KR20130126671A - Hla-binding peptides derived from prostate-associated antigenic molecules and methods of use thereof - Google Patents

Abstract

Methods and compositions for immunotherapy treatment of prostate cancer are disclosed. More specifically, a method of treating prostate cancer patients is disclosed in which a composition containing an HLA binding peptide derived from a prostate related antigen molecule, whether or not an immunoadjuvant is added.

Description

HLA-binding peptide derived from prostate-associated antigen molecule and method of using the same {HLA-BINDING PEPTIDES DERIVED FROM PROSTATE-ASSOCIATED ANTIGENIC MOLECULES AND METHODS OF USE THEREOF}

Methods and compositions for immunotherapy treatment of prostate cancer are disclosed. More specifically, methods of treating prostate cancer patients comprising administering compositions containing HLA binding peptides derived from prostate related antigen molecules, whether or not an immunoadjuvant is added, are disclosed.

Prostate cancer is one of the most common malignant diseases in men, with 346,000 cases in Europe reported in 2006, killing 87,000 people. This is the most commonly diagnosed cancer in the United States, the second leading cause of cancer-related deaths in men. Because of the increased sensitivity of prostate-specific antigen (PSA) monitoring assays, prostate cancer is found in earlier clinical and local stages where radical treatments such as surgery and radiation can be performed. Nevertheless, these patients have a 10 to 60% chance of increasing PSA (known as biochemical relapse) within 10 years without any symptoms. Biochemical recurrence is most often indicative of the onset of metastasis, although locally recurring cancer is hidden or not yet discovered.

Treatment options in these cases include external radiation therapy and androgen deprivation (also called hormonal ablation). However, no treatment approach has proven ineffective, especially in the long-term survival of patients. Moreover, both therapies are associated with a number of side effects (increased risk of cardiovascular disease, osteoporosis, weight gain, brain perception, development of urethral stenosis, loss of libido and infertility, reduced risk of skeletal calcium salts associated with osteoporosis, and a significant increase in the risk of pathological fractures). Restricted Androgen blockade also causes early androgen-neutral neoplasia to occur, which may further accelerate long-term tumor progression. Moreover, the timing of proper initiation of androgen deprivation therapy is controversial, especially for biochemical recurrences characterized by low PSA levels or long-term average doubling times (DT). In view of these risks, androgen deprivation therapy and external radiation therapy are of questionable therapeutic value in patients with early biochemical recurrence.

Tumor-associated antigens (TAAs) have been identified as potential cancer immunotherapy agents. Many TAAs have been identified that are specific to a variety of different tumor and tissue types, including those related to the prostate. The identification of T-cells specific for TAA in the prostate tumor tissue, tumor drainage lymph nodes, and peripheral circulation of cancer patients, as well as an increase in specific T-cell responses after immunotherapy, can be useful for treating prostate cancer.

Various systems for in vivo antigen presentation of prostate specific TAAs have been tested in clinical trials, including: (1) vaccine therapy using autologous or allogeneic tumor cells; (2) autologous tumor cells engineered to express granulocyte-macrophage colony stimulating factor; (3) dissociation with HLA I and II binding peptides ( ex in vivo ) sensitized dendritic cells; (4) cancer-mRNA transfected dendritic cells and (5) recombinant vaccine virus expressing TAA. However, most of these studies have been conducted in patients with androgen-resistant prostate carcinoma. Little information is available on vaccine therapies for androgen-sensitive patients who have undergone biochemical relapses before androgen deprivation therapy.

Therefore, new treatment options are needed for patients with prostate cancer, especially those with early biochemical recurrence.

The present invention relates to a composition for use in immunotherapy and its use. In particular, the present invention relates to the immunotherapy of androgen-sensitive prostate cancer in cancer, more specifically prostate cancer, and even more specifically in early biochemical relapse patients who have never received androgen deprivation therapy. The invention also relates to compositions of HLA binding peptides of HLA class I and class II, wherein the HLA binding peptides are prostate specific antigens, prostate stem cell antigens, prostate specific membrane antigens, survivin, prosteins. And prostate-associated antigen molecules such as transient receptor translocation-p8 (TRP-p8).

In one aspect, the invention relates to a composition containing at least one HLA binding peptide, wherein the HLA binding peptide comprises an epitope derived from prostate-associated antigen molecule.

In another aspect, the present invention relates to a composition containing at least one HLA binding peptide, wherein the HLA binding peptide is a prostate specific antigen, a prostate stem cell antigen, a prostate specific membrane antigen, survivin, prostein epitopes derived from prostate-associated antigen molecules selected from the group consisting of prostein and transient receptor translocation-p8.

Particularly preferred aspects of the invention relate to compositions comprising at least two HLA binding peptides, wherein (a) at least one of said at least two HLA binding peptides comprises an epitope according to SEQ ID NO: 23 Or a fusion protein of SEQ ID NO: 23 comprising 80 N-terminal amino acids of a HLA-DR antigen related constant chain, and (b) at least one of said at least two peptides is SEQ ID NO: 1 to SEQ ID NO: 11 , A peptide comprising an epitope selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 22 and SEQ ID NO: 24 to SEQ ID NO: 42.

Preferably the composition according to the invention comprises at least two peptides consisting of the amino acid sequence according to (b).

The composition according to the invention comprises at least two peptides, preferably at least four peptides and more preferably ten peptides, consisting of an amino acid sequence according to SEQ ID NO: 23, SEQ ID NO: 1, SEQ ID NO: 2, A peptide selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14, and consisting of SEQ ID NO: 15 to SEQ ID NO: 22 and SEQ ID NO: 24 to SEQ ID NO: 42 It is preferred to include at least one peptide selected from the group.

Also preferred are compositions of the invention wherein additional peptides are selected according to the HLA set of subjects to be treated.

The composition according to the invention comprises at least four peptides consisting of the amino acid sequence according to SEQ ID NO: 23, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13 and at least one peptide selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 22 and SEQ ID NO: 24 to SEQ ID NO: 42.

Also preferred are compositions according to the invention wherein at least one of the peptides is a class II peptide.

Another aspect relates to a composition according to the invention further comprising an adjuvant, or a mixture of two or three immunoadjuvant such as, for example, GM-CSF and imiquimod.

In the compositions according to the invention it is preferred that the adjuvant comprises a Toll like receptor agonist, for example a Toll like receptor-7 agonist.

It is also preferred if the composition according to the invention contains at least one antigen presenting cell, for example dendritic cells such as autologous dendritic cells sensitized or mounted with peptides.

Another aspect relates to a composition according to the invention for use in the treatment of prostate cancer.

In the composition according to the invention it is preferred that the prostate cancer is androgen-sensitive and the patient has not been subjected to androgen deprivation therapy.

In the composition according to the invention it is more preferred that the prostate cancer is androgen-insensitive.

Yet another aspect relates to a method of treating prostate cancer comprising administering to a patient an effective dose of a composition according to the invention.

In the method according to the invention it is preferred that said prostate cancer is androgen-sensitive and the patient has not been subjected to androgen deprivation therapy.

In the method according to the invention it is more preferred that the prostate cancer is androgen-insensitive.

In a more specific aspect, the composition of the present invention is SEQ ID NO: 24, SEQ ID NO: 7, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 9, sequence An epitope according to SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15 to SEQ ID NO: 23, or SEQ ID NO: 25 to SEQ ID NO: 40.

In a more specific aspect, the composition comprises at least two HLA binding peptides comprising epitopes derived from at least prostate related antigen molecules, wherein at least one of the at least two HLA binding peptides is HLA class I And at least one of the at least two HLA peptides is an HLA class II peptide.

Another aspect of the invention relates to a composition comprising at least two HLA binding peptides, wherein at least one of the HLA binding peptides is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and HLA class I peptides comprising an epitope selected from the group consisting of SEQ ID NO: 11 and at least one of the HLA binding peptides HLA class II peptides comprising epitopes selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14.

Another aspect of the invention relates to a composition comprising at least two HLA binding peptides, wherein (a) at least one of said at least two HLA binding peptides is SEQ ID NO: 24, SEQ ID NO: 15 to SEQ ID NO: 23 and a peptide comprising an epitope selected from the group consisting of SEQ ID NO: 25 to SEQ ID NO: 37, (b) at least one of said at least two peptides is SEQ ID NO: 7, SEQ ID NO: 1 to SEQ ID NO: 6, sequence It is a peptide comprising an epitope selected from the group consisting of SEQ ID NO: 8 to SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 38 to SEQ ID NO: 42.

Another aspect of the invention relates to a composition comprising at least two HLA binding peptides, wherein at least one of said HLA binding peptides is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, sequence A HLA class I peptide consisting essentially of an epitope selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11 and at least one of the HLA binding peptides The species is an HLA class II peptide consisting essentially of an epitope selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14.

Another aspect of the invention relates to a composition comprising at least two HLA binding peptides, wherein at least one of said HLA binding peptides is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, sequence A HLA class I peptide consisting of an epitope selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, and at least one of the HLA binding peptides is HLA class II peptide consisting of epitopes selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14.

Another aspect of the invention relates to a composition comprising at least two HLA binding peptides, wherein at least one of said HLA binding peptides binds to an allele other than HLA-A * 02. Class I peptides.

Another aspect of the invention relates to a composition comprising at least two HLA binding peptides, wherein at least one of the HLA binding peptides is HLA-A * 24, HLA-A * 11, HLA-B * 41, HLA class I peptides that bind to alleles selected from the group of HLA-B * 51 or HLA-C.

Another aspect of the invention relates to a composition comprising at least six HLA binding peptides, wherein (a) at least one HLA binding peptide comprises a prostate specific antigen derived epitope; (b) at least one HLA binding peptide comprises a protoplast stem cell antigen derived epitope; (c) at least one HLA binding peptide comprises an epitope derived from a prostate specific membrane antigen; (d) at least one HLA binding peptide comprises a survivin derived epitope; (e) at least one HLA binding peptide comprises a prostein derived epitope; (f) At least one HLA binding peptide comprises a transient receptor translocation-p8 derived epitope.

Another aspect of the invention relates to the composition described above, wherein said composition comprises at least two peptides consisting of the amino acid sequence according to (b).

Another aspect of the invention relates to the composition described above, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 , SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, sequence SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 , HLA binding peptides according to SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, and SEQ ID NO: 42.

Another aspect of the invention relates to the composition described above, wherein the composition is in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14 And a peptide consisting of the amino acid sequence according to the invention and optionally comprising at least one peptide selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 42.

Another aspect of the invention relates to a composition described above, wherein the composition is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14 At least two peptides, preferably at least four peptides, more preferably ten peptides, each of which is composed of an amino acid sequence according to Include.

Another aspect of the invention relates to the composition described above, wherein the composition is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14 At least two peptides, preferably at least four peptides, more preferably ten peptides, each of which is composed of an amino acid sequence according to And additional peptides are selected according to the HLA set of subjects to be treated.

Another aspect of the invention relates to the composition described above, wherein the composition is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14 And at least four peptides consisting of the amino acid sequence according to SEQ ID NO: 15 to SEQ ID NO: 42.

Another aspect of the invention relates to any one of the compositions described above, wherein at least one of the peptides is a class II peptide.

Another aspect of the invention relates to any one of the previously described compositions further comprising an adjuvant or a mixture of two or three adjuvant such as, for example, GM-CSF and imiquimod. The adjuvant can be any known adjuvant and includes toll like receptor-7 agonist imiquimod, mucin-1-mRNA / protamine complex and cytokine granulocyte macrophage colony stimulating factor (GM-CSF).

A further aspect of the present invention then relates to a method of treating prostate cancer comprising administering to a patient any of the compositions disclosed herein, with or without the addition of an adjuvant. The prostate cancer may be androgen-sensitive and the patient may not have been subjected to androgen deprivation therapy.

Still another aspect of the present invention relates to a method of treating androgen-sensitive or androgen-insensitive prostate cancer in a patient, the method comprising any of the compositions described above to the patient, whether or not an adjuvant is added. Administering.

FIG. 1 shows the various epitopes included in the HLA binding peptides in the HLA binding peptide cocktail used in Example 1, comprising 13 epitopes derived from prostate-associated antigen molecules. The epitope according to SEQ ID NO: 1 to SEQ ID NO: 12 are HLA class I epitopes, ie HLA-A * 201 restriction epitopes. An epitope according to SEQ ID NO: 13 and SEQ ID NO: 14 is an HLA class II epitope.
2 shows the DT statistics for the study group as a whole. DT is expressed in months. "n" indicates the number of patients whose statistics are included in each category. "%" Represents the percentage of patients in each category. Patient 5's doubling time was not included in the geometric mean or range at the end of this study due to the patient's negative doubling time.
3 shows the DT changes before, during and after treatment of patients with the HLA binding cocktail used in Example 1. FIG. Positive values indicate PSA doubling time, indicating that the patient's PSA levels are increasing. Negative values indicate a PSA half-life, indicating that the patient's PSA level is decreasing.
4 shows the clinical response of treatment divided by type of adjuvant. If there is no clinical response, it is indicated with a "-" sign. If PSA accelerates after the PSA decrease or stabilization in the middle, it is indicated by "+/-" sign. In the case where there is a PSA decrease and a PSA DT increase after the PSA increase in the middle, it is indicated by a "-/ +" sign. The increase in PSA DT is indicated by the "+" sign.
FIG. 5 shows PSA levels in patients receiving peptides in montanide (but not adjuvant or thermotherapy). FIG.
6 shows PSA levels in patients receiving peptides in montanides with mucin-1-mRNA / protamine complex.
7 shows PSA levels in patients receiving peptides in Montanide using thermotherapy.
8 shows PSA levels in patients receiving peptides in montanide using GM-CSF.
9 shows PSA levels in patients receiving peptides in montanide with imiquimod.
10 shows the percentage change in PSA value from baseline at day 84. FIG.
11 shows the percentage change in PSA values during the vaccination period.
12 shows the number of patients with T-cells responding to each peptide after vaccination.
FIG. 13 shows the specificity for PSMA 459-473 epitopes and survivin 97-111 epitopes of CD4 + T-cells derived from PBMCs of Patient No. 15 and Patient No. 26. FIG. PSMA: PSMA 459-473 epitope. Surv: survivin 97-111 epitope.
14 shows peptide reactivity of clone Pro26_1 C. PMA / ionomycin = antigen-independent nonspecific activation; Survivin (II): stimulation with survivin 97-111 epitope; PSMA (II): Stimulated with PSMA 459-473 epitope. All reactive cells are CD4 positive.
15 shows peptide reactivity of clone Pro15_10_O. PMA / ionomycin = antigen independent nonspecific activation; Survivin (II): stimulation with survivin 97-111 epitope; PSMA (II): Stimulated with PSMA 459-473 epitope. All reactive cells are CD4 positive.
Figure 16 shows the characterization of the response of CD4 + survivin specific T-cell clone Pro15_ "D" to dendritic cells developed with full-length survivin or survivin 97-111 epitopes. It is observed by intracellular cytokine staining that immature dendritic cells cultured with recombinant survivin protein or survivin 97-111 epitope are recognized by survivin specific T-cells in vaccinated patients. These results suggest that survivin is naturally processed by proteinases in dendritic cells and that the survivin 97-111 epitope is not destroyed by processing mechanisms. In addition, these CD4 + T-cells are multifunctional because they secrete the cytokines IFN-γ, TNF-α and IL-2, have a surface expression of CD40 ligand (CD154), and a decrease in granulation caused by surface expression of CD107a occurs. . The indicated T-cell response is antigen specific because T-cells are not activated by dendritic cells incubated with the irrelevant protein RAP80, or HIV-001 peptide.
17 shows the response of CD4 + survivin specific T-cell clone Pro26_10_C to dendritic cells developed with full length survivin, survivin 97-111 epitope, or PSMA 459-473 epitope. Intracellular cytokine staining is observed for immature dendritic cells incubated with recombinant Servinbin protein, Servinbin 97-111 epitope and PSMA 459-473 epitope. While responding to both survivin and PSMA epitopes, the T-cell response was stronger in stimulating responses using the survivin epitope. These CD4 + T-cells are multifunctional because they secrete the cytokines IFN-γ, TNF-α, and IL-2, have a surface expression of CD40 ligand (CD154), and a decrease in granulation caused by surface expression of CD107a occurs. The indicated T-cell response is antigen specific because T-cells are not activated by dendritic cells cultured with the irrelevant protein RAP80.
18 shows the response of CD4 + survivin specific T-cell clone Pro26_10_C to dendritic cells developed with full length PSMA protein, PSMA protein treated with proteinase K, or survivin protein treated with proteinase K. .
19 shows the response of CD4 + survivin specific T-cell clone Pro26_10_C to immobilized dendritic cells cultured with full length survivin, survivin 97-111 epitope, full size PSMA, or PSMA 459-473 epitope. It is observed by intracellular cytokine staining that fixed dendritic cells cultured with the survivin 97-111 epitope (but not full-length survivin) are recognized by antigen-specific T-cells of vaccinated patients. This fact confirms that full-length survivin must be cleaved by antigen presenting cells to become an antigen.
FIG. 20 shows the response of CD4 + PSMA specific T-cell clone Pro26_10_D to dendritic cells developed with full length survivin, survivin 97-111 epitope, full length PSMA, or PSMA 459-473 epitope. It is observed by intracellular cytokine staining that immature dendritic cells cultured with PSMA 459-473 epitopes are recognized by antigen specific T-cells of vaccinated patients. These CD4 + T-cells are multifunctional because they secrete the cytokines IFN-γ, TNF-α, and IL-2, have a surface expression of CD40 ligand (CD154), and a decrease in granulation caused by surface expression of CD107a occurs. The indicated T-cell response is antigen specific because T-cells are not activated by dendritic cells cultured with survivin or survivin 97-111 epitopes.
21 shows that several tumor cell lines expressing different HLA-DR alleles are recognized by patient derived PBMCs (observed for Pro26 and Pro15 in patients). Patients produce polyclonal T-cell responses after vaccination with Survivin 97-111. Survivin 97-111 appears to bind freely to several HLA Class II alleles such as: DR1 (see also Wang et al.); DQ5 (not tested by Wang et al.); DR11 (see also King et al.); Or DRB3 (in comparison with Wang et al., 2008, Table 1). Functional presentation of survivin 97-111 is possible in the context of several HLA class II molecules (TNF-α production). For HLA class I (HLA-A, B, C), in principle also three different loci express HLA class II, HLA-DQ, HLA-DP, which express functional class II molecules on the cell surface. And for HLA-DR. Class I molecules are composed of heavy chains (-A, -B, -C) and β-2-microglobulin that are constant in all three genes. Nevertheless, class II molecules are composed of two variable chains (α and β). So sophisticated genotyping is always complicated by class II. The figure shows the so-called serum types based on antibody binding. Thus, for example, "DQ3" is usually composed of different alleles of the HLA-DQ α and β chains that are found together and react with a particular antibody. The cells in the figure are as follows: AL = E418 EBV modified B-cell line (Human Immunology Volume 51, Issue 1, November 1996, Pages 1322); LAM = B lymphoma cell line (Oncogenomics 19 September 2002, Volume 21, Number 42, Pages 6549-6556); HO301 = EBV modified B cell line (The Journal of Immunology, 1998, 160: 3363-3373); BM15 = Dr11 + APC cell line (The Journal of Immunology, 2004, 173: 1876-1886); MGAR = homozygous B-LCL (Gene Therapy, 2004 11, 1408-1415); LG2-EBV = autologous B cell line (Cancer Immunity, Vol. 2, p. 9 (19 July 2002)); EMJ = B lymphoblastic cell line (ECACC NO: 8602103 IHW Number 9097: and Hum Immunol. 1980 Dec; 1 (4): 36-38).

The present invention relates to compositions and methods for use in the manipulation of immune responses against prostate cancer cells through the use of antigenic peptides specific for prostate tissue and / or prostate tumors.

To stimulate the immune response, an antigen must be present that the host immune system recognizes as foreign. In recent years, (1) they are specifically expressed in prostate tissue or prostate tumors; (2) Various antigens recognized by T-cells have been successfully identified. These antigens, when expressed in antigen presenting cells (APCs) as a complex of HLA molecules and peptides, can stimulate T-cells and induce antigen-specific T-cell responses against prostate cancer cells and ultimately lead to tumor cell lysis. For example, various epitopes of protein survivin have been recognized as prostate TAA. The discovery and character of prostate-specific TAA now increases the possibility of using the host's immune system to participate in tumor growth. Various mechanisms of using both humoral and cellular attack weapons of the immune system to treat cancer with immunotherapy are currently being studied.

Specific cellular immune response elements have the ability to specifically recognize and destroy tumor cells. Isolation of CD8 + cytotoxic T-cells (CTLs) from tumor infiltrating cell populations or peripheral blood shows that these cells play an important role in natural immune defense against cancer. CD8 + T-cells, which recognize class I molecules of major histocompatibility complex (MHC) containing peptides usually of 8 to 10 amino acid residues derived from defective ribosome products (DRIP) or proteins located in the cell substrate, are particularly involved in this reaction. Plays an important role (Schubert U, Ant LC, Gibbs J, Norbury CC, Yewdell JW, Bennink JR .; Rapid degradation of a large fraction of newly synthesized proteins by proteasomes; Nature 2000; 404 (6779): 770-774)) .

Antigens are presented through one of two major classes of major histocompatibility complex (MHC) molecules, MHC class I and MHC class II. In humans, MHC molecules are called human leukocyte antigen (HLA) molecules. There are two classes of MHC molecules: MHC class I molecules and larger peptides found in most cells with the nucleus presenting DRIP, a peptide primarily derived from endogenous, cellular substrate, or proteolytic cleavage of nuclear proteins. . However, exogenous peptides derived from endosomal compartments are also frequently found in MHC class I molecules. This non-traditional way of class I presentation is called cross-presentation in the literature. MHC class II molecules can be found predominantly in specialized antigen presenting cells (APCs) and present mainly and treated peptides of the exogenous protein to be processed which are taken up by APC during intracellular influx. In the case of class I, alternative methods are described prior to the antigen handler that allow endogenous peptides to be presented by MHC class II molecules (eg autophagy). Peptide and MHC class I molecular complexes are recognized by CD8 + cytotoxic T-lymphocytes with appropriate TCRs, and peptide and MHC class II molecular complexes are recognized by CD4 positive helper T-cells with appropriate TCRs.

HLA class I molecules are found in all the nucleated cells of the body and function to show cytoplasmic protein fragments to CD8 + cytotoxic T-cells (CTLs). The protein is cleaved from the proteasome, and the resulting peptide is released from the cell substrate and transported to the lumen of the endoplasmic reticulum and bound to HLA class I molecules. The complex between the antigen and the HLA class I molecule is then carried to the cell surface where the antigen can be presented to the CTL. The presentation of antigens to CTLs induces a chain reaction leading to the proliferation of antigen-specific CTLs that directly kill cells, allowing the antigen / HLA complexes to bind to their surface. This process occurs in all nucleated cells, allowing the immune system to accurately monitor whether each individual cell has a heterologous, modified or embryonic protein. Peptides normally presented by HLA class I molecules are derived from endogenous intracellular protein molecules, but some claim that exogenous antigens taken up by cells by macropinocytosis or phagocytosis may also be present. have. Thus antigen specific CTL responses can be induced by immunizing the host directly with a polypeptide comprising a HLA class I specific epitope.

Compared to constitutively expressed HLA class I molecules, almost all HLA class II molecules are found in specialized antigen presenting cells such as macrophages, dendritic cells, B-cells and the like. Professional APCs absorb extracellular proteins by professional APCs, which are digested in lysosomes and bound by HLA class II molecules before the molecules move to the plasma membrane. HLA class II binding peptides are presented in CD4 + T helper cells. T-helper cells do not directly cytotoxic or phagocytize, thus acting to induce or augment other immune system components to respond to or infect infected cells without directly killing infected or failed host cells. For example, activation of CD4 + T-cells can locally increase the level of interferon-γ (IFN-γ).

CD4-positive helper T-cells play an important role in modulating the effector function of anti-tumor T-cell responses, which makes identification of TA4-derived CD4-positive T-cell epitopes an important factor in the development of drugs that produce anti-tumor immune responses. Can be. It has been shown in mammalian models that local secretion of interferon-γ IFN-γ by T-helper cells can inhibit the proliferation of cancer through inhibition of angiogenesis, even without CTL ( see Qin, Z). and T. Blankenstein.CD4 + T-cell-mediated tumor rejection involves inhibition of angiogenesis that is dependent on IFN gamma receptor expression by non-hematopoietic cells.Immunity. 2000, 12: 677-686). In addition, T-helper cells that recognize TAA presented in HLA class II molecules can fight cancer progression by inducing antibody responses (Kennedy, RC, MH Shearer, AM Watts, and RK Bright. CD4 + T lymphocytes play a) critical role in antibody production and tumor immunity against simian virus 40 large tumor antigen.Cancer Res. 2003, 63: 1040-1045).

Compared to HLA Class I Binding TAAs, only HLA Class II Binding TAAs have been described so far (www.cancerimmunity.org, www.syfpeithi.de). Since constitutive expression of HLA class II molecules is usually confined to cells of the immune system, it was not considered possible to directly separate class II peptides from the original tumor. Recently, however, a number of MHC class II epitopes have been identified directly from tumors. Moreover, it has been surprisingly found that tumor cells express MHC class II molecules (EP 1642905, EP 1760088; Dengjel J, Nastke MD, Gouttefangeas C, Gitsioudis G, Schoor O, Altenberend F, Muller M, Kramer B, Missiou). A, Sauter M, Hennenlotter J, Wernet D, Stenzl A, Rammensee HG, Klingel K, Stevanovic S .; Unexpected abundance of HLA class II presented peptides in primary renal cell carcinomas; Clin Cancer Res. 2006; 12: 4163-4170) .

In order for a peptide to induce a cellular immune response, it must bind to an MHC molecule. This process depends on the specific polymorphism of the allele of the MHC molecule and the amino acid sequence of the peptide. MHC class I binding peptides are usually 8 to 10 amino acid residues in length and generally have two conserved residues (anchors) in the sequence that interact with the corresponding binding groove of the MHC molecule. Because of this, each MHC allele has a "binding motif" that determines which peptide can specifically bind to which binding groove (Rammensee HG, Bachmann J, Stevanovic S. MHC ligands and peptide motifs, Landes Bioscience, USA, 1997).

In MHC dependent immune responses, peptides must be able to bind to specific MHC molecules expressed by cancer cells as well as be recognized by T-cells with specific T-cell receptors (TCRs).

Tumor specific T-lymphocytes, ie antigens recognized by epitopes thereof, may be molecules derived from all protein classes such as enzymes, receptors, transcription factors and the like. Moreover, tumor associated antigens may be present only in tumor cells, for example as mutated gene products. Another important class of tumor-associated antigens is tissue-specific antigens, such as cancer testis (CT) antigens, which are expressed in other tumor types or healthy testis tissues.

Numerous TAAs have been successfully identified and characterized in recent years. Moreover, many research efforts have been made to identify additional tumor associated antigens. Several groups of tumor associated antigens, also known in the art as tumor specific antigens, are tissue specific. These examples include, but are not limited to, melanoma tyrosinase, PSA and PSMA in prostate cancer, bcr / abl in lymphoma, and the like. However, many of the tumor associated antigens identified are tumorigenic proteins and / or tumor suppressor genes that occur in multiple tumor types and actually cause modification (tumor suppressor genes are reviewed for example for kidney cancer; Linehan WM, Walther MM, Zbar B. The genetic basis of cancer of the kidney.J Urol. 2003 Dec; 170 (6Pt1): 216-372) occurs in almost all tumor types. For example, normal cell proteins that control cell growth and differentiation, such as p53 (an example of a tumor suppressor gene), ras, c-met, myc, pRB, VHL, HER-2 / neu, etc. Mutations that increase the risk can be accumulated to make them carcinogenic (McCartey et al. Cancer Research, 1998, 15:58 2601-5; Disis et al. Ciba Found. Symp. 1994, 187: 198-211) . These mutant proteins may also be targets of tumor specific immune responses in multiple cancer types.

Whether the presentation is effective or ineffective determines the type and extent of the induced immune response, ranging from immune to resistant. Inactive antigen naïve (na) To stimulate CTLs in an antigen specific method, CTLs must receive two signals from antigen presenting cells: antigen specific T-cell receptors (TCRs) that interact with HLA / peptide complexes. ) And a second signal through co-stimulatory factors (B7 molecules, ICAM-1 and other adhesion molecules) or cytokines (eg IL-2). When T-lymphocytes receive only one of these signals, anergy of T-cells occurs. In general, tumor cells do not function properly because they have no costimulatory molecules and usually show only low HLA class I expression. In addition, malignant cells usually express cytokines or surface molecules that suppress the immune response to them. As such, the manner in which TAA is presented to T-cells is of great importance for the induction of tumor specific immune responses.

Special requirements must be met for proteins to be recognized by CTL as tumor specific or related antigens and used in therapy. Antigens should be expressed mainly by tumor cells and not by normal healthy tissue or in relatively small amounts. More preferred is when the antigen is present in one tumor type as well as in high concentrations (ie, the number of peptides per cell is replicated). Tumor specific and tumor associated antigens are usually derived from proteins that are directly involved in turning normal cells into tumor cells due to function (eg cell cycle control or apoptosis). In addition, subtargets of proteins that are directly responsible for the mutation may also be enhanced and indirectly involved in the tumor. Such indirect tumor associated antigens may also be targeted by vaccination approaches. In both cases the presence of epitopes in the amino acid sequence of the antigen is essential because such peptides (immunogenic peptides) derived from tumor associated antigens must lead to in vitro or in vivo T-cell responses.

Basically any peptide capable of binding an MHC molecule can act as a T-cell epitope. A prerequisite for the induction of an in vitro or in vivo T-cell response is that the T-cell together with the corresponding TCR must be present and not resistant to this particular epitope. T-helper cells play an important role in modulating the effector function of CTLs in antitumor immunity. T-helper cell epitopes that trigger TH1-type T-helper cell responses support the effector function of CD8 + killing T-cells, which display tumor-associated peptide / MHC complexes on the cell surface and counter tumor cells. Toxic functions are also included. In this way, the tumor associated T-helper cell peptide epitope can act as an active pharmaceutical ingredient of a vaccine composition that alone or in combination with other tumor associated peptides stimulates an anti-tumor immune response.

For most tumors, only a small number of TAAs are known or defined for one specific HLA type. In contrast, many prostate-specific antigens and prostate carcinoma-associated antigens and peptides recognized by CTL have been successfully identified. These TAAs can stimulate T-cells and induce antigen specific CTLs when expressed as a complex of HLA molecules and peptides on antigen presenting cells.

In malignant melanoma studies, where most clinical trials using immunotherapy have been conducted, immunization with one or two TAAs leads to the selection of tumor cells, which can lead to disease progression during DC therapy. This was known. Both types of CD8 and CD4 dependent responses co- and synergistically contribute to antitumor effects, so CD8 + CTL (MHC class I molecules) or CD4 + Identification and characterization of tumor associated antigens recognized by CTLs (MHC class II molecules) is important for the development of tumor vaccines.

However, in general, the onset of one type of CTL is insufficient to remove all tumor cells. Tumors are highly mutant and can respond quickly to CTL attacks by altering their protein patterns to avoid recognition by CTLs. Various specific peptides are used for vaccination to counter tumor evasion mechanisms. In this way, extensive simultaneous attacks on tumors can be developed using multiple CTL clones simultaneously. This reduces the likelihood that the tumor will avoid an immune response. This hypothesis has been recently confirmed in clinical studies treating late stage melanoma patients. Although there are very few exceptions, patients with at least three distinct T-cell responses show a target clinical response or steady state (Banchereau et al., 2001), as well as increased survival, while T- The majority of patients with less than three cellular responses were diagnosed with progressive disease.

Similar effects were seen when patients with renal cell carcinoma were treated with a vaccine consisting of 13 different peptides (H. Singh-Jasuja, S. Walter, T. Weinschenk, A. Mayer, PY Dietrich, M. Staehler, A). Stenzl, S. Stevanovic, H. Rammensee, J. Frisch; Correlation of T-cell response, clinical activity and regulatory T-cell levels in renal cell carcinoma patients treated with IMA901, a novel multi-peptide vaccine; ASCO Meeting 2007 Poster # 3017; M. Staehler, A. Stenzl, PY Dietrich, T. Eisen, A. Haferkamp, J. Beck, A. Mayer, S. Walter, H. Singh, J. Frisch, CG Stief; An open label study to evaluate the safety and immunogenicity of the peptide based cancer vaccine IMA901, ASCO meeting 2007; Poster # 3017).

Therefore, the main task of tumor vaccine development is to identify and characterize neotumor-associated antigens and their derived immunogenic T-helper epitopes, as well as to increase the likelihood of response to more than one epitope for each patient. Combining epitopes.

Accordingly, there is provided a composition comprising at least one HLA binding peptide comprising, consisting essentially of, or composed of one epitope derived from a prostate-associated antigen molecule.

As used herein, the phrase “HLA binding peptide” refers to all polypeptides that can be bound by human leukocyte antigen molecules of HLA class I or HLA class II.

As used herein, the term “antigen determinant” refers to an amino acid sequence sufficient to allow a molecule comprising an amino acid to be bound by a human leukocyte antigen molecule of HLA class I or HLA class II. Methods of identifying epitopes are known in the art. These methods include, but are not limited to: (a) Gene expression analysis using T-cells in individual patients, reference: van der Bruggen et al ., A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma, Science 1991; 254 (5038): 1643-1647; (b) Mass spectrometry sequencing of tumor associated peptides, see Cox et. al ., Identification of a peptide recognized by five melanoma-specific human cytotoxic T cell lines, Science 1994; 264 (5159): 716-719; And (c) “Immunoimmunoassay”, in which known TAA is used to predict epitopes according to allele specific peptide motifs, see Stevanovic, Identification of tumor-associated T-cell epitopes for vaccine development, Nat. Rev. Cancer 2002; 2 (7): 514-520; Celis et al ., Induction of anti-tumor cytotoxic T lymphocytes in normal humans using primary cultures and synthetic peptide epitopes, Proc. Nat'l Acad. Sci. USA 1994; 91 (6): 2105-2109.

As used herein, the term "prostate-associated antigenic molecule" refers to a molecule that contains (a) prostate tissue, or (b) all epitopes differentially expressed in prostate cancer cells. Examples of prostate-associated antigen molecules include prostate specific antigens, prostate stem cell antigens, prostate specific membrane antigens, survivin, prosteins, transient receptor translocation-p8, and the like. Examples of epitopes derived from such prostate related antigen molecules are shown in FIG. 1. However, epitopes derived from any prostate-associated antigen molecule can be used.

HLA binding peptides disclosed herein can be altered by substituting one or more residues at different, possibly selective, sites within the peptide chain, unless otherwise noted. Such substitution may, for example, be of a conservative nature, in which one amino acid is substituted by amino acids of similar structure and feature (eg, hydrophobic amino acids are substituted by other hydrophobic amino acids). Amino acid replacements of the same or similar size and chemical nature, such as leucine being replaced with isoleucine, are even more conservative. In the study of sequence changes in naturally occurring homologous proteins, substitutions of certain amino acids are more often tolerated than others, and these substitutions are usually related in size, charge, polarity, and hydrophobicity between the original and replacement amino acids. Correlation with Similarity

Conservative substitutions are defined herein as exchanges within one of the following five groups: group 1—small aliphatic, nonpolar or weakly polar residues (Ala, Ser, Thr, Pro, Gly); Group 2-polar, negatively charged residues and their amides (Asp, Asn, Glu, Gln); Group 3-polar, positively charged residues (His, Arg, Lys); Group 4-large aliphatic, nonpolar residues (Met, Leu, Ile, Val, Cys); And group 5-large aromatic residues (Phe, Tyr, Trp).

Less conservative substitutions may include substitution of an amino acid by one amino acid having similar characteristics but somewhat different size, such as alanine being substituted by an isoleucine residue. Very non-conservative substitutions may include replacing polar amino acids, even basic amino acids, with acidic amino acids. However, such "rapid" substitutions cannot be ignored as ineffective because the chemical effects are not entirely predictable, and abrupt substitutions naturally cause accidental effects that were otherwise unpredictable from simple chemical principles.

Such substitutions may, of course, include structures other than regular L-amino acids. Therefore, D-amino acids may substitute for L-amino acids commonly found in published antigen peptides and should be included in the disclosure herein. Non-standard R groups (ie, R groups other than those found in the twenty general amino acids of natural proteins) can also be used for substitution purposes to produce immunogens and immunogenic peptides according to the invention.

If a substitution at one or more positions is found to be a peptide with significantly equivalent or greater antigenic activity as defined below, to determine if the combined substitution has a secondary or synergistic effect on the antigenicity of the peptide. Combinations of such substituents will be examined. At most fewer than four positions in a peptide are substituted at the same time.

In another aspect there is provided a composition containing at least two HLA binding peptides, wherein at least one of the HLA binding peptides is an HLA class I peptide and at least one of the HLA binding peptides is an HLA class II peptide.

As used herein, the phrase “HLA class I peptide” refers to all polypeptides comprising epitopes that are capable of or are predicted to be bound by HLA class I human leukocyte antigen molecules. For example, "HLA class I peptide" includes an HLA-A2 restriction peptide that binds to a specific allele of the HLA-A2 serotype and includes HLA-A * 0201, * 0202, * 0203, * 0206, or * 0207 α. Including but not limited to HLA-A2 serotypes having chains. Examples of "HLA class I peptides" include SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: Number 11 and they are all HLA-A * 201 restriction peptides.

The invention furthermore derives from antigens associated with tumorigenesis, and is directed to immune responses of human leukocytes, in particular lymphocytes, in particular T-lymphocytes, in particular CD4 + T-lymphocytes, in particular CD4 + T-lymphocytes, which mediate T _H1 type immune responses. Provided are peptides capable of sufficiently binding to the triggering MHC (HLA) class II molecules. As used herein, the phrase “HLA class II peptide” refers to all polypeptides comprising epitopes that can be or are predicted to be bound by HLA class II human leukocyte antigen molecules. Examples of “HLA class II peptides” are shown in FIG. 1 as SEQ ID NO: 13 and SEQ ID NO: 14.

Additional peptides of the invention are as follows:

HLA class II peptides are thought to be independent of certain HLA specific amino acid motifs and N- and / or C-terminal extensions that do not optionally interfere with the function of the core sequence (ie, are not related to peptide-T-cell interactions). Consisting of a "core sequence". N- and / or C-terminal extensions may be, for example, between 1 and 10 amino acids each in length. These peptides can be used to directly mount HLA class II molecules or the sequence can be replicated to a carrier according to the description below. Since these peptides form the final processing product of larger peptides in cells, longer peptides can likewise be used. The HLA class II peptides disclosed herein can be any size, with molecular weight of less than 100,000 Daltons, molecular weight less than 50,000 Daltons, molecular weight less than 10,000 Daltons, molecular weight less than 5,000 Daltons, molecular weight less than 2,500 Daltons, or molecular weights from about 1000 to 2000 Including but not limited to the size of Daltons. Peptides of the invention in terms of number of amino acid residues, by way of example but not limitation, may have less than 1000 residues, less than 500 residues, or less than 100 residues.

Thus, the total length of the peptide and its variants is 8 to 100, 8 to 60 amino acids, 8 to 30, 8 to 17, or the total length of the peptide or variant thereof is 8, 9, 10, 11, 12, 13, 14 Compositions of peptides that are 15 or 16 amino acids and variants thereof are disclosed. In another aspect, the peptide has an extension site of 1 to 10 amino acids at the C-terminus and / or N-terminus in a key sequence selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 14 under these conditions: The total number of amino acids is 1 to 12, 1 to 10, 1 to 8, 1 to 6, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 3 to 12, 3 to 10, 3 to 8, 3 To 6, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 5 to 12, 5 to 10, 5 to 8, 5 to 6, 6 to 12, 6 to 10, 6 to 8, 7 to 12 , 7 to 10, 7 to 8, 8 to 12, 8 to 10, 9 to 12, 9 to 10, or 10 to 12, and the peptide can still bind to the HLA molecule in the same manner as the peptide. The ratio of peripheral amino acids to the C- and N-terminus (eg all peripheral amino acids may be added to one end, or several If the acid that can be distributed both to evenly at the terminal or any ratio may also be added), also.

In addition, MHC class I epitopes, although usually 8 to 10 amino acids in length, can be produced by peptide processing from longer peptides or proteins containing the actual epitopes. Similar to the MHC class II epitopes, the peripheral residues of the precursor peptides extending (lengthened) above and / or below the N- and C-terminus of the actual epitope do not significantly affect the presentation of the peptide to CTL. Nor may it be chosen to not mask the proteolytic cleavage site necessary to make the actual epitope mediated by the processing of the elongated peptide.

Thus, in another aspect, the peptide has a core sequence consisting of SEQ ID NO: 1 to SEQ ID NO: 11 and SEQ ID NO: 15 to SEQ ID NO: 37, extending 1 to 10 peripheral amino acids at the C- and / or N-terminus. do. In another aspect, the total number of these peripheral amino acids is 1 to 12, 1 to 10, 1 to 8, 1 to 6, 2 to 12, 2 to 10, 2 to 8, 2 to 6, 3 to 12, 3 to 10, 3 to 8, 3 to 6, 4 to 12, 4 to 10, 4 to 8, 4 to 6, 5 to 12, 5 to 10, 5 to 8, 5 to 6, 6 to 12, 6 to 10, 6 to 8, 7 to 12, 7 to 10, 7 to 8, 8 to 12, 8 to 10, 9 to 12, 9 to 10, or 10 to 12, and the peptide is SEQ ID NO: 1 to SEQ ID NO: If any of 12 and SEQ ID NO: 15 to SEQ ID NO: 40 can still bind to the HLA molecule in the same manner as the peptide, the proportion of the peripheral amino acid at the C-terminus and the N-terminus (eg all peripheral amino acids) These may be added at one end, or several amino acids may be added at both ends evenly or in any proportion. It can also be distributed as).

In another aspect, the invention provides a total length of 8 to 100 amino acids, 8 to 60 amino acids, 8 to 30 amino acids, 8 to 18 amino acids, or 8, 9, 10, 11, 12, 13, 14, 15 Peptides and variants of the MHC class I epitope that are 16 or 17 amino acids.

Of course, the disclosed peptides or variants will have the ability to bind to molecules of human MHC class I or II. Binding of peptides or variants to MHC complexes can be tested by methods known in the art, such as those described in the Examples of the present invention below or in the literature for other MHC class II alleles. (Eg, Vogt AB, Kropshofer H, Kalbacher H, Kalbus M, Rammensee HG, Coligan JE, Martin R; Ligand motifs of HLA-DRB5 * 0101 and DRB1 * 1501 molecules delineated from self-peptides; J Immunol. 1994; 153 (4): 1665-1673; Malcherek G, Gnau V, Stevanovic S, Rammensee HG, Jung G, Melms A; Analysis of allele-specific contact sites of natural HLA-DR17 ligands; J Immunol. 1994; 153 (3) 1141-1149; Manici S, Sturniolo T, Imro MA, Hammer J, Sinigaglia F, Noppen C, Spagnoli G, Mazzi B, Bellone M, Dellabona P, Protti MP; Melanoma cells present a MAGE3 epitope to CD4 (+) cytotoxic T cells in association with histocompatibility leukocyte antigen DR11; J Exp Med. 1999; 189 (5): 871-876; Hammer J, Gallazzi F, Bono E, Karr RW, Guenot J, Valsasnini P, Nagy ZA, Sinigaglia F; Peptide binding specificity of HLA-DR4 molecules: correlation with rheumatoid arthritis association; .J Exp Med. 1995 181 (5): 1847-1855; Tompkins SM, Rota PA, Moore JC, Jensen PE; A europium fluoro-immunoassay for measuring binding of antigen to class II MHC glycoproteins; J Immunol Methods. 1993; 163 (2): 209-216; Boyton RJ, Lohmann T, Londei M, Kalbacher H, Halder T, Frater AJ, Douek DC, Leslie DG, Flavell RA, Altmann DM; Glutamic acid decarboxylase T lymphocyte responses associated with susceptibility or resistance to type I diabetes: analysis in disease discordant human twins, nonobese diabetic mice and HLA-DQ transgenic mice; Int Immunol. 1998 (12): 1765-1776).

In addition, the expansion of amino acids located at the N- and / or C-terminus does not necessarily constitute part of the peptide that acts as a substantial epitope of the MHC molecule, but is important for effective peptide entry into cells according to the present invention. Can be. In one embodiment, peptides of the invention are described, for example, in NCBI, GenBank Accession No. X00497 (Strubin, M., Mach, B. and Long, EO The complete sequence of the mRNA for the HLA-DR- associated invariant chain reveals a polypeptide with an unusual transmembrane polarity EMBO J. 3 (4), 869-872 (1984)). 80 N- of the HLA-DR antigen-associated constant chain (p33 in "Ii" below)) Is a fusion protein comprising terminal amino acids.

In another aspect, the peptide has a total length of 8 to 100 amino acids, 8 to 60 amino acids, 8 to 30 amino acids, 8 to 17 amino acids, or 9, 10, 11, 12, 13, 14, 15 or 16 amino acids.

In addition, the peptides or variants may be further modified to enhance stability and / or binding to MHC molecules to induce a stronger immune response. Methods of optimizing peptide sequences are well known in the art and include, for example, the introduction of reverse peptide bonds or non-peptide bonds.

Therefore according to another aspect the present invention provides a composition wherein at least one peptide or variant comprises a non-peptide bond.

In reverse peptide bonds, amino acid residues are not bound by peptide (-CO-NH-) linkages and the peptide bonds are reversed. Such retro-inverso peptidomimetic are described by methods known in the art, such as Meziere et al., (1997) J. Immunol. 159, 3230-3237 can be made using the method described. This approach involves preparing peptide mimetics that include changes that include the backbone but not the orientation of the side branches. Meziere et al. (1997) show that such peptide mimetics are useful for MHC and T-helper cell responses. Retroinverse peptides containing NH-CO bonds instead of CO-NH peptide bonds are much more resistant to proteolysis.

Non-peptide bonds include, for example, —CH ₂ —NH, —CH ₂ —S, —CH ₂ CH ₂ —, —CH═CH—, —COCH ₂ —, —CH (OH) CH ₂ —, and —CH ₂ SO-. US Pat. No. 4,897,445 discloses solid phase synthesis of non-peptide bonds synthesized by reacting aminoaldehydes with amino acids when a non-peptide bond (-CH ₂ -NH) and NaCNBH ₃ are present in a polypeptide chain comprising a polypeptide synthesized by standard methods. Provide a way

Peptides consisting of the sequences of the invention described above can be synthesized, for example, with additional chemical groups present at the amino and / or carboxy termini to enhance the stability, bioavailability and / or affinity of the peptide. For example, hydrophobic groups such as carbobenzoxyl, dansyl, or t-butyloxycarbonyl groups can be added to the amino terminus of the peptide. Likewise an acetyl or 9-fluorenylmethyloxy-carbonyl group can be placed at the amino terminus of the peptide. Additionally, for example, a hydrophobic group, t-butyloxycarbonyl, or an amino group may be added to the carboxy terminus of the peptide.

Moreover, all peptides of the present invention can be synthesized to alter their steric configuration. For example, D-isomers of one or more amino acid residues of a peptide may be used instead of the usual L-isomers. Moreover, at least one of the amino acid residues of the peptides of the invention may be substituted with one of the well known, non-naturally occurring amino acids. Such alterations may contribute to increased stability, bioavailability and / or binding activity of the peptides of the invention.

Similarly, peptides or variants of the invention can be chemically modified by reacting certain amino acids before or after synthesis of the peptide. Examples of such alterations are well known in the art and are described, for example, in R. Lundblad, Chemical Reagents for Protein Modification, 3rd ed. Summarized in CRC Press, 2005, which is incorporated herein by reference. Chemical modifications of amino acids include, but are not limited to the following modifications: acylation, amidation, pyridoxylation of lysine, reduction alkylation, of amino groups using 2, 4, 6 trinitrobenzene sulfonic acid (TNBS) Trinitrobenzylation, alteration of the amide of the carboxyl group and oxidation of cysteine to formic acid to cysteinic acid, formation of mercury derivatives, formation of mixed disulfides using other thiol compounds, reaction with maleimide, iodide or iodiacetamide Carboxymethylation and carbamoylation using cyanate at alkaline pH. In this regard, one of ordinary skill in the art would appreciate the following reference for a broad methodology involving chemical modification of proteins: Current Protocols In Protein Science, Eds. See chapter 15 of Coligan et al., (John Wiley & Sons NY 1995-2000).

While crosslinking of proteins with glutaraldehyde, polyethylene glycol (PEG) diacrylate and formaldehyde is used for the preparation of hydrogels, circulatory is usually the case when successful modification of therapeutic proteins and peptides with PEG is successful. half-life also increases. Chemical alteration of allergens for immunotherapy is usually accomplished by carbamylation using potassium cyanate.

In general, peptides and variants thereof (at least those having peptide linkages between amino acid residues) are described, for example, in Lu et al., (1981) J. Org. Chem. 46, 3433, and references therein, may be synthesized using the Fmoc-polyamide mode of solid phase peptide synthesis.

Purification can be performed by one or a combination of techniques such as recrystallization, size exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography, and (usually) reversed phase high performance liquid chromatography.

Analysis of peptides was performed by thin layer chromatography, electrophoresis, in particular capillary electrophoresis, solid phase extraction (CSPE), reverse phase high performance liquid chromatography, amino acid analysis after acid hydrolysis, and high-speed atomic collision (FAB) mass spectrometry, as well as MALDI and It can also be performed by ESI-Q-TOF mass spectrometry.

Another aspect of the invention provides nucleic acids (eg polynucleotides) encoding one of the disclosed peptides or variants. Polynucleotides are, for example, DNA, cDNA, PNA, CNA, RNA, single and / or double stranded, or polynucleotides using natural or stabilized forms of polynucleotides, such as pororotate periods, or combinations thereof. And may or may not include an intron that encodes a peptide. Of course this is just a peptide comprising naturally occurring amino acid residues which are bound by naturally occurring peptide bonds which can be encoded by polynucleotides. Another aspect of the invention provides an expression mediator capable of expressing a polypeptide according to the invention. Expression mediators for different cell types are well known in the art and can be selected without undue experimentation.

In general, DNA is inserted into an expression medium such as a plasmid in an orientation suitable for expression and in an accurate reading frame. If desired, the DNA can be linked to the corresponding transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, although such control is generally in the expression mediator. The mediator is then introduced into the host through standard techniques well known in the art.

In one embodiment, the composition comprises at least two peptides consisting essentially of the amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 11 and SEQ ID NO: 13 to SEQ ID NO: 40.

In another embodiment, the composition comprises at least two species essentially consisting of the amino acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 13 and SEQ ID NO: 40 Peptides.

In another embodiment, the composition comprises at least four species consisting essentially of the amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 13 and SEQ ID NO: 40 Peptides.

In another embodiment, the composition comprises 10 species consisting essentially of the amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 and SEQ ID NO: 13 and SEQ ID NO: 40 Peptides.

In another embodiment, the composition comprises at least two species consisting essentially of the amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14 Peptides, at least 4 peptides, or at least 10 peptides, and SEQ ID NO: 38 BIR-002b FTELTLGEF from Servivin HLA-A1, SEQ ID NO: 39 BIR-002c LMLGEFLKL from Servivin HLA-A2, Servi SEQ ID NO: 40 BIR-002d EPDLAQCFY from empty HLA-B35, BIR-002a SEQ ID NO: 41 TLGEFLKLDRERAKD from survivin HLA-DR and BIR-004 SEQ ID NO: from Servibin HLA-DR and HLA-A * 02 And at least one peptide selected from the group consisting of 42 ELTLGEFLKLDRERAKN.

In another embodiment, the composition comprises at least two species consisting essentially of the amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14 Peptides, at least 4 peptides, or at least 10 peptides, and SEQ ID NO: 38 BIR-002b FTELTLGEF from Servicin HLA-A1, SEQ ID NO: 39 BIR-002c LMLGEFLKL from Servicin HLA-A2, Servi SEQ ID NO: 40 BIR-002d EPDLAQCFY from empty HLA-B35, BIR-002a SEQ ID NO: 41 TLGEFLKLDRERAKD from survivin HLA-DR and BIR-004 SEQ ID NO: derived from survivin HLA-DR and HLA-A * 02 And at least one peptide selected from the group consisting of 42 ELTLGEFLKLDRERAKN, wherein additional peptides are selected according to the HLA combination of the subject.

In one embodiment, the composition comprises at least two peptides consisting of amino acid sequences according to SEQ ID NO: 1 to SEQ ID NO: 11 and SEQ ID NO: 13 to SEQ ID NO: 42.

In another embodiment, the composition comprises at least two amino acids consisting of the amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, and SEQ ID NO: 13 to SEQ ID NO: 42 Peptides.

In another embodiment, the composition comprises at least four amino acids consisting of amino acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, and SEQ ID NO: 13 to SEQ ID NO: 42 Peptides.

In another embodiment, the composition comprises 10 peptides consisting of amino acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, and SEQ ID NO: 13 to SEQ ID NO: 42 It includes.

In another embodiment, the composition comprises at least two amino acids consisting of the amino acid sequence according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14 A peptide, at least four peptides, or at least ten peptides, and SEQ ID NO: 38 BIR-002b FTELTLGEF from Servivin HLA-A1, SEQ ID NO: 39 BIR-002c LMLGEFLKL from Servivin HLA-A2, Survivin SEQ ID NO: 40 BIR-002d EPDLAQCFY from HLA-B35, BIR-002a SEQ ID NO: 41 from TLIVFLKLDRERAKD from Survivin HLA-DR and BIR-004 SEQ ID 42 from Servivin HLA-DR and HLA-A * 02 At least one peptide selected from the group consisting of ELTLGEFLKLDRERAKN.

In another embodiment, the composition comprises at least two peptides consisting of amino acid sequences according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14 , At least 4 peptides, or at least 10 peptides, and SEQ ID NO: 38 BIR-002b FTELTLGEF derived from Servin HLA-A1, SEQ ID NO: 39 BIR-002c LMLGEFLKL from Servin HLA-A2, Servin HLA SEQ ID NO: 40 BIR-002d EPDLAQCFY from B35, BIR-002a SEQ ID NO: 41 from TLBIFLKLDRERAKD from Survivin HLA-DR and BIR-004 SEQ ID NO: 42 ELTLGEFLKLDRERAKN from Survivin HLA-DR and HLA-A * 02 At least one peptide selected from the group consisting of: the additional peptide is selected according to the HLA combination of the subject in need.

In one embodiment, the composition comprises at least two peptides consisting essentially of the amino acid sequence according to SEQ ID NO: 1 to SEQ ID NO: 11 and SEQ ID NO: 13 to SEQ ID NO: 14 and SEQ ID NO: 15 to SEQ ID NO: 31.

In another embodiment, the composition is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 23, SEQ ID NO: 24, sequence At least two peptides consisting essentially of the amino acid sequence according to SEQ ID NO: 25, SEQ ID NO: 20, SEQ ID NO: 31 and SEQ ID NO: 32.

In another embodiment, the composition is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 23, SEQ ID NO: 24, sequence At least four peptides necessarily consisting of the amino acid sequence according to SEQ ID NO: 25, SEQ ID NO: 20, SEQ ID NO: 31 and SEQ ID NO: 32.

In another embodiment, the composition is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 23, SEQ ID NO: 24, sequence 10 peptides consisting essentially of the amino acids according to SEQ ID NO: 25, SEQ ID NO: 20, SEQ ID NO: 31 and SEQ ID NO: 32 and selected from the group consisting of said sequences.

In another embodiment, the composition is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 23, SEQ ID NO: 24, sequence At least two peptides, at least four peptides, or at least ten peptides consisting essentially of the amino acid sequences according to SEQ ID NO: 25, SEQ ID NO: 20, SEQ ID NO: 31 and SEQ ID NO: 32, and selected from the group consisting of said sequences, And SEQ ID NO: 38 BIR-002b FTELTLGEF derived from Servin HLA-A1, SEQ ID NO: 39 BIR-002c derived from Servin HLA-A2, SEQ ID NO: 40 BIR-002d EPDLAQCFY derived from Servin HLA-B35, Servi At least one peptide selected from the group consisting of BIR-002a SEQ ID NO: 41 TLGEFLKLDRERAKD derived from empty HLA-DR and BIR-004 SEQ ID NO: 42 ELTLGEFLKLDRERAKN derived from survivin HLA-DR and HLA-A * 02 .

In another embodiment, the composition is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 23, SEQ ID NO: 24, sequence Derived from at least two peptides, at least four peptides, or at least ten peptides consisting essentially of the amino acid sequences according to SEQ ID NO: 25, SEQ ID NO: 20, SEQ ID NO: 31 and SEQ ID NO: 32, and survivin HLA-A1 SEQ ID NO: 38 BIR-002b FTELTLGEF, SEQ ID NO: 39 BIR-002c derived from Servivin HLA-A2, SEQ ID NO: 40 BIR-002d derived from Survivin HLA-B35, BIR- derived from Survivin HLA-DR 002a SEQ ID NO: 41 TLGEFLKLDRERAKD and BIR-004 SEQ ID NO: 42 derived from survivin HLA-DR and HLA-A * 02, and at least one peptide selected from the group consisting of HLA-A * 02, wherein the additional peptide comprises a HLA combination of subjects to be treated Is selected according to.

In one embodiment of HLA-A * 02, the composition comprises at least one peptide consisting of amino acid sequences according to SEQ ID NO: 23 and SEQ ID NO: 24 and SEQ ID NO: 1 to SEQ ID NO: 11 and SEQ ID NO: 13 to SEQ ID NO: 14 At least one peptide consisting of the amino acid sequence according to.

In one embodiment of HLA-A * 02, the composition comprises SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13 And at least one peptide consisting of the amino acid sequence according to SEQ ID NO: 14.

In one embodiment of HLA-A * 02, the composition comprises SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13 And at least one peptide consisting of the amino acid sequence according to SEQ ID NO: 14.

As another example of an embodiment for HLA-A * 02, the composition comprises SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, sequence 10 peptides consisting of the amino acid sequence according to SEQ ID NO: 13 and SEQ ID NO: 14.

As another example of an embodiment for HLA-A * 02, the composition comprises SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, sequence At least two peptides, at least four peptides, or at least ten peptides consisting of the amino acid sequence according to SEQ ID NO: 13 and SEQ ID NO: 14, and SEQ ID NO: 38 BIR-002b FTELTLGEF, survivin derived from Servin HLA-A1 SEQ ID NO: 39 BIR-002c LMLGEFLKL derived from HLA-A2, SEQ ID NO: 40 BIR-002d derived from HLA-B35, BIR-002a SEQ ID NO: 41 TLGEFLKLDRERAKD derived from Survivin HLA-DR and Servin HLA- At least one peptide selected from the group consisting of BIR-004 SEQ ID NO: 42 ELTLGEFLKLDRERAKN from DR and HLA-A * 02.

As another example of an embodiment for HLA-A * 02, the composition comprises SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, sequence At least two peptides, at least four peptides, or at least ten peptides consisting of the amino acid sequence according to SEQ ID NO: 13 and SEQ ID NO: 14, and SEQ ID NO: 38 BIR-002b FTELTLGEF, survivin derived from Servin HLA-A1 SEQ ID NO: 39 BIR-002c LMLGEFLKL derived from HLA-A2, SEQ ID NO: 40 BIR-002d derived from HLA-B35, BIR-002a SEQ ID NO: 41 TLGEFLKLDRERAKD derived from Survivin HLA-DR and Servin HLA- At least one peptide selected from the group consisting of BIR-004 SEQ ID NO: 42 ELTLGEFLKLDRERAKN, derived from DR and HLA-A * 02, wherein the additional peptide is selected according to the HLA combination of the subject in need.

As an example of an embodiment for HLA-A * 24 in combination with HLA-A * 02, the composition comprises SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 1 to SEQ ID NO: 11, SEQ ID NO: 13 Or at least two peptides consisting essentially of an amino acid sequence according to the group consisting of SEQ ID NO: 14.

As another example of an embodiment for HLA-A * 24 in combination with HLA-A * 02, the composition comprises SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 1, SEQ ID NO: 2, sequence In a group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 20, SEQ ID NO: 31, and SEQ ID NO: 32 At least two different peptides consisting essentially of the corresponding amino acid sequence.

As another example of an embodiment for HLA-A * 24 in combination with HLA-A * 02, the composition comprises SEQ ID NO: 20, SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 1, SEQ ID NO: 2, sequence In a group consisting of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 20, SEQ ID NO: 31, and SEQ ID NO: 32 At least two different peptides consisting essentially of the corresponding amino acid sequence.

In another embodiment, the composition is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 23, SEQ ID NO: 24, sequence 10 peptides consisting essentially of the amino acid sequences according to SEQ ID NO: 25, SEQ ID NO: 20, SEQ ID NO: 31, and SEQ ID NO: 32 and selected from the group consisting of these sequences.

In another embodiment, the composition is SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 23, SEQ ID NO: 24, sequence At least two peptides, at least four peptides, or at least ten peptides consisting essentially of the amino acid sequences according to SEQ ID NO: 25, SEQ ID NO: 20, SEQ ID NO: 31 and SEQ ID NO: 32, and selected from the group consisting of said sequences, And SEQ ID NO: 38 BIR-002b FTELTLGEF derived from Servin HLA-A1, SEQ ID NO: 39 BIR-002c derived from Servin HLA-A2, SEQ ID NO: 40 BIR-002d EPDLAQCFY derived from Servin HLA-B35, Servi At least one peptide selected from the group consisting of BIR-002a SEQ ID NO: 41 TLGEFLKLDRERAKD derived from empty HLA-DR and BIR-004 SEQ ID NO: 42 ELTLGEFLKLDRERAKN derived from survivin HLA-DR and HLA-A * 02 .

In another embodiment, the composition comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: At least two peptides consisting essentially of the amino acid sequences according to SEQ ID NO: 20, SEQ ID NO: 31 and SEQ ID NO: 32, at least 4 peptides, or at least 10 peptides, and sequences derived from Servinbin HLA-A1 No. 38 BIR-002b FTELTLGEF, SEQ ID NO: 39 BIR-002c derived from Survivin HLA-A2, SEQ ID NO: 40 BIR-002d derived from Survivin HLA-B35, BIR-002a derived from Survivin HLA-DR At least one peptide selected from the group consisting of SEQ ID NO: 41 TLGEFLKLDRERAKD and BIR-004 SEQ ID NO: 42 ELTLGEFLKLDRERAKN derived from survivin HLA-DR and HLA-A * 02, wherein the additional peptide is present in the HLA combination of the subject Is selected accordingly.

In one embodiment, the composition comprises at least an amino acid sequence consisting of SEQ ID NO: 20, SEQ ID NO: 23 to SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 1 to SEQ ID NO: 11, and SEQ ID NO: 13 to SEQ ID NO: 14 In comprising two peptides and at least one additional peptide according to SEQ ID NO: 15 to SEQ ID 19, SEQ ID: 21, SEQ ID NO: 22, SEQ ID NO: 26 to SEQ ID NO: 30, and SEQ ID NO: 33, the additional peptide is Depending on the HLA combination of the subject to be treated and optionally at least one peptide is SEQ ID NO: 38 BIR-002b FTELTLGEF from Servicin HLA-A1, SEQ ID NO: 39 BIR-002c LMLGEFLKL from Servicin HLA-A2, SEQ ID NO: 40 BIR-002d EPDLAQCFY from Survivin HLA-B35, BIR-002a SEQ ID NO: 41 TLGEFLKLDRERAKD from Survivin HLA-DR and BIR-004 sequence from Survivin HLA-DR and HLA-A * 02 Number 42 ELTLGEFLKLDRERAKN Is selected from the group consisting of, in which case the additional peptide is selected in accordance with a combination of HLA treated subjects.

In another embodiment, the composition is SEQ ID NO: 20, SEQ ID NO: 23 to SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: At least two peptides consisting of amino acid sequences according to SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 14, and SEQ ID NO: 15 to SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 26 to SEQ ID NO: 30, and SEQ ID NO: 33 In containing at least one additional peptide according to claim 1, wherein the additional peptide is selected according to the HLA combination of the subject of interest and optionally at least one peptide is SEQ ID NO: 38 BIR-002b FTELTLGEF derived from survivin HLA-A1. , SEQ ID NO: 39 BIR-002c LMLGEFLKL from Servicin HLA-A2, SEQ ID NO: 40 BIR-002d EPDLAQCFY from Servicin HLA-B35, BIR-002a SEQ ID NO: 41 TLGEFLKLDRERAKD and Servic from Servivin HLA-DR Empty HLA BIR-004 SEQ ID NO: 42 ELTLGEFLKLDRERAKN, derived from -DR and HLA-A * 02, wherein the additional peptide is selected according to the HLA combination of the subject.

In another embodiment, the composition is SEQ ID NO: 20, SEQ ID NO: 23 to SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: At least four peptides consisting of amino acid sequences according to SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 14, and SEQ ID NO: 15 to SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 26 to SEQ ID NO: 30, and SEQ ID NO: 33 In containing at least one additional peptide according to claim 1, wherein the additional peptide is selected according to the HLA combination of the subject to be treated, optionally the at least one peptide is SEQ ID NO: 38 BIR-002b FTELTLGEF derived from survivin HLA-A1. , SEQ ID NO: 39 BIR-002c LMLGEFLKL from Servicin HLA-A2, SEQ ID NO: 40 BIR-002d EPDLAQCFY from Servicin HLA-B35, BIR-002a SEQ ID NO: 41 TLGEFLKLDRERAKD and Servic from Servivin HLA-DR Empty HLA-DR And BIR-004 SEQ ID NO: 42 ELTLGEFLKLDRERAKN from HLA-A * 02, in which case the additional peptide is selected according to the HLA combination of the subject.

In another embodiment, the composition is SEQ ID NO: 20, SEQ ID NO: 23 to SEQ ID NO: 25, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 10 peptides consisting of amino acid sequences according to SEQ ID NO: 11, SEQ ID NO: 13 and SEQ ID NO: 14, and SEQ ID NO: 15 to SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 26 to SEQ ID NO: 30, and SEQ ID NO: 33 In containing at least one additional peptide according to claim 1, wherein the additional peptide is selected according to the HLA combination of the subject in need and optionally at least one peptide is SEQ ID NO: 38 BIR-002b FTELTLGEF, derived from survivin HLA-A1, SEQ ID NO: 39 BIR-002c LMLGEFLKL from Servicin HLA-A2, SEQ ID NO: 40 BIR-002d EPDLAQCFY from Servicin HLA-B35, BIR-002a SEQ ID NO: 41 TLGEFLKLDRERAKD and Servicin derived from Survivin HLA-DR HLA-DR Selected from the group consisting of BIR-004 SEQ ID NO: 42 ELTLGEFLKLDRERAKN derived from HLA-A * 02 and, in this case, the additional peptide is selected in accordance with a combination of HLA treated subjects.

International Patent Application Publication No. 2004/067023 describes MHC class I restriction peptides derived from tumor-associated antigen survivin, which peptides can bind with high affinity to class I HLA molecules.

The optimal amount and optimal dosage regimen of each peptide to be included in the vaccine can be determined by one skilled in the art without undue experimentation. For example, peptides or variants thereof may be formulated for intravenous (i.v.) injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, intramuscular (i.m.) injection. By way of example, but not limitation, peptide injections can be performed in s.c., i.d., i.p., i.m. And i.v. Can be implemented by route. As an example, but not limited to DNA injection, i.d., i.m., s.c., i.p. And i.v. Can be implemented by route. For example, doses of 1 to 500 mg, 50 μg and 1.5 mg, or 125 to 500 μg of peptide or DNA can be administered and the dose will vary depending on the peptide or DNA in question. Doses in this range have been used successfully in previous clinical trials (Brunsvig PF, Aamdal S, Gjertsen MK, Kvalheim G, Markowski-Grimsrud CJ, Sve I, Dyrhaug M, Trachsel S, Møller M, Eriksen JA, Gaudernack G; Telomerase peptide vaccination: a phase I / II study in patients with non-small cell lung cancer; Cancer Immunol Immunother. 2006; 55 (12): 1553-1564; M. Staehler, A. Stenzl, PY Dietrich, T. Eisen, A. Haferkamp, J. Beck, A. Mayer, S. Walter, H. Singh, J. Frisch, CG Stief; An open label study to evaluate the safety and immunogenicity of the peptide based cancer vaccine IMA901, ASCO meeting 2007; Abstract No 3017).

Compositions disclosed herein can be edited such that the selection, number and / or amount of peptides in the composition is tissue, cancer and / or patient specific. For example, peptides can be correctly selected according to the expression pattern of the parent protein in a given tissue to avoid side effects. Such choices may vary depending on the particular type of cancer the patient being treated with, as well as the condition of the disease, previous treatment method, the patient's immune status, and of course the HLA- haplotype of the patient. Moreover, the vaccines according to the invention may comprise components which are individualized according to the individual needs of a particular patient. For example, the amount of other peptides, unwanted side effects from personal allergies or other therapies, and the adaptation of secondary treatment after the first treatment or treatment plan, depending on the expression of the relevant TAA in a particular patient.

One skilled in the art can, for example, examine the production of T-cells in vitro and their efficacy and overall presence, or by analyzing the proliferation, affinity, and spread of T-cells for particular peptides (eg, by analyzing IFN-γ production). ) May be used to select the desired combination of immunogen peptides (see also the examples below). In general, the most potent peptide after this test is combined into a vaccine for the purposes described above.

Suitable vaccines may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 other peptides. . The peptide used in the cancer vaccine may be of any suitable peptide. In particular, it may be a suitable 9-mer peptide or a suitable 8-ploid or 9-ploid or 10-ploid or 11-ploid peptide or 12-ploid, 13-ploid, 14-ploid or 15-ploid. Longer peptides may also be suitable; 9- or 10-plote peptides are preferred for MHC class I peptides, while 12 to 15-plots are preferred for MHC class II peptides, as described in the accompanying Tables 1 and 2.

Peptides constitute a tumor or cancer vaccine. This may be administered directly to the patient, to the affected organ or systemic, to cells derived from the patient, i.e., in vitro to a human cell line, and then to the patient, or to a subpopulation of immune cells obtained from the patient (again May be used in vitro to select).

Peptides can be highly purified or combined with an immune stimulatory adjuvant (see below), or in combination with an immune stimulating cytokine, or administered in an appropriate delivery system such as, for example, liposomes. Peptides may also be conjugated to suitable carriers such as keyhole clam hemocyanin (KLH) or mannan (International Application No. 95/18145 and Longenecker, et al., (1993) Ann. NY Acad. Sci 690, 276-291). The peptide may be labeled, may be a fusion protein, or may be a hybrid molecule. Peptides conferred sequences are expected to stimulate CD4 T-cells or CD8 CTLs. However, stimulation is more efficient with the help provided by T-cells that are positive for the opposite CD. Therefore, for MHC class II epitopes that stimulate CD4 T-cells, a fusion partner or some section of the hybrid molecule provides appropriate epitopes that stimulate CD8-positive T-cells. On the other hand, for MHC class I epitopes that stimulate CD8 CTL, the fusion partner or some section of the hybrid molecule provides the epitopes that stimulate CD4-positive T-cells as appropriate. CD4- and CD8-stimulating epitopes are well known in the art and include those identified in the present invention.

Pharmaceutically acceptable carriers are well known and are usually formulated in liquid form with the addition of the active therapeutic agent. Carriers provide chemical and / or biological stability, release properties, and the like, but generally do not provide any pharmaceutical activity to the preparation. Preferred preparations are described, for example, in Alfonso R. Gennaro. Remington: The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams & Wilkins, 2000, which may include, but is not limited to, saline, water, buffered water, 0.3% glycine, hyaluronic acid, dextrose, and the like. Recently, it has been discovered that certain fat emulsions, which have been used for years for jugular vein nutrition in human patients, can also act as carriers of peptides. Two examples of such emulsions are commercial fat emulsions known as Intralipid and Lipofundin. "Intralipid" is a registered trademark of Kabi Pharmacia, Sweden, and is a fat emulsion for jugular nutrition described in US Pat. No. 3,169,094. "Lipofundin" is the German beret. Is a registered trademark of B. Braun Melsungen. Both contain soybean oil as fat (100 or 200 g in 10 ml of purified water: 10% or 20%, respectively). Egg yolk phospholipid is used in intralipid (12 g / l purified water) and egg yolk lecithin in lipofundine (12 g / l purified water) as an emulsifier. Isotonicity can be determined by adding glycerol (25 g / l) to both intralipid and lipofundine.

In order to induce an immune response, it is generally necessary to include adjuvant to add immunogenicity to the composition. In another aspect, the composition is provided consisting of at least one HLA binding peptide and one adjuvant, wherein the HLA binding peptide contains an epitope derived from a prostate-associated antigen molecule.

As used herein, the term “immune adjuvant” refers to a substance that, when combined with an antigen molecule, nonspecifically accelerates, prolongs, or otherwise augments an antigen specific immune response. Adjuvants are well known in the art and any adjuvant may be used. Suitable adjuvant include 1018 ISS, aluminum salt, Amplivax®, AS15, BCG, CP870,893, CpG7909, CyaA, Mologen dSLIM®, GM-CSF, IC30, IC31, Imiquimod®, ImuFact IMP321, Interferon-α or -β, IS Patch, ISS, ISCOM, JuvImmune®, LipoVac MF59, monophosphoryl lipid A and other nontoxic LPS derivatives, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174 , OM-197-MP-EC, ONTAK, PepTel® mediator system, PLG particulates, resiquimod®, SRL172, virosomes and other virus like particles, YF- Aquila's QS21 stimulon (Aquila Biotech, Walcester, Miami, USA), derived from 17D, VEGF Trap, R848, β-glucan, Pam3Cys, Saponin Icobacterial extracts and synthetic bacterial cell wall analogs (mimics) and other Ribi patents such as Detox, Quil or Superfos, but not limited to. Adjuvants such as imiquimod, Resimiquimod, incomplete Freund, interferon-α or GM-CSF and the like are preferred. Some adjuvant specific to dendritic cells and their preparations (eg MF59) have been described above (Dupuis M, Murphy TJ, Higgins D, Ugozzoli M, van Nest G, Ott G, McDonald DM; Dendritic cells internalize vaccine adjuvant after intramuscular injection; Cell Immunol. 1998; 186 (1): 18-27; Allison AC; The mode of action of immunological adjuvants; Dev Biol Stand. 1998; 92: 3-11). Cytokines can also be used. Several cytokines bind directly to affect the migration of dendritic cells to lymphoid tissues (e.g., TNF-α), accelerate dendritic cells maturation into efficient antigen presenting cells for T-lymphocytes (e.g., For example, GM-CSF, IL-1 and IL-4) (US Pat. No. 5,849,589, specifically incorporated herein by reference in its entirety) and serve as an adjuvant (eg IL-12) (Gabrilovich DI , Cunningham HT, Carbone DP; IL-12 and mutant P53 peptide-pulsed dendritic cells for the specific immunotherapy of cancer; J Immunother Emphasis Tumor Immunol. 1996 (6): 414-418).

It has also been reported that CpG immunostimulatory oligonucleotides enhance the effect of the adjuvant and act as adjuvant itself in the vaccine environment. Contrary to theory, CpG oligonucleotides work by activating the innate (non-adaptive) immune system via toll like receptors (TLR), mainly TLR9. Initiation of TLR9 activation by CpG results in antigen-specific humoral and cellular responses to a wide range of antigens, including peptides or peptide antigens, live or dead viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates of prophylactic and therapeutic vaccines. Response is enhanced. More importantly, this promotes the maturation and differentiation of dendritic cells, which enhances T _H1 cell activation and greatly increases cytotoxic T-lymphocyte (CTL) production even without the help of CD4 T-cells. TH1 bias induced by TLR9 stimulation is usually maintained even with vaccine adjuvant such as alum or incomplete Freund's adjuvant (IFA) that promote TH2 bias. When administered in combination with other adjuvant, CpG oligonucleotides show greater adjuvant activity when formulated in preparations such as particulates, nanoparticles, lipid emulsions, or similar formulations, which induce a strong response when the antigen is relatively weak. Specially needed. They also promote an immune response and in some experiments have comparable antibody responses against full dose vaccines without CpG, allowing the antigen dose to be reduced to approximately two digits (Arthur M. Krieg, Therapeutic potential of Toll like receptor 9 activation, Nature Reviews, Drug Discovery, 2006, 5, 471-484). According to the specification of US Pat. No. 6,406,705 B1, the combination of CpG oligonucleotides, non-nucleic acid adjuvant and antigen induces an antigen-specific immune response. A commercial CpG TLR9 antagonist is dSLIM® (double stem ring immunomodulator) from Mologen (Berlin, Germany). Other TLR binding molecules can also be used, such as RNA binding TLR 7, TLR 8 and / or TLR 9 and the like.

Other examples of useful adjuvant include, but are not limited to, immunoactive small molecules and antibodies such as imidazoquinoline, cyclophosphamide, sunitinib, bevacizumab, cerebrex, NCX-4016, sildenafil, tata Dalfil, vardenafil, sorafinib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab and SC58175 as well as chemicals CpG (eg CpR, Idera), mucin-1-mRNA / protamine complexes, Poly (I: C) (eg PolyI: C12U), non-CpG bacterial DNA or RNA In turn they may act as therapeutics and / or as adjuvant. The amounts and concentrations of adjuvants and additives useful in the context of the present invention can be readily determined by those skilled in the art without undue experimentation.

In one embodiment, the adjuvant is dSLIM®, BCG, OK432, imiquimod, mucin-1-mRNA / protamine complex, resimiquimod, GM-CSF, interferon-α, PeviTer® Trademarks) and jubilee or combinations thereof.

In another embodiment, the adjuvant is granulocyte macrophage colony stimulating factor (GM-CSF, Sargramostim), imiquimod®, mucin-1-mRNA / protamine complex, resimiquimod and interferon- colony stimulating factors such as α and the like.

In another embodiment of the composition according to the invention, the adjuvant is a mucin-1-mRNA / protamine complex, imiquimod, or resimiquimod.

Such compositions disclosed herein can be used for parenteral or oral administration such as subcutaneous, intradermal, intramuscular, intraperitoneal, or oral administration. To this end, the peptide and optionally other molecules are dissolved or suspended in a pharmaceutically acceptable carrier, preferably an aqueous carrier. The composition may also include excipients such as buffers, binders, explosives, diluents, fragrances, lubricants and the like. The peptide may also be administered with an immune stimulant such as a cytokine. An extensive list of excipients that can be used in this composition is described, for example, in A. Kibbe's Pharmaceutical Excipients Handbook, 3rd Ed. 2000 (American Pharmaceutical Association and pharmaceutical press). The composition can be used for the prevention, prevention and / or treatment of adenomatous or cancerous diseases, preferably CRC.

Cytotoxic T-cells (CTLs) recognize antigens in the form of peptides bound to MHC molecules rather than intact foreign antigens themselves. The MHC molecule itself is located on the cell surface of antigen presenting cells. Therefore, activation of CTL is possible only in the presence of a trimer complex of peptide antigen, MHC molecule and APC. Relatively, this may enhance the immune response not only when the peptide is used for activation of CTL but also when APC is additionally added with the corresponding MHC molecule.

In another embodiment, the composition according to the invention additionally comprises at least one antigen presenting cell.

Antigen presenting cells (or stimulating cells) generally have MHC class I or II molecules on their surface, and as an example of an embodiment, essentially by itself cannot mount MHC class I or II molecules as the selected antigen. As described in more detail below, class I or II molecules may be readily loaded with antigens selected in vitro.

In one embodiment, the mammalian cell lacks or has reduced TAP peptide carrier or reduced function. Suitable cells without a TAP peptide carrier include T2, a human peptide carrying a defective cell line (available as catalog number CRL 1992 from American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, USA); TAP deficient cell lines such as T2 can be used as APCs, and almost all peptides presented by MHC class I due to lack of TAP will be peptides used with caution to externally load empty MHC class I molecules in these cell lines. . So all the effects will certainly be due to the peptide used.

In another embodiment, the antigen presenting cell is a dendritic cell. Suitably, dendritic cells are autologous dendritic cells sensitized with an antigenic peptide. The antigenic peptide may be any antigenic peptide as long as it generates a sufficient T-cell response. T-cell therapy using autologous dendritic cells sensitized with peptides from tumor-associated antigens has been described in Murphy, et al., The Prostate 29, (1996) 371-380, and Tjua, et al., The Prostate t32, (1997 ) Is published at 272-278.

In another embodiment, the composition containing at least one antigen presenting cell is sensitized or mounted with said peptide.

Alternatively, the antigen presenting cell contains an expression construct that encodes a peptide. The polynucleotide is any polynucleotide. In one embodiment, the polynucleotide can introduce dendritic cell traits such that peptides are presented and immunogenicity is induced.

Conveniently, the nucleic acids of the invention may be constructed from viral polynucleotides or viruses. For example, adenovirus transduced dendritic cells have been shown to induce antigen specific anti-tumor immunity with respect to MUC1 (Gong et al., (1997) Gene Ther. 4, 1023-1028). Similarly, adenovirus based systems may be used (eg, Wan et al., (1997) Hum. Gene Ther. 8, 1355-1363); Retrovirus systems may be used (Specht et al., (1997) J. Exp. Med. 186, 1213-1221 and Szabolcs et al., (1997) Blood particle-mediated transfer to dendritic cells may also be used (Tuting et al., (1997) Eur. J. Immunol. 27, 2702-2707); RNA may also be used (Ashley et al., (1997) J. Exp. Med. 186, 1177-1182).

In general, compositions of the invention comprising nucleic acid (s) of the invention may be administered similarly to those containing the peptide of the invention; For example, intravenous, intraarterial, intraperitoneal, intramuscular, intradermal, intratumoral, oral, skin, nasal, buccal, rectal, inhaled, or topical administration.

Due to the avoidance mechanism, tumors usually form resistance to therapeutic drugs. Drug resistance may occur during treatment or may appear in metastatic tissue and in recurred tumors. To avoid such drug resistance, tumors are usually treated with a combination of drugs, and different combinations are usually needed for tumors and metastases that recur after disease free time. Therefore, in one aspect of the invention, the composition is administered in combination with a secondary antigenic agent. Secondary formulations may be administered before, after, or simultaneously with the present disclosure composition. Simultaneous administration can be carried out by mixing the presently disclosed composition with a secondary anticancer agent, for example, if the chemical properties are correct. Another method of simultaneous administration is, for example, by oral administration of a secondary anticancer agent while the composition is administered by injection, in which the composition and the anticancer agent are administered on the same day separately from the route of administration. The composition and the secondary anticancer agent may also be administered within the same course of treatment but on different days and / or in separate courses of treatment.

In another embodiment, in providing a method for treating or preventing cancer in a patient, the method comprises administering to the patient any one of the disclosed patent compositions in a therapeutically effective dose.

The therapeutically effective dose is a dose sufficient to activate an immune response, specifically a subgroup of CTLs. One skilled in the art can easily determine whether a dose is effective using standard immunological methods such as those provided in the Examples herein. Another way to monitor the effect of certain doses of the composition is to observe the growth and / or recurrence of the treated tumor.

In another embodiment, the composition is used as an anticancer vaccine.

Compositions containing peptides or peptide-encoding nucleic acids can also constitute tumor or cancer vaccines. This may be administered directly to the patient, to the affected organ or systemic, to exfoliate to a cell derived from the patient, ie a human cell line, and then to the patient, or to a subpopulation of immune cells derived from the patient (again May be used in vitro to select).

In one aspect, the vaccine is a multi-peptide tumor vaccine for the treatment of prostate cancer. In another aspect, the vaccine contains a tumor associated peptide combination selected from SEQ ID NO: 1 to SEQ ID NO: 11 and SEQ ID NO: 13 to SEQ ID NO: 14 located in primary prostate cells and / or prostate carcinoma. Such combinations include HLA class I and class II peptides. The peptide combination may also include at least one peptide, such as a peptide of the influenza core antigen, where the peptide is used as a positive control peptide to serve as an immune marker to test the effectiveness of intradermal administration. As an example of a particular embodiment, wherein the vaccine consists of 14 individual peptides (according to SEQ ID NOs: 1 to 14), the dose of each peptide is about 1500 to about 75 μg, about 1000 to about 175 μg of each peptide. At a dose selected from about 500-600 μg, about 578 μg, all of which are purified by HPLC and ion exchange chromatography to appear white to off-white powder. The lyophilisate can be dissolved in sodium bicarbonate and used for intradermal injection within 30 minutes after reconstitution at room temperature. The total amount of peptide per 500 μl of solution may vary from about 0.1 to 100 mg, about 0.1 to 1 mg and about 300 μg to 800 μg. The word "about" in this specification means ± 10 percent of a given value unless otherwise specified. One skilled in the art will be able to adjust the actual amount of peptide to be used based on a number of factors, such as, for example, the immune status of an individual patient and / or TUMAP present in a particular cancer type. The peptide may be provided in other suitable form (sterile solution, etc.) instead of lyophilisate.

The composition may contain the peptide in free form or in the form of a pharmaceutically acceptable salt.

As used herein, “pharmaceutically acceptable salts” refers to derivatives of the peptides of this disclosure, where the peptide is modified by making the agent an acid or base salt. For example, acid salts are formulated from free bases that typically contain a reaction with an appropriate acid (typically when the neutral form of the drug has a neutral -NH ₂ group). Acids suitable for preparing acid salts are organic acids (e.g. acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methane) Sulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like) and inorganic acids (eg, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like). In contrast, preparations of basic salts of acid residues that may be present in the peptide are prepared using pharmaceutically acceptable bases such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide, trimethylamine or the like. As an example, but not limited thereto, the composition may contain a peptide as a salt of acetic acid (acetate), ammonium, or hydrochloric acid (chloride).

As another example, the composition may include sugars, sugar alcohols, amino acids (glycine, arginine, glutamic acid, etc.) as a framework former. The sugar may be monosaccharide, disaccharide or trisaccharide. These sugars may be used alone or in combination with sugar alcohols. Examples of sugars include glucose, mannose, galactose, fructose or sorbose, disaccharides such as sucrose, lactose, maltose and trehalose, and trisaccharides include raffinose. Sugar alcohols may include, for example, mannitol. In one embodiment, the composition comprises sucrose, lactose, maltose, trehalose, mannite and / or sorbet. In another embodiment, the composition comprises mannitol.

Furthermore, the compositions are physiologically resistant excipients such as antioxidants such as ascorbic acid or glutathione (see Handbook of Pharmaceutical Excipients, 5 ^th ed., Edited by Raymond Rowe, Paul Sheskey and Sian Owen, Pharmaceutical Press (2006)). Preservatives such as phenol, m-cresol, methyl- or propylparaben, chlorobutanol, thiomersal or benzalkonium chloride, stabilizers, sucrose, lactose, maltose, trehalose, mannose, mannite and And / or frame formers such as sorbet, mannite and / or lactose, etc. and polyethylene glycol (PEG), ie PEG 3000, 3350, 4000 or 6000, or cyclodextrins, ie hydroxypropyl-ß-cyclodextrin, sulfobutylethyl -ß-cyclodextrin or γ cyclodextrin, or dextran or poloxamer (poloxamer), ie poloxamer 407, poloxamer 188, or Tween ) 20, Tween (may include agents such as R) 80. In another aspect, one or more resistant excipients selected from the group consisting of antioxidants, frame formers and stabilizers may be included.

In another aspect, the pH for intravenous and intramuscular administration is selected from pH 2 to pH 12, while the pH for subcutaneous administration is lowered from pH 2.7 to pH as the rate of dilution in the body decreases, increasing the likelihood of radiation therapy at the injection site. selected at pH 9.0. Strickley Robert G., Pharm. Res., 21, NO: 2, 201-230 (2004).

In another aspect, a pharmaceutical preparation comprising a peptide and / or nucleic acid according to the invention is administered to a patient suffering from adenomatous or cancerous diseases associated with the peptide or antigen. This may initiate a T-cell mediated immune response.

In another aspect, the amount of peptide (s), nucleic acid (s), or expression mediator (s) according to the invention (particularly tumor-associated) is present in tissue, cancer and / or patient specific compositions. To the public.

In another embodiment, the vaccine is a nucleic acid vaccine. It is known that inoculation with nucleic acid vaccines such as DNA vaccines encoding polypeptides leads to T-cell responses. This may be administered directly to the patient (in affected organs or whole body) or dislodged into cells taken from the patient. Application, ie, applied to a human cell line and then administered again to the patient, or a subpopulation may be selected from immune cells taken from the patient and used in vitro and then administered again to the patient. When the nucleic acid is administered to the cell in vitro, it may be useful for the cell to be transfected to co-express an immune stimulating cytokine such as interleukin-2 or GM-CSF. The nucleic acid (s) can be administered in a fairly pure or in combination with an immune stimulatory adjuvant or in combination with an immune stimulating cytokine or using an appropriate delivery system such as for example liposomes. The nucleic acid vaccine can also be administered with adjuvant such as those described for the peptide vaccine above. The nucleic acid vaccine can be administered without adjuvant.

Such polynucleotides may be quite pure or may be contained within a suitable mediator or delivery system. Suitable mediators and delivery systems include viral systems such as adenoviruses, vaccine viruses, retroviruses, herpes viruses, adeno-associated viruses, or hybrid based systems comprising one or more viral elements. Nonviral delivery systems include cationic lipids and cationic polymers that are well known in the art of DNA delivery. Physical delivery, such as through a "gene-gun", can also be used. The peptide encoded by the present peptide or nucleic acid may be, for example, a fusion protein having an epitope present in a tetanus toxin that stimulates CD4-positive T-cells.

Suitably, all nucleic acids administered to the patient are sterilized and the pyrogenic material removed. Naked DNA can be administered by intramuscular, intradermal, or subcutaneous routes. Conveniently, the nucleic acid vaccine may contain any suitable nucleic acid delivery means. The nucleic acid may also be delivered in liposomes or as part of a viral mediator delivery system. In one embodiment, nucleic acid vaccines such as DNA vaccines are administered into the muscle. In another embodiment, the peptide vaccine is s.c. Or i.d. Administered by route. In another embodiment, the vaccine is administered into the skin.

It is believed that the uptake of the nucleic acid and the expression of the encoded polypeptide by specialized antigen presenting cells, such as dendritic cells, may be the starting mechanism of the immune response; However, dendritic cells may not be transfected but are still important. Because they can obtain expressed peptides from cells transfected in tissues (cross-start), for example Thomas AM, Santarsiero LM, Lutz ER, Armstrong TD, Chen YC, Huang LQ, Laheru DA, Goggins M, Hruban RH, Jaffee EM.Mesothelin-specific CD8 (+) T cell responses provide evidence of in vivo cross-priming by antigen-presenting cells in vaccinated pancreatic cancer patients.J Exp Med. 2004 Aug 2; 200 (3) : 297-306).

Polynucleotide-mediated immunotherapy for cancer is described by Conry et al., (1996) Seminars in Oncology 23, 135-147; Condon et al., (1996) Nature Medicine 2, 11221127; Gong et al., (1997) Nature Medicine 3, 558-561; Zhai et al., (1996) J. Immunol. 156, 700-710; Graham et al., (1996) Int J. Cancer 65, 664-670; And Burchell et al., (1996) 309-313 In: Breast Cancer, Advances in biology and therapeutics, Calvo et al., (Eds), John Libbey Eurotext, and others, which are incorporated herein by reference in their entirety. Are integrated.

Injection site, targeting agents, and any time by the delivery system or for the vaccine either by selective refining and talche administration of the peptide or nucleic acid of the cell population of the patient specific cell populations may be useful to target to (for example, antigen-presenting cells) Dendritic cells may also be described (eg, as described in documents such as Zhou et al., (1995) Blood 86, 3295-3301; Roth et al., (1996) Scand. J. Immunology 43, 646-651, etc.). Can be screened). For example, the target mediator may contain tissue or tumor-specific promoters that allow the antigen to be expressed at a suitable location.

The vaccine can vary depending on the particular type of cancer the patient being treated with, as well as the condition of the disease, previous treatment method, the patient's immune status, and also the HLA- haplotype of the patient. Moreover, the vaccine may contain components that are tailored to the individual needs of the particular patient. For example, varying amounts of peptides depending on the expression of the relevant TAA in a particular patient, unwanted side effects from personal allergies or other treatments, or coordination of secondary treatment after the first treatment or treatment plan.

In addition to being useful for treating cancer, the peptides disclosed herein are also useful for diagnosis. Since many of the peptides were produced in prostate carcinoma and it was determined that these peptides were not present in normal tissues, these peptides could be used to diagnose the presence of cancer.

The presence of these peptides in tissue biopsies can help diagnose pathology cancer. Detection of some of the peptides published by antibodies, mass spectroscopy or other methods known in the art can inform the pathologist whether the tissue is malignant, inflammatory or a general disease. The presence of peptide groups disclosed herein may allow for the classification or subclassification of diseased tissue.

The detection of the peptides disclosed above in a diseased tissue sample can result in the determination of therapeutic benefit, including the immune system, particularly if T-lymphocytes are known or expected to be involved in the mechanism of action. Loss of MHC expression is a well described mechanism whereby infected malignant cells avoid immunosurveillance. Therefore, the presence of peptides disclosed herein shows that this mechanism is not utilized by the cells analyzed.

Peptides disclosed herein can be used to analyze lymphocyte responses to these peptides, such as T-cell responses or antibody responses to the peptides or peptides complexed to the MHC molecule. These lymphocyte responses can be used as prognostic indicators for the determination of further treatment steps. These responses can also be used as surrogate markers in immunotherapeutic approaches aimed at inducing lymphocyte responses by different means (eg, protein vaccination, nucleic acid, autologous material, protonation of lymphocytes, etc.). In a gene therapy environment, lymphocyte response to peptides disclosed herein can be considered in evaluating side effects. In addition, monitoring of lymphocyte response can be a valuable tool for the follow-up of transplantation therapy, for example for the detection of graft versus host and host to host transplant diseases (GVHD and HVGD).

In another aspect to the above, (a) a container containing the composition described above in solution or lyophilized form; (b) optionally, a second container containing a diluent or a reconstituted solution of a lyophilized formulation; And (c) optionally, instructions for (i) the use of a solution or (ii) the reconstitution and / or lyophilized formulation. The kit may also include one or more (iii) buffers, (iv) dilutions, (v) filters, (vi) needles, or (v) syringes. In one embodiment, the container may be selected from the group consisting of bottles, vials, syringes, test tubes, or multipurpose containers. In another embodiment, the composition is lyophilized.

In one aspect, the kit may consist of a lyophilized formulation and / or vaccine of a composition disclosed herein contained in a suitable container for its reconstitution and / or instructions for use. Examples of suitable containers include bottles, vials (eg, twin chambers), syringes (such as twin chambers), and test tubes. The container may be made of various materials such as glass or plastic. In one embodiment, the kit and / or container includes instructions indicated on or in connection with the container surface indicating instructions for reconstitution and / or use. For example, the label may indicate that the lyophilized formulation should be reconstituted to the peptide concentration described above. The label may also indicate that the formulation is suitable or suitable for use for subcutaneous administration.

The container containing the formulation may use a multi-use vial capable of repeat administration of the reconstituted formulation (eg 2-6 administrations). The kit may also include a secondary container containing a suitable diluent (eg, sodium bicarbonate solution).

In one embodiment, when mixing the diluent with the lyophilized formulation, the final peptide concentration of the reconstituted formulation is at least 0.15 mg / mL / peptide (= 75 μg) and 3 mg / mL / peptide (= 1500 μg). Do not go over. The kit may also include other materials desirable from a commercial or user standpoint, including other buffers, diluents, filters, needles, syringes, instructions for use in packaging.

The kit may comprise a single container containing a composition formulation containing other components (eg, other compounds or compositions of these compounds) or may comprise containers that are independent for each component.

In addition, the kit may be used in combination with a secondary compound (eg, an adjuvant (eg, imiquimod), a chemotherapeutic agent, a natural product, a hormone or antagonist, an anti-angiogenic agent or inhibitor, an apoptosis inducer or chelator, etc.) or a composition thereof. Compositions and / or vaccine formulations disclosed herein packaged for use with administration. The components of the kit may be prepared in advance in a complex or contained in separate individual containers before each component is administered to the patient. The components of the kit may be provided in one or more solutions. In one embodiment, the solution is an aqueous solution. In another embodiment, the solution is a sterile aqueous solution. The components of the kit can also be supplied as a solid that can be converted into a liquid by the addition of a suitable solvent which can be supplied in another separate container.

The container of the treatment kit may be a vial, a test tube, a flask, a bottle, a syringe, or other tool for holding a solid or liquid. Usually when there is more than one component, the kit may include a second vial or other container for separate infusion. The kit may also include another container that can contain a pharmaceutically acceptable liquid. In one embodiment, the treatment kit may comprise an apparatus (eg, one or more needles, syringes, eye drops, pipettes, etc.) that allow for administration of the presently disclosed formulations that are components of the kit.

The pharmaceutical formulation may be suitable for administration of the peptide by acceptable routes such as oral (via intestinal route), nasal, eye, subcutaneous, intradermal, intramuscular, intravenous or transdermal. In one aspect, the administration is in a subcutaneous route and may be performed by an infusion pump.

The term "T-cell response" refers to the specific proliferation and activation of effector action induced by peptides in vitro or in vivo. For MHC class I restricted CTLs, the effector function can be peptide sensitization, peptide precursor sensitization, or lysis of natural peptide presenting target cells, secretion of cytokines, secretion of functional group molecules, or degranulation. In one embodiment, the T-cell response is the secretion of cytokines induced by the peptide, in which the peptide is secreted in the group consisting of interferon-γ, TNF-α, or IL-2. In one embodiment, the T-cell response is the secretion of effector molecules induced by the peptide, in which the effector molecule is secreted in the group consisting of granzyme and perforin. For MHC class II constrained T-helper cells, the effector action may be secretion of cytokines induced by peptides such as IFN-γ, TNF-α, IL-4, IL-5, IL-10 or IL- 2, or but not limited to peptide-derived degranulation. Possible effector functions of CTL and T-helper cells are not limited to this list.

Further disclosed are compositions consisting of a combination of amino acid sequences of peptides capable of binding to molecules of human major histocompatibility complex (MHC) class I (HLA class I) or II (HLA class II).

The compositions can be used as an effective antiprostate cancer vaccine based on a combination of several peptides.

Another aspect is a method of treating prostate cancer comprising administering any composition disclosed herein to a prostate cancer patient. These compositions can be administered with or without the accompanying treatment with an adjuvant. When an adjuvant is used, it may be included in the compositions disclosed herein, or may be administered separately through the same route of administration or through another route of administration.

The compositions and methods disclosed herein can be used as primary, adjuvant, or palliative therapies, either alone or in other therapies including but not limited to surgical therapy (including prostatectomy), radiation therapy, and chemotherapy. Can be used with The methods of this disclosure can also be used for primary tumors, biological relapses, local relapses, or metastatic relapses. The methods disclosed herein may also be used before, with or after the administration of androgen deprivation therapy in both androgen-sensitive and androgen-insensitive cancers.

As used herein, the phrase “androgen-sensitive prostate cancer” refers to all prostate cancers that require androgen for tumor growth.

As used herein, the phrase “androgen-insensitive prostate cancer” refers to all prostate cancers in which tumor growth occurs without androgen.

As used herein, the phrase “androgen deprivation therapy” refers to all the treatments whose primary effect is to inhibit androgen signaling or androgen production.

The following examples illustrate certain aspects and are not intended to limit the invention. All references cited herein are hereby incorporated by reference in their entirety.

Example

Example  One

HLA binding peptide cocktails have no diagnostic evidence of metastasis, but have been tested in patients with biochemical recurrence following radical prostatectomy to determine whether these compositions are effective in stabilizing and / or increasing the PSA doublet time (DT).

Patient selection

All patients showed biochemical recurrence after initial curative treatment with radical prostatectomy. Biological recurrence was defined as an increase of 50% or more from the lowest level of PSA assessed twice, at least 14 days after surgery, surgery and radiation. Other eligibility criteria are: Exclusion of metastatic disease or local tumor recurrence in bone scan, axial imaging, and CT determination, Eastern Oncology Cooperative Group (ECOG) performance status 0 or 1, 45 to 80 Age of age, exclusion of corticosteroids or other immunomodulatory therapies; Exclusion of concurrent therapy of radiation, hormones or chemotherapy; Exclusion of other malignant, epileptic or pulmonary diseases. At the time of entry in this study, all patients were androgen-sensitive and had stopped androgen deprivation therapy for at least 12 months prior to selection. All patients were HLA-A * 02 positive. Patient characteristics are shown in Table 1 below.

[Table 1]

cure

To prevent tumor evasion through genetic variation and antigen loss, multiple recognition peptide compositions were used that target a broad spectrum of specific T-cells. The composition consisted of 13 synthetic HLA binding peptides specific for both HLA class I and class II from prostate associated antigen molecules for activation of cytotoxic CD8 + and CD4 + T-helper cells. Eleven of the peptides (SEQ ID NO: 1 to SEQ ID NO: 11) consisted of HLA-A * 0201 restriction epitopes. Two peptides (SEQ ID NO: 13 and SEQ ID NO: 14) consisted of HLA class II binding epitopes. An additional peptide consisting of HLA-A * 0201 epitope from influenza virus (SEQ ID NO: 12) was added as a marker peptide that activates the memory CD8 + T-cell response. Peptides were emulsified with 500 ml Montanide ISA-51 and then injected subcutaneously at 300 mg per individual peptide.

In addition, patients were randomly selected by (1) adjuvant; (2) receive heat therapy; Or (3) no adjuvant or thermotherapy. The adjuvant used was (1) imiquimod (Aldara, Meda Pharma, Bad Homburg, Germany), (2) GM-CSF (Leukine®), Bayer Healthcare (Bayer Healthcare), Leverkussen, Germany), (3) Schel et al. Therapeutic anti-tumor immunity triggered by injections of immunostimulating single-stranded RNA, Eur. J. Immunology 2006; Mucin-1-mRNA / protamine combination described in 36 (10): 2807-2816.

Three patients (Patients 1, 2 and 5) did not receive adjuvant or thermotherapy.

Four patients (patients 16, 17, 18 and 19) received mucin-1-mRNA / protamine combination as adjuvant. For patients receiving mucin-1-mRNA / protamine combination, adjuvant 110 was emulsified with peptide and Montanide ISA-51. All ingredients were administered in one injection.

Four patients (patients 3, 7, 8 and 11) received imiquimod as an adjuvant. For four patients, imiquimod ointment was applied topically to the injection site following peptide treatment and then dressing. Patients were asked to wash the imiquimodized area with water after 8 hours.

Six patients (patients 4, 6, 9, 12, 14 and 15) received GM-CSF as adjuvant. For these patients, the adjuvant was injected at a dose of 225 μg, shallow subcutaneous at the injection site.

Two patients (patients 10 and 13) underwent thermotherapy. For these patients, the heat source maintained at 41 ° C. was directed to the exposed 20 cm ² abdominal skin in situ immediately after injection and held for 20 minutes.

Treatment using the peptide composition, adjuvant and / or thermotherapy was repeated at 7, 14, 28, 42 and 56 days after the initial treatment using the same site, respectively. Therefore, if PSA levels returned to normal or showed stabilization, treatment continued every 4 weeks up to 420 days.

Response evaluation

Treatment response was evaluated using the PSA values measured at each visit as surrogate parameters. Hematology and blood chemistry tests were repeated every three months after the first six treatments (8 parkings). Clinical examination and rectal balance examination (DRE) were performed on a similar schedule for evaluation of clinical course.

Data Analysis

Responses were evaluated by calculating the doubling time geometric mean. The logarithm of PSA levels for each patient was adjusted to a straight line with varying slopes. The intersection between the two slopes, the other slopes and the initial values were estimated by the least squares method using a nonlinear fitting routine. Complete response was defined as the case where no PSA levels were measured; Some reactions resulted in a 50% drop in PSA levels; Steady state is a decrease of less than 50% or an increase of less than 10%; And progression was defined as an increase of at least 10% above baseline PSA levels. These measurements had to be confirmed after 4 weeks. The biochemical response of the patients who ended the study was followed until further treatment with local radiation or androgen deprivation therapy.

result

The mean PSA doubling time (DT) before treatment was 8.4 months in all patients and 11.2 months at the end of treatment. Four patients (21%) showed biochemical therapeutic benefit. Clinical tumor recurrence was detected by an anal resin test in patients with advanced PSA levels and confirmed by PET-CT. Treatment response, or PSA levels, varied among patients and could be categorized into five response groups. The data is summarized in FIG.

PSA  Stability and DT Increase

Two patients (patients 3 and 8; 11%) showed PSA stability (FIG. 3) during the treatment period and at 14 and 16 months follow-up after the last treatment. The mean stability duration after initiation of treatment was 29.5 months with data cutoff in an average of 17 treatment runs (14 and 20). Patient 3 had some PSA response (> 50%) for 9 months and then slowly increased PSA levels to 20.5 months compared to 9.8 months of doubling time before treatment. Initial PSA recurrences before the start of the study began 18 months after prostatectomy (pT2pN0 GS 5). Patient 3 stopped participating due to allergic reaction at the 20th treatment point. Patient 8 showed a steady state pattern after the start of treatment. The patient discontinued treatment due to an allergic reaction at the 14th treatment point 10 months later. The patient was a pT3b Gleason 3 + 4 tumor with a PSA nadir of 0.6 ng / ml after radical prostatectomy. DT was increased from 6.6 months to 148.0 months. Both patients received intradermal imiquimod as an adjuvant (FIG. 4).

PSA  Without stability PSA DT Increase

Two patients (patients 11 and 16, 11%) had increased PSA DT during the treatment period and increased PSA levels at the same time. PSA DT in patient 11 increased from 1.5 months to 10.1 months after the first 6 months of treatment. The patient began treatment with a PSA level of 10.8 ng / ml and increased to 17.8 ng / ml 6 months after treatment. The study ended and hormone replacement therapy began. In this patient, no gross malignant lesions were observed on PETCT. Imiquimod was used as an adjuvant. For patient 16, the DT was 6.1 months at the start of the study. PSA levels in this patient decreased and the DT changed to 2.7 months after the first 5 months of treatment. The patient's DT then increased to 14.4 months and this condition persisted for 16 months after the start of treatment. At the start of treatment, the patient's PSA level was 0.29 ng / ml and 0.41 ng / ml at the end of follow-up. This patient received mucin-1-mRNA / protamine combination as an adjuvant (FIGS. 3 and 4).

Treatment medium PSA  After rise PSA  Decrease and PSA DT Increase

One patient (5, 5%) had increased PSA regardless of treatment after initiation of treatment and discontinued treatment after eleventh peptide administration when the PSA level was 1.31 ng / ml. Thereafter, the patient's PSA decreased and DT increased to 20.2 months until the end of follow-up; This patient did not receive any further treatment during this period. For this patient no adjuvant was used and the peptide was emulsified alone in Montanide (Figures 3 and 4).

for a bit PSA  After reduction or stabilization PSA Rapidly rising

Three patients (Patients 7, 15 and 17, 16%) remained or decreased PSA levels, which then rose rapidly. Patient 7's DT was 3.7 months at the start of treatment and increased to 21.5 months for 4 months of treatment, after which the DT continued to decrease to 2.8 months. This patient had pT2b stage cancer that was positive for surgical resection. This patient refused local radiotherapy (FIG. 3). DT of patient 15 was 1.3 months at the beginning of the study. Prior to the first treatment, the last two consecutive PSA levels were measured at our hospital for a four month period. As a result of the laboratory differences, the PSA DT changed from 1.3 months to 25.8 months. During the treatment with GM-CSF, DT decreased to 9.9 months and remained stable for 6 months. Since then, the PSA has progressed and the DT has reached 7.4 months. Interpretation of these PSA patterns was hampered by short-term changes in baseline PSA DT prior to initiation of treatment. For patient 17, PSA DT decreased from 10.2 months to 4.8 months during treatment, after decreasing to 1.9 months of PSA half-life within the treatment period, and this condition persisted for two months and then increased PSA (FIG. 3).

PSA Progress of

PSA levels in 11 patients (58%) progressed to a constant PSA DT state during the study, leading to early termination of the study (FIG. 4). On average 13 treatments (range 718) were performed.

Impact of Adjuvant

Of the eight patients with elevated PSA DT or decreased PSA levels, four received imiquimod as an adjuvant. One patient received GM-CSF, two received mucin-1-mRNA / protamine combination, and one received no adjuvant. None of the two patients treated with topical thermotherapy received a response, ie clinical benefit (FIG. 4).

Example  2

The reactivity of specific HLA class I binding peptides included in the multi-peptide cocktail was tested after treatment in Example 1.

In vitro amplification of specific T-cells

Peripheral blood mononuclear cells from patients with prostate cancer were collected at various time points during vaccination and cryopreserved with liquefied nitrogen in 90% fetal bovine serum and 10% DMSO. After thawing, about 5 × 10 ⁶ cells were treated with 50 U / ml penicillin, 50 μg / ml streptomycin (both from Biowhittaker (Bechbeey, Belgium)), 10% heat inactivated human serum (cc pro, Cultured at 37 ° C. and 7.5% CO ₂ in IMDM medium with 50 μM β-mercaptoethanol. Mixed synthetic HLA class I or HLA class II binding peptides were added on day 1 at a concentration of 1 μg / ml for HLA class I and 5 μg / ml for HLA class II. The solution was prepared for recombinant human IL-2 (r-hIL-2, R & D Systems GmbH, Vesbaden, Germany) at the 3, 5, 7 and 9 days of T-cell stimulation for HLA class I, Promokine and IL-4 and 7 were added at day 0, and for HLA class II, recombinant human IL-2 (r-hIL- at time 3, 5, 7 and 9 at T-cell stimulation 2, R & D Systems GmbH and Promokin were added.

Enzyme-linked immunosorbent sites ( ELISAPOT ) Analysis (enzyme immunoassay)

Functionality of the expanded T-cells was examined by standard interferon-γ ELISPOT assay according to the Immunoguiding Program (CIP) of the Cancer Immunotherapy Association. In summary, cells were harvested on day 12 of culture, washed, counted and then seeded in culture medium on ELISPOT plates (Millipore, Schwarbach, Germany). About 0.20 to 0.40 x 10 ⁶ cells are (i) with the peptide-presenting cell line K562A2 and each individual HLA class I binding peptide at a concentration of 1 μg / ml, or (ii) each individual HLA class II at a concentration of 2.5 μg / ml The test was repeated 2-3 times in the presence of the binding peptide. PHA (10 μg / ml) or SEB (1 μg / ml) was used as a positive control stimulant. IFN- [gamma] production was produced by a pair of specific monoclonal antibodies (1D1-k and 7-B6-1, both from Mabtech, Nakka Strand, Sweden) after 26 hours of incubation at 37 ° C. and 7.5% CO ₂ . ). Extraavidinalkaline phosphatase and BCIP / NBT substrates (both from SigmaAldrich) were added for 1 hour and 10 minutes respectively. ELISPOT analysis was performed using an Equipped spot reader (Series 3A and 5, Cellular Technology Ltd, Allen, Germany).

Tables were recorded for each peptide of each patient for the presence of T-cells producing IFN-γ. As shown in FIG. 13, eight of the eleven HLA class I peptides elicited a response in at least one patient. The most common response was induced by PSMA 711 (SEQ ID NO: 7), which induced peptide reactivity in 25 of the 29 patients analyzed.

Example  3

The reactivity of specific HLA class II binding peptides included in the multi-peptide cocktail was also tested after treatment in Example 1.

Synthetic Peptides and Stimulants

Synthetic peptides used for stimulation and functional testing were HIV derived epitopes (HIV gag 164-181: YVDRFYKTLRAEQASQEV (SEQ ID NO: 15), negative controls; PSMA 459-473: NYTLRVDCTPLMYSL (SEQ ID NO: 13) and Servibin 97-111: TLGEFLKLDRERAKN) (SEQ ID NO: 14).

In vitro amplification of specific T-cells

Peripheral blood mononuclear cells from prostate cancer patients 15 and 26 were harvested at various time points during vaccination and cryopreserved with liquefied nitrogen in 90% fetal bovine serum and 10% DMSO. After thawing, approximately 5 x 10 ⁶ cells contained 50 U / ml penicillin, 50 μg / ml streptomycin (all from BioWitacker), 10% heat inactivated human serum (cc pro) and 50 μM β-mercaptoethanol. Incubated at 37 ° C. and 7.5% CO ₂ in added IMDM medium (24-well cell culture plate, Grainer BioOne, Freikenhausen, Germany). Mixed synthetic HLA class II binding peptides were added at the first day time point, 5 μg / ml each, and the culture medium was recombinant human IL-2 (r-hIL) at the 3rd, 5th, 7th and 9th day of T-cell stimulation. -2, R & D Systems GmbH. After 12 days of stimulation, cells were harvested, washed, counted and then stimulated again with peptide (10 μg / ml) for 6 hours. IFN-γ secreting cells were labeled with IFN-γ capture reagents and IFN-γ PE antibodies according to the MACS IFN-γ secretion assay protocol (Miltenyi Biotech, Bergheim Gladbach, Germany), followed by FACSAria IMDM 10 with 150 U / ml IL-2, 1 μg / ml PHA and irradiated allogenic feeder (PBMC + LG2-EBV) using Biosciences, Heidelberg, Germany 96 well plates containing% HS were sorted. IL-2 (150 U / ml) was added every 4 days and feeder cells every 3 weeks.

Intracellular  Cytokine Staining

The effectors were harvested, washed and then HIV, PSMA and SG in the presence of Golgi-STOP (BD Biosaise) and Brefeldin A (10 μg / ml, Sigma Aldrich) according to the manufacturer's instructions. 5 μg / ml of survivin peptide, or PMA and ionomycin (50 ng / ml and 1 μM, respectively) were stimulated by standard assays. After 4-6 hours of incubation, cells were washed in PBS, 2% FCS, 0.02% NaN ₃ and then monoclonal antibody (MoAb) CD4-APC-Cy7 (BD Biosciences) and CD8-PE-Cy7 (Burkman Coulter). (Beckman Coulter) for 20 minutes in the dark at 4 ℃. After the washing step, cells were permeabilized with cytofix / cytofirm reagent (BD Biosciences) and then stained for intracellular IFN-γ using monoclonal IFN-γ-FITC antibody (BD Biosciences). Was done. Cell acquisition was performed on Cytometer Canto II using software Diva and FlowJo (BD Biosciences) assays. In the multifunctional test, CD4-APC-Cy7 and CD8-PerCP were used for cell membrane staining and IFN-g PE-Cy7, TNF-Pacific Blue, IL-2-PE were used for intracellular staining (TNF-Pacific Blue, All products except Bioolegend. During the stimulation period 1.5 μl of CD107a-FITC (BD Biosciences) was added per test.

Functional Testing of T-Cell Clones Using Autologous Dendritic Cells

Monocyte-derived, immature autologous DCs were generated by incubating monocytes for 7 days in 50 μM β-mercaptoethanol supplemented with IMDM 10% HS, 1% PenStrep, 1000 U / ml IL-4 and 800 U / ml GM-CSF. . For functional experiments, DCs were loaded with the relevant HLA class II binding peptide (HIC, Survivin or PSMA, 10 μg / ml) or sensitized with recombinant protein (Servibin, PSMA or RAP-80, 20 μg / ml) The cells were harvested, washed several times, and incubated with specific CD4 + T-cell clones for 12 hours with a 1: 5 ratio of DC: effector before intracellular cytokine staining.

For control experiments, 1 μg of recombinant protein was used with 100 μg PBS, pH 7.2 using 10 μg proteinase K (Macherey-Nagel, Germany) in the presence of 1 mM CaCl _2. Pretreated at 37 ° C. for 2 hours. Alternatively, DC was fixed for 15 seconds in 0.05% final glutaraldehyde (Sigma). The reaction was stopped using 0.2 M L-lysine (Sigma) and DC was washed twice before loading the peptide or protein.

HLA  class II  Determination of restrictions

Peptide presentation assays used PBMCs pre-sensitized with peptides for 12 days in patients 15 and 26 as effector cells and stimulated EBV-modified cell lines loaded with peptide (10 μg / ml peptide, overnight mounted at IMDM 2% FCS). (See HLA-DR, DP and DQ alleles in FIG. 21) and performed at a ratio of one effector to one peptide presenting cell. After 5 hours co-culture, intracellular cytokine staining was completed as described previously.

                          <110> Immatics Biotechnologies GmbH   <120> HLA-BINDING PEPTIDES DERIVED FROM PROSTATE-ASSOCIATED        ANTIGENIC MOLECULES AND METHODS OF USE THEREOF <130> I32214PCT <140> PCT / EP2011 / 070024 <141> 2011-11-14 <150> PCT / EP2010 / 069675 <151> 2010-12-14 <160> 42 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Homo sapiens <400> 1 Phe Leu Thr Pro Lys Lys Leu Gln Cys Val 1 5 10 <210> 2 <211> 9 <212> PRT <213> Homo sapiens <400> 2 Lys Leu Gln Cys Val Asp Leu His Val 1 5 <210> 3 <211> 10 <212> PRT <213> Homo sapiens <400> 3 Val Ile Ser Asn Asp Val Cys Ala Gln Val 1 5 10 <210> 4 <211> 9 <212> PRT <213> Homo sapiens <400> 4 Ala Leu Gln Pro Gly Thr Ala Leu Leu 1 5 <210> 5 <211> 9 <212> PRT <213> Homo sapiens <400> 5 Ala Ile Leu Ala Leu Leu Pro Ala Leu 1 5 <210> 6 <211> 9 <212> PRT <213> Homo sapiens <400> 6 Leu Leu His Glu Thr Asp Ser Ala Val 1 5 <210> 7 <211> 9 <212> PRT <213> Homo sapiens <400> 7 Ala Leu Phe Asp Ile Glu Ser Lys Val 1 5 <210> 8 <211> 10 <212> PRT <213> Homo sapiens <400> 8 Glu Leu Thr Leu Gly Glu Phe Leu Lys Leu 1 5 10 <210> 9 <211> 10 <212> PRT <213> Homo sapiens <400> 9 Thr Leu Pro Pro Ala Trp Gln Pro Phe Leu 1 5 10 <210> 10 <211> 9 <212> PRT <213> Homo sapiens <400> 10 Gly Leu Met Lys Tyr Ile Gly Glu Val 1 5 <210> 11 <211> 9 <212> PRT <213> Homo sapiens <400> 11 Cys Leu Ala Ala Gly Ile Thr Tyr Val 1 5 <210> 12 <211> 9 <212> PRT <213> Homo sapiens <400> 12 Gly Ile Leu Gly Phe Val Phe Thr Leu 1 5 <210> 13 <211> 15 <212> PRT <213> Homo sapiens <400> 13 Asn Tyr Thr Leu Arg Val Asp Cys Thr Pro Leu Met Tyr Ser Leu 1 5 10 15 <210> 14 <211> 15 <212> PRT <213> Homo sapiens <400> 14 Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Lys Asn 1 5 10 15 <210> 15 <211> 14 <212> PRT <213> Homo sapiens <400> 15 Asp Phe Ile Ala Thr Leu Gly Lys Leu Ser Gly Leu His Gly 1 5 10 <210> 16 <211> 9 <212> PRT <213> Homo sapiens <400> 16 Ala Thr Val Leu Phe Gly Ile Ala Arg 1 5 <210> 17 <211> 10 <212> PRT <213> Homo sapiens <400> 17 Ala Val Cys Gly Gly Val Leu Val His Pro 1 5 10 <210> 18 <211> 11 <212> PRT <213> Homo sapiens <400> 18 Ala Val Cys Gly Gly Val Leu Val His Pro Gln 1 5 10 <210> 19 <211> 9 <212> PRT <213> Homo sapiens <400> 19 Asp Gln Leu Asx Phe Leu Glu Arg Ala 1 5 <210> 20 <211> 9 <212> PRT <213> Homo sapiens <400> 20 Asp Tyr Asn Phe Val Phe Thr Ser Phe 1 5 <210> 21 <211> 11 <212> PRT <213> Homo sapiens <400> 21 Glu Val Ile Gly His Tyr Pro Gly Ser Ser Phe 1 5 10 <210> 22 <211> 9 <212> PRT <213> Homo sapiens <400> 22 Glu Val Ile Thr Gly Ile Arg Ile Ile 1 5 <210> 23 <211> 9 <212> PRT <213> Homo sapiens <400> 23 Leu Leu Pro Pro Pro Pro Leu Leu Ala 1 5 <210> 24 <211> 9 <212> PRT <213> Homo sapiens <400> 24 Asn Ala Asp Pro Gln Ala Val Thr Met 1 5 <210> 25 <211> 9 <212> PRT <213> Homo sapiens <400> 25 Asn Tyr Glu Glu Thr Phe Pro His Ile 1 5 <210> 26 <211> 9 <212> PRT <213> Homo sapiens <400> 26 Ser Glu Ser Asp Thr Ile Arg Ser Ile 1 5 <210> 27 <211> 10 <212> PRT <213> Homo sapiens <400> 27 Ser Val Val Gly Gly Phe Val Ser His Tyr 1 5 10 <210> 28 <211> 9 <212> PRT <213> Homo sapiens <400> 28 Ser Tyr Pro Tyr Tyr Pro Tyr Leu Tyr 1 5 <210> 29 <211> 11 <212> PRT <213> Homo sapiens <400> 29 Thr Ile Ile Asp Ser Asp Lys Ile Met Val Leu 1 5 10 <210> 30 <211> 9 <212> PRT <213> Homo sapiens <400> 30 Thr Tyr Asp Phe Ala His Cys Thr Phe 1 5 <210> 31 <211> 10 <212> PRT <213> Homo sapiens <400> 31 Val Phe Asp Thr Ala Ile Ala His Leu Phe 1 5 10 <210> 32 <211> 9 <212> PRT <213> Homo sapiens <400> 32 Val Tyr Asn Pro Thr Pro Asn Ser Leu 1 5 <210> 33 <211> 10 <212> PRT <213> Homo sapiens <400> 33 Tyr Thr Ile Gly Leu Gly Leu His Ser Leu 1 5 10 <210> 34 <211> 9 <212> PRT <213> Homo sapiens <400> 34 Ala Pro Leu Leu Leu Ala Arg Ala Ala 1 5 <210> 35 <211> 9 <212> PRT <213> Homo sapiens <400> 35 Lys Arg Ala Thr Gln Ile Pro Ser Tyr 1 5 <210> 36 <211> 8 <212> PRT <213> Homo sapiens <400> 36 Met Arg Ala Ala Pro Leu Leu Leu 1 5 <210> 37 <211> 9 <212> PRT <213> Homo sapiens <400> 37 Met Arg Ala Ala Pro Leu Leu Leu Ala 1 5 <210> 38 <211> 9 <212> PRT <213> Homo sapiens <400> 38 Phe Thr Glu Leu Thr Leu Gly Glu Phe 1 5 <210> 39 <211> 9 <212> PRT <213> Homo sapiens <400> 39 Leu Met Leu Gly Glu Phe Leu Lys Leu 1 5 <210> 40 <211> 9 <212> PRT <213> Homo sapiens <400> 40 Glu Pro Asp Leu Ala Gln Cys Phe Tyr 1 5 <210> 41 <211> 15 <212> PRT <213> Homo sapiens <400> 41 Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Lys Asp 1 5 10 15 <210> 42 <211> 17 <212> PRT <213> Homo sapiens <400> 42 Glu Leu Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Lys 1 5 10 15 Asn     

Claims (15)

In a composition comprising at least two HLA binding peptides:
(a) at least one of said at least two HLA binding peptides comprises a fusion protein of SEQ ID NO: 23 comprising an 80 N-terminal amino acid of an epitope or HLA-DR antigen related constant chain according to SEQ ID NO: 23; It is a peptide to include,
(b) at least one of the at least two peptides is a peptide comprising an epitope selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11, SEQ ID NO: 13 to SEQ ID NO: 22, and SEQ ID NO: 24 to SEQ ID NO: 42,
Composition. The method of claim 1,
A composition comprising at least two peptides consisting of the amino acid sequence according to (b). The method of claim 1,
At least two peptides consisting of an amino acid sequence according to SEQ ID NO: 23, preferably at least four peptides, more preferably 10 peptides and SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, sequence At least one peptide selected from the group consisting of SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14 and at least one peptide selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 22 and SEQ ID NO: 24 to SEQ ID NO: 42 Comprising; The method of claim 3, wherein
Wherein said additional peptide is selected according to the HLA set of the patient to be treated. The method of claim 1,
At least four peptides consisting of an amino acid sequence according to SEQ ID NO: 23 and in the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9 to SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14 A composition comprising one peptide selected and at least one peptide selected from the group consisting of SEQ ID NO: 15 to SEQ ID NO: 22 and SEQ ID NO: 24 to SEQ ID NO: 42. The method of claim 5, wherein
At least one of said peptides is a class II peptide. 7. The method according to any one of claims 1 to 6,
An adjuvant, or a mixture of two or three adjuvant such as, for example, GM-CSF and imiquimod. The method of claim 7, wherein
Wherein said adjuvant comprises a toll like receptor agonist, for example Toll like receptor-7 agonist. 7. The method according to any one of claims 1 to 6,
A composition comprising at least one antigen presenting cell, for example dendritic cells such as autologous dendritic cells sensitized or mounted with a peptide. 10. The method according to any one of claims 1 to 9,
Compositions used in the treatment of prostate cancer. 11. The method of claim 10,
Wherein the prostate cancer is androgen-sensitive and the patient has not been subjected to androgen deprivation therapy. 11. The method of claim 10,
Wherein the prostate cancer is androgen-insensitive. A method of treating prostate cancer, comprising administering to a patient an effective dose of a composition according to any one of claims 1 to 9. The method of claim 13,
Wherein the prostate cancer is androgen-sensitive and the patient has not received androgen deprivation therapy. 15. The method of claim 14,
Wherein the prostate cancer is androgen-insensitive.

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