Combination Therapy for Cancer

Abstract

To provide methods for the prevention or treatment of cancer in subjects, and immunotherapeutic compositions for use in the methods for the prevention or treatment of cancer in subjects.SOLUTION: This method comprises the step for administering to the subject (i) an immunotherapeutic composition comprising a component of an immune system checkpoint, or an immunogenic fragment of the component; and (ii) an immunomodulatory agent which blocks or inhibits an immune system checkpoint, which may be the same as or different from the checkpoint of which the (i) composition comprises a component.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method of preventing or treating cancer in a subject, the method comprising administering to the subject an immunotherapeutic composition comprising a component of an immune system checkpoint, or an immunogenic fragment of said component, and an immunomodulatory agent that blocks or inhibits an immune system checkpoint, which may be the same as or different from the checkpoint of which the composition comprises the component. The present invention also relates to said immunotherapeutic composition and said agent, as well as kits comprising them. [Background technology]

[0002] The human immune system has the ability to mount a response against cancerous tumors. Harnessing this response is increasingly recognized as one of the most promising routes to treating or preventing cancer. The key effector cells for long-lasting antitumor immune responses are activated tumor-specific effector T cells. However, although cancer patients typically possess T cells specific for tumor antigens, the activity of these T cells is frequently suppressed by inhibitory factors and pathways, and cancer remains a leading cause of premature death in developed countries.

[0003] Over the past decade, therapies that specifically target immune system checkpoints have emerged. An example of this is ipilimumab, a fully human IgG1 antibody specific for CTLA-4. Treatment of metastatic melanoma with ipilimumab was associated with an overall response rate of 10.9% and a clinical benefit rate of approximately 30% in a large phase III trial, and subsequent analyses suggested that responses could be durable and long-lasting. However, these figures still suggest that a large proportion of patients do not benefit from treatment, leaving room for improvement.

[0004] Therefore, there is a need for methods of preventing or treating cancer that enhance T cell anti-tumor responses in a greater proportion of patients, but do not induce undesirable effects such as autoimmune disease. Summary of the Invention

[0005] The inventors have demonstrated that immunotherapeutic compositions comprising immune system checkpoint components or immunogenic fragments thereof can be safely combined with the administration of additional immunomodulatory agents to provide effective treatment or prevention of cancer.

[0006] The present invention provides a method for preventing or treating cancer in a subject, comprising: (i) an immunotherapeutic composition comprising a component of an immune system checkpoint, or an immunogenic fragment of said component; and (ii) an immunomodulatory agent that blocks or inhibits an immune system checkpoint, which may be the same as or different from the checkpoint that the composition of (i) comprises a component of. to said subject.

[0007] The present invention provides (i) an immunotherapeutic composition comprising a component of an immune system checkpoint, or an immunogenic fragment thereof; and / or (ii) immunomodulators and optionally, (i) and (ii) are provided in separate, sealed containers.

[0008] The present invention also provides said immunotherapeutic composition and / or said immunomodulatory agent independently of each other.

[0009] The present invention provides a method for preventing or treating cancer in a subject, comprising: (i) an immunotherapeutic composition comprising a component of an immune system checkpoint, or an immunogenic fragment of said component; and (ii) a composition comprising a tumor antigen or an immunogenic fragment thereof to said subject.

[0010] The present invention provides an immunotherapeutic composition comprising an adjuvant and an immunogenic fragment of IDO consisting of up to 25 consecutive amino acids of the sequence of SEQ ID NO: 1, wherein the consecutive amino acids include the sequence of ALLEIASCL (SEQ ID NO: 2) or the sequence of DTLLKALLEIASCLEKALQVF (SEQ ID NO: 3).

[0011] A brief explanation of sequence listings SEQ ID NO: 1 is the amino acid sequence of indoleamine 2,3-dioxygenase (IDO1). SEQ ID NO: 2 is the amino acid sequence of a fragment of IDO1, referred to herein as IO101 or IDO5. SEQ ID NO: 3 is the amino acid sequence of a fragment of IDO1, referred to herein as IO102. SEQ ID NOs: 4 to 13 are the amino acid sequences of other fragments of IDO1 disclosed in the present specification. SEQ ID NO: 14 is the amino acid sequence of PD-L1. SEQ ID NOs: 15 to 31 and 32 are the amino acid sequences of fragments of PD-L1 disclosed herein. SEQ ID NOs: 33 and 34 are the amino acid sequences of fragments of mouse IDO1, referred to herein as IDO-Pep1 and IDO-EP2, respectively. SEQ ID NO: 35 is a fragment of the E7 oncoprotein of HPV. [Brief explanation of the drawings]

[0012] [Figure 1] Figure 1 shows serum cytokine concentrations in a patient (#10) treated according to the present invention. Levels of seven different cytokines in serum are shown at several different time points during treatment. IL: Interleukin. TNF: Tumor necrosis factor. IFN: Interferon. Wk: Weeks after the first series of ipilimumab. [Figure 2-1]Figure 1 shows vaccine responses in patients treated according to the present invention. Vaccine-induced responses within patients were assessed by direct interferon-gamma ELISpot and intracellular cytokine staining. a) Response to IO102 peptide. Bars represent the number of specific spots, i.e., the number of negative controls subtracted. b) IO102 reactivity in six patients treated with ipilimumab without IDO peptide vaccine. None of these patients showed any measurable response to IO102. c) Intracellular cytokine staining of IO102-stimulated T cell cultures after 4 weeks of in vitro IO102 peptide stimulation in patients #02, #05, and #07. d) Intracellular cytokine staining of IO102-stimulated T cell cultures as shown in 2c, but after additional TNF-alpha capture and rapid expansion (see Methods). TNF: tumor necrosis factor. IFN: interferon. Wk: number of weeks after the first series of ipilimumab. [Figure 2-2] This is a continuation of Figure 2-1. [Figure 3-1] Figure 1 shows regulatory cells in the peripheral blood of patients treated according to the present invention. Flow cytometry analysis of the frequencies of T cells, T helper cells, regulatory T cells (Treg), and myeloid-derived suppressor cells (MDSC). a+b) Gating strategy for Treg and MDSC (including singlet gate and live cell gate, not shown here). c) Percentage of Treg among CD4+ T cells during treatment. d) Percentage of MDSC among live singlet PBMC. e) Percentage of T cells within the lymphocyte gate. f) Percentage of CD4+ cells among T cells. [Figure 3-2] This is a continuation of Figure 3-1. [Figure 4]Figure 1 shows clinical responses in patients treated according to the present invention. Change in target lesion diameter measured according to RECIST 1.1. Patients were evaluated by PET-CT before treatment initiation (baseline), after 12 weeks, and then every 8-12 weeks until progression. Change in target lesion diameter was calculated as the percentage change from baseline by summing the target lesion diameters. Lighter triangles indicate the appearance of new lesions. PD: progressive disease. PR: partial response. [Figure 5] Figure 1 shows that IO102 is superior to IDO5 in inducing a boost of specific T cells. Flow cytometry dot plots show the boosting effect on the number of CMV-specific T cells when IDO5 or IO102 is added to cells stimulated with a CMV peptide. Stimulation with an irrelevant HIV peptide was included as a control. Results from two different PBMC batches (A and B) are shown. Percentages indicate the proportion of cells specific for CMV. NLV-PE = CMV tetramer conjugated to phycoerythrin (PE); NLV-APC = CMV tetramer conjugated to allophycocyanin (APC). [Figure 6] Figure 1 shows that IO102 enhances the IDO SMI-induced boost of specific T cells. Flow cytometry dot plots show the boosting effect on the number of CMV-specific T cells when IO102 is added to cells stimulated with a CMV peptide and the IDO small molecule inhibitor (SMI) 1-MT. Percentages indicate the proportion of cells specific for CMV. NLV-PE = CMV tetramer conjugated to phycoerythrin (PE), NLV-APC = CMV tetramer conjugated to allophycocyanin (APC). [Figure 7] Figure 1 shows the percent lysis of THP-1 target cells induced by PBMCs (effector cells) cultured at the indicated effector:target ratios in the presence of IDO-scrambled peptide (black bars) or IO102 (gray bars). [Figure 8]FIG. 1 shows the percent lysis of THP-1 target cells induced by PBMCs (effector cells) cultured at the indicated effector:target ratios in the presence of control (IDO-scrambled, black bars), IDO5 (light gray bars), or IO102 (dark gray bars). [Figure 9] Figure 1 shows the percent lysis of THP-1 target cells induced by PBMCs (effector cells) cultured in the presence of IDO-scrambled plus anti-PD-1 antibody (control) or IO102 plus anti-PD-1 antibody. Bars represent lysis induced by IO102 plus anti-PD-1 antibody, minus the control, at the indicated effector:target ratios. [Figure 10] Figure 1 shows the change in tumor volume over time (A) and percent survival (B) for C57BL / 6 mice bearing TC-1 tumors treated with IDO peptide (IDO-Pep1) or E7 peptide (E7-Vax). Untreated mice are shown as controls. [Figure 11] Figure 1 shows the change in percent survival over time for C57BL / 6 mice bearing TC-1 tumors treated with IDO peptide (Pep1), E7 peptide (E7-Vax), or both (Pep1+E7-Vax). Untreated mice serve as controls. [Figure 12] Figure 1 shows the change in percent survival over time for C57BL / 6 mice bearing TC-1 tumors treated with IDO peptide (Pep1), 1-MT, or both (Pep1+1-MT). Untreated mice serve as controls. [Figure 13] 1 shows the change in tumor volume over time for BALB / c mice bearing CT26 tumors treated with IDO peptide (EP2) or montanide alone (vehicle), with untreated mice shown as controls. DETAILED DESCRIPTION OF THE INVENTION

[0013] It is understood that different applications may be devised for the disclosed products and methods depending on the particular needs of the art, and that the terminology used herein is for the purpose of describing particular embodiments of the invention and is not intended to be limiting.

[0014] Furthermore, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to an "inhibitor" includes two or more such inhibitors or reference to an "oligonucleotide" includes two or more such oligonucleotides, etc.

[0015] A "subject," as used herein, includes any mammal, preferably a human.

[0016] "Polypeptide" is used broadly herein to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. The term "polypeptide" therefore includes short peptide sequences and longer polypeptides and proteins. As used herein, the term "amino acid" refers to natural and / or unnatural amino acids, or synthetic amino acids, including both D- or L-optical isomers, as well as amino acid analogs and peptidomimetics.

[0017] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0018] Immune System Checkpoints Activation of effector T cells is typically triggered by T cell receptors that recognize antigenic peptides presented by the MHC complex. The type and level of activation achieved is then determined by the balance between signals that stimulate and inhibit effector T cell responses. The term "immune system checkpoint" is used herein to refer to any molecular interaction that shifts the balance in favor of inhibiting effector T cell responses. That is, a molecular interaction that, when it occurs, negatively regulates effector T cell activation. Such an interaction can be direct, such as the interaction between a ligand and a cell surface receptor that transmits an inhibitory signal to the effector T cell. Alternatively, it can be indirect, such as the interaction between a ligand and a cell surface receptor that otherwise transmits an activating signal to the effector T cell, or blocking or inhibiting an interaction that promotes upregulation of an inhibitory molecule or cell, or enzymatic depletion of a metabolite required by the effector T cell, or any combination thereof.

[0019] Examples of immune system checkpoints include: a) The interaction between indoleamine-2,3-dioxygenase (IDO1) and its substrates; b) the interaction between PD1 and PDL1, and / or PD1 and PDL2; c) interactions between CTLA4 and CD86, and / or CTLA4 and CD80; d) interactions between B7-H3 and / or B7-H4 and their respective ligands; e) the interaction between HVEM and BTLA; f) the interaction between GAL9 and TIM3; g) Interaction between MHC class I or II and LAG3, and h) Interaction between MHC class I or II and KIR Examples include:

[0020] Checkpoint (a), i.e., the interaction between IDO1 and its substrate, is a preferred checkpoint for purposes of the present invention. This checkpoint is an intracellular metabolic pathway of the immune system that requires the essential amino acid tryptophan. Tryptophan deficiency results in a general suppression of effector T cell function and promotes the conversion of naive T cells into regulatory (i.e., immunosuppressive) T cells (Tregs). The protein IDO1 is upregulated in many tumor cells and is associated with decreased tryptophan levels. IDO1 is an enzyme that catalyzes the conversion of L-tryptophan to N-formylkynurenine and is therefore the first rate-limiting enzyme in tryptophan catabolism via the kynurenine pathway. Therefore, IDO1 is a component of an immune system checkpoint that may be a suitable target in the methods of the present invention.

[0021] Another preferred checkpoint for the purposes of the present invention is checkpoint (b), i.e., the interaction between PD1 and its ligands PD-L1 and PD-L2. PD1 is expressed on effector T cells. Binding to either ligand results in a signal that downregulates activation. The ligands are expressed by several tumors. PD-L1, in particular, is expressed by many solid tumors, including melanoma. Such tumors may downregulate immune-mediated antitumor effects through activation of the inhibitory PD-1 receptor on T cells. Blocking the interaction between PD1 and one or both of its ligands can ablate the immune response checkpoint and enhance antitumor T cell responses. Thus, PD1 and its ligands are examples of immune system checkpoint components that may be suitable targets in the methods of the present invention.

[0022] Another preferred checkpoint for purposes of the present invention is checkpoint (c), i.e., the interaction between the T cell receptor CTLA-4 and its ligands, the B7 proteins (B7-1 and B7-2). CTLA-4 is normally upregulated on the surface of T cells after initial activation, and ligand binding results in a signal that inhibits further / continuous activation. CTLA-4 competes with the receptor CD28, which is also expressed on the surface of T cells and upregulates activation, for binding to the B7 protein. Thus, blocking the interaction between CTLA-4 and B7 proteins, but not between CD28 and B7 proteins, can eliminate one of the normal checkpoints of the immune response, resulting in an enhanced antitumor T cell response. CTLA4 and its ligands are therefore examples of immune system checkpoint components that may be suitable targets in the methods of the present invention.

[0023] Methods for preventing or treating cancer The methods of the present invention can target any component of any of the checkpoints described in the preceding section.

[0024] The methods of the present invention relate to the prevention or treatment of cancer.

[0025] The cancer may be prostate cancer, brain cancer, breast cancer, colorectal cancer, pancreatic cancer, ovarian cancer, lung cancer, cervical cancer, liver cancer, head / neck / throat cancer, skin cancer, bladder cancer, or blood cancer. The cancer may take the form of a tumor or a cancer of blood origin. The tumor may be solid. The tumor is typically malignant and may be metastatic. The tumor may be an adenoma, adenocarcinoma, blastoma, carcinoma, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, germ cell tumor, lymphoma, leukemia, sarcoma, Wilms' tumor, lung tumor, colon tumor, lymphatic tumor, breast tumor, or melanoma.

[0026] Types of blastoma include hepatoblastoma, glioblastoma, neuroblastoma, or retinoblastoma. Types of carcinoma include colorectal carcinoma or hepatocellular carcinoma, carcinoma of the pancreas, prostate, stomach, esophagus, cervix, and head and neck, and adenocarcinoma. Types of sarcoma include Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, or any other soft tissue sarcoma. Types of melanoma include lentigo maligna, lentigo maligna melanoma, superficial spreading melanoma, acral lentiginous melanoma, mucosal malignant melanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic malignant melanoma, soft tissue melanoma, melanoma with small nevus-like cells, melanoma with Spitz nevus features, and uveal melanoma. Lymphoma and leukemia types include precursor T-cell leukemia / lymphoma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia / lymphoma, MALT lymphoma, Burkitt lymphoma, mycosis fungoides, peripheral T-cell lymphoma, nodular sclerosing Hodgkin lymphoma, and mixed cellularity subtype Hodgkin lymphoma. Lung tumor types include non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma, and large cell carcinoma) and small cell lung cancer.

[0027] The methods of the invention function by activating or enhancing a T cell anti-cancer response in a subject. This is accomplished by blocking or inhibiting one or more immune checkpoints to enhance the activation of cancer- or tumor-specific effector T cells. The methods of the invention utilize at least two different approaches to blocking or inhibiting said one or more immune checkpoints.

[0028] The first approach is to block or inhibit a checkpoint by administering an immunotherapeutic composition that induces an immune response in a subject against a checkpoint component, thereby blocking or inhibiting the activity of the checkpoint. Therefore, the immunotherapeutic composition can alternatively be described as a vaccine against the checkpoint component. The checkpoint component targeted by the immune response is preferably expressed by tumor cells, but can also be expressed by normal cells, where it has an immunosuppressive effect. Thus, the immune response has a dual effect in that it both blocks and inhibits the activity of the checkpoint and also directly attacks the tumor.

[0029] A second approach is to block or inhibit checkpoints by administering an immunomodulatory agent that binds to or otherwise modifies a checkpoint component, thereby blocking or inhibiting checkpoint activity. The agent can be an antibody or small molecule inhibitor that binds to a checkpoint component. Multiple such agents can be administered, each targeting a different checkpoint or a different component of the same checkpoint.

[0030] By utilizing different approaches to block or inhibit immune system checkpoints, the methods of the present invention induce greater anti-tumor responses while producing fewer side effects or complications than alternative methods. The anti-tumor responses typically exceed those expected when using only a single approach. Furthermore, the first approach (vaccine) actively benefits from such responses and may also provide long-lasting effects, making it less likely to experience reduced efficacy due to anti-drug responses. Such benefits are achieved even if both approaches target the same immune system checkpoints.

[0031] An example of this embodiment includes immunotherapeutic compositions that target IDO1 and use an antibody or small molecule inhibitor of IDO1 as an immunomodulator. Another example includes immunotherapeutic compositions that target PD-L1 and use an antibody or small molecule inhibitor of PD1 that binds PD-L1 and / or PD-L2 as an immunomodulator.

[0032] The methods of the invention may also target two different immune system checkpoints, each using a different approach. In such embodiments, similar benefits are achieved, except that the anti-tumor response also typically exceeds that expected when each checkpoint is targeted using the same type of approach. An example of this embodiment includes the use of an immunotherapeutic composition that targets IDO1 (checkpoint (a)) and an antibody or small molecule inhibitor that targets PD1 or CTLA4 (checkpoints (b) and (c)). Another example of this embodiment includes the use of an immunotherapeutic composition that targets PD-L1 (checkpoint (b)) and an antibody or small molecule inhibitor that targets IDO1 or CTLA4 (checkpoints (a) and (c)).

[0033] In other words, the present invention provides a method of preventing or treating cancer in a subject, comprising: (i) an immunotherapeutic composition comprising a component of an immune system checkpoint, or an immunogenic fragment of said component; and (ii) an immunomodulatory agent that blocks or inhibits an immune system checkpoint, which may be the same as or different from the checkpoint that the composition of (i) comprises a component of. to said subject.

[0034] The invention also provides immunotherapeutic compositions for use in the methods of the invention, i.e., for preventing or treating cancer in a subject, wherein the immunotherapeutic composition comprises an immune system checkpoint component or an immunogenic fragment thereof, and the method comprises: (i) the immunotherapeutic composition, and (ii) an immunomodulatory agent that blocks or inhibits an immune system checkpoint, which may be the same as or different from the checkpoint that the composition of (i) comprises a component of. to said subject.

[0035] The invention also provides an immunomodulatory agent for use in the methods of the invention, i.e., for preventing or treating cancer in a subject, wherein the immunomodulatory agent blocks or inhibits an immune system checkpoint, and the method comprises: (i) the immunomodulatory agent, and (ii) an immunotherapeutic composition comprising a component of an immune system checkpoint, or an immunogenic fragment thereof, which may be the same as or different from the checkpoint blocked or inhibited by (i). to said subject.

[0036] Alternatively, the present invention provides a method of preventing or treating cancer in a subject, comprising: (i) an immunotherapeutic composition comprising a component of an immune system checkpoint, or an immunogenic fragment of said component; and (ii) a composition comprising a tumor antigen or an immunogenic fragment thereof to said subject.

[0037] In this embodiment, the method functions by activating or enhancing a T cell anticancer response in a subject against the specific tumor antigen of the composition (ii). This is accomplished by administering the antigen or an immunogenic fragment thereof so that it is presented to the subject's T cells, and simultaneously or sequentially using an immunotherapeutic composition that blocks or inhibits an immune checkpoint that otherwise reduces activation of the T cells to enhance activation of tumor antigen-specific effector T cells. In the context of this method, a composition comprising a tumor antigen or an immunogenic fragment thereof can alternatively be described as a vaccine against the tumor antigen, and administration with an immunotherapeutic composition of the invention can be described as enhancing the vaccine.

[0038] The invention also provides immunotherapeutic compositions for use in the methods of the invention, i.e., for preventing or treating cancer in a subject, wherein the immunotherapeutic composition comprises an immune system checkpoint component or an immunogenic fragment thereof, and the method comprises: (i) the immunotherapeutic composition, and (ii) a composition comprising a tumor antigen or an immunogenic fragment thereof to said subject.

[0039] The invention also provides a composition comprising a tumor antigen or an immunogenic fragment thereof for use in a method of the invention, i.e., for preventing or treating cancer in a subject, the method comprising: (i) the composition, and (ii) an immunotherapeutic composition comprising an immune system checkpoint component or an immunogenic fragment thereof; to said subject.

[0040] The invention also provides the use of an immunotherapeutic composition in the manufacture of a medicament for preventing or treating cancer in a subject, wherein the immunotherapeutic composition comprises an immune system checkpoint component or an immunogenic fragment thereof and is formulated for administration before, concurrently with, and / or after an immunomodulatory agent or a composition comprising a tumor antigen or an immunogenic fragment thereof.

[0041] The invention also provides the use of an immunomodulatory agent that blocks or inhibits an immune system checkpoint in the manufacture of a medicament for preventing or treating cancer in a subject, wherein the agent is formulated for administration before, simultaneously with, and / or after an immunotherapeutic composition comprising a component of an immune system checkpoint or an immunogenic fragment thereof.

[0042] The present invention also provides the use of a composition comprising a tumor antigen or an immunogenic fragment thereof in the manufacture of a medicament for preventing or treating cancer in a subject, wherein the medicament is formulated for administration before, simultaneously with, and / or after an immunotherapeutic composition comprising an immune system checkpoint component or an immunogenic fragment thereof.

[0043] immunotherapy composition The immunotherapeutic compositions of the present invention induce an immune response against a component of an immune system checkpoint. The component is typically a polypeptide. Thus, the immunotherapeutic composition may comprise said component or an immunogenic fragment thereof. The term "immunogenic fragment" is used herein to mean a polypeptide that is shorter than said immune system checkpoint component but that retains the ability to induce an immune response against said component.

[0044] The ability of a fragment to elicit an immune response against a component of an immune system checkpoint ("immunogenicity") can be assessed by any suitable method. Typically, the fragment has the ability to induce in vitro proliferation and / or cytokine release in T cells specific for said component, where said cells may be present in a lymphocyte sample taken from a cancer patient. Proliferation and / or cytokine release can be assessed by any suitable method, including ELISA and ELISPOT. Exemplary methods are described in the Examples. Preferably, the fragment induces proliferation of component-specific T cells and / or induces the release of interferon gamma from such cells.

[0045] In order to induce proliferation and / or cytokine release in T cells specific for said component, the fragment must be capable of binding to an MHC molecule so that it can be presented to T cells. In other words, the fragment comprises or consists of at least one MHC-binding epitope of said component. Said epitope may be an MHC class I-binding epitope or an MHC class II-binding epitope. When the fragment comprises two or more MHC-binding epitopes, it is particularly preferred that each of said epitopes binds to an MHC molecule expressed from a different HLA-allele, thereby broadening the coverage of subjects taken from an outbred human population.

[0046] MHC binding can be evaluated by any suitable method, including the use of in silico methods. Preferred methods include competitive inhibition assays, in which binding is measured in comparison with a reference peptide. The reference peptide is typically a peptide known to be a strong binder for a given MHC molecule. In such an assay, if a peptide has an IC50 for a given HLA molecule that is less than 1 / 100 of that of the reference peptide, the peptide is a weak binder. If a peptide has an IC50 for a given HLA molecule that is at least 1 / 100 but less than 1 / 20 of that of the reference peptide, the peptide is a moderate binder. If a peptide has an IC50 for a given HLA molecule that is at least 1 / 20 of that of the reference peptide, the peptide is a strong binder.

[0047] Preferably, the fragment comprising an MHC class I epitope binds to an MHC class I HLA species selected from the group consisting of HLA-A1, HLA-A2, HLA-A3, HLA-A11, and HLA-A24, more preferably HLA-A3 or HLA-A2. Alternatively, the fragment may bind to an MHC class I HLA-B species selected from the group consisting of HLA-B7, HLA-B35, HLA-B44, HLA-B8, HLA-B15, HLA-B27, and HLA-B51.

[0048] Preferably, the fragment containing an MHC class II epitope binds to an MHC class II HLA species selected from the group consisting of HLA-DPA-1, HLA-DPB-1, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB, and all alleles of this group as well as HLA-DM and HLA-DO.

[0049] An immunotherapeutic composition can include one immunogenic fragment of an immune system checkpoint component, or a combination of two or more such fragments, each specifically interacting with at least one different HLA molecule, to cover a larger proportion of the target population. Thus, by way of example, a composition can include a combination of peptides restricted by HLA-A molecules and peptides restricted by HLA-B molecules, including, for example, HLA-A and HLA-B molecules corresponding to predominant HLA phenotypes in the target population, such as HLA-A2 and HLA-B35. Additionally, a composition can include peptides restricted by HLA-C molecules.

[0050] A preferred immunotherapeutic composition of the present invention elicits an immune response against the polypeptide indoleamine-2,3-dioxygenase (IDO1). In other words, the method of the present invention preferably includes a step of administering an immunotherapeutic composition that elicits an immune response against IDO1. The immunotherapeutic composition may therefore alternatively be described as a vaccine against IDO1. Vaccines against IDO1 that can be used as immunotherapeutic compositions of the present invention are described in WO 2009 / 143843; Andersen and Svane (2015), Oncoimmunology, Vol. 4, No. 1, e983770; and Iversen et al. (2014), Clin Cancer Res, Vol. 20, No. 1, pp. 221-32. The immunotherapeutic composition of the present invention may comprise IDO1 or an immunogenic fragment thereof. The fragment may consist of at least 8, preferably at least 9, consecutive amino acids of IDO1 (SEQ ID NO: 1). The fragment may consist of up to 40 consecutive amino acids of IDO1 (SEQ ID NO: 1), up to 30 consecutive amino acids of IDO1 (SEQ ID NO: 1), preferably up to 25 consecutive amino acids of IDO1 (SEQ ID NO: 1). Thus, the fragment may comprise or consist of 8 to 40, 8 to 30, 8 to 25, 9 to 40, 9 to 30, or 9 to 25 consecutive amino acids of IDO1 (SEQ ID NO: 1). Preferably, the fragment comprises or consists of 9 to 25 consecutive amino acids of IDO1 (SEQ ID NO: 1).

[0051] The fragment may comprise or consist of any one of the following sequences: IO101: ALLEIASCL [199-207] (SEQ ID NO: 2); IO102: DTLLKALLEIASCLEKALQVF [194-214] (SEQ ID NO: 3); IOx1: QLRERVEKL [54-62] (SEQ ID NO: 4); IOx2: FLVSLLVEI [164-172] (SEQ ID NO: 5); IOx3: TLLKALLEI [195-203] (SEQ ID NO: 6); IOx4: FIAKHLPDL [41-49] (SEQ ID NO: 7); IOx6: VLSKGDAGL [320-328] (SEQ ID NO: 8); IOx7: DLMNFLKTV [383-391] (SEQ ID NO: 9); IOx8: VLLGIQQTA [275-283] (SEQ ID NO: 10); IOx9: KVLPRNIAV [101-109] (SEQ ID NO: 11); IOx10: KLNMLSIDHL [61-70] (SEQ ID NO: 12); IOx11: SLRSYHLQIV [341-350] (SEQ ID NO: 13)

[0052] Numbers in square brackets [ ] indicate the corresponding positions in the IDO polypeptide of SEQ ID NO:1, counting from the N-terminus towards the C-terminus.

[0053] The fragment preferably comprises or consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3, and most preferably comprises or consists of the amino acid sequence of SEQ ID NO: 3. Peptides comprising or consisting of SEQ ID NO: 2 bind well to HLA-A2, a particularly common HLA species. Peptides consisting of SEQ ID NO: 3 bind well to at least one of the specific class I and class II HLA species described above. Fragments comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3 are advantageous in that they are effective in a high proportion of the outbred human population.

[0054] Another preferred immunotherapeutic composition of the present invention elicits an immune response against the polypeptide programmed death-ligand 1 (PD-L1). In other words, the methods of the present invention preferably comprise the step of administering an immunotherapeutic composition that elicits an immune response against PD-L1. The immunotherapeutic composition may therefore alternatively be described as a vaccine against PD-L1. Vaccines against PD-L1 can be used as immunotherapeutic compositions of the present invention and are described in WO 2013 / 056716. The immunotherapeutic compositions of the present invention may comprise PD-L1 or an immunogenic fragment thereof. The fragment may consist of at least 8, preferably at least 9, consecutive amino acids of PD-L1 (SEQ ID NO: 14). The fragment may consist of up to 40 consecutive amino acids of PD-L1 (SEQ ID NO: 14), up to 30 consecutive amino acids of PD-L1 (SEQ ID NO: 14), and preferably up to 25 consecutive amino acids of PD-L1 (SEQ ID NO: 14). Thus, a fragment may comprise or consist of 8-40, 8-30, 8-25, 9-40, 9-30, or 9-25 consecutive amino acids of PD-L1 (SEQ ID NO: 14). Preferably, the fragment comprises or consists of 9-25 consecutive amino acids of PD-L1 (SEQ ID NO: 14).

[0055] The fragment may comprise or consist of any one of the following sequences:

[0056] TIFF0007785817000001.tif135129

[0057] Preferably, the fragment comprises or consists of the sequence of one of SEQ ID NOs: 15, 25, 28 or 32.

[0058] Immunotherapeutic compositions may preferably comprise an adjuvant and / or a carrier. Particularly preferred immunotherapeutic compositions provided by the present invention comprise an adjuvant and, as an active ingredient, a polypeptide of up to 25 amino acids in length comprising or consisting of the amino acid sequence of SEQ ID NO: 3. The composition may be provided for use in the methods of the present invention or for use in any other method of preventing or treating cancer that involves administration of the composition.

[0059] An adjuvant is any substance that, when mixed with a composition, enhances or otherwise modifies the immune response elicited by the composition. Broadly defined, an adjuvant is a substance that promotes an immune response. Adjuvants may also preferably have a depot effect, in that they also cause a slow, sustained release of the active agent from the site of administration. A general discussion of adjuvants is presented in Goding, Monoclonal Antibodies: Principles & Practice (2nd ed., 1986), pp. 61-63.

[0060] Adjuvants include AlK(SO4)2, AlNa(SO4)2, AlNH4(SO4), silica, alum, Al(OH)3, Ca3(PO4)2, kaolin, carbon, aluminum hydroxide, muramyl dipeptide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nornuramyl-L-alanyl-D-isoglutamine (CGP 11687, also known as nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1',2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (CGP 11687). 19835A, also known as MTP-PE), RIBI in 2% squalene / Tween-80 RTM emulsion (MPL+TDM+CWS), lipopolysaccharides and their various derivatives including lipid A, Freund's complete adjuvant (FCA), Freund's incomplete adjuvant, Merck adjuvant 65, polynucleotides (e.g., polyIC acid and polyAU acid), wax D from Mycobacterium tuberculosis, substances found in Corynebacterium parvum, Bordetella pertussis, and members of the genus Brucella, Titermax, ISCOMS, Quil A, ALUN (see U.S. Patent Nos. 58767 and 5,554,372), lipid A derivatives, cholera toxin derivatives, HSP derivatives, LPS derivatives, synthetic peptide matrices or GMDP, interleukin 1, interleukin 2, montanide ISA-51, and QS-21. Various saponin extracts have also been suggested to be useful as adjuvants in immunogenic compositions. Granulocyte-macrophage colony-stimulating factor (GM-CSF) can also be used as an adjuvant.

[0061] Preferred adjuvants for use in the present invention include oil / surfactant-based adjuvants, such as Montanide adjuvant (available from Seppic, Belgium), preferably Montanide ISA-51. Other preferred adjuvants are bacterial DNA-based adjuvants, such as adjuvants containing CpG oligonucleotide sequences. Still other preferred adjuvants are viral dsRNA-based adjuvants, such as poly I:C. GM-CSF and imidazolinone are also examples of preferred adjuvants.

[0062] The adjuvant is most preferably a Montanide ISA adjuvant. The Montanide ISA adjuvant is preferably Montanide ISA 51 or Montanide ISA 720.

[0063] Goding, Monoclonal Antibodies: Principles & Practice (2nd ed., 1986), pp. 61-63, also points out that when the antigen of interest is low molecular weight or poorly immunogenic, it is recommended to conjugate it to an immunogenic carrier. The polypeptide or fragment of the immunotherapeutic composition of the present invention can be conjugated to a carrier. The carrier can exist independently of an adjuvant. The function of the carrier can be, for example, to increase the molecular weight of the polypeptide fragment to enhance activity or immunogenicity, confer stability, enhance biological activity, or extend serum half-life. Furthermore, the carrier may be useful for presenting the polypeptide or its fragment to T cells. Thus, in an immunogenic composition, the polypeptide or its fragment can be associated with a carrier, such as those described below.

[0064] The carrier can be any suitable carrier known to those skilled in the art, such as 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. Alternatively, the carrier protein can be tetanus toxoid or diphtheria toxoid. Alternatively, the carrier can be dextran, such as sepharose. The carrier must be physiologically acceptable and safe for humans.

[0065] Immunotherapeutic compositions may optionally contain pharmaceutically acceptable excipients. An excipient must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not toxic to the recipient. Auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in the excipient. Such excipients and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition and may be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, and ethanol. Pharmaceutically acceptable salts, such as salts of mineral acids, e.g., hydrochlorides, hydrobromides, phosphates, sulfates, and the like, and salts of organic acids, e.g., acetates, propionates, malonates, benzoates, and the like, may also be included. A comprehensive discussion of pharmaceutically acceptable excipients, vehicles, and adjuvants is available in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0066] Immunotherapeutic compositions may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable compositions may be prepared, packaged, or sold in unit-dosage form, such as 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. In one embodiment of the composition, the active ingredient is provided in a dry form (e.g., as a powder or granules) for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) before administration of the reconstituted composition. The composition may be prepared, packaged, or sold in the form of an injectable sterile aqueous or oily suspension or solution. The suspension or solution may be formulated according to known techniques and may contain additional ingredients in addition to the active ingredient, such as adjuvants, excipients, and auxiliary agents described herein. Such injectable sterile formulations can be prepared using a nontoxic, parenterally acceptable diluent or solvent, such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic saline, and fixed oils, such as synthetic mono- or diglycerides. Other useful compositions include those comprising the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system. Sustained-release or implantable compositions can include pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion exchange resins, sparingly soluble polymers, or sparingly soluble salts. Alternatively, the active ingredient of the composition can be encapsulated, adsorbed to, or associated with particulate carriers. Suitable particulate carriers include those derived from polymethyl methacrylate polymers and PLG microparticles derived from poly(lactide) and poly(lactide-co-glycolide). See, e.g., Jeffery et al. (1993) Pharm. Res. 10:362-368. Other particulate systems and polymers, such as polymers of polylysine, polyarginine, polyornithine, spermine, spermidine, and the like, as well as conjugates of such molecules, can also be used.

[0067] immunomodulators "Immunomodulatory agent" is used herein to mean any agent that, when administered to a subject, blocks or inhibits the action of an immune system checkpoint, resulting in upregulation of an immune effector response, typically a T cell effector response, including preferably an anti-tumor T cell effector response, in the subject.

[0068] The immunomodulatory agents used in the methods of the present invention may block or inhibit any of the above immune system checkpoints. The agent may be an antibody or any other suitable agent that causes said blocking or inhibition. The agent may therefore be generally referred to as an inhibitor of said checkpoint.

[0069] As used herein, "antibody" includes all antibodies and any antigen-binding fragment (i.e., "antigen-binding portion"), or single chain thereof. Antibodies may be polyclonal or monoclonal, and may be produced by any suitable method. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include Fab fragments, F(ab')2 fragments, Fab' fragments, Fd fragments, Fv fragments, dAb fragments, and isolated complementarity-determining regions (CDRs). Single-chain antibodies, such as scFvs, and heavy-chain antibodies, such as VHHs and camelid antibodies, are also intended to be encompassed by the term "antigen-binding portion" of an antibody.

[0070] Preferred antibodies that block or inhibit the interaction of CTLA-4 with B7 protein include ipilimumab, tremelimumab, or any of the antibodies disclosed in WO 2014 / 207063. Other molecules include polypeptides or soluble mutant CD86 polypeptides.

[0071] Preferred antibodies that block or inhibit the interaction between PD1 and PD-L1 include nivolumab, pembrolizumab, lambrolizumab, pidilzumab, and AMP-224. Anti-PD-L1 antibodies include MEDI-4736 and MPDL3280A.

[0072] Other suitable inhibitors include small molecule inhibitors (SMIs), which are typically small organic molecules.

[0073] Preferred inhibitors of IDO1 include epacadostat (INCB24360), indoximod, GDC-0919 (NLG919), and F001287. Other inhibitors of IDO1 include 1-methyltryptophan (1MT).

[0074] The immunomodulatory agents of the invention, such as antibodies or SMIs, can be formulated with a pharmaceutically acceptable excipient for administration to a subject. Suitable excipients and adjuvants are described above for the immunotherapeutic compositions of the invention, and can also be used with the immunomodulatory agents of the invention. Suitable forms for preparing, packaging, and selling the immunotherapeutic compositions are also described above. Similar considerations apply to the immunomodulatory agents of the invention.

[0075] Dosing regimen To treat cancer, the immunotherapeutic composition and the immunomodulatory agent are each administered to a subject in a therapeutically effective amount. A "therapeutically effective amount" of a substance means that a given substance is administered to a subject suffering from cancer in an amount sufficient to cure, alleviate, or partially prevent the cancer or one or more of its symptoms. Such therapeutic treatment may result in a decrease in the severity of disease symptoms or an increase in the frequency or duration of symptom-free periods. Such treatment may result in a decrease in solid tumor volume.

[0076] To prevent cancer, the immunotherapeutic composition and immunomodulatory agent are each administered to a subject in a prophylactically effective amount. A "prophylactically effective amount" of an agent means that a given agent is administered to a subject in an amount sufficient to prevent the occurrence or recurrence of one or more symptoms associated with cancer over the long term.

[0077] The effective amount for a given purpose and for a given composition or agent will depend on the severity of the disease, as well as the weight and general state of the subject, and can be readily determined by a physician.

[0078] The immunotherapeutic composition and immunomodulatory agent may be administered simultaneously or sequentially in any order. The appropriate route of administration and dosage of each may be determined by a physician, and the composition and agent will be formulated accordingly.

[0079] Immunotherapeutic compositions are typically administered parenterally, typically by injection. Administration can be preferably via subcutaneous, intradermal, intramuscular, or intratumoral routes. The injection site can be pre-treated with, for example, imiquimod or a similar topical adjuvant to enhance immunogenicity. The total amount of polypeptide present as the active agent in a single administration of the immunotherapeutic composition of the invention typically ranges from 10 μg to 1000 μg, preferably from 10 μg to 150 μg.

[0080] When the immunomodulator is an antibody, it is typically administered as a systemic infusion, for example, intravenously. When the immunomodulator is an SMI, it is typically administered orally. The appropriate dosage of the antibody and SMI can be determined by a physician. The appropriate dosage of the antibody is typically proportional to the subject's body weight.

[0081] A typical regimen for the methods of the present invention involves multiple independent administrations of both the immunotherapeutic composition and the immunomodulatory agent. Each may be administered independently two or more times, e.g., 2, 3, 4, 5, 6, 7 or more times. When the immunotherapeutic composition is administered two or more times, repeated administration may provide increased benefit, particularly since it may enhance the resulting immune response. Individual administrations of the composition or agent may be separated by an appropriate interval, as determined by the physician, but the interval is typically one to two weeks. The interval between administrations is typically shorter at the beginning of the treatment course and increases toward the end of the treatment course.

[0082] An exemplary dosing regimen involves administering an immunomodulatory agent at a dose of 3 milligrams per kilogram of body weight every three weeks for a total of about four series, with an immunotherapeutic composition (typically including an adjuvant) also administered subcutaneously in the back of the arm or front of the thigh, alternating between the right and left sides. Administration of the immunotherapeutic composition can begin simultaneously with the first series of agents, with the composition delivered a total of about seven times, initially weekly for a total of four doses, followed by three booster doses every two weeks. This type of regimen is described in Example 1.

[0083] Another exemplary dosing regimen involves treating a subject with an immunotherapy composition (typically including an adjuvant) administered subcutaneously every two weeks (induction) for 2.5 months, then monthly (maintenance). Imiquimod ointment (Aldara, Meda AS, www.meda.se) may optionally be administered 8 hours before administration of the composition, and the skin is covered with a patch until it is administered to the same area of ​​skin.

[0084] Hereinafter, an embodiment of the present invention will be described. (1) A method for preventing or treating cancer in a subject, comprising: (i) an immunotherapeutic composition comprising a component of an immune system checkpoint or an immunogenic fragment of said component; and (ii) an immunomodulatory agent that blocks or inhibits an immune system checkpoint, which may be the same as or different from the checkpoint that the composition of (i) comprises a component of. to the subject. (2) at least one of the checkpoints is: a) The interaction between IDO1 and its substrates; b) the interaction between PD1 and PDL1, and / or PD1 and PDL2; c) interactions between CTLA4 and CD86, and / or CTLA4 and CD80; d) interactions between B7-H3 and / or B7-H4 and their respective ligands; e) the interaction between HVEM and BTLA; f) the interaction between GAL9 and TIM3; g) Interaction between MHC class I or II and LAG3, and h) Interaction between MHC class I or II and KIR The method according to (1), wherein the method is selected from the group consisting of: (3) (A)(i) the composition comprises a component of checkpoint (a) or an immunogenic fragment thereof, and (ii) the agent blocks or inhibits the same or a different checkpoint, optionally the different checkpoint is checkpoint (b) or (c); or (B) (i) the composition comprises a component of checkpoint (b) or an immunogenic fragment thereof, and (ii) the agent blocks or inhibits the same or a different checkpoint, optionally the different checkpoint is checkpoint (a) or (c); (2) The method described above. (4) The method according to any one of (1) to (3), wherein the component of the immune system checkpoint is IDO1 (SEQ ID NO: 1), the immunogenic fragment of IDO consists of up to 25 consecutive amino acids of the sequence of SEQ ID NO: 1, and the consecutive amino acids include the sequence of ALLEIASCL (SEQ ID NO: 2) or the sequence of DTLLKALLEIASCLEKALQVF (SEQ ID NO: 3). (5) The method according to (4), wherein the immunogenic fragment comprises or consists of the sequence ALLEIASCL (SEQ ID NO: 2) or the sequence DTLLKALLEIASCLEKALQVF (SEQ ID NO: 3). (6) The method of any one of (1) to (3), wherein the component of the immune system checkpoint is PD-L1 (SEQ ID NO: 14), and the immunogenic fragment of PD-L1 consists of up to 25 consecutive amino acids of the sequence of SEQ ID NO: 14, wherein the consecutive amino acids comprise any one of the sequences of SEQ ID NOs: 15 to 31, preferably any one of the sequences of SEQ ID NOs: 15, 25, or 28. (7) The method of any one of (1) to (6), wherein the immunomodulator is an antibody or small molecule inhibitor (SMI) that binds to the component of the immune system checkpoint. (8) The method of (7), wherein the agent is a small molecule inhibitor of IDO1, optionally wherein the inhibitor is epacadostat (INCB24360), indoximod, GDC-0919 (NLG919) or F001287, or wherein the agent is an antibody that binds to CTLA4 or PD1, optionally wherein the antibody that binds to CTLA4 is ipilimumab and the antibody that binds to PD1 is pembrolizumab. (9) The method according to any one of (1) to (8), wherein the composition (i) comprises an adjuvant or a carrier, and optionally the adjuvant is selected from the group consisting of a bacterial DNA adjuvant, an oil / surfactant adjuvant, a viral dsRNA adjuvant, an imidazoquinoline, and GM-CSF. (10) The method according to (9), wherein the adjuvant is a Montanide ISA adjuvant, and optionally selected from Montanide ISA 51 or Montanide ISA 720. (11) An immunotherapeutic composition for use in a method for preventing or treating cancer in a subject, the immunotherapeutic composition comprising an immune system checkpoint component or an immunogenic fragment thereof, the method comprising: (i) the immunotherapeutic composition, and (ii) immunomodulators to said subject. The immunotherapeutic composition. (12) Use of an immunotherapeutic composition in the manufacture of a medicament for preventing or treating cancer in a subject, wherein the immunotherapeutic composition comprises a component of an immune system checkpoint or an immunogenic fragment thereof and is formulated for administration before, simultaneously with, and / or after an immunomodulatory agent. (13)(i) an immunotherapeutic composition comprising a component of an immune system checkpoint or an immunogenic fragment thereof, and / or (ii) immunomodulators wherein optionally (i) and (ii) are provided in separate sealed containers. (14) An immunotherapeutic composition comprising an adjuvant and an immunogenic fragment of IDO consisting of up to 25 consecutive amino acids of the sequence of SEQ ID NO: 1, wherein the consecutive amino acids comprise the sequence of ALLEIASCL (SEQ ID NO: 2) or the sequence of DTLLKALLEIASCLEKALQVF (SEQ ID NO: 3). (15) The immunotherapeutic composition according to (14), wherein the immunogenic fragment of IDO consists of the sequence DTLLKALLEIASCLEKALQVF (SEQ ID NO: 3). (16) The immunotherapeutic composition according to (14) or (15) for use in a method for preventing or treating cancer in a subject, which is optionally a method according to any one of (1) to (10). (17) Use of the immunotherapeutic composition according to (14) or (15) in the manufacture of a medicament for preventing or treating cancer in a subject. The invention is illustrated by the following examples. [Example]

[0085] [Example 1] Phase I trials method Study design Patients were enrolled in a first-in-human phase I clinical trial conducted between March 2014 and August 2014 at the Department of Oncology, Herlev Hospital, University of Copenhagen (Herlev, Denmark). The trial was initially designed as a two-arm phase I study combining IDO1-derived peptide vaccination with standard treatment with ipilimumab or vemurafenib. Only data from patients treated with ipilimumab in combination with the vaccine are reported. Patients had to have histologically confirmed unresectable stage III or stage IV malignant melanoma, be aged 18 years or older, have an Eastern Cooperative Oncology Group (ECOG) performance status of ≤2, have had at least 21 days since their last systemic treatment for melanoma and have fully recovered since then, and have adequate hematologic, renal, and hepatic function. Previous anti-CTLA-4 therapy was permitted, provided the treatment was not ineffective or discontinued due to toxicity.

[0086] Key exclusion criteria included concurrent systemic immunosuppressive therapy, known chronic infection, a history of cancer within 3 years, concurrent serious medical illness, major abdominal surgery within 28 days, pregnant or breastfeeding women, severe psychiatric illness affecting compliance, known intolerance to the vaccine adjuvants montanide or imiquimod, a history of autoimmunity, or recipients who had received a prophylactic vaccine within 28 days. The study was approved by the Danish Health and Medicines Agency and the local Ethics Committee, Capital Region of Denmark. The study was conducted in accordance with the Declaration of Helsinki II and Good Clinical Practice (GCP). All subjects provided written informed consent before any study-related procedures were performed. The study is registered at www.clinicaltrials.gov (NCT02077114) and https: / / eudract.ema.europa.eu / (EudraCT# 2013-000365-37).

[0087] The primary endpoint was toxicity. Additionally, the immune response to the vaccine peptide and the clinical benefit of treatment were evaluated. Patients received ipilimumab treatment at a dose of 3 milligrams per kilogram of body weight every three weeks for a total of four series. Additionally, patients received a vaccine containing the IO102 peptide in a phosphate-buffered saline (PBS)-Montanide ISA-51 emulsion delivered subcutaneously in the back of the arm or the front of the thigh, alternating between the right and left sides. Vaccination began simultaneously with the first series of ipilimumab and included seven vaccines: four weekly doses, followed by three additional doses every two weeks.

[0088] IO102 vaccine The vaccine consists of a 21-amino acid peptide corresponding to residues 194-214 of indoleamine-2,3-dioxygenase. The peptide is referred to herein as IO102. The peptide has the amino acid sequence DTLLKALLEIASCLEKALQVF (SEQ ID NO: 3). It was produced according to GMP standards by JPT Peptides Technologies GmbH, Berlin, Germany. For administration, a hospital pharmacy (Capital Region of Denmark) dissolved the peptide in 2% dimethyl sulfoxide and 98% PBS and mixed it with Montanide ISA-51 (a product of Seppic, a healthcare division of the Air Liquide Group, Paris-La Défence, France). Topical 5% imiquimod cream (Medea, Allerod, Denmark) was applied to the vaccine site and protected with an occlusive dressing for 6-12 hours before injection.

[0089] The peptide has a large number of different HLA class I epitopes nested within its sequence and was predicted by in silico analysis to bind to some of the most common HLA-alleles (http: / / www.cbs.dtu.dk / services / NetMHC / ).

[0090] Clinical evaluation criteria Safety of study treatment was assessed for the occurrence of adverse events and graded according to the Common Terminology Criteria for Adverse Events (CTCAE), version 4.0. Antitumor activity was assessed using positron emission tomography-computed tomography (PET-CT) scans obtained at baseline (before treatment initiation, up to 28 days), after 12 weeks, and every 8 to 12 weeks thereafter until progression. CT scans were evaluated according to the Response Evaluation Criteria in Solid Tumors (RECIST), version 1.1. Response classifications were complete response (CR), partial response (PR), progressive disease (PD), and stable disease (SD). Patients who received at least five doses of the vaccine were considered eligible for evaluation of the clinical and immunological endpoints of this study.

[0091] Blood sample processing PBMCs were purified from heparinized blood using lymphoprep™ (StemCell Technologies) density gradient centrifugation in LeucoSep™ tubes (Greiner Bio-One). After processing, cells were frozen in Nunc® 1.8 ml CryoTubes (Thermo Scientific) and stored at -150°C in 90% human AB serum (Sigma-Aldrich) and 10% dimethyl sulfoxide (Herlev Hospital Pharmacy). Patient-treatment times were kept as short as possible, and treatment generally began within 4 hours. Blood for serum collection was collected into 8 ml Vacuette® gel tubes (Greiner Bio-One) containing a clot activator. Serum was aliquoted into Nunc® 1.8 ml CryoTubes (Thermo Scientific) and stored at -80°C until analysis. PBMCs were processed from 100 ml of heparinized blood per sampling, and serum was processed from 8 ml of whole blood. Blood samples were obtained at baseline, weeks 4, 8, and 12. In non-progressing patients, additional blood samples were obtained every 8 to 12 weeks, and radiological evaluations were performed at the same time.

[0092] In vitro validation of IO102 immunogenicity ELISpot analysis was performed according to the CIMT Immuno Guiding Program (CIP) guidelines (http: / / cimt.eu / cimt / files / dl / cip_guidelines.pdf). Vaccine responses were assessed directly ex vivo in PBMC samples obtained before, during, and after therapy. Cryopreserved samples were thawed and placed in 24-well plates overnight in X-vivio culture medium (Lonza) containing 5% human AB serum (Sigma). Nitrocellulose-bottom 96-well plates (MultiScreen MSIPN4W, Millipore) were coated overnight with IFN-γ capture antibody (Mabtech). Wells were washed with PBS (Sigma-Aldrich), blocked for 2 hours ex-vivo at 37°C in a humidified atmosphere, and PBMCs were added at 5 × 10 5 / well and 2 x 10 5The plate was added at a concentration of 1000 μM / well, and IO102 peptide (purchased from KJ Ross-Petersen, Klampenborg, Denmark) was added at a concentration of 5 μM. An unrelated HIV-derived peptide (ILKEPVHGV, purchased from KJ Ross-Petersen, Klampenborg, Denmark) was used as a negative control. After peptide addition, the plate was incubated overnight at 37°C in a humidified atmosphere supplemented with 5% CO2. The next day, the medium was discarded, and after washing the wells, a biotinylated IFN-γ secondary antibody (Mabtech) was added. The plate was incubated for 2 hours at room temperature, washed, and an avidin-enzyme conjugate (AP-avidin, Calbiochem / Invitrogen Life Technologies) was added to each well. The plate was incubated for 1 hour at room temperature, and the enzyme substrate NBT / BCIP (Invitrogen Life Technologies) was added to each well and incubated for 1-10 minutes at room temperature. Upon the appearance of dark purple spots as judged by visual inspection, the reaction was terminated by washing with demineralized water. Spot numbers were counted using an ImmunoSpotSeries 2.0 analyzer (CTL Analyzers). ELISpot responses were defined as those with at least two-fold greater numbers of IFN-γ-secreting cells than negative controls and a minimum of 50 spots (5 × 10 PBMCs). 5 A positive result was observed when ≥ 1000 cells / ml were detected. All experiments were repeated three times. Results are presented after subtracting the mean background value.

[0093] Establishment of IDO-specific T cell cultures PBMCs were stimulated in 24-well plates (Nunc, Fischer Scientific) with IO102 peptide in X-vivo 15 containing 5% human AB serum and 120 U / ml IL2 (Novartis, Denmark). Initially, 20 μM peptide was used, and cultures were restimulated every 7 days with a log10 decrease in peptide concentration each week. Cell cultures were replenished with fresh IL2 every 7–10 days. Cells were cultured at 3–4 × 106 The peptides were maintained at a concentration of 1000 μg / well. For enrichment and expansion of IDO-specific T cells, cultured cells were restimulated with 20 μM peptide after 4 weeks of culture and enriched using a TNF-α secretion assay (Miltenyi Biotec) according to the manufacturer's instructions. Cells were stained with primary antibodies after 2.5 hours of peptide stimulation. The enriched cells were expanded in a modified rapid expansion protocol (see below).

[0094] Rapid Growth Protocol T cells enriched for IDO-reactivity were expanded and cultured at approximately 1-2 x 10 5 Cells were initially used in 20 ml of X-vivo 15 (Lonza) supplemented with 10% human AB serum, 0.6 μg of anti-CD3 (OKT3, Janssen-Cilag), 6000 U / ml of IL2 (Proleukin, Novartis), 1.25 μg / ml of Fungizone (Squibb), 100 + 100 U / ml of PenStrep (Gribco, Life Technologies), and 2 × 10 7 Cultures were initiated in upright T25 flasks (Nunc) containing 1000 feeder cells. Allogeneic PBMCs mixed from at least three different donors were used as feeder cells. Immediately before use, feeder cells were thawed in RPMI (Gribco, Life Technologies) containing 0.025 mg / ml Pulmozyme (Roche) and γ-irradiated at 30 Gy. After 5 days of culture, 10 ml of medium was carefully aspirated, and the bottles were replenished with fresh medium containing 10% human AB serum, 6000 U / ml IL2, 1.25 μg / ml Fungizone, and 100 + 100 U / ml PenStrep. Cultures were periodically assessed for cell number and medium color, and, if appropriate, transferred to T80 or T175 flasks with additional fresh medium. After a total of 14 days of culture, cells were harvested and either analyzed directly or cryopreserved in 90% human AB serum and 10% dimethyl sulfoxide.

[0095] Intracellular cytokine staining Cells were plated in 96-well plates (Nunc, Fischer Scientific) at 2–3 × 10 6 Cells were plated at a concentration of 1000 cells / ml. IO102 peptide was added at a concentration of 5 μM, and after 1 hour, GolgiPlug™ (BD Biosciences) was added at a concentration of 1 μl per ml of medium. After 5 hours, cells were harvested and further processed. Cells were stained for surface antigens and dead cells using the following antibodies / stains: anti-CD3-PerCP, anti-CD4-Horizon V500, anti-CD8-FITC, and LIVE / DEADR Fixable Near-IR Dead Cell Stain (Life Technologies). Cells were fixed and permeabilized using the BD Cytofix / Cytoperm Kit (BD Biosciences) according to the manufacturer's instructions. Cells were stained for intracellular cytokines using the following antibodies: anti-IL2-PE, anti-IFN-γ-APC (BD Biosciences), and anti-TNF-α-PE-Cy7 (Biolegend). Cells were acquired on a FACSCanto II (BD Biosciences) using FACSDiva software, version 6.1.3 (BD ​​Biosciences). FlowJo software, version 10 (Tree Star, Ashland, USA) was used to determine the frequency of cytokine-positive cells.

[0096] Analysis of regulatory cells in peripheral blood PBMC samples were thawed in RPMI 1640 medium (Lonza) supplemented with 2.5 ml of DNAse-containing Pulmozyme (Roche) and 0.26 mmol of MgCl (Herlev Hospital Pharmacy) per 100 ml of buffer at 37°C. All staining was performed in phosphate-buffered saline (PBS) (Lonza) containing 0.5% bovine serum albumin (Sigma-Aldrich). The following antibodies were used: FoxP3-PE, HLA-DR-HV500, CD3-PE-Cy7, CD19-PE-Cy7, CD56-PE-Cy7, CD4-HV500, CD11b-APC, CD3-APC (purchased from BD Bioscience), CD33-FITC, CD124-PE (purchased from BD Pharmigen), HELIOS-PerCP-Cy5.5, CD14-BV421, CD25-BV421 (all purchased from Biolegend), CD127-FITC, and CD39-PE-Cy7 (purchased from eBioscience). Additionally, all samples were stained with the LIVE / DEADR Fixable Near-IR Dead Cell Stain Kit (Life Technologies). For intracellular staining of transcription factors, the Foxp3 / Transcription Factor Staining Buffer Set (eBioscience) was used according to the manufacturer's instructions. Cells were acquired on a FACSCanto II using FACSDiva software, version 6.1.3 (BD ​​Biosciences). Flow data analysis was performed using FlowJo software, version 10 OSX (TreeStar Inc., Ashland, OR). Data analysis was performed after filtering for dead cells and doublets / triplets.

[0097] Cytokine bead array Interleukin (IL) 2, IL4, IL6, IL10, IL17A, TNF-α, and IFN-γ concentrations in serum obtained from patients before and during treatment were measured using the BD™ Human Th1 / Th2 / Th17 CBA Kit (BD Biosciences) according to the manufacturer's instructions. Cytokine concentrations were measured in thawed, undiluted serum samples. Cytokine concentrations were calculated using FCAP Array™ software, version 3.0 (BD SoftFlow).

[0098] Reference patient cohort For comparison of clinical and immunological data, we used a cohort of patients treated with ipilimumab at our institution (patient data are presented in Manuscript II). These patients participated in a biomarker study approved by the local Ethics Committee for The Capital Region of Denmark (approval number H-2-2012-058), and all patients provided informed consent. The inclusion criteria for this protocol were similar to those for the vaccine study, except that known intolerance to the vaccine adjuvants montanide or imiquimod was not assessed. Patients were selected as controls based on age (+ / - 5 years) and M-phase. For comparison of T cell reactivity to IO102 during ipilimumab therapy, IFN-γ secretion was measured in an ELISpot assay as described above. Reactivity was assessed in PBMC samples obtained before treatment initiation and after three series of treatment.

[0099] statistics All statistical calculations were performed using GraphPad Prism (GraphPad Software, La Jolla, CA). Scatter plots are presented with the median (horizontal line). All tests were two-sided, and a p-value of ≤ 0.05 was considered significant. Where applicable, differences over time were assessed using the Wilcoxon paired signed-rank test.

[0100] result demographics Thirteen patients were screened for inclusion in the study, 12 were enrolled, and one was unwilling to participate. Two patients were diagnosed with clinically significant brain metastases before receiving their first treatment and were excluded from the study. Ten patients received protocol-defined treatment, and all were deemed eligible for evaluation of the study's clinical and immunological endpoints. The patient characteristics of these 10 patients are presented in Table 1. Eight patients received four protocol-defined series of ipilimumab and seven peptide vaccine doses. One patient developed symptomatic brain metastases after three series of ipilimumab and five vaccine doses and was excluded from the protocol because he required high-dose corticosteroid treatment. All 10 patients received ipilimumab as first-line therapy. One patient received interferon-α as adjuvant therapy before entering the study. None of the patients had received any prior systemic therapy for metastatic disease.

[0101] [Table 1]

[0102] toxicity The results are presented in Table 2. No CTCAE grade III or IV adverse reactions were observed related to the vaccine. In general, treatment was well tolerated and associated with limited and manageable toxicity.

[0103] Most patients experienced mild to moderate injection site reactions, including itching, swelling, and erythema, at the vaccine administration site. In most cases, symptoms responded to oral antihistamines, and all patients experienced symptom resolution after completing vaccine treatment. Interestingly, many patients showed increased PET signal in the vaccine administration area.

[0104] Several patients experienced adverse reactions most likely related to ipilimumab treatment. Two patients experienced grade II diarrhea that responded to treatment with loperamide. One patient (Patient #10) was admitted to our institution for treatment of grade III diarrhea, and a diagnosis of ipilimumab-induced colitis was made by colonoscopy. The condition was initially refractory to high-dose oral corticosteroids but resolved after intravenous methylprednisolone administration. One week after discharge, the patient was readmitted to a local hospital with recurrent diarrhea, peripheral edema, and general fatigue. At this time, laboratory investigations were performed on the patient and were notable for hyponatremia, hypokalemia, hypoalbuminemia, and thrombocytopenia. The patient was treated with electrolyte and fluid replacement therapy, but died one week after admission. The exact cause of death was not entirely clear at this time. A PET-CT scan performed 4 weeks before this patient's death revealed only cutaneous and subcutaneous metastases and stabilization of the disease, making disease progression highly unlikely. The patient most likely developed accelerated colitis due to corticosteroid non-adherence or steroid-refractory disease. While ipilimumab-induced colitis is fatal in itself, it is possible that the patient developed sepsis during his hospital stay. Analysis of inflammatory cytokines in serum samples using cytokine bead arrays revealed that this patient had significantly increased levels of IL17A and IL6 by week 12, coinciding with the onset of fulminant colitis (Figure 1).

[0105] Additionally, two patients experienced a grade II maculopapular rash on the trunk and extremities, which responded to topical steroid treatment and completely resolved after treatment. One patient experienced a flare-up of pre-existing rosacea after the first dose of ipilimumab, but resolved during subsequent therapy. One patient experienced grade II choroiditis, possibly related to treatment, 10 weeks after the last dose of ipilimumab, which resolved after topical corticosteroid treatment. Finally, one patient was diagnosed with multiple small, clinically asymptomatic pulmonary emboli evident on PET-CT scan 2 months after receiving the last dose of ipilimumab. This patient had a history of pulmonary metastases and early-stage chronic obstructive pulmonary disease, and the event was highly likely unrelated to either ipilimumab or vaccine treatment.

[0106] [Table 2]

[0107] T cell responses to vaccine epitopes Spontaneous T cell responses against IO102 (SEQ ID NO: 3) and the nested HLA-A2 epitope IDO5 (SEQ ID NO: 2, also called IO101) have been previously reported, and both the clinical efficacy and immunogenicity of IDO5 as a vaccine epitope have been demonstrated in a previous phase I clinical trial for lung cancer.

[0108] In this study, initial screening of IFN-γ release in IO102-stimulated PBMC samples was performed using direct ELISpot. All experiments were performed using two different cell concentrations (5 × 10 5 pcs and 2 x 10 5The vaccine was repeated three times using 1000 vaccinia immunizations (1000 vaccinia immunizations). Responses were considered specific if the spot count was at least twice above background and at least 50 spots higher than the corresponding negative control (HIV peptide). As seen in Figure 2a, none of the patients had a detectable pretreatment response to IO102. However, three patients demonstrated responses exceeding the experimental threshold of 50 spots above the negative control during therapy, which appeared to be somewhat enhanced by repeated vaccination.

[0109] Neither the presence nor the degree of vaccine response appeared to correlate with the clinical efficacy or type / grade of toxicity of the treatment. To determine whether IDO-specific responses were induced by the vaccine or rather a general phenomenon during ipilimumab treatment, we measured IDO reactivity in PBMCs obtained from melanoma patients treated with ipilimumab without the vaccine (see Methods). Reactivity was assessed before and after three series of treatment. As seen in Figure 2b, no induction of IDO-specific cells was observed during ipilimumab treatment without the IDO vaccine. Therefore, the observed IDO responses were induced by the vaccine. To further investigate the induced vaccine-specific T cells, cytokine production was assessed in IO102-stimulated PBMC samples both directly ex vivo and 4 weeks after in vitro stimulation with the IO102 peptide using intracellular cytokine staining and flow cytometry. This was attempted in three patients selected based on the results of ELISpot analysis.

[0110] As seen in Figure 2c, stimulation with IO102 demonstrated enrichment of cytokine-positive T cells, primarily CD4+ cells. These cells primarily produced TNF-α, with some also secreting IFN-γ. Furthermore, IDO-reactive T cells were purified based on the extracellular capture of TNF-α and subsequent nonspecific proliferation (see Methods). The results of intracellular cytokine staining after this enrichment are presented in Figure 2d. As shown, CD4+ IDO-reactive T cells were still present in all three patients. Furthermore, enrichment of IDO-reactive CD8+ T cells was observed in patients #07 and #02, although only a few CD8+ T cells were observed in the cultures obtained from patient #02. None of the subsets exhibited any significant production of IL2 (data not shown).

[0111] Regulatory cell dynamics throughout treatment IDO is a key mediator of immunosuppression and may have important effects on the dynamics of regulatory immune cells. In mice, IDO expression and metabolites produced by IDO-catalyzed tryptophan degradation have been shown to induce the generation of Tregs. Furthermore, IDO can be expressed in MDSCs (myeloid-derived suppressor cells), thereby representing one of several effector mechanisms for this cell type. Levels of Tregs and MDSCs in peripheral blood were measured before, during, and after therapy.

[0112] Tregs were defined as CD3+CD4+CD25highCD127-FOXP3+.

[0113] MDSCs were defined as PBMCs negative for lineage markers CD3, CD19, and CD56, and also HLADR- / lowCD14+CD11b+CD33+CD124-.

[0114] The gating strategy is presented in Figure 3a+b. The results are presented in Figure 3c-f.

[0115] As shown, we observed an increase in Treg cell frequency after 4 weeks (two ipilimumab series and four vaccines) and 8 weeks (three ipilimumab series and five vaccines). After the full treatment course was completed by week 12, levels were still elevated compared to baseline, but not as markedly. The change only reached significance at week 8 (p = 0.008). Furthermore, we closely examined Tregs for expression of the surface marker CD39 and the transcription factor Helios, which can distinguish activated / rested naive natural Tregs from Tregs derived from pooled effector T cells. No consistent changes were observed in any of these Treg subsets (data not shown). There was no change in the percentage of CD3+ lymphocytes, and only minor changes in the frequency of CD4+ T cells were observed (Figure 3e+f).

[0116] Regarding MDSCs, we observed a striking mirror image of Treg levels, i.e., lower levels compared to baseline. However, the changes did not reach significance at any of the time points assessed (baseline to week 8, p = 0.13). Comparing Tregs and MDSCs, we found no convincing correlation between their levels and changes (data not shown).

[0117] Clinical efficacy Ten of the 12 patients enrolled were treated as part of the protocol, and all received at least five doses of the vaccine as per the protocol defined for evaluable patients. Thus, seven of the 10 patients are still alive 10 months after completing treatment.

[0118] The results are presented in Figure 4. At the first evaluation, one patient (#03) had a partial response (PR) with a 44% reduction in target lesion (TL) diameter, and four patients were within the limits of stable disease (SD). Five patients progressed and were referred for other treatments. Of these, two patients (#02 and #13) were diagnosed with brain metastases after receiving the third series of ipilimumab, and one patient was diagnosed with brain metastases shortly after completing the fourth series and seventh vaccination of ipilimumab. Because brain imaging was not routinely performed at baseline in asymptomatic patients, it is unclear whether patients #02 and #07 developed brain metastases during therapy or whether the lesions were already present. However, in the case of patient #13, a magnetic resonance scan was performed immediately before enrollment and showed no signs of central nervous system metastasis.

[0119] Of the four patients who showed SD at the first evaluation, one (#01) showed clear progression at the second evaluation 8 weeks later, with a 100% increase in the sum of the TL diameters. One patient (#10) died 6 weeks after receiving final treatment (see the "Toxicity" section). Two patients maintained SD. One of these patients was treated with argon beam irradiation between evaluation scans for lung metastases, but it is impossible to determine which treatment was more responsible for disease stabilization. Patient #03, who showed an initial (unconfirmed) PR, showed a further slight decrease in TL diameter compared to baseline, to -57%. Unfortunately, PET-CT revealed new lesions in the liver and subcutaneous tissue, and this patient's best response was SD. Currently, two patients have continued SD and await further evaluation.

[0120] In summary, the overall objective response rate, i.e., complete response plus partial response (CR+PR), was 0%, as no patient achieved a response better than SD. Thus, 5 patients (50%) were in SD by the first evaluation, of which 2 were confirmed, 2 had progressed by the second evaluation, and 1 patient died between the first and second evaluations.

[0121] Consideration Ipilimumab targets the immunoinhibitory molecule CTLA-4 and has been shown to extend overall survival in patients with metastatic melanoma. This treatment can induce durable responses, with dramatic reductions in tumor burden and, in some patients, complete responses. Despite this optimism, only a small percentage of patients actually respond to therapy, leaving significant room for improvement. In an attempt to achieve this, numerous trials have focused on combination therapy as a means of hitting more than one target at a time. Several trials have tested the combination of ipilimumab with other interventions, and to date, the most promising data have been based on its combination with the PD-1-targeting antibody nivolumab. However, prior to the study reported here, no attempts had been made to combine it with a vaccine against IDO1.

[0122] Combination treatment was generally safe and well tolerated, although it was associated with mild to moderate toxicity in most patients. Most patients experienced some degree of local reaction at the vaccine administration site, including erythema, edema, and a non-tender lump in the subcutaneous tissue. The latter is a common and transient side effect of peptide vaccines containing montanide, an oil-based adjuvant used in this study.

[0123] Several patients experienced reactions commonly associated with ipilimumab treatment, including diarrhea and a maculopapular rash. One patient was diagnosed 10 weeks after the last treatment with monocular choroiditis, a previously reported rare side effect of ipilimumab. Whether this was treatment-related is difficult to prove, given the temporal dissociation between treatment and symptoms.

[0124] One patient developed ipilimumab-associated colitis during therapy, which was initially managed with parenteral high-dose prednisolone during hospitalization at our institution. Due to unfortunate circumstances, this patient was subsequently admitted to a local hospital with no experience treating this type of patient and died one week after admission. At the time of admission, clinical chemistry test results and presentation suggested the possibility of accelerated colitis and sepsis. The appearance of colitis coincided with marked increases in the levels of the cytokines IL17A and IL6, as demonstrated in serum samples obtained from this patient. Importantly, both cytokines may play a role in autoimmune colitis. Similar increases were not demonstrated in any of the other treated patients, suggesting that elevated cytokine levels may mediate colitis or represent a downstream response to intestinal inflammation. Because IDO is highly expressed in the gastrointestinal tract, it is theoretically possible that vaccines could cause or enhance ipilimumab-induced diarrhea or colitis. No association was found between the presence or degree of vaccine-induced IDO-reactive T cells and the occurrence of toxicity. In a previous study conducted at the same institution, however, the lowest dose epitope vaccine targeting IDO in lung cancer was associated with grade I / II diarrhea in 27% of patients. Adverse reactions were otherwise generally manageable.

[0125] All participating patients were tested for responses to the IO102 peptide using IFN-γ ELISpot, and vaccine-induced responses above our experimental threshold were found in 3 of 10 treated patients. None of the age- and stage-matched patients who received ipilimumab without the vaccine showed any signs of IDO reactivity during therapy, so the responses in these patients were highly likely induced by the vaccine. None of the patients had a detectable response at baseline. Screening for IDO reactivity was performed directly ex vivo to increase the specificity of the assay. However, this procedure traded off sensitivity, and it is certainly possible that an indirect approach would have demonstrated more immune responses.

[0126] To further characterize the reactive T cells, we performed in vitro boosting of IDO responses in patients who responded in the initial screening. This revealed that the reactive cells were primarily CD4+ T cells producing TNF-α, but after enrichment and rapid expansion, IDO-reactive cytotoxic T cells could be detected. IDO-reactive T cells were not immediately demonstrated ex vivo by intracellular cytokine staining. Conflicting data have been published regarding the benefits of tumor-specific CD4+ T cells. Extensive evidence regarding the function of T helper cells in tumor immunity suggests that the benefits depend on the dominant cell subtype, resulting in the establishment of a cytokine milieu within the tumor. Preclinical studies have shown that both Th1 and Th2 cells can exert antitumor activity, either directly or via supportive substances, and chemotactic effects on other cells, while Th17 and Treg cells can exert adverse effects depending on the tumor type. Vaccination trials using long peptides derived from telomerase demonstrated CD4 T cell responses with clear cytotoxic potential and signs of clinical efficacy.

[0127] Analysis performed in this study revealed that expanded IDO-reactive CD4+ T cells produced primarily TNF-α and minimal IFN-γ, whereas reactive CD8+ T cells were primarily double-positive. This suggests an inflammatory phenotype in both cell subsets and, in the case of T helper cells, a more exhausted state, possibly induced by the ex vivo extensive culture of the cells. As all three patients experienced disease progression, there was no good correlation between treatment efficacy and vaccine response. Previous IDO vaccination trials in lung cancer found a significant correlation between pre-existing IDO responses and treatment response, but not vaccine-induced responses, theoretically due to migration of tumor-reactive T cells to the tumor site upon vaccination. Several other studies have attempted to correlate clinical responses with immune responses, but have yielded conflicting results, often due to the small number of responders in vaccination trials.

[0128] As previously described, IDO is expressed in several different tissues, including several MDSC subtypes, and IDO-targeted therapy may affect MDSC frequency. Furthermore, MDSC frequency has been shown to decrease with ipilimumab treatment. Consistent with this, a decrease in monocytic MDSC frequency was observed during treatment. Interestingly, this mirrored the increase in Treg levels, and although no proportional relationship was found between MDSC versus Treg levels / changes, this may represent a counter-regulatory mechanism preventing autoimmunity. While several publications have previously reported that ipilimumab can increase Treg frequency in peripheral blood, other reports have demonstrated the opposite.

[0129] A decrease in Treg levels but no change in MDSCs was observed in patients treated with ipilimumab without vaccination using the same gating and marker panel used in this study (data not shown). Assuming that the inverse changes in Tregs and MDSCs are not due to random biological variation, this could suggest a specific effect of the vaccine, possibly targeting IDO-expressing cells, e.g., MDSCs.

[0130] Ten months after the end of treatment, seven of the ten patients are still alive, and two patients still have stable disease. Given the modest sample size of the current study, it is expected that the number of responders may differ substantially from larger clinical trials using ipilimumab as monotherapy, regardless of the vaccine, due primarily to biological variability. Response to ipilimumab is strongly associated with the number of nonsynonymous mutations that give rise to novel epitopes, and such epitopes have recently been shown to be present in approximately half of scrutinized patient samples, providing a significant opportunity for unequal distribution of this and other prognostic factors in small studies. In the study cohort, the objective response rate was very modest; none of the patients had a confirmed objective response. One patient had a highly significant reduction in tumor burden at the first evaluation, and at the second confirmatory evaluation, despite ongoing responses to target lesions, the patient progressed and developed new lesions at multiple sites. In this patient, a malignant clone with poor or no response to treatment expanded, while genetic tumor heterogeneity may have allowed some lesions to continue to respond. Five of the 10 treated patients were considered to have a possible SD state at the first evaluation, two of which were confirmed at the second evaluation, one patient died, and two patients progressed. It should be understood that the response rate was not very high, as the number of patients who developed clinically evident brain metastases was quite high (3 patients), which always poses a poor prognosis.

[0131] In conclusion, administration of the IDO peptide vaccine, when combined with ipilimumab, was safe and associated with minimal toxicity. The vaccine induced IDO T cell responses that were directly detectable ex vivo.

[0132] [Example 2] Further characterization of IDO peptide vaccine components (1) Comparison of the boosting effects of IDO5 and IO102 on CMV peptide-induced stimulation in T cells method The overall scheme of the method is summarized in Table X. Day 1: - Buffy coat samples were thawed, washed twice, and resuspended in culture medium. Cell counts were performed. - The cells were then cultured at 5 x 10 6 / 0.5 ml was added to the wells of a cell culture plate. - The wells were stimulated with a CMV peptide for 2 hours at room temperature. The CMV peptide used was HCMV pp65 495-504 (NLVPMVATV). After 2 hours, IDO5, IO102, or an irrelevant HIV peptide used as a control was added according to Table X, and the plates were incubated for another 2 hours at room temperature. - 1500 microL of X-VIVO / 5% HS was added to each well and the plate was placed in a 37°C incubator. Days 2 and 9: - IL2 was added (120 U / μl). Day 8: - Wells were restimulated with IDO5, IO102 or HIV peptides, added according to Table X, and then incubated for 2 hours at room temperature. After restimulation, the contents of each well were divided into two portions, and 1.5 ml of X-VIVO+5% HS was added to each well. - The plates were placed in a 37°C incubator. Day 15: - Cells were harvested and analyzed by flow cytometry. A -CD8 monoclonal antibody (mAb) and the tetrameric complex HLA-A2 / CMV pp65 495-504 were used to identify the percentage of CMV-specific CD8 T cells in each well. - As a control, cells were additionally stained with the tetrameric complex HLA-A2 / HIV-1 pol476-484 and CD8 mAb (data not shown).

[0133] TIFF0007785817000004.tif59116

[0134] result The results (see Figure 5) show that the addition of IO102 to CMV-stimulated cell cultures results in the induction of a greater proportion of CMV-specific T cells compared to the addition of IDO5 / IO101, thus demonstrating that IO102 is a better inducer of specific T cells than IDO5 / IO101.

[0135] [Example 3] Further characterization of IDO peptide vaccine components (2) Examining the effects of IO102 in combination with the IDO small molecule inhibitor (SMI), 1-methyltryptophan (1MT), when added to PBMCs stimulated with CMV peptides method The overall scheme of the method is summarized in Table Y. Day 1: - Buffy coat samples were thawed, washed twice, and resuspended in culture medium. Cell counts were performed. - The cells were then cultured at 5 x 10 6 1 / 2 ml was added to the wells of a cell culture plate. The wells were stimulated with peptides and SMI as shown in Table Y. The CMV peptide used was HCMV pp65 495-504 (NLVPMVATV). - The plates were placed in a 37 degree incubator. Days 2 and 9: - IL2 was added (100 U / μl). Day 8: - 1 ml was removed from each well and 1 ml of fresh medium was added. - Wells were restimulated with peptides and SMI according to Table Y. - The plates were placed in a 37°C incubator. Day 15: - Cells were harvested and analyzed by flow cytometry. - CD8 monoclonal antibody (mAb) as well as the tetrameric complex HLA-A2 / CMV pp65 495-504 were used to identify the percentage of CMV-specific CD8 T cells in each well. - As a control, cells were additionally stained with the tetrameric complex HLA-A2 / HIV-1 pol476-484 and CD8 mAb (data not shown).

[0136] TIFF0007785817000005.tif7799

[0137] result The results (see Figure 6) show that IO102 in combination with the IDO small molecule inhibitor 1MT boosts CMV-specific T cell stimulation more potently than 1MT alone. Thus, simultaneously targeting IDO with both IO102 and SMI is more effective than targeting IDO alone with an IDO inhibitor, such as 1MT. IO102 enhances the IDO SMI-induced boost of specific T cells.

[0138] [Example 4] Further characterization of IDO peptide vaccine components (3) Addition of IO102, but not a peptide containing amino acids identical to IO102 in a scrambled sequence, enhances allogenic killing of the acute monocytic leukemia cell line THP-1 by PBMCs. method The overall scheme of the method is summarized in Table Z4.

[0139] Buffy coats were thawed (day 0), washed twice, resuspended in medium, counted, and plated into wells as described in Table Z4. THP-1 cells were irradiated at 30 GY and washed twice in medium. The number of THP-1 cells was counted and added to wells containing PBMCs as described in Table Z4 (total volume 2 ml). Plates were placed in a 37° incubator. Peptides and cytokines were added at intervals, with concentrations shown in Table Z5. On days 7 and 14, 1 ml of supernatant was removed from each well. Fresh irradiated THP-1 cells were prepared and counted as above, and 1 ml was added to wells as shown in Table Z4. On day 17, cells were harvested and analyzed for cytotoxic activity. Standard 51 A Cr-release assay was used to measure the cytotoxic potential of peptide-treated PBMCs. 51 Cr-labeled THP-1 cells were used as target cells, and peptide-treated PBMCs were used as effector cells. Triton-X-treated wells represented the maximum measurable lysis, and medium alone represented the minimum lysis. Specific lysis%=((cpm サンプル - cpm 最低 ) / (cpm 最大 - cpm 最低 ))×100%

[0140] TIFF0007785817000006.tif7892

[0141] result Addition of IO102 to cultures of PMBC and THP-1 cancer cells enhanced allogenic killing of THP-1 cells. The boosted cytotoxicity is specific to the IO102 peptide, since a peptide containing the same amino acid sequence as IO102 but with a scrambled sequence (CILDSKLEVEALAQLLTFALK (SEQ ID NO: 15), Figure 7, black bars) induced less lysis of THP-1 cells compared with IO102 (Figure 7, gray bars) at a range of effector-to-target (E / T) ratios.

[0142] [Example 5] Further characterization of the IDO peptide vaccine component (4) IO102 induces better PBMC-mediated cytotoxicity in THP-1 cells than a peptide containing identical amino acids to IO102 in the scrambled sequence (control, IDO-scrambled). IO102 also causes better killing of THP-1 cells at low effector-to-target (E / T) ratios than IDO5. method The overall scheme of the method is summarized in Table Z5.

[0143] Buffy coats were thawed (day 0), washed twice, resuspended in medium, counted, and plated into wells as described in Table Z5. THP-1 cells were irradiated at 30 GY and washed twice in medium. The number of THP-1 cells was counted and added to wells containing PBMCs as described in Table Z5 (total volume 2 ml). The plates were placed in a 37° incubator. Peptides and cytokines were added at intervals and the concentrations are shown in Table Z5. On day 7, 1 ml of supernatant was removed from each well. Fresh irradiated THP-1 cells were prepared and counted as above, and 1 ml was added to wells as shown in Table Z5, cells were harvested, and analyzed for cytotoxic activity on day 23. Standard 51 A Cr-release assay was used to measure the cytotoxic potential of peptide-treated PBMCs. 51 Cr-labeled THP-1 cells were used as target cells and peptide-treated PBMCs as effector cells. Triton-X-treated wells were used as the maximum measurable lysis and medium alone as the minimum lysis. % specific lysis = ((cpm サンプル - cpm 最低 ) / (cpm 最大 - cpm 最低 ))×100%

[0144] TIFF0007785817000007.tif83119

[0145] result Addition of IO102 (Figure 8, dark gray bars) to cultures of PBMC and THP-1 cancer cells enhanced allogenic killing of THP-1 cells at all E / T ratios tested (2:1 to 60:1) compared with the control peptide, IDO-scrambled (Figure 8, black bars). Furthermore, addition of IO102 to PBMC cultures induced more efficient lysis of THP-1 cells at low E / T ratios compared with the addition of IDO5 peptide (Figure 8, light gray bars). This suggests that IO102-specific T cells more efficiently support allogenic anticancer T cell responses.

[0146] [Example 6] Characterization of an IDO peptide vaccine in combination with additional checkpoint inhibitors IO102 plus anti-PD1 induces better PBMC-mediated cytotoxicity in THP-1 cells compared to control peptide plus anti-PD-1. The overall scheme of the method is summarized in Table Z6.

[0147] Buffy coats were thawed (day 0), washed twice, resuspended in medium, counted, and plated into wells as described in Table Z6. THP-1 cells were irradiated at 30 GY and washed twice in medium. THP-1 cells were counted and added to wells containing PBMCs (total volume 2 ml) as described in Table Z5. Plates were placed in a 37°C incubator. Peptides, antibodies (pembrolizumab, Merck), and cytokines were added at intervals and at the concentrations shown in Table Z6. On day 7, 1 ml of supernatant was removed from each well. Fresh irradiated THP-1 cells were prepared and counted as described above, and 1 ml was added to wells as shown in Table Z6. On day 23, cells were harvested and analyzed for cytotoxic activity. Standard 51 A Cr-release assay was used to measure the cytotoxic potential of peptide-treated PBMCs. 51Cr-labeled THP-1 cells were used as target cells, and peptide-treated PBMCs were used as effector cells. Triton-X-treated wells represented the maximum measurable lysis, and medium alone represented the minimum lysis. Specific lysis%=((cpm サンプル - cpm 最低 ) / (cpm 最大 - cpm 最低 ))×100%

[0148] TIFF0007785817000008.tif83148

[0149] result Addition of a combination of IO102 and anti-PD-1 antibody to cultures of PBMCs enhanced allogenic killing of THP-1 target cells at all E / T ratios (0.7:1 to 20:1) compared with lysis by PBMCs cultured with a combination of IDO-scrambled control peptide and anti-PD-1 antibody ( Figure 9 ).

[0150] [Example 7] Mouse model study of IDO peptide vaccine C57BL / 6 mice bearing TC-1 tumors were treated with the TC-1-specific E7-peptide (RAHYNIVTF, SEQ ID NO: 35) or IDO-Pep1 (MTYENMDIL, SEQ ID NO: 33), and the efficacy of the peptide vaccine was evaluated by tumor burden and survival. Because the sequence of mouse IDO1 differs from that of human IDO1, the mouse IDO peptide sequence (IDO-Pep1) was chosen to provide a mouse analog of the human IDO peptide tested in the above examples.

[0151] method Four- to six-week-old C57BL / 6 mice were injected with TC-1 cells at 70,000 cells / mouse. Treatment within each group began when tumors reached an average size of approximately 0.075 cm3 (approximately 10 days after tumor inoculation). Peptides used for vaccination included the TC1-specific E7-peptide (RAHYNIVTF, SEQ ID NO: 35) and IDO-Pep1 (MTYENMDIL, SEQ ID NO: 33). The murine IDO peptide was designed based on an MHC class I and II binding algorithm. Mice were subcutaneously vaccinated with a combination of a pan-HLA DR-binding epitope (PADRE, aK-Cha-VAAWTLKAAa, 20 μg / mouse) and QuilA (10 μg / mouse) at a dose of 100 μg / mouse. Mice were vaccinated two to three times at one-week intervals, and the efficacy of the peptide vaccine was evaluated by measuring tumor volume and / or overall survival.

[0152] result Vaccination with murine IDO peptide demonstrated antitumor protective effects in the TC-1 tumor model, providing superior protection to vaccination with the tumor-specific peptide antigen, E7 peptide (see Figure 10).

[0153] [Example 8] Testing IDO peptide vaccines in combination with tumor antigen vaccines in a mouse model C57BL / 6 mice bearing TC-1 tumors were treated with the TC-1-specific E7-peptide (RAHYNIVTF, SEQ ID NO: 35), or IDO-Pep1 (MTYENMDIL, SEQ ID NO: 33), or a combination of both E7 and IDO-Pep1. Vaccine efficacy was assessed by overall survival. The method was identical to that in Example 7, except for the inclusion of peptide combinations.

[0154] result Mice vaccinated with E7 peptide have therapeutic efficacy in the TC-1 model as expected (and shown in Figure 10). However, when administered together with IDO-Pep1, E7 peptide provides superior protection than E7 alone or IDO-peptide alone. See Figure 11.

[0155] [Example 9] Testing an IDO peptide vaccine in a mouse model in combination with additional checkpoint inhibitors C57BL / 6 mice bearing TC-1 tumors were treated with IDO-Pep1 (MTYENMDIL, SEQ ID NO: 33) alone, 1-methyltryptophan (1-MT) alone, or a combination of both IDO-Pep1 and 1-MT. Vaccine efficacy was assessed by overall survival.

[0156] method Four to six week old C57BL / 6 mice were injected with TC-1 cells at 70,000 cells / mouse. Tumors were approximately 0.075 cm. 3 Treatment within each group began when tumors reached an average size (approximately 10 days after tumor inoculation). Mice were vaccinated using the IDO epitope, IDO-Pep1. Mice were subcutaneously vaccinated with IDO-Pep1 at a dose of 100 μg / mouse, combining a pan-HLADR-binding epitope (PADRE, aK-Cha-VAAWTLKAAa, 20 μg / mouse) and QuilA (10 μg / mouse). Mice were vaccinated two to three times at one-week intervals, and the efficacy of the peptide vaccine was assessed by measuring tumor volume and / or survival rate. One group of mice was treated with 1-methyltryptophan (1-MT) (2 mg / ml) dissolved in drinking water for the study period, and an additional group of mice was treated with both IDO-Pep1 and 1-MT.

[0157] result When TC-1-bearing mice were treated with oral a-MT, modest therapeutic efficacy was achieved. Co-administration of IDO Pep-1 to these mice enhanced efficacy, as demonstrated by prolonged survival. See Figure 12.

[0158] [Example 10] Alternative mouse model testing of IDO peptide vaccines BALB / c mice were prophylactically vaccinated with the murine IDO peptide IDO-EP2 (LPTLSTDGL, SEQ ID NO: 34), and challenged with CT26 tumors 7 days later. The efficacy of the peptide vaccine was assessed by tumor burden. Because the sequence of murine IDO1 differs from that of human IDO1, the murine IDO peptide sequence (IDO-EP2) was chosen to provide a murine analog of the human IDO peptide tested in the above examples.

[0159] method Six- to ten-week-old BALB / c mice were immunized subcutaneously at the base of the tail with 100 μL of Montanide adjuvant (Montanide ISA 51 VG (Seppic)) containing 100 μg of peptide IDO-EP2 + 30 μg of CpG, prepared as a 1:1 emulsion. Montanide alone (vehicle) was used as a control. Some mice were left untreated as additional controls. Immunizations were performed 7 days before tumor challenge. For CT26 tumor cell implantation, each mouse received 1 × 10 5 Each mouse received two subcutaneous injections (one in each flank) of 100 μL of CT26 in serum-free DMEM. Caliper measurements of tumor length and width were recorded every 3–4 days starting on day 6. Tumor volume was calculated using the formula 0.52(L × W) 2 ) was used to calculate the total tumor volume. 3 Mice were euthanized when they reached a defined endpoint of 10 days.

[0160] result Prophylactic vaccination with murine IDO peptide demonstrated anti-tumor protective effects in the CT26 tumor model. See Figure 13.

[0161] [Example 11] Clinical trial of PD-L1 / IDO peptide vaccine combined with anti-PD1 antibody The primary objective is to evaluate the tolerability and safety of a peptide vaccine containing the peptides IDO long (DTLLKALLEIASCLEKALQVF, SEQ ID NO: 3) and PD-L1 long 1 (FMTYWHLLNAFTVTVPKDL, SEQ ID NO: 32) adjuvanted with montanide ISA 51 when administered in combination with the immune checkpoint blocking antibody nivolumab (specific for human PD1) to patients with metastatic malignant melanoma (MM). The endpoint is adverse events (AEs) assessed by CTCAE 4.0.

[0162] The secondary objective is to evaluate immune responses before, during, and after treatment. Blood samples will be collected before treatment and then every 3 months for up to 5 years. Antigen-specific immune reactivity will be tested using a panel of relevant immunological assays, including ELISPOT, proliferation assays, cytotoxicity assays, intracellular staining (ICS), and multimer staining of PD-L1- and IDO-specific CD8 T cells. Efforts will be made to obtain biopsy samples from available tumor lesions or involved lymph nodes before the first vaccination and after the sixth vaccination. The objective is to evaluate each patient's tumor immune microenvironment. Immunohistochemistry, quantification of gene expression in different immune genes, and whole-exome sequencing to evaluate the initial mutation status will be performed.

[0163] The third objective is to evaluate the clinical efficacy of the treatment. The endpoints are objective response (OR), progression-free survival (PFS), and overall survival (OS).

[0164] The study is designed as an open-label Phase I / II trial. Six patients with MM will be treated in Phase I. Before Phase II can begin, all six patients must receive the first four doses of treatment without any grade 3-4 adverse events, excluding those expected for nivolumab. If three or more patients experience grade 3-4 AEs related to vaccination in Phase I, the study will be stopped. An additional 26 patients will be enrolled in Phase II.

[0165] Patients enrolled in the trial will be treated with nivolumab according to the standard regimen, currently including an outpatient IV infusion of 3 mg / kg every 2 weeks as long as clinical response exists. The PD-L1 / IDO vaccine will be administered every 2 weeks for the first 6 vaccines, then every 4 weeks for up to 47 weeks from the start of nivolumab. A total of 15 vaccines will be administered. At the end of vaccination, patients who are not excluded from the protocol due to progression will continue treatment with nivolumab according to standard guidelines.

[0166] Full-length protein sequence TIFF0007785817000009.tif116140TIFF0007785817000010.tif252143TIFF0007785817000011.tif18137 SEQUENCE LISTING <110> IO Biotech ApS <120> Combination therapy against cancer <130> PA23-674 <150> GB 1603805.1 <151> 2016-03-04 <150> GB 1610018.2 <151> 2016-06-08 <160> 35 <170> PatentIn version 3.5 <210> 1 <211> 403 <212> PRT <213> Homo sapiens <400> 1 Met Ala His Ala Met Glu Asn Ser Trp Thr Ile Ser Lys Glu Tyr His 1 5 10 15 Ile Asp Glu Glu Val Gly Phe Ala Leu Pro Asn Pro Gln Glu Asn Leu 20 25 30 Pro Asp Phe Tyr Asn Asp Trp Met Phe Ile Ala Lys His Leu Pro Asp 35 40 45 Leu Ile Glu Ser Gly Gln Leu Arg Glu Arg Val Glu Lys Leu Asn Met 50 55 60 Leu Ser Ile Asp His Leu Thr Asp His Lys Ser Gln Arg Leu Ala Arg 65 70 75 80 Leu Val Leu Gly Cys Ile Thr Met Ala Tyr Val Trp Gly Lys Gly His 85 90 95 Gly Asp Val Arg Lys Val Leu Pro Arg Asn Ile Ala Val Pro Tyr Cys 100 105 110 Gln Leu Ser Lys Lys Leu Glu Leu Pro Pro Ile Leu Val Tyr Ala Asp 115 120 125 Cys Val Leu Ala Asn Trp Lys Lys Lys Asp Pro Asn Lys Pro Leu Thr 130 135 140 Tyr Glu Asn Met Asp Val Leu Phe Ser Phe Arg Asp Gly Asp Cys Ser 145 150 155 160 Lys Gly Phe Phe Leu Val Ser Leu Leu Val Glu Ile Ala Ala Ala Ser 165 170 175 Ala Ile Lys Val Ile Pro Thr Val Phe Lys Ala Met Gln Met Gln Glu 180 185 190 Arg Asp Thr Leu Leu Lys Ala Leu Leu Glu Ile Ala Ser Cys Leu Glu 195 200 205 Lys Ala Leu Gln Val Phe His Gln Ile His Asp His Val Asn Pro Lys 210 215 220 Ala Phe Phe Ser Val Leu Arg Ile Tyr Leu Ser Gly Trp Lys Gly Asn 225 230 235 240 Pro Gln Leu Ser Asp Gly Leu Val Tyr Glu Gly Phe Trp Glu Asp Pro 245 250 255 Lys Glu Phe Ala Gly Gly Ser Ala Gly Gln Ser Ser Val Phe Gln Cys 260 265 270 Phe Asp Val Leu Leu Gly Ile Gln Gln Thr Ala Gly Gly Gly His Ala 275 280 285 Ala Gln Phe Leu Gln Asp Met Arg Arg Tyr Met Pro Pro Ala His Arg 290 295 300 Asn Phe Leu Cys Ser Leu Glu Ser Asn Pro Ser Val Arg Glu Phe Val 305 310 315 320 Leu Ser Lys Gly Asp Ala Gly Leu Arg Glu Ala Tyr Asp Ala Cys Val 325 330 335 Lys Ala Leu Val Ser Leu Arg Ser Tyr His Leu Gln Ile Val Thr Lys 340 345 350 Tyr Ile Leu Ile Pro Ala Ser Gln Gln Pro Lys Glu Asn Lys Thr Ser 355 360 365 Glu Asp Pro Ser Lys Leu Glu Ala Lys Gly Thr Gly Gly Thr Asp Leu 370 375 380 Met Asn Phe Leu Lys Thr Val Arg Ser Thr Thr Glu Lys Ser Leu Leu 385 390 395 400 Lys Glu Gly <210> 2 <211> 9 <212> PRT <213> Homo sapiens <400> 2 Ala Leu Leu Glu Ile Ala Ser Cys Leu 1 5 <210> 3 <211> 21 <212> PRT <213> Homo sapiens <400> 3 Asp Thr Leu Leu Lys Ala Leu Leu Glu Ile Ala Ser Cys Leu Glu Lys 1 5 10 15 Ala Leu Gln Val Phe 20 <210> 4 <211> 9 <212> PRT <213> Homo sapiens <400> 4 Gln Leu Arg Glu Arg Val Glu Lys Leu 1 5 <210> 5 <211> 9 <212> PRT <213> Homo sapiens <400> 5 Phe Leu Val Ser Leu Leu Val Glu Ile 1 5 <210> 6 <211> 9 <212> PRT <213> Homo sapiens <400> 6 Thr Leu Leu Lys Ala Leu Leu Glu Ile 1 5 <210> 7 <211> 9 <212> PRT <213> Homo sapiens <400> 7 Phe Ile Ala Lys His Leu Pro Asp Leu 1 5 <210> 8 <211> 9 <212> PRT <213> Homo sapiens <400> 8 Val Leu Ser Lys Gly Asp Ala Gly Leu 1 5 <210> 9 <211> 9 <212> PRT <213> Homo sapiens <400> 9 Asp Leu Met Asn Phe Leu Lys Thr Val 1 5 <210> 10 <211> 9 <212> PRT <213> Homo sapiens <400> 10 Val Leu Leu Gly Ile Gln Gln Thr Ala 1 5 <210> 11 <211> 9 <212> PRT <213> Homo sapiens <400> 11 Lys Val Leu Pro Arg Asn Ile Ala Val 1 5 <210> 12 <211> 10 <212> PRT <213> Homo sapiens <400> 12 Lys Leu Donkey Met Leu Ser Ile Asp His Leu 1 5 10 <210> 13 <211> 10 <212> PRT <213> Homo sapiens <400> 13 Ser Leu Arg Ser Tyr His Leu Gln Ile Val 1 5 10 <210> 14 <211> 290 <212> PRT <213> Homo sapiens <400> 14 Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40 45 Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55 60 Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser 65 70 75 80 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn 85 90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val 115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val 130 135 140 Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175 Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185 190 Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195 200 205 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210 215 220 Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His 225 230 235 240 Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr 245 250 255 Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys 260 265 270 Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu 275 280 285 Glu Thr 290 <210> 15 <211> 9 <212> PRT <213> Homo sapiens <400> 15 Leu Leu Asn Ala Phe Thr Val Thr Val 1 5 <210> 16 <211> 9 <212> PRT <213> Homo sapiens <400> 16 Ile Leu Leu Cys Leu Gly Val Ala Leu 1 5 <210> 17 <211> 9 <212> PRT <213> Homo sapiens <400> 17 Ile Leu Gly Ala Ile Leu Leu Cys Leu 1 5 <210> 18 <211> 9 <212> PRT <213> Homo sapiens <400> 18 Ala Leu Gln Ile Thr Asp Val Lys Leu 1 5 <210> 19 <211> 9 <212> PRT <213> Homo sapiens <400> 19 Lys Leu Phe Asn Val Thr Ser Thr Leu 1 5 <210> 20 <211> 9 <212> PRT <213> Homo sapiens <400> 20 Arg Leu Leu Lys Asp Gln Leu Ser Leu 1 5 <210> 21 <211> 9 <212> PRT <213> Homo sapiens <400> 21 Gln Leu Ser Leu Gly Asn Ala Ala Leu 1 5 <210> 22 <211> 9 <212> PRT <213> Homo sapiens <400> 22 Lys Ile Asn Gln Arg Ile Leu Val Val 1 5 <210> 23 <211> 9 <212> PRT <213> Homo sapiens <400> 23 His Leu Val Ile Leu Gly Ala Ile Leu 1 5 <210> 24 <211> 9 <212> PRT <213> Homo sapiens <400> 24 Arg Ile Asn Thr Thr Thr Asn Glu Ile 1 5 <210> 25 <211> 9 <212> PRT <213> Homo sapiens <400> 25 Cys Leu Gly Val Ala Leu Thr Phe Ile 1 5 <210> 26 <211> 9 <212> PRT <213> Homo sapiens <400> 26 Gln Leu Asp Leu Ala Ala Leu Ile Val 1 5 <210> 27 <211> 9 <212> PRT <213> Homo sapiens <400> 27 Ser Leu Gly Asn Ala Ala Leu Gln Ile 1 5 <210> 28 <211> 10 <212> PRT <213> Homo sapiens <400> 28 Val Ile Leu Gly Ala Ile Leu Leu Cys Leu 1 5 10 <210> 29 <211> 10 <212> PRT <213> Homo sapiens <400> 29 His Thr Ala Glu Leu Val Ile Pro Glu Leu 1 5 10 <210> 30 <211> 10 <212> PRT <213> Homo sapiens <400> 30 Phe Ile Phe Met Thr Tyr Trp His Leu Leu 1 5 10 <210> 31 <211> 10 <212> PRT <213> Homo sapiens <400> 31 Val Ile Trp Thr Ser Ser Asp His Gln Val 1 5 10 <210> 32 <211> 19 <212> PRT <213> Homo sapiens <400> 32 Phe Met Thr Tyr Trp His Leu Leu Asn Ala Phe Thr Val Thr Val Pro 1 5 10 15 Lys Asp Leu <210> 33 <211> 9 <212> PRT <213> Mus musculus <400> 33 Met Thr Tyr Glu Asn Met Asp Ile Leu 1 5 <210> 34 <211> 9 <212> PRT <213> Homo sapiens <400> 34 Leu Pro Thr Leu Ser Thr Asp Gly Leu 1 5 <210> 35 <211> 9 <212> PRT <213> Artificial <220> <223> Fragment of the E7 oncoprotein of HPV <400> 35 Arg Ala His Tyr Asn Ile Val Thr Phe 1 5

Claims

1. 1. A pharmaceutical composition for use in a method of preventing or treating cancer in a subject, comprising: (i) a peptide of the sequence FMTYWHLLNAFTVTVPKDL (SEQ ID NO: 32), and (ii) Anti-PD1 antibody that binds to PD1 and administering to said subject a pharmaceutical composition for said use comprising:

2. 2. The pharmaceutical composition for use according to claim 1, wherein the anti-PD1 antibody is pembrolizumab or nivolumab.

3. A pharmaceutical composition for use according to claim 1 or 2, comprising an adjuvant or carrier, optionally wherein the adjuvant is selected from a bacterial DNA adjuvant, an oil / surfactant adjuvant, a viral dsRNA adjuvant, an imidazoquinoline and GM-CSF.

4. 4. The pharmaceutical composition for use according to claim 3, wherein the adjuvant is a Montanide ISA adjuvant, optionally selected from Montanide ISA 51 or Montanide ISA 720.

5. 5. The pharmaceutical composition for use according to any one of claims 1 to 4, wherein the method for preventing or treating cancer further comprises administering to the subject an additional immunotherapeutic composition comprising a peptide of the sequence DTLLKALLEIASCLEKALQVF (SEQ ID NO: 3).

6. An immunotherapeutic composition for use in a method for preventing or treating cancer in a subject, comprising: comprising a peptide of the sequence FMTYWHLLNAFTVTVPKDL (SEQ ID NO: 32), An immunotherapeutic composition for said use, administered to said subject in combination with an immunomodulatory agent comprising an anti-PD1 antibody that binds to PD1.

7. An immunotherapy composition for use as described in claim 6, wherein the anti-PD1 antibody is pembrolizumab or nivolumab.

8. An immunotherapy composition for use according to claim 6 or 7, comprising an adjuvant or carrier, optionally wherein the adjuvant is selected from a bacterial DNA adjuvant, an oil / surfactant adjuvant, a viral dsRNA adjuvant, an imidazoquinoline and GM-CSF.

9. An immunotherapy composition for use as described in claim 8, wherein the adjuvant is a Montanide ISA adjuvant, optionally selected from Montanide ISA 51 or Montanide ISA 720.

10. An immunotherapeutic composition for use according to any one of claims 6 to 9, wherein the method for preventing or treating cancer further comprises administering to the subject a further immunotherapeutic composition comprising a peptide of the sequence DTLLKALLEIASCLEKALQVF (sequence number 3).

11. An immunomodulator for use in a method for preventing or treating cancer in a subject, comprising: an anti-PD1 antibody that binds to PD1, An immunomodulatory agent for said use, administered to said subject in combination with an immunotherapeutic composition comprising a peptide of the sequence FMTYWHLLNAFTVTVPKDL (SEQ ID NO: 32).

12. An immunomodulator for use as described in claim 11, wherein the anti-PD1 antibody is pembrolizumab or nivolumab.

13. An immunomodulator for use according to claim 11 or 12, wherein the immunotherapy composition comprises an adjuvant or carrier, and optionally the adjuvant is selected from a bacterial DNA adjuvant, an oil / surfactant adjuvant, a viral dsRNA adjuvant, an imidazoquinoline and GM-CSF.

14. An immunomodulator for use according to claim 13, wherein the adjuvant is a Montanide ISA adjuvant, optionally selected from Montanide ISA 51 or Montanide ISA 720.

15. An immunomodulator for use according to any one of claims 11 to 14, wherein the method for preventing or treating cancer further comprises administering to the subject a further immunotherapeutic composition comprising a peptide of the sequence DTLLKALLEIASCLEKALQVF (SEQ ID NO: 3).

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