selected from the lists above or otherwise provided herein.
  • An immunogenic composition described herein e.g., an immunogenic composition comprising one or more stimulatory antigens described herein
  • treatment methods described herein are performed on subjects for which other treatments of the medical condition have failed or have had less success in treatment through other means, e.g., in subjects having a cancer refractory to standard-of-care treatment.
  • the treatment methods described herein can be performed in conjunction with one or more additional treatments of the medical condition, e.g., in addition to or in combination with standard-of-care treatment.
  • the method can comprise administering a cancer regimen, e.g., nonmyeloablative chemotherapy, surgery, hormone therapy, and/or radiation, prior to, substantially simultaneously with, or after the administration of an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein).
  • a subject to which an immunogenic composition described herein e.g., an immunogenic composition comprising one or more stimulatory antigens described herein
  • an immunogenic composition described herein can also be treated with antibiotics and/or one or more additional pharmaceutical agents.
  • All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.
  • the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.
  • NSCLC Non-small cell lung cancer
  • SCCHN Squamous cell carcinoma of the head and neck
  • GEN 009 is an investigational, personalized adjuvanted vaccine that is being developed for the treatment of patients with solid tumors.
  • a system as described above, and herein called ATLAS (Antigen Lead Acquisition System) was used to identify neoantigens in each patient’s tumor that are recognized by their CD4+ and/or CD8+ T cells.
  • SLPs synthetic long peptides
  • the SLPs were divided into 4 pools, with each pool containing 1 to 5 SLPs.
  • the 4 pools were administered subcutaneously (SC) in each of the patient’s limbs.
  • SC subcutaneously
  • these pools of SLPs are the GEN 009 drug product. If fewer than 4 pools were available due to manufacturing, stability, or other issues, the patient was vaccinated with the available drug product.
  • Each pool of GEN 009 drug product consisted of 100 to 1500 pg total peptide administered with 0.45 mg poly-ICLC adjuvant (Hiltonol) per injection.
  • Part A Schedule Evaluation of GEN 009 Monotherapy in Patients with No Evidence of Disease
  • a 5-dose schedule was evaluated (Schedule 1; Days 1, 22, 43, 85 [12 weeks after Day 1], and 169 [24 weeks after Day 1]).
  • PBMC peripheral blood mononuclear cells
  • NGS next-generation sequencing
  • each patient was reevaluated.
  • a sufficient number of stimulatory antigens for SLP manufacture must have been identified from the ATLAS process, and the patient must continue to meet study eligibility criteria, including NED on a radiographic disease assessment performed within 8 weeks prior to the reevaluation.
  • Part B GEN 009 in Patients with Advanced or Metastatic Solid Tumors
  • Part B included patients with one of 5 tumor types (NSCLC, SCCHN, cutaneous melanoma, urothelial carcinoma or RCC) who were enrolled in a disease-specific expansion cohort (up to 15 response evaluable patients each).
  • NSCLC tumor type-specific treatments identified below (i.e., PD-1 inhibitor monotherapy or PD-1 inhibitor in combination, per disease-specific standard of care and USPI).
  • GEN 009 dosing was initiated (starting as soon as the vaccine was available and at the schedule selected in Part A) in combination with the PD-1 inhibitor:
  • NSCLC pembrolizumab with chemotherapy (pemetrexed and platinum chemotherapy for non-squamous histologies; carboplatin and either paclitaxel or nab-paclitaxel for squamous NSCLC) during the screening period, followed by pembrolizumab and GEN 009 during the treatment period;
  • SCCHN pembrolizumab monotherapy during the screening period, followed by pembrolizumab and GEN 009 during the treatment period;
  • Cutaneous melanoma nivolumab monotherapy or nivolumab in combination with ipilimumab during the screening period, followed by nivolumab and GEN 009 during the treatment period;
  • Urothelial carcinoma pembrolizumab monotherapy during the screening period, followed by pembrolizumab and GEN 009 during the treatment period;
  • RCC nivolumab monotherapy or nivolumab in combination with ipilimumab during the screening period, followed by nivolumab and GEN 009 during the treatment period.
  • PBMCs peripheral blood mononuclear cells
  • samples of tumor obtained after the patient’s most recent systemic cancer therapy, if applicable, prior to initiation of the PD-1 inhibitor, and not from a previously irradiated lesion
  • saliva PBMCs from leukapheresis was used for SCCHN patients due to potential malignant contamination in saliva
  • DA #1 A baseline radiographic disease assessment was performed within 4 weeks prior to initiation of the PD-1 inhibitor ( ⁇ chemotherapy or ipilimumab, as applicable); scans performed within this timeframe according to standard of care were acceptable.
  • nivolumab as monotherapy or with ipilimumab for cutaneous melanoma and RCC
  • pembrolizumab as monotherapy for SCCHN and urothelial carcinoma, or with chemotherapy for NSCLC.
  • Patients who had progressive disease (PD) on their PD-1 inhibitor-containing regimen prior to vaccination and required alternate (e.g., salvage) therapy were allowed to continue in the study during their alternate therapy and were vaccinated at an appropriate time in their disease course in the opinion of the Investigator and the Medical Monitor, if the patient continued to meet performance and laboratory eligibility criteria. For example, the patient was vaccinated at flexible 3- to 4-week intervals to fit into the patient’s alternate treatment regimen, or after a pause. These patients (i.e., the relapsed/refractory cohort, Part B Refractory Study) were assessed separately for objective response rate (ORR).
  • ORR objective response rate
  • the dosing schedules are outlined in Table 4 and depicted visually in Figure 1 Panel A.
  • a 5-dose schedule was evaluated (Schedule 1; Days 1, 22, 43, 85 [12 weeks after Day 1], and 169 [24 weeks after Day 1]).
  • PBMCs peripheral blood mononuclear cells
  • IFN- y interferon-gamma
  • GrB granzyme B
  • TNF-a ZFN-y/tumor necrosis factor-alpha
  • CD4+ and CD8+ polyfunctional T cell responses in PBMCs were assessed by immune assays such as intracellular cytokine staining.
  • Phenotypes of PBMC cell populations before and after vaccination were assessed by assays such as flow cytometry-based immunophenotyping panels examining regulatory T cells, activation/inhibition markers, and potentially other cell populations.
  • Part A Disease-free survival (DFS). Disease assessment (radiological imaging and for patients with urothelial carcinoma who had not undergone cystectomy, urine cytology; and for patients with tumor potentially visible by cystoscopy [e.g., of the urethra, bladder, ureterovesical junction], cystoscopy) was performed during the screening period (as per study eligibility), then every 12 weeks (starting 12 weeks after Day 1) through Day 337 (i.e., 4 assessments post-Day 1), then every 26 weeks until disease recurrence, initiation of another systemic anticancer therapy, or study closure. Note: PET/CT was used instead of CT or MRI per agreement of the Medical Monitor and Investigator for patients in Part A.
  • DFS Disease-free survival
  • Part B Since the GEN 009 vaccine was administered after 3 to 4 months of known active therapy, a traditional response rate and duration of response was difficult to evaluate. In general, the great majority of patients will have defined the course of their disease within those 3 to 4 months, so that any significant change in trajectory after addition of the vaccine likely represents an impact of the vaccine, noting that pseudoprogression could be responsible for a small percentage of responses. In this setting, each patient served as their own control in an exploratory analysis of relative intensity ratio (RIR) on imaging, duration of response (DoR), and progression-free survival (PFS).
  • RIR relative intensity ratio
  • DoR duration of response
  • PFS progression-free survival
  • Study-specific disease assessments were obtained during screening within 4 weeks prior to initiation of PD-1 inhibitor therapy, 6 to 10 weeks after initiation of PD-1 inhibitor therapy, and within 14 days prior to the first dose of GEN 009. Post first dose, study-specific disease assessments occured at Day 50 ( ⁇ 3 days) and Day 92 ( ⁇ 3 days). Additionally, throughout the study, standard of care disease assessments were recorded until disease progression, initiation of another systemic anticancer therapy, or study closure. Antitumor activity was also assessed by improvement in tumor growth kinetics (i.e., increase in tumor shrinkage rate or decrease in tumor growth rate) with GEN 009 vs projected rate without GEN 009. The overall study design is depicted visually in Figure 1, Panel B.
  • Part A The primary categorization for data summary and analysis consisted of the separate parts of the study. Within Part A, additional categories for summarization consisted of all schedules studied, as well as overall for certain data presentations. For Part B, data were analyzed separately for patients with PD prior to GEN 009 dosing. Further categories for data summarization for Part B consisted of data for each tumor type, data for those with relapsed/refractory disease, and overall. Select safety presentations used an overall pool across parts for summarization, as appropriate.
  • Part A DFS was summarized using Kaplan- Meier methods.
  • Part B RIR was tabulated by frequency distribution, with 2-sided exact 90% Cis. Median time to response and DoR were summarized for those patients with confirmed responses, using Kaplan Meier methods. PFS and overall survival (OS) were similarly summarized. In addition, the rate of patients with PFS and OS of at least 12 months duration was presented with 2-sided 90% Cis.
  • RIR in Part B was summarized as categorical data and by use of shift tables. Improvement in tumor growth kinetics, which was measured by comparing observed tumor growth rate with GEN 009 vs projected tumor growth rate without GEN 009 for each period from Day 1 was summarized by period for each patient.
  • Observed tumor growth rate for each period was calculated as the average percent change in the sum of the longest diameters from earlier time points when imaging was collected; and projected tumor growth rate post Day 1 was a weighted average of observed tumor growth rates prior to Day 1, where time points closer to Day 1 were assigned with heavier weighting.
  • Clinical activity analyses were descriptive; statistical tests were used as appropriate to compare changes before and after vaccination or between tumor types. Subgroup analysis of various immunologic parameters, as well as rate of response and time to event endpoints, based on demographic and baseline disease characteristics were performed as well as exploratory analyses, as appropriate.
  • GEN-009-101 is a first-in-human Phase l/2a study testing ATLAS platform feasibility, safety, immunogenicity and clinical activity in selected solid tumors.
  • SLPs stimulatory synthetic long peptides
  • neoantigens patient-specific stimulatory antigens
  • GEN 009 was administered with poly-ICLC on Day 1 (week 0), Day 22 (week 3), Day 43 (week 6) with booster vaccinations on Day 85 (week 12 after Day 1 vaccination) and Day 169 (week 24 after Day 1 vaccination).
  • PBMCs were collected for immunogenicity assessments from whole blood drawn at Day 1 vaccination (just prior to vaccination) and at Day 29, Day 92, and Day 176 (z.e., one week following vaccinations on Day 22, Day 85, and Day 169), and also via a leukapheresis procedure at initial patient screening (baseline) and at either Day 50, Day 92, or Day 176 (z.e., one week following vaccinations on Day 43, Day 85, or Day 169). Plasma was also obtained from these samples.
  • the cellular immune response to GEN 009 was monitored by examining T cell responses using a dual-color FluoroSpot assay.
  • the ex vivo FluoroSpot assay simultaneously detects release of interferon gamma (IFN-y) and granzyme B (GrB) from PBMCs, or T cell subsets enriched from PBMCs, following stimulation with peptide antigens for a duration of approximately 2 days.
  • This method varies the traditional Enzyme-linked ImmunoSpot (ELISpot) assay by replacing the colorimetric detection with fluorescence detection, enabling quantification of individual, peptide-reactive T cells that secrete multiple analytes of interest in a high- throughput format.
  • ELISpot Enzyme-linked ImmunoSpot
  • the method detects effector T cell responses.
  • complete PBMC populations, or CD4+ or CD8+ T cells enriched from PBMCs were stimulated with overlapping peptides spanning either the unique individual SLPs or all SLPs included in each of the 4 pools of SLPs for that patient to determine the frequency of antigen-specific T cells.
  • PBMCs or T cells enriched from PBMCs were combined with overlapping peptides spanning patient-specific SLPs in pre-conditioned, poly vinylidene difluoride membrane-bound 96-well plates, and incubated at 37°C for 44 ⁇ 4 hours.
  • Development of immune response-induced fluorescent spots was facilitated by addition of detection antibodies (anti-IFN-y monoclonal antibody and biotinylated anti-granzyme B monoclonal antibody) followed by anti-BAM-490 and SA-550 fluorescent antibodies.
  • the in vitro stimulated (IVS) FluoroSpot assay simultaneously detects release of interferon gamma (IFN-y) and tumor necrosis factor alpha (TNFa) from T cell subsets enriched from PBMCs, following in vitro stimulation (IVS) with peptide antigens for a duration of approximately 10 days in culture.
  • IFN-y interferon gamma
  • TNFa tumor necrosis factor alpha
  • the method is aimed at generating increased polyfunctional, peptide-reactive T cells over the course of the culture period. In general, the method detects memory T cell responses.
  • CD4+ or CD8+ T cells enriched from PBMCs were expanded in culture for 10 days with overlapping peptides spanning the unique individual SLPs or all SLPs included in each of the 4 pools of SLPs for that patient in the presence of IL-7.
  • cells were rested in fresh culture media without cytokines.
  • the expanded T cells were then combined with fresh antigen presenting cells and their respective overlapping peptides spanning patientspecific SLPs in pre-conditioned, polyvinylidene difluoride membrane-bound 96-well plates, and incubated at 37°C for 20 ⁇ 4 hours.
  • Development of immune response-induced fluorescent spots was facilitated by addition of detection antibodies (anti-IFN-y monoclonal antibody and biotinylated anti-TNF ⁇ monoclonal antibody) followed by anti-BAM-490 and SA-550 fluorescent antibodies.
  • TMB tumor mutational burden
  • patient-specific antigens eliciting stimulatory or inhibitory T cell responses as measured by IFN- y and/or TNF-a secretion
  • prior therapies for each patient selected for GEN 009 vaccination. All values were generated prior to GEN 009 vaccination.
  • the FluoroSpot assays are indicated as: ex vivo assay (complete PBMCs), ex vivo assay (enriched T cell subsets), and IVS assay (enriched T cell subsets). Both CD8+ and CD4+ T cell responses were observed. Ten-day in vitro stimulated FluoroSpot assays resulted in a greater proportion of positive and/or broader immune responses than the ex vivo FluoroSpot assays. In aggregate, all patient-specific peptide antigens (SLPs corresponding to ATLAS- identified, patient-specific stimulatory antigens) from the combined patients elicited:
  • Memory T cell responses for combined 8 patients A, B, C, E, F, G, H, and K, as detected by IVS assays (enriched T cell subsets):
  • Total PBMC (combined CD4+ and CD8+ T cell) responses for combined 8 patients A, B, C, E, F, G, H, and K, as detected by:
  • Tables 6-7 show GEN 009 immunogenicity assays against patient-specific peptide antigens (SLPs), after priming series of three vaccinations at Day 1, Day 22 and Day 43.
  • Figure 2 shows representative results of in vitro stimulated FluoroSpot assays on CD4+ and CD8+ T cells enriched from PBMCs collected at baseline (prior to vaccination) and at Day 50 from each of 5 patients (patients A, B, C, E, and F). Data are represented as the mean IFN-y spot forming cells (SFC) +/- SEM per 10,000 or 20,000 T cells, as indicated, for a given patient, for each of the 4 pools of SLPs (each pool comprising 1-5 SLPs) included in that patient’s vaccine.
  • SFC mean IFN-y spot forming cells
  • FIG. 3 shows representative results of ex vivo FluoroSpot assays and in vitro stimulated FluoroSpot assays on CD4+ and CD8+ T cells enriched from PBMCs collected at baseline (prior to vaccination) and at Day 50 from a representative patient (patient E).
  • Panels A and B ex vivo FluoroSpot assays. Data are represented as the mean cytokine spot forming cells (SFC) per million T cells, for each SLP included in the patient’s vaccine, as indicated.
  • Panel C in vitro stimulated FluoroSpot assays. Data are represented as the mean cytokine spot forming cells (SFC) per 20,000 T cells, for each SLP included in the patient’s vaccine, as indicated.
  • SFC mean cytokine spot forming cells
  • Figure 4 shows representative summary results of ex vivo FluoroSpot assays and in vitro stimulated FluoroSpot assays on total PBMC or PBMCs depleted of CD4+ or CD8+ T cells collected at baseline (prior to vaccination) and at Day 50 from patients A-H and K. Data are reported as the proportion of peptides positive by the DFR(eq) test. Circles represent baseline, squares represent D50 time point.
  • PBMCs depleted of CD4+ or CD8+ T cells were stimulated with overlapping peptides (OLPs) spanning each immunized SLP in an IFN-y and granzyme B (GrB) dual- color ex vivo FluoroSpot assay.
  • OLPs overlapping peptides
  • Panel B PBMCs depleted of CD4+ or CD8+ T cells were stimulated with OLPs for 10 days followed by an overnight IFN-y and TNF ⁇ dual- color FluoroSpot assay.
  • Panel C shows the proportion of SLPs scored positive by any assay for patients A-H, and K.
  • Data from patients A-L are shown in Figure 5, including for each patient, the tumor type, stage of cancer at diagnosis, period of time from diagnosis, prior therapies the patient received, the patients calculated tumor mutational burden (TMB), the number of stimulatory and inhibitory neoantigens identified for each patient, and the number of peptides in the vaccine administered.
  • the graph indicates the status of each patient at different points within the example vaccination regimen.
  • the timing of vaccination is indicated by a vertical arrows.
  • the color of the horizontal bars indicate the stage of cancer at diagnosis.
  • a blue horizontal arrow indicates that the patient has not yet completed the vaccination regimen (i.e., is within the dosign period).
  • a black horizontal arrow indicates that the patient has completed the vaccination regimen (i.e., is past the treatment period or post vaccination schedule).
  • a black circle indicates a status of “NED” or no evidence of disease.
  • the graph shows that all patients post vaccination experienced recurrence-free survival for at least 4 months.
  • Figure 6 shows representative results of ex vivo dual-analyte FluoroSpot assays on CD4+ and CD8+ T cells enriched from PBMCs of three representative patients (patients A and E; low response patient H).
  • Bulk PBMCs were isolated from the patients at baseline (prior to vaccination) and at the indicated timepoints over the course of their treatment.
  • the secretion of IFN-y and granzyme B (GrB) was quantified via ex vivo dual-analyte FluoroSpot after stimulation with overlapping peptide pools (OLPs) spanning the patient-specific SLPs used for immunization.
  • OTPs overlapping peptide pools
  • Panel A data are expressed as mean ( ⁇ SEM) spot forming cells (SFC) per million PBMCs to each of the four pools.
  • Panel B shows the number of positive pools for each time point.
  • GEN-009-101 is a first-in-human Phase l/2a study testing personalized vaccines designed using the ATLAS platform for feasibility, safety, immunogenicity and clinical activity in selected solid tumors.
  • eligible patients with advanced cancer received standard-of-care PD-1 checkpoint inhibitor therapy, with or without chemotherapy, during the period of manufacture of their GEN 009 vaccine.
  • Patients then received five doses of GEN 009 vaccine over six months, along with continuation with PD- 1 checkpoint inhibitor therapy.
  • Patients who progressed prior to vaccination received appropriate alternate therapy followed by GEN 009 vaccine combined with PD-1 checkpoint inhibitor therapy.
  • Cellular immune responses were measured by dual-analyte (IFN-y and granzyme B) FluoroSpot assays, assessing ex vivo CD4+ and CD8+ T cell responses to each SLP pool, and by IFN-y and TNF ⁇ FluoroSpot assays, assessing in vitro stimulated (IVS) T cell responses to each SLP.
  • the FluoroSpot assays were performed as described in Example 2. Immunophenotyping of PBMC populations was performed by flow cytometry. Further CD4+ and CD8+ polyfunctional responses were assessed by intracellular cytokine staining.
  • Antitumor activity was also assessed by improvement in tumor growth kinetics (/. ⁇ ., increase in tumor shrinkage rate or decrease in tumor growth rate) with GEN 009 vs projected rate without GEN 009.
  • the tumor micro-environment and infiltrating cells were evaluated by RNA sequencing and immunofluorescence.
  • Figure 7 shows a spider plot for an idealized patient, illustrating the method of assessing GEN-009 vaccine-specific contribution to clinical responses in Part B of the study.
  • Each point on the plot represents assessment of the idealized patient’s target lesions.
  • the X axis shows time and treatment.
  • the left side of the graph shows the period of the idealized patient’s initial assessment and standard-of-care (SOC) treatment.
  • the right side of the graph shows the period of standard-of-care treatment combined with GEN 009 vaccination (SOC & Vaccine).
  • the Y axis shows percent change in the sum, or diameter, of the idealized patient’s target lesions.
  • Baseline is defined as the patient’s lesions at initial assessment.
  • Progressive disease (PD), partial response (PR), and complete response (CR) are defined relative to baseline as +20%, -30% and -100% change, respectively (RECIST criteria).
  • the dotted line in the right side of the graph shows the expected course of disease, that is, for the majority of patients, the course set during the previous 3 to 4 months of active therapy.
  • the expected course of the idealized patient’s disease, on standard-of-care therapy alone is shown as approximately - 35% change in target lesions, in the lower range of partial response.
  • the post-vaccination response is shown as approximately -50% change in target lesions, in a higher range of partial response than the expected course on standard-of-care therapy alone.
  • the shift in trajectory is attributable to vaccination with GEN 009.
  • Table 8 shows detailed clinical history and GEN-009 vaccine composition for selected patients.
  • Table 9 shows summary clinical history and GEN-009 vaccine composition for all patients enrolled in the Main Part B Study and in the Part B Refractory Study.
  • Figures 8 through 15 show representative results for selected checkpoint inhibitor responsive patients enrolled in the Main Part B Study.
  • Figure 8 shows results for patient -058 (Main Part B Study).
  • Panel A Spider plot, as described for Figure 7, showing a post-vaccination drop from approximately -50% to - 70% change in target lesions.
  • Panel B Results of in vitro stimulated FluoroSpot assays performed at baseline and at Day 50 post- initial vaccination. The height of each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN-y and TNFa, for each SLP included in the patient’s vaccine, as indicated.
  • SFC cytokine spot forming cells
  • Panel C Aggregate results of in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ⁇ SD per 20,000 T cells (IVS). The dark bars indicate total IFNy responses, the lighter bars indicate total TNF ⁇ responses.
  • Panel D Radiographic imaging of mediastinal tumor, showing tumor mass at 3 timepoints (Baseline, Pre-vaccine, Postvaccine).
  • Panel E Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 10 6 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
  • Figure 9 shows results for patient -105 (Main Part B Study).
  • Panel A Spider plot, as described for Figure 7, showing a post-vaccination drop from approximately -37% to - 70% change in target lesions.
  • Panel B, top Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed with PBMCs collected at baseline and at the indicated days after initial vaccination with GEN-009. The height of each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine.
  • SFU spot forming units
  • Panel B bottom: Results of in vitro stimulated FluoroSpot assays performed on CD4+ and CD8+ T cells enriched from PBMC at baseline and at Day 50 after initial vaccination.
  • the height of each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN-y (left axis) and TNF ⁇ (right axis), for each SLP included in the patient’s vaccine, as indicated.
  • SFC cytokine spot forming cells
  • IFN-y left axis
  • TNF ⁇ right axis
  • CD4+ and CD8+ T cell responses are presented in the left and right panels, respectively.
  • Panel C Summary results of ATLAS screens performed at baseline and at Day 50 after initial vaccination with GEN 009.
  • Each pie chart shows percentages of the patient’s tumor mutations that elicit stimulatory T cell responses (neoantigens) or inhibitory T cell responses (inhibigens). For this patient, a slight proportional reduction in inhibigens was observed following GEN 009 vaccination.
  • Panel D Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ⁇ SD per 10 6 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel).
  • Panel E Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 10 6 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
  • Figure 10 shows results for patient -001 (Main Part B Study).
  • Panel A Spider plot, as described for Figure 7, showing a post-vaccination drop from approximately -60% to - 100% change in target lesions. This patient achieved a partial response (PR) on checkpoint inhibitor therapy, followed by a complete response (CR) after vaccination with GEN-009.
  • Panel B, top Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed on PBMCs collected at baseline and at the indicated days after initial vaccination with GEN-009. The height of each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine.
  • SFU cytokine spot forming units
  • the DI time point represents post-checkpoint inhibitor therapy, pre-vaccine responses; therefore, increases in frequency of PBMC responses at subsequent time points can be attributed to GEN 009 vaccination.
  • Panel B, bottom Results of in vitro stimulated FluoroSpot assays performed on CD4+ and CD8+ T cell enriched from PBMC at baseline and at Day 50 after initial vaccination. The height of each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN-y (left axis) and TNF ⁇ (right axis), for each SLP included in the patient’s vaccine, as indicated.
  • CD4+ and CD8+ T cell responses are presented in the left and right panels, respectively.
  • Panel C Summary results of ATLAS screens performed at baseline and at Day 50 after initial vaccination with GEN 009. Each pie chart shows percentages of the patient’s expressed tumor mutations that elicit stimulatory T cell responses (neoantigens) or inhibitory T cell responses (inhibigens). For this patient, proportional increases in both neoantigens and inhibigens were observed following GEN 009 vaccination.
  • Panel D Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown.
  • Panel E Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 10 6 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
  • Figure 11 shows results for patient -057 (Main Part B Study).
  • Panel A Spider plot, as described for Figure 7, showing a drop to -100% change in target lesions, i.e., complete response (CR), prior to vaccination with GEN-009. The patient sustained a complete response for the duration of the study.
  • Panel B, top Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed on PBMCs collected at baseline and at the indicated days after initial vaccination with GEN 009. The height of each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine.
  • SFU cytokine spot forming units
  • the DI time point represents post-checkpoint inhibitor therapy, pre-vaccine responses; therefore, increases in frequency of PBMC responses at subsequent time points can be attributed to GEN 009 vaccination.
  • Panel B, bottom Results of in vitro stimulated FluoroSpot assays performed on CD4+ and CD8+ T cells enriched from PBMC at baseline and at Day 50 after initial vaccination. The height of each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN-y (left axis) and TNF ⁇ (right axis), for each SLP included in the patient’s vaccine, as indicated.
  • CD4+ and CD8+ T cell responses are presented in the left and right panels, respectively.
  • Panel C Summary results of ATLAS screens performed at baseline and at Day 50 after initial vaccination with GEN 009. Each pie chart shows percentages of the patient’s tumor mutations that elicit stimulatory T cell responses (neoantigens) or inhibitory T cell responses (inhibigens). For this patient, both a proportional decrease in neoantigens and proportional increase in inhibigens were observed following GEN 009 vaccination.
  • Panel D Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown.
  • Panel E Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 10 6 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
  • Figure 12 shows results for patient -031 (Main Part B Study).
  • Panel A Spider plot, as described for Figure 7, showing a slight drop from approximately -55% to - 60% change in target lesions.
  • Panel B, top Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed on PBMCs collected at baseline and at the indicated days after initial vaccination with GEN 009. The height of each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine.
  • SFU spot forming units
  • Panel B Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ⁇ SD per 10 6 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNF ⁇ responses.
  • Panel C Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume.
  • Values for IFNy are reported on the right axis as mean background subtracted SFC per 10 6 cells.
  • the corresponding patient tumor scans are indicated on the left axis (open triangles).
  • the dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%).
  • the grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
  • Figure 13 shows results of all ex vivo dual-color FluoroSpot assays plotted over time.
  • Peripheral blood mononuclear cells were collected from patients participating in the Main Part B Study at the indicated time points (day 1 is the day of the first GEN-009 vaccination) and viably frozen.
  • Thawed PBMCs were stimulated with pools containing the vaccine antigens (or controls).
  • Results are shown as the mean cytokine spot forming cells (SFC) per million PBMC (+/- SEM) for each subject contributing data at each time point. Not all patients contributed data for each time point: the N at each time point is indicated.
  • Figure 14 shows summary results for all patients enrolled in the Main Part B Study.
  • Panel A is a composite of all spider plots, as described for Figure 7, showing the percent target tumor lesion change for each patient, from the time of consent and initial assessment.
  • Panel B is a composite of all spider plots showing the percent target tumor lesion change for each patient from the time the patient received GEN-009 dose 1 (i.e., baseline is defined at day 0 of the study).
  • Panel C is a swimmers’ plot showing timelines of PD-1 standard-of-care therapy, GEN-009 dosing, and outcomes for each patient.
  • Panel D shows aggregate results for all patients of dual-analyte immunogenicity assays performed at screening and at Day 50 post-initial vaccination.
  • PBMC, CD4+ and CD8+ T cell responses are shown. Values presented are the means of background subtracted SFCs ⁇ SEM per 20,000 T cells. The dark bars indicate total IFNy responses, the lighter bars indicate total TNF ⁇ responses.
  • Figures 15 through 20 show representative results for selected checkpoint inhibitor refractory patients enrolled in the Part B Refractory Study.
  • Figure 15 shows results for patient -106 (Part B Refractory Study).
  • Panel A Spider plot, as described for Figure 7. This patient progressed rapidly on the standard-of-care checkpoint inhibitor therapy and was treated with chemotherapy from days approximately -110 to -20, after which they continued on checkpoint inhibitor therapy and received GEN 009 vaccine on day 0. The spider plot shows maintenance of the PR achieved on chemotherapy, with a slight post-vaccination drop then a slight rise from approximately -50% to -55% change in target lesions.
  • Panel B, top Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed on PBMCs collected at baseline and at the indicated days after initial vaccination with GEN 009.
  • IFN-y and granzyme B results of ex vivo dual analyte
  • each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine.
  • the DI time point represents postcheckpoint inhibitor therapy and post-chemotherapy, pre-vaccine responses; therefore, increases in frequency of PBMC responses at subsequent time points can be interpreted as attributable to GEN 009 vaccination.
  • Panel B, bottom Results of in vitro stimulated FluoroSpot assays on CD4+ and CD8+ T cells enriched from PBMCs performed at baseline and at Day 50 after initial vaccination.
  • each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN-y (left axis) and TNF ⁇ (right axis), for each SLP included in the patient’s vaccine, as indicated.
  • CD4+ and CD8+ T cell responses are presented in the left and right panels, respectively.
  • Panel C Summary results of ATLAS screens performed at baseline and at Day 50 after initial vaccination with GEN 009. Each pie chart shows percentages of the patient’s expressed tumor mutations that elicit stimulatory T cell responses (neoantigens) or inhibitory T cell responses (inhibigens). For this patient, a slight proportional increase in inhibigens was observed following GEN 009 vaccination.
  • Panel D Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ⁇ SD per 10 6 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNF ⁇ responses.
  • Panel E Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume.
  • Values for IFNy are reported on the right axis as mean background subtracted SFC per 10 6 cells.
  • the corresponding patient tumor scans are indicated on the left axis (open triangles).
  • the dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%).
  • the grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
  • Figure 16 shows results for patient -109 (Part B Refractory Study).
  • Panel A Spider plot, as described for Figure 7. This patient progressed rapidly on standard-of-care checkpoint inhibitor therapy and was treated with chemotherapy, after which they continued on checkpoint inhibitor therapy and received GEN 009 vaccine on day 0. The spider plot shows maintenance of the PR achieved on chemotherapy, with a slight post-vaccination drop from approximately -70% to -75% change in target lesions.
  • Panel B Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown.
  • Figure 17 shows results for patient -027 (Part B Refractory Study).
  • Panel A Spider plot, as described for Figure 7. This patient responded to standard-of-care checkpoint inhibitor therapy and then had a new tumor mass emerge (arrow around D-100). The patient was further treated with monoclonal antibody, after which they received GEN 009 vaccine on day 0. The spider plot shows maintenance of the PR, with no additional change to date in target lesions post-vaccination.
  • Panel B Results of ex vivo ZFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for ZFNy are reported on the right axis as mean background subtracted SFC per 10 6 cells.
  • the corresponding patient tumor scans are indicated on the left axis (open triangles).
  • the dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%).
  • the grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
  • Figure 18 shows results for patient -110 (Part B Refractory Study).
  • Panel A Spider plot, as described for Figure 7. This patient progressed rapidly on standard-of-care checkpoint inhibitor therapy and was treated with chemotherapy, after which they continued on checkpoint inhibitor therapy and received GEN 009 vaccine on day 0.
  • the spider plot shows reversal of the PR (beginning just prior to vaccination), with a sharp post-vaccination increase from approximately -50% to -5% change in target lesions.
  • the lesion appearance change is being followed as a possible pseudo-progression, suggestive of massive immune cell infiltration post-treatment rather than a failure of therapy.
  • Panel B Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 postinitial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ⁇ SD per 10 6 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNF ⁇ responses.
  • Figure 19 shows results for patient -060 (Part B Refractory Study).
  • Panel A Spider plot, as described for Figure 7. This patient responded to standard-of-care checkpoint inhibitor therapy, but experienced a therapy -related adverse event. The patient was treated for the adverse event, after which they received GEN 009 vaccine on day 0. The spider plot shows reversal of the PR (beginning just prior to vaccination), with a slight post-vaccination increase from approximately -25% to -20% change in target lesions.
  • Figure 20 shows summary results for all patients enrolled in the Main Part B Refractory Study.
  • Panel A is a composite of all spider plots, as described for Figure 7, showing the percent target tumor lesion change for each patient, from the time of consent and initial assessment.
  • Panel B is a composite of all spider plots showing the percent target tumor lesion change for each patient from the time the patient received GEN-009 dose 1 (i.e., baseline is defined at day 0 of the study).
  • Panel C is a swimmers’ plot showing timelines of PD-1 standard- of-care therapy, GEN-009 dosing, and outcomes for each patient.
  • Panel D shows aggregate results for all patients of dual-analyte immunogenicity assays performed at screening and at Day 50 post-initial vaccination.
  • PBMC, CD4+ and CD8+ T cell responses are shown. Values presented are the means of background subtracted SFCs ⁇ SEM per 20,000 T cells. The dark bars indicate total IFNy responses, the lighter bars indicate total TNF ⁇ responses.
  • Figure 21 shows representative summary results for CD4+ and CD8+ T cell responsiveness, prior to and after vaccination with GEN 009.
  • the bars labeled “ATLAS” indicate the overall percentages of antigens that elicited robust CD4+ or CD8+ T cell responses in initial ATLAS screening of the selected Part B patients and were consequently included in the GEN 009 vaccine compositions administered to these patients.
  • the bars labeled “Post GEN-009 Treatment” indicate the overall percentages of antigens that elicited CD4+ or CD8+ T cell responses in ATLAS screening of the selected Part B patients following their vaccination with GEN 009. The comparison shows changes in the breadth of T cell responses.
  • Panel B is a graph showing aggregate IFNy responses (ex vivo FluoroSpot assays) as SFCs ⁇ SEM per million PBMCs over several timepoints, from screening to 6 months after patients received GEN 009 dose 3.
  • Panel C shows the average number of neoantigens that elicited positive CD4+ and CD8+ T cell responses, as determined by ATLAS, at the time of screening and at Day 50 post-initial vaccination, broken out for Main Part B (Controlled) and Part B Refractory (Refractory) patients.
  • Panel D shows the total number of non-synonymous mutations (tumor mutational burden) for each cohort at the time of screening.
  • the checkpoint inhibitor-controlled cohort had a significantly greater overall number of neoantigens eliciting T cell responses than the checkpoint inhibitory-refractive cohort.
  • the controlled cohort showed an increase in the number of neoantigens to which CD8+ T cells responded after GEN 009 vaccination (D50), indicating epitope spread.
  • the refractory cohort showed a decrease in the number of neoantigens to which either CD4+ or CD8+ T cells responded after GEN 009 vaccination (D50), indicating contraction of the breadth of response.
  • Figure 22 shows overall characterization of stimulatory antigens (neoantigens), inhibigens, and non-responsive antigens identified in 37 patients screened for inclusion in the Part A Study, the Main Part B Study, and the Part B Refractory Study of GEN-009.
  • Panel B Total proportion of stimulatory antigens (neoantigens), inhibigens, and non-responsive antigens. Inhibigens accounted for 16% (range 1.8%-47.5%) of total ATLAS-identified responses.
  • Figure 23 shows the overall proportion of CD4 + and CD8 + stimulatory antigens (neoantigens), inhibigens, and non-responsive antigens identified in 37 patients screened for inclusion in the Part A Study, the Main Part B Study, and the Part B Refractory Study of GEN- 009. Results are expressed as a percentage of total CD4 + and CD8 + responses. Inhibigen- specific T cell responses were identified in both CD4 + and CD8 + T cell subsets, with more detected in the CD4 + T cell subset (mean 10.3%; 0.5%-42%) as compared to CD8 + (mean 6.1%; 1.2%-23%).
  • Figure 24 shows overall characterization, against total mutations screened, of somatic mutations, stimulatory antigens (neoantigens), and inhibigens identified in 37 patients screened for inclusion in the Part A Study, the Main Part B Study, and the Part B Refractory Study of GEN-009.
  • Panel A Number of mutations screened ( ⁇ SEM) by tumor type. The highest number of mutations were found in melanoma and NSCLC as compared to bladder, RCC, and HNSCC.
  • Figure 25 shows the proportion of stimulatory antigens and inhibigens identified by ATLAS screens for each patient at initial screening at D50 post-initiation of vaccinations, broken out by T cell subset, tumor type and checkpoint inhibition responsiveness vs. non-responsiveness. For each set of bars, the top bar shows results obtained at initial screening, and the bottom bar shows results obtained at Day 50.
  • Middle rows individual patient responses, with patient ID indicated to the left of each set.
  • Bottom row pooled responses by tumor type.
  • Figure 26 shows the results of 16-plex patient-specific somatic assays for detection of circulating tumor DNA (ctDNA). Assays were performed on plasma samples taken at initial screening (Baseline) and at D50 post initiation of vaccination (Post-Tx). Two patients enrolled in the Main Part B Study are represented, one in each graph. Results are expressed as the percent frequency of variant alleles in plasma, and show disappearance of ctDNA postvaccination.
  • non-antigenic, tumor-specific mutations circles
  • triangles neoantigens not included in the GNE-009 vaccine
  • squares neoantigens included in the GEN-009 vaccine
  • a cohort of C57BL/6J mice bearing B16F10 tumors were euthanized and their tumors and spleens harvested.
  • DNA obtained from pooled tumors was sequenced and analyzed for non-synonymous mutations. Over 1600 such mutations were identified, and these were synthesized as 399 bp DNA fragments centered upon the base pair change and transformed individually into E. coll bacteria expressing cLLO to build a candidate neoantigen library.
  • Splenocytes frozen from pooled spleens of the tumor-bearing mice were thawed, and CD8 + T cells were sorted using a negative selection bead kit.
  • Mouse APCs (RAW309 Cr. l macrophage cell line) were cultured overnight, washed with PBS, then co-cultured with the bacterial library for 2 hours, washed with PBS, and then cultured overnight with the non-specifically expanded and rested CD8 + T cells.
  • Harvested supernatant from the co-culture was tested for fFNy and TNF ⁇ by a custom mouse 384-well Meso Scale Discovery (MSD) electrochemiluminescence assay.
  • MSD Meso Scale Discovery
  • the top ten ranked antigens (stimulatory) and eight of the bottom ten ranked antigens (inhibitory) were each synthesized as 27mer synthetic long peptides (SLPs) for use in mouse vaccination and B16F10 melanoma challenge studies.
  • M27 CD8 neoantigen
  • M30 CD4 neoantigen
  • Trp2 CD8 tumor-associated antigen, TAA
  • SLPs 27mer synthetic long peptides
  • the top ranked and known B16F10 antigens were also each synthesized as four 15mer overlapping peptides (OLPs) for use in ex vivo assays.
  • OHPs 15mer overlapping peptides
  • Example 5 Mouse cancer vaccine studies: Inhibigen-specific suppression of immune responses (therapeutic vaccination ⁇
  • SLPs corresponding to inhibigens identified by mATLAS and to three known B16F10 antigens were synthesized according to Example 4. Lyophilized SLPs were reconstituted in 50% ACN in H2O and pre-mixed, then frozen and lyophilized for 21h and subsequently frozen again as lyophilized pools. These were reconstituted on the day of immunization in either PBS/DMSO or PBS/adjuvants/DMSO (final DMSO concentration: 4%).
  • B16F10 tumor-bearing mice were vaccinated on the following schedule: cancer cells were injected subcutaneously on the right flank on dO (ATCC-passage 7, 100K cells in lOOpl of 20% Matrigel); vaccine was injected subcutaneously either at the tail base or scruff of the neck on d3, dlO, dl7.
  • the experimental groups of the second study were injected with: 1) a variant of the above-described protective vaccine comprising a pool of two known (published) stimulatory B16F10 antigens: M30 (CD4 neoantigen) and Trp2 (CD8 tumor-associated antigen, TAA), plus adjuvant (also labeled Protective Vaccine), or 2) the same protective vaccine plus inhibitory antigen MMP9FS (Protective Vaccine + Inhibigen MMP9FS), or 3) inhibitory antigen MMP9FS alone plus adjuvant (MMP9FS + adj).
  • the experimental groups of the third study were injected with: 1) a further variant of the above-described protective vaccine comprising a pool of one ATLAS-identified stimulatory antigen, Gal3Stl (CD8 neoantigen) plus two known (published) stimulatory B16F10 antigens: M30 (CD4 neoantigen) and Trp2 (CD8 tumor-associated antigen), plus adjuvant (also labeled Protective Vaccine), or 2) the same protective vaccine plus inhibitory antigen MMP9FS (Protective Vaccine + Inhibigen MMP9FS), or 3) inhibitory antigen MMP9FS alone plus adjuvant (MMP9FS + adj).
  • Control groups were injected with adjuvant alone (Adjuvant Only; triple adjuvant comprising CpG, 3D-PHAD, QS-21). SLP dosage was 50pg per SLP/mouse/day.
  • Splenocytes were collected on dl6 of the study (i.e., 6 days after vaccine injection #2) and resuspended in OpTmizer media. In some instances, splenocytes were collected on d4 and dl l, or on d21-d26 of the study. Cells were normalized to one cell (4xl0 5 cells/well) concentration and seeded into an IFNy ELISPOT plate with stimulants for overnight culture. Cells from each individual mouse were split into 2 wells: well 1 contained media alone, well 2 contained pooled OLPs (4pg/ml or lug/mL/OLP) specific to the vaccine that the mouse received. For example, for a mouse immunized with peptides 1-4, the cells were stimulated with OLPs la-d, 2a-d, 3a-d and 4a-d (16 individual 15mers overlapping by 11 amino acids total) across different wells.
  • Tumor size was measured 3x/week and subsequently on a daily basis after reaching a specified size threshold (1000mm 3 ).
  • Statistical analysis was performed by one-way ANOVA with multiple comparisons. Mice were euthanized when tumors reached maximum size, or tumors became ulcerated and did not heal within 24 hours. No mice in this study were euthanized for other health reasons.
  • B16F10 tumors were collected at one timepoint on d21-28 of the study, formalin- fixed, paraffin-embedded, and sections stained by chromogenic immunohistochemistry (IHC). In some instances, early-stage tumors were collected on dl 1 of the study. For assessment of infiltrating immune cell populations, sections were stained with antibodies specific for mouse CD4 or CD8a (T cells), FoxP3 (Tregs), F4/80 (macrophages), or CD11c (dendritic cells), in pink/red, and counterstained for nuclei with hematoxylin (blue). For assessment of tumor vasculature, sections were stained with antibodies specific for the endothelial marker CD31. For assessment of metabolic status, sections were stained with antibodies specific for the metabolic markers LDHA, ARG1, and CD73.
  • IHC immunohistochemistry
  • FIG 28 Panel A shows results of ZFNy ELISPOT performed on splenocytes from tumor-bearing and vaccinated mice of the first study (top graph) and of the third study (bottom graph) after in vitro stimulation with overlapping peptides spanning the vaccine antigenss. Statistical analysis was by multiple T-tests (top graph) and one-way ANOVA (bottom graph; p ⁇ 0.0001).
  • Figure 28, Panel B shows mean tumor growth kinetics following therapeutic vaccination with adjuvant only, protective vaccine, or protective vaccine plus inhibigen (first study).
  • Panel D shows mean tumor growth kinetics following therapeutic vaccination with adjuvant only, or inhibigen (MMP9FS) plus adjuvant (second study).
  • MMP9FS inhibigen plus adjuvant
  • Panel A shows representative sections of immunohistochemically labeled tumors from mice therapeutically vaccinated with adjuvant alone (Adjuvant, top row), protective vaccine (Protective, middle row), or protective vaccine plus inhibigen (MMP9FS) (+ Inhibigen, bottom row).
  • Tumor sections were labeled with antibodies specific for CD4 or CD8a (T cells), FoxP3 (Tregs), F4/80 (macrophages), or CD11c (dendritic cells). Endogenous melanin deposits in tumors are visible (brown) and specific labeling is indicated in red.
  • Panel B shows quantification of Panel A images.
  • Panel C shows representative tumor sections immuno-histochemically stained for the endothelial marker CD31, from tumor-bearing mice vaccinated with protective vaccine (Protective, top row), or protective vaccine plus inhibigen MMP9FS (+ Inhibigen, bottom row).
  • Panel D shows representative tumor sections immuno-histochemically stained for metabolic markers LDHA, ARG1, and CD73, from tumor-bearing mice vaccinated with protective vaccine plus inhibigen MMP9FS. LDHA was predominantly expressed in tumor tissue, ARG1 was predominantly expressed in tumorinfiltrating immune cells, and CD73 was predominantly expressed in vascular tissue.
  • Panel E shows quantification of Panel D images, together with quantification of images (not shown) of tumor sections from mice vaccinated with adjuvant alone, or with protective vaccine.
  • Panel F shows quantification of representative tumor sections immuno-histochemically stained for CD8 molecules on T cells, from tumor-bearing mice therapeutically vaccinated with adjuvant only, protective vaccine, or MMP9FS only (top graph), or tumor-bearing mice vaccinated with adjuvant only, protective vaccine, or protective vaccine plus MMP9FS (bottom graph).
  • top graph tumor-bearing mice vaccinated with adjuvant only, protective vaccine, or protective vaccine plus MMP9FS
  • mice of Example 5 (therapeutically vaccinated with adjuvant only, protective vaccine, or the protective vaccine plus inhibigen MMP9FS) and incubated with the pulsed APCs above (Together) were further restimulated with respective antigen peptides, stained for intracellular cytokines, and gated by flow cytometry on live, CD45 + , CD19 nes /NKl.l neS , CD3 + , CD8 + T cells. Data shown are pooled splenocytes from four mice from each vaccination group. In Figure 32, CD8+ IFNy expressing T cells are represented as a percentage of total CD3 + T cells. Results show that restimulation with a peptide pool containing an inhibigen fails to decrease IFNY responses from T cells of mice immunized with the protective vaccine.
  • Example 7 Mouse cancer vaccine study: combination of vaccine and checkpoint inhibitor (CPI ⁇ (therapeutic vaccination ⁇
  • SLPs corresponding to antigens identified by mATLAS and to three known B16F10 antigens were synthesized according to Example 4. Lyophilized SLPs were reconstituted in 50% ACN in H2O and pre-mixed, then frozen and lyophilized for 21h and subsequently frozen again as lyophilized pools. These were reconstituted on the day of immunization in either PBS/DMSO or PBS/adjuvants/DMSO (final DMSO concentration: 4%).
  • B16F10 tumor-bearing mice were vaccinated on the following schedule: cancer cells were injected subcutaneously on the right flank on dO (ATCC-passage 6, 100K cells in lOOpl of 20% Matrigel in DMEM); vaccine was injected subcutaneously at the tail base on d3, dlO, d!7.
  • the protective vaccine of Example 5 comprising a pool of one ATLAS-identified stimulatory antigen Gal3Stl (CD8 neoantigen) plus two previously known (published) stimulatory B16F10 antigens: M30 (CD4 neoantigen), and Trp2 (CD8 tumor-associated antigen, TAA), plus adjuvant, or 2) the same protective vaccine plus the inhibitory antigen MMP9FS.
  • the experimental groups of the second study were injected with: 1) the protective vaccine of Example 5, comprising a pool of two previously known (published) stimulatory B16F10 antigens: M30 (CD4 neoantigen), and Trp2 (CD8 tumor-associated antigen, TAA), plus adjuvant, or 2) inhibitory antigen MMP9FS alone plus adjuvant.
  • SLP dosage was 50pg per SLP/mouse/day.
  • Checkpoint inhibitors anti-PD-1, anti-PD-Ll, anti-CTLA-4, or anti-PD-1 plus anti-CTLA-4 were administered on d7, dlO, dl4, dl7, and d21 (4 days after each of the 3 vaccinations and on the days of 2 nd and 3 rd vaccinations).
  • Control groups received adjuvant alone (triple adjuvant comprising CpG, 3D-PHAD, QS-21), or no checkpoint inhibitor.
  • the workflow for Bl 6F 10 melanoma challenge and vaccination in conjunction with checkpoint inhibitor is shown in Figure 33.
  • Splenocytes were collected on d23 of the study (i.e., 2 days after final dose of checkpoint inhibitor) and resuspended in OpTmizer media. Cells were normalized to one cell concentration (2xl0 5 cells/well) and seeded into an IFNy ELISPOT plate with stimulants for overnight culture. Cells from each individual mouse were split into 2 wells: well 1 contained media alone, well 2 contained pooled OLPs (4pg/ml or lug/mL/peptide) specific to the vaccine that the mouse received. For example, for a mouse immunized with peptides 1-4, the cells were stimulated with OLPs la-d, 2a-d, 3a-d and 4a-d (16 individual 15mers overlapping by 11 amino acids total).
  • FIG. 34 shows results of IFNy ELISPOT performed on splenocytes from therapeutically vaccinated mice, with and without checkpoint inhibition, after in vitro stimulation with overlapping peptides spanning vaccine components.
  • Figure 35 shows mean tumor growth kinetics following therapeutic vaccination.
  • Panel A shows tumor growth for groups therapeutically vaccinated with protective vaccine, or with protective vaccine plus inhibigen, and treated with anti-PD-1 checkpoint inhibitor (first study).
  • Panel B shows tumor growth for groups therapeutically vaccinated with protective vaccine, or with protective vaccine plus inhibigen, and treated with anti-CTLA-4 checkpoint inhibitor (first study).
  • Panel C shows tumor growth for groups therapeutically vaccinated with protective vaccine, or with inhibigen plus adjuvant, and treated with an anti-PD-1, anti-PD-Ll, or anti-CTLA-4 checkpoint inhibitor (second study).
  • Panel D shows tumor growth for groups therapeutically vaccinated with protective vaccine, or with inhibigen plus adjuvant, and treated with a combination of anti-PD-1 and anti-CTLA-4 checkpoint inhibitors (second study).
  • Statistical analyses were performed by one-way ANOVA with multiple comparisons. The results of the first study show that anti-PD-1 checkpoint inhibition did not prevent accelerated tumor growth seen in mice vaccinated with an inhibigen, while anti-CTLA-4 checkpoint inhibition partially ameliorated such tumor growth.
  • results of the second study similarly show that anti-PD-1 and anti-PDL-1 did not prevent accelerated tumor growth in mice vaccinated with an inhibigen, while anti-CTLA-4 checkpoint inhibition partially ameliorated such accelerated tumor growth.
  • results of the second study show that combination checkpoint inhibition (anti-PD-1 plus anti-CTLA-4) partially ameliorated tumor growth, but the effect did not appear to be additive or synergistic compared to checkpoint inhibition with anti-CTLA-4 alone.
  • B16F10 tumors were collected at one timepoint on d21-28 of the study, formalin- fixed, paraffin-embedded, and sections were stained by chromogenic immunohistochemistry (H4C).
  • the different panels of Figure 36 show tumor sections from mice vaccinated with adjuvant alone (Adjuvant, top row), the protective vaccine (Protective, middle row), or the protective vaccine plus the MMP9FS inhibigen (+ Inhibigen, bottom row), with and without anti- PD-1, anti-CTLA-4, or anti-PD-1 plus anti-CTLA-4 checkpoint therapy.
  • the tumor sections were stained with an anti-mouse CD8a antibody (pink/red) or CD4 antibody (not pictured) and counterstained with hematoxylin (blue). Endogenous melanin deposits in tumors are visible (brown).
  • Image quantification shown on Figure 37, was determined by ImageJ software thresholding on positive stain (pink) divided by tissue area (total image area - white non-tissue).
  • the top panel shows results for CD8 + tumor infiltrating lymphocytes (TILs); the bottom panel shows results for CD4 + TILs.
  • TILs tumor infiltrating lymphocytes
  • Each symbol in the graph represents data from one mouse, the horizontal line indicates the mean ⁇ SD.
  • Statistical analyses were performed by one-way ANOVA with multiple comparisons.
  • CD8 + and CD4 + TILs were present in increased numbers in mouse Bl 6F 10 tumor tissue after vaccination with a protective vaccine (and greatly increased in combination with anti-PD-1 checkpoint inhibitor), and were reduced to baseline levels when the protective vaccine included an inhibigen. Treatment with anti-PD-1 and/or anti-CTLA-4 check-point inhibitor did not rescue the reduced T cell infiltration into the tumor tissue associated with vaccination with an inhibigen-containing formulation.
  • B16F10 tumor sections were immunohistochemically stained with antibodies specific for checkpoint markers CTLA-4 and PD-L1. Representative results are shown in Figure 38.
  • Panel A shows representative tumor sections stained for CTLA-4 (top) and PD-L1 (bottom).
  • Panel B shows image quantification of tumor sections stained for CTLA-4 and PD-L1, from mice vaccinated with adjuvant alone (None), protective vaccine (Protective), or protective vaccine plus inhibigen MMP9FS (+ Inhibigen), with and without anti-PD-1 or anti- CTLA-4 checkpoint therapy. Quantification of all immunohistochemical images was performed using ImageJ assessment of pink staining area.
  • Example 8 Mouse cancer vaccine studies: Inhibisen-specific suppression of immune responses (therapeutic vaccination ⁇
  • Synthetic long peptides corresponding to inhibigens identified by mATLAS and to known Bl 6F 10 antigens were synthesized according to Example 4. Lyophilized SLPs were reconstituted in 50% ACN in H2O and pre-mixed, then frozen and lyophilized for 21h and subsequently frozen again as lyophilized pools. These were reconstituted on the day of immunization in either PBS/DMSO or PBS/adjuvants/DMSO (final DMSO concentration: 4%).
  • B16F10 tumor-bearing mice were vaccinated on the following schedule: cancer cells were injected subcutaneously on the right flank on dO (ATCC-passage 7, 100K cells in lOOpl of 20% Matrigel); vaccine was injected subcutaneously either at the tail base or scuff of the neck on d3, dlO, dl7.
  • the experimental groups of the second study were injected with: 1) a protective vaccine of Example 5, comprising a pool of one ATLAS-identified stimulatory antigen Gal3Stl (CD8 neoantigen) plus two previously known (published) stimulatory B16F10 antigens: M30 (CD4 neoantigen), and Trp2 (CD8 tumor-associated antigen, TAA), plus adjuvant, or 2) the same protective vaccine plus inhibitory antigen MMP9FS, or 3) inhibitory antigen MMP9FS alone plus adjuvant (MMP9FS + adj).
  • Control groups were injected with adjuvant alone (Adjuvant Only; triple adjuvant comprising CpG, 3D-PHAD, QS-21).
  • SLP dosage was 50pg per SLP/mouse/day.
  • Splenocytes were collected on dl6 (i.e., 6 days after vaccine injection #2) and resuspended in OpTmizer media. In some instances, splenocytes were harvested on d4, dl 1, or d21-d28 of the study. Cells were normalized to one cell (IxlO 6 cells/well) concentration and stimulated in an overnight IFNy ELISPOT assay. For vaccine-specific stimulation, cells from each individual mouse were split into 2 wells: well 1 contained media alone, well 2 contained pooled OLPs (4pg/ml or lug/mL/OLP) specific to the vaccine that the mouse received.
  • well 1 contained media alone
  • well 2 contained pooled OLPs (4pg/ml or lug/mL/OLP) specific to the vaccine that the mouse received.
  • the cells were stimulated with OLPs la-d, 2a-d, 3a-d and 4a-d (16 individual 15mers overlapping by 11 amino acids total) across different wells.
  • OLPs la-d, 2a-d, 3a-d and 4a-d (16 individual 15mers overlapping by 11 amino acids total) across different wells.
  • OLPs la-d, 2a-d, 3a-d and 4a-d (16 individual 15mers overlapping by 11 amino acids total) across different wells.
  • OLPs la-d, 2a-d, 3a-d and 4a-d (16 individual 15mers overlapping by 11 amino acids total) across different wells.
  • an anti-CD3 antibody positive control
  • PMA/ionomycin to evaluate a non-specific defect in ability to proliferate
  • T cells expressing IFNy- and markers CD44, CD69, PD-1, 4- IBB (CD 137), and CD62L were then quantified by flow cytometry (gated on live lymphocytes, CD3 + T cells
  • FIG 39 Panel A shows results of an IFNy ELISPOT assay performed on splenocytes from vaccinated mice after in vitro stimulation with overlapping peptides spanning vaccine components, at day 21 of the first study.
  • Panel B shows a time course of IFNy ELISPOT at days 4, 8, and 21 of the first study. Statistical analysis was by multiple T-tests.
  • Panel C shows flow cytometry results for IFNy-expressing cells (left), and cells expressing CD44 or CD69 molecules (right) from splenocytes of therapeutically vaccinated mice of the first study, after non-specific stimulation with PMA/ionomycin or anti-CD3 antibody.
  • Panel D shows flow-cytometry results for IFNy- expressing cells from splenocytes of vaccinated mice harvested at day 4 of the second study. Cells were re-stimulated with PMA/ionomycin (with Golgi plug and Golgi stop).
  • Panel E shows flow-cytometry results for cells expressing markers CD69, PD-1, 4-1BB (CD137), or CD62L, from the same collection of splenocytes harvested at day 4 of the second study.
  • Results are shown as the proportion of viable CD3 + T cells from a pool of 4 mice per group, for tumorbearing mice therapeutically vaccinated with adjuvant alone (Adjuvant), protective vaccine (Protective vaccine), or protective vaccine plus inhibigen MMP9FS (Protective vaccine + Inhibigen).
  • Panel F shows flow-cytometry results for myeloid populations (Ml and M2 macrophages, myeloid-derived suppressor cells [MDSC’s]) from splenocytes of vaccinated mice of the second study. Suppressive myeloid populations did not expand upon vaccination with an inhibigen.
  • splenocytes from tumor-bearing and therapeutically vaccinated mice were stained with cell trace violet and cultured with or without IL-2 (0.17 lU/ l) and anti-CD3 antibody. Medium was exchanged every 3 days. After 7 days, cells were harvested, labeled with fluorescently conjugated antibodies specific for T cell markers, and analyzed by flow cytometry.
  • Panel A shows total CD3 + T cell proliferation in the presence of IL-2 and anti-CD3 antibody.
  • Panel B shows specific proliferation of CD8 + T cells in response to anti-CD3 stimulation by quantitation of cell trace violet.
  • splenocytes were harvested from tumor-bearing baccinated mice and non-specifically stimulated with a dose titration of anti-CD3 antibody, starting at a concentration 5-fold higher than what is normally used, and analyzed by flow cytometry for cleaved Caspase-3 in conjunction with expression of surface markers.
  • a dose titration of the mitogen PMA/ionomycin, starting at a 5- fold greater concentration than standardly used served as a positive AICD control.
  • Figure 42 shows the proportion of CD3 + T cells expressing cleaved Caspase-3.
  • Figure 43 shows RNA counts per million reads for markers CD44, CTLA-4 and LDHA (Panel A) and PD-1, NK1 and Tox2 (Panel B) at day 21-28.
  • Tumor-bearing mice were vaccinated with adjuvant only (Adjuvant), protective vaccine plus inhibigen MMP9FS (Inhibitory), or protective vaccine (Protective), as indicated on the x-axis.
  • Panel C shows volcano plots, comparing representative groups of differentially expressed genes at day 4 and day 8; significantly upregulated genes are shown in red, downregulated genes are shown in blue. Table 11 below summarizes global T cell transcriptional patterns at day 4 and day 8.
  • BMDCs bone marrow dendritic cells
  • splenic T cells derived from B16F10 tumor-bearing mice or OT-I/OT-II mice.
  • the B16F10 antigen was Gal3stl; the OT-I antigen was SIFNFEKL; the OT-II antigen was ISQ.
  • Panel A shows results for BMDCs from B16F10 tumor-bearing mice.
  • Panel B shows results for CD4 + T cells from B16F10 tumor-bearing mice and OT-II mice.
  • Panel C shows results for CD8 + T cells from B16F10 tumor-bearing mice and OT-I mice.
  • Incubation with MMP9FS peptide did not impact cell viability in vitro.
  • BMDC functional expression was further characterized by flow cytometry.
  • OT-I and OT-II T cells were incubated ⁇ BMDC’s, with PMA/ionomycin ⁇ inhibitory antigen peptide (MMP9FS), and T cell activation was assessed 5 days later by flow cytometry.
  • Figure 45 shows flow-cytometry results for the indicated cell markers. Incubation with MMP9FS peptide did not impact functional expression of cell markers in vitro.
  • B16F10 cancer cells were injected subcutaneously in the right flank on dO (ATCC-passage 6, 100K cells/mouse in lOOpl of 20% Matrigel in DMEM); a bolus of 10M purified T cells was injected intravenously in the tail vein also on dO. Tumor volume was measured on d5, d7, d9, and dl2, and euthanasia was performed on dl4. Statistical analysis was performed by one-way ANOVA with multiple comparisons.
  • Figure 46 panel A is a schematic representation of the experiment.
  • Figure 46, panel B shows mean tumor growth kinetics following adoptive transfer of T cells. The results show that adoptive transfer of T cells from mice vaccinated with protective vaccine were significantly protected. In contrast, adoptive transfer of T cell from mice vaccinated with protective vaccine plus inhibigen conferred no protection against tumor growth. This shows that the detrimental effect of inhibigens can be passively transferred.
  • Example 11 Mouse cancer vaccine study: combination of vaccine and checkpoint inhibitor (CPI ⁇ (therapeutic vaccination ⁇
  • SLPs corresponding to antigens identified by mATLAS and to three known B16F10 antigens were synthesized according to Example 4. Lyophilized SLPs were reconstituted in 50% ACN in H2O and pre-mixed, then frozen and lyophilized for 21h and subsequently frozen again as lyophilized pools. These were reconstituted on the day of immunization in either PBS/DMSO or PBS/adjuvants/DMSO (final DMSO concentration: 4%).
  • B16F10 tumor-bearing mice were vaccinated on the following schedule: cancer cells were injected subcutaneously on the right flank on dO (ATCC-passage 6, 100K cells in lOOpl of 20% Matrigel in DMEM); vaccine was injected subcutaneously at the tail base on d3, dlO, dl7.
  • SLP dosage was 50pg per SLP/mouse/day.
  • Checkpoint inhibitors anti-PD-1, anti-PD-Ll, anti-CTLA- 4, or anti-PD-1 plus anti-CTLA-4 were administered on d5, d9, dl2, and dl6.
  • Control groups received adjuvant alone (triple adjuvant comprising CpG, 3D-PHAD, QS-21).
  • the workflow for B16F10 melanoma challenge and vaccination in conjunction with checkpoint inhibitor is shown in Figure 33.
  • Splenocytes were collected on dl 8 of the study (i.e., 2 days after final dose of checkpoint inhibitor) and resuspended in OpTmizer media. Cells were normalized to one cell concentration (2xl0 5 cells/well) and seeded into an IFNy ELISPOT plate with stimulants for overnight culture. Cells from each individual mouse were split into 2 wells: well 1 contained media alone, well 2 contained pooled OLPs (4pg/ml or lug/mL/peptide) specific to the vaccine that the mouse received. For example, for a mouse immunized with peptides 1-4, the cells were stimulated with OLPs la-d, 2a-d, 3a-d and 4a-d (16 individual 15mers overlapping by 11 amino acids total).
  • Tumor size was measured 3x/week and subsequently on a daily basis after reaching a specified size threshold (1000mm 3 ).
  • Statistical analysis was performed by one-way ANOVA with multiple comparisons. Mice were euthanized when tumors reached maximum size, or tumors became ulcerated and did not heal within 24 hours. No mice in this study were euthanized for other health reasons.
  • Figure 47 shows mean tumor growth kinetics following therapeutic vaccination and treatment with a combination of anti-PD-1 and CTLA-4 checkpoint inhibitors. Groups were vaccinated as indicated with adjuvant only (control), protective vaccine, protective vaccine plus inhibigen MMP9FS, or inhibigen MMP9FS plus adjuvant. As in Example 7, results of this study show that combination checkpoint inhibition partially ameliorated accelerated tumor growth in mice vaccinated with an inhibigen, but the effect did not appear to be additive or synergistic compared to checkpoint inhibition with anti-CTLA-4 alone (data not shown).
  • Figure 48 show results of ZFNy ELISPOT performed on splenocytes from therapeutically vaccinated mice, with and without checkpoint inhibition, after in vitro stimulation with overlapping peptides spanning vaccine components.
  • Peptides used for in vitro stimulation are indicated in the keys on the right side of each panel: M30 andTrp2 peptides correspond to stimulatory antigens of the same name, S10 peptides correspond to stimulatory antigen Gal3Stl, and In21 peptides correspond to inhibigen MMP9FS.
  • Statistical analyses were performed by one- way ANOVA with multiple comparisons.
  • Figure 48, panel A shows representative results for mice receiving protective vaccine and the checkpoint inhibitors indicated on the x axis.
  • T cells from mice receiving protective vaccine plus checkpoint inhibitors produced robust IFNy responses, with an average of 2000 spots per lxlO A 6 cells.
  • Figure 48, panel B shows representative results for mice receiving protective vaccine plus inhibigen and the checkpoint inhibitors indicated on the x axis.
  • T cells from mice receiving protective vaccine plus inhibigen showed 10-fold lower immunogenicity, with an average of 200 spots per lxlO A 6 cells.
  • Checkpoint inhibition did not restore immunogenicity of T cells from these mice.
  • the presence or absence of inhibigens at any given time in a cancer patient’s tumor may correlate with that patient’s response to different checkpoint inhibitors, cellbased cancer therapies, or other cancer therapies.
  • a patient’s tumor includes numerous inhibigens, one or more dominant inhibigens, and/or an unfavorable ratio of inhibigens to stimulatory antigens
  • anti-PD-1 or anti-PD-Ll checkpoint inhibition may be less preferred than checkpoint inhibition by an agent such as anti-CTLA-4 that counteracts deleterious effects of inhibigens.
  • a patient’s tumor includes fewer inhibigens, less dominant inhibigens, and/or a more favorable ratio of inhibigens to stimulatory antigens, it may be inferred that treatment improved the immune profile of the patient’s tumor.
  • Rapoport AP Aqui NA, Stadtmauer EA, Vogl DT, Xu YY, Kalos M, et al. Combination immunotherapy after ASCT for multiple myeloma using MAGE-A3/Poly-ICLC immunizations followed by adoptive transfer of vaccine-primed and costimulated autologous T cells. Clin Cancer Res. 2014;20(5): 1355-65.

Abstract

Methods and compositions for identifying tumor antigens of human lymphocytes, and for identifying subjects for cancer therapy, are provided herein.

Description

TREATMENT METHODS
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional Application No. 63/078,313, filed September 14, 2020, U.S. Provisional Application No. 63/111,505, filed November 9, 2020, U.S. Provisional Application No. 63/140,721, filed January 22, 2021, U.S. Provisional Application No. 63/173,148, filed April 9, 2021, U.S. Provisional Application No. 63/186,541, filed May 10, 2021, and U.S. Provisional Application No. 63/197,101, filed June 4, 2021, the contents of each of which are hereby incorporated by reference herein in their entirety.
BACKGROUND
[0002] Cancer is characterized by proliferation of abnormal cells. Many treatments include costly and painful surgeries and chemotherapies. Although there is a growing interest in cancer therapies that target cancerous cells using a patient’s own immune system, such therapies have had limited success.
SUMMARY
[0003] The present invention features, inter alia, methods of identifying and/or selecting antigens that improve, increase and/or stimulate immune control of a tumor or cancer and methods of administering the same. Additionally, methods described herein include obtaining a subject response profile and selecting a subject for a cancer therapy based on a subject response profile.
[0004] Accordingly, one aspect the disclosure features a method of selecting a subject for cancer therapy. In some embodiments, the method comprises: a) obtaining a first response profile from the subject, the first response profile comprising a representation of (i) a first number of identified stimulatory tumor antigens and (ii) a first number of identified inhibitory tumor antigens, at a first time; b) obtaining a second response profile from the subject, the second response profile comprising a representation of (i) a second number of identified stimulatory tumor antigens and (ii) a second number of identified inhibitory tumor antigens, at a second time; c) comparing the first response profile to the second response profile; and if (i) the first number of stimulatory tumor antigens is different from the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is different from the second number of inhibitory tumor antigens, then selecting the subject as a candidate subject for initiation, continuation, modification, discontinuation or non-initiation of a cancer therapy or sequence of cancer therapies (e.g., a regimen of the same or different cancer therapies, e.g., in a specific sequence or order).
[0005] In some embodiments, the first time is prior to initiation of a cancer therapy. In some embodiments, the second time is after initiation of a cancer therapy. In some embodiments, the first time and second time are after initiation of a cancer therapy.
[0006] In some embodiments, stimulatory antigens and/or inhibitory antigens are identified by: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators; f) identifying as one or more stimulatory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) increase level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that decrease immune response; and g) identifying as one or more inhibitory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) increase level of expression and/or secretion of one or more immune mediators that decrease immune response.
[0007] In some embodiments, the cancer therapy comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor (CPI) therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
[0008] In some embodiments, if (i) the first number of stimulatory tumor antigens is greater than the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is less than the second number of inhibitory tumor antigens, then the method comprises selecting the subject as a candidate subject for modification or discontinuation of the cancer therapy. In some embodiments, the method further comprises modifying or discontinuing the cancer therapy for the subject.
[0009] In some embodiments, if (i) the first number of stimulatory tumor antigens is less than the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is greater than the second number of inhibitory tumor antigens, then the method comprises selecting the subject as a candidate subject for continuation of the cancer therapy. In some embodiments, the method further comprises continuing the cancer therapy for the subject.
[0010] Another aspect of the disclosure features a method of selecting a subject for cancer therapy, where the method comprises: a) obtaining a first response profile from the subject, the first response profile comprising a representation of (i) a first number of identified stimulatory tumor antigens and (ii) a first number of identified inhibitory tumor antigens, at a first time; b) obtaining a second response profile from the subject, the second response profile comprising a representation of (i) a second number of identified stimulatory tumor antigens and (ii) a second number of identified inhibitory tumor antigens, at a second time; c) comparing the first response profile to the second response profile; and if (i) the first number of stimulatory tumor antigens is greater than the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is less than the second number of inhibitory tumor antigens, then selecting the subject as a candidate subject for modification or discontinuation of a cancer therapy.
[0011] In some embodiments, the first time is prior to initiation of a cancer therapy. In some embodiments, the second time is after initiation of a cancer therapy. In some embodiments, the first time and second time are after initiation of a cancer therapy.
[0012] In some embodiments, stimulatory antigens and/or inhibitory antigens are identified by: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators; f) identifying as one or more stimulatory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) increase level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that decrease immune response; and g) identifying as one or more inhibitory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) increase level of expression and/or secretion of one or more immune mediators that decrease immune response.
[0013] In some embodiments, the cancer therapy comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
[0014] Another aspect of the disclosure provides a method of selecting a subject for cancer therapy, where the method comprises: a) obtaining a first response profile from the subject, the first response profile comprising a representation of (i) a first number of identified stimulatory tumor antigens and (ii) a first number of identified inhibitory tumor antigens, at a first time; b) obtaining a second response profile from the subject, the second response profile comprising a representation of (i) a second number of identified stimulatory tumor antigens and (ii) a second number of identified inhibitory tumor antigens, at a second time; c) comparing the first response profile to the second response profile; and if (i) the first number of stimulatory tumor antigens is less than the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is greater than the second number of inhibitory tumor antigens, then selecting the subject as a candidate subject for continuation of a cancer therapy.
[0015] In some embodiments, the method further comprises continuing the cancer therapy for the subject. In some embodiments, the first time is prior to initiation of a cancer therapy. In some embodiments, the second time is after initiation of a cancer therapy. In some embodiments, the first time and second time are after initiation of a cancer therapy.
[0016] In some embodiments, stimulatory antigens and/or inhibitory antigens are identified by: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators; f) identifying as one or more stimulatory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) increase level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that decrease immune response; and g) identifying as one or more inhibitory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) increase level of expression and/or secretion of one or more immune mediators that decrease immune response.
[0017] In some embodiments, the cancer therapy comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
[0018] In another aspect, the disclosure provides a method of selecting a subject for cancer therapy, comprising: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) obtained from a subject at a first time, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes obtained from the subject at the first time, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators; f) identifying as one or more stimulatory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) increase level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that decrease immune response; g) identifying as one or more inhibitory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) increase level of expression and/or secretion of one or more immune mediators that decrease immune response; and h) obtaining a first response profile from the subject, the first response profile comprising a representation of (i) a first number of identified stimulatory tumor antigens and (ii) a first number of identified inhibitory tumor antigens, at the first time; i) repeating steps a) through g) with APCs and/or lymphocytes obtained from the subject at a second time; j) obtaining a second response profile from the subject, the second response profile comprising a representation of (i) a second number of identified stimulatory tumor antigens and (ii) a second number of identified inhibitory tumor antigens, at the second time; k) comparing the first response profile to the second response profile; and if (i) the first number of stimulatory tumor antigens is different from the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is different from the second number of inhibitory tumor antigens, then selecting the subject as a candidate subject for initiation, continuation, modification, discontinuation or non-initiation of a cancer therapy or sequence of cancer therapies (e.g., a regimen of the same or different cancer therapies, e.g., in a specific sequence or order).
[0019] In some embodiments, the first time is prior to initiation of a cancer therapy. In some embodiments, the second time is after initiation of a cancer therapy. In some embodiments, the first time and second time are after initiation of a cancer therapy. In some embodiments, the cancer therapy comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cellbased therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing. [0020] In some embodiments, if (i) the first number of stimulatory tumor antigens is greater than the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is less than the second number of inhibitory tumor antigens, then the method comprises selecting the subject as a candidate subject for modification or discontinuation of the cancer therapy.
[0021] In some embodiments, the method further comprises modifying or discontinuing the cancer therapy for the subject. In some embodiments, if (i) the first number of stimulatory tumor antigens is less than the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is greater than the second number of inhibitory tumor antigens, then the method comprises selecting the subject as a candidate subject for continuation of the cancer therapy. In some embodiments, the method further comprises continuing the cancer therapy for the subject.
[0022] In another aspect, the present disclosure provides a method of selecting a subject for cancer therapy, comprising: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) obtained from a subject at a first time, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes obtained from the subject at the first time, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators; f) identifying as one or more stimulatory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) increase level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that decrease immune response; g) identifying as one or more inhibitory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) increase level of expression and/or secretion of one or more immune mediators that decrease immune response; and h) obtaining a first response profile from the subject, the first response profile comprising a representation of (i) a first number of identified stimulatory tumor antigens and (ii) a first number of identified inhibitory tumor antigens, at the first time; i) repeating steps a) through g) with APCs and/or lymphocytes obtained from the subject at a second time; j) obtaining a second response profile from the subject, the second response profile comprising a representation of (i) a second number of identified stimulatory tumor antigens and (ii) a second number of identified inhibitory tumor antigens, at the second time; k) comparing the first response profile to the second response profile; and if (i) the first number of stimulatory tumor antigens is greater than the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is less than the second number of inhibitory tumor antigens, then selecting the subject as a candidate subject for modification or discontinuation of a cancer therapy.
[0023] In some embodiments, the method further comprises modifying or discontinuing the cancer therapy for the subject. In some embodiments, the first time is prior to initiation of a cancer therapy. In some embodiments, the second time is after initiation of a cancer therapy. In some embodiments, the first time and second time are after initiation of a cancer therapy. In some embodiments, the cancer therapy comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
[0024] In another aspect, the present disclosure describes a method of selecting a subject for cancer therapy, comprising: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) obtained from a subject at a first time, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes obtained from the subject at the first time, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators; f) identifying as one or more stimulatory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) increase level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) inhibit and.or suppress level of expression and/or secretion of one or more immune mediators that decrease immune response; g) identifying as one or more inhibitory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) increase level of expression and/or secretion of one or more immune mediators that decrease immune response; and h) obtaining a first response profile from the subject, the first response profile comprising a representation of (i) a first number of identified stimulatory tumor antigens and (ii) a first number of identified inhibitory tumor antigens, at the first time; i) repeating steps a) through g) with APCs and/or lymphocytes obtained from the subject at a second time; j) obtaining a second response profile from the subject, the second response profile comprising a representation of (i) a second number of identified stimulatory tumor antigens and (ii) a second number of identified inhibitory tumor antigens, at the second time; k) comparing the first response profile to the second response profile; and if (i) the first number of stimulatory tumor antigens is less than the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is greater than the second number of inhibitory tumor antigens, then selecting the subject as a candidate subject for continuation of a cancer therapy. [0025] In some embodiments, the method further comprises continuing the cancer therapy for the subject. In some embodiments, the first time is prior to initiation of a cancer therapy. In some embodiments, the second time is after initiation of a cancer therapy. In some embodiments, the first time and second time are after initiation of a cancer therapy. In some embodiments, the cancer therapy comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
[0026] In another aspect, the present disclosure describes a method of selecting a cancer therapy for a subject, the method comprising: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators; f) identifying as one or more stimulatory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) increase level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that decrease immune response; g) identifying as one or more inhibitory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) increase level of expression and/or secretion of one or more immune mediators that decrease immune response; and h) selecting for the subject, based on the number and/or characteristics of the identified stimulatory and inhibitory antigens, a cancer therapy or sequence of cancer therapies (e.g., a regimen of the same or different cancer therapies, e.g., in a specific sequence or order).
[0027] In some embodiments, the cancer therapy or sequence of cancer therapies comprises
(i) an immunogenic composition comprising one or more of the identified stimulatory antigens,
(ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
[0028] In some embodiments, the checkpoint inhibitor therapy comprises one or more agents directed to PD-1, PD-L1, CTLA4, Lag-3, Tim-3, TIGIT, BTLA, CBLB, CISH, VISTA, B7-H3, CSF-1R, or GITR. In some embodiments, the checkpoint inhibitor therapy comprises two or more agents directed to PD-1, PD-L1, CTLA4, Lag-3, Tim-3, TIGIT, BTLA, CBLB, CISH, VISTA, B7-H3, CSF-1R, or GITR. In some embodiments, the checkpoint inhibitor therapy comprises one or more of pembrolizumab, nivolumab. atezolizumab, avelumab, durvalumab, ipilimumab, cemiplimab, spartalizumab, camrelizumab, cabiralizumab, dostarlimab, sintilmab, tisleliumab, or toripalimab. In some embodiments, the method further comprises administering the selected cancer therapy or sequence of cancer therapies to the subject.
[0029] In some embodiments, the cancer therapy or sequence of cancer therapies comprises
(i) an immunogenic composition comprising one or more of the identified stimulatory antigens,
(ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
[0030] Another aspect of the disclosure features a method of treating cancer in a subject, the method comprising: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators; f) identifying as one or more stimulatory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) increase level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that decrease immune response; g) identifying as one or more inhibitory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) increase level of expression and/or secretion of one or more immune mediators that decrease immune response; h) selecting for the subject, based on the number and/or characteristics of the identified stimulatory and inhibitory antigens, a cancer therapy or sequence of cancer therapies; and i) administering the selected cancer therapy or sequence of cancer therapies to the subject.
[0031] In some embodiments, the cancer therapy or sequence of cancer therapies comprises
(i) an immunogenic composition comprising one or more of the identified stimulatory antigens,
(ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
[0032] In some embodiments, the checkpoint inhibitor therapy comprises one or more agents directed to PD-1, PD-L1, CTLA4, Lag-3, Tim-3, TIGIT, BTLA, CBLB, CISH, VISTA, B7-H3, CSF-1R, or GITR. In some embodiments, the checkpoint inhibitor therapy comprises two or more agents directed to PD-1, PD-L1, CTLA4, Lag-3, Tim-3, TIGIT, BTLA, CBLB, CISH, VISTA, B7-H3, CSF-1R, or GITR. In some embodiments, the checkpoint inhibitor therapy comprises one or more of pembrolizumab, nivolumab. atezolizumab, avelumab, durvalumab, ipilimumab, cemiplimab, spartalizumab, camrelizumab, cabirali umab, dostarlimab, sintilmab, tisleliumab, or toripalimab. [0033] In some embodiments, the one or more immune mediators are selected from the group consisting of cytokines, soluble mediators, and cell surface markers expressed by the lymphocytes. In some embodiments, the one or more immune mediators are cytokines. In some embodiments, the one or more cytokines are selected from the group consisting of TRAIL, IFN- gamma, IL-12p70, IL-2, TNF-alpha, MIP1 -alpha, MIPl-beta, CXCL9, CXCL10, MCP1, RANTES, IL-1 beta, IL-4, IL-6, IL-8, IL-9, IL-10, IL-13, IL-15, CXCL11, IL-3, IL-5, IL-17, IL- 18, IL-21, IL-22, IL-23 A, IL-24, IL-27, IL-31, IL-32, TGF-beta, CSF, GM-CSF, TRANCE (also known as RANK L), MIP3 -alpha, fractalkine, aldesleukin, IFN-alpha-2a, IFN-alpha-2b, and pegIFN-alpha-2b.
[0034] In some embodiments, the one or more immune mediators are soluble mediators. In some embodiments, the one or more soluble mediators are selected from the group consisting of granzyme A, granzyme B, sFas, sFasL, perforin, and granulysin. In some embodiments, the one or more immune mediators are cell surface markers. In some embodiments, the one or more cell surface markers are selected from the group consisting of CD107a, CD107b, CD25, CD69, CD45RA, CD45RO, CD137 (4-1BB), CD44, CD62L, CD27, CCR7, CD154 (CD40L), KLRG-1, CD71, HLA-DR, CD122 (IL-2RB), CD28, IL7Ra (CD127), CD38, CD26, CD134 (OX-40), CTLA-4 (CD 152), LAG-3, TIM-3 (CD366), CD39, PD-1 (CD279), FoxP3, TIGIT, CD 160, BTLA, 2B4 (CD244), and KLRG1.
[0035] In some embodiments, the lymphocytes comprise CD4+ T cells. In some embodiments, the lymphocytes comprise CD8+ T cells. In some embodiments, lymphocyte inhibition and/or suppression is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, or 200% higher or lower than a control level.
[0036] In some embodiments, lymphocyte inhibition and/or suppression is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 1, 2, or 3 standard deviations greater or lower than the mean of a control level. In some embodiments, lymphocyte inhibition and/or suppression is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) greater or lower than a median response level to a control.
[0037] In some embodiments, lymphocyte stimulation is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, or 200% higher or lower than a control level. In some embodiments, lymphocyte stimulation is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 1, 2, or 3 standard deviations higher or lower than the mean of a control level. In some embodiments, lymphocyte stimulation is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) greater or lower than a median response level to a control.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] The present teachings described herein will be more fully understood from the following description of various illustrative embodiments, when read together with the accompanying drawings. It should be understood that the drawings described below are for illustration purposes only and are not intended to limit the scope of the present teachings in any way.
[0039] Figure 1 shows exemplary elements of GEN-009-101, a first-in-human Phase l/2a study testing personalized vaccines designed using the ATLAS platform for feasibility, safety, immunogenicity and clinical activity in selected solid tumors. Panel A shows Schedule 1, an exemplary dosing regimen. Panel B shows a schematic of the overall design of the clinical study.
[0040] Figure 2 shows representative results of in vitro stimulated FluoroSpot assays on CD4+ and CD8+ T cells enriched from PBMCs collected at baseline (prior to vaccination) and at Day 50 from each of 5 patients (patients A, B, C, E, and F). [0041] Figure 3 shows representative results of ex vivo FluoroSpot assays and in vitro stimulated FluoroSpot assays on CD4+ and CD8+ T cells enriched from PBMCs collected at baseline (prior to vaccination) and at Day 50 from a representative patient (patient E). Panels A and B: ex vivo FluoroSpot assays. Panel C: in vitro stimulated FluoroSpot assays.
[0042] Figure 4 shows representative summary results of ex vivo FluoroSpot assays and in vitro stimulated FluoroSpot assays on total PBMC or PBMCs depleted of CD4+ or CD8+ T cells collected at baseline (prior to vaccination) and at Day 50 from patients A-H and K. Data are reported as the proportion of peptides positive by the DFReq test. Circles represent baseline, squares represent D50 time point. Panel A shows ex vivo FluoroSpot assays for patients A-H, and K. Panel B shows in vitro stimulated FluoroSpot assays for patients A-H, and K. Panel C shows the proportion of SLPs scored positive by any assay for patients A-H, and K.
[0043] Figure 5 shows data for and the status of each patient and includes, for each patient, the tumor type, stage of cancer at diagnosis, period of time from diagnosis, prior therapies the patient received, the patient’s calculated tumor mutational burden (TMB), the number of stimulatory and inhibitory neoantigens identified for each patient, and the number of peptides in the example vaccine administered. The graph indicates the status of each patient at different time points within the example vaccination regimen. The timing of example vaccination is indicated by the vertical arrows. The color of the horizontal bars indicates the stage of cancer at diagnosis. A blue horizontal arrow indicates that the patient has not yet completed the vaccination regimen (i.e., is within the dosing period). A black horizontal arrow indicates that the patient has completed the vaccination regimen (i.e., is past the treatment period or post vaccination schedule). A black circle indicates a status of “NED” or no evidence of disease.
[0044] Figure 6 shows representative results of ex vivo dual-analyte FluoroSpot assays on CD4+ and CD8+ T cells enriched from PBMCs of three representative patients (patients A and E; low response patient H). Bulk PBMCs were isolated from the patients at baseline (prior to vaccination) and at the indicated timepoints over the course of their treatment. The secretion of IFN-y and granzyme B (GrB) was quantified via ex vivo dual-analyte FluoroSpot after stimulation with overlapping peptide pools (OLPs) spanning the patient-specific SLPs used for immunization. In Panel A, data are expressed as mean (± SEM) spot forming cells (SFC) per million PBMCs to each of the four pools. Panel B shows the number of positive pools for each time point. The value above each bar represents the number of subjects contributing data. Grey = prior to vaccination; Blue = post-vaccination. Responses were determined by DFR(eq) test (P<0.05) and SFC greater than the assay LOD.
[0045] Figure 7 shows a spider plot for an idealized patient, illustrating the method of assessing GEN 009 vaccine-specific contribution to clinical responses in Part B of the study. Each point on the plot represents assessment of the idealized patient’s target lesions. The X axis shows time and treatment. The left side of the graph shows the period of the idealized patient’s initial assessment and standard-of-care (SOC) treatment. The right side of the graph shows the period of standard-of-care treatment combined with GEN 009 vaccination (SOC & Vaccine). The Y axis shows percent change in the sum, or diameter, of the idealized patient’s target lesions. Baseline is defined as the patient’s lesions at initial assessment. Progressive disease (PD), partial response (PR), and complete response (CR) are defined relative to baseline as +20%, -30% and -100% change, respectively (RECIST criteria). The dotted line on the right side of the graph represents the course of disease, that is, for the majority of patients, the course set during the previous 3 to 4 months of active therapy.
[0046] Figure 8 shows results for patient -058, enrolled in the Main Part B Study. Panel A: Spider plot, as described for Figure 7. Panel B: Results of in vitro stimulated FluoroSpot assays performed at baseline and at Day 50 post-initial vaccination. The height of each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN- y and TNF-a, for each SLP included in the patient’s vaccine, as indicated. CD4+ and CD8+ T cell responses are presented in the left and right panels, respectively. Panel C: Aggregate results of in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ± SD per 20,000 T cells (IVS). The dark bars indicate total IFNy responses, the lighter bars indicate total TNFα responses. Panel D: Radiographic imaging of mediastinal tumor, showing tumor mass at 3 timepoints (Baseline, Pre-vaccine, Postvaccine). Panel E: Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 106 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
[0047] Figure 9 shows results for patient -105, enrolled in the Main Part B Study. Panel A: Spider plot, as described for Figure 7. Panel B, top: Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed with PBMCs collected at baseline and at the indicated days after initial vaccination with GEN 009. The height of each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine. The DI time point represents post-checkpoint inhibitor therapy, pre-vaccine responses; therefore, increases in frequency of PBMC responses at subsequent time points can be attributed to GEN 009 vaccination. Panel B, bottom: Results of in vitro stimulated FluoroSpot assays performed on CD4+ and CD8+ T cells enriched from PBMC at baseline and at Day 50 after initial vaccination. The height of each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN-y (left axis) and TNF-a (right axis), for each SLP included in the patient’s vaccine, as indicated. CD4+ and CD8+ T cell responses are presented in the left and right panels, respectively. Panel C: Summary results of ATLAS screens performed at baseline and at Day 50 after initial vaccination with GEN 009. Each pie chart shows percentages of the patient’s tumor mutations that elicit stimulatory T cell responses (stimulatory antigens) or inhibitory T cell responses (inhibigens). Panel D: Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ± SD per 106 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNFα responses. Panel E: Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 106 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN- 009 dosing, with black triangles representing days of vaccination.
[0048] Figure 10 shows results for patient -001, enrolled in the Main Part B Study. Panel A: Spider plot, as described for Figure 7. Panel B, top: Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed on PBMCs collected at baseline and at the indicated days after initial vaccination with GEN 009. The height of each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine. The DI time point represents post-checkpoint inhibitor therapy, pre-vaccine responses; therefore, increases in frequency of PBMC responses at subsequent time points can be attributed to GEN 009 vaccination. Panel B, bottom: Results of in vitro stimulated FluoroSpot assays performed on CD4+ and CD8+ T cell enriched from PBMC at baseline and at Day 50 after initial vaccination. The height of each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN-y (left axis) and TNF-a (right axis), for each SLP included in the patient’s vaccine, as indicated. CD4+ and CD8+ T cell responses are presented in the left and right panels, respectively. Panel C: Summary results of ATLAS screens performed at baseline and at Day 50 after initial vaccination with GEN 009. Each pie chart shows percentages of the patient’s expressed tumor mutations that elicit stimulatory T cell responses (stimulatory antigens) or inhibitory T cell responses (inhibigens). Panel D: Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown.
Values presented are the means of background subtracted SFCs ± SD per 106 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNFα responses. Panel E: Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 106 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
[0049] Figure 11 shows results for patient -057, enrolled in the Main Part B Study.
Panel A: Spider plot, as described for Figure 7. Panel B, top: Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed on PBMCs collected at baseline and at the indicated days after initial vaccination with GEN 009. The height of each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine. The DI time point represents post-checkpoint inhibitor therapy, pre-vaccine responses; therefore, increases in frequency of PBMC responses at subsequent time points can be attributed to GEN 009 vaccination. Panel B, bottom: Results of in vitro stimulated FluoroSpot assays performed on CD4+ and CD8+ T cells enriched from PBMC at baseline and at Day 50 after initial vaccination. The height of each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN-y (left axis) and TNF-a (right axis), for each SLP included in the patient’s vaccine, as indicated. CD4+ and CD8+ T cell responses are presented in the left and right panels, respectively. Panel C: Summary results of ATLAS screens performed at baseline and at Day 50 after initial vaccination with GEN 009. Each pie chart shows percentages of the patient’s tumor mutations that elicit stimulatory T cell responses (stimulatory antigens) or inhibitory T cell responses (inhibigens). Panel D: Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ± SD per 106 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNFα responses. Panel E: Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 106 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN- 009 dosing, with black triangles representing days of vaccination.
[0050] Figure 12 shows results for patient -031, enrolled in the Main Part B Study. Panel A: Spider plot, as described for Figure 7. Panel B, top: Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed on PBMCs collected at baseline and at the indicated days after initial vaccination with GEN 009. The height of each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine. The DI time point represents post-checkpoint inhibitor therapy, pre-vaccine responses; therefore, increases in frequency of PBMC responses at subsequent time points can be attributed to GEN 009 vaccination. Panel B: Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ± SD per 106 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNFa responses. Panel C: Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 106 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
[0051] Figure 13 shows results of all ex vivo dual-color FluoroSpot assays plotted over time. Peripheral blood mononuclear cells were collected from patients participating in the Main Part B Study at the indicated time points (day 1 is the day of the first GEN-009 vaccination) and viably frozen. Thawed PBMCs were stimulated with pools containing the vaccine antigens (or controls). Results are shown as the mean cytokine spot forming cells (SFC) per million PBMC (+/- SEM) for each subject contributing data at each time point. Not all patients contributed data for each time point: the N at each time point is indicated.
[0052] Figure 14 shows summary results for all patients enrolled in the Main Part B Study. Panel A is a composite of all spider plots, as described for Figure 7, showing the percent target tumor lesion change for each patient, from the time of consent and initial assessment. Panel B is a composite of all spider plots showing the percent target tumor lesion change for each patient from the time the patient received GEN-009 dose 1 (i.e., baseline is defined at day 0 of the study). Panel C is a swimmers’ plot showing timelines of PD-1 standard-of-care therapy, GEN-009 dosing, and outcomes for each patient. Panel D shows aggregate results for all patients of dual-analyte immunogenicity assays performed at screening and at Day 50 post-initial vaccination. PBMC, CD4+ T cell and CD8+ T cell responses are shown. Values presented are the means of background subtracted SFCs ± SEM per 20,000 T cells. The dark bars indicate total IFNy responses, the lighter bars indicate total TNFα responses.
[0053] Figure 15 shows results for patient -106, enrolled in the Part B Refractory Study. Panel A: Spider plot, as described for Figure 7. Panel B, top: Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed on PBMCs collected at baseline and at the indicated days after initial vaccination with GEN 009. The height of each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine. The DI time point represents post-checkpoint inhibitor therapy and post-chemotherapy, pre-vaccine responses; therefore, increases in frequency of PBMC responses at subsequent time points can be interpreted as attributable to GEN 009 vaccination. Panel B, bottom: Results of in vitro stimulated FluoroSpot assays on CD4+ and CD8+ T cells enriched from PBMCs performed at baseline and at Day 50 after initial vaccination. The height of each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN-y (left axis) and TNF-a (right axis), for each SLP included in the patient’s vaccine, as indicated. CD4+ and CD8+ T cell responses are presented in the left and right panels, respectively. Panel C: Summary results of ATLAS screens performed at baseline and at Day 50 after initial vaccination with GEN 009. Each pie chart shows percentages of the patient’s expressed tumor mutations that elicit stimulatory T cell responses (stimulatory antigens) or inhibitory T cell responses (inhibigens). Panel D: Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ± SD per 106 T cells (ex vivo, top panel) or 20,000 T cells (in vitro stimulated, bottom panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNFα responses. Panel E: Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 106 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
[0054] Figure 16 shows results for patient -109, enrolled in the Part B Refractory Study. Panel A: Spider plot, as described for Figure 7. Panel B: Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 postinitial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ± SD per 106 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNFα responses.
[0055] Figure 17 shows results for patient -027, enrolled in the Part B Refractory Study. Panel A: Spider plot, as described for Figure 7. Panel B: Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 106 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN- 009 dosing, with black triangles representing days of vaccination.
[0056] Figure 18 shows results for patient -110, enrolled in the Part B Refractory Study. Panel A: Spider plot, as described for Figure 7. Panel B: Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 postinitial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ± SD per 106 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNFα responses.
[0057] Figure 19 shows results for patient -060, enrolled in the Part B Refractory Study. Panel A: Spider plot, as described for Figure 7.
[0058] Figure 20 shows summary results for all patients enrolled in the Main Part B Refractory Study. Panel A is a composite of all spider plots, as described for Figure 7, showing the percent target tumor lesion change for each patient, from the time of consent and initial assessment. Panel B is a composite of all spider plots showing the percent target tumor lesion change for each patient from the time the patient received GEN-009 dose 1 (i.e., baseline is defined at day 0 of the study). Panel C is a swimmers’ plot showing timelines of PD-1 standard- of-care therapy, GEN-009 dosing, and outcomes for each patient. Panel D shows aggregate results for all patients of dual-analyte immunogenicity assays performed at screening and at Day 50 post-initial vaccination. PBMC, CD4+ T cell and CD8+ T cell responses are shown. Values presented are the means of background subtracted SFCs ± SEM per 20,000 T cells. The dark bars indicate total IFNy responses, the lighter bars indicate total TNFα responses.
[0059] Figure 21 shows representative summary results for CD4+ and CD8+ T cell responsiveness, prior to and after vaccination with GEN 009. In Panel A, the bars labeled “ATLAS” indicate the overall percentages of antigens that elicited robust CD4+ or CD8+ T cell responses in initial ATLAS screening of the selected Part B patients and were consequently included in the GEN 009 vaccine compositions administered to these patients. The bars labeled “Post GEN-009 Treatment” indicate the overall percentages of antigens that elicited CD4+ or CD8+ T cell responses in ATLAS screening of the selected Part B patients following their vaccination with GEN 009. The comparison shows changes in the breadth of T cell responses. Panel B is a graph showing aggregate IFNy responses (ex vivo FluoroSpot assays) as SFCs ± SEM per million PBMCs over several timepoints, from screening to 6 months after patients received GEN 009 dose 3. Panel C shows the average number of neoantigens that elicited positive CD4+ and CD8+ T cell responses, as determined by ATLAS, at the time of screening and at Day 50 post-initial vaccination, broken out for Main Part B (Controlled) and Part B Refractory (Refractory) patients. Panel D shows the total number of non-synonymous mutations (tumor mutational burden) for each cohort at the time of screening.
[0060] Figure 22 shows overall characterization of stimulatory antigens (neoantigens), inhibigens, and non-responsive antigens identified in 37 patients screened for inclusion in the Part A Study, the Main Part B Study, and the Part B Refractory Study of GEN-009. Panel A: Total proportion of patients in which CD4+ and CD8+ T cells responded with an inhibitory phenotype (n=33). Panel B: Total proportion of stimulatory antigens (neoantigens), inhibigens, and non-responsive antigens.
[0061] Figure 23 shows the overall proportion of CD4+ and CD8+ stimulatory antigens (neoantigens), inhibigens, and non-responsive antigens identified in 37 patients screened for inclusion in the Part A Study, the Main Part B Study, and the Part B Refractory Study of GEN- 009. Results are expressed as a percentage of total CD4+ and CD8+ responses.
[0062] Figure 24 shows overall characterization, against total mutations screened, of somatic mutations, stimulatory antigens (neoantigens), and inhibigens identified in 37 patients screened for inclusion in the Part A Study, the Main Part B Study, and the Part B Refractory Study of GEN-009. Panel A: Number of mutations screened (± SEM) by tumor type. Panel B: Number of inhibigens detected, by tumor type, as a percent of total mutations screened. Panel C: Correlation analysis between the number of inhibigens detected and the size of the mutanome screened (linear regression, R2 = 0.005).
[0063] Figure 25 shows the proportion of stimulatory antigens and inhibigens identified by ATLAS screens for each patient at initial screening at D50 post-initiation of vaccinations, broken out by T cell subset, tumor type and checkpoint inhibition responsiveness vs. nonresponsiveness. For each set of bars, the top bar shows results obtained at initial screening, and the bottom bar shows results obtained at Day 50. Top rows: pooled data for the cohort of checkpoint inhibitor-responsive patients (left: CPI responsive, N=6) or -refractory (right: CPI refractory, N=2) patients. Middle rows: individual patient responses, with patient ID indicated to the left of each set. Bottom row: pooled responses by tumor type. NSCLC N = 2, RCC N = 1, UC N = 1, SCCHN N = 4.
[0064] Figure 26 shows the results of 16-plex patient-specific somatic assays for detection of circulating tumor DNA (ctDNA). Assays were performed on plasma samples taken at initial screening (Baseline) and at D50 post initiation of vaccination (Post-Tx). Two patients enrolled in the Main Part B Study are represented, one in each graph. Results are expressed as the percent frequency of variant alleles in plasma, and show disappearance of ctDNA postvaccination. The data are stratified by non-antigenic, tumor-specific mutations (circles) or ATLAS-identified antigens (diamonds = inhibigens, triangles = neoantigens not included in the GNE-009 vaccine, and squares = neoantigens included in the GEN-009 vaccine).
[0065] Figure 27 shows a representative workflow and representative results for mouse ATLAS (m ATLAS) screening, preliminary to Bl 6F 10 melanoma challenge and vaccination studies. Panel A: m ATLAS workflow. Panel B: Representative results of mATLAS screening for IFNy only.
[0066] Figure 28, Panel A shows results of IFNy ELISPOT performed on splenocytes from vaccinated mice of the first study (top graph) and of the third study (bottom graph) after in vitro stimulation with overlapping peptides spanning vaccine components. Statistical analysis was by multiple T-tests (top graph) and one-way ANOVA (bottom graph; p <0.0001). Panel B: Mean tumor growth kinetics following vaccination with adjuvant only, protective vaccine, or protective vaccine plus inhibigen (first study). Panel C: Mean tumor growth kinetics following vaccination with adjuvant only, protective vaccine, or protective vaccine plus inhibigen (second study). Panel D: Mean tumor growth kinetics following vaccination with adjuvant only, or inhibigen plus adjuvant (third study). [0067] Figure 29 shows immunohistochemically stained sections and quantitation of tumors from vaccinated mice. Panel A shows representative tumor sections stained with antibodies specific for mouse CD4 or CD8a (T cells), FoxP3 (Tregs), F4/80 (macrophages), or CD11c (dendritic cells), in pink/red, and counterstained for nuclei with hematoxylin (blue), for assessment of infiltrating immune cell populations. Mice were vaccinated with adjuvant alone (Adjuvant, top row), protective vaccine (Protective, middle row), or protective vaccine plus inhibigen (MMP9FS) (+ Inhibigen, bottom row). For assessment of metabolic status, tumor sections were stained with antibodies specific for metabolic markers LDHA, ARG1, and CD73. Panel B shows quantification of Panel A images. Panel C shows representative tumor sections stained for the endothelial marker CD31, for assessment of tumor vasculature. Mice were vaccinated with protective vaccine (Protective, top row), or protective vaccine plus inhibigen MMP9FS (+ Inhibigen, bottom row). Panel D shows tumor sections stained for metabolic markers LDHA, ARG1, and CD73, for assessment of metabolic status. Mice were vaccinated with protective vaccine plus inhibigen MMP9FS. Panel E shows quantification of Panel D images, together with quantification of images (not shown) of tumor sections from mice vaccinated with adjuvant alone, or with protective vaccine. Panel F shows quantification of representative tumor sections immuno-histochemically stained for CD8 T cell markers, from mice vaccinated with adjuvant only, protective vaccine, or MMP9FS only (top graph), or mice vaccinated with adjuvant only, protective vaccine, or protective vaccine plus MMP9FS (bottom graph). In the bottom graph, tumors were resected on day 11 of the study (early-stage tumors). Image quantification in Panels B, E, and F was determined by Imaged software thresholding on positive stain (pink) divided by tissue area (total image area - white non-tissue). Statistical analysis was performed by one-way ANOVA with multiple comparisons.
[0068] Figure 30 is a schematic showing workflow for MHC competition experiments.
[0069] Figure 31 shows results of MHC competition experiments. Splenocytes collected from mice vaccinated with adjuvant only, protective vaccine, or the protective vaccine plus inhibigen MMP9FS were incubated with pulsed APCs (Together or Separate). After incubation, an IFNy ELISPOT was performed. Panel A: Mean IFNy Spot Forming Cells (SFC) per IxlO5 APCs and 5xl05 splenocytes (± SEM) pooled from three mice per vaccination group. Panel B: Representative wells.
[0070] Figure 32 shows results of flow cytometry experiments. Splenocytes collected from mice vaccinated with adjuvant only, protective vaccine, or the protective vaccine plus inhibigen MMP9FS and incubated with the pulsed APCs (Together) were further restimulated with respective antigen peptides, stained for intracellular cytokines, and gated by flow cytometry on live, CD45+, CD19nes/NKl.lnes, CD3+, CD8+ T cells. Splenocytes were pooled from four mice from each vaccination group. IFNy expressing T cells are shown graphed as a percentage of CD3+ T cells.
[0071] Figure 33 is a schematic showing workflow for B 16F10 melanoma challenge and vaccination studies in conjunction with checkpoint inhibitor.
[0072] Figure 34 shows results of IFNy ELISPOT performed on splenocytes from vaccinated mice, with and without checkpoint inhibition, after in vitro stimulation with overlapping peptides spanning vaccine components. As indicated, the experimental groups received protective vaccine or protective vaccine plus inhibigen MMP9FS, in each case combined with anti-PD-1, anti-CTLA-4, or no checkpoint inhibitor.
[0073] Figure 35 shows mean tumor growth kinetics following vaccination in conjunction with checkpoint inhibitors. Panel A: Tumor growth for groups vaccinated with protective vaccine, or with protective vaccine plus inhibigen, and treated with anti-PD-1 checkpoint inhibitor (first study). Panel B: Tumor growth for groups vaccinated with protective vaccine or protective vaccine plus inhibigen, and treated with anti-CTLA-4 checkpoint inhibitor (first study). Panel C: Tumor growth for groups vaccinated with protective vaccine, or with inhibigen plus adjuvant, and treated with anti-PD-1, anti-PD-Ll, or anti-CTLA-4 checkpoint inhibitor (second study). Panel D: Tumor growth for groups vaccinated with protective vaccine, or with inhibigen plus adjuvant, and treated with a combination of anti-PD-1 plus anti-CTLA-4 checkpoint inhibitors (second study). Statistical analyses were performed by one-way ANOVA with multiple comparisons. [0074] Figure 36 shows representative immunohistochemically stained tumor sections from mice vaccinated with adjuvant alone (Adjuvant, top row), the protective vaccine (Protective, middle row), or the protective vaccine plus inhibigen (+ Inhibigen, bottom row), with and without anti-PD-1, anti-CTLA-4, or anti-PD-1 plus anti-CTLA-4 checkpoint therapy. The tumor sections were stained with an anti-mouse CD8a antibody (pink) or CD4 antibody (not pictured) and counterstained with hematoxylin (blue). Endogenous melanin deposits in tumors are visible (brown).
[0075] Figure 37 shows image quantification of tumor sections by ImageJ software thresholding on positive stain (pink) divided by tissue area (total image area - white non-tissue). The top panel shows results for CD8+ tumor infiltrating lymphocytes (TILs); the bottom panel shows results for CD4+ TILs. Each symbol in the graph represents data from one mouse, the horizontal line indicates the mean ± SD. Statistical analyses were performed by one-way ANOVA with multiple comparisons.
[0076] Figure 38 shows immunohistochemically stained sections and quantitation of tumors from vaccinated mice. Tumor sections were stained with antibodies specific for checkpoint markers CTLA-4 and PD-L1. Panel A shows representative tumor sections stained for CTLA-4 (top) and PD-L1 (bottom). Panel B shows image quantification of tumor sections stained for CTLA-4 and PD-L1, from mice vaccinated with adjuvant alone (None), protective vaccine (Protective), or protective vaccine plus inhibigen MMP9FS (+ Inhibigen), with and without anti-PD-1 and/or anti-CTLA-4 checkpoint therapy. Quantification of all immunohistochemical images was performed using ImageJ assessment of pink staining area. Graphs depict mean and standard deviation; statistical analysis is ANOVA with multiple comparisons, * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[0077] Figure 39, Panel A shows results of IFNy ELISPOT performed on splenocytes from vaccinated mice after in vitro stimulation with overlapping peptides spanning vaccine components, at day 21 of the study. Panel B shows a time course of IFNy ELISPOT at days 4, 8, and 21 of the study. Statistical analysis was by multiple T-tests. Panel C shows flow-cytometry results for IFNy-expressing cells (left), and CD44- or CD69-expressing cells (right) from splenocytes of vaccinated mice of the first study, after non-specific stimulation with PMA/ionomycin or anti-CD3 antibody and treatment with Golgi stop and Golgi plug. Mice were vaccinated with adjuvant alone (Adj only), protective vaccine (Protective), or protective vaccine plus inhibigen MMP9FS (+ Inhibigen). Panel D shows flow-cytometry results for IFNv- expressing cells from splenocytes of vaccinated mice harvested at day 4 of the second study. Cells were re-stimulated with PMA/ionomycin. Panel E shows flow-cytometry results for cells expressing markers CD69, PD-1, 4- IBB (CD 137), or CD62L, from splenocytes harvested at day 4 of the second study. Results are shown as the proportion of viable CD3+ T cells from a pool of 4 mice per group, for mice vaccinated with adjuvant alone (Adjuvant), protective vaccine (Protective vaccine), or protective vaccine plus inhibigen MMP9FS (Protective vaccine + Inhibigen). Responses were recalled with specific peptide stimulation from the vaccination. Panel F shows flow-cytometry results for myeloid populations (Ml and M2 macrophages, and myeloid-derived suppressor cells [MDSC’s]) from splenocytes of vaccinated mice.
[0078] Figure 40 shows the effect of inhibigens on T cell proliferation in splenocytes of vaccinated mice from Figure 39. Panel A shows total CD3+ T cell proliferation in the presence of IL-2 and anti-CD3 antibody. Panel B shows specific proliferation of CD8+ T cells in response to anti-CD3 antibodies by quantitation of cell trace violet.
[0079] Figure 41 shows the effect of inhibigens on TCR signal strength in splenocytes of vaccinated mice from Figure 39. Splenocytes were non-specifically stimulated with excess anti- CD3, at a concentration 5-fold higher than what is normally used, stained for activation markers CD69 and 4-1BB (CD137), and analyzed by flow cytometry. Results are expressed as the percentage of CD3+ T cells expressing the indicated marker, and as Mean Fluoresence Intensity (MFI) on CD3+ T cells.
[0080] Figure 42 shows the effect of inhibigens on activation-induced cell death (AICD) in splenocytes of vaccinated mice from Figure 39. Splenocytes were non-specifically stimulated with excess aCD3, at a concentration 5-fold higher than what is normally used, stained for cleaved Caspase-3 in conjunction with surface markers, and analyzed by flow cytometry. PMA/ionomycin at 5-fold greater concentrations was used as a positive AICD control. Results are expressed as the percentage of CD3+ T cells expressing cleaved Caspase-3.
[0081] Figure 43 shows gene expression of selected markers CD44, CTLA-4 and LDHA (Panel A) and PD-1, NK1 and Tox2 (Panel B) in draining lymph nodes collected from vaccinated mice. Gene expression was assessed by RNAseq. Results are expressed as RNA counts per million reads. Mice were vaccinated with adjuvant only (Adjuvant), protective vaccine plus inhibigen (Inhibitory or MMP9FS), or protective vaccine (Protective), as indicated on the x-axis. Panel C shows volcano plots, comparing representative groups of differentially expressed genes; significantly upregulated genes are shown in red, downregulated genes are shown in blue.
[0082] Figure 44 shows viability of cells incubated for 24 hrs in vitro with peptide corresponding to inhibigen MMP9FS. Panel A shows results for BMDCs from B16F10 tumorbearing mice. Panel B shows results for CD4+T cells from B16F10 tumor-bearing mice and OT- II mice. Panel C shows results for CD8+T cells from B16F10 tumor-bearing mice and OT-I mice.
[0083] Figure 45 shows functional expression of the indicated markers, characterized by flow cytometry. OT-I and OT-II T cells were incubated ± bone marrow dendritic cells (BMDC’s), with PMA/ionomycin ± inhibigen (MMP9FS). T cell activation was assessed 5 days later by flow cytometry.
[0084] Figure 46 shows representative results for adoptive transfer of T cells into a mouse B16F10 melanoma model. Panel A is a schematic representation of the experiment. Panel B shows mean tumor growth kinetics following adoptive transfer of T cells.
[0085] Figure 47 shows mean tumor growth kinetics following vaccination in conjunction with a combination of checkpoint inhibitors. Groups were vaccinated with protective vaccine, protective vaccine plus inhibigen, or inhibigen plus adjuvant, or adjuvant only (control), and treated with a combination of anti -PD-1 and CTLA-4 checkpoint inhibitors. Statistical analyses were performed by one-way ANOVA with multiple comparisons. [0086] Figure 48 shows results of IFNy ELISPOT performed on splenocytes from vaccinated mice, with and without checkpoint inhibition, after in vitro stimulation with overlapping peptides spanning vaccine components. Peptides used for in vitro stimulation are indicated in the keys on the right side of each panel: M30 and Trp2 peptides correspond to stimulatory antigens of the same name, S10 peptides correspond to stimulatory antigen Gal3Stl, and In21 peptides correspond to inhibigen MMP9FS. Panel A shows representative results for mice receiving protective vaccine and the checkpoint inhibitors indicated on the x axis. Panel B shows representative results for mice receiving protective vaccine plus inhibigen and the checkpoint inhibitors indicated on the x axis.
DEFINITIONS
[0087] Activate-. As used herein, a peptide presented by an antigen presenting cell (APC) “activates” a lymphocyte if lymphocyte activity is detectably modulated after exposure to the peptide presented by the APC under conditions that permit antigen-specific recognition to occur. Any indicator of lymphocyte activity can be evaluated to determine whether a lymphocyte is activated, e.g., T cell proliferation, phosphorylation or dephosphorylation of a receptor, calcium flux, cytoskeletal rearrangement, increased or decreased expression and/or secretion of immune mediators such as cytokines or soluble mediators, increased or decreased expression of one or more cell surface markers.
[0088] Administration'. As used herein, the term “administration” typically refers to the administration of a composition to a subject or system. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be systemic or local. In some embodiments, administration may be enteral or parenteral. In some embodiments, administration may be by injection (e.g., intramuscular, intravenous, or subcutaneous injection). In some embodiments, injection may involve bolus injection, drip, perfusion, or infusion. In some embodiments administration may be topical. Those skilled in the art will be aware of appropriate administration routes for use with particular therapies described herein, for example from among those listed on www.fda.gov, which include auricular (otic), buccal, conjunctival, cutaneous, dental, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, interstitial, intra-abdominal, intra-amniotic, intraarterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebral, intraci sternal, intracorneal, intracoronal, intracorporus cavemosum, intradermal, intranodal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastic, intragingival, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathecal, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intraventricular, intravitreal, laryngeal, nasal, nasogastric, ophthalmic, oral, oropharyngeal, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (e.g., inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, ureteral, urethral, or vaginal. In some embodiments, administration may involve electro-osmosis, hemodialysis, infiltration, iontophoresis, irrigation, and/or occlusive dressing. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing.
[0089] Antigen'. The term “antigen”, as used herein, refers to a molecule (e.g., a polypeptide) that elicits a specific immune response. Antigen-specific immunological responses, also known as adaptive immune responses, are mediated by lymphocytes (e.g., T cells, B cells, NK cells) that express antigen receptors (e.g., T cell receptors, B cell receptors). In certain embodiments, an antigen is a T cell antigen, and elicits a cellular immune response. In certain embodiments, an antigen is a B cell antigen, and elicits a humoral (/.< ., antibody) response. In certain embodiments, an antigen is both a T cell antigen and a B cell antigen. As used herein, the term “antigen” encompasses both a full-length polypeptide as well as a portion or immunogenic fragment of the polypeptide, and a peptide epitope within the polypeptides (e.g., a peptide epitope bound by a Major Histocompatibility Complex (MHC) molecule (e.g., MHC class I, or MHC class II)).
[0090] Antigen presenting cell'. An “antigen presenting cell” or “APC” refers to a cell that presents peptides on MHC class I and/or MHC class II molecules for recognition by T cells. APC include both professional APC (e.g., dendritic cells, macrophages, B cells), which have the ability to stimulate naive lymphocytes, and non-professional APC (e.g., fibroblasts, epithelial cells, endothelial cells, glial cells). In certain embodiments, APC are able to internalize (e.g., endocytose) members of a library (e.g., cells of a library of bacterial cells) that express heterologous polypeptides as candidate antigens.
[0091] Autolysin polypeptide. An “autolysin polypeptide” is a polypeptide that facilitates or mediates autolysis of a cell (e.g., a bacterial cell) that has been internalized by a eukaryotic cell. In some embodiments, an autolysin polypeptide is a bacterial autolysin polypeptide. Autolysin polypeptides include, and are not limited to, polypeptides whose sequences are disclosed in GenBank® under Acc. Nos. NP_388823.1, NP_266427.1, and P0AGC3.1.
[0092] Cancer'. As used herein, the term “cancer” refers to a disease, disorder, or condition in which cells exhibit relatively abnormal, uncontrolled, and/or autonomous growth, so that they display an abnormally elevated proliferation rate and/or aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, a cancer may be characterized by one or more tumors. Those skilled in the art are aware of a variety of types of cancer including, for example, adrenocortical carcinoma, astrocytoma, basal cell carcinoma, carcinoid, cardiac, cholangiocarcinoma, chordoma, chronic myeloproliferative neoplasms, craniopharyngioma, ductal carcinoma in situ, ependymoma, intraocular melanoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), gestational trophoblastic disease, glioma, histiocytosis, leukemia (e.g., acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), hairy cell leukemia, myelogenous leukemia, myeloid leukemia), lymphoma (e.g., Burkitt lymphoma [non-Hodgkin lymphoma], cutaneous T cell lymphoma, Hodgkin lymphoma, mycosis fungoides, Sezary syndrome, AIDS-related lymphoma, follicular lymphoma, diffuse large B-cell lymphoma), melanoma, merkel cell carcinoma, mesothelioma, myeloma (e.g., multiple myeloma), myelodysplastic syndrome, papillomatosis, paraganglioma, pheochromacytoma, pleuropulmonary blastoma, retinoblastoma, sarcoma (e.g., Ewing sarcoma, Kaposi sarcoma, osteosarcoma, rhabdomyosarcoma, uterine sarcoma, vascular sarcoma), Wilms’ tumor, and/or cancer of the adrenal cortex, anus, appendix, bile duct, bladder, bone, brain, breast, bronchus, central nervous system, cervix, colon, endometrium, esophagus, eye, fallopian tube, gall bladder, gastrointestinal tract, germ cell, head and neck, heart, intestine, kidney (e.g., Wilms’ tumor), larynx, liver, lung (e.g., non-small cell lung cancer, small cell lung cancer), mouth, nasal cavity, oral cavity, ovary, pancreas, rectum, skin, stomach, testes, throat, thyroid, penis, pharynx, peritoneum, pituitary, prostate, rectum, salivary gland, ureter, urethra, uterus, vagina, or vulva.
[0093] Cytolysin polypeptide'. A “cytolysin polypeptide” is a polypeptide that has the ability to form pores in a membrane of a eukaryotic cell. A cytolysin polypeptide, when expressed in host cell (e.g., a bacterial cell) that has been internalized by a eukaryotic cell, facilitates release of host cell components (e.g., host cell macromolecules, such as host cell polypeptides) into the cytosol of the internalizing cell. In some embodiments, a cytolysin polypeptide is bacterial cytolysin polypeptide. In some embodiments, a cytolysin polypeptide is a cytoplasmic cytolysin polypeptide. Cytolysin polypeptides include, and are not limited to, polypeptides whose sequences are disclosed in U.S. Pat. No. 6,004,815, and in GenBank® under Acc. Nos. NP_463733.1, NP_979614, NP_834769, YP_084586, YP_895748, YP_694620, YP_012823, NP_346351, YP_597752, BAB41212.2, NP_561079.1, YP 001198769, and NP_359331.1.
[0094] Cytoplasmic cytolysin polypeptide'. A “cytoplasmic cytolysin polypeptide” is a cytolysin polypeptide that has the ability to form pores in a membrane of a eukaryotic cell, and that is expressed as a cytoplasmic polypeptide in a bacterial cell. A cytoplasmic cytolysin polypeptide is not significantly secreted by a bacterial cell. Cytoplasmic cytolysin polypeptides can be provided by a variety of means. In some embodiments, a cytoplasmic cytolysin polypeptide is provided as a nucleic acid encoding the cytoplasmic ccytolysin polypeptide. In some embodiments, a cytoplasmic cytolysin polypeptide is provided attached to a bead. In some embodiments, a cytoplasmic cytolysin polypeptide has a sequence that is altered relative to the sequence of a secreted cytolysin polypeptide (e.g., altered by deletion or alteration of a signal sequence to render it nonfunctional). In some embodiments, a cytoplasmic cytolysin polypeptide is cytoplasmic because it is expressed in a secretion-incompetent cell. In some embodiments, a cytoplasmic cytolysin polypeptide is cytoplasmic because it is expressed in a cell that does not recognize and mediate secretion of a signal sequence linked to the cytolysin polypeptide. In some embodiments, a cytoplasmic cytolysin polypeptide is a bacterial cytolysin polypeptide.
[0095] Heterologous'. The term “heterologous”, as used herein to refer to genes or polypeptides, refers to a gene or polypeptide that does not naturally occur in the organism in which it is present and/or being expressed, and/or that has been introduced into the organism by the hand of man. In some embodiments, a heterologous polypeptide is a tumor antigen described herein.
[0096] Immune mediator'. As used herein, the term “immune mediator” refers to any molecule that affects the cells and processes involved in immune responses. Immune mediators include cytokines, chemokines, soluble proteins, and cell surface markers.
[0097] Improve, increase, inhibit, stimulate, suppress, or reduce'. As used herein, the terms “improve”, “increase”, “inhibit”, “stimulate”, “suppress”, “reduce”, or grammatical equivalents thereof, indicate values that are relative to a baseline or other reference measurement. In some embodiments, an appropriate reference measurement may be or comprise a measurement in a particular system (e.g., in a single individual) under otherwise comparable conditions absent presence of (e.g., prior to and/or after) a particular agent or treatment, or in presence of an appropriate comparable reference agent. The effect of a particular agent or treatment may be direct or indirect. In some embodiments, an appropriate reference measurement may be or may comprise a measurement in a comparable system known or expected to respond in a particular way, in presence of the relevant agent or treatment. In some embodiments, a peptide presented by an antigen presenting cell (APC) “stimulates” or is “stimulatory” to a lymphocyte if the lymphocyte is activated to a phenotype associated with beneficial responses, after exposure to the peptide presented by the APC under conditions that permit antigen-specific recognition to occur, as observed by, e.g., T cell proliferation, phosphorylation or dephosphorylation of a receptor, calcium flux, cytoskeletal rearrangement, increased or decreased expression and/or secretion of immune mediators such as cytokines or soluble mediators, increased or decreased expression of one or more cell surface markers, relative to a control. In some embodiments, a peptide presented by an antigen presenting cell “suppresses”, “inhibits” or is “inhibitory” to a lymphocyte if the lymphocyte is activated to a phenotype associated with deleterious or non-beneficial responses, after exposure to the peptide presented by the APC under conditions that permit antigen-specific recognition to occur, as observed by, e.g., phosphorylation or dephosphorylation of a receptor, calcium flux, cytoskeletal rearrangement, increased or decreased expression and/or secretion of immune mediators such as cytokines or soluble mediators, increased or decreased expression of one or more cell surface markers, relative to a control.
[0098] Inhibitory Antigen: An “inhibitory antigen” is an antigen that inhibits, suppresses, impairs and/or reduces immune control of a tumor or cancer. In some embodiments, an inhibitory antigen promotes tumor growth, enables tumor growth, increases and/or enables tumor metastasis, and/or accelerates tumor growth. In some embodiments, an inhibitory antigen stimulates one or more lymphocyte responses that are deleterious or non-beneficial to a subject; and/or inhibits and/or suppresses one or more lymphocyte responses that are beneficial to a subject. In some embodiments, an inhibitory antigen is the target of one or more lymphocyte responses that are deleterious or non-beneficial to a subject; and/or inhibits and/or suppresses one or more lymphocyte responses that are beneficial to a subject.
[0099] Invasin polypeptide'. An “invasin polypeptide” is a polypeptide that facilitates or mediates uptake of a cell (e.g., a bacterial cell) by a eukaryotic cell. Expression of an invasin polypeptide in a noninvasive bacterial cell confers on the cell the ability to enter a eukaryotic cell. In some embodiments, an invasin polypeptide is a bacterial invasin polypeptide. In some embodiments, an invasin polypeptide is a Yersinia invasin polypeptide (e.g., a Yersinia invasin polypeptide comprising a sequence disclosed in GenBank® under Acc. No. YP 070195.1). [0100] Listeriolysin O (LLO)'. The terms “listeriolysin O” or “LLO” refer to a listeriolysin O polypeptide of Listeria monocytogenes and truncated forms thereof that retain pore-forming ability (e.g., cytoplasmic forms of LLO, including truncated forms lacking a signal sequence). In some embodiments, an LLO is a cytoplasmic LLO. Exemplary LLO sequences are shown in Table 1, below.
[0101] Polypeptide'. The term “polypeptide”, as used herein, generally has its art- recognized meaning of a polymer of at least three amino acids. Those of ordinary skill in the art will appreciate, however, that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having the complete sequence recited herein (or in a reference or database specifically mentioned herein), but also to encompass polypeptides that represent functional fragments (/.< ., fragments retaining at least one activity) and immunogenic fragments of such complete polypeptides. Moreover, those of ordinary skill in the art understand that protein sequences generally tolerate some substitution without destroying activity. Thus, any polypeptide that retains activity and shares at least about 30-40% overall sequence identity, often greater than about 50%, 60%, 70%, or 80%, and further usually including at least one region of much higher identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99% in one or more highly conserved regions, usually encompassing at least 3-4 and often up to 20 or more amino acids, with another polypeptide of the same class, is encompassed within the relevant term “polypeptide” as used herein. Other regions of similarity and/or identity can be determined by those of ordinary skill in the art by analysis of the sequences of various polypeptides.
[0102] Primary cells'. As used herein, “primary cells” refers to cells from an organism that have not been immortalized in vitro. In some embodiments, primary cells are cells taken directly from a subject (e.g., a human). In some embodiments, primary cells are progeny of cells taken from a subject (e.g., cells that have been passaged in vitro). Primary cells include cells that have been stimulated to proliferate in culture.
[0103] Response: As used herein, in the context of a subject (a patient or experimental organism), “response”, “responsive”, or “responsiveness” refers to an alteration in a subject’s condition that occurs as a result of, or correlates with, treatment. In certain embodiments, a response is a beneficial response. In certain embodiments, a beneficial response can include stabilization of a subject’s condition (e.g., prevention or delay of deterioration expected or typically observed to occur absent the treatment), amelioration (e.g., reduction in frequency and/or intensity) of one or more symptoms of the condition, and/or improvement in the prospects for cure of the condition, etc. In certain embodiments, for a subject who has cancer, a beneficial response can include: the subject has a positive clinical response to cancer therapy or a combination of therapies; the subject has a spontaneous response to a cancer; the subject is in partial or complete remission from cancer; the subject has cleared a cancer; the subject has not had a relapse, recurrence or metastasis of a cancer; the subject has a positive cancer prognosis; the subject has not experienced toxic responses or side effects to a cancer therapy or combination of therapies. In certain embodiments, for a subject who had cancer, the beneficial responses occurred in the past, or are ongoing.
[0104] In certain embodiments, a response is a deleterious or non-beneficial response. In certain embodiments, a deleterious or non-beneficial response can include deterioration of a subject’s condition, lack of amelioration (e.g., no reduction in frequency and/or intensity) of one or more symptoms of the condition, and/or degradation in the prospects for cure of the condition, etc. In certain embodiments, for a subject who has cancer, a deleterious or non-beneficial response can include: the subject has a negative clinical response to cancer therapy or a combination of therapies; the subject is not in remission from cancer; the subject has not cleared a cancer; the subject has had a relapse, recurrence or metastasis of a cancer; the subject has a negative cancer prognosis; the subject has experienced toxic responses or side effects to a cancer therapy or combination of therapies. In certain embodiments, for a subject who had cancer, the deleterious or non-beneficial responses occurred in the past, or are ongoing.
[0105] As used herein, in the context of a cell, organ, tissue, or cell component, e.g., a lymphocyte, “response”, “responsive”, or “responsiveness” refers to an alteration in cellular activity that occurs as a result of, or correlates with, administration of or exposure to an agent, e.g. a tumor antigen. In certain embodiments, a beneficial response can include increased expression and/or secretion of immune mediators associated with positive clinical responses or outcomes in a subject. In certain embodiments, a beneficial response can include decreased expression and/or secretion of immune mediators associated with negative clinical response or outcomes in a subject. In certain embodiments, a deleterious or non-beneficial response can include increased expression and/or secretion of immune mediators associated with negative clinical responses or outcomes in a subject. In certain embodiments, a deleterious or non- beneficial response can include decreased expression and/or secretion of immune mediators associated with positive clinical responses or outcomes in a subject. In certain embodiments, a response is a clinical response. In certain embodiments, a response is a cellular response. In certain embodiments, a response is a direct response. In certain embodiments, a response is an indirect response. In certain embodiments, “non-response”, “non-responsive”, or “nonresponsiveness” mean minimal response or no detectable response. In certain embodiments, a “minimal response” includes no detectable response. In certain embodiments, presence, extent, and/or nature of response can be measured and/or characterized according to particular criteria. In certain embodiments, such criteria can include clinical criteria and/or objective criteria. In certain embodiments, techniques for assessing response can include, but are not limited to, clinical examination, positron emission tomography, chest X-ray, CT scan, MRI, ultrasound, endoscopy, laparoscopy, presence or level of a particular marker in a sample, cytology, and/or histology. Where a response of interest is a response of a tumor to a therapy, ones skilled in the art will be aware of a variety of established techniques for assessing such response, including, for example, for determining tumor burden, tumor size, tumor stage, etc. Methods and guidelines for assessing response to treatment are discussed in Therasse et al., J. Natl. Cancer Inst., 2000, 92(3):205-216; and Seymour et al., Lancet Oncol., 2017, 18:el43-52. The exact response criteria can be selected in any appropriate manner, provided that when comparing groups of tumors, patients or experimental organism, and/or cells, organs, tissues, or cell components, the groups to be compared are assessed based on the same or comparable criteria for determining response rate. One of ordinary skill in the art will be able to select appropriate criteria.
[0106] Stimulatory Antigen'. A “stimulatory antigen” is an antigen that improves, increases and/or stimulates immune control of a tumor or cancer. In some embodiments, a stimulatory antigen is the target of an immune response that reduces, kills, shrinks, resorbs, and/or eradicates tumor growth; does not enable tumor growth; decreases tumor metastasis, and/or decelerates tumor growth. In some embodiments, a stimulatory antigen inhibits and/or suppresses one or more lymphocyte responses that are deleterious or non-beneficial to a subject; and/or stimulates one or more lymphocyte responses that are beneficial to a subject.
[0107] Tumor'. As used herein, the term “tumor” refers to an abnormal growth of cells or tissue. In some embodiments, a tumor may comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic. In some embodiments, a tumor is associated with, or is a manifestation of, a cancer. In some embodiments, a tumor may be a disperse tumor or a liquid tumor. In some embodiments, a tumor may be a solid tumor.
DETAILED DESCRIPTION
[0108] Recent advances in immune checkpoint inhibitor (CPI) therapies such as ipilimumab, nivolumab, and pembrolizumab for cancer immunotherapy have resulted in dramatic efficacy in subjects suffering from NSCLC, among other indications. Nivolumab and pembroluzimab have been approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) for use in patients with advanced NSCLC who have previously been treated with chemotherapy. They have solidified the importance of T cell responses in control of tumors. Neoantigens, potential cancer rejection antigens that are entirely absent from the normal human genome, are postulated to be relevant to tumor control; however, attempts to define them and their role in tumor clearance has been hindered by the paucity of available tools to define them in a biologically relevant and unbiased way (Schumacher and Schreiber, 2015 Science 348:69-74, Gilchuk et al., 2015 Curr Opin Immunol 34:43-51)
[0109] Taking non-small cell lung carcinoma (NSCLC) as an example, whole exome sequencing of NSCLC tumors from patients treated with pembrolizumab showed that higher non-synonymous mutation burden in tumors was associated with improved objective response, durable clinical benefit, and progression-free survival (Rizvi et al., (2015) Science 348(6230): 124-8). In this study, the median non-synonymous mutational burden of the discovery cohort was 209 and of the validation cohort was 200. However, simply because a mutation was identified by sequencing, does not mean that the epitope it creates can be recognized by a T cell or serves as a protective antigen for T cell responses (Gilchuk et al., 2015 Curr Opin Immunol 34:43-51 making the use of the word neoantigen somewhat of a misnomer. With 200 or more potential targets of T cells in NSCLC, it is not feasible to test every predicted epitope to determine which of the mutations serve as neoantigens, and which neoantigens are associated with clinical evidence of tumor control. Recently, a study by McGranahan et al., showed that clonal neoantigen burden and overall survival in primary lung adenocarcinomas are related. However, even enriching for clonal neoantigens results in potential antigen targets ranging from 50 to approximately 400 (McGranahan et al., 2016 Science 351 : 1463-69). Similar findings have been described for melanoma patients who have responded to ipilimumab therapy (Snyder et al., 2015 NEJM; Van Allen et al., 2015 Science) and in patients with mismatch-repair deficient colorectal cancer who were treated with pembrolizumab (Le et al., 2015 NEJM).
[0110] In well-established tumors, activation of endogenous anti-tumor T cell responses is often insufficient to result in complete tumor regression. Moreover, T cells that have been educated in the context of the tumor microenvironment sometimes are sub-optimally activated, have low avidity, and ultimately fail to recognize the tumor cells that express antigen. In addition, tumors are complex and comprise numerous cell types with varying degrees of expression of mutated genes, making it difficult to generate polyclonal T cell responses that are adequate to control tumor growth. As a result, researchers in the field have proposed that it is important in cancer subjects to identify the mutations that are “potential tumor antigens” in addition to those that are confirmed in the cancer subject to be recognized by their T cells.
[OHl] There are currently no reliable methods of identifying potential tumor antigens in a comprehensive way. Computational methods have been developed in an attempt to predict what is an antigen, however there are many limitations to these approaches. First, modeling epitope prediction and presentation needs to take into account the greater than 12,000 HLA alleles encoding MHC molecules, with each subject expressing as many as 14 of them, all with different epitope affinities. Second, the vast majority of predicted epitopes fail to be found presented by tumors when they are evaluated using mass spectrometry. Third, the predictive algorithms do not take into account T cell recognition of the antigen, and the majority of predicted epitopes are incapable of eliciting T cell responses even when they are present. Finally, the second arm of cellular immunity, the CD4+ T cell subset, is often overlooked; the majority of in silico tools focus on MHC class I binders. The tools for predicting MHC class II epitopes are under-developed and more variable.
[0112] Cancer immune therapies boost immune responses, mainly T cell responses, to kill cancer cells while sparing normal cells. The success of checkpoint blockade immunotherapies in producing durable remission in a significant subset of cancer patients has reinforced that immunotherapeutic interventions can result in tumor control. Additionally, they have demonstrated the importance of tumor reactive T cells in antitumor efficacy. Despite significant progress, however, checkpoint inhibitor therapy is effective in only 20%-30% of treated patients. Therefore, there remains a large unmet need for safe and effective immune therapies that might be applicable to a broader range of tumor types.
[0113] A hallmark of tumorigenesis is the accumulation of mutations in cancer cells. These mutations are found as both driver and passenger events and they are exclusively present in tumor but not in normal tissue. The mutated protein fragments that are presented by the peptide human leukocyte antigen (pHLA) complexes on the cell surface and recognizable by the immune system are known as neoantigens. Neoantigens may induce reactive T cells that can mediate the killing of cancer cells by the host immune system (1, 2). There is a substantial body of evidence supporting a critical role for neoantigens in anti tumor control by marking the cancer cells as non-self, which leads to immune system targeting for destruction:
• Neoantigens represent dominant targets in tumor-infiltrating lymphocyte populations in patients benefiting from adoptive T cell therapy, and a neoantigen specific T cell population was sufficient to induce tumor regression in mouse and man (3, 4).
• The widespread detection of spontaneously occurring neoantigen-specific T cells demonstrates that processing and presentation of multiple neoantigens on tumors occurs despite the current insensitivity of biochemical detection (5-7) • Checkpoint blockade therapy has revealed new and amplified neoantigen-specific T cell responses which, in the mouse, are central to disease control (7, 8).
• A retrospective meta-analysis of 6 tumor types showed that overall survival was improved in patients predicted to have at least 1 immunogenic neoantigen epitope (9).
• Memory cytotoxic T lymphocyte responses to mutated antigens are generated in patients with unexpected long-term survival or those who have undergone effective immunotherapy (10, 11).
• A neoantigen-specific CD4 T cell product caused regression of a metastatic cholangiocarcinoma (12).
[0114] Because of the tumor exclusivity of neoantigens, they can serve as tumor-specific targets for T cell-mediated recognition and destruction of tumor cells. Cancer vaccines targeting neoantigens are expected to be effective in activating T cells that can recognize and kill tumors. Several clinical trials have been initiated to directly test neoantigen vaccines. Most solid tumors harbor over 100 non-synonymous mutations; however, not all mutated proteins are processed and presented to T cells by pHLA. So far, most neoantigen vaccines depend on algorithms to identify which mutations detected in tumors are the appropriate neoantigens for inclusion in the vaccine. The present disclosure provides methods and systems for the rapid identification of tumor antigens (e.g., tumor specific antigens (TSAs, or neoantigens), tumor associated antigens (TAAs), or cancer/testis antigens (CTAs)) that elicit T cell responses and particularly that elicit human T cell responses, as well as polypeptides that are potential tumor antigens. For purposes of this disclosure, “tumor antigens” includes both tumor antigens and potential tumor antigens. As described herein, methods of the present disclosure identified stimulatory tumor antigens that were not identified by known algorithms. Further, methods of the present disclosure identified suppressive and/or inhibitory tumor antigens that are not identifiable by known algorithms.
Methods of the present disclosure also identified polypeptides that are potential tumor antigens, z.e., polypeptides that activate T cells of non-cancerous subjects, but not T cells of subjects suffering from cancer. The present disclosure also provides methods of selecting tumor antigens and potential tumor antigens, methods of using the selected tumor antigens and potential tumor antigens, immunogenic compositions comprising the selected tumor antigens and potential tumor antigens, and methods of manufacturing immunogenic compositions.
[0115] The present disclosure further provides methods for identifying stimulatory and/or inhibitory antigens in a particular subject suffering from cancer. Generally, potential tumor antigens may be identified from a tumor sample from the subject; a library of bacterial cells or beads comprising a plurality of tumor antigens may be generated, where each bacterial cell or bead of the library comprises a different tumor antigen; APCs from the patient can then be contacted with and internatize the bacterial cells or beads. The subject’s T cells are then exposed to APCs expressing the potential antigens. Stimulatory and inhibitory antigens may then be identified based on the measuring T cell response to the different antigens.
[0116] The present disclosure also provides methods for monitoring disease progression, and effectiveness or suitability of therapies in a particular subject suffering from cancer, based on comparison of stimulatory and/or inhibitory antigen profiles obtained at different times. Also provided are methods for selecting a cancer therapy for a subject, and methods of treating cancer in a subject.
Library generation
[0117] A library is a collection of members (e.g., cells or non-cellular particles, such as virus particles, liposomes, or beads (e.g., beads coated with polypeptides, such as in vitro translated polypeptides, e.g., affinity beads, e.g., antibody coated beads, or NTA-Ni beads bound to polypeptides of interest). According to the present disclosure, members of a library include (e.g., internally express or carry) polypeptides of interest described herein. In some embodiments, members of a library are cells that internally express polypeptides of interest described herein. In some embodiments, members of a library which are particles carry, and/or are bound to, polypeptides of interest. Use of a library in an assay system allows simultaneous evaluation in vitro of cellular responses to multiple candidate antigens. According to the present disclosure, a library is designed to be internalized by human antigen presenting cells so that peptides from library members, including peptides from internally expressed polypeptides of interest, are presented on MHC molecules of the antigen presenting cells for recognition by T cells.
[0118] Libraries can be used in assays that detect peptides presented by human MHC class I and MHC class II molecules. Polypeptides expressed by the internalized library members are digested in intracellular endocytic compartments (e.g., phagosomes, endosomes, lysosomes) of the human cells and presented on MHC class II molecules, which are recognized by human CD4+ T cells. In some embodiments, library members include a cytolysin polypeptide, in addition to a polypeptide of interest. In some embodiments, library members include an invasin polypeptide, in addition to the polypeptide of interest. In some embodiments, library members include an autolysin polypeptide, in addition to the polypeptide of interest. In some embodiments, library members are provided with cells that express a cytolysin polypeptide (i.e., the cytolysin and polypeptide of interest are not expressed in the same cell, and an antigen presenting cell is exposed to members that include the cytolysin and members that include the polypeptide of interest, such that the antigen presenting cell internalizes both, and such that the cytolysin facilitates delivery of polypeptides of interest to the MHC class I pathway of the antigen presenting cell). A cytolysin polypeptide can be constitutively expressed in a cell, or it can be under the control of an inducible expression system (e.g., an inducible promoter). In some embodiments, a cytolysin is expressed under the control of an inducible promoter to minimize cytotoxicity to the cell that expresses the cytolysin.
[0119] Once internalized by a human cell, a cytolysin polypeptide perforates intracellular compartments in the human cell, allowing polypeptides expressed by the library members to gain access to the cytosol of the human cell. Polypeptides released into the cytosol are presented on MHC class I molecules, which are recognized by CD8+ T cells.
[0120] A library can include any type of cell or particle that can be internalized by and deliver a polypeptide of interest (and a cytolysin polypeptide, in applications where a cytolysin polypeptide is desirable) to, antigen presenting cells for use in methods described herein. Although the term “cell” is used throughout the present specification to refer to a library member, it is understood that, in some embodiments, the library member is a non-cellular particle, such as a virus particle, liposome, or bead. In some embodiments, members of the library include polynucleotides that encode the polypeptide of interest (and cytolysin polypeptide), and can be induced to express the polypeptide of interest (and cytolysin polypeptide) prior to, and/or during internalization by antigen presenting cells.
[0121] In some embodiments, the cytolysin polypeptide is heterologous to the library cell in which it is expressed, and facilitates delivery of polypeptides expressed by the library cell into the cytosol of a human cell that has internalized the library cell. Cytolysin polypeptides include bacterial cytolysin polypeptides, such as listeriolysin O (LLO), streptolysin O (SLO), and perfringolysin O (PFO). Additional cytolysin polypeptides are described in U.S. Pat. 6,004,815. In certain embodiments, library members express LLO. In some embodiments, a cytolysin polypeptide is not significantly secreted by the library cell (e.g., less than 20%, 10%, 5%, or 1% of the cytolysin polypeptide produced by the cell is secreted). For example, the cytolysin polypeptide is a cytoplasmic cytolysin polypeptide, such as a cytoplasmic LLO polypeptide (e.g., a form of LLO which lacks the N-terminal signal sequence, as described in Higgins et al., Mol. Microbiol. 31(6): 1631-1641, 1999). Exemplary cytolysin polypeptide sequences are shown in Table 1. The listeriolysin O (A3-25) sequence shown in the second row of Table 1 has a deletion of residues 3-25, relative to the LLO sequence in shown in the first row of Table 1, and is a cytoplasmic LLO polypeptide. In some embodiments, a cytolysin is expressed constitutively in a library host cell. In other embodiments, a cytolysin is expressed under the control of an inducible promoter. Cytolysin polypeptides can be expressed from the same vector, or from a different vector, as the polypeptide of interest in a library cell.
Table 1. Exemplary Cytolysin Polypeptides
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
[0122] In some embodiments, a library member (e.g., a library member which is a bacterial cell) includes an invasin that facilitates uptake by the antigen presenting cell. In some embodiments, a library member includes an autolysin that facilitates autolysis of the library member within the antigen presenting cell. In some embodiments, a library member includes both an invasin and an autolysin. In some embodiments, a library member which is an E. coll cell includes an invasin and/or an autolysin. In various embodiments, library cells that express an invasin and/or autolysin are used in methods that also employ non-professional antigen presenting cells or antigen presenting cells that are from cell lines. Isberg et al. (Cell, 1987, 50:769-778), Sizemore et al. (Science, 1995, 270:299-302) and Courvalin et al. (C.R. Acad. Sci. Paris, 1995, 318: 1207-12) describe expression of an invasin to effect endocytosis of bacteria by target cells. Autolysins are described by Cao et al., Infect. Immun. 1998, 66(6): 2984-2986; Margot et al., J. Bacteriol. 1998, 180(3): 749-752; Buist et al., Appl. Environ. Microbiol., 1997, 63(7):2722-2728; Yamanaka et al. , FEMS Microbiol. Lett., 1997, 150(2): 269-275; Romero et al., FEMS Microbiol. Lett., 1993, 108(l):87-92; Betzner and Keck, Mol. Gen. Genet., 1989, 219(3): 489-491; Lubitz et al., J. Bacteriol., 1984, 159(l):385-387; and Tomasz et al., J. Bacteriol., 1988, 170(12): 5931-5934. In some embodiments, an autolysin has a feature that permits delayed lysis, e.g., the autolysin is temperature-sensitive or time-sensitive (see, e.g., Chang et al., 1995, J. Bact. 177, 3283-3294; Raab et al., 1985, J. Mol. Biol. 19, 95-105; Gerds et al., 1995, Mol. Microbiol. 17, 205-210). Useful cytolysins also include addiction (poison/antidote) autolysins, (see, e.g., Magnuson R, et al., 1996, J. Biol. Chem. 271(31), 18705- 18710; Smith A S, et al., 991, Mol. Microbiol. 26(5), 961-970).
[0123] In some embodiments, members of the library include bacterial cells. In certain embodiments, the library includes non-pathogenic, non-virulent bacterial cells. Examples of bacteria for use as library members include E. coli, mycobacteria, Listeria monocytogenes, Shigella flexneri, Bacillus subtilis, or Salmonella.
[0124] In some embodiments, members of the library include eukaryotic cells (e.g., yeast cells). In some embodiments, members of the library include viruses (e.g., bacteriophages). In some embodiments, members of the library include liposomes. Methods for preparing liposomes that include a cytolysin and other agents are described in Kyung-Dall et al., U.S. Pat. No. 5,643,599. In some embodiments, members of the library include beads. Methods for preparing libraries comprised of beads are described, e.g., in Lam et al., Nature 354: 82-84, 1991, U.S. Pat. Nos. 5,510,240 and 7,262,269, and references cited therein.
[0125] In certain embodiments, a library is constructed by cloning polynucleotides encoding polypeptides of interest, or portions thereof, into vectors that express the polypeptides of interest in cells of the library. The polynucleotides can be synthetically synthesized. The polynucleotides can be cloned by designing primers that amplify the polynucleotides. Primers can be designed using available software, such as Primer3Plus (available the following URL: bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi; see Rozen and Skaletsky, In: Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology. Humana Press, Totowa, NJ, pp. 365-386, 2000). Other methods for designing primers are known to those of skill in the art. In some embodiments, primers are constructed so as to produce polypeptides that are truncated, and/or lack hydrophobic regions (e.g., signal sequences or transmembrane regions) to promote efficient expression. The location of predicted signal sequences and predicted signal sequence cleavage sites in a given open reading frame (ORF) sequence can be determined using available software, see, e.g., Dyrlov et al, J. Mol. Biol., 340:783-795, 2004, and the following URL: cbs.dtu.dk/services/SignalP/). For example, if a signal sequence is predicted to occur at the N-terminal 20 amino acids of a given polypeptide sequence, a primer is designed to anneal to a coding sequence downstream of the nucleotides encoding the N-terminal 20 amino acids, such that the amplified sequence encodes a product lacking this signal sequence.
[0126] Primers can also be designed to include sequences that facilitate subsequent cloning steps. ORFs can be amplified directly from genomic DNA (e.g., genomic DNA of a tumor cell), or from polynucleotides produced by reverse transcription (RT-PCR) of mRNAs expressed by the tumor cell. RT-PCR of mRNA is useful, e.g., when the genomic sequence of interest contains intronic regions. PCR-amplified ORFs are cloned into an appropriate vector, and size, sequence, and expression of ORFs can be verified prior to use in immunological assays.
[0127] In some embodiments, a polynucleotide encoding a polypeptide of interest is linked to a sequence encoding a tag (e.g., an N-terminal or C-terminal epitope tag) or a reporter protein (e.g., a fluorescent protein). Epitope tags and reporter proteins facilitate purification of expressed polypeptides, and can allow one to verify that a given polypeptide is properly expressed in a library host cell, e.g., prior to using the cell in a screen. Useful epitope tags include, for example, a polyhistidine (His) tag, a V5 epitope tag from the P and V protein of paramyxovirus, a hemagglutinin (HA) tag, a myc tag, and others. In some embodiments, a polynucleotide encoding a polypeptide of interest is fused to a sequence encoding a tag which is a known antigenic epitope (e.g., an MHC class I- and/or MHC class Il-restricted T cell epitope of a model antigen such as an ovalbumin), and which can be used to verify that a polypeptide of interest is expressed and that the polypeptide-tag fusion protein is processed and presented in antigen presentation assays. In some embodiments a tag includes a T cell epitope of a murine T cell (e.g., a murine T cell line). In some embodiments, a polynucleotide encoding a polypeptide of interest is linked to a tag that facilitates purification and a tag that is a known antigenic epitope. Useful reporter proteins include naturally occurring fluorescent proteins and their derivatives, for example, Green Fluorescent Protein (Aequorea Victoria) and Neon Green (Branchiostoma lanceolatum). Panels of synthetically derived fluorescent and chromogenic proteins are also available from commercial sources. [0128] Polynucleotides encoding a polypeptide of interest are cloned into an expression vector for introduction into library host cells. Various vector systems are available to facilitate cloning and manipulation of polynucleotides, such as the Gateway® Cloning system (Invitrogen). As is known to those of skill in the art, expression vectors include elements that drive production of polypeptides of interest encoded by a polynucleotide in library host cells (e.g., promoter and other regulatory elements). In some embodiments, polypeptide expression is controlled by an inducible element (e.g., an inducible promoter, e.g., an IPTG- or arabinose- inducible promoter, or an IPTG-inducible phage T7 RNA polymerase system, a lactose (lac) promoter, a tryptophan (trp) promoter, a tac promoter, a trc promoter, a phage lambda promoter, an alkaline phosphatase (phoA) promoter, to give just a few examples; see Cantrell, Meth, in Mol. Biol., 23525 -2 6, Humana Press, Casali and Preston, Eds.). In some embodiments, polypeptides are expressed as cytoplasmic polypeptides. In some embodiments, the vector used for polypeptide expression is a vector that has a high copy number in a library host cell. In some embodiments, the vector used for expression has a copy number that is more than 25, 50, 75, 100, 150, 200, or 250 copies per cell. In some embodiments, the vector used for expression has a ColEl origin of replication. Useful vectors for polypeptide expression in bacteria include pET vectors (Novagen), Gateway® pDEST vectors (Invitrogen), pGEX vectors (Amersham Biosciences), pPRO vectors (BD Biosciences), pB AD vectors (Invitrogen), pLEX vectors (Invitrogen), pMAL™ vectors (New England BioLabs), pGEMEX vectors (Promega), and pQE vectors (Qiagen). Vector systems for producing phage libraries are known and include Novagen T7Select® vectors, and New England Biolabs Ph D.™ Peptide Display Cloning System.
[0129] In some embodiments, library host cells express (either constitutively, or when induced, depending on the selected expression system) a polypeptide of interest to at least 10%, 20%, 30%, 40%, 50%, 60%, or 70% of the total cellular protein. In some embodiments, the level a polypeptide available in or on a library member (e.g., cell, virus particle, liposome, bead) is such that antigen presenting cells exposed to a sufficient quantity of the library members are presented on MHC molecules polypeptide epitopes at a density that is comparable to the density presented by antigen presenting cells pulsed with purified peptides. [0130] Methods for efficient, large-scale production of libraries are available. For example, site-specific recombinases or rare-cutting restriction enzymes can be used to transfer polynucleotides between expression vectors in the proper orientation and reading frame (Walhout et al., Meth. EnzymoL 328:575-592, 2000; Marsischky et al., Genome Res. 14:2020-202, 2004; Blommel et al., Protein Expr. Purif 47:562-570, 2006).
[0131] For production of liposome libraries, expressed polypeptides (e.g., purified or partially purified polypeptides) can be entrapped in liposomal membranes, e.g., as described in Wassef et al., U.S. Pat. No. 4,863,874; Wheatley et al., U.S. Pat. No. 4,921,757; Huang et al., U.S. Pat. No. 4,925,661; or Martin et al., U.S. Pat. No. 5,225,212.
[0132] A library can be designed to include full length polypeptides and/or portions of polypeptides. Expression of full length polypeptides maximizes epitopes available for presentation by a human antigen presenting cell, thereby increasing the likelihood of identifying an antigen. However, in some embodiments, it is useful to express portions of polypeptides, or polypeptides that are otherwise altered, to achieve efficient expression. For example, in some embodiments, polynucleotides encoding polypeptides that are large (e.g., greater than 1,000 amino acids), that have extended hydrophobic regions, signal peptides, transmembrane domains, or domains that cause cellular toxicity, are modified (e.g., by C-terminal truncation, N-terminal truncation, or internal deletion) to reduce cytotoxicity and permit efficient expression a library cell, which in turn facilitates presentation of the encoded polypeptides on human cells. Other types of modifications, such as point mutations or codon optimization, may also be used to enhance expression.
[0133] The number of polypeptides included in a library can be varied. For example, in some embodiments, a library can be designed to express polypeptides from at least 5%, 10%, 15%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of ORFs in a target cell (e.g., tumor cell). In some embodiments, a library expresses at least 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 2500, 5000, 10,000, or more different polypeptides of interest, each of which may represent a polypeptide encoded by a single full length polynucleotide or portion thereof.
[0134] In some embodiments, assays may focus on identifying antigens that are secreted polypeptides, cell surface-expressed polypeptides, or virulence determinants, e.g., to identify antigens that are likely to be targets of both humoral and cell mediated immune responses.
[0135] In addition to polypeptides of interest, libraries can include tags or reporter proteins that allow one to easily purify, analyze, or evaluate MHC presentation, of the polypeptide of interest. In some embodiments, polypeptides expressed by a library include C- terminal tags that include both an MHC class I and an MHC class Il-restricted T cell epitope from a model antigen, such as chicken ovalbumin (OVA). Library protein expression and MHC presentation is validated using these epitopes. In some embodiments, the epitopes are OVA247- 265 and OVA258-265 respectfully, corresponding to positions in the amino acid sequence found in GenBank® under Acc. No. NP 990483. Expression and presentation of linked ORFs can be verified with antigen presentation assays using T cell hybridomas (e.g., B3Z T hybridoma cells, which are H2-Kb restricted, and KZO T hybridoma cells, which are H2-Ak restricted) that specifically recognize these epitopes.
[0136] Sets of library members (e.g., bacterial cells) can be provided on an array (e.g., on a solid support, such as a 96-well plate) and separated such that members in each location express a different polypeptide of interest, or a different set of polypeptides of interest.
[0137] Methods of using library members for identifying T cell antigens are described in detail below. In addition to these methods, library members also have utility in assays to identify B cell antigens. For example, lysate prepared from library members that include polypeptides of interest can be used to screen a sample comprising antibodies (e.g, a serum sample) from a subject (e.g, a subject who has been exposed to an infectious agent of interest, a subject who has cancer, and/or a control subject), to determine whether antibodies present in the subject react with the polypeptide of interest. Suitable methods for evaluating antibody reactivity are known and include, e.g., ELISA assays. Polypeptides of Interest
[0138] In some embodiments, methods and compositions described herein can be used to identify and/or detect immune responses to a polypeptide of interest. In some embodiments, a polypeptide of interest is encoded by an ORF from a target tumor cell, and members of a library include (e.g., internally express or carry) ORFs from a target tumor cell. In some such embodiments, a library can be used in methods described herein to assess immune responses to one or more polypeptides of interest encoded by one or more ORFs. In some embodiments, methods of the disclosure identify one or more polypeptides of interest as stimulatory antigens (e.g., that stimulate an immune response, e.g., a T cell response, e.g., expression and/or secretion of one or more immune mediators). In some embodiments, methods of the disclosure identify one or more polypeptides of interest as antigens or potential antigens that have minimal or no effect on an immune response (e.g., expression and/or secretion of one or more immune mediators). In some embodiments, methods of the disclosure identify one or more polypeptides of interest as inhibitory and/or suppressive antigens (e.g., that inhibit, suppress, down-regulate, impair, and/or prevent an immune response, e.g., a T cell response, e.g., expression and/or secretion of one or more immune mediators). In some embodiments, methods of the disclosure identify one or more polypeptides of interest as tumor antigens or potential tumor antigens, e.g., tumor specific antigens (TSAs, or neoantigens), tumor associated antigens (TAAs), or cancer/testis antigens (CTAs).
[0139] In some embodiments, a polypeptide of interest is a putative tumor antigen, and methods and compositions described herein can be used to identify and/or detect immune responses to one or more putative tumor antigens. For example, members of a library include (e.g., internally express or carry) putative tumor antigens (e.g., a polypeptide previously identified (e.g., by a third party) as a tumor antigen, e.g., identified as a tumor antigen using a method other than a method of the present disclosure). In some embodiments, a putative tumor antigen is a tumor antigen described herein. In some such embodiments, such libraries can be used to assess whether and/or the extent to which such putative tumor antigen mediates an immune response. In some embodiments, methods of the disclosure identify one or more putative tumor antigens as stimulatory antigens. In some embodiments, methods of the disclosure identify one or more putative tumor antigens as antigens that have minimal or no effect on an immune response. In some embodiments, methods of the disclosure identify one or more putative tumor antigens as inhibitory and/or suppressive antigens.
[0140] In some embodiments, a polypeptide of interest is a pre-selected tumor antigen, and methods and compositions described herein can be used to identify and/or detect immune responses to one or more pre-selected tumor antigens. For example, in some embodiments, members of a library include (e.g., internally express or carry) one or more polypeptides identified as tumor antigens using a method of the present disclosure and/or using a method other than a method of the present disclosure. In some such embodiments, such libraries can be used to assess whether and/or the extent to which such tumor antigens mediate an immune response by an immune cell from one or more subjects (e.g., a subject who has cancer and/or a control subject) to obtain one or more response profiles described herein. In some embodiments, methods of the disclosure identify one or more pre-selected tumor antigens as stimulatory antigens for one or more subjects. In some embodiments, methods of the disclosure identify one or more pre-selected tumor antigens as antigens that have minimal or no effect on an immune response for one or more subjects. In some embodiments, methods of the disclosure identify one or more pre-selected tumor antigens as inhibitory and/or suppressive antigens for one or more subjects.
[0141] In some embodiments, a polypeptide of interest is a known tumor antigen, and methods and compositions described herein can be used to identify and/or detect immune responses to one or more known tumor antigens. For example, in some embodiments, members of a library include (e.g., internally express or carry) one or more polypeptides identified as a tumor antigen using a method of the present disclosure and/or using a method other than a method of the present disclosure. In some such embodiments, such libraries can be used to assess whether and/or the extent to which such tumor antigens mediate an immune response by an immune cell from one or more subjects (e.g., a subject who has cancer and/or a control subject) to obtain one or more response profiles described herein. In some embodiments, methods of the disclosure identify one or more known tumor antigens as stimulatory antigens for one or more subjects. In some embodiments, methods of the disclosure identify one or more known tumor antigens as antigens that have minimal or no effect on an immune response for one or more subjects. In some embodiments, methods of the disclosure identify one or more known tumor antigens as inhibitory and/or suppressive antigens for one or more subjects.
[0142] In some embodiments, a polypeptide of interest is a potential tumor antigen, and methods and compositions described herein can be used to identify and/or detect immune responses to one or more potential tumor antigens. For example, in some embodiments, members of a library include (e.g., internally express or carry) one or more polypeptides identified as being of interest, e.g., encoding mutations associated with a tumor, using a method of the present disclosure and/or using a method other than a method of the present disclosure. In some such embodiments, such libraries can be used to assess whether and/or the extent to which such polypeptides mediate an immune response by an immune cell from one or more subjects (e.g., a subject who has cancer and/or a control subject) to obtain one or more response profiles described herein. In some embodiments, methods of the disclosure identify one or more polypeptides as stimulatory antigens for one or more subjects. In some embodiments, methods of the disclosure identify one or more polypeptides as antigens that have minimal or no effect on an immune response for one or more subjects. In some embodiments, methods of the disclosure identify one or more polypeptides as inhibitory and/or suppressive antigens for one or more subjects.
Tumor Antigens
[0143] Polypeptides of interest used in methods and systems described herein include tumor antigens and potential tumor antigens, e.g., tumor specific antigens (TSAs, or neoantigens), tumor associated antigens (TAAs), and/or cancer/testis antigens (CTAs). Exemplary tumor antigens include, e.g., MART-l/MelanA (MART -I or MLANA), gplOO (Pmel 17 or SILV), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3 (also known as HIP8), BAGE, GAGE-1, GAGE-2, pl 5, Calcitonin, Calretinin, Carcinoembryonic antigen (CEA), Chromogranin, Cytokeratin, Desmin, Epithelial membrane protein (EMA), Factor VIII, Glial fibrillary acidic protein (GFAP), Gross cystic disease fluid protein (GCDFP-15), HMB-45, Human chorionic gonadotropin (hCG), inhibin, lymphocyte marker, MART-1 (Melan-A), Myo DI, muscle-specific actin (MSA), neurofilament, neuron-specific enolase (NSE), placental alkaline phosphatase (PLAP), prostate-specific antigen, PTPRC (CD45), SI 00 protein, smooth muscle actin (SMA), synaptophysin, thyroglobulin, thyroid transcription factor- 1, Tumor M2 - PK, vimentin, p53, Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens (e.g., EBNA1), human papillomavirus (HPV) antigen E6 or E7 (HPV_E6 or HPV_E7), TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO-1 (also known as CTAG1B), erbB, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein (AFP), beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, MUC16, IL13Ra2, FRa, VEGFR2, Lewis Y, FAP, EphA2, CEACAM5, EGFR, CA6, CA9, GPNMB, EGP1, FOLR1, endothelial receptor, STEAP1, SLC44A4, Nectin-4, AGS- 16, guanalyl cyclase C, MUC-1, CFC1B, integrin alpha 3 chain (of a3bl, a laminin receptor chain), TPS, CD 19, CD20, CD22, CD30, CD31, CD72, CD 180, CD171 (LI CAM), CD 123, CD 133, CD138, CD37, CD70, CD79a, CD79b, CD56, CD74, CD166, CD71, CD34, CD99, CD117, CD80, CD28, CD13, CD15, CD25, CD10, CLL-1/CLEC12A, ROR1, Glypican 3 (GPC3), Mesothelin, CD33/IL3Ra, c-Met, PSCA, PSMA, Glycolipid F77, EGFRvIII, BCMA, GD-2, PSAP, prostein (also known as P501S), PSMA, Survivin (also known as BIRC5), and MAGE- A3, MAGEA2, MAGEA4, MAGEA6, MAGEA9, MAGEA10, MAGEA12, BIRC5, CDH3, CEACAM3, CGB_isoform2, ELK4, ERBB2, HPSE1, HPSE2, KRAS isoforml, KRAS_isoform2, MUC1, SMAD4, TERT, 2. TERT.3, TGFBR2, EGAG9_isoforml, TP53, CGB isoforml, IMPDH2, LCK, angiopoietin-1 (Angl) (also known as ANGPT1), XIAP (also known as BIRC4), galectin-3 (also known as LGALS3), VEGF-A (also known as VEGF), ATP6S1 (also known as ATP6AP1), MAGE- Al, cIAP-1 (also known as BIRC2), macrophage migration inhibitory factor (MIF), galectin-9 (also known as LGALS9), progranulin PGRN (also known as granulin), OGFR, MLIAP (also known as BIRC7), TBX4 (also known as ICPPS, SPS or T-Box4), secretory leukocyte protein inhibitor (Slpi) (also known as antileukoproteinase), Ang2 (also known as ANGPT2), galectin-1 (also known as LGALS1), TRP-2 (also known as DCT), hTERT (telomerase reverse transcriptase) tyrosinase-related protein 1 (TRP-1, TYRP1), NOR-90/UBF-2 (also known as UBTF), LGMN, SPA17, PRTN3, TRRAP l, TRRAP 2, TRRAP 3, TRRAP 4, MAGEC2, PRAME, SOXIO, RAC1, HRAS, GAGE4, AR, CYP1B1, MMP8, TYR, PDGFRB, KLK3, PAX3, PAX5, ST3GAL5, PLAC1, RhoC, MYCN, REG3A, CSAG2, CTAG2-la, CTAG2-lb, PAGE4, BRAF, GRM3, ERBB4, KIT, MAPK1, MFI2, SART3, ST8SIA1, WDR46, AKAP-4, RGS5, FOSL1, PRM2, ACRBP, CTCFL, CSPG4, CCNB1, MSLN, WT1, SSX2, KDR, ANKRD30A, MAGED1, MAP3K9, XAGE1B, PREX2, CD276, TEK, AIM1, ALK, FOLH1, GRIN2A MAP3K5 and one or more isoforms of any preceding tumor antigens. Exemplary tumor antigens are provided in the accompanying list of sequences.
[0144] Tumor specific antigens (TSAs, or neoantigens) are tumor antigens that are not encoded in normal host genome (see, e.g., Yarchoan et al., Nat. Rev. Cancer. 2017 Feb 24. doi: 10.1038/nrc.2016.154; Gubin et al., J. Clin. Invest. 125:3413-3421 (2015)). In some embodiments, TSAs arise from somatic mutations and/or other genetic alterations. In some embodiments, TSAs arise from missense or in-frame mutations. In some embodiments, TSAs arise from frame-shift mutations or loss-of-stop-codon mutations. In some embodiments, TSAs arise from insertion or deletion mutations. In some embodiments, TSAs arise from duplication or repeat expansion mutations. In some embodiments, TSAs arise from splice variants or improper splicing. In some embodiments, TSAs arise from gene fusions. In some embodiments, TSAs arise from translocations. In some embodiments, TSAs include oncogenic viral proteins. For example, as with Merkel cell carcinoma (MCC) associated with the Merkel cell polyomavirus (MCPy V) and cancers of the cervix, oropharynx and other sites associated with the human papillomavirus (HPV), TSAs include proteins encoded by viral open reading frames. For purposes of this disclosure, the terms “mutation” and “mutations” encompass all mutations and genetic alterations that may give rise to an antigen encoded in the genome of a cancer or tumor cell of a subject, but not in a normal or non-cancerous cell of the same subject. In some embodiments, TSAs are specific (personal) to a subject. In some embodiments, TSAs are shared by more than one subject, e.g., less than 1%, 1-3%, 1-5%, 1-10% , or more of subjects suffering from a cancer. In some embodiments, TSAs shared by more than one subject may be known or pre-selected.
[0145] In some embodiments, a TSA is encoded by an open reading frame from a virus. For example, a library can be designed to express polypeptides from one of the following viruses: an immunodeficiency virus (e.g., a human immunodeficiency virus (HIV), e.g., HIV-1, HIV-2), a hepatitis virus (e.g., hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis A virus, non-A and non-B hepatitis virus), a herpes virus (e.g., herpes simplex virus type I (HSV- 1), HSV-2, Varicella-zoster virus, Epstein Barr virus, human cytomegalovirus, human herpesvirus 6 (HHV-6), HHV-7, HHV-8), a poxvirus (e.g., variola, vaccinia, monkeypox, Molluscum contagiosum virus), an influenza virus, a human papilloma virus, adenovirus, rhinovirus, coronavirus, respiratory syncytial virus, rabies virus, coxsackie virus, human T cell leukemia virus (types I, II and III), parainfluenza virus, paramyxovirus, poliovirus, rotavirus, rhinovirus, rubella virus, measles virus, mumps virus, adenovirus, yellow fever virus, Norwalk virus, West Nile virus, a Dengue virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), bunyavirus, Ebola virus, Marburg virus, Eastern equine encephalitis virus, Venezuelan equine encephalitis virus, Japanese encephalitis virus, St. Louis encephalitis virus, Junin virus, Lassa virus, and Lymphocytic choriomeningitis virus. Libraries for other viruses can also be produced and used according to methods described herein.
[0146] Tumor specific antigens are known in the art, any of which can be used in methods described herein. In some embodiments, gene sequences encoding polypeptides that are potential or putative neoantigens are determined by sequencing the genome and/or exome of tumor tissue and healthy tissue from a subject having cancer using next generation sequencing technologies. In some embodiments, genes that are selected based on their frequency of mutation and ability to encode a potential or putative neoantigen are sequenced using next- generation sequencing technology. Next-generation sequencing applies to genome sequencing, genome resequencing, transcriptome profiling (RNA-Seq), DNA-protein interactions (ChlP- sequencing), and epigenome characterization (de Magalhaes et al. (2010) Ageing Research Reviews 9 (3): 315-323; Hall N (2007) J. Exp. Biol. 209 (Pt 9): 1518-1525; Church (2006) Sci. Am. 294 (1): 46-54; ten Bosch et al. (2008) Journal of Molecular Diagnostics 10 (6): 484-492; Tucker T et al. (2009) The American Journal of Human Genetics 85 (2): 142-154). Nextgeneration sequencing can be used to rapidly reveal the presence of discrete mutations such as coding mutations in individual tumors, e.g., single amino acid changes (e.g., missense mutations, in-frame mutations) or novel stretches of amino acids generated by frame-shift insertions, deletions, gene fusions, read-through mutations in stop codons, duplication or repeat expansion mutations, and translation of splice variants or improperly spliced introns, and translocations (e.g., “neoORFs”).
[0147] Another method for identifying potential or putative neoantigens is direct protein sequencing. Protein sequencing of enzymatic digests using multidimensional MS techniques (MSn) including tandem mass spectrometry (MS/MS)) can also be used to identify neoantigens. Such proteomic approaches can be used for rapid, highly automated analysis (see, e.g., Gevaert et al., Electrophoresis 21 : 1145-1154 (2000)). High-throughput methods for de novo sequencing of unknown proteins can also be used to analyze the proteome of a subject’s tumor to identify expressed potential or putative neoantigens. For example, meta shotgun protein sequencing may be used to identify expressed potential or putative neoantigens (see e.g., Guthals et al. (2012) Molecular and Cellular Proteomics 11(10): 1084-96).
[0148] Potential or putative neoantigens may also be identified using MHC multimers to identify neoantigen-specific T cell responses. For example, high-throughput analysis of neoantigen-specific T cell responses in patient samples may be performed using MHC tetramerbased screening techniques (see e.g., Hombrink et al. (2011) PLoS One; 6(8): e22523; Hadrup et al. (2009) Nature Methods, 6(7):520-26; van Rooij et al. (2013) Journal of Clinical Oncology, 31 : 1-4; and Heemskerk et al. (2013) EMBO Journal, 32(2): 194-203). [0149] In some embodiments, one or more known or pre-selected tumor specific antigens, or one or more potential or putative tumor specific antigens identified using one of these methods, can be included in a library described herein.
[0150] Tumor associated antigens (TAAs) include proteins encoded in a normal genome (see, e.g., Ward et al., Adv. Immunol. 130:25-74 (2016)). In some embodiments, TAAs are either normal differentiation antigens or aberrantly expressed normal proteins. Overexpressed normal proteins that possess growth/ survival -promoting functions, such as Wilms tumor 1 (WT1) (Ohminami et al., Blood 95:286-293 (2000)) or Her2/neu (Kawashima et al., Cancer Res. 59:431-435 (1999)), are TAAs that directly participate in the oncogenic process. Post- translational modifications, such as phosphorylation, of proteins may also lead to formation of TAAs (Doyle, J. Biol. Chem. 281 :32676-32683 (2006); Cobbold, Sci. Transl. Med. 5:203ral25 (2013)). TAAs are generally shared by more than one subject, e.g., less than 1%, 1-3%, 1-5%, 1- 10%, 1-20%, or more of subjects suffering from a cancer. In some embodiments, TAAs are known or pre-selected tumor antigens. In some embodiments, with respect to an individual subject, TAAs are potential or putative tumor antigens. Cancer/testis antigens (CTAs) are expressed by various tumor types and by reproductive tissues (for example, testes, fetal ovaries and trophoblasts) but have limited or no detectable expression in other normal tissues in the adult and are generally not presented on normal reproductive cells, because these tissues do not express MHC class I molecules (see, e.g., Coulie et al., Nat. Rev. Cancer 14: 135-146 (2014); Simpson et al., Nat. Rev. Cancer 5:615-625 (2005); Scanlan et al., Immunol. Rev. 188:22-32 (2002)).
Human Cells for Antigen Presentation
[0151] Methods of the present disclosure utilize human antigen presenting cells. Human antigen presenting cells express ligands for antigen receptors and other immune activation molecules on human lymphocytes. Given differences in MHC peptide binding specificities and antigen processing enzymes between species, antigens processed and presented by human cells are more likely to be physiologically relevant human antigens in vivo than antigens identified in non-human systems. Accordingly, methods of identifying these antigens employ human cells to present candidate tumor antigen polypeptides. Any human cell that internalizes library members and presents polypeptides expressed by the library members on MHC molecules can be used as an antigen presenting cell according to the present disclosure. In some embodiments, human cells used for antigen presentation are primary human cells. The cells can include peripheral blood mononuclear cells (PBMC) of a human. In some embodiments, peripheral blood cells are separated into subsets (e.g., subsets comprising dendritic cells, macrophages, monocytes, B cells, or combinations thereof) prior to use in an antigen presentation assay. In some embodiments, a subset of cells that expresses MHC class II is selected from peripheral blood. In one example, a cell population including dendritic cells is isolated from peripheral blood. In some embodiments, a subset of dendritic cells is isolated (e.g., plasmacytoid, myeloid, or a subset thereof). Human dendritic cell markers include CDlc, CDla, CD303, CD304, CD141, and CD209. Cells can be selected based on expression of one or more of these markers (e.g., cells that express CD303, CDlc, and CD141).
[0152] Dendritic cells can be isolated by positive selection from peripheral blood using commercially available kits (e.g., from Miltenyi Biotec Inc.). In some embodiments, the dendritic cells are expanded ex vivo prior to use in an assay. Dendritic cells can also be produced by culturing peripheral blood cells under conditions that promote differentiation of monocyte precursors into dendritic cells in vitro. These conditions typically include culturing the cells in the presence of cytokines such as GM-CSF and IL-4 (see, e.g., Inaba et al., Isolation of dendritic cells, Curr. Protoc. Immunol. May; Chapter 3: Unit 3.7, 2001). Procedures for in vitro expansion of hematopoietic stem and progenitor cells (e.g., taken from bone marrow or peripheral blood), and differentiation of these cells into dendritic cells in vitro, is described in U.S. Pat. No. 5,199,942, and U.S. Pat. Pub. 20030077263. Briefly, CD34+ hematopoietic stem and progenitor cells are isolated from peripheral blood or bone marrow and expanded in vitro in culture conditions that include one or more of Flt3-L, IL-1, IL-3, and c-kit ligand.
[0153] In some embodiments, immortalized cells that express human MHC molecules (e.g., human cells, or non-human cells that are engineered to express human MHC molecules) are used for antigen presentation. For example, assays can employ COS cells transfected with human MHC molecules or HeLa cells.
[0154] In some embodiments, both the antigen presenting cells and immune cells used in the method are derived from the same subject (e.g., autologous T cells and APC are used). In these embodiments, it can be advantageous to sequentially isolate subsets of cells from peripheral blood of the subject, to maximize the yield of cells available for assays. For example, one can first isolate CD4+ and CD8+ T cell subsets from the peripheral blood. Next, dendritic cells (DC) are isolated from the T cell-depleted cell population. The remaining T- and DC- depleted cells are used to supplement the DC in assays, or are used alone as antigen presenting cells. In some embodiments, DC are used with T- and DC-depleted cells in an assay, at a ratio of 1 :2, 1 :3, 1 :4, or 1 :5. In some embodiments, the antigen presenting cells and immune cells used in the method are derived from different subjects (e.g., heterologous T cells and APC are used).
[0155] Antigen presenting cells can be isolated from sources other than peripheral blood. For example, antigen presenting cells can be taken from a mucosal tissue (e.g., nose, mouth, bronchial tissue, tracheal tissue, the gastrointestinal tract, the genital tract (e.g., vaginal tissue), or associated lymphoid tissue), peritoneal cavity, lymph nodes, spleen, bone marrow, thymus, lung, liver, kidney, neuronal tissue, endocrine tissue, or other tissue, for use in screening assays. In some embodiments, cells are taken from a tissue that is the site of an active immune response (e.g, an ulcer, sore, or abscess). Cells may be isolated from tissue removed surgically, via lavage, or other means.
[0156] Antigen presenting cells useful in methods described herein are not limited to “professional” antigen presenting cells. In some embodiments, non-professional antigen presenting cells can be utilized effectively in the practice of methods of the present disclosure. Non-professional antigen presenting cells include fibroblasts, epithelial cells, endothelial cells, neuronal/glial cells, lymphoid or myeloid cells that are not professional antigen presenting cells (e.g, T cells, neutrophils), muscle cells, liver cells, and other types of cells.
[0157] Antigen presenting cells are cultured with library members that express a polypeptide of interest (and, if desired, a cytolysin polypeptide) under conditions in which the antigen presenting cells internalize, process and present polypeptides expressed by the library members on MHC molecules. In some embodiments, library members are killed or inactivated prior to culture with the antigen presenting cells. Cells or viruses can be inactivated by any appropriate agent (e.g., fixation with organic solvents, irradiation, freezing). In some embodiments, the library members are cells that express ORFs linked to a tag (e.g., a tag which comprises one or more known T cell epitopes) or reporter protein, expression of which has been verified prior to the culturing.
[0158] In some embodiments, antigen presenting cells are incubated with library members at 37°C for between 30 minutes and 5 hours (e.g., for 45 min. to 1.5 hours). After the incubation, the antigen presenting cells can be washed to remove library members that have not been internalized. In certain embodiments, the antigen presenting cells are non-adherent, and washing requires centrifugation of the cells. The washed antigen presenting cells can be incubated at 37°C for an additional period of time (e.g., 30 min. to 2 hours) prior to exposure to lymphocytes, to allow antigen processing. In some embodiments, it is desirable to fix and kill the antigen presenting cells prior to exposure to lymphocytes (e.g., by treating the cells with 1% paraformaldehyde).
[0159] The antigen presenting cell and library member numbers can be varied, so long as the library members provide quantities of polypeptides of interest sufficient for presentation on MHC molecules. In some embodiments, antigen presenting cells are provided in an array, and are contacted with sets of library cells, each set expressing a different polypeptide of interest. In certain embodiments, each location in the array includes 1 x 103 - 1 x 106 antigen presenting cells, and the cells are contacted with 1 x 103 - 1 x 108 library cells which are bacterial cells.
[0160] In any of the embodiments described herein, antigen presenting cells can be freshly isolated, maintained in culture, and/or thawed from frozen storage prior to incubation with library cells, or after incubation with library cells.
Human Lymphocytes [0161] In methods of the present disclosure, human lymphocytes are tested for antigenspecific reactivity to antigen presenting cells, e.g., antigen presenting cells that have been incubated with libraries expressing polypeptides of interest as described above. The methods of the present disclosure permit rapid identification of human antigens using pools of lymphocytes isolated from an individual, or progeny of the cells. The detection of antigen-specific responses does not rely on laborious procedures to isolate individual T cell clones. In some embodiments, the human lymphocytes are primary lymphocytes. In some embodiments, human lymphocytes are NKT cells, gamma-delta T cells, or NK cells. Just as antigen presenting cells may be separated into subsets prior to use in antigen presentation assays, a population of lymphocytes having a specific marker or other feature can be used. In some embodiments, a population of T lymphocytes is isolated. In some embodiments, a population of CD4+ T cells is isolated. In some embodiments, a population of CD8+ T cells is isolated. CD8+ T cells recognize peptide antigens presented in the context of MHC class I molecules. Thus, in some embodiments, the CD8+ T cells are used with antigen presenting cells that have been exposed to library host cells that co-express a cytolysin polypeptide, in addition to a polypeptide of interest. T cell subsets that express other cell surface markers may also be isolated, e.g., to provide cells having a particular phenotype. These include CLA (for skin-homing T cells), CD25, CD30, CD69, CD 154 (for activated T cells), CD45RO (for memory T cells), CD294 (for Th2 cells), y/5 TCR- expressing cells, CD3 and CD56 (for NK T cells). Other subsets can also be selected.
[0162] Lymphocytes can be isolated, and separated, by any means known in the art (e.g., using antibody -based methods such as those that employ magnetic bead separation, panning, or flow cytometry). Reagents to identify and isolate human lymphocytes and subsets thereof are well known and commercially available.
[0163] Lymphocytes for use in methods described herein can be isolated from peripheral blood mononuclear cells, or from other tissues in a human. In some embodiments, lymphocytes are taken from tumors, lymph nodes, a mucosal tissue (e.g., nose, mouth, bronchial tissue, tracheal tissue, the gastrointestinal tract, the genital tract (e.g., vaginal tissue), or associated lymphoid tissue), peritoneal cavity, spleen, thymus, lung, liver, kidney, neuronal tissue, endocrine tissue, peritoneal cavity, bone marrow, or other tissues. In some embodiments, cells are taken from a tissue that is the site of an active immune response (e.g., an ulcer, sore, or abscess). Cells may be isolated from tissue removed surgically, via lavage, or other means.
[0164] Lymphocytes taken from an individual can be maintained in culture or frozen until use in antigen presentation assays. In some embodiments, freshly isolated lymphocytes can be stimulated in vitro by antigen presenting cells exposed to library cells as described above. In some embodiments, these lymphocytes exhibit detectable stimulation without the need for prior non-antigen specific expansion. However, primary lymphocytes also elicit detectable antigenspecific responses when first stimulated non-specifically in vitro. Thus, in some embodiments, lymphocytes are stimulated to proliferate in vitro in a non-antigen specific manner, prior to use in an antigen presentation assay. Lymphocytes can also be stimulated in an antigen-specific manner prior to use in an antigen presentation assay. In some embodiments, cells are stimulated to proliferate by a library (e.g., prior to use in an antigen presentation assay that employs the library). Expanding cells in vitro provides greater numbers of cells for use in assays. Primary T cells can be stimulated to expand, e.g., by exposure to a polyclonal T cell mitogen, such as phytohemagglutinin or concanavalin, by treatment with antibodies that stimulate proliferation, or by treatment with particles coated with the antibodies. In some embodiments, T cells are expanded by treatment with anti-CD2, anti-CD3, and anti-CD28 antibodies. In some embodiments, T cells are expanded by treatment with interleukin-2. In some embodiments, lymphocytes are thawed from frozen storage and expanded (e.g., stimulated to proliferate, e.g., in a non-antigen specific manner or in an antigen-specific manner) prior to contacting with antigen presenting cells. In some embodiments, lymphocytes are thawed from frozen storage and are not expanded prior to contacting with antigen presenting cells. In some embodiments, lymphocytes are freshly isolated and expanded (e.g., stimulated to proliferate, e.g., in a non-antigen specific manner or in an antigen-specific manner) prior to contacting with antigen presenting cells.
Antigen Presentation Assays [0165] In antigen presentation assays, T cells are cultured with antigen presenting cells prepared according to the methods described above, under conditions that permit T cell recognition of peptides presented by MHC molecules on the antigen presenting cells. In some embodiments, T cells are incubated with antigen presenting cells at 37°C for between 12-48 hours (e.g., for 24 hours). In some embodiments, T cells are incubated with antigen presenting cells at 37°C for 3, 4, 5, 6, 7, or 8 days. Numbers of antigen presenting cells and T cells can be varied. In some embodiments, the ratio of T cells to antigen presenting cells in a given assay is 1 : 10, 1 :5, 1 :2, 1 : 1, 2: 1, 5: 1, 10: 1, 20: 1, 25: 1, 30: 1, 32: 1, 35:1 or 40: 1. In some embodiments, antigen presenting cells are provided in an array (e.g., in a 96-well plate), wherein cells in each location of the array have been contacted with sets of library cells, each set including a different polypeptide of interest. In certain embodiments, each location in the array includes 1 x 103 - 1 x 106 antigen presenting cells, and the cells are contacted with 1 X 103 - 1 X 106 T cells.
[0166] After T cells have been incubated with antigen presenting cells, cultures are assayed for activation. Lymphocyte activation can be detected by any means known in the art, e.g., T cell proliferation, phosphorylation or dephosphorylation of a receptor, calcium flux, cytoskeletal rearrangement, increased or decreased expression and/or secretion of immune mediators such as cytokines or soluble mediators, increased or decreased expression of one or more cell surface markers. In some embodiments, culture supernatants are harvested and assayed for increased and/or decreased expression and/or secretion of one or more polypeptides associated with activation, e.g., a cytokine, soluble mediator, cell surface marker, or other immune mediator. In some embodiments, the one or more cytokines are selected from TRAIL, IFN-gamma, IL-12p70, IL-2, TNF-alpha, MIP1 -alpha, MIPl-beta, CXCL9, CXCL10, MCP1, RANTES, IL-1 beta, IL-4, IL-6, IL-8, IL-9, IL-10, IL-13, IL-15, CXCL11, IL-3, IL-5, IL-17, IL- 18, IL-21, IL-22, IL-23 A, IL-24, IL-27, IL-31, IL-32, TGF-beta, CSF, GM-CSF, TRANCE (also known as RANK L), MIP3 -alpha, and fractalkine. In some embodiments, the one or more soluble mediators are selected from granzyme A, granzyme B, sFas, sFasL, perforin, and granulysin. In some embodiments, the one or more cell surface markers are selected from CD107a, CD107b, CD25, CD69, CD45RA, CD45RO, CD137 (4-1BB), CD44, CD62L, CD27, CCR7, CD 154 (CD40L), KLRG-1, CD71, HLA-DR, CD 122 (IL-2RB), CD28, IL7Ra (CD 127), CD38, CD26, CD134 (OX-40), CTLA-4 (CD152), LAG-3, TIM-3 (CD366), CD39, PD-1 (CD279), FoxP3, TIGIT, CD160, BTLA, 2B4 (CD244), and KLRG1. Cytokine secretion in culture supernatants can be detected, e.g., by ELISA, bead array, e.g., with a Luminex® analyzer. Cytokine production can also be assayed by RT-PCR of mRNA isolated from the T cells, or by ELISPOT analysis of cytokines released by the T cells. In some embodiments, proliferation of T cells in the cultures is determined (e.g., by detecting 3H thymidine incorporation). In some embodiments, target cell lysis is determined (e.g., by detecting T cell dependent lysis of antigen presenting cells labeled with Na2 51CrO4). Target cell lysis assays are typically performed with CD8+ T cells. Protocols for these detection methods are known. See, e.g., Current Protocols In Immunology, John E. Coligan et al. (eds), Wiley and Sons, New York, N.Y., 2007. One of skill in the art understands that appropriate controls are used in these detection methods, e.g, to adjust for non-antigen specific background activation, to confirm the presenting capacity of antigen presenting cells, and to confirm the viability of lymphocytes.
[0167] In some embodiments, antigen presenting cells and lymphocytes used in the method are from the same individual. In some embodiments, antigen presenting cells and lymphocytes used in the method are from different individuals.
[0168] In some embodiments, antigen presentation assays are repeated using lymphocytes from the same individual that have undergone one or more previous rounds of exposure to antigen presenting cells, e.g, to enhance detection of responses, or to enhance weak initial responses. In some embodiments, antigen presentation assays are repeated using antigen presenting cells from the same individual that have undergone one or more previous rounds of exposure to a library, e.g, to enhance detection of responses, or to enhance weak initial responses. In some embodiments, antigen presentation assays are repeated using lymphocytes from the same individual that have undergone one or more previous rounds of exposure to antigen presenting cells, and antigen presenting cells from the same individual that have undergone one or more previous rounds of exposure to a library, e.g., to enhance detection of responses, or to enhance weak initial responses. In some embodiments, antigen presentation assays are repeated using antigen presenting cells and lymphocytes from different individuals, e.g., to identify antigens recognized by multiple individuals, or compare reactivities that differ between individuals.
Methods of Identifying Tumor Antigens
[0169] One advantage of methods described herein is their ability to identify clinically relevant human antigens. Humans that have cancer may have lymphocytes that specifically recognize tumor antigens, which are the product of an adaptive immune response arising from prior exposure. In some embodiments, these cells are present at a higher frequency than cells from an individual who does not have cancer, and/or the cells are readily reactivated when reexposed to the proper antigenic stimulus (e.g., the cells are “memory” cells). Thus, humans that have or have had cancer are particularly useful donors of cells for identifying antigens in vitro. The individual may be one who has recovered from cancer. In some embodiments, the individual has been recently diagnosed with cancer (e.g., the individual was diagnosed less than one year, three months, two months, one month, or two weeks, prior to isolation of lymphocytes and/or antigen presenting cells from the individual). In some embodiments, the individual was first diagnosed with cancer more than three months, six months, or one year prior to isolation of lymphocytes and/or antigen presenting cells.
[0170] In some embodiments, lymphocytes are screened against antigen presenting cells that have been contacted with a library of cells whose members express or carry polypeptides of interest, and the lymphocytes are from an individual who has not been diagnosed with cancer. In some embodiments, such lymphocytes are used to determine background (i.e., non-antigen- specific) reactivities. In some embodiments, such lymphocytes are used to identify antigens, reactivity to which exists in non-cancer individuals.
[0171] Cells from multiple donors (e.g., multiple subjects who have cancer) can be collected and assayed in methods described herein. In some embodiments, cells from multiple donors are assayed in order to determine if a given tumor antigen is reactive in a broad portion of the population, or to identify multiple tumor antigens that can be later combined to produce an immunogenic composition that will be effective in a broad portion of the population.
[0172] Antigen presentation assays are useful in the context of both infectious and non- infectious diseases. The methods described herein are applicable to any context in which a rapid evaluation of human cellular immunity is beneficial. In some embodiments, antigenic reactivity to polypeptides that are differentially expressed by neoplastic cells (e.g., tumor cells) is evaluated. Sets of nucleic acids differentially expressed by neoplastic cells have been identified using established techniques such as subtractive hybridization. Methods described herein can be used to identify antigens that were functional in a subject in which an anti -tumor immune response occurred. In other embodiments, methods are used to evaluate whether a subject has lymphocytes that react to a tumor antigen or set of tumor antigens.
[0173] In some embodiments, antigen presentation assays are used to examine reactivity to autoantigens in cells of an individual, e.g., an individual predisposed to, or suffering from, an autoimmune condition. Such methods can be used to provide diagnostic or prognostic indicators of the individual’s disease state, or to identify autoantigens. For these assays, in some embodiments, libraries that include an array of human polypeptides are prepared. In some embodiments, libraries that include polypeptides from infectious agents which are suspected of eliciting cross-reactive responses to autoantigens are prepared. For examples of antigens from infectious agents thought to elicit cross-reactive autoimmune responses, see Barzilai et al.. Curr Opin Rheumatol. , 19(6):636-43, 2007; Ayada et al.. Ann N Y Acad Sci., 1108:594-602, 2007;
Drouin et al., Mol ImmunoL, 45(1): 180-9, 2008; and Bach, J Autoimmun., 25 Suppl:74-80, 2005.
[0174] As discussed, the present disclosure includes methods in which polypeptides of interest are included in a library (e.g., expressed in library cells or carried in or on particles or beads). After members of the library are internalized by antigen presenting cells, the polypeptides of interest are proteolytically processed within the antigen presenting cells, and peptide fragments of the polypeptides are presented on MHC molecules expressed in the antigen presenting cells. The identity of the polypeptide that stimulates a human lymphocyte in an assay described herein can be determined from examination of the set of library cells that were provided to the antigen presenting cells that produced the stimulation. In some embodiments, it is useful to map the epitope within the polypeptide that is bound by MHC molecules to produce the observed stimulation. This epitope, or the longer polypeptide from which it is derived (both of which are referred to as an “antigen” herein) can form the basis for an immunogenic composition, or for an antigenic stimulus in future antigen presentation assays.
[0175] Methods for identifying peptides bound by MHC molecules are known. In some embodiments, epitopes are identified by generating deletion mutants of the polypeptide of interest and testing these for the ability to stimulate lymphocytes. Deletions that lose the ability to stimulate lymphocytes, when processed and presented by antigen presenting cells, have lost the peptide epitope. In some embodiments, epitopes are identified by synthesizing peptides corresponding to portions of the polypeptide of interest and testing the peptides for the ability to stimulate lymphocytes (e.g., in antigen presentation assays in which antigen presenting cells are pulsed with the peptides). Other methods for identifying MHC bound peptides involve lysis of the antigen presenting cells that include the antigenic peptide, affinity purification of the MHC molecules from cell lysates, and subsequent elution and analysis of peptides from the MHC (Falk, K. et al. Nature 351 :290, 1991, and U.S. Pat. No. 5,989,565).
[0176] In other embodiments, it is useful to identify the clonal T cell receptors that have been expanded in response to the antigen. Clonal T cell receptors are identified by DNA sequencing of the T cell receptor repertoire (Howie et al, 2015 Sci Trans Med 7:301). By identifying TCR specificity and function, TCRs can be transfected into other cell types and used in functional studies or for novel immunotherapies. In other embodiments, it is useful to identify and isolate T cells responsive to a tumor antigen in a subject. The isolated T cells can be expanded ex vivo and administered to a subject for cancer therapy or prophylaxis.
Methods of Identifying an Immune Response in a Subject
[0177] The disclosure provides methods of identifying one or more immune responses of a subject. In some embodiments, one or more immune responses of a subject are determined by a) providing a library described herein that includes a panel of tumor antigens (e.g., known tumor antigens, tumor antigens described herein, or tumor antigens, potential tumor antigens, and/or other polypeptides of interest identified using a method described herein); b) contacting the library with antigen presenting cells from the subject; c) contacting the antigen presenting cells with lymphocytes from the subject; and d) determining whether one or more lymphocytes are stimulated by, inhibited and/or suppressed by, activated by, or non-responsive to one or more tumor antigens presented by one or more antigen presenting cells. In some embodiments, the library includes about 1, 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or more tumor antigens.
[0178] In some embodiments, a subject is (i) a cancer subject who has not received a cancer therapy; (ii) a cancer subject who has not responded and/or is not responding and/or has responded negatively, clinically to a cancer therapy; or (iii) a subject who has not been diagnosed with a cancer.
[0179] In some embodiments, lymphocyte stimulation, non-stimulation, inhibition and/or suppression, activation, and/or non-responsiveness is determined by assessing levels of one or more expressed or secreted cytokines or other immune mediators described herein. In some embodiments, levels of one or more expressed or secreted cytokines that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, higher than a control level indicates lymphocyte stimulation. In some embodiments, a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 standard deviations greater than the mean of a control level indicates lymphocyte stimulation. In some embodiments, a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) greater than a median response level to a control indicates lymphocyte stimulation. In some embodiments, a control is a negative control, for example, a clone expressing Neon Green (NG). In some embodiments, a level of one or more expressed or secreted cytokines that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, lower than a control level indicates lymphocyte inhibition and/or suppression. In some embodiments, a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 standard deviations lower than the mean of a control level indicates lymphocyte inhibition and/or suppression. In some embodiments, a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) lower than a median response level to a control indicates lymphocyte inhibition and/or suppression. In some embodiments, a control is a negative control, for example, a clone expressing Neon Green (NG). In some embodiments, levels of one or more expressed or secreted cytokines that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, higher or lower than a control level indicates lymphocyte activation. In some embodiments, a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 standard deviations greater or lower than the mean of a control level indicates lymphocyte activation. In some embodiments, a level of one or more expressed or secreted cytokines that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) greater or lower than a median response level to a control indicates lymphocyte activation. In some embodiments, a control is a negative control, for example, a clone expressing Neon Green (NG). In some embodiments, a level of one or more expressed or secreted cytokines that is within about 20%, 15%, 10%, 5%, or less, of a control level indicates lymphocyte non-responsiveness or nonstimulation. In some embodiments, a level of one or more expressed or secreted cytokines that is less than 1 or 2 standard deviations higher or lower than the mean of a control level indicates lymphocyte non-responsiveness or non-stimulation. In some embodiments, a level of one or more expressed or secreted cytokines that is less than 1 or 2 median absolute deviations (MADs) higher or lower than a median response level to a control indicates lymphocyte non- responsiveness or non-stimulation. In some embodiments, a subject response profile can include a quantification, identification, and/or representation of a panel of different cytokines (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more cytokines) and of the total number of tumor antigens (e.g., of all or a portion of different tumor antigens from the library) that stimulate, do not stimulate, inhibit and/or suppress, activate, or have no or minimal effect on production, expression or secretion of each member of the panel of cytokines. Method of Obtaining a Subject Response Profile
[0180] The disclosure provides methods for obtaining a subject response profile from a test subject (a “subject response profile”).
[0181] In some embodiments, the subject response profile of a test subject is obtained by a) providing a library described herein that includes a panel of tumor antigens (e.g., known tumor antigens, tumor antigens described herein, or tumor antigens, potential tumor antigens, and/or other polypeptides of interest identified using a method described herein); b) contacting the library with antigen presenting cells from the test subject; c) contacting the antigen presenting cells with lymphocytes from the test subject; and d) determining whether one or more lymphocytes are stimulated by, inhibited and/or suppressed by, activated by, or non-responsive to one or more tumor antigens presented by one or more antigen presenting cells, to obtain the subject response profile. In some embodiments, the library includes about 1, 3, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 500, 1000, or more tumor antigens.
[0182] The subject response profile can include a quantification, identification, and/or representation of all or a portion of the panel of tumor antigens, identified by the methods of the disclosure, that stimulate lymphocytes, that do not stimulate lymphocytes, that inhibit and/or suppress lymphocytes, that activate lymphocytes, or to which lymphocytes are non-responsive. In some embodiments, the subject response profile further includes a quantification, identification, and/or representation of the level of expression or secretion of one or more immune mediators, e.g., one or more cytokines.
[0183] In some embodiments, the subject response profile includes a quantification, identification, and/or representation of all or a portion of the panel of tumor antigens, identified by the methods of the disclosure, that stimulate expression or secretion of one or more immune mediators, that inhibit and/or suppress expression or secretion of one or more immune mediators, and/or which do not, or minimally, affect expression or secretion of immune mediators. In some embodiments, the subject response profile further includes a quantification, identification, and/or representation of the level of expression or secretion of one or more immune mediators, e.g., one or more cytokines. In some embodiments, the subject response profile includes a quantification, identification, and/or representation of all or a portion of the panel of stimulatory and/or inhibitory antigens, identified by methods of the disclosure.
[0184] In some embodiments, a subject response profile is obtained for a subject at one or more times before initiation of treatment a cancer therapy, such as (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, or (iv) any combination of the foregoing. In some embodiments, a subject response profile is a representation of (i) a number of identified stimulatory tumor antigens and (ii) a number of identified inhibitory tumor antigens, at a given time, for the subject.
[0185] In some embodiments, a subject response profile is obtained for a subject at one or more times after initiation of treatment a cancer therapy, such as (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, or (iv) any combination of the foregoing. In some embodiments, a subject response profile is a representation of (i) a number of identified stimulatory tumor antigens and (ii) a number of identified inhibitory tumor antigens, at a given time, for the subject.
Methods of Determining a Change in Subject Response Profile
[0186] As described herein, the present disclosure encompasses the recognition that a subject response profile can change over a period of time, e.g., over a course of cancer therapy. A change in subject response profile can be used to predict responsiveness to a cancer therapy and/or determine suitability or efficacy of a cancer therapy. In some embodiments, change in subject response profile can be used to determine a course of treatment for a subject (e.g., initiation, continuation, modification, discontinuation or non-initiation of a cancer therapy, or of a sequence of cancer therapies).
[0187] In some embodiments, a first subject response profile is obtained before or after a subject is treated with a cancer therapy described herein. In some embodiments, a first subject response profile is obtained after a subject is treated with a cancer therapy described herein. In some embodiments, a second subject response profile is obtained after a subject is treated with a cancer therapy described herein. In some embodiments, a second subject response profile is obtained after a subject has ceased treatment with a cancer therapy described herein.
[0188] In some embodiments, a second subject response profile is obtained from the subject about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or about 7 days, about 1 week, 2 week, 3 week, 4 week, 5 week, 6 weeks, 7 weeks, or about 8 weeks, about 1 month, 2 months, 3 months, 4 months, 5 months, or about 6 months, about 1 year, 2 years, 3 years, 4 years, or about 5 years, after the first subject response profile is obtained.
[0189] In some embodiments, subject response profile is obtained from a subject at least once per week during treatment with a cancer therapy. In some embodiments, subject response profile is obtained from a subject at least once per month during treatment with a cancer therapy.
[0190] In some embodiments, a first subject response profile is compared to a second subject response profile. In some embodiments, a change between a first subject response profile and a second subject response profile is determined by a change in number of stimulatory antigens identified in a subject. In some embodiments, a change between a first subject response profile and a second subject response profile is determined by a change in number of inhibitory antigens identified in a subject.
Lymphocytes
[0191] In some embodiments, a first subject response profile does not differ from a second subject response profile if the identified tumor antigens that stimulate lymphocytes in the first subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that stimulate lymphocytes in the second subject response profile; if the identified tumor antigens that do not stimulate lymphocytes in the first subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that do not stimulate lymphocytes in the second subject response profile; if the identified tumor antigens that inhibit and/or suppress lymphocytes in the first subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that inhibit and/or suppress lymphocytes in the second subject response profile; if the identified tumor antigens that activate lymphocytes in the first subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens that activate lymphocytes in the second subjectresponse profile; and/or if the identified tumor antigens that do not stimulate lymphocytes or to which lymphocytes are non-responsive in the first subject response profile differ by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the identified tumor antigens to which lymphocytes are not, or are minimally, responsive in the second subjectresponse profile.
[0192] In some embodiments, a first subject response profile differs from a second subject response profile if the identified tumor antigens that stimulate lymphocytes in the first subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that stimulate lymphocytes in the second subject response profile; if the identified tumor antigens that do not stimulate lymphocytes in the first subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that do not stimulate lymphocytes in the second subject response profile; if the identified tumor antigens that inhibit and/or suppress lymphocytes in the first subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that inhibit and/or suppress lymphocytes in the second subject response profile; if the identified tumor antigens that activate lymphocytes in the first subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens that activate lymphocytes in the second subjectresponse profile; and/or if the identified tumor antigens that do not stimulate lymphocytes or to which lymphocytes are non-responsive in the first subject response profile differ by more than 5, 6, 7, 8, 9, 10, 20, or more, from the identified tumor antigens to which lymphocytes are not, or are minimally, responsive in the second subject response profile.
[0193] In some embodiments, a first subject response profile does not differ from a second subject response profile if the identified tumor antigens that stimulate lymphocytes in the first subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that stimulate lymphocytes in the second subject response profile; if the identified tumor antigens that do not stimulate lymphocytes in the first subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that do not stimulate lymphocytes in the second subject response profile; if the identified tumor antigens that inhibit and/or suppress lymphocytes in the first subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that inhibit and/or suppress lymphocytes in the second subject response profile; if the identified tumor antigens that activate lymphocytes in the first subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens that activate lymphocytes in the second subject response profile; and/or if the identified tumor antigens that do not stimulate lymphocytes or to which lymphocytes are non-responsive in the first subject response profile differ by no more than 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, or 25% from the identified tumor antigens to which lymphocytes are not, or are minimally, responsive in the second subject response profile.
[0194] In some embodiments, a first subject response profile differs from a second subject response profile if the identified tumor antigens that stimulate lymphocytes in the first subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%, or more, from the identified tumor antigens that stimulate lymphocytes in the second subject response profile if the identified tumor antigens that do not stimulate lymphocytes in the first subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20% ,or more, from the identified tumor antigens that do not stimulate lymphocytes in the second subject response profile; and/or if the identified tumor antigens that inhibit and/or suppress lymphocytes in the first subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%, or more, from the identified tumor antigens that inhibit and/or suppress lymphocytes in the second subject response profile; if the identified tumor antigens that activate lymphocytes in the first subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%, or more, from the identified tumor antigens that activate lymphocytes in the second subject response profile; and/or if the identified tumor antigens that do not stimulate lymphocytes or to which lymphocytes are non-responsive in the first subject response profile differ by more than 5%, 6%, 7%, 8%, 9%, 10%, 20%, or more, from the identified tumor antigens to which lymphocytes are not, or are minimally, responsive in the second subject response profile.
Cytokines
[0195] In some embodiments, a second subject response profile can include a quantification, identification, and/or representation of one or more cytokines and the total number of tumor antigens (e.g., of the same tumor antigens included in a first subject response profile) that stimulate, do not stimulate, inhibit and/or suppress, or have no or minimal effect on cytokine production, expression and/or secretion. In some embodiments, the second subject response profile can include a quantification, identification, and/or representation of a panel of different cytokines (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, or more (e.g., all) of the cytokines included in the first subject response profile) and the total number of tumor antigens (e.g., of the same tumor antigens included in thefirst subject response profile) that stimulate, do not stimulate, inhibit and/or suppress, or have no or minimal effect on production, expression and/or secretion of the panel of cytokines.
[0196] In some embodiments, a first subject response profile is not different from a second subject response profile if the total number of antigens that stimulate expression and/or secretion of one or more cytokines included in the first subject response profile differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the total number of antigens that stimulate the same one or more cytokines included in second subject response profile; if the total number of antigens that do not stimulate expression and/or secretion of one or more cytokines included in the first subject response profile differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the total number of antigens that do not stimulate the same one or more cytokines included in the second subject response profile; if the total number of antigens that inhibit and/or suppress one or more cytokines included in the first subject response profile differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the total number of antigens that inhibit and/or suppress expression and/or secretion of the same one or more cytokines included in the second subject response profile; and/or if the total number of antigens that have no or minimal effect on expression and/or secretion of one or more cytokines included in the first subject response profile differs by no more than 1, 2, 3, 4, 5, 10, 15, 20, or 25 from the total number of antigens that that have no or minimal effect on the same one or more cytokines included in the second subject response profile.
[0197] In some embodiments, a first subject response profile differs from a second response profile if the total number of antigens that stimulate expression and/or secretion of one or more cytokines included in the first subject response profile differs by more than 5, 6, 7, 8, 9, 10, 20, or more, from the total number of antigens that stimulate the same one or more cytokines included in the second subject response profile; if the total number of antigens that do not stimulate expression and/or secretion of one or more cytokines included in the first subject response profile differs by more than 5, 6, 7, 8, 9, 10, 20, or more, from the total number of antigens that do not stimulate the same one or more cytokines included in the second subject response profile ; if the total number of antigens that inhibit and/or suppress expression and/or secretion of one or more cytokines included in the first subject response profile differs by more than 5, 6, 7, 8, 9, 10, 20, or more, from the total number of antigens that inhibit and/or suppress the same one or more cytokines included in the second subject response profile; and/or if the total number of antigens that have no or minimal effect on expression and/or secretion of one or more cytokines included in the first subject response profile differs by more than 5, 6, 7, 8, 9, 10, 20, or more, from the total number of antigens that that have no or minimal effect on the same one or more cytokines included in the second subject response profile.
[0198] The foregoing methods apply to subject response profiles obtained with libraries encoding polypeptides that are potential tumor antigens, as well as tumor antigens.
Methods of Identifying/Selecting Subjects for Cancer Therapy
[0199] The disclosure provides methods of using a subject response profile, as described herein, from a subject, e.g., a cancer subject, to determine a course of cancer therapy. [0200] In some embodiments, determining a course of therapy includes initiation, continuation, modification, and/or discontinuation or in some cases non-initiation of a cancer therapy (e.g., a cancer therapy described herein), or of a course of cancer therapies. In some embodiments, methods include, e.g., comparing at least two subject response profiles obtained from a cancer subject who has not received a cancer therapy, who has not responded and/or is not responding and/or has responded negatively, clinically to a cancer therapy. In some embodiments, a first subject response profile is obtained at a first time (e.g., before initiation of a cancer therapy or after initiation of a cancer therapy) and a second subject response profile is obtained at a second time (e.g., after initiation of a cancer therapy), as described herein.
[0201] As described herein, in some embodiments, a change in subject response profile comprises a change in number of stimulatory antigens and/or inhibitory antigens detected in a subject. In some embodiments, a change in subject response profile is measured as a change in a subject’s lymphocytes level of expression or secretion of one or more immune mediators, e.g., one or more cytokines to a particular tumor antigen. In some embodiments, a change in subject response profile is identified when a lymphocyte would no longer be activated (i.e., activated to secrete/express a marker associated with a deleterious or beneficial immune response). In some embodiments, a change in subject response profile is identified when a lymphocyte initially is not activated (i.e., activated to secrete/express a marker associated with a deleterious or beneficial immune response) by a particular tumor antigen developes a recognition/response to that particular tumor antigen.
[0202] In some embodiments, (i) an increase in number of stimulatory antigens in a second subject response profile relative to a first subject response profile and/or (ii) a decrease in number of inhibitory antigens in a second subject response profile relative to a first subject response profile indicates that the subject should continue a course of cancer therapy.
[0203] In some embodiments, (i) a decrease in number of stimulatory antigens in a second subject response profile, relative to a first subject response profile, and/or (ii) an increase in number of inhibitory in a second subject response profile, relative to a first subject response profile, indicates that the subject should modify the course of cancer therapy or should initiate a new course of cancer therapy (e.g., combine with one or more other modalities).
[0204] In some embodiments, determining the course of therapy and/or sequence of cancer therapies is based on the number and/or characteristics of the identified stimulatory and inhibitory antigens in a subject response profile.
[0205] For example, in some embodiments, a cancer therapy and/or sequence of cancer therapies is selected based on identification of one or more inhibitory antigens that reduce and/or inhibit an immune response by, e.g., at least 50%, 60%, 70%, 80%, 90%, or 100%, relative to control. In some such embodiments, a cancer therapy (e.g., an immunogenic composition comprising one or more of the identified stimulatory antigens, checkpoint inhibitor therapy, chemotherapy, cell-based therapy, immunotherapy, cytokine therapy, and/or kinase inhibitors) is selected for the subject and/or is administered to the subject based on identification of such inhibitory antigens.
[0206] In some embodiments, a cancer therapy and/or sequence of cancer therapies, is selected based on identification of one or more stimulatory antigens that increase an immune response by, e.g., at least 50%, 60%, 70%, 80%, 90%, or 100%, relative to control. In some such embodiments, a cancer therapy (e.g., an immunogenic composition comprising one or more of the identified stimulatory antigens, checkpoint inhibitor therapy, chemotherapy, cell-based therapy, immunotherapy, cytokine therapy, and/or kinase inhibitors) is selected for the subject and/or is administered to the subject based on identification of such stimulatory antigens.
[0207] In some embodiments, a cancer therapy and/or sequence of cancer therapies is selected based on the presence or absence of identified dominant stimulatory antigens in a subject response profile. In some embodiments, a cancer therapy and/or sequence of cancer therapies is based on the presence or absence of identified dominant inhibitory antigens in a subject response profile. A dominant inhibitory antigen is one that abrogates the effects of a protective cancer vaccine and results in, e.g., hyperprogresssion of a tumor. A sub-dominant inhibitory antigen is one that partially abrogates the effects of a protective cancer vaccine. [0208] In some embodiments, a cancer therapy and/or sequence of cancer therapies is selected based on the number of identified stimulatory and/or inhibitory antigens in a subject response profile. In some embodiments, a cancer therapy and/or sequence of cancer therapies is based on the ratio of identified stimulatory and/or inhibitory antigens in a subject response profile.
Methods of Identifying and Selecting Stimulatory and Inhibitory Tumor Antigens
[0209] In general, immune responses can be usefully defined in terms of their integrated, functional end-effects. Dhabar et al. (2014) have proposed that immune responses can be categorized as being immunoprotective, immunopathological, and immunoregulatory/inhibitory. While these categories provide useful constructs with which to organize ideas, an overall in vivo immune response is likely to consist of several types of responses with varying amounts of dominance from each category. Immunoprotective or beneficial responses are defined as responses that promote efficient wound healing, eliminate infections and cancer, and mediate vaccine-induced immunological memory. These responses are associated with cytokines and mediators such as IFN-gamma, IL-12, IL-2, granzyme B, CD107, etc. Immunopathological or deleterious responses are defined as those that are directed against self (autoimmune disease like multiple sclerosis, arthritis, lupus) or innocuous antigens (asthma, allergies) and responses involving chronic, non-resolving inflammation. These responses can also be associated with molecules that are implicated in immunoprotective responses, but also include immune mediators such as TNF-alpha, IL-10, IL-13, IL-17, IL-4, IgE, histamine, etc. Immunoregulatory responses are defined as those that involve immune cells and factors that regulate (mostly down- regulate) the function of other immune cells. Recent studies suggest that there is an arm of the immune system that functions to inhibit immune responses. For example, regulatory CD4+CD25+FoxP3+ T cells, IL-10, and TGF-beta, among others have been shown to have immunoregulatory/inhibitory functions. The physiological function of these factors is to keep pro-inflammatory, allergic, and autoimmune responses in check, but they may also suppress antitumor immunity and be indicative of negative prognosis for cancer. In the context of tumors, the expression of co-stimulatory molecules often decreases, and the expression of co-inhibitory ligands increases. MHC molecules are often down-regulated on tumor cells, favoring their escape. The tumor micro-environment, including stromal cells, tumor associated immune cells, and other cell types, produce many inhibitory factors, such as, IL-10, TGF-P, and IDO. Inhibitory immune cells, including T regs, Tri cells, immature DCs (iDCs), pDCs, and MDSC can be found in the tumor microenvironment. (Y Li UT GSBS Thesis 2016). Examples of mediators and their immune effects are shown in Table 2.
Table 2: Immune Mediators
Figure imgf000086_0001
Figure imgf000087_0001
Figure imgf000088_0001
Figure imgf000089_0001
Figure imgf000090_0001
Figure imgf000091_0001
ID = Infectious disease
I A = Autoimmune disease
[0210] In some embodiments, a stimulatory antigen is a tumor antigen (e.g., a tumor antigen described herein) that stimulates one or more lymphocyte responses that are beneficial to the subject. In some embodiments, a stimulatory antigen is a tumor antigen (e.g., a tumor antigen described herein) that inhibits and/or suppresses one or more lymphocyte responses that are deleterious or non-beneficial to the subject. Examples of immune responses that may lead to beneficial anti -tumor responses (e.g., that may enhance immune control of a tumor) include but are not limited to 1) cytotoxic CD8+ T cells which can effectively kill cancer cells and release the mediators perforin and/or granzymes to drive tumor cell death; and 2) CD4+ Thl T cells which play an important role in host defense and can secrete IL-2, IFN-gamma and TNF-alpha. These are induced by IL- 12, IL-2, and IFN gamma among other cytokines.
[0211] In some embodiments, an inhibitory antigen is a tumor antigen (e.g., a tumor antigen described herein) that stimulates one or more lymphocyte responses that are deleterious or non-beneficial to the subject. In some embodiments, an inhibitory antigen is a tumor antigen (e.g., a tumor antigen described herein) that inhibits and/or suppresses one or more lymphocyte responses that are beneficial to the subject. Examples of immune responses that may lead to deleterious or non-beneficial anti -tumor responses (e.g., that may impair or reduce control of a tumor) include but are not limited to 1) T regulatory cells which are a population of T cells that can suppress an immune response and secrete immunosuppressive cytokines such as TGF-beta and IL-10 and express the molecules CD25 and FoxP3; and 2) Th2 cells which target responses against allergens but are not productive against cancer. These are induced by increased IL-4 and IL- 10 and can secrete IL-4, IL-5, IL-6, IL-9 and IL-13.
[0212] The disclosure provides methods and systems for identifying and selecting tumor antigens, e.g., stimulatory and/or inhibitory antigens. In some embodiments, one or more selected antigen is a stimulatory antigen. A stimulatory antigen may be selected based on the measured immune response to the antigen using a method of the disclosure. A stimulatory antigen may be selected if the antigen produces an immune response that stimulates the expression and/or release of one or more of any cytokine associated with a beneficial response, as shown for example, in Table 2. In some embodiments, the cytokine comprises one or more of IL-2, IFN-gamma and TNF-alpha. A stimulatory antigen may be selected if the antigen produces an immune response that inhibits the expression and/or release of one or more of any of the cytokines associated with a deleterious response, as shown for example, in Table 2. In some embodiments, the cytokine comprises one or more of TGF-beta and IL-10.
[0213] In some embodiments, a composition comprising the one or more selected tumor antigens is administered to a cancer subject before, during, and/or after administration of a cancer therapy.
[0214] The disclosure provides methods for selecting tumor antigens identified by the methods herein based on comparison of a subject response profile to a subsequently obtained subject response profile from the same subject. The disclosure also provides methods for selecting (or de-selecting) tumor antigens identified by the methods herein, based on association with desirable or beneficial responses. The disclosure also provides methods for selecting (or deselecting) tumor antigens identified by the methods herein, based on association with undesirable, deleterious or non-beneficial responses. In some embodiments, the methods for selecting tumor antigens are combined. The methods may be combined in any order, e.g. selection may be carried out by comparison of a subject response profile to a subsequently obtained subject response profile, followed by selection based on association with a desirable (or undesirable) response; or, selection may be carried out based on association with a desirable (or undesirable) response, followed by comparison of the subject response profile to a subsequently obtained subject response profile.
[0215] Methods for identifying tumor antigens and potential tumor antigens are provided herein. Methods for generating or obtaining a subject response profile are provided herein. Methods for generating or obtaining as subsequent subject response profile, e.g. a subject response profile obtained before and during treatment, are provided herein. Methods for comparison of a subject response profile to a subsequently obtained subject response profile are provided herein. Methods for determining whether a subject response profile has changed over time are provided herein.
[0216] In some embodiments, a first subject response profile and a second subject response profile are generated or obtained using the same plurality of polypeptides of interest. In some embodiments, a first subject response profile and a second subject response profile are generated or obtained using the same plurality of tumor antigens.
[0217] The subject response profile includes a quantification, identification, and/or representation of one or more tumor antigens that stimulate lymphocytes, that do not stimulate lymphocytes, that inhibit and/or suppress lymphocytes, that activate lymphocytes, and/or to which lymphocytes are non-responsive.
[0218] In some embodiments, one or more tumor antigens are identified as inhibiting and/or suppressing lymphocytes in the subject (e.g., identified from a first subject response profile), and the same one or more tumor antigens are identified as stimulating lymphocytes in the subject at a later timepoint (e.g., identified from a second subject response profile). In some embodiments, one or more tumor antigens are identified as stimulating lymphocytes in the subject (e.g., identified from a first subject response profile) and the same one or more tumor antigens are identified as inhibiting and/or suppressing lymphocytes in the subject at a later point in time (e.g., identified in a second subject response profile). In some embodiments, one or more tumor antigens or potential tumor antigens are identified as eliciting minimal or no response from lymphocytes in the subject (e.g., identified from a first subject response profile), and the same one or more tumor antigens are identified as stimulating, or inhibiting and/or suppressing lymphocytes in the subject at a later point in time (e.g., identified from a second subject response profile). In some embodiments, one or more tumor antigens are identified as stimulating, or inhibiting and/or suppressing, lymphocytes in the subject (e.g., identified from a first subject response profile), and the same one or more tumor antigens are identified as eliciting minimal or no response from lymphocytes in the subject at a later point in time (e.g., identified from a second subject response profile).
[0219] Tumor antigens may be identified and/or selected (or de-selected) based on association with desirable or beneficial responses. Tumor antigens may be identified and/or selected (or de-selected) based on association with undesirable, deleterious or non-beneficial responses. Tumor antigens may be identified and/or selected (or de-selected) based on a combination of the preceding methods, applied in any order.
[0220] In some embodiments, a stimulatory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a beneficial response is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, higher than a control level indicates lymphocyte stimulation. In some embodiments, a stimulatory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a beneficial response is at least 1, 2, 3, 4 or 5 standard deviations greater than the mean of a control level indicates lymphocyte stimulation. In some embodiments, a stimulatory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a beneficial response is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) greater than a median response level to a control indicates lymphocyte stimulation. In some embodiments, a control is a negative control, for example, a clone expressing Neon Green (NG).
[0221] In some embodiments, a stimulatory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a deleterious response is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, lower than a control level indicates lymphocyte inhibition and/or suppression. In some embodiments, a stimulatory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a deleterious response is at least 1, 2, 3, 4 or 5 standard deviations lower than the mean of a control level indicates lymphocyte inhibition and/or suppression. In some embodiments, a stimulatory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a deleterious response is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) lower than a median response level to a control that indicates lymphocyte inhibition and/or suppression. In some embodiments, a control is a negative control, for example, a clone expressing Neon Green (NG).
[0222] In some embodiments, one or more selected antigen is an inhibitory antigen. An inhibitory antigen may be de-selected based on a measured immune response to the antigen using a method of the disclosure. An inhibitory antigen may be selected if the antigen produces an immune response that stimulates the expression and/or release of one or more cytokines associated with a deleterious response, as shown for example, in Table 2. In some embodiments, the cytokine comprises one or more of TGF-beta and IL-10. An inhibitory antigen may be selected if the antigen produces an immune response that inhibits the expression and/or release of one or more of any cytokine associated with a beneficial response, as shown for example, in Table 2. In some embodiments, the cytokine comprises one or more of IL-2, IFN-gamma and TNF-alpha.
[0223] In some embodiments, an inhibitory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a deleterious response is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, higher than a control level indicates lymphocyte stimulation. In some embodiments, an inhibitory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a deleterious response is at least 1, 2, 3, 4 or 5 standard deviations greater than the mean of a control level indicates lymphocyte stimulation. In some embodiments, an inhibitory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a deleterious response is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) greater than a median response level to a control that indicates lymphocyte stimulation. In some embodiments, a control is a negative control, for example, a clone expressing Neon Green (NG).
[0224] In some embodiments, an inhibitory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a beneficial response is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, 200% or more, lower than a control level that indicates lymphocyte inhibition and/or suppression. In some embodiments, an inhibitory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a beneficial response is at least 1, 2, 3, 4 or 5 standard deviations lower than the mean of a control level that indicates lymphocyte inhibition and/or suppression. In some embodiments, an inhibitory antigen is selected if the level of one or more of the expressed or secreted cytokines associated with a beneficial response is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) lower than a median response level to a control that indicates lymphocyte inhibition and/or suppression. In some embodiments, a control is a negative control, for example, a clone expressing Neon Green (NG).
Production of Tumor Antigens
[0225] A tumor antigen suitable for use in any method or composition of the disclosure may be produced by any available means, such as recombinantly or synthetically (see, e.g., Jaradat Amino Acids 50:39-68 (2018); Behrendt et al., J. Pept. Sci. 22:4-27 (2016)). For example, a tumor antigen may be recombinantly produced by utilizing a host cell system engineered to express a tumor antigen-encoding nucleic acid. Alternatively or additionally, a tumor antigen may be produced by activating endogenous genes. Alternatively or additionally, a tumor antigen may be partially or fully prepared by chemical synthesis.
[0226] Where proteins are recombinantly produced, any expression system can be used. To give but a few examples, known expression systems include, for example, E. coll. egg, baculovirus, plant, yeast, or mammalian cells. [0227] In some embodiments, recombinant tumor antigen suitable for the present invention are produced in mammalian cells. Non-limiting examples of mammalian cells that may be used in accordance with the present invention include BALB/c mouse myeloma line (NSO/1, ECACC No: 85110503); human retinoblasts (PER.C6, CruCell, Leiden, The Netherlands); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (HEK293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59,1977); human fibrosarcoma cell line (e.g., HT1080); baby hamster kidney cells (BHK21, ATCC CCL 10); Chinese hamster ovary cells +/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
[0228] In some embodiments, the present disclosure provides recombinant tumor antigen produced from human cells. In some embodiments, the present disclosure provides recombinant tumor antigen produced from CHO cells or HT1080 cells.
[0229] Typically, cells that are engineered to express a recombinant tumor antigen may comprise a transgene that encodes a recombinant tumor antigen described herein. It should be appreciated that the nucleic acids encoding recombinant tumor antigen may contain regulatory sequences, gene control sequences, promoters, non-coding sequences and/or other appropriate sequences for expressing the recombinant tumor antigen. Typically, the coding region is operably linked with one or more of these nucleic acid components.
[0230] The coding region of a transgene may include one or more silent mutations to optimize codon usage for a particular cell type. For example, the codons of a tumor antigen transgene may be optimized for expression in a vertebrate cell. In some embodiments, the codons of a tumor antigen transgene may be optimized for expression in a mammalian cell. In some embodiments, the codons of a tumor antigen transgene may be optimized for expression in a human cell.
Immunogenic Compositions and Uses Thereof
[0231] The present disclosure provides compositions (e.g., immunogenic compositions) that include a tumor antigen or tumor antigens identified or selected by methods described herein, nucleic acids encoding the tumor antigens, and methods of using the compositions. In some embodiments, a composition includes tumor antigens that are peptides 8-40 amino acids, 8- 60 amino acids, 8-100, 8-150, or 8-200 amino acids in length (e.g., MHC binding peptides, e.g., peptides 23-29, 24-28, 25-27, 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24, 8-23, 8-22, 8-21, 8-20, 8- 15, 8-12 amino acids in length). In some embodiments, a composition includes one or more tumor antigens that are about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the length of the full-length polypeptides. In some embodiments, a composition includes one or more tumor antigens that are truncated by about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids, relative to the full-length polypeptides. The compositions can include tumor antigens that are, or that comprise, MHC class I-binding peptides, MHC class Il-binding peptides, or both MHC class I and MHC class Il-binding peptides. Compositions can include a single tumor antigen, or multiple tumor antigens. In some embodiments, a composition includes a set of two, three, four, five, six, seven, eight, nine, ten, or more tumor antigens. In some embodiments, a composition includes ten, fifteen, twenty, twenty -five, thirty, or more tumor antigens. In some embodiments, the tumor antigens or peptides are provided as one or more fusion proteins. In some embodiments, a composition comprises nucleic acids encoding the tumor antigens or peptides. In some embodiments, the nucleic acids encoding the tumor antigens or peptides are provided as one or more fusion constructs. In some embodiments, an immunogenic composition includes a tumor antigen linked to a carrier protein. Examples of carrier proteins include, e.g., toxins and toxoids (chemical or genetic), which may or may not be mutant, such as anthrax toxin, PA and DNI (PharmAthene, Inc.), diphtheria toxoid (Massachusetts State Biological Labs; Serum Institute of India, Ltd.) or CRM 197, tetanus toxin, tetanus toxoid (Massachusetts State Biological Labs; Serum Institute of India, Ltd.), tetanus toxin fragment Z, exotoxin A or mutants of exotoxin A of Pseudomonas aeruginosa, bacterial flagellin, pneumolysin, an outer membrane protein of Neisseria meningitidis (strain available from the ATCC (American Type Culture Collection, Manassas, Va.)), Pseudomonas aeruginosa Hcpl protein, E. coli heat labile enterotoxin, shiga-like toxin, human LTB protein, a protein extract from whole bacterial cells, and any other protein that can be cross-linked by a linker. Other useful carrier proteins include high density lipoprotein (HDL), bovine serum albumin (BSA), P40, and chicken riboflavin. Many carrier proteins are commercially available (e.g., from Sigma Aldrich.).
[0232] The disclosure also provides nucleic acids encoding the tumor antigens. The nucleic acids can be used to produce expression vectors, e.g., for recombinant production of the tumor antigens, or for nucleic acid-based administration in vivo (e.g., DNA vaccination).
[0233] In some embodiments, an immunogenic composition may be suitable for administration to a human patient, and vaccine preparation may conform to USFDA guidelines. In some embodiments, an immunogenic composition is suitable for administration to a nonhuman animal. I n some embodiments, an immunogenic composition is substantially free of either endotoxins or exotoxins. Endotoxins include pyrogens, such as lipopolysaccharide (LPS) molecules. An immunogenic composition may also be substantially free of inactive protein fragments. In some embodiments, an immunogenic composition has lower levels of pyrogens than industrial water, tap water, or distilled water. Other components of the immunogenic composition may be purified using methods known in the art, such as ion-exchange chromatography, ultrafiltration, or distillation. In other embodiments, the pyrogens may be inactivated or destroyed prior to administration to a patient. Raw materials for immunogenic compositions, such as water, buffers, salts and other chemicals may also be screened and depyrogenated. All materials in a immunogenic composition may be sterile, and each lot of the composition may be tested for sterility. Thus, in certain embodiments the endotoxin levels in the immunogenic composition fall below the levels set by the USFDA, for example 0.2 endotoxin (EU)/kg of product for an intrathecal injectable composition; 5 EU/kg of product for a non- intrathecal injectable composition, and 0.25-0.5 EU/ml for sterile water.
[0234] In some embodiments, an immunogenic composition (e.g., a vaccine and/or a vaccine formulation) comprising a polypeptide contains less than 5%, 2%, 1%, 0.5%, 0.2%, 0.1% of other, undesired unpolypeptides, relative to the amount of desired polypeptides. In some embodiments, an immunogenic composition contains less than 5%, less than 2%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% DNA and/or RNA.
[0235] Immunogenic compositions can be prepared as formulations suitable for route of administration. Formulations suitable for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, intranasal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described.
Adjuvants
[0236] Immunogenic compositions described herein may include an adjuvant. Adjuvants can be used as vaccine delivery systems and/or for their immunostimulatory properties. Vaccine delivery systems are often particulate formulations, e.g., emulsions, microparticles, immune- stimulating complexes (ISCOMs), which may be, for example, particles and/or matrices, and liposomes. Immunostimulatory adjuvants include ISCOMS or may be derived from pathogens and can represent pathogen associated molecular patterns (PAMP), e.g., lipopolysaccharides (LPS), monophosphoryl lipid (MPL), or CpG-containing DNA, which activate cells of the innate immune system. An exemplary adjuvant is Poly-ICLC (Hiltonol, Oncovir Inc). [0237] Adjuvants may also be classified as organic and inorganic. Inorganic adjuvants include aluminum salts such as aluminum phosphate, amorphous aluminum hydroxyphosphate sulfate, and aluminum hydroxide, which are commonly used in human vaccines. Organic adjuvants comprise organic molecules including macromolecules. An example of an organic adjuvant is cholera toxin.
[0238] Adjuvants may also be classified by the response they induce, and adjuvants can activate more than one type of immunostimulatory response. In some embodiments, the adjuvant induces the activation of CD4+ T cells. The adjuvant may induce activation of TH1 cells and/or activation of TH17 cells and/or activation of TH2 cells. Alternately, the adjuvant may induce activation of TH1 cells and/or TH17 cells but not activation of TH2 cells, or vice versa. In some embodiments, the adjuvant induces activation of CD8+ T cells. In further embodiments, the adjuvant may induce activation of Natural Killer T (NKT) cells. In some embodiments, the adjuvant induces the activation of TH1 cells or TH17 cells or TH2 cells. In other embodiments, the adjuvant induces the activation of B cells. In yet other embodiments, the adjuvant induces the activation of APCs. These categories are not mutually exclusive; in some cases, an adjuvant activates more than one type of cell.
[0239] In certain embodiments, an adjuvant stimulates an immune response by increasing the numbers or activity of APCs such as dendritic cells. In certain embodiments, an adjuvant promotes the maturation of APCs such as dendritic cells. In some embodiments, the adjuvant is or comprises a saponin. In some embodiments, a saponin adjuvant is immunostimulatory. Typically, a saponin is a triterpene glycoside, such as those isolated from the bark of the Quillaja saponaria tree. A saponin extract from a biological source can be further fractionated (e.g., by chromatography) to isolate the portions of the extract with the best adjuvant activity and with acceptable toxicity. Typical fractions of extract from Quillaja saponaria tree used as adjuvants are known as fractions A and C. An exemplary saponin adjuvant is QS-21 (fraction C), which is available from Antigenics. QS-21 is an oligosaccharide-conjugated small molecule. Optionally, QS-21 may be admixed with a lipid such as 3D-MPL or cholesterol. [0240] A particular form of saponins that may be used in vaccine formulations described herein is immunostimulating complexes (ISCOMs). ISCOMs are an art-recognized class of adjuvants, that generally comprise Quillaja saponin fractions and lipids (e.g., cholesterol and phospholipids such as phosphatidyl choline). In certain embodiments, an ISCOM is assembled together with a polypeptide or nucleic acid of interest. However, different saponin fractions may be used in different ratios. In addition, the different saponin fractions may either exist together in the same particles or have substantially only one fraction per particle (such that the indicated ratio of fractions A and C are generated by mixing together particles with the different fractions). In this context, "substantially" refers to less than 20%, 15%, 10%, 5%, 4%, 3%, 2% or even 1%. Such adjuvants may comprise fraction A and fraction C mixed into a ratio of 70-95 A: 30-5 C, such as 70 A : 30 C to 75 A : 25 C; 75 A : 25 C to 80 A : 20 C; 80 A : 20 C to 85 A : 15 C; 85 A : 15 C to 90 A : 10 C; 90 A : 10 C to 95 A : 5 C; or 95 A : 5 C to 99 A : 1 C. ISCOMatrix, produced by CSL, and AblSCO 100 and 300, produced by Isconova, are ISCOM matrices comprising saponin, cholesterol and phospholipid (lipids from cell membranes), which form cage-like structures typically 40-50 nm in diameter. Posintro, produced by Nordic Vaccines, is an ISCOM matrix where the immunogen is bound to the particle by a multitude of different mechanisms, e.g., electrostatic interaction by charge modification, incorporation of chelating groups, or direct binding.
[0241] In some embodiments, the adjuvant is Matrix-M2 (MM2). In some embodiments, the Matrix-M2 adjuvant comprises saponin fractions purified from Quillaja saponaria (soapbark tree) bark, phosphatidylcholine and cholesterol. In some embodiments, the adjuvant is diluted in normal saline, for example 0.9% saline.
[0242] In some embodiments, the adjuvant is a TLR ligand. TLRs are proteins that may be found on leukocyte membranes, and recognize foreign antigens (including microbial antigens). An exemplary TLR ligand is IC-31, which is available from Intercell. IC-31 comprises an anti -microbial peptide, KLK, and an immunostimulatory oligodeoxynucleotide, ODNla. IC- 31 has TLR9 agonist activity. Another example is CpG-containing DNA. Different varieties of CpG-containing DNA are available from Prizer (Coley): Vaxlmmune is CpG 7909 (a (CpG)- containing oligodeoxy-nucleotide), and Actilon is CpG 10101 (a (CpG)-containing oligodeoxynucleotide).
[0243] In some embodiments, the adjuvant is a nanoemulsion. One exemplary nanoemulsion adjuvant is Nanostat Vaccine, produced by Nanobio. This nanoemulsion is a high- energy, oil-in-water emulsion. This nanoemulsion typically has a size of 150-400 nanometers, and includes surfactants to provide stability. More information about Nanostat can be found in US Patents 6,015,832, 6,506,803, 6,559,189, 6,635,676, and 7,314,624.
[0244] In some embodiments, an adjuvant includes a cytokine. In some embodiments, the cytokine is an interleukin such as IL-1, IL-6, IL-12, IL-17 and IL-23. In some embodiments, the cytokine is granulocyte-macrophage colony-stimulating factor (GM-CSF). The adjuvant may include cytokine as a purified polypeptide. Alternatively, the adjuvant may include nucleic acids encoding the cytokine.
[0245] Adjuvants may be covalently bound to antigens (e.g., the polypeptides described above). In some embodiments, the adjuvant may be a protein which induces inflammatory responses through activation of APCs. In some embodiments, one or more of these proteins can be recombinantly fused with an antigen of choice, such that the resultant fusion molecule promotes dendritic cell maturation, activates dendritic cells to produce cytokines and chemokines, and ultimately, enhances presentation of the antigen to T cells and initiation of T cell responses (see Wu et al., Cancer Res 2005; 65(11), pp 4947-4954). Other exemplary adjuvants that may be covalently bound to antigens comprise polysaccharides, synthetic peptides, lipopeptides, and nucleic acids.
[0246] The adjuvant can be used alone or in combination of two or more kinds. Adjuvants may be directly conjugated to antigens. Adjuvants may be administered in therapeutically effective amounts, for example, an amount that produces the desired effect (e.g., immunostimulatory effect) for which it is administered. Adjuvants may also be combined to increase the magnitude of the immune response to the antigen. Typically, the same adjuvant or mixture of adjuvants is present in each dose of an immunogenic composition (e.g., a vaccine and/or a vaccine formulation). Optionally, however, an adjuvant may be administered with a first dose of an immunogenic composition and not with subsequent doses (e.g., additional dose(s) or maintenance dose(s)). Alternatively, a strong adjuvant may be administered with the first dose of an immunogenic composition and a weaker adjuvant or lower dose of the strong adjuvant may be administered with subsequent doses. The adjuvant can be administered before the administration of the antigen, concurrent with the administration of the antigen or after the administration of the antigen to a subject (sometimes within 1, 2, 6, or 12 hours, and sometimes within 1, 2, or 5 days). Certain adjuvants are appropriate for human patients, non-human animals, or both.
Additional Components
[0247] In addition to the antigens and the adjuvants described above, an immunogenic composition, e.g., a vaccine, a vaccine formulation and/or a pharmaceutical composition, may include one or more additional components.
[0248] In certain embodiments, an immunogenic composition may include one or more stabilizers such as sugars (such as sucrose, glucose, or fructose), phosphate (such as sodium phosphate dibasic, potassium phosphate monobasic, dibasic potassium phosphate, or monosodium phosphate), glutamate (such as monosodium L-glutamate), gelatin (such as processed gelatin, hydrolyzed gelatin, or porcine gelatin), amino acids (such as arginine, asparagine, histidine, L-histidine, alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and the alkyl esters thereof), inosine, or sodium borate.
[0249] In certain embodiments, an immunogenic composition includes one or more buffers such as a mixture of sodium bicarbonate and ascorbic acid. In some embodiments, an immunogenic composition may be administered in saline (e.g., 0.9% saline), such as phosphate buffered saline (PBS), or distilled water.
[0250] In certain embodiments, an immunogenic composition includes one or more surfactants such as polysorbate 20 (Tween 20), polysorbate 80 (Tween 80), Polyethylene glycol tert-octylphenyl ether t-Octylphenoxypolyethoxyethanol 4-(l,l,3,3-Tetramethylbutyl)phenyl- polyethylene glycol (TRITON X-100); Polyoxy ethylenesorbitan monolaurate Polyethylene glycol sorbitan monolaurate (TWEEN 20); and 4-(l,l,3,3-Tetramethylbutyl)phenol polymer with formaldehyde and oxirane (TYLOXAPOL). A surfactant can be ionic or nonionic.
[0251] In certain embodiments, an immunogenic composition includes one or more salts such as sodium chloride, ammonium chloride, calcium chloride, or potassium chloride.
[0252] In certain embodiments, a preservative is included in an immunogenic composition. In other embodiments, no preservative is used. A preservative is most often used in multi-dose vaccine vials, and is less often needed in single-dose vaccine vials. In certain embodiments, the preservative is 2-phenoxyethanol, methyl and propyl parabens, benzyl alcohol, and/or sorbic acid.
[0253] In certain embodiments, an immunogenic composition is a controlled-release formulation.
Dosing Regimens
[0254] In some embodiments, an immunogenic composition is administered to a subject according to a dosing regimen or dosing schedule. The amount of antigen in each immunogenic composition dose (e.g., a vaccine, vaccine formulation and/or pharmaceutical composition) is selected to be a therapeutically effective amount, which induces a prophylactic or therapeutic response, as described above, in either a single dose or over multiple doses. Preferably, a dose is without significant adverse side effects in typical immunogenic compositions. Such amount will vary depending upon which specific antigen is employed. Generally, it is expected that a single dose will comprise about 100 to about 1500 pg total peptide. In some embodiments, a total volume of a single dose is 0.5 mL to 1.0 mL. In some embodiments, a single dose will comprise more than one antigen, for example, 2, 3, 4, 5 or more.
[0255] In some embodiments, a dosing regimen comprises an initial dose of an immunogenic composition and at least one additional dose of the immunogenic composition. In some embodiments, after an initial dose is administered, an additional dose is administered about 3 weeks following the initial dose. In some embodiments, an additional dose is administered about 6 weeks following the initial dose. In some embodiments, an additional dose is administered about 12 weeks following the initial dose. In some embodiments, and an additional dose is administered about 24 weeks following the initial dose.
[0256] In some embodiments, the dosing regimen comprises administration of different immunogenic compositions, e.g., 2, 3, 4, 5, 6, 7, 8, or more different immunogenic compositions comprising antigens. In some embodiments, each dose comprises administering different immunogenic compositions, e.g., in succession. In some embodiments, each dose comprises administering the same set of different immunogenic compostions. For example, a dosing regimen can include an initial dose of 2, 3, 4, 5, 6, 7, 8, or more different immunogenic compositions, and at least one additional dose of the 2, 3, 4, 5, 6, 7, 8, or more different immunogenic compositions. In some embodiments, an immunogenic composition comprises one antigen. In some embodiments, an immunogenic composition comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.
[0257] In some embodiments, a dosing regimen can include an initial dose of 2, 3, 4, 5, 6, 7, 8, or more different immunogenic compositions (e.g., where each immunogenic composition can separately include 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens), and at least one additional dose of the 2, 3, 4, 5, 6, 7, 8, or more different immunogenic compositions. For example, a dosing regimen can include an initial dose of 4 different immunogenic compositions, where each immunogenic composition comprises 1, 2, 3, 4 or 5 different antigens. In some embodiments, such dosing regimen further includes at least 1 (e.g., at least 2, 3, 4, 5, 6, or more) additional doses of the 4 different immunogenic compositions. In some embodiments, a second dose is administered about 1, 2, 3, 4, or 5 weeks after the initial dose; a third dose is administered about 1, 2, 3, 4, or 5 weeks after the second dose; a fourth dose is administered about 2, 4, 6, 8, 10, 12, 14, 16, or 18 weeks after the third dose; and a fifth dose is administered about 2, 4, 6, 8, 10, 12, 14, 16, or 18 weeks after the fourth dose. Uses
[0258] In some embodiments, tumor antigens are used in diagnostic assays. For these assays, compositions including the tumor antigens can be provided in kits, e.g., for detecting antibody reactivity, or cellular reactivity, in a sample from an individual.
[0259] In some embodiments, tumor antigen compositions are used to induce an immune response in a subject. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The tumor antigen compositions can be used to raise antibodies (e.g., in a non-human animal, such as a mouse, rat, hamster, or goat), e.g., for use in diagnostic assays, and for therapeutic applications. For an example of a therapeutic use, a tumor antigen discovered by a method described herein may be a potent T cell and/or B cell antigen.
Preparations of antibodies may be produced by immunizing a subject with the tumor antigen and isolating antiserum from the subject. Methods for eliciting high titers of high affinity, antigenspecific antibodies, and for isolating the tumor antigen-specific antibodies from antisera, are known in the art. In some embodiments, the tumor antigen compositions are used to raise monoclonal antibodies, e.g., human monoclonal antibodies.
[0260] In some embodiments, a tumor antigen composition is used to induce an immune response in a human subject to provide a therapeutic response. In some embodiments, a tumor antigen composition is used to induce an immune response in a human subject that redirects an undesirable immune response. In some embodiments, a tumor antigen composition elicits an immune response that causes the subject to have a positive clinical response described herein, e.g., as compared to a subject who has not been administered the tumor antigen composition. In some embodiments, a tumor antigen composition elicits an immune response that causes the subject to have an improved clinical response, e.g., as compared to a subject who has not been administered the tumor antigen composition. In some embodiments, a tumor antigen composition is used to induce an immune response in a human subject for palliative effect. The response can be complete or partial therapy. [0261] In some embodiments, a tumor antigen composition is used to induce an immune response in a human subject to provide a prophylactic response. The response can be complete or partial protection.
[0262] In some embodiments, immunogenicity of a tumor antigen is evaluated in vivo. In some embodiments, humoral responses to a tumor antigen are evaluated (e.g., by detecting antibody titers to the administered tumor antigen). In some embodiments, cellular immune responses to a tumor antigen are evaluated, e.g., by detecting the frequency of antigen-specific cells in a sample from the subject (e.g., by staining T cells from the subject with MHC/peptide tetramers containing the antigenic peptide, to detect antigen-specific T cells, or by detecting antigen-specific cells using an antigen presentation assay such as an assay described herein). In some embodiments, the ability of a tumor antigen or antigens to elicit protective or therapeutic immunity is evaluated in an animal model. In some embodiments, the ability of a tumor antigen or antigens to stimulate or to suppress and/or inhibit immunity is evaluated in an animal model.
Cancer and Cancer Therapy
[0263] The present disclosure provides methods and systems related to subjects having or diagnosed with cancer, such as a tumor. In some embodiments, a tumor is or comprises a hematologic malignancy, including but not limited to, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, AIDS-related lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, Langerhans cell histiocytosis, multiple myeloma, or myeloproliferative neoplasms.
[0264] In some embodiments, a tumor is or comprises a solid tumor, including but not limited to breast carcinoma, a squamous cell carcinoma, a colon cancer, a head and neck cancer, ovarian cancer, a lung cancer, mesothelioma, a genitourinary cancer, a rectal cancer, a gastric cancer, or an esophageal cancer.
[0265] In some particular embodiments, a tumor is or comprises an advanced tumor, and/or a refractory tumor. In some embodiments, a tumor is characterized as advanced when certain pathologies are observed in a tumor (e.g., in a tissue sample, such as a biopsy sample, obtained from a tumor) and/or when cancer patients with such tumors are typically considered not to be candidates for conventional chemotherapy. In some embodiments, pathologies characterizing tumors as advanced can include tumor size, altered expression of genetic markers, invasion of adjacent organs and/ or lymph nodes by tumor cells. In some embodiments, a tumor is characterized as refractory when patients having such a tumor are resistant to one or more known therapeutic modalities (e.g., one or more conventional chemotherapy regimens) and/or when a particular patient has demonstrated resistance (e.g., lack of responsiveness) to one or more such known therapeutic modalities.
[0266] In some embodiments, the present disclosure provides methods and systems related to cancer therapy. The present disclosure is not limited to any specific cancer therapy, and any known or developed cancer therapy is encompassed by the present disclosure. Known cancer therapies include, e.g., administration of chemotherapeutic agents, radiation therapy, surgical excision, chemotherapy following surgical excision of tumor, adjuvant therapy, localized hypothermia or hyperthermia, anti-tumor antibodies, and anti-angiogenic agents. In some embodiments, cancer and/or adjuvant therapy includes a TLR agonist (e.g., CpG, Poly I:C, etc., see, e.g., Wittig et al., Crit. Rev. Oncol. Hematol. 94:31-44 (2015); Huen et al., Curr. Opin. Oncol. 26:237-44 (2014); Kaczanowska et al., J. Leukoc. Biol. 93:847-863 (2013)), a STING agonist (see, e.g., US20160362441; US20140329889; Fu et al., Sci. Transl. Med. 7:283ra52 (2015); and WO2014189805), a non-specific stimulus of innate immunity, and/or dendritic cells, or administration of GM-CSF, Interleukin- 12, Interleukin-7, Flt-3, or other cytokines. In some embodiments, the cancer therapy is or comprises oncolytic virus therapy, e.g., talimogene leherparepvec. (see, e.g., Fukuhara et al., Cancer Sci. 107: 1373-1379 (2016)). In some embodiments, the cancer therapy is or comprises bi-specific antibody therapy (e.g., Choi et al., 2011 Expert Opin Biol Ther, Huehls et al., 2015, Immunol and Cell Biol). In some embodiments, the cancer therapy is or comprises cellular therapy such as chimeric antigen receptor T (CAR-T) cells, TCR-transduced T cells, dendritic cells, tumor infiltrating lymphocytes (TIL), or natural killer (NK) cells (e.g., as reviewed in Sharpe and Mount, 2015, Dis Model Meeh 8:337-50). [0267] Anti-tumor antibody therapies (i.e., therapeutic regimens that involve administration of one or more anti-tumor antibody agents) are rapidly becoming the standard of care for treatment of many tumors. Antibody agents have been designed or selected to bind to tumor antigens, particularly those expressed on tumor cell surfaces. Various review articles have been published that describe useful anti-tumor antibody agents (see, for example, Adler et al., Hematol. Oncol. Clin. North Am. 26:447-81 (2012); Li et al., Drug Discov. Ther. 7: 178-84 (2013); Scott et al., Cancer Immun. 12: 14 (2012); and Sliwkowski et al., Science 341 : 1192-1198 (2013)). The below Table 3 presents a non-comprehensive list of certain human antigens targeted by known, available antibody agents, and notes certain cancer indications for which the antibody agents have been proposed to be useful:
Table 3: Human Antigens Targeted by Antibody Agents
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
Figure imgf000113_0001
Immunotherapy
[0268] In some embodiments, a cancer therapy is or comprises an immunotherapy. In some embodiments, an immunotherapy is or comprises immune checkpoint blockade therapy (see, e.g., Martin-Liberal et al., Cancer Treat. Rev. 54:74-86 (2017); Menon et al., Cancers (Basel) 8: 106 (2016)), or immune suppression blockade therapy. Certain cancer cells thrive by taking advantage of immune checkpoint pathways as a major mechanism of immune resistance, particularly with respect to T cells that are specific for tumor antigens. For example, certain cancer cells may overexpress one or more immune checkpoint proteins responsible for inhibiting a cytotoxic T cell response. Thus, immune checkpoint blockade therapy may be administered to overcome the inhibitory signals and permit and/or augment an immune attack against cancer cells. Immune checkpoint blockade therapy may facilitate immune cell responses against cancer cells by decreasing, inhibiting, or abrogating signaling by negative immune response regulators (e.g., CTLA-4). In some embodiments, a cancer therapy or may stimulate or enhance signaling of positive regulators of immune response (e.g., CD28).
[0269] Examples of immune checkpoint blockade and immune suppression blockade therapy include agents targeting one or more of A2AR, B7-H4, BTLA, CTLA-4, CD28, CD40, CD137, GITR, IDO, KIR, LAG-3, PD-1, PD-L1, 0X40, TIM-3, TIGIT, CBLB, CISH, B7-H3, CSF-1R, VISTA, . Specific examples of immune checkpoint blockade agents include the following monoclonal antibodies: ipilimumab (targets CTLA-4); tremelimumab (targets CTLA- 4); atezolizumab (targets PD-L1); pembrolizumab (targets PD-1); nivolumab (targets PD-1); avelumab; durvalumab; spartalizumab; camrelizumab; cabiralizumab; cemiplimab; dostarlimab; sintilmab; tislelizumab; and toripalirnab.
[0270] The best known of these are monoclonal antibodies that block CTLA-4 and PD-1 pathways, representing critical inhibitory checkpoints that restrain T cells from full and persistent activation and proliferation under normal physiologic conditions. Blockade of CTLA- 4 and/or PD-1 pathways can result in durable regressions for patients with a widening spectrum of malignancies. In some embodiments, an immunotherapy is or comprises administration of one or more of PD-1 or PD-L1 blockade therapies. In some embodiments, an immunotherapy is or comprises administration of one or more of CTLA-4 blockade therapies. In some embodiments, an immunotherapy can include any of ipilumimab and tremelimumab which target CTLA-4; pembrolizumab, nivolumab, avelumab, durvalumab, and atezoluzumab, which target PD-1; or combinations thereof.
[0271] In some embodiments, immunotherapy (e.g. immune checkpoint blockade therapy) involves administration of an agent that acts as a blockade of cytotoxic T-lymphocyte- associated protein 4 (CTLA-4). In certain embodiments, immunotherapy involves treatment with an agent that interferes with an interaction involving CTLA-4 (e.g., with CD80 or CD86). In some embodiments, immunotherapy involves administration of one or more of tremelimumab and/or ipilimumab. In some embodiments, immunotherapy (e.g. immune checkpoint blockade therapy) involves administration of an agent (e.g. antibody agent) that acts as a blockade of programmed cell death 1 (PD-1). In certain embodiments, immunotherapy involves treatment with an agent that interferes with an interaction involving PD-1 (e.g., with PD-L1). In some embodiments, immunotherapy involves administration of an agent (e.g. antibody agent) that specifically interacts with PD-1 or with PD-L1. In some embodiments, immunotherapy (e.g. immune checkpoint blockade therapy) involves administration of one or more of nivolumab, pembrolizumab, atezolizumab, avelumab, and/or durvalumab.
[0272] Specific examples of immune suppression blockade agents include: Vista (B7- H5, v-domain Ig suppressor of T cell activation) inhibitors; Lag-3 (lymphocyte-activation gene 3, CD223) inhibitors; IDO (indolemamine-pyrrole-2,3,-dioxygenase-l,2) inhibitors; KIR receptor family (killer cell immunoglobulin-like receptor) inhibitors; CD47 inhibitors; and TIGIT (T cell immunoreceptor with Ig and ITIM domain) inhibitors.
PD-1
[0273] PD-1 is expressed on T cells, B cells, and certain myeloid cells; however, its role is best characterized in T cells. PD-1 expression on T cells is induced by antigen stimulation. Unlike CTLA-4, which limits early T-cell activation, PD-1 mainly exerts its inhibitory effect on T cells in the periphery where T cells encounter PD-1 ligands. Two ligands of PD-1 have been identified so far, PD-L1 and PD-L2, which are expressed by a large range of cell types, including tumor cells, monocyte-derived myeloid dendritic cells, epithelial cells, T cells, and B cells. In cancer, tumor cells and myeloid cells are thought to be main cell types mediating T-cell suppression through PD-1 ligation. It is still unclear whether effects of PD-L1 and PD-L2 on PD-1 downstream signaling are dependent on cell type that expresses a given ligand. Moreover, there are differences between PD-L1- versus PD-L2-induced effects, which remain to be fully elucidated.
[0274] Several mechanisms of PD-1 -mediated T cell suppression have been proposed. One mechanism suggests that PD-1 ligation inhibits T cell activation only upon T cell receptor engagement. PD-1 has an intracellular “immunoreceptor tyrosine-based inhibition motif’ or (ITIM) and an immunoreceptor tyrosine-based switch motif. It has been shown that PD-1 ligation leads to recruitment of phosphatases called “src homology 2 domain-containing tyrosine phosphatases,” or SHP-1 and SHP-2, to immunoreceptor tyrosine-based switch motif.
Moreover, PD-1 ligation has been shown to interfere with signaling molecules, such as phosphatidylinositol-4,5-bisphosphate 3-kinase and Ras, which are important for T-cell proliferation, cytokine secretion, and metabolism. Analysis of human immunodeficiency virus (HlV)-specific T cells has also demonstrated PD-1 -dependent basic leucine zipper transcription factor upregulation, which inhibits T cell function. Ligation of PD-1 has also been shown to induce metabolic alterations in T cells. Metabolic reprogramming of T cells from glycolysis to lipolysis is a consequence of PD-1 -mediated impairment of T-cell effector function.
Furthermore, PD-l-induced defects in mitochondrial respiration and glycolysis leads to impaired T-cell effector function that could be reversed by a mammalian target of rapamycin inhibition. Since most of the identified mechanisms of PD-1 -mediated T-cell suppression are based on in vitro or ex vivo experiments, it remains to be demonstrated that these same mechanisms are responsible for T-cell exhaustion in vivo.
[0275] PD-1 is a type I membrane protein of 288 amino acids and is a member of the extended CD28/CTLA-4 family of T cell regulators. PD-1 protein structure includes an extracellular IgV domain followed by a transmembrane region and an intracellular tail, which contains two phosphorylation sites located in an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif, which suggests that PD-1 negatively regulates T cell receptor signals. This is consistent with binding of SHP-1 and SHP-2 phosphatases to PD-1 cytoplasmic tail upon ligand binding. In addition, PD-1 ligation up- regulates E3 -ubiquitin ligases CBL-b and c-CBL that trigger T cell receptor down-modulation. PD-1 is expressed on the surface of activated T cells, B cells, and macrophages, suggesting that compared to CTLA-4, PD-1 more broadly negatively regulates immune responses.
CTLA-4
[0276] CTLA-4 is a member of the immunoglobulin superfamily that is expressed by activated T cells and transmits an inhibitory signal to T cells. CTLA-4 is structurally similar to T cell co-stimulatory protein, CD28, and both molecules bind to CD80 and CD86 on antigen- presenting cells. CTLA-4 binds CD80 and CD86 with greater affinity than CD28, thus enabling it to outcompete CD28 for its ligands. CTLA-4 transmits an inhibitory signal to T cells, whereas CD28 transmits a stimulatory signal. T cell activation through T cell receptor and CD28 leads to increased expression of CTLA-4.
[0277] The mechanism by which CTLA-4 acts in T cells remains somewhat elusive. Biochemical evidence suggests that CTLA-4 recruits a phosphatase to a T cell receptor, thus attenuating the signal. It has also been suggested that CTLA-4 may function in vivo by capturing and removing CD80 and CD86 from membranes of antigen-presenting cells, thus making these antigens unavailable for triggering of CD28.
[0278] CTLA-4 protein contains an extracellular V domain, a transmembrane domain, and a cytoplasmic tail. CTLA-4 has an intracellular domain that is similar to that of CD28, in that it has no intrinsic catalytic activity and contains one YVKM motif able to bind PI3K, PP2A and SHP-2, as well as one proline-rich motif able to bind SH3 -containing proteins. One role of CTLA-4 in inhibiting T cell responses seems to directly involve SHP-2 and PP2A dephosphorylation of T cell receptor-proximal signaling proteins, such as CD3 and LAT. CTLA- 4 can also affect signaling indirectly, via competition with CD28 for CD80 and/or CD86 binding. [0279] The first clinical evidence that modulation of T cell activation could result in effective anti-cancer therapy came from development of CTLA-4 blockade antibody ipilimumab. In some embodiments, ipilimumab is a human IgGl antibody with specificity for CTLA-4. In some embodiments, another CTLA-4 blockade therapy, tremelimumab, is a human IgG2 antibody.
[0280] In some embodiments, an immunotherapyis or comprises immune activation therapy. Specific examples of immune activators include: CD40 agonists; GITR (glucocorticoid-induced TNF-R-related protein, CD357) agonists; 0X40 (CD134) agonists; 4- 1BB (CD137) agonists; ICOS (inducible T cell stimulator); CD278 agonists; IL-2 (interleukin 2) agonists; and interferon agonists.
[0281] In some embodiments, an immunotherapy is or comprises a combination of one or more immune checkpoint blockade agents, immune suppression blockade agents, and/or immune activators, or a combination of one or more immune checkpoint blockade agents, immune suppression blockade agents, and/or immune activators, and other cancer therapies.
Administration of Immune Checkpoint Blockade Therapy
[0282] In accordance with certain methods of the invention, an immune checkpoint blockade therapy is and/or has been administered to an individual.
[0283] Those of ordinary skill in the art will appreciate that appropriate formulations, indications, and dosing regimens are typically analyzed and approved by government regulatory authorities such as the Food and Drug Administration in the United States. For example, Examples 4 and 5 present certain FDA-approved dosing information for PD-1 and CTLA-4 blockade regimens, respectively. In some embodiments an immune checkpoint blockade therapy is administered in accordance with the present invention according to such an approved protocol.
[0284] In some embodiments an immune checkpoint blockade therapy is administered in a pharmaceutical composition that also comprises a physiologically acceptable carrier or excipient. In some embodiments, a pharmaceutical composition is sterile. In many embodiments, a pharmaceutical composition is formulated for a particular mode of administration.
[0285] In some embodiments, suitable pharmaceutically acceptable carriers include but are not limited to water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, sugars such as mannitol, sucrose, or others, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, etc., as well as combinations thereof. In some embodiments, a pharmaceutical preparation can, if desired, comprise one or more auxiliary agents (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, flavoring and/or aromatic substances and the like) which do not deleteriously react with the active compounds or interference with their activity. In some embodiments, a water-soluble carrier suitable for intravenous administration is used.
[0286] In some embodiments, a pharmaceutical composition or medicament, if desired, can contain an amount (typically a minor amount) of wetting or emulsifying agents, and/or of pH buffering agents. In some embodiments, a pharmaceutical composition can be a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. In some embodiments, a pharmaceutical composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. In some embodiments, oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharine, cellulose, magnesium carbonate, etc.
[0287] In some embodiments, a pharmaceutical composition can be formulated in accordance with routine procedures as a pharmaceutical composition adapted for administration to human beings. For example, in some embodiments, a composition for intravenous administration typically is a solution in sterile isotonic aqueous buffer. In some embodiments, where necessary, a composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. In some embodiments, generally, ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachet indicating the quantity of active agent. In some embodiments, where a composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. In some embodiments, where a composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
[0288] In some embodiments, an immune checkpoint blockade therapy can be formulated in a neutral form; in some embodiments it may be formulated in a salt form. In some embodiments, pharmaceutically acceptable salts include those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
[0289] Pharmaceutical compositions for use in accordance with the present invention may be administered by any appropriate route. In some embodiments, a pharmaceutical composition is administered intravenously. In some embodiments, a pharmaceutical composition is administered subcutaneously. In some embodiments, a pharmaceutical composition is administered by direct administration to a target tissue, such as heart or muscle (e.g., intramuscular), or nervous system (e.g., direct injection into the brain; intraventricularly; intrathecally). Alternatively or additionally, in some embodiments, a pharmaceutical composition is administered parenterally, transdermally, or transmucosally (e.g., orally or nasally). More than one route can be used concurrently, if desired.
[0290] In some embodiments, an immune checkpoint blockade therapy, can be administered alone, or in conjunction with other immunomodulatory agents. The term, “in conjunction with,” indicates that a first an immune checkpoint blockade therapy is administered prior to, at about the same time as, or following another an immune checkpoint blockade therapy. In some embodiments, a first an immune checkpoint blockade therapy can be mixed into a composition containing one or more different an immune checkpoint blockade therapies, and thereby administered contemporaneously; alternatively, in some embodiments, the agent can be administered contemporaneously, without mixing (e.g., by “piggybacking” delivery of the agent on the intravenous line by which the an immune checkpoint blockade therapy is also administered, or vice versa). In some embodiments, an immune checkpoint blockade therapy can be administered separately (e.g., not admixed), but within a short time frame (e.g., within 24 hours) of administration of another an immune checkpoint blockade therapy.
[0291] In some embodiments, subjects treated with an immune checkpoint blockade therapy are administered one or more immunosuppressants. In some embodiments, one or more immunosuppressants are administered to decrease, inhibit, or prevent an undesired autoimmune response (e.g., enterocolitis, hepatitis, dermatitis (including toxic epidermal necrolysis), neuropathy, and/or endocrinopathy), for example, hypothyroidism. In some embodiments, exemplary immunosuppressants include steroids, antibodies, immunoglobulin fusion proteins, and the like. In some embodiments, an immunosuppressant inhibits B cell activity (e.g. rituximab). In some embodiments, an immunosuppressant is a decoy polypeptide antigen.
[0292] In some embodiments, an immune checkpoint blockade therapy is administered in a therapeutically effective amount (e.g., a dosage amount and/or according to a dosage regimen that has been shown, when administered to a relevant population, to be sufficient to treat cancer, such as by ameliorating symptoms associated with the cancer, preventing or delaying the onset of the cancer, and/or also lessening the severity or frequency of symptoms of cancer). In some embodiments, long term clinical benefit is observed after treatment with an immune checkpoint blockade therapy, including, for example, PD-1 blockade such as pembrolizumab, CTLA-4 blockade such as ipilimumab, and/or other agents. Those of ordinary skill in the art will appreciate that a dose which will be therapeutically effective for the treatment of cancer in a given patient may depend, at least to some extent, on the nature and extent of cancer, and can be determined by standard clinical techniques. In some embodiments, one or more in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. In some embodiments, a particular dose to be employed in the treatment of a given individual may depend on the route of administration, the extent of cancer, and/or one or more other factors deemed relevant in the judgment of a practitioner in light of patient’s circumstances. In some embodiments, effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems (e.g., as described by the U.S. Department of Health and Human Services, Food and Drug Administration, and Center for Drug Evaluation and Research in “Guidance for Industry: Estimating Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers”, Pharmacology and Toxicology, July 2005).
[0293] In some embodiments, a therapeutically effective amount of an immune checkpoint blockade therapy can be, for example, more than about 0.01 mg/kg, more than about 0.05 mg/kg, more than about 0.1 mg/kg, more than about 0.5 mg/kg, more than about 1.0 mg/kg, more than about 1.5 mg/kg, more than about 2.0 mg/kg, more than about 2.5 mg/kg, more than about 5.0 mg/kg, more than about 7.5 mg/kg, more than about 10 mg/kg, more than about 12.5 mg/kg, more than about 15 mg/kg, more than about 17.5 mg/kg, more than about 20 mg/kg, more than about 22.5 mg/kg, or more than about 25 mg/kg body weight. In some embodiments, a therapeutically effective amount can be about 0.01-25 mg/kg, about 0.01-20 mg/kg, about 0. QI- 15 mg/kg, about 0.01-10 mg/kg, about 0.01-7.5 mg/kg, about 0.01-5 mg/kg, about 0.01-4 mg/kg, about 0.01-3 mg/kg, about 0.01-2 mg/kg, about 0.01-1.5 mg/kg, about 0.01-1.0 mg/kg, about 0.01-0.5 mg/kg, about 0.01-0.1 mg/kg, about 1-20 mg/kg, about 4-20 mg/kg, about 5-15 mg/kg, about 5-10 mg/kg body weight. In some embodiments, a therapeutically effective amount is about 0.01 mg/kg, about 0.05 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1.0 mg/kg, about 1.1 mg/kg, about 1.2 mg/kg, about 1.3 mg/kg about 1.4 mg/kg, about 1.5 mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, about 1.9 mg/kg, about 2.0 mg/kg, about 2.5 mg/kg, about 3.0 mg/kg, about 4.0 mg/kg, about 5.0 mg/kg, about 6.0 mg/kg, about 7.0 mg/kg, about 8.0 mg/kg, about 9.0 mg/kg, about 10.0 mg/kg, about 11.0 mg/kg, about 12.0 mg/kg, about 13.0 mg/kg, about 14.0 mg/kg, about 15.0 mg/kg, about 16.0 mg/kg, about 17.0 mg/kg, about 18.0 mg/kg, about 19.0 mg/kg, about 20.0 mg/kg, body weight, or more. In some embodiments, the therapeutically effective amount is no greater than about 30 mg/kg, no greater than about 20 mg/kg, no greater than about 15 mg/kg, no greater than about 10 mg/kg, no greater than about 7.5 mg/kg, no greater than about 5 mg/kg, no greater than about 4 mg/kg, no greater than about 3 mg/kg, no greater than about 2 mg/kg, or no greater than about 1 mg/kg body weight or less.
[0294] In some embodiments, the administered dose for a particular individual is varied (e.g., increased or decreased) over time, depending on the needs of the individual.
[0295] In some embodiments, a loading dose (e.g., an initial higher dose) of a therapeutic composition may be given at the beginning of a course of treatment, followed by administration of a decreased maintenance dose (e.g., a subsequent lower dose) of the therapeutic composition. Without wishing to be bound by any theories, it is contemplated that a loading dose may clear out an initial and, in some cases massive, accumulation of undesirable materials (e.g., fatty materials and/or tumor cells, etc) in tissues (e.g., in the liver), and maintenance dosing may delay, reduce, or prevent buildup of fatty materials after initial clearance.
[0296] In some embodiments, it will be appreciated that a loading dose and maintenance dose amounts, intervals, and duration of treatment may be determined by any available method, such as those exemplified herein and those known in the art. In some embodiments, a loading dose amount is about 0.01-1 mg/kg, about 0.01-5 mg/kg, about 0.01-10 mg/kg, about 0.1-10 mg/kg, about 0.1-20 mg/kg, about 0.1-25 mg/kg, about 0.1-30 mg/kg, about 0.1-5 mg/kg, about 0.1-2 mg/kg, about 0.1-1 mg/kg, or about 0.1-0.5 mg/kg body weight. In some embodiments, a maintenance dose amount is about 0-10 mg/kg, about 0-5 mg/kg, about 0-2 mg/kg, about 0-1 mg/kg, about 0-0.5 mg/kg, about 0-0.4 mg/kg, about 0-0.3 mg/kg, about 0-0.2 mg/kg, about 0- 0.1 mg/kg body weight. In some embodiments, a loading dose is administered to an individual at regular intervals for a given period of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months) and/or a given number of doses (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or more doses), followed by maintenance dosing. In some embodiments, a maintenance dose ranges from 0 - 2 mg/kg, about 0-1.5 mg/kg, about 0-1.0 mg/kg, about 0-0.75 mg/kg, about 0-0.5 mg/kg, about 0-0.4 mg/kg, about 0-0.3 mg/kg, about 0-0.2 mg/kg, or about 0-0.1 mg/kg body weight. In some embodiments, a maintenance dose is about 0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 1.8, or 2.0 mg/kg body weight. In some embodiments, maintenance dosing is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months. In some embodiments, maintenance dosing is administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more years. In some embodiments, maintenance dosing is administered indefinitely (e.g., for life time).
[0297] In some embodiments, a therapeutically effective amount of an immune checkpoint blockade therapy may be administered as a one-time dose or administered at intervals, depending on the nature and extent of the cancer, and on an ongoing basis. Administration at an “interval,” as used herein indicates that the therapeutically effective amount is administered periodically (as distinguished from a one-time dose). In some embodiments, an interval can be determined by standard clinical techniques. In some embodiments, an immunomodulatory agent (e.g. immune checkpoint modulator) is administered bimonthly, monthly, twice monthly, triweekly, biweekly, weekly, twice weekly, thrice weekly, or daily. In some embodiments, the administration interval for a single individual need not be a fixed interval, but can be varied over time, depending on the needs and rate of recovery of the individual.
[0298] As used herein, those of skill in the art are familiar with certain terms commonly used to describe dosing regimens. For example, the term “bimonthly” has its art understood meaning, referring to administration once per two months (i.e., once every two months); the term “monthly” means administration once per month; the term “triweekly” means administration once per three weeks (i.e., once every three weeks); the term “biweekly” means administration once per two weeks (i.e., once every two weeks); the term “weekly” means administration once per week; and the term “daily” means administration once per day.
[0299] The invention, among other things, additionally pertains to a pharmaceutical composition comprising an immune checkpoint blockade therapy, as described herein; in some embodiments in a container (e.g., a vial, bottle, bag for intravenous administration, syringe, etc.) with a label containing instructions for administration of the composition for treatment of cancer. Patients
[0300] As discussed herein, in some embodiments, the present disclosure provides methods and systems related to subjects who do not respond and/or have not responded; or respond and/or have responded (e.g., clinically responsive, e.g., clinically positively responsive or clinically negatively responsive) to a cancer therapy. In some embodiments, subjects respond and/or have responded positively clinically to a cancer therapy. In some embodiments, subjects respond and/or have responded negatively clinically to a cancer therapy. In some embodiments, subjects do not respond and/or have not responded (e.g., clinically non-responsive or refractory) to a cancer therapy.
[0301] Whether a subject responds positively, responds negatively, and/or fails to respond to a cancer therapy can be measured and/or characterized according to particular criteria. In certain embodiments, such criteria can include clinical criteria and/or objective criteria. In certain embodiments, techniques for assessing response can include, but are not limited to, clinical examination, positron emission tomography, chest X-ray, CT scan, MRI, ultrasound, endoscopy, laparoscopy, presence or level of a particular marker in a sample, cytology, and/or histology. A positive response, a negative response, and/or no response, of a tumor to a therapy can be assessed by ones skilled in the art using a variety of established techniques for assessing such response, including, for example, for determining one or more of tumor burden, tumor size, tumor stage, etc. Methods and guidelines for assessing response to treatment are discussed in Therasse et al., J. Natl. Cancer Inst., 2000, 92(3):205-216; and Seymour et al., Lancet Oncol., 2017, 18:el43-52.
[0302] In some embodiments, a responsive subject exhibits a decrease in tumor burden, tumor size, and/or tumor stage upon administration of a cancer therapy. In some embodiments, a non-responsive or refractory subject does not exhibit a decrease in tumor burden, tumor size, or tumor stage upon administration of a cancer therapy. In some embodiments, a non-responsive or refractory subject exhibits an increase in tumor burden, tumor size, or tumor stage upon administration of a cancer therapy. [0303] In some embodiments, a cancer subject is identified and/or selected for administration of a cancer therapy as described herein. In some embodiments, the cancer therapy is administered to the subject. In some embodiments, upon administration of the cancer therapy, the subject exhibits a positive clinical response to the cancer therapy, e.g., exhibits an improvement based on one or more clinical and/or objective criteria (e.g., exhibits a decrease in tumor burden, tumor size, and/or tumor stage). In some embodiments, the clinical response is more positive than a clinical response to the cancer therapy administered to a cancer subject who is identified (using a method described herein) as a cancer subject who should not initiate, and/or should modify (e.g., reduce and/or combine with one or more other modalities), and/or should discontinue the cancer therapy, and/or should initiate an alternative cancer therapy.
[0304] Methods described herein can include preparing and/or providing a report, such as in electronic, web-based, or paper form. The report can include one or more outputs from a method described herein, e.g., a set of stimulatory and/or inhibitory antigens described herein. In some embodiments, a report is generated, such as in paper or electronic form, which identifies the presence or absence of one or more tumor antigens (e.g., one or more stimulatory and/or inhibitory and/or suppressive tumor antigens, or tumor antigens to which lymphocytes are not responsive, described herein) for a cancer patient, and optionally, a recommended course of cancer therapy. In some embodiments, the report includes an identifier for the cancer patient. In one embodiment, the report is in web-based form.
[0305] In some embodiments, additionally or alternatively, a report includes information on prognosis, resistance, or potential or suggested therapeutic options. The report can include information on the likely effectiveness of a therapeutic option, the acceptability of a therapeutic option, or the advisability of applying the therapeutic option to a cancer patient, e.g., identified in the report. For example, the report can include information, or a recommendation, on the administration of a cancer therapy, e.g., the administration of a pre-selected dosage or in a preselected treatment regimen, e.g., in combination with one or more alternative cancer therapies, to the patient. The report can be delivered, e.g., to an entity described herein, within 7, 14, 21, 30, or 45 days from performing a method described herein. In some embodiments, the report is a personalized cancer treatment report.
[0306] In some embodiments, a report is generated to memorialize each time a cancer subject is tested using a method described herein. The cancer subject can be reevaluated at intervals, such as every month, every two months, every six months or every year, or more or less frequently, to monitor the subject for responsiveness to a cancer therapy and/or for an improvement in one or more cancer symptoms, e.g., described herein. In some embodiments, the report can record at least the treatment history of the cancer subject.
[0307] In one embodiment, the method further includes providing a report to another party. The other party can be, for example, the cancer subject, a caregiver, a physician, an oncologist, a hospital, clinic, third-party payor, insurance company or a government office.
Combination Therapy
[0308] In some embodiments, an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein) is administered in combination with one or more cancer therapies. Combination therapy refers to those situations in which a subject or population of subjects is simultaneously exposed to two or more therapeutic agents (e.g., an immunogenic composition and a cancer therapy). In some embodiments, the two or more therapies may be administered simultaneously (e.g., concurrently). In some embodiments, such therapies may be administered sequentially (e.g., all “doses” of a first therapeutic agent are administered prior to administration of any doses of a second therapeutic agent). In some embodiments, “administration” of combination therapy may involve administration of one or more agents or modalities to a subject receiving the other agents or modalities in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition, or even in a combination compound (e.g., as part of a single chemical complex or covalent entity).
[0309] In some embodiments, an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein) is administered in combination with one or more cancer therapies, such as an immunotherapy.
[0310] In some embodiments, an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein) is administered to a patient that has received or is recieving an immunotherapy.
[0311] In some embodiments, immunotherapy (e.g. immune checkpoint blockade therapy) involves administration of an agent that acts as a blockade of cytotoxic T-lymphocyte- associated protein 4 (CTLA-4). In certain embodiments, immunotherapy involves treatment with an agent that interferes with an interaction involving CTLA-4 (e.g., with CD80 or CD86). In some embodiments, immunotherapy involves administration of one or more of tremelimumab and/or ipilimumab. In some embodiments, immunotherapy (e.g. immune checkpoint blockade therapy) involves administration of an agent (e.g. antibody agent) that acts as a blockade of programmed cell death 1 (PD-1). In certain embodiments, immunotherapy involves treatment with an agent that interferes with an interaction involving PD-1 (e.g., with PD-L1). In some embodiments, immunotherapy involves administration of an agent (e.g. antibody agent) that specifically interacts with PD-1 or with PD-L1. In some embodiments, immunotherapy (e.g. immune checkpoint blockade therapy) involves administration of one or more of nivolumab, pembrolizumab, atezolizumab, avelumab, and/or durvalumab.
[0312] In some embodiments, a patient is receiving or has received an immunotherapy for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 12 weeks, or longer prior to receiving an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein). In some embodiments, a patient is receiving or has received an immunotherapy for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 12 months, or longer prior to receiving an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein). In sime embodiments, the immunotherapy treatment is continued which the subject receives the immunogenic composition. In some embodiments, the immunotherapy treatment and immunogenic composition is administered concurrently to the subject.
[0313] In some embodiments, the subject exhibits no cancer progression after 12 months after treatment with the immunogenic composition. In some embodiments, the efficacy of the immunogenic composition and/or the immunotherapy is improved in the subject over a specified period of time related to the subject receiving only the immunogenic composition or the immunotherapy alone. In some embodiments, the efficacy of the immunogenic composition and/or the immunotherapy is measured or indicated by a decrease in target lesion sum or diameter based on a cancer patient’s tumor size. In some embodiments, the decrease in target lesion sum or diameter comprises at least a 10%, 20%, 30%, 40%, or 50% or more decrease in target lesion sum or diameter compared to the control level.
[0314] In some embodiments, the efficacy of the immunogenic composition and/or the immunotherapy is measured or indicated by a decrease in cancer progression. In some embodiments, the efficacy of the immunogenic composition is measured or indicated by an increase in humoral response and/or an increase in cellular response.
[0315] In some embodiments, an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein) is administered to a patient that has received or is recieving one or more additional therapeutic agents, such as a cancer therapeutic agent, e.g., a chemotherapeutic agent or a biological agent. [0316] For example, in some embodiments, an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein) is administered to a subject who has received, is receiving, and/or will receive therapy with another therapeutic agent or modality (e.g., with a chemotherapeutic agent, surgery, radiation, or a combination thereof).
[0317] Some embodiments of combination therapy modalities provided by the present disclosure provide, for example, administration of an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein) and additional agent(s) in a single pharmaceutical formulation. Some embodiments provide administration of an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein) and administration of an additional therapeutic agent in separate pharmaceutical formulations.
[0318] Examples of chemotherapeutic agents that can be used in combination with an immunomodulatory agent described herein include platinum compounds (e.g., cisplatin, carboplatin, and oxaliplatin), alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, nitrogen mustard, thiotepa, melphalan, busulfan, procarbazine, streptozocin, temozolomide, dacarbazine, and bendamustine), antitumor antibiotics (e.g., daunorubicin, doxorubicin, idarubicin, epirubicin, mitoxantrone, bleomycin, mytomycin C, plicamycin, and dactinomycin), taxanes (e.g., paclitaxel and docetaxel), antimetabolites (e.g., 5-fluorouracil, cytarabine, premetrexed, thioguanine, floxuridine, capecitabine, and methotrexate), nucleoside analogues (e.g., fludarabine, clofarabine, cladribine, pentostatin, and nelarabine), topoisomerase inhibitors (e.g., topotecan and irinotecan), hypomethylating agents (e.g., azacitidine and decitabine), proteosome inhibitors (e.g., bortezomib), epipodophyllotoxins (e.g., etoposide and teniposide), DNA synthesis inhibitors (e.g., hydroxyurea), vinca alkaloids (e.g., vicristine, vindesine, vinorelbine, and vinblastine), tyrosine kinase inhibitors (e.g., imatinib, dasatinib, nilotinib, sorafenib, sunitinib, and pexidartinib), nitrosoureas (e.g., carmustine, fotemustine, and lomustine), hexamethylmelamine, mitotane, angiogenesis inhibitors (e.g., thalidomide and lenalidomide), steroids (e.g., prednisone, dexamethasone, and prednisolone), hormonal agents (e.g., tamoxifen, raloxifene, leuprolide, bicaluatmide, granisetron, and flutamide), aromatase inhibitors (e.g., letrozole and anastrozole), arsenic trioxide, tretinoin, nonselective cyclooxygenase inhibitors (e.g., nonsteroidal anti-inflammatory agents, salicylates, aspirin, piroxicam, ibuprofen, indomethacin, naprosyn, diclofenac, tolmetin, ketoprofen, nabumetone, and oxaprozin), selective cyclooxygenase-2 (COX-2) inhibitors, or any combination thereof. [0319] Examples of biological agents that can be used in the compositions and methods described herein include monoclonal antibodies (e.g., rituximab, cetuximab, panetumumab, tositumomab, trastuzumab, alemtuzumab, gemtuzumab ozogamicin, bevacizumab, catumaxomab, denosumab, obinutuzumab, ofatumumab, ramucirumab, pertuzumab, ipilimumab, nivolumab, nimotuzumab, lambrolizumab, pidilizumab, siltuximab, BMS-936559, RG7446/MPDL3280A, MEDI4736, tremelimumab, dostarlimab, siniilmab, tisleliumab, or toripalimab, trastuzumab, or others known in the art), enzymes (e.g., L-asparaginase), cytokines (e.g., interferons and interleukins), growth factors (e.g., colony stimulating factors and erythropoietin), bi-specific antibodies, cell-based therapies, immunotherapies, cancer vaccines, gene therapy vectors, or any combination thereof.
[0320] In some embodiments, an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein) is administered to a subject in need thereof in combination with another agent for the treatment of cancer, either in the same or in different pharmaceutical compositions. In some embodiments, the additional agent is an anticancer agent. In some embodiments, the additional agent affects (e.g, inhibits) histone modifications, such as histone acetylation or histone methylation. In certain embodiments, an additional anticancer agent is selected from the group consisting of chemotherapeutics (such as 2CdA, 5-FU, 6-Mercaptopurine, 6-TG, Abraxane™, Accutane®, Actinomycin-D, Adriamycin®, Alimta®, all-trans retinoic acid, amethopterin, Ara-C, Azacitadine, BCNU, Blenoxane®, Camptosar®, CeeNU®, Clofarabine, Clolar™, Cytoxan®, daunorubicin hydrochloride, DaunoXome®, Dacogen®, DIC, Doxil®, Ellence®, Eloxatin®, Emcyt®, etoposide phosphate, Fludara®, FUDR®, Gemzar®, Gleevec®, hexamethylmelamine, Hycamtin®, Hydrea®, Idamycin®, Ifex®, ixabepilone, Ixempra®, L-asparaginase, Leukeran®, liposomal Ara-C, L-PAM, Lysodren, Matulane®, mithracin, Mitomycin-C, Myleran®, Navelbine®, Neutrexin®, nilotinib, Nipent®, Nitrogen Mustard, Novantrone®, Oncaspar®, Panretin®, Paraplatin®, Platinol®, prolifeprospan 20 with carmustine implant, Sandostatin®, Targretin®, Tasigna®, Taxotere®, Temodar®, TESPA, Trisenox®, Valstar®, Velban®, Vidaza™, vincristine sulfate, VM 26, Xeloda® and Zanosar®); biologies (such as Alpha Interferon, Bacillus Calmette-Guerin, Bexxar®, Campath®, Ergamisol®, Erlotinib, Herceptin®, Interleukin-2, Iressa®, lenalidomide, Mylotarg®, Ontak®, Pegasys®, Revlimid®, Rituxan®, Tarceva™, Thalomid®, Velcade® and Zevalin™); small molecules (such as Tykerb®); corticosteroids (such as dexamethasone sodium phosphate, DeltaSone® and Delta-Cortef®); hormonal therapies (such as Arimidex®, Aromasin®, Casodex®, Cytadren®, Eligard®, Eulexin®, Evista®, Faslodex®, Femara®, Halotestin®, Megace®, Nilandron®, Nolvadex®, Plenaxis™ and Zoladex®); and radiopharmaceuticals (such as lodotope®, Metastron®, Phosphocol® and Samarium SM-153).
[0321] The additional agents that can be used in combination with an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein) as set forth above are for illustrative purposes and not intended to be limiting. The combinations embraced by this disclosure, include, without limitation, an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein) and at least one additional agent selected from the lists above or otherwise provided herein. An immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein) can also be used in combination with one or with more than one additional agent, e.g., with two, three, four, five, or six, or more, additional agents.
[0322] In some embodiments, treatment methods described herein are performed on subjects for which other treatments of the medical condition have failed or have had less success in treatment through other means, e.g., in subjects having a cancer refractory to standard-of-care treatment. Additionally, the treatment methods described herein can be performed in conjunction with one or more additional treatments of the medical condition, e.g., in addition to or in combination with standard-of-care treatment. For instance, the method can comprise administering a cancer regimen, e.g., nonmyeloablative chemotherapy, surgery, hormone therapy, and/or radiation, prior to, substantially simultaneously with, or after the administration of an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein). In certain embodiments, a subject to which an immunogenic composition described herein (e.g., an immunogenic composition comprising one or more stimulatory antigens described herein) is administered can also be treated with antibiotics and/or one or more additional pharmaceutical agents. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.
[0323] The disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the disclosure in any way.
EXAMPLES
Example 1. Clinical Evaluation of GEN 009
[0324] A Phase l/2a Study to Evaluate the Safety, Tolerability, Immunogenicity, and Antitumor Activity of GEN 009 Adjuvanted Vaccine in Adult Patients with Selected Solid Tumors
Study Phase: l/2a
Number of Patients: Up to 99 evaluable patients.
Study Design Overview
[0325] This first-in-human, open-label, multicenter, Phase l/2a study of GEN 009 was conducted in adult patients with the following tumor types:
• Melanoma (cutaneous)
• Non-small cell lung cancer (NSCLC)
• Squamous cell carcinoma of the head and neck (SCCHN) (oral, oropharyngeal, hypopharyngeal, or laryngeal)
• Urothelial carcinoma (bladder, ureter, urethra, or renal pelvis)
• Renal cell carcinoma (RCC) with a clear cell component (Part B only) [0326] GEN 009 is an investigational, personalized adjuvanted vaccine that is being developed for the treatment of patients with solid tumors. A system as described above, and herein called ATLAS (Antigen Lead Acquisition System), was used to identify neoantigens in each patient’s tumor that are recognized by their CD4+ and/or CD8+ T cells. ATLAS-identified neoantigens that are recognized by CD4+ and/or CD8+ T cells, and are shown to be stimulatory antigens, were incorporated into a patient’s personalized vaccine in the form of synthetic long peptides (SLPs). A personalized vaccine, consisting of 4 to 20 SLPs, was generated for each patient. The SLPs were divided into 4 pools, with each pool containing 1 to 5 SLPs. The 4 pools were administered subcutaneously (SC) in each of the patient’s limbs. Collectively, these pools of SLPs are the GEN 009 drug product. If fewer than 4 pools were available due to manufacturing, stability, or other issues, the patient was vaccinated with the available drug product. Each pool of GEN 009 drug product consisted of 100 to 1500 pg total peptide administered with 0.45 mg poly-ICLC adjuvant (Hiltonol) per injection.
[0327] This study was conducted in 2 parts as follows:
Part A: Schedule Evaluation of GEN 009 Monotherapy in Patients with No Evidence of Disease
[0328] In Part A, the safety and immunogenicity of GEN 009 monotherapy was evaluated in patients with cutaneous melanoma, NSCLC, SCCHN, or urothelial carcinoma who completed treatment with curative intent for their disease (e.g., surgical resection, neoadjuvant and/or adjuvant chemotherapy and/or radiation therapy) and had no evidence of disease (NED) by the time of initiating vaccination with GEN 009.
[0329] A 5-dose schedule was evaluated (Schedule 1; Days 1, 22, 43, 85 [12 weeks after Day 1], and 169 [24 weeks after Day 1]).
Patient Screening/Vaccine Manufacture
[0330] After informed consent was provided, each potential Part A patient underwent leukapheresis for collection of peripheral blood mononuclear cells (PBMC). PBMC, along with samples of tumor and saliva (PBMCs from leukapheresis were used for SCCHN patients due to potential malignant contamination), were subjected to next-generation sequencing (NGS) and the ATLAS process to identify potential neoantigens.
[0331] Following completion of the ATLAS process, each patient was reevaluated. In order to proceed with vaccine manufacture, a sufficient number of stimulatory antigens for SLP manufacture must have been identified from the ATLAS process, and the patient must continue to meet study eligibility criteria, including NED on a radiographic disease assessment performed within 8 weeks prior to the reevaluation.
[0332] Following completion of vaccine manufacture and prior to Study Day 1 (the first dose of GEN 009), the patient was reevaluated again. A disease assessment performed within 12 weeks prior to the second reevaluation and no more than 4 weeks prior to Day 1 vaccination needed to show NED, and patients needed to continue to meet eligibility criteria, including recovery from any clinically significant toxicity from prior therapies, in order to receive GEN 009.
[0333] Any patient with a recurrence of disease during the screening period for Part A was considered for Part B if they met the eligibility criteria for Part B when this arm is actively enrolling.
Part B: GEN 009 in Patients with Advanced or Metastatic Solid Tumors
[0334] Part B included patients with one of 5 tumor types (NSCLC, SCCHN, cutaneous melanoma, urothelial carcinoma or RCC) who were enrolled in a disease-specific expansion cohort (up to 15 response evaluable patients each). During the screening period, patients received the tumor type-specific treatments identified below (i.e., PD-1 inhibitor monotherapy or PD-1 inhibitor in combination, per disease-specific standard of care and USPI). Following completion of this screening period therapy and for patients who continued to meet study eligibility and would continue on PD-1 inhibitor therapy, GEN 009 dosing was initiated (starting as soon as the vaccine was available and at the schedule selected in Part A) in combination with the PD-1 inhibitor:
• NSCLC: pembrolizumab with chemotherapy (pemetrexed and platinum chemotherapy for non-squamous histologies; carboplatin and either paclitaxel or nab-paclitaxel for squamous NSCLC) during the screening period, followed by pembrolizumab and GEN 009 during the treatment period;
• SCCHN: pembrolizumab monotherapy during the screening period, followed by pembrolizumab and GEN 009 during the treatment period;
• Cutaneous melanoma: nivolumab monotherapy or nivolumab in combination with ipilimumab during the screening period, followed by nivolumab and GEN 009 during the treatment period;
• Urothelial carcinoma: pembrolizumab monotherapy during the screening period, followed by pembrolizumab and GEN 009 during the treatment period;
• RCC: nivolumab monotherapy or nivolumab in combination with ipilimumab during the screening period, followed by nivolumab and GEN 009 during the treatment period.
[0335] In addition, approximately 15 patients enrolled in one of the above diseasespecific cohorts but whose disease progressed during the screening period therapy were enrolled into a separate relap sed/refractory disease cohort.
[0336] Each cohort in Part B was evaluated for safety, immunogenicity, and antitumor activity.
Patient Screening/Vaccine Manufacture
[0337] After informed consent was provided, each potential Part B patient underwent leukapheresis for collection of PBMCs. PBMCs, along with samples of tumor (obtained after the patient’s most recent systemic cancer therapy, if applicable, prior to initiation of the PD-1 inhibitor, and not from a previously irradiated lesion) and saliva (PBMCs from leukapheresis was used for SCCHN patients due to potential malignant contamination in saliva), were subjected to NGS and the ATLAS process. A baseline radiographic disease assessment (DA #1) was performed within 4 weeks prior to initiation of the PD-1 inhibitor (± chemotherapy or ipilimumab, as applicable); scans performed within this timeframe according to standard of care were acceptable.
[0338] After collection of tumor and PBMC samples and baseline radiographic assessment, patients in Part B initiated therapy consisting of nivolumab (as monotherapy or with ipilimumab for cutaneous melanoma and RCC) or pembrolizumab (as monotherapy for SCCHN and urothelial carcinoma, or with chemotherapy for NSCLC).
[0339] Repeat radiographic disease assessments were performed 6 to 10 weeks after initiation of the PD-1 inhibitor (DA #2) and within 14 days prior to Day 1 of GEN 009 dosing (DA #3); scans performed within these timeframes according to standard of care were acceptable. Patients who had adequate disease control (potentially including patients with minimal disease progression per RECIST vl.l) during these time frames and did not need alternate therapy in the opinion of the investigator and the patient, and who continued to meet study eligibility criteria were dosed with GEN 009. Patients who had progressive disease (PD) on their PD-1 inhibitor-containing regimen prior to vaccination and required alternate (e.g., salvage) therapy were allowed to continue in the study during their alternate therapy and were vaccinated at an appropriate time in their disease course in the opinion of the Investigator and the Medical Monitor, if the patient continued to meet performance and laboratory eligibility criteria. For example, the patient was vaccinated at flexible 3- to 4-week intervals to fit into the patient’s alternate treatment regimen, or after a pause. These patients (i.e., the relapsed/refractory cohort, Part B Refractory Study) were assessed separately for objective response rate (ORR). Patients with a complete response (CR) prior to vaccination were dosed with GEN 009 at the discretion of the Investigator and with agreement from the Sponsor/Medical Monitor pending GEN 009 availability. These patients did not count toward the 15 response-evaluable patients in each cohort. Patients receiving ipilimumab or chemotherapy along with the PD-1 inhibitor needed to complete these therapies at least 14 days prior to Day 1 of GEN 009 dosing. Treatment Period - Parts A and B
[0340] The dosing schedules are outlined in Table 4 and depicted visually in Figure 1 Panel A. A 5-dose schedule was evaluated (Schedule 1; Days 1, 22, 43, 85 [12 weeks after Day 1], and 169 [24 weeks after Day 1]).
[0341] Patients continued to receive GEN 009 through Day 169 as long as they were tolerating treatment without recurrence (Part A) or progression of disease (Part B), and did not meet another treatment withdrawal criterion. Patients in Part B with evidence of progression continued treatment beyond RECIST vl.l progression if continued treatment was consistent with iRECIST principles, and if the patient and treating investigator believed that alternate treatment was not immediately necessary, and only upon Sponsor/Medical Monitor approval.
Table 4: Administration Schedule of GEN 009
Figure imgf000138_0001
Post Treatment Period - Parts A and B
[0342] All patients returned 30 days after their last dose of GEN 009 for an end of treatment evaluation. All patients who were alive, not lost to follow-up, and/or who had not withdrawn consent were followed for safety for 1 year after their last dose of GEN 009. Data regarding subsequent therapy and response to those therapies were collected during this followup period. Patients who demonstrated immunogenicity at Day 366 were asked to provide an additional blood sample for immunogenicity testing at approximately Day 547 (18 months following Day 1). On-study disease assessments by imaging continued until disease recurrence (Part A), disease progression (Part B), initiation of another systemic anticancer therapy, or study closure. Criteria for Evaluation - Parts A and B
Immunogenicity:
[0343] In all parts, patient blood samples were drawn to evaluate the immunogenicity of GEN 009 on Day 29, Day 50, Day 92, Day 176, Day 366 (z.e., after 1 year) and Day 547 (z.e., after 18 months if the Day 366 sample demonstrated immunogenicity). T cell responses in peripheral blood mononuclear cells (PBMCs) were assessed by interferon-gamma (IFN- y)/granzyme B (GrB) FluoroSpot assay or by ZFN-y/tumor necrosis factor-alpha (TNF-a) FluoroSpot assay (mean spot-forming cells, fold change, and responder rate per SLP). CD4+ and CD8+ polyfunctional T cell responses in PBMCs were assessed by immune assays such as intracellular cytokine staining. Phenotypes of PBMC cell populations before and after vaccination were assessed by assays such as flow cytometry-based immunophenotyping panels examining regulatory T cells, activation/inhibition markers, and potentially other cell populations.
Clinical Activity:
[0344] Patients were assessed by CT or MRI for the following clinical endpoints:
[0345] Part A: Disease-free survival (DFS). Disease assessment (radiological imaging and for patients with urothelial carcinoma who had not undergone cystectomy, urine cytology; and for patients with tumor potentially visible by cystoscopy [e.g., of the urethra, bladder, ureterovesical junction], cystoscopy) was performed during the screening period (as per study eligibility), then every 12 weeks (starting 12 weeks after Day 1) through Day 337 (i.e., 4 assessments post-Day 1), then every 26 weeks until disease recurrence, initiation of another systemic anticancer therapy, or study closure. Note: PET/CT was used instead of CT or MRI per agreement of the Medical Monitor and Investigator for patients in Part A.
[0346] Part B: Since the GEN 009 vaccine was administered after 3 to 4 months of known active therapy, a traditional response rate and duration of response was difficult to evaluate. In general, the great majority of patients will have defined the course of their disease within those 3 to 4 months, so that any significant change in trajectory after addition of the vaccine likely represents an impact of the vaccine, noting that pseudoprogression could be responsible for a small percentage of responses. In this setting, each patient served as their own control in an exploratory analysis of relative intensity ratio (RIR) on imaging, duration of response (DoR), and progression-free survival (PFS). Study-specific disease assessments (radiological imaging) were obtained during screening within 4 weeks prior to initiation of PD-1 inhibitor therapy, 6 to 10 weeks after initiation of PD-1 inhibitor therapy, and within 14 days prior to the first dose of GEN 009. Post first dose, study-specific disease assessments occured at Day 50 (± 3 days) and Day 92 (± 3 days). Additionally, throughout the study, standard of care disease assessments were recorded until disease progression, initiation of another systemic anticancer therapy, or study closure. Antitumor activity was also assessed by improvement in tumor growth kinetics (i.e., increase in tumor shrinkage rate or decrease in tumor growth rate) with GEN 009 vs projected rate without GEN 009. The overall study design is depicted visually in Figure 1, Panel B.
Statistical Methods:
[0347] The primary categorization for data summary and analysis consisted of the separate parts of the study. Within Part A, additional categories for summarization consisted of all schedules studied, as well as overall for certain data presentations. For Part B, data were analyzed separately for patients with PD prior to GEN 009 dosing. Further categories for data summarization for Part B consisted of data for each tumor type, data for those with relapsed/refractory disease, and overall. Select safety presentations used an overall pool across parts for summarization, as appropriate.
[0348] All statistics were descriptive and included number of patients and number of SLPs, mean, median, standard deviation (SD), and minimum/maximum for continuous variables. Categorical variables were tabulated by number of observations and proportions. Time to event distribution was estimated using Kaplan-Meier techniques. When appropriate, the median along with CI was provided. [0349] For Immunogenicity Analyses: A positive cellular immune response for a given SLP was determined using statistical and/or empirical criteria. Cellular immune responses to GEN 009 were summarized for each patient by magnitude of response and/or fold change from baseline for each time point. Immune responses were summarized for each tumor type and for all patients combined.
[0350] Statistical tests such as Wilcoxon rank sum test were used to compare magnitude of response between tumor types or changes before and after vaccination when applicable.
[0351] For Clinical Activity Analyses: For Part A, DFS was summarized using Kaplan- Meier methods. For Part B, RIR was tabulated by frequency distribution, with 2-sided exact 90% Cis. Median time to response and DoR were summarized for those patients with confirmed responses, using Kaplan Meier methods. PFS and overall survival (OS) were similarly summarized. In addition, the rate of patients with PFS and OS of at least 12 months duration was presented with 2-sided 90% Cis. RIR in Part B was summarized as categorical data and by use of shift tables. Improvement in tumor growth kinetics, which was measured by comparing observed tumor growth rate with GEN 009 vs projected tumor growth rate without GEN 009 for each period from Day 1 was summarized by period for each patient. Observed tumor growth rate for each period was calculated as the average percent change in the sum of the longest diameters from earlier time points when imaging was collected; and projected tumor growth rate post Day 1 was a weighted average of observed tumor growth rates prior to Day 1, where time points closer to Day 1 were assigned with heavier weighting.
[0352] Clinical activity analyses were descriptive; statistical tests were used as appropriate to compare changes before and after vaccination or between tumor types. Subgroup analysis of various immunologic parameters, as well as rate of response and time to event endpoints, based on demographic and baseline disease characteristics were performed as well as exploratory analyses, as appropriate.
[0353] For Interim Analysis: For each cohort in Part B (tumor-specific and relapsed/refractory), an ongoing non-binding interim analysis was planned for the initial response evaluable patients’ responses. Example 2. Immunogenicity of GEN 009 vaccine. Part A of Phase l/2a Study
[0354] As described in Example 1, GEN-009-101 is a first-in-human Phase l/2a study testing ATLAS platform feasibility, safety, immunogenicity and clinical activity in selected solid tumors. After next-generation sequencing of patient tumors and cytokine-based ATLAS assessment using autologous T cells and APCs, up to 20 stimulatory synthetic long peptides (SLPs), corresponding to ATLAS-identified, patient-specific stimulatory antigens (neoantigens), were used in each personalized vaccine. For each patient, SLPs were divided into 4 pools, each comprising 1 to 5 SLPs. For each patient, the GEN 009 vaccine comprised the 4 pools of SLPs. GEN 009 was administered with poly-ICLC on Day 1 (week 0), Day 22 (week 3), Day 43 (week 6) with booster vaccinations on Day 85 (week 12 after Day 1 vaccination) and Day 169 (week 24 after Day 1 vaccination). PBMCs were collected for immunogenicity assessments from whole blood drawn at Day 1 vaccination (just prior to vaccination) and at Day 29, Day 92, and Day 176 (z.e., one week following vaccinations on Day 22, Day 85, and Day 169), and also via a leukapheresis procedure at initial patient screening (baseline) and at either Day 50, Day 92, or Day 176 (z.e., one week following vaccinations on Day 43, Day 85, or Day 169). Plasma was also obtained from these samples.
Ex vivo FluoroSpot assay
[0355] The cellular immune response to GEN 009 was monitored by examining T cell responses using a dual-color FluoroSpot assay. The ex vivo FluoroSpot assay simultaneously detects release of interferon gamma (IFN-y) and granzyme B (GrB) from PBMCs, or T cell subsets enriched from PBMCs, following stimulation with peptide antigens for a duration of approximately 2 days. This method varies the traditional Enzyme-linked ImmunoSpot (ELISpot) assay by replacing the colorimetric detection with fluorescence detection, enabling quantification of individual, peptide-reactive T cells that secrete multiple analytes of interest in a high- throughput format. In general, the method detects effector T cell responses. [0356] For each patient, complete PBMC populations, or CD4+ or CD8+ T cells enriched from PBMCs, were stimulated with overlapping peptides spanning either the unique individual SLPs or all SLPs included in each of the 4 pools of SLPs for that patient to determine the frequency of antigen-specific T cells. PBMCs or T cells enriched from PBMCs were combined with overlapping peptides spanning patient-specific SLPs in pre-conditioned, poly vinylidene difluoride membrane-bound 96-well plates, and incubated at 37°C for 44 ± 4 hours. Development of immune response-induced fluorescent spots was facilitated by addition of detection antibodies (anti-IFN-y monoclonal antibody and biotinylated anti-granzyme B monoclonal antibody) followed by anti-BAM-490 and SA-550 fluorescent antibodies.
In vitro stimulated FluoroSpot assay
[0357] The in vitro stimulated (IVS) FluoroSpot assay simultaneously detects release of interferon gamma (IFN-y) and tumor necrosis factor alpha (TNFa) from T cell subsets enriched from PBMCs, following in vitro stimulation (IVS) with peptide antigens for a duration of approximately 10 days in culture. The method is aimed at generating increased polyfunctional, peptide-reactive T cells over the course of the culture period. In general, the method detects memory T cell responses.
[0358] For each patient, CD4+ or CD8+ T cells enriched from PBMCs were expanded in culture for 10 days with overlapping peptides spanning the unique individual SLPs or all SLPs included in each of the 4 pools of SLPs for that patient in the presence of IL-7. On days 2, 4 and 7, half the culture media was changed and IL-2, IL- 15 and IL-21 were added. On day 9, cells were rested in fresh culture media without cytokines. The expanded T cells were then combined with fresh antigen presenting cells and their respective overlapping peptides spanning patientspecific SLPs in pre-conditioned, polyvinylidene difluoride membrane-bound 96-well plates, and incubated at 37°C for 20 ± 4 hours. Development of immune response-induced fluorescent spots was facilitated by addition of detection antibodies (anti-IFN-y monoclonal antibody and biotinylated anti-TNFα monoclonal antibody) followed by anti-BAM-490 and SA-550 fluorescent antibodies. Results
[0359] Repeated dosing with GEN 009 was well tolerated with only mild local discomfort and no dose-limiting toxicity. ATLAS screening results show inter-patient variability in the number of stimulatory and inhibitory antigens and immune profile. Table 5 summarizes the tumor mutational burden (TMB; mutations/Mb of DNA), the number of ATLAS-identified, patient-specific antigens eliciting stimulatory or inhibitory T cell responses as measured by IFN- y and/or TNF-a secretion, the number of patient-specific SLPs included in each vaccine, and prior therapies for each patient selected for GEN 009 vaccination. All values were generated prior to GEN 009 vaccination.
Table 5. Patients screened and selected for GEN 009 vaccination
Figure imgf000144_0001
[0360] As shown in Table 6 below, vaccination with GEN 009 resulted, after the priming series of three vaccinations at Day 1, Day 22 and Day 43, in detectable immune responses (i) in 100% of patients, and (ii) against 90% or more of individual patient-specific peptide antigens (SLPs corresponding to ATLAS-identified, patient-specific stimulatory antigens) across all patients and all FluoroSpot assays. Each number in Table 6 represents aggregate immunogenicity at Day 50 for all patient-specific peptide antigens (SLPs) for a given patient, by a given assay. The FluoroSpot assays are indicated as: ex vivo assay (complete PBMCs), ex vivo assay (enriched T cell subsets), and IVS assay (enriched T cell subsets). Both CD8+ and CD4+ T cell responses were observed. Ten-day in vitro stimulated FluoroSpot assays resulted in a greater proportion of positive and/or broader immune responses than the ex vivo FluoroSpot assays. In aggregate, all patient-specific peptide antigens (SLPs corresponding to ATLAS- identified, patient-specific stimulatory antigens) from the combined patients elicited:
Overall response rate: 99% of peptides positive
[0361] Effector T cell responses (for combined 8 patients A, B, C, E, F, G, H, and K), as detected by ex vivo assays (enriched T cell subsets):
CD4+ = 51%
CD8+ = 41%
[0362] Memory T cell responses (for combined 8 patients A, B, C, E, F, G, H, and K), as detected by IVS assays (enriched T cell subsets):
CD4+ = 87%
CD8+ = 59%
Total CD8+ T cell responses (for combined 8 patients A, B, C, E, F, G, H, and K), as detected by ex vivo and/or IVS assays (enriched T cell subsets) = 74%
Total CD4+ T cell responses (for combined 8 patients A, B, C, E, F, G, H, and K), as detected by ex vivo and/or IVS assays (enriched T cell subsets) = 92%
Total PBMC (combined CD4+ and CD8+ T cell) responses (for combined 8 patients A, B, C, E, F, G, H, and K), as detected by:
Any assay: 91% • ex vivo assays = 45%
• IVS: 88%
[0363] Tables 6-7 show GEN 009 immunogenicity assays against patient-specific peptide antigens (SLPs), after priming series of three vaccinations at Day 1, Day 22 and Day 43.
Table 6.
Figure imgf000146_0001
Table 7.
Figure imgf000146_0002
Data are presented as the proportion of peptides defined as positive by the DFR(eq) test (p<0.05) at the indicated time point for each cell type. *“Day 50” indicates 50 days post initial vaccination.
[0364] Figure 2 shows representative results of in vitro stimulated FluoroSpot assays on CD4+ and CD8+ T cells enriched from PBMCs collected at baseline (prior to vaccination) and at Day 50 from each of 5 patients (patients A, B, C, E, and F). Data are represented as the mean IFN-y spot forming cells (SFC) +/- SEM per 10,000 or 20,000 T cells, as indicated, for a given patient, for each of the 4 pools of SLPs (each pool comprising 1-5 SLPs) included in that patient’s vaccine.
[0365] Figure 3 shows representative results of ex vivo FluoroSpot assays and in vitro stimulated FluoroSpot assays on CD4+ and CD8+ T cells enriched from PBMCs collected at baseline (prior to vaccination) and at Day 50 from a representative patient (patient E). Panels A and B: ex vivo FluoroSpot assays. Data are represented as the mean cytokine spot forming cells (SFC) per million T cells, for each SLP included in the patient’s vaccine, as indicated. Panel C: in vitro stimulated FluoroSpot assays. Data are represented as the mean cytokine spot forming cells (SFC) per 20,000 T cells, for each SLP included in the patient’s vaccine, as indicated.
[0366] Figure 4 shows representative summary results of ex vivo FluoroSpot assays and in vitro stimulated FluoroSpot assays on total PBMC or PBMCs depleted of CD4+ or CD8+ T cells collected at baseline (prior to vaccination) and at Day 50 from patients A-H and K. Data are reported as the proportion of peptides positive by the DFR(eq) test. Circles represent baseline, squares represent D50 time point. For results shown in Panel A, 200,000 total PBMC or PBMCs depleted of CD4+ or CD8+ T cells were stimulated with overlapping peptides (OLPs) spanning each immunized SLP in an IFN-y and granzyme B (GrB) dual- color ex vivo FluoroSpot assay. For results as shown in Panel B, PBMCs depleted of CD4+ or CD8+ T cells were stimulated with OLPs for 10 days followed by an overnight IFN-y and TNFα dual- color FluoroSpot assay. Panel C shows the proportion of SLPs scored positive by any assay for patients A-H, and K. [0367] Data from patients A-L are shown in Figure 5, including for each patient, the tumor type, stage of cancer at diagnosis, period of time from diagnosis, prior therapies the patient received, the patients calculated tumor mutational burden (TMB), the number of stimulatory and inhibitory neoantigens identified for each patient, and the number of peptides in the vaccine administered. The graph indicates the status of each patient at different points within the example vaccination regimen. The timing of vaccination is indicated by a vertical arrows. The color of the horizontal bars indicate the stage of cancer at diagnosis. A blue horizontal arrow indicates that the patient has not yet completed the vaccination regimen (i.e., is within the dosign period). A black horizontal arrow indicates that the patient has completed the vaccination regimen (i.e., is past the treatment period or post vaccination schedule). A black circle indicates a status of “NED” or no evidence of disease. The graph shows that all patients post vaccination experienced recurrence-free survival for at least 4 months.
[0368] In subsequent follow-up, 7 of 8 vaccinated patients had no disease progression after median follow-up of 14 months (range 8 to 17 months). This outcome compares favorably to the expected relapse rates in these malignancies. The patient who progressed (H) had low immune responses (see Figure 6, Panel A), but exceeded previous remissions.
[0369] Figure 6 shows representative results of ex vivo dual-analyte FluoroSpot assays on CD4+ and CD8+ T cells enriched from PBMCs of three representative patients (patients A and E; low response patient H). Bulk PBMCs were isolated from the patients at baseline (prior to vaccination) and at the indicated timepoints over the course of their treatment. The secretion of IFN-y and granzyme B (GrB) was quantified via ex vivo dual-analyte FluoroSpot after stimulation with overlapping peptide pools (OLPs) spanning the patient-specific SLPs used for immunization. In Panel A, data are expressed as mean (± SEM) spot forming cells (SFC) per million PBMCs to each of the four pools. Panel B shows the number of positive pools for each time point.
[0370] These results demonstrate that immune responses developed early and were seen at the first sampling post initial vaccination at day 29, and were also found as far out as 12 months, 6 months after completion of vaccination. Peak ex vivo T cell responses occurred at 3 months, after 3 vaccinations.
Example 3. GEN 009 vaccine: Preliminary Results of Phase l/2a Study, Part B
[0371] As described in Example 1, GEN-009-101 is a first-in-human Phase l/2a study testing personalized vaccines designed using the ATLAS platform for feasibility, safety, immunogenicity and clinical activity in selected solid tumors. In Part B of the study, eligible patients with advanced cancer received standard-of-care PD-1 checkpoint inhibitor therapy, with or without chemotherapy, during the period of manufacture of their GEN 009 vaccine. Patients then received five doses of GEN 009 vaccine over six months, along with continuation with PD- 1 checkpoint inhibitor therapy. Patients who progressed prior to vaccination received appropriate alternate therapy followed by GEN 009 vaccine combined with PD-1 checkpoint inhibitor therapy.
[0372] Since the GEN 009 vaccine was administered after 3 to 4 months of known active therapy, a vaccine-atrributable response rate and duration of response was complex to evaluate. In general, the great majority of patients will have defined the course of their disease within those 3 to 4 months, so that any significant change in trajectory after addition of the GEN 009 vaccine likely represents an impact of the vaccine,. In this setting, each patient served as their own control.
[0373] Cellular immune responses were measured by dual-analyte (IFN-y and granzyme B) FluoroSpot assays, assessing ex vivo CD4+ and CD8+ T cell responses to each SLP pool, and by IFN-y and TNFα FluoroSpot assays, assessing in vitro stimulated (IVS) T cell responses to each SLP. The FluoroSpot assays were performed as described in Example 2. Immunophenotyping of PBMC populations was performed by flow cytometry. Further CD4+ and CD8+ polyfunctional responses were assessed by intracellular cytokine staining.
[0374] Antitumor activity was also assessed by improvement in tumor growth kinetics (/.< ., increase in tumor shrinkage rate or decrease in tumor growth rate) with GEN 009 vs projected rate without GEN 009. The tumor micro-environment and infiltrating cells were evaluated by RNA sequencing and immunofluorescence.
[0375] Figure 7 shows a spider plot for an idealized patient, illustrating the method of assessing GEN-009 vaccine-specific contribution to clinical responses in Part B of the study. Each point on the plot represents assessment of the idealized patient’s target lesions. The X axis shows time and treatment. The left side of the graph shows the period of the idealized patient’s initial assessment and standard-of-care (SOC) treatment. The right side of the graph shows the period of standard-of-care treatment combined with GEN 009 vaccination (SOC & Vaccine). The Y axis shows percent change in the sum, or diameter, of the idealized patient’s target lesions. Baseline is defined as the patient’s lesions at initial assessment. Progressive disease (PD), partial response (PR), and complete response (CR) are defined relative to baseline as +20%, -30% and -100% change, respectively (RECIST criteria). The dotted line in the right side of the graph shows the expected course of disease, that is, for the majority of patients, the course set during the previous 3 to 4 months of active therapy. In this example, the expected course of the idealized patient’s disease, on standard-of-care therapy alone, is shown as approximately - 35% change in target lesions, in the lower range of partial response. The post-vaccination response is shown as approximately -50% change in target lesions, in a higher range of partial response than the expected course on standard-of-care therapy alone. The shift in trajectory is attributable to vaccination with GEN 009.
[0376] Table 8 shows detailed clinical history and GEN-009 vaccine composition for selected patients.
Figure imgf000150_0001
Figure imgf000151_0001
Figure imgf000152_0001
Table 9 shows summary clinical history and GEN-009 vaccine composition for all patients enrolled in the Main Part B Study and in the Part B Refractory Study.
Table 9.
Figure imgf000152_0002
Figure imgf000153_0001
Main Part B Study
[0377] Figures 8 through 15 show representative results for selected checkpoint inhibitor responsive patients enrolled in the Main Part B Study.
[0378] Figure 8 shows results for patient -058 (Main Part B Study). Panel A: Spider plot, as described for Figure 7, showing a post-vaccination drop from approximately -50% to - 70% change in target lesions. Panel B: Results of in vitro stimulated FluoroSpot assays performed at baseline and at Day 50 post- initial vaccination. The height of each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN-y and TNFa, for each SLP included in the patient’s vaccine, as indicated. CD4+ and CD8+ T cell responses are presented in the left and right panels, respectively. Panel C: Aggregate results of in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ± SD per 20,000 T cells (IVS). The dark bars indicate total IFNy responses, the lighter bars indicate total TNFα responses. Panel D: Radiographic imaging of mediastinal tumor, showing tumor mass at 3 timepoints (Baseline, Pre-vaccine, Postvaccine). The tumor mass is observed to have shrunk from 7.4 cm diameter at baseline (prior to checkpoint inhibitor therapy) to 3.7 cm diameter during standard-of-care checkpoint inhibitor therapy (prior to vaccination), then to 2.3 cm following vaccination with GEN 009 and concurrent checkpoint inhibitor therapy. This result suggests possible additive antitumor activity of GEN 009 vaccine in combination with PD-1 inhibitors. Panel E: Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 106 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
[0379] Figure 9 shows results for patient -105 (Main Part B Study). Panel A: Spider plot, as described for Figure 7, showing a post-vaccination drop from approximately -37% to - 70% change in target lesions. Panel B, top: Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed with PBMCs collected at baseline and at the indicated days after initial vaccination with GEN-009. The height of each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine. The DI time point represents post-checkpoint inhibitor therapy, pre-vaccine responses; therefore, increases in frequency of PBMC responses at subsequent time points can be attributed to GEN 009 vaccination. Panel B, bottom: Results of in vitro stimulated FluoroSpot assays performed on CD4+ and CD8+ T cells enriched from PBMC at baseline and at Day 50 after initial vaccination. The height of each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN-y (left axis) and TNFα (right axis), for each SLP included in the patient’s vaccine, as indicated. CD4+ and CD8+ T cell responses are presented in the left and right panels, respectively. Panel C: Summary results of ATLAS screens performed at baseline and at Day 50 after initial vaccination with GEN 009. Each pie chart shows percentages of the patient’s tumor mutations that elicit stimulatory T cell responses (neoantigens) or inhibitory T cell responses (inhibigens). For this patient, a slight proportional reduction in inhibigens was observed following GEN 009 vaccination. Panel D: Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ± SD per 106 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNFα responses. Panel E: Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 106 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
[0380] Figure 10 shows results for patient -001 (Main Part B Study). Panel A: Spider plot, as described for Figure 7, showing a post-vaccination drop from approximately -60% to - 100% change in target lesions. This patient achieved a partial response (PR) on checkpoint inhibitor therapy, followed by a complete response (CR) after vaccination with GEN-009. Panel B, top: Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed on PBMCs collected at baseline and at the indicated days after initial vaccination with GEN-009. The height of each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine. The DI time point represents post-checkpoint inhibitor therapy, pre-vaccine responses; therefore, increases in frequency of PBMC responses at subsequent time points can be attributed to GEN 009 vaccination. Panel B, bottom: Results of in vitro stimulated FluoroSpot assays performed on CD4+ and CD8+ T cell enriched from PBMC at baseline and at Day 50 after initial vaccination. The height of each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN-y (left axis) and TNFα (right axis), for each SLP included in the patient’s vaccine, as indicated. CD4+ and CD8+ T cell responses are presented in the left and right panels, respectively. Panel C: Summary results of ATLAS screens performed at baseline and at Day 50 after initial vaccination with GEN 009. Each pie chart shows percentages of the patient’s expressed tumor mutations that elicit stimulatory T cell responses (neoantigens) or inhibitory T cell responses (inhibigens). For this patient, proportional increases in both neoantigens and inhibigens were observed following GEN 009 vaccination. Panel D: Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ± SD per 106 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNFα responses. Panel E: Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 106 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
[0381] Figure 11 shows results for patient -057 (Main Part B Study). Panel A: Spider plot, as described for Figure 7, showing a drop to -100% change in target lesions, i.e., complete response (CR), prior to vaccination with GEN-009. The patient sustained a complete response for the duration of the study. Panel B, top: Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed on PBMCs collected at baseline and at the indicated days after initial vaccination with GEN 009. The height of each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine. The DI time point represents post-checkpoint inhibitor therapy, pre-vaccine responses; therefore, increases in frequency of PBMC responses at subsequent time points can be attributed to GEN 009 vaccination. Panel B, bottom: Results of in vitro stimulated FluoroSpot assays performed on CD4+ and CD8+ T cells enriched from PBMC at baseline and at Day 50 after initial vaccination. The height of each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN-y (left axis) and TNFα (right axis), for each SLP included in the patient’s vaccine, as indicated. CD4+ and CD8+ T cell responses are presented in the left and right panels, respectively. Panel C: Summary results of ATLAS screens performed at baseline and at Day 50 after initial vaccination with GEN 009. Each pie chart shows percentages of the patient’s tumor mutations that elicit stimulatory T cell responses (neoantigens) or inhibitory T cell responses (inhibigens). For this patient, both a proportional decrease in neoantigens and proportional increase in inhibigens were observed following GEN 009 vaccination. Panel D: Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ± SD per 106 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNFα responses. Panel E: Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 106 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
[0382] Figure 12 shows results for patient -031 (Main Part B Study). Panel A: Spider plot, as described for Figure 7, showing a slight drop from approximately -55% to - 60% change in target lesions. Panel B, top: Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed on PBMCs collected at baseline and at the indicated days after initial vaccination with GEN 009. The height of each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine. The DI time point represents postcheckpoint inhibitor therapy, pre-vaccine responses; therefore, increases in frequency of PBMC responses at subsequent time points can be attributed to GEN 009 vaccination. Panel B: Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ± SD per 106 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNFα responses. Panel C: Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 106 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
[0383] Figure 13 shows results of all ex vivo dual-color FluoroSpot assays plotted over time. Peripheral blood mononuclear cells were collected from patients participating in the Main Part B Study at the indicated time points (day 1 is the day of the first GEN-009 vaccination) and viably frozen. Thawed PBMCs were stimulated with pools containing the vaccine antigens (or controls). Results are shown as the mean cytokine spot forming cells (SFC) per million PBMC (+/- SEM) for each subject contributing data at each time point. Not all patients contributed data for each time point: the N at each time point is indicated. These interim results indicate that the magnitude of response peaked at D92, six weeks after the third dose of vaccine.
[0384] Figure 14 shows summary results for all patients enrolled in the Main Part B Study. Panel A is a composite of all spider plots, as described for Figure 7, showing the percent target tumor lesion change for each patient, from the time of consent and initial assessment. Panel B is a composite of all spider plots showing the percent target tumor lesion change for each patient from the time the patient received GEN-009 dose 1 (i.e., baseline is defined at day 0 of the study). Panel C is a swimmers’ plot showing timelines of PD-1 standard-of-care therapy, GEN-009 dosing, and outcomes for each patient. Panel D shows aggregate results for all patients of dual-analyte immunogenicity assays performed at screening and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ T cell responses are shown. Values presented are the means of background subtracted SFCs ± SEM per 20,000 T cells. The dark bars indicate total IFNy responses, the lighter bars indicate total TNFα responses.
Part B Refractory Study
[0385] Figures 15 through 20 show representative results for selected checkpoint inhibitor refractory patients enrolled in the Part B Refractory Study.
[0386] Figure 15 shows results for patient -106 (Part B Refractory Study). Panel A: Spider plot, as described for Figure 7. This patient progressed rapidly on the standard-of-care checkpoint inhibitor therapy and was treated with chemotherapy from days approximately -110 to -20, after which they continued on checkpoint inhibitor therapy and received GEN 009 vaccine on day 0. The spider plot shows maintenance of the PR achieved on chemotherapy, with a slight post-vaccination drop then a slight rise from approximately -50% to -55% change in target lesions. Panel B, top: Results of ex vivo dual analyte (IFN-y and granzyme B) assays performed on PBMCs collected at baseline and at the indicated days after initial vaccination with GEN 009. The height of each bar indicates mean cytokine spot forming units (SFU) per million T cells for all SLPs included in the patient’s vaccine. The DI time point represents postcheckpoint inhibitor therapy and post-chemotherapy, pre-vaccine responses; therefore, increases in frequency of PBMC responses at subsequent time points can be interpreted as attributable to GEN 009 vaccination. Panel B, bottom: Results of in vitro stimulated FluoroSpot assays on CD4+ and CD8+ T cells enriched from PBMCs performed at baseline and at Day 50 after initial vaccination. The height of each colored bar represents the mean of cytokine spot forming cells (SFC) per 20,000 T cells, for each of IFN-y (left axis) and TNFα (right axis), for each SLP included in the patient’s vaccine, as indicated. CD4+ and CD8+ T cell responses are presented in the left and right panels, respectively. Panel C: Summary results of ATLAS screens performed at baseline and at Day 50 after initial vaccination with GEN 009. Each pie chart shows percentages of the patient’s expressed tumor mutations that elicit stimulatory T cell responses (neoantigens) or inhibitory T cell responses (inhibigens). For this patient, a slight proportional increase in inhibigens was observed following GEN 009 vaccination. Panel D: Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ± SD per 106 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNFα responses. Panel E: Results of ex vivo IFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for IFNy are reported on the right axis as mean background subtracted SFC per 106 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
[0387] Figure 16 shows results for patient -109 (Part B Refractory Study). Panel A: Spider plot, as described for Figure 7. This patient progressed rapidly on standard-of-care checkpoint inhibitor therapy and was treated with chemotherapy, after which they continued on checkpoint inhibitor therapy and received GEN 009 vaccine on day 0. The spider plot shows maintenance of the PR achieved on chemotherapy, with a slight post-vaccination drop from approximately -70% to -75% change in target lesions. Panel B: Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ± SD per 106 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNFα responses.
[0388] Figure 17 shows results for patient -027 (Part B Refractory Study). Panel A: Spider plot, as described for Figure 7. This patient responded to standard-of-care checkpoint inhibitor therapy and then had a new tumor mass emerge (arrow around D-100). The patient was further treated with monoclonal antibody, after which they received GEN 009 vaccine on day 0. The spider plot shows maintenance of the PR, with no additional change to date in target lesions post-vaccination. Panel B: Results of ex vivo ZFNy FluoroSpot assays plotted over time (x axis) against percent change in the patient’s tumor volume. Values for ZFNy are reported on the right axis as mean background subtracted SFC per 106 cells. The corresponding patient tumor scans are indicated on the left axis (open triangles). The dotted horizontal lines represent the RECIST criteria for progressive disease (PD, +20%) and partial response (PR, -30%). The grey shading on right side of the plot represents the initiation of GEN-009 dosing, with black triangles representing days of vaccination.
[0389] Figure 18 shows results for patient -110 (Part B Refractory Study). Panel A: Spider plot, as described for Figure 7. This patient progressed rapidly on standard-of-care checkpoint inhibitor therapy and was treated with chemotherapy, after which they continued on checkpoint inhibitor therapy and received GEN 009 vaccine on day 0. The spider plot shows reversal of the PR (beginning just prior to vaccination), with a sharp post-vaccination increase from approximately -50% to -5% change in target lesions. The lesion appearance change is being followed as a possible pseudo-progression, suggestive of massive immune cell infiltration post-treatment rather than a failure of therapy. Panel B: Aggregate results of ex vivo and in vitro stimulated FluoroSpot assays for all SLPs, performed at baseline (SC) and at Day 50 postinitial vaccination. PBMC, CD4+ and CD8+ responses are shown. Values presented are the means of background subtracted SFCs ± SD per 106 T cells (ex vivo, left panel) or 20,000 T cells (in vitro stimulated, right panel). The dark bars indicate total IFNy responses, the lighter bars indicate total TNFα responses.
[0390] Figure 19 shows results for patient -060 (Part B Refractory Study). Panel A: Spider plot, as described for Figure 7. This patient responded to standard-of-care checkpoint inhibitor therapy, but experienced a therapy -related adverse event. The patient was treated for the adverse event, after which they received GEN 009 vaccine on day 0. The spider plot shows reversal of the PR (beginning just prior to vaccination), with a slight post-vaccination increase from approximately -25% to -20% change in target lesions.
[0391] Figure 20 shows summary results for all patients enrolled in the Main Part B Refractory Study. Panel A is a composite of all spider plots, as described for Figure 7, showing the percent target tumor lesion change for each patient, from the time of consent and initial assessment. Panel B is a composite of all spider plots showing the percent target tumor lesion change for each patient from the time the patient received GEN-009 dose 1 (i.e., baseline is defined at day 0 of the study). Panel C is a swimmers’ plot showing timelines of PD-1 standard- of-care therapy, GEN-009 dosing, and outcomes for each patient. Panel D shows aggregate results for all patients of dual-analyte immunogenicity assays performed at screening and at Day 50 post-initial vaccination. PBMC, CD4+ and CD8+ T cell responses are shown. Values presented are the means of background subtracted SFCs ± SEM per 20,000 T cells. The dark bars indicate total IFNy responses, the lighter bars indicate total TNFα responses.
[0392] Figure 21 shows representative summary results for CD4+ and CD8+ T cell responsiveness, prior to and after vaccination with GEN 009. In Panel A, the bars labeled “ATLAS” indicate the overall percentages of antigens that elicited robust CD4+ or CD8+ T cell responses in initial ATLAS screening of the selected Part B patients and were consequently included in the GEN 009 vaccine compositions administered to these patients. The bars labeled “Post GEN-009 Treatment” indicate the overall percentages of antigens that elicited CD4+ or CD8+ T cell responses in ATLAS screening of the selected Part B patients following their vaccination with GEN 009. The comparison shows changes in the breadth of T cell responses. As shown, the relative proportion of antigens recognized by CD8+ T cells remained essentially the same, while the relative proportion of antigens recognized by CD4+ T cells increased approximately 3 -fold post-vaccination. These results suggest that vaccination with GEN-009 induces de novo CD4+ T cell responses, and expands existing CD8+ T cell responses. Panel B is a graph showing aggregate IFNy responses (ex vivo FluoroSpot assays) as SFCs ± SEM per million PBMCs over several timepoints, from screening to 6 months after patients received GEN 009 dose 3. As can be seen, ex vivo responses to GEN 009 antigens were present at the time of screening (Baseline) and did not change significantly after checkpoint blockade therapy (Predose); continued increase in responses was observed after each GEN 009 dose and was maintained to the last time point (6 months). Panel C shows the average number of neoantigens that elicited positive CD4+ and CD8+ T cell responses, as determined by ATLAS, at the time of screening and at Day 50 post-initial vaccination, broken out for Main Part B (Controlled) and Part B Refractory (Refractory) patients. Panel D shows the total number of non-synonymous mutations (tumor mutational burden) for each cohort at the time of screening. These results show that, at screening, the checkpoint inhibitor-controlled cohort had a significantly greater overall number of neoantigens eliciting T cell responses than the checkpoint inhibitory-refractive cohort. In addition, the controlled cohort showed an increase in the number of neoantigens to which CD8+ T cells responded after GEN 009 vaccination (D50), indicating epitope spread. In contrast, the refractory cohort showed a decrease in the number of neoantigens to which either CD4+ or CD8+ T cells responded after GEN 009 vaccination (D50), indicating contraction of the breadth of response. This cannot be explained simply by greater tumor mutational burden: while there was a trend towards greater overall number of mutations (independently of whether or not they were defined as neoantigens) in the controlled cohort, the tumor mutational burden in the controlled and refractory cohorts were not statistically different.
[0393] Together, these results show a trend toward disease reduction in Part B patients that is attributable to vaccination with GEN 009, additive to standard-of-care checkpoint inhibitor therapy. Overall, 89% of the SLPs selected for inclusion in GEN 009 vaccines following the initial ATLAS screen drove immune responses in patients by Day 50 following vaccination. Of these, 65% elicited CD8+ T cell responses; 83% elicited CD4+ responses; and 80% elicited PBMC responses. ATLAS screening also revealed the evolution of patient T cell responsiveness to expressed tumor mutations during the course of treatment. In some cases, expansion of stimulatory antigen (neoantigen) or inhibitory antigen (inhibigen) breadth was observed. Reduction of stimulatory antigen (neoantigen) or inhibitory antigen (inhibigen) breadth was also observed.
[0394] Figure 22 shows overall characterization of stimulatory antigens (neoantigens), inhibigens, and non-responsive antigens identified in 37 patients screened for inclusion in the Part A Study, the Main Part B Study, and the Part B Refractory Study of GEN-009. Panel A: Total proportion of patients in which CD4+ and CD8+ T cells responded with an inhibitory phenotype (n=33). Panel B: Total proportion of stimulatory antigens (neoantigens), inhibigens, and non-responsive antigens. Inhibigens accounted for 16% (range 1.8%-47.5%) of total ATLAS-identified responses.
[0395] Figure 23 shows the overall proportion of CD4+ and CD8+ stimulatory antigens (neoantigens), inhibigens, and non-responsive antigens identified in 37 patients screened for inclusion in the Part A Study, the Main Part B Study, and the Part B Refractory Study of GEN- 009. Results are expressed as a percentage of total CD4+ and CD8+ responses. Inhibigen- specific T cell responses were identified in both CD4+ and CD8+ T cell subsets, with more detected in the CD4+ T cell subset (mean 10.3%; 0.5%-42%) as compared to CD8+ (mean 6.1%; 1.2%-23%).
[0396] Figure 24 shows overall characterization, against total mutations screened, of somatic mutations, stimulatory antigens (neoantigens), and inhibigens identified in 37 patients screened for inclusion in the Part A Study, the Main Part B Study, and the Part B Refractory Study of GEN-009. Panel A: Number of mutations screened (± SEM) by tumor type. The highest number of mutations were found in melanoma and NSCLC as compared to bladder, RCC, and HNSCC. Panel B: Number of inhibigens detected, by tumor type, as a percent of total mutations screened. Stratification of patients by cancer type revealed no significant difference in the proportion of inhibigens identified (one-way ANOVA p= 0.140). Panel C: Correlation analysis between the number of inhibigens detected and the size of the mutanome screened (linear regression, R2 = 0.005). These results show that inhibigens are found in the majority of cancer patients at a frequency similar to stimulatory antigens (neoantigens). Figure 25 shows the proportion of stimulatory antigens and inhibigens identified by ATLAS screens for each patient at initial screening at D50 post-initiation of vaccinations, broken out by T cell subset, tumor type and checkpoint inhibition responsiveness vs. non-responsiveness. For each set of bars, the top bar shows results obtained at initial screening, and the bottom bar shows results obtained at Day 50. Top rows: pooled data for the cohort of checkpoint inhibitor-responsive patients (left: CPI responsive, N=6) or -refractory (right: CPI refractory, N=2) patients. Middle rows: individual patient responses, with patient ID indicated to the left of each set. Bottom row: pooled responses by tumor type. NSCLC N = 2, RCC N = 1, UC N = 1, SCCHN N = 4. These results suggest epitope spread in checkpoint inhibitor-responsive patients, and epitope contraction in checkpoint inhibitor refractory patients.
[0397] Figure 26 shows the results of 16-plex patient-specific somatic assays for detection of circulating tumor DNA (ctDNA). Assays were performed on plasma samples taken at initial screening (Baseline) and at D50 post initiation of vaccination (Post-Tx). Two patients enrolled in the Main Part B Study are represented, one in each graph. Results are expressed as the percent frequency of variant alleles in plasma, and show disappearance of ctDNA postvaccination. The data are stratified by non-antigenic, tumor-specific mutations (circles) or ATLAS-identified antigens (diamonds = inhibigens, triangles = neoantigens not included in the GNE-009 vaccine, and squares = neoantigens included in the GEN-009 vaccine). In both patients, there was a complete disappearance of ctDNA post-treatment, irrespective of the antigenic profile of the mutations or whether the patient was initially checkpoint-responsive or - refractory. These data support the clearance of tumors as a result of GEN-009 + anti-PD-1 combination therapy and corroborate spider plots showing primary tumor reduction and/or control in both patients post-GEN-009 treatment.
Example 4. Identification of stimulatory and inhibitory antisens using mouse ATLAS (mATLAS} screens
[0398] Methods for mATLAS screen
[0399] A cohort of C57BL/6J mice bearing B16F10 tumors were euthanized and their tumors and spleens harvested. DNA obtained from pooled tumors was sequenced and analyzed for non-synonymous mutations. Over 1600 such mutations were identified, and these were synthesized as 399 bp DNA fragments centered upon the base pair change and transformed individually into E. coll bacteria expressing cLLO to build a candidate neoantigen library. Splenocytes frozen from pooled spleens of the tumor-bearing mice were thawed, and CD8+ T cells were sorted using a negative selection bead kit. These were subsequently expanded with CD3/CD28 beads and IL-2 for 7 days followed by 1 day of rest after removal of beads and cytokine. Mouse APCs (RAW309 Cr. l macrophage cell line) were cultured overnight, washed with PBS, then co-cultured with the bacterial library for 2 hours, washed with PBS, and then cultured overnight with the non-specifically expanded and rested CD8+ T cells. Harvested supernatant from the co-culture was tested for fFNy and TNFα by a custom mouse 384-well Meso Scale Discovery (MSD) electrochemiluminescence assay.
[0400] Results
[0401] Sixty-eight antigens were identified as stimulatory (exceeding a statistical threshold above the negative control, a 399 bp fragment of the mouse actin gene) and 57 antigens were identified as inhibitory (reduced beyond a statistical threshold below the negative control), for either IFNy, TNFa, or both. Overall, only 2% (6 of 283) of NetMHCpan (Nielsen et al., PLoS One. 2007 Aug 29;2(8):e796) predicted binding antigens were empirically identified by mATLAS as stimulatory antigens. 6% (17 of 283) of NetMHCpan predicted antigens were identified by mATLAS as inhibitory antigens.
[0402] The top 50 stimulatory and 50 inhibitory antigens, and approximately 50 antigens closest to the negative control (non-responses), were used in two additional repeat mATLAS screens with increased replicates. Each antigen was ranked by its fFNy signal across all three screens, as well as a separate rank for its TNFα signal across all three screens. The top ten ranked antigens (stimulatory) and eight of the bottom ten ranked antigens (inhibitory) were each synthesized as 27mer synthetic long peptides (SLPs) for use in mouse vaccination and B16F10 melanoma challenge studies. Additionally, three published stimulatory Bl 6F 10 antigens: M27 (CD8 neoantigen), M30 (CD4 neoantigen), and Trp2 (CD8 tumor-associated antigen, TAA), were each synthesized as 27mer synthetic long peptides (SLPs). These three antigens were previously shown to have both immunogenicity and efficacy in treating the Bl 6F 10 tumor model (Castle JC, Kreiter S et al (2012). Exploiting the Mutanome for Tumor Vaccination. Cancer Research 72(5); Kreiter S et al (2015). Mutant MHC class II epitopes drive therapeutic immune responses to cancer. Nature 520(7549)). The top ranked and known B16F10 antigens were also each synthesized as four 15mer overlapping peptides (OLPs) for use in ex vivo assays. [0403] The m ATLAS workflow, preliminary to Bl 6F 10 melanoma challenge and vaccination studies (Example 5, below), is shown in Figure 27, Panel A. Representative results of m ATLAS screening for ZFNy only are shown in Figure 27, Panel B.
Example 5. Mouse cancer vaccine studies: Inhibigen-specific suppression of immune responses (therapeutic vaccination}
[0404] These therapeutic vaccination studies examined whether inhibitory antigens can compete or interfere with previously known stimulatory antigens.
[0405] Methods
[0406] SLPs corresponding to inhibigens identified by mATLAS and to three known B16F10 antigens were synthesized according to Example 4. Lyophilized SLPs were reconstituted in 50% ACN in H2O and pre-mixed, then frozen and lyophilized for 21h and subsequently frozen again as lyophilized pools. These were reconstituted on the day of immunization in either PBS/DMSO or PBS/adjuvants/DMSO (final DMSO concentration: 4%).
[0407] B16F10 tumor-bearing mice were vaccinated on the following schedule: cancer cells were injected subcutaneously on the right flank on dO (ATCC-passage 7, 100K cells in lOOpl of 20% Matrigel); vaccine was injected subcutaneously either at the tail base or scruff of the neck on d3, dlO, dl7.
[0408] The experimental groups (n =12) of the first study were injected with: 1) a pool of three previously known (published) stimulatory Bl 6F 10 antigens: M27 (CD8 neoantigen), M30 (CD4 neoantigen), and Trp2 (CD8 tumor-associated antigen, TAA), plus adjuvant (Protective Vaccine), or 2) the same protective vaccine plus inhibitory antigen MMP9FS (Inhibigen + Protective Vaccine).
[0409] The experimental groups of the second study were injected with: 1) a variant of the above-described protective vaccine comprising a pool of two known (published) stimulatory B16F10 antigens: M30 (CD4 neoantigen) and Trp2 (CD8 tumor-associated antigen, TAA), plus adjuvant (also labeled Protective Vaccine), or 2) the same protective vaccine plus inhibitory antigen MMP9FS (Protective Vaccine + Inhibigen MMP9FS), or 3) inhibitory antigen MMP9FS alone plus adjuvant (MMP9FS + adj).
[0410] The experimental groups of the third study were injected with: 1) a further variant of the above-described protective vaccine comprising a pool of one ATLAS-identified stimulatory antigen, Gal3Stl (CD8 neoantigen) plus two known (published) stimulatory B16F10 antigens: M30 (CD4 neoantigen) and Trp2 (CD8 tumor-associated antigen), plus adjuvant (also labeled Protective Vaccine), or 2) the same protective vaccine plus inhibitory antigen MMP9FS (Protective Vaccine + Inhibigen MMP9FS), or 3) inhibitory antigen MMP9FS alone plus adjuvant (MMP9FS + adj). Control groups were injected with adjuvant alone (Adjuvant Only; triple adjuvant comprising CpG, 3D-PHAD, QS-21). SLP dosage was 50pg per SLP/mouse/day.
[0411] Splenocytes were collected on dl6 of the study (i.e., 6 days after vaccine injection #2) and resuspended in OpTmizer media. In some instances, splenocytes were collected on d4 and dl l, or on d21-d26 of the study. Cells were normalized to one cell (4xl05 cells/well) concentration and seeded into an IFNy ELISPOT plate with stimulants for overnight culture. Cells from each individual mouse were split into 2 wells: well 1 contained media alone, well 2 contained pooled OLPs (4pg/ml or lug/mL/OLP) specific to the vaccine that the mouse received. For example, for a mouse immunized with peptides 1-4, the cells were stimulated with OLPs la-d, 2a-d, 3a-d and 4a-d (16 individual 15mers overlapping by 11 amino acids total) across different wells.
[0412] Tumor size was measured 3x/week and subsequently on a daily basis after reaching a specified size threshold (1000mm3). Statistical analysis was performed by one-way ANOVA with multiple comparisons. Mice were euthanized when tumors reached maximum size, or tumors became ulcerated and did not heal within 24 hours. No mice in this study were euthanized for other health reasons.
[0413] B16F10 tumors were collected at one timepoint on d21-28 of the study, formalin- fixed, paraffin-embedded, and sections stained by chromogenic immunohistochemistry (IHC). In some instances, early-stage tumors were collected on dl 1 of the study. For assessment of infiltrating immune cell populations, sections were stained with antibodies specific for mouse CD4 or CD8a (T cells), FoxP3 (Tregs), F4/80 (macrophages), or CD11c (dendritic cells), in pink/red, and counterstained for nuclei with hematoxylin (blue). For assessment of tumor vasculature, sections were stained with antibodies specific for the endothelial marker CD31. For assessment of metabolic status, sections were stained with antibodies specific for the metabolic markers LDHA, ARG1, and CD73.
[0414] Figure 28, Panel A shows results of ZFNy ELISPOT performed on splenocytes from tumor-bearing and vaccinated mice of the first study (top graph) and of the third study (bottom graph) after in vitro stimulation with overlapping peptides spanning the vaccine antigenss. Statistical analysis was by multiple T-tests (top graph) and one-way ANOVA (bottom graph; p <0.0001). Figure 28, Panel B shows mean tumor growth kinetics following therapeutic vaccination with adjuvant only, protective vaccine, or protective vaccine plus inhibigen (first study). Figure 28, Panel C shows mean tumor growth kinetics following therapeutic vaccination with adjuvant only, protective vaccine, or protective vaccine plus inhibigen (MMP9FS; second study). Figure 28, Panel D shows mean tumor growth kinetics following therapeutic vaccination with adjuvant only, or inhibigen (MMP9FS) plus adjuvant (second study). The results demonstrate that inclusion of an inhibigen broadly reduces immunogenicity and leads to accelerated tumor growth, abrogating the efficacy of an otherwise protective vaccine. Vaccination with an inhibigen plus adjuvant leads to more sharply accelerated tumor growth.
[0415] The different panels of Figure 29, Panel A shows representative sections of immunohistochemically labeled tumors from mice therapeutically vaccinated with adjuvant alone (Adjuvant, top row), protective vaccine (Protective, middle row), or protective vaccine plus inhibigen (MMP9FS) (+ Inhibigen, bottom row). Tumor sections were labeled with antibodies specific for CD4 or CD8a (T cells), FoxP3 (Tregs), F4/80 (macrophages), or CD11c (dendritic cells). Endogenous melanin deposits in tumors are visible (brown) and specific labeling is indicated in red. Panel B shows quantification of Panel A images. Panel C shows representative tumor sections immuno-histochemically stained for the endothelial marker CD31, from tumor-bearing mice vaccinated with protective vaccine (Protective, top row), or protective vaccine plus inhibigen MMP9FS (+ Inhibigen, bottom row). Panel D shows representative tumor sections immuno-histochemically stained for metabolic markers LDHA, ARG1, and CD73, from tumor-bearing mice vaccinated with protective vaccine plus inhibigen MMP9FS. LDHA was predominantly expressed in tumor tissue, ARG1 was predominantly expressed in tumorinfiltrating immune cells, and CD73 was predominantly expressed in vascular tissue. Panel E shows quantification of Panel D images, together with quantification of images (not shown) of tumor sections from mice vaccinated with adjuvant alone, or with protective vaccine. Panel F shows quantification of representative tumor sections immuno-histochemically stained for CD8 molecules on T cells, from tumor-bearing mice therapeutically vaccinated with adjuvant only, protective vaccine, or MMP9FS only (top graph), or tumor-bearing mice vaccinated with adjuvant only, protective vaccine, or protective vaccine plus MMP9FS (bottom graph). In the bottom graph, tumors were resected on day 11 of the study (early-stage tumors).
[0416] Image quantification in Panels B, E, and F was determined by ImageJ software thresholding on positive stain (pink) divided by tissue area (total image area - white non-tissue). Statistical analysis was performed by one-way ANOVA with multiple comparisons. Graphs depict mean and standard deviation; * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, NS=not significant.
[0417] These results demonstrate that inclusion of an inhibigen in an otherwise protective vaccine alters the tumor microenvironment. Specifically, inclusion of an inhibigen abrogates beneficial immune cell (CD8+ T cells, F4/80 macrophages, CDl lc dendritic cells) infiltration into the tumor (Panels A, B, and F); correlates with gross alterations in tumor vasculature, suggesting uncontrolled growth and lack of vascular maturation consistent with an aggressive tumor microenvironment (Panel C); and downregulates expression of metabolic markers (Panels D and E). Example 6. MHC competition experiments: peptide-pulsed antisen presentins cells (APCs)
[0418] MHC competition experiments were performed according to the schematic shown on Figure 30. Peptides corresponding to inhibigen MMP9FS and two known protective Bl 6F 10 antigens (M30, Trp2) were either pulsed on the same APCs (Together), or separately pulsed on different APCs before combining (Separate). In the former, peptides may compete for MHC binding and presentation; in the latter, separated pulsing precludes MHC competition.
[0419] Splenocytes collected from tumor-bearing mice therapeutically vaccinated with adjuvant only, protective vaccine, or the protective vaccine plus inhibigen MMP9FS of Example 5 were incubated with the pulsed APCs above (Together or Separate) in an IFNy ELISPOT assay. Results are shown in Figure 31, Panel A as the mean IFNy Spot Forming Cells (SFC) per 5xl05 splenocytes (± SEM) pooled from three mice per vaccination group. Figure 31, Panel B shows representative wells. The results demonstrate that recall IFNy T cell responses are not affected by combined inhibigen MMP9FS +M30+Trp2 peptide pulsing to the same APCs in vitro. This in turn suggests that inhibigens do not interfere with immunogenicity through competition for binding to MHC.
[0420] The MHC restrictions of the known protective antigens and inhibigen MMP9FS used in this study, shown in Table 10 below, provide further support. Since theantigens are presented by different MHC molecules, MHC competition is unlikely.
[0421] Table 10. MHC Restrictions
Figure imgf000171_0001
[0422] *MHC restriction published [0423] * *MHC restriction determined by Applicant
[0424] The splenocytes collected from mice of Example 5 (therapeutically vaccinated with adjuvant only, protective vaccine, or the protective vaccine plus inhibigen MMP9FS) and incubated with the pulsed APCs above (Together) were further restimulated with respective antigen peptides, stained for intracellular cytokines, and gated by flow cytometry on live, CD45+, CD19nes/NKl.lneS, CD3+, CD8+ T cells. Data shown are pooled splenocytes from four mice from each vaccination group. In Figure 32, CD8+ IFNy expressing T cells are represented as a percentage of total CD3+ T cells. Results show that restimulation with a peptide pool containing an inhibigen fails to decrease IFNY responses from T cells of mice immunized with the protective vaccine.
Example 7. Mouse cancer vaccine study: combination of vaccine and checkpoint inhibitor (CPI} (therapeutic vaccination}
[0425] These therapeutic vaccination studies included study arms with and without checkpoint inhibitors anti-PD-1, anti-PD-Ll, and/or anti-CTLA-4. The effects of each checkpoint inhibitor in conjunction with a protective vaccine or a protective vaccine plus inhibigen MMP9FS were assessed.
[0426] Methods
[0427] SLPs corresponding to antigens identified by mATLAS and to three known B16F10 antigens were synthesized according to Example 4. Lyophilized SLPs were reconstituted in 50% ACN in H2O and pre-mixed, then frozen and lyophilized for 21h and subsequently frozen again as lyophilized pools. These were reconstituted on the day of immunization in either PBS/DMSO or PBS/adjuvants/DMSO (final DMSO concentration: 4%).
[0428] B16F10 tumor-bearing mice were vaccinated on the following schedule: cancer cells were injected subcutaneously on the right flank on dO (ATCC-passage 6, 100K cells in lOOpl of 20% Matrigel in DMEM); vaccine was injected subcutaneously at the tail base on d3, dlO, d!7. [0429] The experimental groups (n =12) of the first study were injected with: 1) the protective vaccine of Example 5, comprising a pool of one ATLAS-identified stimulatory antigen Gal3Stl (CD8 neoantigen) plus two previously known (published) stimulatory B16F10 antigens: M30 (CD4 neoantigen), and Trp2 (CD8 tumor-associated antigen, TAA), plus adjuvant, or 2) the same protective vaccine plus the inhibitory antigen MMP9FS.
[0430] The experimental groups of the second study were injected with: 1) the protective vaccine of Example 5, comprising a pool of two previously known (published) stimulatory B16F10 antigens: M30 (CD4 neoantigen), and Trp2 (CD8 tumor-associated antigen, TAA), plus adjuvant, or 2) inhibitory antigen MMP9FS alone plus adjuvant. SLP dosage was 50pg per SLP/mouse/day. Checkpoint inhibitors anti-PD-1, anti-PD-Ll, anti-CTLA-4, or anti-PD-1 plus anti-CTLA-4 were administered on d7, dlO, dl4, dl7, and d21 (4 days after each of the 3 vaccinations and on the days of 2nd and 3rd vaccinations). Control groups received adjuvant alone (triple adjuvant comprising CpG, 3D-PHAD, QS-21), or no checkpoint inhibitor. The workflow for Bl 6F 10 melanoma challenge and vaccination in conjunction with checkpoint inhibitor is shown in Figure 33.
[0431] Splenocytes were collected on d23 of the study (i.e., 2 days after final dose of checkpoint inhibitor) and resuspended in OpTmizer media. Cells were normalized to one cell concentration (2xl05 cells/well) and seeded into an IFNy ELISPOT plate with stimulants for overnight culture. Cells from each individual mouse were split into 2 wells: well 1 contained media alone, well 2 contained pooled OLPs (4pg/ml or lug/mL/peptide) specific to the vaccine that the mouse received. For example, for a mouse immunized with peptides 1-4, the cells were stimulated with OLPs la-d, 2a-d, 3a-d and 4a-d (16 individual 15mers overlapping by 11 amino acids total).
[0432] Tumor size was measured 3x/week and subsequently on a daily basis after reaching a specified size threshold (1000mm3). Statistical analysis was performed by one-way ANOVA with multiple comparisons. Mice were euthanized when tumors reached maximum size, or tumors became ulcerated and did not heal within 24 hours. No mice in this study were euthanized for other health reasons. [0433] Figure 34 shows results of IFNy ELISPOT performed on splenocytes from therapeutically vaccinated mice, with and without checkpoint inhibition, after in vitro stimulation with overlapping peptides spanning vaccine components. As indicated, the experimental groups received protective vaccine or protective vaccine plus inhibigen MMP9FS, in each case combined with anti-PD-1, anti-CTLA-4, or no checkpoint inhibitor. Figure 35 shows mean tumor growth kinetics following therapeutic vaccination. Panel A shows tumor growth for groups therapeutically vaccinated with protective vaccine, or with protective vaccine plus inhibigen, and treated with anti-PD-1 checkpoint inhibitor (first study). Panel B shows tumor growth for groups therapeutically vaccinated with protective vaccine, or with protective vaccine plus inhibigen, and treated with anti-CTLA-4 checkpoint inhibitor (first study). Panel C shows tumor growth for groups therapeutically vaccinated with protective vaccine, or with inhibigen plus adjuvant, and treated with an anti-PD-1, anti-PD-Ll, or anti-CTLA-4 checkpoint inhibitor (second study). Panel D shows tumor growth for groups therapeutically vaccinated with protective vaccine, or with inhibigen plus adjuvant, and treated with a combination of anti-PD-1 and anti-CTLA-4 checkpoint inhibitors (second study). Statistical analyses were performed by one-way ANOVA with multiple comparisons. The results of the first study show that anti-PD-1 checkpoint inhibition did not prevent accelerated tumor growth seen in mice vaccinated with an inhibigen, while anti-CTLA-4 checkpoint inhibition partially ameliorated such tumor growth. The results of the second study similarly show that anti-PD-1 and anti-PDL-1 did not prevent accelerated tumor growth in mice vaccinated with an inhibigen, while anti-CTLA-4 checkpoint inhibition partially ameliorated such accelerated tumor growth. In addition, results of the second study show that combination checkpoint inhibition (anti-PD-1 plus anti-CTLA-4) partially ameliorated tumor growth, but the effect did not appear to be additive or synergistic compared to checkpoint inhibition with anti-CTLA-4 alone.
[0434] B16F10 tumors were collected at one timepoint on d21-28 of the study, formalin- fixed, paraffin-embedded, and sections were stained by chromogenic immunohistochemistry (H4C). The different panels of Figure 36 show tumor sections from mice vaccinated with adjuvant alone (Adjuvant, top row), the protective vaccine (Protective, middle row), or the protective vaccine plus the MMP9FS inhibigen (+ Inhibigen, bottom row), with and without anti- PD-1, anti-CTLA-4, or anti-PD-1 plus anti-CTLA-4 checkpoint therapy. The tumor sections were stained with an anti-mouse CD8a antibody (pink/red) or CD4 antibody (not pictured) and counterstained with hematoxylin (blue). Endogenous melanin deposits in tumors are visible (brown). Image quantification, shown on Figure 37, was determined by ImageJ software thresholding on positive stain (pink) divided by tissue area (total image area - white non-tissue). The top panel shows results for CD8+ tumor infiltrating lymphocytes (TILs); the bottom panel shows results for CD4+ TILs. Each symbol in the graph represents data from one mouse, the horizontal line indicates the mean ± SD. Statistical analyses were performed by one-way ANOVA with multiple comparisons. CD8+ and CD4+ TILs were present in increased numbers in mouse Bl 6F 10 tumor tissue after vaccination with a protective vaccine (and greatly increased in combination with anti-PD-1 checkpoint inhibitor), and were reduced to baseline levels when the protective vaccine included an inhibigen. Treatment with anti-PD-1 and/or anti-CTLA-4 check-point inhibitor did not rescue the reduced T cell infiltration into the tumor tissue associated with vaccination with an inhibigen-containing formulation.
[0435] Additionally, B16F10 tumor sections were immunohistochemically stained with antibodies specific for checkpoint markers CTLA-4 and PD-L1. Representative results are shown in Figure 38. Panel A shows representative tumor sections stained for CTLA-4 (top) and PD-L1 (bottom). Panel B shows image quantification of tumor sections stained for CTLA-4 and PD-L1, from mice vaccinated with adjuvant alone (None), protective vaccine (Protective), or protective vaccine plus inhibigen MMP9FS (+ Inhibigen), with and without anti-PD-1 or anti- CTLA-4 checkpoint therapy. Quantification of all immunohistochemical images was performed using ImageJ assessment of pink staining area. Graphs depict mean and standard deviation; statistical analysis is ANOVA with multiple comparisons, * p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. These results show that reduced expression of CTLA-4 checkpoint marker correlates with partial failure of anti-CTLA-4 checkpoint therapy to control tumor growth in mice vaccinated with an inhibigen-containing formulation. Similarly, reduced expression of PD- L1 checkpoint marker correlates with failure of anti-PD-1 checkpoint therapy to control tumor growth in mice vaccinated with an inhibigen-containing formulation. [0436] Taken together, these results indicate that checkpoint inhibitors are not equally effective in overcoming the deleterious effects of vaccination with an inhibigen. This suggests that tumors burdened with numerous inhibigens, one or more dominant inhibigens, and/or an unfavorable ratio of inhibigens to stimulatory antigens may be treatable with certain checkpoint inhibitors, but not with others.
Example 8. Mouse cancer vaccine studies: Inhibisen-specific suppression of immune responses (therapeutic vaccination}
[0437] These therapeutic vaccination studies examined whether inhibitory antigens dampen T cell immunity.
Methods
[0438] Synthetic long peptides (SLPs) corresponding to inhibigens identified by mATLAS and to known Bl 6F 10 antigens were synthesized according to Example 4. Lyophilized SLPs were reconstituted in 50% ACN in H2O and pre-mixed, then frozen and lyophilized for 21h and subsequently frozen again as lyophilized pools. These were reconstituted on the day of immunization in either PBS/DMSO or PBS/adjuvants/DMSO (final DMSO concentration: 4%).
[0439] B16F10 tumor-bearing mice were vaccinated on the following schedule: cancer cells were injected subcutaneously on the right flank on dO (ATCC-passage 7, 100K cells in lOOpl of 20% Matrigel); vaccine was injected subcutaneously either at the tail base or scuff of the neck on d3, dlO, dl7.
[0440] The experimental groups (n =12) of the first study were injected with: 1) a pool of two previously known (published) stimulatory B16F10 antigens: M30 (CD4 neoantigen) and Trp2 (CD8 tumor-associated antigen, TAA), plus adjuvant (Protective Vaccine), or 2) the same protective vaccine plus inhibitory antigen MMP9FS (Inhibigen + Protective Vaccine).
[0441] The experimental groups of the second study were injected with: 1) a protective vaccine of Example 5, comprising a pool of one ATLAS-identified stimulatory antigen Gal3Stl (CD8 neoantigen) plus two previously known (published) stimulatory B16F10 antigens: M30 (CD4 neoantigen), and Trp2 (CD8 tumor-associated antigen, TAA), plus adjuvant, or 2) the same protective vaccine plus inhibitory antigen MMP9FS, or 3) inhibitory antigen MMP9FS alone plus adjuvant (MMP9FS + adj). Control groups were injected with adjuvant alone (Adjuvant Only; triple adjuvant comprising CpG, 3D-PHAD, QS-21). SLP dosage was 50pg per SLP/mouse/day.
[0442] Splenocytes were collected on dl6 (i.e., 6 days after vaccine injection #2) and resuspended in OpTmizer media. In some instances, splenocytes were harvested on d4, dl 1, or d21-d28 of the study. Cells were normalized to one cell (IxlO6 cells/well) concentration and stimulated in an overnight IFNy ELISPOT assay. For vaccine-specific stimulation, cells from each individual mouse were split into 2 wells: well 1 contained media alone, well 2 contained pooled OLPs (4pg/ml or lug/mL/OLP) specific to the vaccine that the mouse received. For example, for a mouse immunized with peptides 1-4, the cells were stimulated with OLPs la-d, 2a-d, 3a-d and 4a-d (16 individual 15mers overlapping by 11 amino acids total) across different wells. For non-specific stimulation, to evaluate a non-specific defect in ability to proliferate, cells were incubated with an anti-CD3 antibody (positive control), or with PMA/ionomycin. T cells expressing IFNy- and markers CD44, CD69, PD-1, 4- IBB (CD 137), and CD62L were then quantified by flow cytometry (gated on live lymphocytes, CD3+ T cells).
[0443] Figure 39, Panel A shows results of an IFNy ELISPOT assay performed on splenocytes from vaccinated mice after in vitro stimulation with overlapping peptides spanning vaccine components, at day 21 of the first study. Panel B shows a time course of IFNy ELISPOT at days 4, 8, and 21 of the first study. Statistical analysis was by multiple T-tests. Panel C shows flow cytometry results for IFNy-expressing cells (left), and cells expressing CD44 or CD69 molecules (right) from splenocytes of therapeutically vaccinated mice of the first study, after non-specific stimulation with PMA/ionomycin or anti-CD3 antibody. Tumor-bearing mice were vaccinated with adjuvant alone (Adj only), protective vaccine (Protective), or protective vaccine plus inhibigen MMP9FS (+ Inhibigen). Panel D shows flow-cytometry results for IFNy- expressing cells from splenocytes of vaccinated mice harvested at day 4 of the second study. Cells were re-stimulated with PMA/ionomycin (with Golgi plug and Golgi stop). Panel E shows flow-cytometry results for cells expressing markers CD69, PD-1, 4-1BB (CD137), or CD62L, from the same collection of splenocytes harvested at day 4 of the second study. Results are shown as the proportion of viable CD3+ T cells from a pool of 4 mice per group, for tumorbearing mice therapeutically vaccinated with adjuvant alone (Adjuvant), protective vaccine (Protective vaccine), or protective vaccine plus inhibigen MMP9FS (Protective vaccine + Inhibigen). Panel F shows flow-cytometry results for myeloid populations (Ml and M2 macrophages, myeloid-derived suppressor cells [MDSC’s]) from splenocytes of vaccinated mice of the second study. Suppressive myeloid populations did not expand upon vaccination with an inhibigen.
[0444] To further evaluate the effect of inhibigens on cell proliferation, splenocytes from tumor-bearing and therapeutically vaccinated mice were stained with cell trace violet and cultured with or without IL-2 (0.17 lU/ l) and anti-CD3 antibody. Medium was exchanged every 3 days. After 7 days, cells were harvested, labeled with fluorescently conjugated antibodies specific for T cell markers, and analyzed by flow cytometry. Figure 40, Panel A shows total CD3+ T cell proliferation in the presence of IL-2 and anti-CD3 antibody. Panel B shows specific proliferation of CD8+ T cells in response to anti-CD3 stimulation by quantitation of cell trace violet. These result show that T cells isolated from tumor-bearing inhibigen- vaccinated mice proliferate similarly after non-specific expansion to T cells from mice therapeutically vaccinated with protective vaccine only or adjuvant only.
[0445] To evaluate the effect of inhibigens on TCR signal strength, splenocytes from tumor-bearing and vaccinated mice were non-specifically stimulated with a dose titration of anti- CD3 antibody, starting at a concentration 5-fold higher than what is normally used, land evaluated for upregulation of activation markers CD69 (top) and 4-1BB (CD137; bottom) by flow cytometry. Figure 41 shows results expressed as the percentage of CD3+ T cells expressing the indicated marker (top left), and as Mean Fluoresence Intensity (MFI) on CD3+ T cells (top right, bottom). These results indicate that T cells isolated from inhibigen-vaccinated mice have similar activation potential to T cells from mice vaccinated with protective vaccine only or adjuvant only.
[0446] To evaluate the effect of inhibigens on activation-induced cell death (AICD), splenocytes were harvested from tumor-bearing baccinated mice and non-specifically stimulated with a dose titration of anti-CD3 antibody, starting at a concentration 5-fold higher than what is normally used, and analyzed by flow cytometry for cleaved Caspase-3 in conjunction with expression of surface markers. A dose titration of the mitogen PMA/ionomycin, starting at a 5- fold greater concentration than standardly used served as a positive AICD control. Figure 42 shows the proportion of CD3+ T cells expressing cleaved Caspase-3. These results indicate that T cells isolated from inhibigen-vaccinated mice do not undergo AICD more readily that T cells from mice vaccinated with protective vaccine only or adjuvant only.
[0447] Draining lymph nodes from tumor-bearing and vaccinated mice were collected at one time point on day 4, day 8, and day 21-28 of the second study and enriched for T cells. Gene expression of selected markers, including CD44, CTLA-4, and LDHA, was assessed by RNAseq. All samples were analyzed using Illumina HiSeq at -350 million bp per read. Quality of the data yielded -20x106 reads per sample, with >90% bases read and mean quality score >35.
[0448] Figure 43 shows RNA counts per million reads for markers CD44, CTLA-4 and LDHA (Panel A) and PD-1, NK1 and Tox2 (Panel B) at day 21-28. Tumor-bearing mice were vaccinated with adjuvant only (Adjuvant), protective vaccine plus inhibigen MMP9FS (Inhibitory), or protective vaccine (Protective), as indicated on the x-axis. Panel C shows volcano plots, comparing representative groups of differentially expressed genes at day 4 and day 8; significantly upregulated genes are shown in red, downregulated genes are shown in blue. Table 11 below summarizes global T cell transcriptional patterns at day 4 and day 8.
Table 11. T cell Transcriptional Patterns
Figure imgf000180_0001
Example 9. Impact of Incubation with Inhibitory Antisen Peptide
[0449] To assess the direct impact on viability, cells were incubated in vitro with peptide corresponding to inhibitory antigen MMP9FS for 24h. Cells were naive bone marrow dendritic cells (BMDCs) or splenic T cells derived from B16F10 tumor-bearing mice or OT-I/OT-II mice. The B16F10 antigen was Gal3stl; the OT-I antigen was SIFNFEKL; the OT-II antigen was ISQ. Figure 44, Panel A shows results for BMDCs from B16F10 tumor-bearing mice. Panel B shows results for CD4+ T cells from B16F10 tumor-bearing mice and OT-II mice. Panel C shows results for CD8+T cells from B16F10 tumor-bearing mice and OT-I mice. Incubation with MMP9FS peptide did not impact cell viability in vitro.
[0450] BMDC functional expression was further characterized by flow cytometry. OT-I and OT-II T cells were incubated ± BMDC’s, with PMA/ionomycin ± inhibitory antigen peptide (MMP9FS), and T cell activation was assessed 5 days later by flow cytometry. Figure 45 shows flow-cytometry results for the indicated cell markers. Incubation with MMP9FS peptide did not impact functional expression of cell markers in vitro.
Example 10. Adoptive transfer of T cells and the inhibigen phenotype
[0451] This study evaluated the effect of adoptive transfer of T cells on the inhibigen phenotype of B 1610 melanoma mice. C57BL/6 donor tumor-bearing mice were vaccinated with adjuvant only (control), protective vaccine, or protective vaccine plus inhibigen (inhibitory antigen) MMP9FS of Example 5. Splenocytes were collected on dl4 of the study (i.e. 4 days after vaccine injection #2), and CD3+ T cells were isolated from respective groups by negative selection.
[0452] Recipient athymic, Nude mice were treated on the following schedule: B16F10 cancer cells were injected subcutaneously in the right flank on dO (ATCC-passage 6, 100K cells/mouse in lOOpl of 20% Matrigel in DMEM); a bolus of 10M purified T cells was injected intravenously in the tail vein also on dO. Tumor volume was measured on d5, d7, d9, and dl2, and euthanasia was performed on dl4. Statistical analysis was performed by one-way ANOVA with multiple comparisons.
[0453] Figure 46, panel A is a schematic representation of the experiment. Figure 46, panel B shows mean tumor growth kinetics following adoptive transfer of T cells. The results show that adoptive transfer of T cells from mice vaccinated with protective vaccine were significantly protected. In contrast, adoptive transfer of T cell from mice vaccinated with protective vaccine plus inhibigen conferred no protection against tumor growth. This shows that the detrimental effect of inhibigens can be passively transferred.
Example 11. Mouse cancer vaccine study: combination of vaccine and checkpoint inhibitor (CPI} (therapeutic vaccination}
[0454] These therapeutic vaccination studies included study arms with and without checkpoint inhibitors anti-PD-1, anti-PD-Ll, and/or anti-CTLA-4. The effects of each checkpoint inhibitor in conjunction with a protective vaccine or a protective vaccine plus inhibigen MMP9FS were assessed.
[0455] Methods
[0456] SLPs corresponding to antigens identified by mATLAS and to three known B16F10 antigens were synthesized according to Example 4. Lyophilized SLPs were reconstituted in 50% ACN in H2O and pre-mixed, then frozen and lyophilized for 21h and subsequently frozen again as lyophilized pools. These were reconstituted on the day of immunization in either PBS/DMSO or PBS/adjuvants/DMSO (final DMSO concentration: 4%).
[0457] B16F10 tumor-bearing mice were vaccinated on the following schedule: cancer cells were injected subcutaneously on the right flank on dO (ATCC-passage 6, 100K cells in lOOpl of 20% Matrigel in DMEM); vaccine was injected subcutaneously at the tail base on d3, dlO, dl7.
[0458] The experimental groups (n =8) were injected with: 1) the protective vaccine of Example 5, comprising a pool of one ATLAS-identified stimulatory antigen Gal3Stl (CD8 neoantigen) plus two previously known (published) stimulatory B16F10 antigens: M30 (CD4 neoantigen), and Trp2 (CD8 tumor-associated antigen, TAA), plus adjuvant; 2) the same protective vaccine plus inhibigen MMP9FS; or 3) inhibigen MMP9FS alone plus adjuvant. SLP dosage was 50pg per SLP/mouse/day. Checkpoint inhibitors anti-PD-1, anti-PD-Ll, anti-CTLA- 4, or anti-PD-1 plus anti-CTLA-4 were administered on d5, d9, dl2, and dl6. Control groups received adjuvant alone (triple adjuvant comprising CpG, 3D-PHAD, QS-21). The workflow for B16F10 melanoma challenge and vaccination in conjunction with checkpoint inhibitor is shown in Figure 33.
[0459] Splenocytes were collected on dl 8 of the study (i.e., 2 days after final dose of checkpoint inhibitor) and resuspended in OpTmizer media. Cells were normalized to one cell concentration (2xl05 cells/well) and seeded into an IFNy ELISPOT plate with stimulants for overnight culture. Cells from each individual mouse were split into 2 wells: well 1 contained media alone, well 2 contained pooled OLPs (4pg/ml or lug/mL/peptide) specific to the vaccine that the mouse received. For example, for a mouse immunized with peptides 1-4, the cells were stimulated with OLPs la-d, 2a-d, 3a-d and 4a-d (16 individual 15mers overlapping by 11 amino acids total).
[0460] Tumor size was measured 3x/week and subsequently on a daily basis after reaching a specified size threshold (1000mm3). Statistical analysis was performed by one-way ANOVA with multiple comparisons. Mice were euthanized when tumors reached maximum size, or tumors became ulcerated and did not heal within 24 hours. No mice in this study were euthanized for other health reasons.
[0461] Figure 47 shows mean tumor growth kinetics following therapeutic vaccination and treatment with a combination of anti-PD-1 and CTLA-4 checkpoint inhibitors. Groups were vaccinated as indicated with adjuvant only (control), protective vaccine, protective vaccine plus inhibigen MMP9FS, or inhibigen MMP9FS plus adjuvant. As in Example 7, results of this study show that combination checkpoint inhibition partially ameliorated accelerated tumor growth in mice vaccinated with an inhibigen, but the effect did not appear to be additive or synergistic compared to checkpoint inhibition with anti-CTLA-4 alone (data not shown).
[0462] Figure 48 show results of ZFNy ELISPOT performed on splenocytes from therapeutically vaccinated mice, with and without checkpoint inhibition, after in vitro stimulation with overlapping peptides spanning vaccine components. Peptides used for in vitro stimulation are indicated in the keys on the right side of each panel: M30 andTrp2 peptides correspond to stimulatory antigens of the same name, S10 peptides correspond to stimulatory antigen Gal3Stl, and In21 peptides correspond to inhibigen MMP9FS. Statistical analyses were performed by one- way ANOVA with multiple comparisons. Figure 48, panel A shows representative results for mice receiving protective vaccine and the checkpoint inhibitors indicated on the x axis. T cells from mice receiving protective vaccine plus checkpoint inhibitors produced robust IFNy responses, with an average of 2000 spots per lxlOA6 cells. Figure 48, panel B shows representative results for mice receiving protective vaccine plus inhibigen and the checkpoint inhibitors indicated on the x axis. In contrast to mice that received protective vaccine, T cells from mice receiving protective vaccine plus inhibigen showed 10-fold lower immunogenicity, with an average of 200 spots per lxlOA6 cells. Checkpoint inhibition did not restore immunogenicity of T cells from these mice.
[0463] These results demonstrate that if an inhibigen is present in an otherwise protective vaccine, immune responses to the protective antigens are not rescued equally by all checkpoint inhibition therapies. Specifically, anti-PD-1 or anti-PD-Ll checkpoint inhibition did not ameliorate the detrimental effects of a vaccine containing an inhibigen, including accelerated tumor growth, decreased immunogenicity, and decreased tumor infiltration of immune cells, while anti-CTLA-4 checkpoint inhibition led to partial improvement in some of these measures. The combination of anti-PD-1 and anti-CTLA-4 checkpoint inhibition appeared to be neither additive nor synergistic compared to anti-CTLA-4 alone.
[0464] By extension, the presence or absence of inhibigens at any given time in a cancer patient’s tumor may correlate with that patient’s response to different checkpoint inhibitors, cellbased cancer therapies, or other cancer therapies. For example, where upon initial evaluation a patient’s tumor includes numerous inhibigens, one or more dominant inhibigens, and/or an unfavorable ratio of inhibigens to stimulatory antigens, anti-PD-1 or anti-PD-Ll checkpoint inhibition may be less preferred than checkpoint inhibition by an agent such as anti-CTLA-4 that counteracts deleterious effects of inhibigens. Additionally, where upon subsequent evaluation, following a course of treatment with checkpoint inhibitors, cell-based therapies, cytokine therapy, or other cancer therapies, a patient’s tumor includes fewer inhibigens, less dominant inhibigens, and/or a more favorable ratio of inhibigens to stimulatory antigens, it may be inferred that treatment improved the immune profile of the patient’s tumor. REFERENCES
1. Hacohen N, Fritsch EF, Carter TA, Lander ES, Wu CJ. Getting personal with neoantigenbased therapeutic cancer vaccines. Cancer Immunol Res. 2013; 1(1): 11-5.
2. Heemskerk B, Kvistborg P, Schumacher TN. The cancer antigenome. EMBO J. 2013;32(2): 194-203.
3. Castle JC, Kreiter S, Diekmann J, Lower M, van de Roemer N, de Graaf J, et al. Exploiting the mutanome for tumor vaccination. Cancer Res. 2012;72(5): 1081-91.
4. Lawrence MS, Stojanov P, Polak P, Kryukov GV, Cibulskis K, Sivachenko A, et al. Mutational heterogeneity in cancer and the search for new cancer-associated genes. Nature. 2013;499(7457):214-8
5. Berd D, Murphy G, Maguire HC, Jr., Mastrangelo MJ. Immunization with haptenized, autologous tumor cells induces inflammation of human melanoma metastases. Cancer Res. I991;51(10):273I-4.
6. Randazzo M, Temess P, Opelz G, Kleist C. Active-specific immunotherapy of human cancers with the heat shock protein Gp96-revisited. Int J Cancer. 2012;130(10):2219-31.
7. Senzer NN, Kaufman HL, Amatruda T, Nemunaitis M, Reid T, Daniels G, et al. Phase II clinical trial of a granulocyte-macrophage colony-stimulating factor-encoding, second-generation oncolytic herpesvirus in patients with unresectable metastatic melanoma. J Clin Oncol.
2009;27(34):5763-71.
8. Kvistborg P, Philips D, Kelderman S, Hageman L, Ottensmeier C, Joseph-Pietras D, et al. Anti-CTLA-4 therapy broadens the melanoma-reactive CD8+ T cell response. Sci Transl Med. 2014;6(254):254ral28.
9. Bystryn JC, Zeleniuch-Jacquotte A, Oratz R, Shapiro RL, Harris MN, Roses DF. Doubleblind trial of a polyvalent, shed-antigen, melanoma vaccine. Clin Cancer Res. 2001;7(7): 1882-7.
10. Lennerz V, Fatho M, Gentilini C, Frye RA, Lifke A, Ferel D, et al. The response of autologous T cells to a human melanoma is dominated by mutated neoantigens. Proc Natl Acad Sci U S A. 2005;102(44): 16013-8.
11. Zhou J, Dudley ME, Rosenberg SA, Robbins PF. Persistence of multiple tumor-specific T-cell clones is associated with complete tumor regression in a melanoma patient receiving adoptive cell transfer therapy. J Immunother. 2005;28(l):53-62. 12. Tran E, Turcotte S, Gros A, Robbins PF, Lu YC, Dudley ME, et al. Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer. Science. 2014;344(6184):641-5.
13. Abelin JG, Keskin DB, Sarkizova S, Hartigan CR, Zhang W, Sidney J, et al. Mass Spectrometry Profiling of HLA-Associated Peptidomes in Mono-allelic Cells Enables More Accurate Epitope Prediction. Immunity. 2017;46(2):315-26.
14. Slingluff CL, Jr. The present and future of peptide vaccines for cancer: single or multiple, long or short, alone or in combination? Cancer J. 2011 ; 17(5):343-50.
15. Ammi R, De Waele J, Willemen Y, Van Brussel I, Schrijvers DM, Lion E, et al. Poly(LC) as cancer vaccine adjuvant: knocking on the door of medical breakthroughs. Pharmacol Ther. 2015;146: 120-31.
16. Okada H, Butterfield LH, Hamilton RL, Hoji A, Sakaki M, Ahn BJ, et al. Induction of robust type-I CD8+ T-cell responses in WHO grade 2 low-grade glioma patients receiving peptide-based vaccines in combination with poly-ICLC. Clin Cancer Res. 2015;21(2):286-94.
17. Rapoport AP, Aqui NA, Stadtmauer EA, Vogl DT, Xu YY, Kalos M, et al. Combination immunotherapy after ASCT for multiple myeloma using MAGE-A3/Poly-ICLC immunizations followed by adoptive transfer of vaccine-primed and costimulated autologous T cells. Clin Cancer Res. 2014;20(5): 1355-65.
18. Sabbatini P, Tsuji T, Ferran L, Ritter E, Sedrak C, Tuballes K, et al. Phase I trial of overlapping long peptides from a tumor self-antigen and poly-ICLC shows rapid induction of integrated immune response in ovarian cancer patients. Clin Cancer Res. 2012;18(23):6497-508.
19. Sharma S, Zhu L, Davoodi M, Harris-White M, Lee JM, St John M, et al. TLR3 agonists and proinflammatory antitumor activities. Expert Opin Ther Targets. 2013 ; 17(5):481 -3.
20. Kimura T, McKolanis JR, Dzubinski LA, Islam K, Potter DM, Salazar AM, et al. MUC1 vaccine for individuals with advanced adenoma of the colon: a cancer immunoprevention feasibility study. Cancer Prev Res (Phila). 2013;6(l): 18-26.
21. Kobayashi Y, Sakura T, Miyawaki S, Toga K, Sogo S, Heike Y. A new peptide vaccine OCV-501 : in vitro pharmacology and phase 1 study in patients with acute myeloid leukemia. Cancer Immunol Immunother. 2017;66(7):851-63.
22. Obara W, Sato F, Takeda K, Kato R, Kato Y, Kanehira M, et al. Phase I clinical trial of cell division associated 1 (CDCA1) peptide vaccination for castration resistant prostate cancer. Cancer Sci. 2017; 108(7): 1452-7. 23. Suekane S, Ueda K, Nishihara K, Sasada T, Yamashita T, Koga N, et al. Personalized peptide vaccination as second-line treatment for metastatic upper tract urothelial carcinoma. Cancer Sci. 2017;108(12):2430-7.
24. Ott PA, Hu Z, Keskin DB, Shukla SA, Sun J, Bozym DJ, et al. An immunogenic personal neoantigen vaccine for patients with melanoma. Nature. 2017;547(7662):217-21.
LISTING OF SEQUENCES
Figure imgf000188_0001
Figure imgf000189_0001
Figure imgf000190_0001
Figure imgf000191_0001
Figure imgf000192_0001
Figure imgf000193_0001
Figure imgf000194_0001
Figure imgf000195_0001
Figure imgf000196_0001
Figure imgf000197_0001
Figure imgf000198_0001
Figure imgf000199_0001
Figure imgf000200_0001
Figure imgf000201_0001
Figure imgf000202_0001
Figure imgf000203_0001
Figure imgf000204_0001
Figure imgf000205_0001
Figure imgf000206_0001
Figure imgf000207_0001
Figure imgf000208_0001
Figure imgf000209_0001
Figure imgf000210_0001
Figure imgf000211_0001
Figure imgf000212_0001
Figure imgf000213_0001
Figure imgf000214_0001
Figure imgf000215_0001
Figure imgf000216_0001
Figure imgf000217_0001
Figure imgf000218_0001
Figure imgf000219_0001
Figure imgf000220_0001
Figure imgf000221_0001
Figure imgf000222_0001
Figure imgf000223_0001
Figure imgf000224_0001
Figure imgf000225_0001
Figure imgf000226_0001
Figure imgf000227_0001
Figure imgf000228_0001
Figure imgf000229_0001
Figure imgf000230_0001
Figure imgf000231_0001
Figure imgf000232_0001
Figure imgf000233_0001
Figure imgf000234_0001
Figure imgf000235_0001
Figure imgf000236_0001
Figure imgf000237_0001
Figure imgf000238_0001
Figure imgf000239_0001
Figure imgf000240_0001
Figure imgf000241_0001
Figure imgf000242_0001
Figure imgf000243_0001
Figure imgf000244_0001
Figure imgf000245_0001
Figure imgf000246_0001
Figure imgf000247_0001
Figure imgf000248_0001
Figure imgf000249_0001
Figure imgf000250_0001
Figure imgf000251_0001
Figure imgf000252_0001
Figure imgf000253_0001
Figure imgf000254_0001
Figure imgf000255_0001
Figure imgf000256_0001
Figure imgf000257_0001
Figure imgf000258_0001
Figure imgf000259_0001
Figure imgf000260_0001
Figure imgf000261_0001
Figure imgf000262_0001
Figure imgf000263_0001
Figure imgf000264_0001
Figure imgf000265_0001
Figure imgf000266_0001
Figure imgf000267_0001
Figure imgf000268_0001
Figure imgf000269_0001
Figure imgf000270_0001
Figure imgf000271_0001
Figure imgf000272_0001
Figure imgf000273_0001
Figure imgf000274_0001
Figure imgf000275_0001
Figure imgf000276_0001
Figure imgf000277_0001
Figure imgf000278_0001
Figure imgf000279_0001
Figure imgf000280_0001
Figure imgf000281_0001
Figure imgf000282_0001
Figure imgf000283_0001
Figure imgf000284_0001
Figure imgf000285_0001
Figure imgf000286_0001
EQUIVALENTS
[0465] It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims:

Claims

We claim:
1. A method of selecting a subject for cancer therapy, the method comprising: a) obtaining a first response profile from the subject, the first response profile comprising a representation of (i) a first number of identified stimulatory tumor antigens and (ii) a first number of identified inhibitory tumor antigens, at a first time; b) obtaining a second response profile from the subject, the second response profile comprising a representation of (i) a second number of identified stimulatory tumor antigens and (ii) a second number of identified inhibitory tumor antigens, at a second time; c) comparing the first response profile to the second response profile; and if (i) the first number of stimulatory tumor antigens is different from the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is different from the second number of inhibitory tumor antigens, then selecting the subject as a candidate subject for initiation, continuation, modification, discontinuation, or non-initiation of a cancer therapy or sequence of cancer therapies.
2. The method of claim 1, wherein the first time is prior to initiation of a cancer therapy.
3. The method of claim 1 or 2, wherein the second time is after initiation of a cancer therapy.
4. The method of claim 1, wherein the first time and second time are after initiation of a cancer therapy.
5. The method of any one of claims 1-4, wherein stimulatory antigens and/or inhibitory antigens are identified by: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators; f) identifying as one or more stimulatory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) increase level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that decrease immune response; and g) identifying as one or more inhibitory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) increase level of expression and/or secretion of one or more immune mediators that decrease immune response.
6. The method of any one of claims 1-5, wherein the cancer therapy comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
7. The method of any one of claims 1-6, wherein if (i) the first number of stimulatory tumor antigens is greater than the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is less than the second number of inhibitory tumor antigens, then the method comprises selecting the subject as a candidate subject for modification or discontinuation of the cancer therapy.
8. The method of claim 7, further comprising modifying or discontinuing the cancer therapy for the subject.
9. The method of any one of claims 1-6, wherein if (i) the first number of stimulatory tumor antigens is less than the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is greater than the second number of inhibitory tumor antigens, then the method comprises selecting the subject as a candidate subject for continuation of the cancer therapy.
10. The method of claim 9, further comprising continuing the cancer therapy for the subject.
11. A method of selecting a subject for cancer therapy, the method comprising: a) obtaining a first response profile from the subject, the first response profile comprising a representation of (i) a first number of identified stimulatory tumor antigens and (ii) a first number of identified inhibitory tumor antigens, at a first time; b) obtaining a second response profile from the subject, the second response profile comprising a representation of (i) a second number of identified stimulatory tumor antigens and (ii) a second number of identified inhibitory tumor antigens, at a second time; c) comparing the first response profile to the second response profile; and if (i) the first number of stimulatory tumor antigens is greater than the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is less than the second number of inhibitory tumor antigens, then selecting the subject as a candidate subject for modification or discontinuation of a cancer therapy.
12. The method of claim 11, wherein the first time is prior to initiation of a cancer therapy.
13. The method of claim 11 or 12, wherein the second time is after initiation of a cancer therapy.
14. The method of claim 11, wherein the first time and second time are after initiation of a cancer therapy.
15. The method of any one of claims 11-14, wherein stimulatory antigens and/or inhibitory antigens are identified by: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators; f) identifying as one or more stimulatory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) increase level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that decrease immune response; and g) identifying as one or more inhibitory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) increase level of expression and/or secretion of one or more immune mediators that decrease immune response.
16. The method of any one of claims 11-15, wherein the cancer therapy comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
17. A method of selecting a subject for cancer therapy, the method comprising: a) obtaining a first response profile from the subject, the first response profile comprising a representation of (i) a first number of identified stimulatory tumor antigens and (ii) a first number of identified inhibitory tumor antigens, at a first time; b) obtaining a second response profile from the subject, the second response profile comprising a representation of (i) a second number of identified stimulatory tumor antigens and (ii) a second number of identified inhibitory tumor antigens, at a second time; c) comparing the first response profile to the second response profile; and if (i) the first number of stimulatory tumor antigens is less than the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is greater than the second number of inhibitory tumor antigens, then selecting the subject as a candidate subject for continuation of a cancer therapy.
18. The method of claim 17, further comprising continuing the cancer therapy for the subject.
19. The method of claim 17 or 18, wherein the first time is prior to initiation of a cancer therapy.
20. The method of any one of claims 17-19, wherein the second time is after initiation of a cancer therapy.
21. The method of claim 17, wherein the first time and second time are after initiation of a cancer therapy.
22. The method of any one of claims 17-21, wherein stimulatory antigens and/or inhibitory antigens are identified by: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators; f) identifying as one or more stimulatory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) increase level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that decrease immune response; and g) identifying as one or more inhibitory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) increase level of expression and/or secretion of one or more immune mediators that decrease immune response.
23. The method of any one of claims 17-22, wherein the cancer therapy comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
24. A method of selecting a subject for cancer therapy, comprising: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) obtained from a subject at a first time, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes obtained from the subject at the first time, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators; f) identifying as one or more stimulatory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) increase level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that decrease immune response; g) identifying as one or more inhibitory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) increase level of expression and/or secretion of one or more immune mediators that decrease immune response; and h) obtaining a first response profile from the subject, the first response profile comprising a representation of (i) a first number of identified stimulatory tumor antigens and (ii) a first number of identified inhibitory tumor antigens, at the first time; i) repeating steps a) through g) with APCs and/or lymphocytes obtained from the subject at a second time; j) obtaining a second response profile from the subject, the second response profile comprising a representation of (i) a second number of identified stimulatory tumor antigens and (ii) a second number of identified inhibitory tumor antigens, at the second time; k) comparing the first response profile to the second response profile; and if (i) the first number of stimulatory tumor antigens is different from the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is different from the second number of inhibitory tumor antigens, then selecting the subject as a candidate subject for initiation, continuation, modification, discontinuation, or non-initiation of a cancer therapy or sequence of cancer therapies.
25. The method of claim 24, wherein the first time is prior to initiation of a cancer therapy.
26. The method of claim 24 or 25, wherein the second time is after initiation of a cancer therapy.
27. The method of claim 24, wherein the first time and second time are after initiation of a cancer therapy.
28. The method of any one of claims 24-27, wherein the cancer therapy comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
29. The method of any one of claims 24-28, wherein if (i) the first number of stimulatory tumor antigens is greater than the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is less than the second number of inhibitory tumor antigens, then the method comprises selecting the subject as a candidate subject for modification or discontinuation of the cancer therapy.
30. The method of claim 29, further comprising modifying or discontinuing the cancer therapy for the subject.
31. The method of any one of claims 24-28, wherein if (i) the first number of stimulatory tumor antigens is less than the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is greater than the second number of inhibitory tumor antigens, then the method comprises selecting the subject as a candidate subject for continuation of the cancer therapy.
32. The method of claim 31, further comprising continuing the cancer therapy for the subject.
33. A method of selecting a subject for cancer therapy, comprising: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) obtained from a subject at a first time, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes obtained from the subject at the first time, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators; f) identifying as one or more stimulatory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) increase level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that decrease immune response; g) identifying as one or more inhibitory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) increase level of expression and/or secretion of one or more immune mediators that decrease immune response; and h) obtaining a first response profile from the subject, the first response profile comprising a representation of (i) a first number of identified stimulatory tumor antigens and (ii) a first number of identified inhibitory tumor antigens, at the first time; i) repeating steps a) through g) with APCs and/or lymphocytes obtained from the subject at a second time; j) obtaining a second response profile from the subject, the second response profile comprising a representation of (i) a second number of identified stimulatory tumor antigens and (ii) a second number of identified inhibitory tumor antigens, at the second time; k) comparing the first response profile to the second response profile; and if (i) the first number of stimulatory tumor antigens is greater than the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is less than the second number of inhibitory tumor antigens, then selecting the subject as a candidate subject for modification or discontinuation of a cancer therapy.
34. The method of claim 33, further comprising modifying or discontinuing the cancer therapy for the subject.
35. The method of claim 33 or 34, wherein the first time is prior to initiation of a cancer therapy.
36. The method of any one of claims 33-35, wherein the second time is after initiation of a cancer therapy.
37. The method of claim 33 or 34, wherein the first time and second time are after initiation of a cancer therapy.
38. The method of any one of claims 33-37, wherein the cancer therapy comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
39. A method of selecting a subject for cancer therapy, comprising: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) obtained from a subject at a first time, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes obtained from the subject at the first time, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators; f) identifying as one or more stimulatory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) increase level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that decrease immune response; g) identifying as one or more inhibitory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) increase level of expression and/or secretion of one or more immune mediators that decrease immune response; and h) obtaining a first response profile from the subject, the first response profile comprising a representation of (i) a first number of identified stimulatory tumor antigens and (ii) a first number of identified inhibitory tumor antigens, at the first time; i) repeating steps a) through g) with APCs and/or lymphocytes obtained from the subject at a second time; j) obtaining a second response profile from the subject, the second response profile comprising a representation of (i) a second number of identified stimulatory tumor antigens and (ii) a second number of identified inhibitory tumor antigens, at the second time; k) comparing the first response profile to the second response profile; and if (i) the first number of stimulatory tumor antigens is less than the second number of stimulatory tumor antigens, and/or (ii) the first number of inhibitory tumor antigens is greater than the second number of inhibitory tumor antigens, then selecting the subject as a candidate subject for continuation of a cancer therapy.
40. The method of claim 39, further comprising continuing the cancer therapy for the subject.
41. The method of claim 39 or 40, wherein the first time is prior to initiation of a cancer therapy.
42. The method of any one of claims 39-41, wherein the second time is after initiation of a cancer therapy.
43. The method of claim 39 or 40, wherein the first time and second time are after initiation of a cancer therapy.
44. The method of any one of claims 39-43, wherein the cancer therapy comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
45. A method of selecting a cancer therapy for a subject, the method comprising: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators; f) identifying as one or more stimulatory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) increase level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that decrease immune response; g) identifying as one or more inhibitory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) increase level of expression and/or secretion of one or more immune mediators that decrease immune response; and h) selecting for the subject, based on the number and/or characteristics of the identified stimulatory and inhibitory antigens, a cancer therapy or sequence of cancer therapies.
46. The method of claim 45, wherein the cancer therapy or sequence of cancer therapies comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cell-based therapy, or (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, (viii) any combination of the foregoing.
47. The method of any one of claims 6, 16, 23, 28, 38, and 46, wherein the checkpoint inhibitor therapy comprises one or more agents directed to PD-1, PD-L1, CTLA-4, LAG-3, TIM- 3, TIGIT, BTLA, CBLB, CISH, VISTA, B7-H3, CSF-1R, or GITR.
48. The method of any one of claims 6, 16, 23, 28, 38, and 46, wherein the checkpoint inhibitor therapy comprises two or more agents directed to PD-1, PD-L1, CTLA4, LAG-3, TIM- 3, TIGIT, BTLA, CBLB, CISH, VISTA, B7-H3, CSF-1R, or GITR.
49. The method of any one of claims 46-48, wherein the checkpoint inhibitor therapy comprises one or more of pembrolizumab, nivolumab. atezolizumab, avelumab, durvalumab, ipilimumab, cemiplimab, spartalizumab, camrelizumab, cabiralizumab, dostarlimab, sintilmab, tisleliumab, or toripalimab.
50. The method of any one of claims 45-49, further comprising administering the selected cancer therapy or sequence of cancer therapies to the subject.
51. A method of treating cancer in a subject, the method comprising: a) obtaining, providing, or generating a library comprising bacterial cells or beads comprising a plurality of tumor antigens, wherein each bacterial cell or bead of the library comprises a different tumor antigen; b) contacting the bacterial cells or beads with antigen presenting cells (APCs) from the subject, wherein the APCs internalize the bacterial cells or beads; c) contacting the APCs with lymphocytes from the subject, under conditions suitable for activation of lymphocytes by a tumor antigen presented by one or more APCs; d) determining whether one or more lymphocytes are activated by, or not responsive to, one or more tumor antigens presented by one or more APCs, e.g., by assessing (e.g., detecting or measuring) a level (e.g., an increased level or decreased level, relative to a control) of expression and/or secretion of one or more immune mediators; e) identifying one or more tumor antigens that stimulate, inhibit and/or suppress, and/or have a minimal effect on a level of expression and/or secretion of one or more immune mediators; f) identifying as one or more stimulatory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) increase level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that decrease immune response; g) identifying as one or more inhibitory antigens, from among the identified tumor antigens, one or more tumor antigens that (i) inhibit and/or suppress level of expression and/or secretion of one or more immune mediators that increase immune response and/or that (ii) increase level of expression and/or secretion of one or more immune mediators that decrease immune response; h) selecting for the subject, based on the number and/or characteristics of the identified stimulatory and inhibitory antigens, a cancer therapy or sequence of cancer therapies; and i) administering the selected cancer therapy or sequence of cancer therapies to the subject.
52. The method of claim 51, wherein the cancer therapy or sequence of cancer therapies comprises (i) an immunogenic composition comprising one or more of the identified stimulatory antigens, (ii) checkpoint inhibitor therapy, (iii) chemotherapy, (iv) cell-based therapy, (v) immunotherapy, (vi) cytokine therapy, (vii) kinase inhibitors, or (viii) any combination of the foregoing.
53. The method of claim 52, wherein the checkpoint inhibitor therapy comprises one or more agents directed to PD-1, PD-L1, CTLA4, Lag-3, Tim-3, TIGIT, BTLA, CBLB, CISH, VISTA, B7-H3, CSF-1R, or GITR.
54. The method of claim 52, wherein the checkpoint inhibitor therapy comprises two or more agents directed to PD-1, PD-L1, CTLA4, Lag-3, Tim-3, TIGIT, BTLA, CBLB, CISH, VISTA, B7-H3, CSF-1R, or GITR.
55. The method of any one of claims 52-54, wherein the checkpoint inhibitor therapy comprises one or more of pembrolizumab, nivolumab. atezolizumab, avelumab, durvalumab, ipilimumab, cemiplimab, spartalizumab, camrelizumab, cabirali umab, dosiarlimab, sintilmab, tisleliumab, or toripalimab.
56. The method of any one of claims 1-55, wherein the one or more immune mediators are selected from the group consisting of cytokines, soluble mediators, and cell surface markers expressed by the lymphocytes.
57. The method of any one of claims 1-56, wherein the one or more immune mediators are cytokines.
58. The method of claim 57, wherein the one or more cytokines are selected from the group consisting of TRAIL, IFN-gamma, IL-12p70, IL-2, TNF-alpha, MIPl-alpha, MIPl-beta, CXCL9, CXCL10, MCP1, RANTES, IL-1 beta, IL-4, IL-6, IL-8, IL-9, IL-10, IL-13, IL-15, CXCL11, IL-3, IL-5, IL-17, IL-18, IL-21, IL-22, IL-23 A, IL-24, IL-27, IL-31, IL-32, TGF-beta, CSF, GM-CSF, TRANCE (also known as RANK L), MIP3-alpha, and fractalkine.
59. The method of any one of claims 1-55, wherein the one or more immune mediators are soluble mediators.
60. The method of claim 59, wherein the one or more soluble mediators are selected from the group consisting of granzyme A, granzyme B, sFas, sFasL, perforin, and granulysin.
61. The method of any one of claims 1-55, wherein the one or more immune mediators are cell surface markers.
62. The method of claim 61, wherein the one or more cell surface markers are selected from the group consisting of CD 107a, CD 107b, CD25, CD69, CD45RA, CD45RO, CD137 (4-1BB), CD44, CD62L, CD27, CCR7, CD154 (CD40L), KLRG-1, CD71, HLA-DR, CD122 (IL-2RB), CD28, IL7Ra (CD127), CD38, CD26, CD134 (OX-40), CTLA-4 (CD152), LAG-3, TIM-3 (CD366), CD39, PD-1 (CD279), FoxP3, TIGIT, CD 160, BTLA, 2B4 (CD244), and KLRGl.
63. The method of any one of claims 1-62, wherein the lymphocytes comprise CD4+ T cells.
64. The method of any one of claims 1-63, wherein the lymphocytes comprise CD8+ T cells.
65. The method of any one of claims 1-64, wherein lymphocyte inhibition and/or suppression is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, or 200% higher or lower than a control level.
66. The method of any one of claims 1-65, wherein lymphocyte inhibition and/or suppression is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 1, 2, or 3 standard deviations higher or lower than the mean of a control level.
67. The method of any one of claims 1-66, wherein lymphocyte inhibition and/or suppression is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) greater or lower than a median response level to a control.
68. The method of any one of claims 1-67, wherein lymphocyte stimulation is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 20%, 40%, 60%, 80%, 100%, 120%, 140%, 160%, 180%, or 200% higher or lower than a control level.
69. The method of any one of claims 1-68, wherein stimulation is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 1, 2, or 3 standard deviations higher or lower than the mean of a control level.
70. The method of any one of claims 1-69, wherein lymphocyte stimulation is determined by assessing a level of one or more expressed or secreted immune mediators that is at least 1, 2, 3, 4 or 5 median absolute deviations (MADs) greater or lower than a median response level to a control.
PCT/US2021/050332 2020-09-14 2021-09-14 Treatment methods WO2022056491A1 (en)

Applications Claiming Priority (12)

Application Number Priority Date Filing Date Title
US202063078313P 2020-09-14 2020-09-14
US63/078,313 2020-09-14
US202063111505P 2020-11-09 2020-11-09
US63/111,505 2020-11-09
US202163140721P 2021-01-22 2021-01-22
US63/140,721 2021-01-22
US202163173148P 2021-04-09 2021-04-09
US63/173,148 2021-04-09
US202163186541P 2021-05-10 2021-05-10
US63/186,541 2021-05-10
US202163197101P 2021-06-04 2021-06-04
US63/197,101 2021-06-04

Publications (1)

Publication Number Publication Date
WO2022056491A1 true WO2022056491A1 (en) 2022-03-17

Family

ID=80629964

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/050332 WO2022056491A1 (en) 2020-09-14 2021-09-14 Treatment methods

Country Status (1)

Country Link
WO (1) WO2022056491A1 (en)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018175505A1 (en) * 2017-03-20 2018-09-27 Genocea Biosciences, Inc. Treatment methods

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018175505A1 (en) * 2017-03-20 2018-09-27 Genocea Biosciences, Inc. Treatment methods

Similar Documents

Publication Publication Date Title
US20210199644A1 (en) Treatment methods
Kroon et al. Radiotherapy and cisplatin increase immunotherapy efficacy by enabling local and systemic intratumoral T-cell activity
Wang et al. Novel exosome-targeted T-cell-based vaccine counteracts T-cell anergy and converts CTL exhaustion in chronic infection via CD40L signaling through the mTORC1 pathway
US20200323905A1 (en) Methods and compositions for modulating the immune system
KR20210091137A (en) treatment method
US20220211832A1 (en) Treatment methods
CN114206357A (en) Compositions and methods for TCR reprogramming using fusion proteins
US20230057310A1 (en) Treatment methods
US20210338725A1 (en) Treatment methods
WO2022056491A1 (en) Treatment methods
JP6654207B2 (en) Method of inducing an early T memory response using a short peptide anti-tumor vaccine.
JP2022513072A (en) Methods for Improving the Efficacy of Survivin Therapeutic Agents in the Treatment of Tumors
US20220412979A1 (en) Treatment methods
WO2023086969A2 (en) Treatment methods
KR20240042414A (en) Compositions and methods for treating melanoma

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21867836

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21867836

Country of ref document: EP

Kind code of ref document: A1