US20200339659A1 - Pdl1 peptides for use in cancer vaccines - Google Patents

Pdl1 peptides for use in cancer vaccines Download PDF

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US20200339659A1
US20200339659A1 US16/310,908 US201716310908A US2020339659A1 US 20200339659 A1 US20200339659 A1 US 20200339659A1 US 201716310908 A US201716310908 A US 201716310908A US 2020339659 A1 US2020339659 A1 US 2020339659A1
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Mads Hald Andersen
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Herlev Hospital Region Hovedstaden
IO Biotech ApS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
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Definitions

  • the present invention relates to novel PD-L1 peptide fragments, as well as compositions, uses, and kit-of-parts comprising these peptide fragments. Furthermore, the invention concerns PD-L1 peptide fragments for use in a method for treatment or prevention of a cancer, when administered simultaneously or sequentially with an additional cancer therapy.
  • the immune system has the capacity to recognize and destroy neoplastic cells; nevertheless, despite the fact that neoplastic transformation is associated with the expression of immunogenic antigens, the immune system often fails to respond effectively to these antigens. The immune system becomes tolerant towards these antigens. When this happens, the neoplastic cells proliferate uncontrollably leading to the formation of malignant cancers with poor prognosis for the affected individuals. The acquired state of tolerance must be overcome for cancer immunotherapy to succeed.
  • Programmed death-1 is a regulatory surface molecule delivering inhibitory signals important to maintain T-cell functional silence against their cognate antigens. Its ligands, known as PD-L1 and PD-L2, or B7-H1 and B7-H2 are expressed on APCs, tumor cells, placental, and nonhematopoietic cells found in an inflammatory microenvironment. Interference with PD-1 or its ligand PD-L1 increases antitumor immunity. It appears that upregulation of PD-L1 is a mechanism that cancers can employ to evade the host immune system.
  • PD-L1 on tumors correlates with poor clinical outcome for a number of cancers including pancreas, renal cell, ovarian, head and neck, and melanoma (Hamanishi et al., 2007, Proc. Natl. Acad. Sci. U. S. A. 104:3360-3365; Nomi et al., 2007, Clin. Cancer Res. 13:2151-2157; Hino et al., 2010, Cancer. 116:1757-1766.
  • analysis of 196 tumor specimens from patients with renal cell carcinoma found that high tumor expression of PD-L1 was associated with increased tumor aggressiveness and a 4.5-fold increased risk of death (Thompson et al., 2004, Proc. Natl. Acad.
  • DCs Dendritic cells
  • 1 DC vaccines are generally composed of peripheral blood monocytes that are matured into DCs and pulsed with antigens in vitro before they are injected.
  • DC-based cancer vaccines have received much attention over the last decade.
  • the induced T cell frequencies are not impressive and additional measures are needed to help increase T cell response.
  • further research is required to optimize the generation and phenotype of DCs to enhance their capacity to induce “fully armored” T cells, to determine the best route of administration, and to identify ideal combinations with additional therapies.
  • Programmed death 1 is an inhibitory molecule that is expressed on the surface of T cells.
  • the PD-1 ligand PD-L1 (also known as CD274 or B7-H1) is constitutively expressed on lymphoid cells such as monocytes, DCs, and T cells, and it is also present on nonhematopoietic cells, such as endothelial and epithelial cells.
  • 2,3 PD-L1 can be upregulated by type I and II interferons (IFNs) through IFN regulatory factor-1 (IRF-1) in what appears to be a JAK/STAT-dependent manner. 4
  • IFNs type I and II interferons
  • IRF-1 IFN regulatory factor-1
  • PD-L1 is a critical negative regulator of self-reactive T cells during both the induction and effector phases of autoimmune disease, and it exerts its inhibitory function in multiple ways.
  • PD-L1 binds B7-1 (CD80), preventing B7-1 co-stimulation.
  • IL-10 is produced upon ligation of PD-L1 and possibly augments apoptosis of activated T cells. 6
  • the immune system is continually looking for foreign pathogens and irregular cells, such as cancer cells. Consequently, in order for cancer to continue to grow, it must hide from the immune system to avoid destruction.
  • PD-1 and its ligands play a central role in maintaining peripheral tolerance and preventing autoimmunity, and cancer cells can exploit this system to create a suppressing microenvironment, thus protecting themselves from immune-mediated killing.
  • PD-L1 expression has been found to be high in multiple cancers, 7,8 and PD-L1 expression was first described as an indicator of tumor aggressiveness in renal cell carcinoma. 9
  • PD-L1 expression on tumor cells has been suggested as a prognostic factor in a number of solid cancers, including ovarian and pancreatic cancer. 10,11
  • these PD-L1-specific T cells were found to lyse PD-L1-expressing cells, including melanoma cells and nonmalignant DCs. 14,16 In addition, activation of PD-L1-specific T cells boosts the immune response toward viral antigens. 17 These findings suggest an autoreactive function for PD-L1-specific T cells in immune homeostasis. Furthermore, it implies that stimulation with PD-L1-derived peptides can boost a previously existing or vaccine-generated immune response by pushing the immune balance in the microenvironment toward less immune inhibition.
  • DCvacc peripheral blood mononuclear cells
  • the present inventors have identified new fragments of human PD-L1 (SEQ ID NO:1) which fragments have good solubility, do not aggregate, are not prone for beta-sheet formation and as such are suitable for, for instance, vaccines together with an adjuvant.
  • the PD-L1 peptide fragment of SEQ ID NO. 91 (which is PDlong2 described in WO2013056716, incorporated herein by reference) is quite hydrophobic and very prone for beta-sheet formation and therefore has low solubility. Furthermore, this peptide contains free SH, and must be handled at low pH to prevent dimer formation.
  • PD-L1 peptide fragments SEQ ID NO. 91 and 89 co-stimulation increases immunogenicity of a dendritic cell-based cancer vaccine.
  • activation of PD-L1-specific T cells by any one of these two PD-L1 peptide fragments may directly modulate immunogenicity of DC vaccines.
  • Addition of PD-L1 epitopes may thus be an easily applicable and attractive option to augment the effectiveness of cancer vaccines and other immunotherapeutic agents.
  • a PD-L1 peptide fragment consisting of SEQ ID NO. 91 or a PD-L1 peptide fragment consisting of SEQ ID NO. 89, as well as longer sequences comprising these will have the effect as shown herein.
  • the present invention relates to a PD-L1 peptide fragment, or pharmaceutically acceptable sale thereof, having the formula:
  • amino acid form of the C terminal residue may be indicated herein by the notation X—OH, whereas if the amide form is present this may be indicated by the notation X—NH 2 . If neither notation is used, it will be understood that both amino acid and amide forms of the C terminal residue are encompassed.
  • the peptide of the invention, or the pharmaceutically acceptable salt thereof may therefore comprise or consist of any one of the amino acid sequences set out in Table A, optionally wherein the C terminal amino acid is replaced with the corresponding amide form.
  • the “Start pos” and “End pos” columns indicate the starting position and the ending position of each peptide within the sequence of SEQ ID NO: 1.
  • the peptides of the invention comprise or consists of between 17 and 33 consecutive amino acids of the PD-L1 sequence of SEQ ID NO: 1.
  • additional residues may be added at the N and/or C termini to improve stability.
  • the consecutive amino acids of SEQ ID NO: 1 preferably comprise at least the amino acids corresponding to positions 242 to 258 of SEQ ID NO: 1, with upto 10 additional amino acids at the C terminal end corresponding to positions 259 to 268 of SEQ ID NO: 1; and/or upto 6 additional amino acids at the N terminal end corresponding to positions 236 to 241 of SEQ ID NO: 1.
  • Particularly preferred is the peptide which comprises or consists of the amino acid sequence RTHLVILGAILLCLGVALTFIFRLRKGR (SEQ ID NO: 52), which corresponds to positions 238 to 265 of SEQ ID NO: 1.
  • This peptide may be referred to herein as IO104.1.
  • the C terminal residue of this sequence may be replaced with the corresponding amide form and be equally preferred.
  • the fragment with C terminal amino acid may be referred to as IO104.1-OH.
  • the fragment with C terminal amide may be referred to herein as IO104.1-NH 2 .
  • One, two, three, four or five conservative substitutions may be made to any one of the sequences of Table A and the resulting sequence still be considered a peptide of the invention, although said peptide is preferably capable of recognition by T cells specific for the HLA-A2 epitope entitled PDL111 (sequence provided as SEQ NO: 92). Most preferably, said conservative substitutions do not alter the amino acids corresponding to positions 250 to 258 of SEQ ID NO: 1, which are the amino acid sequence of the PDL111 epitope.
  • the present invention relates to a composition
  • a composition comprising the PD-L1 peptide fragment of the present invention; optionally together with a pharmaceutically acceptable additive.
  • an immunotherapeutic composition such as a vaccine, comprising
  • the immunotherapeutic composition of the present invention is for use in a method for treatment or prevention of a disease, disorder or condition selected from cancer, such as a tumor forming cancer disease; an infection, such as an infectious disease, e.g. an intracellular infection, for example an intracellular infection with a pathogen selected from the group consisting of L. monocytogenes and plasmodium, a viral infection, for example an infection with a virus selected from the group consisting of HIV and hepatitis; an autoimmune disease, such as diabetes, SLE and sclerosis.
  • a disease, disorder or condition selected from cancer such as a tumor forming cancer disease
  • an infection such as an infectious disease, e.g. an intracellular infection, for example an intracellular infection with a pathogen selected from the group consisting of L. monocytogenes and plasmodium, a viral infection, for example an infection with a virus selected from the group consisting of HIV and hepatitis
  • an autoimmune disease such as diabetes, SLE and sclerosis.
  • the adjuvant is selected from the group consisting of bacterial DNA based adjuvants, oil/surfactant based adjuvants, viral dsRNA based adjuvants, imidazochinilines, a Montanide ISA adjuvant.
  • kit-of-parts comprising:
  • the present invention relates to a method of treating a clinical condition characterized by expression of PD-L1, the method comprising administering to an individual suffering from said clinical condition an effective amount of the peptide fragment of the present invention, the composition of the present invention, or the kit-of-parts of the present invention.
  • the present invention relates to use of a peptide fragment of the present invention for the manufacture of a medicament, such as an immunotherapeutic composition or vaccine, for the treatment or prevention of a clinical condition characterized by expression of PD-L1.
  • a clinical condition to be treated is a cancer disease where PD-L1 is expressed.
  • the clinical condition is selected from the group consisting of infectious diseases and autoimmune diseases.
  • the present invention relates to a PD-L1 peptide fragment having the formula:
  • the PD-L1 peptide fragment may be selected from any of those disclosed in Table A, or a pharmaceutically acceptable salt thereof, optionally wherein the C terminal amino acid is replaced with the corresponding amide form. In an embodiment the PD-L1 peptide fragment is selected from
  • the present invention relates to a PD-L1 peptide fragment comprising the formula:
  • FMTYWHLLNAFTVTVPKDL (SEQ ID NO: 89) wherein the C-terminal amino acid also comprises the amide, or a pharmaceutically acceptable salt thereof;
  • FIG. 1 Costimulation with PD-L1-derived epitopes enhance the frequency of T cells reactive against dendritic cell vaccine.
  • PBMCs (5 ⁇ 10 6 ) from patients were stimulated twice in vitro with DCvacc. The following day, the cultures were costimulated with one or two long PD-L1 epitopes or incubated with an irrelevant HIV control peptide. All cultures were stimulated with IL2 the day after peptide stimulation. Cultures were examined for DCvacc-reactive T cells after 16-20 days by intracellular TNF ⁇ /INF ⁇ staining.
  • B-E Examples of PBMC cultures from three melanoma patients in which co-activation of PD-L1 specific T cells significantly boosted T cell immunity toward DCvacc, as measured by intracellular TNF ⁇ /INF ⁇ staining.
  • B CD4 + T cells released only TNF ⁇ in response to DCvacc.
  • C PD-L1 peptide costimulation induced TNF ⁇ /INF ⁇ double-positive CD4 + T cells in response to DCvacc.
  • D, E Costimulation with PD-L1 epitopes increased the number of both CD4 + and CD8 + cells that reacted against DCvacc.
  • FIG. 2 Costimulation of PBMCs with PDLong1 plus dendritic cell vaccine.
  • A Percentages of DCvacc-reactive CD4 + T cells (A) and CD8 + T cells (B) in cultures of PBMCs taken from eight melanoma patients before vaccination (baseline) and after four vaccinations.
  • FIG. 3 Natural T cell responses to PDLong1 and PDLong2.
  • IFN ⁇ ELISPOT was used to measure T cell response towards PDLong1 and PDLong2 in tumor-infiltrating lymphocytes from melanoma patients. In 5 ⁇ 10 4 cells from 12 melanoma patients, the average number of IFN ⁇ -releasing cells in response to either PDLong1 or PDLong2 was measured.
  • (B) Example of ELISPOT wells performed with TILs from two melanoma patients either without or with PDLong1 or PDLong2 peptides
  • (C) Tumor-infiltrating lymphocytes were cultured for 5 hours either without or with PDLong1 or PDLong2 before being analyzed for intracellular IFN ⁇ /TNF ⁇ staining.
  • (D) Example of IFN ⁇ /TNF ⁇ staining of tumor-infiltrating lymphocytes from a melanoma patient in response to either without peptide or with PDLong1 or PDLong2.
  • FIG. 6 Measurement of IFN ⁇ releasing cells in tumor infiltrating lymphocytes of melanoma patients in response to IO104.1-OH and IO104.1-NH 2 versus an irrelevant control peptide
  • FIG. 7 ELISPOT comparison of PDL111-specific CD8-T cell responses to PDLong2, IO104.1-OH, IO104.1-NH 2 . PDL111 and an irrelevant control peptide.
  • FIG. 8 T cells specific for murine PDL1 are naturally occurring in mice.
  • A) shows the timeline for the experiment described in Example 3;
  • B) Elispot results for splenocytes stimulated with one of 5 peptide candidates ex vivo;
  • C) Representative Elispot wells and results for splenocytes stimulated with the most effective peptide (mLong1). n 5-10 mice/group.
  • FIG. 9 Local inflammation caused by the allergen 2,4-dinitrofluorobenzene (DNFB) elicits a PD-L1-specific T cell response in mice.
  • A) shows the timeline for the experiment described in Example 4;
  • B) Elispot results for cells from the spleen and dLNs was stimulated with mPD-L1long1 or mPD-L1short ex vivo;
  • C) Representative Elispot wells of one of the DNFB treated mice with highest response compared to a control. n 12 mice/group
  • FIG. 10 Vaccination with mPD-L1long1 expands the population of PD-L1-specific T cells in mice.
  • A) shows the timeline for the experiment described in Example 5;
  • B) Elispot results for splenocytes stimulated with mPD-L1long1 or mPD-L1short ex vivo;
  • FIG. 11 Vaccination with mPD-L1long1 shows anti-tumor effect in mice.
  • A) shows the timeline for the experiment described in Example 6;
  • D) Change in mean tumor volume over time for each group. n 3 mice/group.
  • SEQ ID NO: 1 is the full length amino acid sequence of human (h)PD-L1
  • SEQ ID NOs: 2 to 77 are the amino acid sequences of exemplary peptides of the invention, all of which are fragments of hPD-L1.
  • SEQ ID NO: 78 is an amino acid sequence representing a general formula corresponding to the peptides of the invention.
  • SEQ ID Nos: 79 to 81 are various N terminal amino acid sequences which may be added to the formula of SEQ ID NO: 78.
  • SEQ ID NOs: 82 to 88 are various C terminal amino acid sequences which may be added to the formula of SEQ ID NO: 78.
  • SEQ ID NO: 89 is another exemplary peptide of the invention, being a fragment of hPD-L1
  • SEQ ID NO: 90 is a T cell epitope sequence comprised within SEQ ID NO: 89.
  • SEQ ID NO: 91 is the amino acid sequence of a fragment of hPDL1 which is disclosed herein
  • SEQ ID NO: 92 is the amino acid sequence of the HLA-A2 epitope entitled PDL111 (corresponds to positions 250-258 of hPDL1),
  • SEQ ID NOs: 93 and 94 are the amino acid sequence of certain peptides used as controls in the Examples.
  • SEQ ID NO: 95 is the full length amino acid sequence of murine (m)PD-L1.
  • SEQ ID NOs: 96 to 100 are the amino acid sequences of peptides derived from mPD-L1 which are used as analogs for the peptides of the invention in the mouse model experiments described in the Examples.
  • SEQ ID NO: 101 is a T cell epitope sequence comprised within SEQ ID NO: 96.
  • PD-L1 fragments of human PD-L1 full length (SEQ ID NO. 1) was based on the surprising finding by the inventors of spontaneous cytotoxic immune responses against PD-L1 expressing cells in cancer patients.
  • SEQ ID NO. 1 The problem of cancer immunosuppression was solved in WO2013056716 wherein PD-L1 fragments of human PD-L1 full length (SEQ ID NO. 1) was based on the surprising finding by the inventors of spontaneous cytotoxic immune responses against PD-L1 expressing cells in cancer patients.
  • SEQ ID NO. 1 The problem of cancer immunosuppression was solved in WO2013056716 wherein PD-L1 fragments of human PD-L1 full length (SEQ ID NO. 1) was based on the surprising finding by the inventors of spontaneous cytotoxic immune responses against PD-L1 expressing cells in cancer patients.
  • the invention disclosed in WO2013056716 targets the cancer disease by killing the PD-L1 expressing cancer cells directly and by killing the PD-L1 expressing regulatory cells. This is done by enabling the T cells to recognize the PD-L1 expressing cells. Likewise, when the clinical condition is an infection, T cells are enabled to kill PD-L1 expressing APCs /DCs. Thus, the expression of the immune suppressing enzyme PD-L1 in cancer cells and APCs is positive in conjunction with the application of the method of the present invention, which targets these PD-L1 expressing cells.
  • T-cell response specific for PD-L1 demonstrates that cancer patients are capable of generating T-cell responses to PD-L1 in vivo in response to the presence of PD-L1 peptides.
  • the T cells are present in the cancer patient and they have the ability to expand, which are shown in the application as filed. It follows from the general knowledge in the field of immunology that providing additional PD-L1 protein or PD-L1 peptides will lead to generation of PD-L1 specific T-cell responses.
  • the T-cells recognizes a complex ligand, comprising an antigenic peptide bound to a protein called the major histocompatibility complex (MHC).
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • Class I HLA molecules sample peptides from protein-degradation inside the cell and present these at the cell surface to T cells. Hence, this enables T-cells to scan for cellular alterations.
  • the PD-L1 peptide fragment of SEQ ID NO.91 (described in WO2013056716 as PDLong2) is quite hydrophobic and very prone for beta-sheet formation and therefore has low solubility. Furthermore, this peptide contains free SH, and must be handled at low pH to prevent dimer formation. Hence, there is a need for a more soluble and easy to handle PD-L1 peptide fragment comprising the amino acid sequence of SEQ ID NO. 91 or at least a part of that sequence lacking up to 6 amino acids from the C-terminal.
  • the present invention relates to a PD-L1 peptide fragment having the formula:
  • the PD-L1 peptide fragment may be selected from any of those disclosed in Table A, or a pharmaceutically acceptable salt thereof, optionally wherein the C terminal amino acid is replaced with the corresponding amide form.
  • any amino acid sequence shown may be modified at the C-terminal amino acid to be on amide form (—CONH 2 ) or may be on acid form (—COOH), thus any one of these are preferred embodiments, and it is intended that any C-terminal amino acid, such as I, F, R, L, K, G, M, D comprises both amide and acid form unless specified by —NH 2 or —OH.
  • X 2 is selected from the group consisting of FRLRKGROH (SEQ ID NO: 85), FRLRKGR-NH 2 (SEQ ID NO: 85 with C terminal amide) and FRLRKGRMMD-OH (SEQ ID NO: 88).
  • the present invention relates to a composition
  • a composition comprising the PD-L1 peptide fragment of the present invention; optionally together with a pharmaceutically acceptable additive.
  • the PD-L1 peptide fragment of the present invention is selected form any one of the above in relation to the broad aspect.
  • a pharmaceutically acceptable additive is present.
  • the composition is a vaccine composition.
  • the additive is selected from carriers, excipients, diluents, and adjuvants, typically adjuvants.
  • Such adjuvants may be selected form the group consisting of bacterial DNA based adjuvants, oil/surfactant based adjuvants, viral dsRNA based adjuvants, imidazochinilines, a Montanide ISA adjuvant.
  • the present invention relates to an immunotherapeutic composition
  • an immunotherapeutic composition comprising
  • the PD-L1 peptide fragment of the present invention is selected form any one of the above in relation to the broad aspect.
  • the adjuvants may be selected form the group consisting of bacterial DNA based adjuvants, oil/surfactant based adjuvants, viral dsRNA based adjuvants, imidazochinilines, a Montanide ISA adjuvant. Each of these adjuvants or group of adjuvants constitute an individual embodiment.
  • the immunotherapeutic composition of the present invention is for use in a method for treatment or prevention of a disease, disorder or condition selected from cancer, such as a tumor forming cancer disease; an infection, such as an infectious disease, e.g. an intracellular infection, for example an intracellular infection with a pathogen selected from the group consisting of L. monocytogenes and plasmodium, a viral infection, for example an infection with a virus selected from the group consisting of HIV and hepatitis; an autoimmune disease, such as diabetes, SLE and sclerosis.
  • a disease, disorder or condition selected from cancer such as a tumor forming cancer disease
  • an infection such as an infectious disease, e.g. an intracellular infection, for example an intracellular infection with a pathogen selected from the group consisting of L. monocytogenes and plasmodium
  • a viral infection for example an infection with a virus selected from the group consisting of HIV and hepatitis
  • an autoimmune disease such as diabetes, SLE and sclerosis.
  • kit-of-parts comprising:
  • composition comprising at least one second active ingredient, selected from an immunostimulating compound, such as an interleukin, e.g. IL-2 and or IL-21, an anti-cancer agent, such as a chemotherapeutic agent, e.g.
  • Actimide Azacitidine, Azathioprine, Bleomycin, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Irinotecan, Lenalidomide, Leucovorin, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitoxantrone, nivolumab, Oxaliplatin, Paclitaxel, pembrolizumab, Pemetrexed, Revlimid, Temozolomide, Teniposide, Thioguanine, Valrubicin, Vinblastine, Vincristine, Vindesine and Vinorelbine.
  • the provided compositions are to be administered simultaneously. In another embodiment the provided compositions are to be administered sequentially.
  • the immunotherapeutic composition under a) further embodiments of the PD-L1 peptide fragment of the present invention is selected form any one of the above in relation to the broad aspect.
  • the adjuvants may be selected form the group consisting of bacterial DNA based adjuvants, oil/surfactant based adjuvants, viral dsRNA based adjuvants, imidazochinilines, a Montanide ISA adjuvant. Each of these adjuvants or group of adjuvants constitute an individual embodiment.
  • the second active ingredient under b) further embodiments of the second active ingredient is selected form any one of the above which constitutes individual embodiments.
  • kit-of-parts comprising:
  • an immunomodulatory agent which blocks or inhibits an immune system checkpoint
  • which checkpoint may be the same as, or different from, the checkpoint of which the composition of (a) comprises a component. In other words, it may be the same as, or different from, the checkpoint which comprises the interaction between PD1 and PDL1.
  • the immunomodulatory agent is an antibody or small molecule inhibitor (SMI) which binds to a component of a said immune system checkpoint.
  • SI small molecule inhibitor
  • the agent is a small molecule inhibitor of IDO1, optionally wherein said inhibitor is Epacadostat (INCB24360), Indoximod, GDC-0919 (NLG919) or F001287, or wherein said agent is an antibody which binds to CTLA4 or PD1, optionally wherein said antibody which binds to CTLA4 is ipilimumab and said antibody which binds to PD1 is pembrolizumab.
  • the present invention relates to a method of treating a clinical condition characterized by expression of PD-L1, the method comprising administering to an individual suffering from said clinical condition an effective amount of the peptide fragment of the present invention, the composition of the present invention, or the kit-of-parts of the present invention.
  • the present invention relates to a method of treating a clinical condition characterized by expression of PD-L1, the method comprising administering to an individual suffering from said clinical condition an effective amount of a PD-L1 peptide fragment having the formula:
  • the present invention relates to use of a PD-L1 peptide fragment of the present invention for the manufacture of a medicament, such as an immunotherapeutic composition or vaccine, for the treatment or prevention of a clinical condition characterized by expression of PD-L1.
  • a medicament such as an immunotherapeutic composition or vaccine
  • the medicament is an immunotherapeutic composition.
  • the medicament is vaccine.
  • the clinical condition to be treated is a cancer disease where PD-L1 is expressed.
  • the clinical condition is selected from the group consisting of infectious diseases and autoimmune diseases.
  • the PD-L1 peptide fragment has the formula:
  • the present invention relates to a PD-L1 peptide fragment having the formula:
  • the present invention relates to a PD-L1 peptide fragment comprising the formula:
  • FMTYWHLLNAFTVTVPKDL (SEQ ID NO: 89) wherein the C-terminal amino acid also comprises the amide, or a pharmaceutically acceptable salt thereof;
  • the PD-L1 peptide fragment consists of up to 35 consecutive amino acids, such 30, or 25, of the sequence of SEQ ID NO. 1 and comprises the PD-L1 fragment having the formula: FMTYWHLLNAFTVTVPKDL (SEQ ID NO: 89).
  • the PD-L1 peptide fragment is selected from the PD-L1 fragment having the formula: FMTYWHLLNAFTVTVPKDL (SEQ ID NO: 89) wherein the C-terminal amino acid also comprises the amide.
  • the additional cancer therapy is selected from an immune system checkpoint inhibitor, wherein the inhibitor is a checkpoint blocking antibody selected from Actimide, Azacitidine, Azathioprine, Bleomycin, Carboplatin, Capecitabine, Cisplatin, Chlorambucil, Cyclophosphamide, Cytarabine, Daunorubicin, Docetaxel, Doxifluridine, Doxorubicin, Epirubicin, Etoposide, Fludarabine, Fluorouracil, Gemcitabine, Hydroxyurea, Idarubicin, Irinotecan, Lenalidomide, Leucovorin, Mechlorethamine, Melphalan, Mercaptopurine, Methotrexate, Mitoxantron
  • the PD-L1 peptide fragments disclosed herein are made by standard peptide synthesis, such as solid-phase peptide synthesis (SPPS).
  • SPPS is a standard method for synthesizing peptides in the lab. SPPS allows for the synthesis of natural peptides which are difficult to express in bacteria, the incorporation of unnatural amino acids, peptide/protein backbone modification, and the synthesis of D-proteins, which consist of D-amino acids. Small porous beads are treated with functional units ('linkers') on which peptide chains can be built. The peptide will remain covalently attached to the bead until cleaved from it by a reagent such as anhydrous hydrogen fluoride or trifluoroacetic acid.
  • a reagent such as anhydrous hydrogen fluoride or trifluoroacetic acid.
  • the peptide is thus ‘immobilized’ on the solid-phase and can be retained during a filtration process while liquid-phase reagents and by-products of synthesis are flushed away.
  • the general principle of SPPS is one of repeated cycles of deprotection-wash-coupling-wash.
  • the free N-terminal amine of a solid-phase attached peptide is coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which a further amino acid may be attached.
  • the superiority of this technique partially lies in the ability to perform wash cycles after each reaction, removing excess reagent with all of the growing peptide of interest remaining covalently attached to the insoluble resin.
  • a therapeutically effective amount of at least one compound is administered to a mammal in need of said treatment.
  • amino acids are identified by the one or three letter code known to the person skilled in the art and shown in the table below for convenience:
  • treatment means the management and care of a patient for the purpose of combating a condition, such as a disease or a disorder.
  • the term is intended to include the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of the active compound to alleviate the symptoms or complications, to delay the progression of the disease, disorder or condition, to alleviate or relief the symptoms and complications, and/or to cure or eliminate the disease, disorder or condition as well as to prevent the condition, wherein prevention is to be understood as the management and care of a patient for the purpose of combating the disease, condition, or disorder and includes the administration of the active compounds to prevent the onset of the symptoms or complications.
  • the treatment may either be performed in an acute or in a chronic way.
  • the patient to be treated is preferably a mammal; in particular a human being, but it may also include animals, such as dogs, cats, cows, sheep and pigs.
  • a therapeutically effective amount of a PD-L1 peptide fragment of the present invention or a peptide fragment as disclosed herein, as used herein means an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of a given disease and its complications. An amount adequate to accomplish this is defined as “therapeutically effective amount”. Effective amounts for each purpose will depend on the severity of the disease or injury as well as the weight and general state of the subject. It will be understood that determining an appropriate dosage may be achieved using routine experimentation, by constructing a matrix of values and testing different points in the matrix, which is all within the ordinary skills of a trained physician or veterinary.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the PD-L1 peptide fragment of the present invention and optionally a pharmaceutically acceptable additive, such as a carrier or an excipient.
  • pharmaceutically acceptable additive is intended without limitation to include carriers, excipients, diluents, adjuvant, colorings, aroma, preservatives etc. that the skilled person would consider using when formulating a compound of the present invention in order to make a pharmaceutical composition.
  • the adjuvants, diluents, excipients and/or carriers that may be used in the composition of the invention must be pharmaceutically acceptable in the sense of being compatible with the compound of formula (1) and the other ingredients of the pharmaceutical composition, and not deleterious to the recipient thereof. It is preferred that the compositions shall not contain any material that may cause an adverse reaction, such as an allergic reaction.
  • the adjuvants, diluents, excipients and carriers that may be used in the pharmaceutical composition of the invention are well known to a person within the art.
  • Adjuvants are any substance whose admixture into the composition increases or otherwise modifies the immune response elicited by the composition.
  • Adjuvants broadly defined, are substances which promote immune responses. Adjuvants may also preferably have a depot effect, in that they also result in a slow and sustained release of an active agent from the administration site. A general discussion of adjuvants is provided in Goding, Monoclonal Antibodies: Principles & Practice (2nd edition, 1986) at pages 61-63.
  • lipid A lipid A
  • FCA Freund's Complete Adjuvant
  • FCA Freund's Incomplete Adjuvant
  • Merck Adjuvant 65 polynucleotides (for example, poly IC and poly AU acids), wax D from Mycobacterium, tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, Titermax, ISCOMS, Quil A, ALUN (see US 58767 and U.S. Pat. Nos.
  • GM-CSF Granulocyte-macrophage colony stimulating factor
  • Preferred adjuvants to be used with the invention include oil/surfactant based adjuvants such as Montanide adjuvants (available from Seppic, Belgium), preferably Montanide ISA-51.
  • Other preferred adjuvants are bacterial DNA based adjuvants, such as adjuvants including CpG oligonucleotide sequences.
  • Yet other preferred adjuvants are viral dsRNA based adjuvants, such as poly I:C. GM-CSF and Imidazochinilines are also examples of preferred adjuvants.
  • the adjuvant is most preferably a Montanide ISA adjuvant.
  • the Montanide ISA adjuvant is preferably Montanide ISA 51 or Montanide ISA 720.
  • the carrier may be any suitable carrier known to a person skilled in the art, for example a protein or an antigen presenting cell, such as a dendritic cell (DC).
  • Carrier proteins include keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid.
  • the carrier protein may be tetanus toxoid or diphtheria toxoid.
  • the carrier may be a dextran such as sepharose. The carrier must be physiologically acceptable to humans and safe.
  • the immunotherapeutic composition may optionally comprise a pharmaceutically acceptable excipient.
  • the excipient must be ‘acceptable’ in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.
  • Auxiliary substances such as wetting or emulsifying agents, pH buffering substances and the like, may be present in the excipient.
  • These excipients and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition, and which may be administered without undue toxicity.
  • Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethyleneglycol, hyaluronic acid, glycerol and ethanol.
  • Pharmaceutically acceptable salts can also be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • the immunotherapeutic composition may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration.
  • injectable compositions may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative.
  • Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations.
  • the active ingredient is provided in dry (for e.g., a powder or granules) form for reconstitution with a suitable vehicle (e. g., sterile pyrogen-free water) prior to administration of the reconstituted composition.
  • a suitable vehicle e. g., sterile pyrogen-free water
  • the composition may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution.
  • This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the adjuvants, excipients and auxiliary substances described herein.
  • Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example.
  • Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides.
  • compositions which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems.
  • Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.
  • the active ingredients of the composition may be encapsulated, adsorbed to, or associated with, particulate carriers. Suitable particulate carriers include those derived from polymethyl methacrylate polymers, as well as PLG microparticles derived from poly(lactides) and poly(lactide-co-glycolides).
  • particulate systems and polymers can also be used, for example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, as well as conjugates of these molecules.
  • compositions and particularly immunotherapetic compositions as herein disclosed may, in addition to the compounds herein disclosed, further comprise at least one pharmaceutically acceptable adjuvant, diluent, excipient and/or carrier.
  • the pharmaceutical compositions comprise from 1 to 99 weight % of said at least one pharmaceutically acceptable adjuvant, diluent, excipient and/or carrier and from 1 to 99 weight % of a compound as herein disclosed.
  • the combined amount of the active ingredient and of the pharmaceutically acceptable adjuvant, diluent, excipient and/or carrier may not constitute more than 100% by weight of the composition, particularly the pharmaceutical composition.
  • compositions, vaccine, or kit as described herein may additionally comprise a preservative, which may improve the stability of the component peptide fragments of the invention when stored in solution or as a lyophilisate.
  • a preservative which may improve the stability of the component peptide fragments of the invention when stored in solution or as a lyophilisate.
  • Suitable preservatives are well known in the art and are preferably pharmaceutically acceptable.
  • stability of the peptide fragments may be increased by the incorporation of additional terminal residues, at the N terminus, at the C terminus, or at both termini. Such residues would typically be hydrophilic amino acid residues or corresponding amides.
  • the peptide fragments may include an additional 1, 2 or 3 such residues at the N and/or C termini
  • two or more of the compound as herein disclosed are used in combination for the purposes discussed above.
  • composition particularly immunotherapetic composition comprising a compound set forth herein may be adapted for oral, intravenous, topical, intraperitoneal, nasal, buccal, sublingual, or subcutaneous administration, or for administration via the respiratory tract in the form of, for example, an aerosol or an air-suspended fine powder. Therefore, the pharmaceutical composition may be in the form of, for example, tablets, capsules, powders, nanoparticles, crystals, amorphous substances, solutions, transdermal patches or suppositories.
  • PBMC Peripheral Blood Mononuclear Cells
  • DCvacc dendritic cell vaccine
  • PBMC peripheral blood Mononuclear Cells
  • PBMC peripheral blood Mononuclear Cells
  • DCvacc dendritic cell vaccine
  • PBMC peripheral blood mononuclear Cells
  • PBMC dendritic cell vaccine
  • DC vaccines were generated as previously described 24 and all procedures were performed according to Good Manufacturing Practice (GMP) as approved by the Danish Medicines Agency.
  • GMP Good Manufacturing Practice
  • autologous PBMCs were isolated by leukapheresis, and monocytes were further isolated and cultured for 8 days.
  • maturation of DCs was performed using IL-1 ⁇ , TNF ⁇ , IL-6, and PGE2.
  • Aliquots of 1 ⁇ 10 7 DCs were frozen using automated cryopreservation.
  • the matured DCs were transfected with mRNA encoding the tumor associated antigens p53, survivin and hTERT to generate DCvac
  • PDLong1 included sequence of 9mer HLA-A2 restricted peptide (here entitled “PD-L101”) PDL1 15-23 ; (LLNAFTVTV—SEQ ID NO: 90) identified and analyzed using the epitope prediction Database “SYFPEITHI” available on the internet 25 .
  • PD-L101 scored 30 by the SYFPEITHI algorithm and came out as the top candidate epitope.
  • a 20-mer long peptide (here entitled ‘irrelevant control’) GARVERVDFGNFVFNISVLW—SEQ ID NO: 93- was used as control peptide as well as the HLA-A2 high affinity binding epitope HIV-1 pol 476 -484 (ILKEPVHGV—SEQ ID NO: 94) was used as irrelevant controls.
  • PBMCs from malignant melanoma patients were stimulated with autologous DCvacc with ratio 1:10 DCvacc:PBMCs.
  • a day after stimulation cultures were divided and co-stimulated with peptides either with 25 ⁇ g/mL of PDLong1: PDL19-28, [FMTYWHLLNAFTVTVPKDL SEQ ID NO: 89] or PDLong2: PDL1242-264, [VILGAILLCLGVALTFIFRLRKG—SEQ ID NO: 91] or irrelevant long peptide [GARVERVDFGNFVFNISVLW—SEQ ID NO: 93] as control co-stimulation.
  • Second stimulation with DCvacc was performed on day 7 and followed by peptide co-stimulation on day 8.
  • IL-2 120 U/mL was added a day after each peptide costimulations.
  • a week after second peptide co-stimulation the cultures were analyzed for DCvacc response using intracellular cytokine staining.
  • PBMCs that were stimulated with DCvacc and co-stimulated with peptides for two weeks, were stimulated with DCvacc (ratio 1:10 DCvacc:PBMCs) for 5 hours at 37° C. with 5% CO 2 .
  • GolgiPlug (BD) was added at a dilution of 1:200 after the first hour of incubation. After 4 additional hours cells were washed twice with PBS, stained fluorochrome conjugated antibodies for surface markers (CD3-Amcyan, CD4-PerCP and CD8-Pacific Blue, all from BD).
  • Cells were washed one additional time and thereafter were fixed and permeabilized with Fixation/Permeabilization and Permeabilization Buffer (eBioscience), according to manufacturer's instructions. Cells were subsequently stained with fluorochrome-conjugated antibodies for intracellular cytokines. The following combinations were used: IFN- ⁇ -PE-CY7 (BD), TNF- ⁇ -APC (eBioscience). Relevant isotype controls were used to enable correct compensation and confirm antibody specificity. Stained cells were analyzed using a BD FACSCanto II flow cytometer and further analysis was performed with BD FacsDiva Software.
  • cytokine secretion in DCvacc stimulated and peptide co-stimulated (PDLong1 and PDLong2 or HIV peptide) cultures cell culture supernatants were analyzed using BDTM Cytometric Bead Array (CBA) Flex Sets for IFN- ⁇ , TGF- ⁇ 1, TNF- ⁇ , IL-6, IL-10 and IL-17A. Flex sets for IFN- ⁇ , TNF- ⁇ , IL-6 and IL-10 were combined, whereas IL-17A and TGF- ⁇ 1 were analyzed separately. Analysis was performed according to the manufacturer's recommendations. Samples were acquired on FACSCANTO II (BD Biosciences) and data was analyzed using FCAP ArrayTM Software v 3.0.1 (BD Biosciences).
  • CBA Cytometric Bead Array
  • PBMCs were isolated from patients with melanoma at baseline before vaccination as well as after four and, for some patients, after six vaccinations with DCvacc. PBMCs were stimulated twice with DCvacc in combination with either a control HIV epitope or PD-L1 peptides, as shown in FIG. 1A . Overall, we found that DCvacc mainly stimulates CD4 + T cells.
  • PDLong1 [PDL1 9-27, FMTYWHLLNAFTVTVPKDL] SEQ ID NO: 89).
  • 18 PDLong1 includes an HLA-A2-restricted, PD-L1-derived CD8 + T cell epitope (PDL1 15-23 , LLNAFTVTV—SEQ ID NO: 90).
  • FIG. 1B-E shows the results of cultures from three donors in which coactivation of PD-L1-specific T cells significantly boosted T cell immunity toward DCvacc.
  • CD4 + T cells released TNF ⁇ alone in response to DCvacc without co-stimulation ( FIG. 1B ).
  • TNF ⁇ /INF ⁇ double-positive CD4 + T cells were induced by DCvacc with PD-L1 peptide co-stimulation ( FIG. 1C ).
  • IFN ⁇ ELISPOT assay To further investigate whether the immune response against PD-L1 could be augmented by co-stimulation, we used the IFN ⁇ ELISPOT assay to examine tumor-infiltrating lymphocytes from 12 melanoma patients for T cell reactivity against an additional PD-L1 derived epitope (PDLong2 [PDL1 242-264 , VILGAILLCLGVALTFIFRLRKG (SEQ ID NO: 91)]). The IFN ⁇ ELISPOT assay revealed an increased T cell response in tumor-infiltrating lymphocytes cultured with PDLong2 ( FIG. 3A and B). This increased response was confirmed by intracellular cytokine staining ( FIG. 3C and D).
  • CBA Cytometric Bead Array
  • PD-L1 epitopes to a cancer vaccine could strengthen immune responses against the vaccine in vivo. These measures may boost effector T cells by coactivation of proinflammatory PD-L1-specific T cells, which are attracted to the tumor microenvironment due to local expression of PD-L1.
  • Previous studies have reported that exposure of regulatory T cells (Tregs) to IL-6 and other proinflammatory cytokines induces reprogramming of mature Tregs to acquire a phenotype resembling proinflammatory Th17 cells. 21-23 In the present study, IL-6 levels were significantly higher in cultures that had been co-stimulated with PD-L1 epitopes.
  • PD-L1-specific T cells may effectively boost the effector phase of the immune response by both direct and indirect release of proinflammatory cytokines, as well as by direct removal of PD-L1-expressing regulatory immune cells that inhibit PD-1-positive T cells.
  • PD-L1-specific T cells may inhibit other routes of immune suppression mediated by their cognate target cells.
  • Combined PD-1 pathway blockade with vaccination is a promising alternative approach, as vaccines have been shown to recruit immune effector cells into the tumor microenvironment.
  • Targeting immune regulation by induction of PD-L1-specific T cells is an attractive option to boost the immunogenicity of immunotherapeutic agents, as boosting PD-L1-specific T cells may directly modulate immune regulation and alter tolerance.
  • the combination of vaccination with PD-1 pathway blockade should be easily implementable and synergistic, since PD-L1 blockade by antibodies would make the PD-L1-expressing target cells more vulnerable targets for vaccine-induced T cells. Future investigations are required to confirm the safety, tolerability, and effectiveness of a regimen that includes co-stimulation of PD-L1-specific T cells with PD-L1-derived epitopes.
  • IO104.1-OH Arg-Thr-His-Leu-Val-Ile-Leu-Gly-Ala- Ile-Leu-Leu-Cys-Leu-Gly-Val-Ala-Leu-Thr-Phe-Ile- Phe-Arg-Leu-Arg-Lys-Gly-Arg-OH (C-terminus acid) (SEQ ID NO: 52 with C terminal amide)
  • IO104.1-NH 2 Arg-Thr-His-Leu-Val-Ile-Leu-Gly-Ala- Ile-Leu-Leu-Cys-Leu-Gly-Val-Ala-Leu-Thr-Phe-Ile- Phe-Arg-Leu-Arg-Lys-Gly-Arg-Arg-OH (C-terminus acid) (SEQ ID NO: 52 with C terminal amide)
  • IO104.1-NH 2 Arg-Thr-His-Leu-Val-Ile-Leu-Gly-Ala- Ile-
  • the ELISPOT technique enabled screening a high number of peptide antigens for T-cell recognition, despite the availability of relatively few T-cells.
  • CIP cancer immunotherapy immunoguiding program
  • nitrocellulose-bottomed 96-well plates MultiScreen MSIPN4W; Millipore
  • TILs Tumor Infiltrating Lymphoctyes
  • PD-L1 peptides or with control peptide were added at different cell concentrations in triplicate wells, with PD-L1 peptides or with control peptide, and incubated overnight. The following day, the wells were washed, and the relevant biotinylated secondary antibody (Mabtech) was added, followed by the avidin-enzyme conjugate (AP-Avidin; Calbiochem/Invitrogen Life Technologies); finally, we added the enzyme substrate, NBT/BCIP (Invitrogen Life Technologies) for visualization. The spots on the developed ELISPOT plates were analyzed on a CTL ImmunoSpot S6 Ultimate-V analyzer with Immunospot software, v5.1.
  • the IFN ⁇ ELISPOT assay revealed a T cell response in tumor-infiltrating lymphocytes cultured with IO104.1 peptides from three of the patients. See FIG. 6 .
  • IO104.1 specific T cells are naturally present among tumor infiltrating lymphocytes (TILs) of human melanoma patients.
  • TILs tumor infiltrating lymphocytes
  • IO104.1 peptides are also recognized by CD8+ T cells specific for a known epitope of PD-L1 which is comprised within the sequence of IO104.1.
  • mice were injected with 1 ⁇ g IFN ⁇ in 200 ⁇ l PBS i.p (or no injection for control) two days apart (day 0+2) to simulate inflammation. On day 5 the mice were sacrificed and a single cell solution of the removed spleen was made for further analysis by IFN ⁇ -Elispot.
  • the peptides from PDL1 were selected based on the following reasoning.
  • mLong1 and 2 are overlapping sequences from the region of mPDL1 shown in bold above; mLong3, 4 and 5 are overlapping sequences from the region of mPDL1 shown underlined above.
  • mLong1 is considered to be the closest mouse equivalent to the human peptide of PDlong1.
  • mLong3 is considered to be the closest mouse equivalent to the human peptide of PDlong2.
  • Example 3 Given the result in Example 3, we hypothesized PD-L1 specific T cells should also activate and expand in response to local stimulation, such as the inflammatory response to an allergen.
  • mice were vaccinated subcutaneously (s.c) on the lower back with 100 ⁇ g mPD-L1long1 (mLong1 from Example 3) with or without Montanide as an adjuvant (only Montanide as a control) at day 0. On day 7 the mice were sacrificed and a single cell solution of the removed spleen was made for further analysis by IFNy-Elispot.
  • Results of the Elispot are shown in FIG. 10 .
  • a strong PD-L1-specific T cell response was seen in the spleens and draining lymph nodes (DLNs) of the peptide-vaccinated mice compared to mice vaccinated with the adjuvant alone.
  • Ex vivo stimulation with both mPD-L1long and the shorter version mPD-L1short showed increased PD-L1-specific response in mPD-L1 long vaccinated mice seen by IFNy Elispot.
  • mice C56BL/6 mice were inoculated with 2 ⁇ 10 5 B16F10 tumor cells subcutaneously (s.c) on one side of lower back (day 0).
  • the tumor cells were pre-stimulated with IFNy in vitro for 24h before inoculation.
  • mice were vaccinated s.c on the other side of lower back with 100 ⁇ g mPD-L1long1 with Montanide as an adjuvant (only Montanide as a control). Tumor size was measured 3x/week and mice were sacrificed when tumors got too big.
  • the immune checkpoint regulator PD-L1 is a specific target for naturally occurring CD4+T cells. Oncoimmunology 2013; 2:e23991.

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