HK1112367B

HK1112367B - Survivin peptide vaccine

Info

Publication number: HK1112367B
Application number: HK08107684.8A
Authority: HK
Inventors: Mads Hald Andersen
Original assignee: 萨瓦克公司
Priority date: 2005-02-04
Filing date: 2006-02-03
Publication date: 2013-12-06

Description

Survivin peptide vaccine

All patent and non-patent references cited in this patent application are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to therapeutic vaccines comprising one or more survivin polypeptide fragments. The vaccines can be used for prophylactic, ameliorating and/or therapeutic treatment of, for example, cancer diseases. The invention further relates to methods of combination therapy.

Background

The mammalian immune system recognizes and reacts with foreign or heterologous materials. An important aspect of this system is the T cell response. This response requires that the T cells recognize and interact with a complex of cell surface molecules, known as Human Leukocyte Antigens (HLA) that make up the human Major Histocompatibility Complex (MHC), and peptides. The peptide is derived from a larger molecule, which is processed by the cell, which also presents HLA/MHC molecules. The interaction of T cells and HLA/peptide complexes is restricted, requiring that T cells be specific for a particular combination of HLA molecules and peptides. If no specific T cell is present, there is no T cell response even if its partner complex is present. Similarly, if the specific complex is not present and the T cell is present, there is no response.

It is well established that peptide epitopes derived from human Tumor Associated Antigens (TAAs) are recognized by Cytotoxic T Lymphocytes (CTLs) through MHC molecules, and that most, if not all, tumors express the antigen. Therefore, in strategies such as immunization and adoptive T cell therapy, exciting clinical efforts will target these TAAs to generate an effective anti-tumor CTL response in the patient.

However, immunoselection of antigen-depriving variants is a serious obstacle to the therapeutic potential of most known CTL epitopes in clinical oncology, and selection of antigen-deficient mutant tumors is a recognized limitation in therapeutic strategies when targeting antigens not involved in cancer growth. The reason is that most of the characterized peptides are derived from polypeptides that are not essential for the survival of tumor cells. Thus, if a potent CTL response against these peptide antigens is induced by therapeutic means (e.g., immunization), tumor cells lacking expression of the target antigens are highly likely to evade the resulting immune response.

There is a need for more effective therapeutic vaccines and improved methods of treatment for cancerous diseases.

The existence of mechanisms for the recognition of cellular abnormalities by T cells has also been implicated in cancer. In WO92/20356, a gene family is disclosed which can be processed into peptides which are then expressed on the cell surface and allow the lysis of tumor cells by specific CTLs. These genes are referred to as the MAGE family and are said to encode "tumor rejection antigen precursors" or "TRAP" molecules, and the peptides derived therefrom are referred to as "tumor rejection antigens" or "TRAs".

However, although it is generally accepted that most, if not all, tumors are antigenic, only some are truly immunogenic, i.e., the immune system can readily control tumor progression.

To overcome this deficiency, several immunotherapeutic trials were initiated, which were immunized with TAA-derived peptides. For melanoma (tumors that have been characterized to have the greatest number of TAAs defined by CTLs), immunization induced a potent CTL response against the antigen, and some patients had complete regression of the disease. However, most of the peptide epitopes used in these immunization experiments are melanocyte specific, and these peptides cannot be applied to tumors of non-melanocyte origin. Furthermore, these TAAs are expressed heterologously in tumors of different patients and even in tumor metastases obtained from the same patient. However, in recent years, a number of tumour specific peptide antigens have been identified which are expressed in a number of different cancers, namely HER-2, Muc-1 and telomerase.

It has also been shown that by appropriate treatment, tumor antigens present in tumors can be exposed to the immune system. Studies have shown that the CD8+ CTL branch of the immune response, alone or in combination with CD4+ Th cells, constitutes the primary anti-tumor effector branch of the adaptive immune response. Until now, the focus has mainly been on the CTL branch of the immune response. However, it becomes more clear that the CD4T cell response plays a necessary role in tumor rejection, especially in the induction phase in vivo or in the expansion of CTL responses. Thus, combining class 1 restricted tumor antigens into an effective tumor immunization protocol may increase the effectiveness of the vaccine.

Apoptosis is a genetic program of cell suicide, and apoptosis inhibition is implicated as an important mechanism involved in carcinogenesis, by extending the life span of cells, favoring the accumulation of transforming mutations. Survivin (survivin) is a recently identified member of the Inhibitor of Apoptosis Proteins (IAP) family. In a comprehensive gene expression analysis of about 4 million transcripts, survivin was identified as one of the first genes, which was consistently up-regulated in many cancers, but not in normal tissues. Solid malignancies overexpressing survivin include lung, colon, breast, pancreas, and prostate cancers as well as malignant hematopoietic tumors. In addition, a range of melanoma and non-melanoma skin cancers have been reported to be always survivin positive. Overexpression of survivin in most human cancers suggests a general role for apoptosis inhibition in tumor progression, which is confirmed by the following observations: for colorectal and bladder cancers, as well as neuroblastoma, survivin expression correlates with an adverse prognostic outcome. Survivin is expressed in endothelial cells during tumor angiogenesis as another inhibitor of apoptosis, and antisense targeting of survivin during angiogenesis causes endothelial cell apoptosis that promotes rapid collapse of capillary-like vessels in vitro. In contrast, survivin could not be detected in normal adult tissues. Since survivin is overexpressed in most human cancers and inhibition of its function can lead to increased apoptosis, this protein may be a target for therapeutic CTL responses. Survivin and its potential diagnostic and therapeutic applications are disclosed in US6,245,523, which is incorporated herein by reference. Survivin is a 16.5kDa cytoplasmic protein containing a single BIR and a multiply charged carboxy-terminal coiled-region replacing the RING finger structure, which inhibits apoptosis induced by withdrawal of growth factor (IL-3) when transferred into B-cell precursors. The gene encoding survivin almost corresponds to the sequence of the effector cell protease receptor-1 (EPR-1), but points in the opposite direction, thus suggesting that there are 2 isolated genes repeated in a head-to-head configuration. Survivin may therefore be described as an antisense EPR-1 product. Since the orientation of the 2 genes is reversed, the encoded amino acid sequences of the proteins are different.

Functionally, inhibition of survivin expression by up-regulating its natural antisense EPR-1 transcript causes massive apoptosis and decreases cell growth.

US6,245,523 discloses isolated and purified survivin and it provides nucleic acid molecules encoding survivin protein, and antibodies and other molecules that bind survivin. US6,245,523 also discloses anti-apoptotic active fragments of survivin protein and variants thereof, wherein amino acid residues are inserted at the N-or C-terminus of the disclosed survivin protein sequences, or within them. It is specifically disclosed that the peptide should contain key functional residues required for apoptosis, namely Trp at position 67, Pro at position 73 and Cys at position 84.

It has been previously disclosed that a load can be utilized as measured by the ELISPOT testDendritic cells of survivin peptide were immunized to generate a weakly specific T cell response (WO2004/067023 and Otto, k. et al, j. vaccine (2004)). The response is every 10⁴Individual cells were less than 30, and subsequent assessment of clinical outcome showed disease progression. The vaccine has only very limited use. It is therefore of great importance to develop vaccines that induce very strong specific T cell responses, especially clinical responses in which the disease progression is inhibited.

Brief description of the invention

The present invention is based on the finding that MHC class I restricted peptides derived from survivin protein are able to bind to MHC class I HLA molecules and thereby elicit CTL immune responses both ex vivo and in situ in patients with a wide range of cancers. Before use in therapy, the immune response must be analyzed in combination with clinical outcomes to assess the effectiveness of the vaccine composition in treating cancer. The specific T cell response after immunization may be a useful test against a potential antigen for use in a vaccine, preferably a vaccine composition capable of eliciting a very strong T cell response, e.g. every 10⁴There are more than 50 cells releasing INF- γ per PBMC as this may increase the success rate of vaccine compositions for the treatment of cancer. The present invention discloses particularly effective vaccine compositions that induce partial or complete tumor regression. It is evident that these findings open the way to new therapeutic approaches that are generally useful for the control of cancerous diseases, as survivin shows widespread expression in tumor cells.

The vaccine composition or immunogenic composition described herein can be used as a pharmaceutical composition because the peptide can generate a CTL response against neoplasia. Therefore, the vaccine composition can be used as a therapeutic vaccine or a therapeutic or pharmaceutical composition.

In one aspect, the invention relates to a vaccine composition comprising one or more survivin peptide or survivin peptide variants (wherein the sequence of the peptide variant is at least 85% identical over the entire length to the contiguous amino acid sequence of SEQ id no: 23) and an adjuvant formulated for a water-in-oil emulsion comprising a mineral oil and a surfactant, wherein the adjuvant contains up to 14.5 volume percent of the surfactant), for use as a medicament.

One aspect of the present invention relates to survivin peptide variants of up to 50 amino acid residues, capable of binding HLA-B7, comprising a peptide selected from the group consisting of: APPAWQPFL (SEQ ID NO: 13) and RPPAWQPFL (SEQ ID NO: 14).

Other aspects relate to vaccine compositions comprising one or more survivin peptides or peptide variants, wherein the sequence of the peptide variant is identical over the entire length to the sequence of SEQ ID NO: 23, and wherein the composition comprises:

HLA-B7 binding peptides and/or

HLA-A1 and HLA-A2 restricted peptides and/or

HLA-A1 and HLA-B35 restricted peptides

And an adjuvant.

One aspect relates to a vaccine composition comprising three or more survivin peptides or survivin peptide variants, wherein the sequence of the peptide variants is identical over the entire length to the sequence of SEQ ID NO: 23, and wherein,

i. at least one peptide or peptide variant is selected from the group of HLA-A1 binding peptides, and wherein

At least one peptide or peptide variant is selected from the group of HLA-A2 binding peptides, and wherein,

at least one peptide or peptide variant is selected from the group of HLA-B35 binding peptides,

and an adjuvant.

Another aspect of the invention relates to a vaccine composition comprising seven or more survivin peptides or survivin peptide variants, wherein the sequence of the peptide variants is identical over the entire length to the sequence of SEQ ID NO: 23 are at least 85% identical,

i. and wherein at least one peptide or peptide variant is selected from the group of HLA-A1 binding peptides,

and wherein at least one peptide or peptide variant is selected from the group of HLA-A2 binding peptides,

and wherein at least one peptide or peptide variant is selected from the group of HLA-A3 binding peptides

And wherein at least one peptide or peptide variant is selected from the group of HLA-A24 binding peptides

v. and wherein at least one peptide or peptide variant is selected from the group of HLA-a11 binding peptides

And wherein at least one peptide or peptide variant is selected from the group of HLA-B35 binding peptides

And wherein at least one peptide or peptide variant is selected from the group of HLA-B7 binding peptides and adjuvants.

The development of solid tumors is dependent on angiogenesis. Inhibition of angiogenesis can therefore prevent the development of solid tumors. Survivin, bcl-2 and Mcl-1 are expressed in endothelial cells during tumor angiogenesis, suggesting that immunization with the peptides described herein may have an anti-angiogenic effect.

One aspect of the invention relates to a vaccine comprising one or more survivin peptides or peptide variants (wherein the sequence of the peptide variant is at least 85% identical over the entire length to the contiguous amino acid sequence of SEQ ID NO: 23) and an adjuvant capable of inducing infiltration of antigen-specific T cells in the tumor stroma of a subject for use as a medicament.

In another aspect, the present invention relates to a vaccine composition comprising:

i. a nucleic acid encoding:

a) survivin polypeptide (SEQ ID NO: 23),

b) survivin peptide or

c) A survivin peptide variant, wherein the sequence of the peptide variant is identical over the entire length to SEQ ID NO: 23 is at least 85% identical in consecutive amino acid sequences, and

an adjuvant.

One aspect of the invention relates to the use of a vaccine composition comprising one or more survivin peptides or survivin peptide variants and an adjuvant, which vaccine composition is capable of inducing a very strong specific cytotoxic T-cell response in a subject, for the manufacture of a medicament. This is of great significance, since a very strong specific T-cell response can be associated with a high good clinical response rate. In this regard, a very strong specific T cell response is equivalent to every 10 when measured by the ELISPOT assay before and after administration of the vaccine composition⁴One PBMC cell responded with more than 50 peptide-specific spots,

one aspect of the present invention relates to kits (kit in parts) comprising components, including;

i. a vaccine composition comprising;

a) one or more survivin peptides or survivin peptide variants, wherein the sequence of the peptide variant is identical over the entire length to the sequence of SEQ ID NO: 23 are at least 85% identical,

b) and an adjuvant, wherein the adjuvant is a mixture of,

and

a second drug.

One aspect of the invention describes a method of stimulating a strong specific T cell response against survivin in a subject, the method comprising:

a) there is provided a vaccine composition according to the invention,

b) administering the vaccine composition to a subject, wherein the vaccine composition can be administered more than once; and

c) thereby stimulating a strong specific T cell response in the subject, wherein the strong specific T cellThe cellular response is every 10 as measured by the ELISPOT assay before and after administration of the vaccine composition⁴One PBMC cell has more than 50 peptide-specific spots,

d) obtaining a strong specific T cell response in a subject

Another aspect of the invention relates to a method of treating or preventing a disease, comprising;

a) there is provided a vaccine composition according to the invention,

b) administering the vaccine composition to a subject, wherein the vaccine composition can be administered more than once.

In yet another aspect, the present invention relates to a method of treating or preventing a disease, comprising;

a) there is provided a vaccine composition according to the invention,

c) Thereby stimulating a strong specific T cell response in the subject, wherein said strong specific T cell response is every 10 as measured by an ELISPOT assay before and after administration of the vaccine composition⁴One PBMC cell has more than 50 peptide-specific spots,

d) a clinical response is obtained in the subject.

In certain aspects, the invention relates to a method of inducing antigen-specific T-cell infiltration in a tumor stroma of a subject or inhibiting angiogenesis in a subject, comprising;

a) there is provided a vaccine according to the present invention,

b) administering to the subject the vaccine composition to the subject,

c) antigen-specific T-cell infiltration or inhibition of angiogenesis is achieved in the tumor stroma.

A method of combination therapy comprising the separate administration simultaneously, sequentially or in any order:

a) the vaccine composition according to the present invention and,

b) a second drug.

Defining:

AA: see "amino acids".

Adjuvant: vaccine adjuvants are components that enhance a specific immune response against an antigen.

Amino acids: an entity comprising amino terminal portions (NH) separated by a central portion₂) And a carboxyl terminal moiety (COOH), the central moiety comprising a carbon atom, or a chain of carbon atoms, the chain of carbon atoms comprising at least one side chain or one functional group. NH (NH)₂The amino group present at the amino terminus of the amino acid or peptide is indicated, while COOH indicates the carboxyl group present at the carboxyl terminus of the amino acid or peptide. The generic term amino acid includes both natural and unnatural amino acids. Listed in j.biol.chem., 243: the standard nomenclature adopted in sections 3552-59(1969) and 37c.f.r. 1.822(b) (2) for natural amino acids falls within the group of amino acids set forth in table 1 herein below. Unnatural amino acids are those not listed in Table 1. Examples of unnatural amino acids are those listed, for example, in section 1.822(b) (4) of 37c.f.r., which is incorporated by reference herein in its entirety. The amino acid residues described herein may be in the "D" or "L" isomeric form.

Table 1. natural amino acids and their respective codes.

Amino acid residues: the term "amino acid residue" is meant to cover amino acids which react with at least one other species, such as with 2, e.g. with 3, such as with more than 3 further species, whether standard, non-standard or pseudo-amino acids. In particular, the amino acid residue may comprise an amide bond instead of a free carboxyl group, and/or an amine bond and/or an amide bond instead of a free amine group. Furthermore, the reacted amino acid residue may comprise an ester or thioester bond instead of an amide bond.

Antibody: are immunoglobulin molecules and active portions of immunoglobulin molecules. For example, an antibody is an intact immunoglobulin molecule or a fragment thereof that retains immunological activity.

Antigen: a molecule recognized by an antibody. Typically a peptide, polypeptide or multimeric polypeptide. The antigen is preferably capable of eliciting an immune response.

A.p.i. gravity: the term used by the oil industry refers to the relative density of petroleum liquids as determined by API (american petroleum institute). API gravity can be measured by a liquid gravimeter with a scale that measures API degrees.

CTL: cytotoxic T lymphocytes. A subset of T cells expressing CD8 and carrying a T cell receptor, so that they are able to respond to antigens presented by class I molecules.

Emulsion: a suspension of globules of the first liquid in a second liquid, wherein the second liquid is immiscible with the first liquid.

Emulsifier: a surfactant to promote emulsion formation.

HLA: human leukocyte antigens, also known as MHC. Humans are able to synthesize three different MHC class I molecules-HLA-A, HLA-B and HLA-C. Human MHC class II molecules are referred to as HLA-D.

Immunoglobulin: serum antibodies including IgG, IgM, IgA, IgE and IgD

Separating: are used to describe any of the various secretagogues, polypeptides and nucleotides disclosed herein, which are identified and isolated and/or recovered from a component of their natural environment. Contaminant components of their natural environment are materials that generally interfere with diagnostic or therapeutic applications of polypeptides, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In a preferred embodiment, the polypeptide will be purified.

Ligand: molecules (e.g., peptides) that specifically bind to one or more related receptors. For example, an antigen is a ligand for its cognate antibody.

MHC: major histocompatibility complexes, also known as HLA. There are two major MHC subclasses, class I and class II.

Mineral oil: oils derived from mineral sources (e.g., petroleum), as opposed to oils derived from plants and animals. Hydrocarbon mixtures of varying composition.

Montanide ISA (Montanide incorporated Seppic add): preparing an oily adjuvant for the water-in-oil emulsion. Montanide ISA adjuvants are a group of oil/surfactant based adjuvants in which a non-metabolizable and/or metabolizable oil is combined with a surfactant (available from Seppic, belgium).

PBMC: peripheral blood mononuclear cells.

PBL: peripheral blood leukocytes.

Peptide: a plurality of covalently linked amino acid residues defining a sequence and linked by amide bonds. The term is used analogously to oligopeptides and polypeptides. The natural and/or unnatural amino acids can be linked by peptide or non-peptide bonds. The term peptide also includes post-translational modifications introduced by chemical or enzymatic reactions, as is known in the art. Surface tension: surface tension is the energy required to increase the surface area. Between liquid moleculesAdhesion ofThe force causes surface tension, andliquid surface moleculesStrongly adhere to each other. Surfactant (b): a surface active agent capable of reducing the surface tension of a liquid in which it is dissolved. Surfactants are compounds containing hydrophilic polar groups and hydrophobic non-polar groups, and generally comprise a fatty chain.

TAA: tumor associated antigens

Detailed Description

The present invention contemplates vaccine compositions comprising survivin peptides and survivin peptide variants as described below.

Vaccine composition

The vaccine composition according to the invention can be formulated according to known methods, such as by mixing one or more pharmaceutically acceptable excipients or carriers with the active agent, preferably it is suitable for administration to humans. Examples of such excipients, carriers and formulation methods can be found, for example, in Remington's pharmaceutical sciences (Maack Publishing Co, Easton, Pa.). In order to formulate a pharmaceutically acceptable composition suitable for effective administration, according to the present invention, the composition will comprise an effective amount of a survivin polypeptide, survivin peptide or survivin peptide variant as described herein.

The vaccine composition according to the invention may be administered to an individual in a therapeutically effective amount. The effective amount may vary depending on various factors, such as the condition, weight, sex and age of the individual. Other factors include the mode of administration.

In the following, vaccine compositions are meant to cover compositions for therapeutic use, including those in which administration of the composition stimulates an immune response in the patient, such as a strong specific cytotoxic T cell response, and in particular those in which a clinical response is induced against a target lesion. It is further contemplated that the vaccine composition of the present invention does not induce any systemic or local toxic response or any other side effects.

To obtain a vaccine or immunogenic composition, it may be desirable to combine the relatively small survivin peptide and survivin peptide variant molecules described herein with various materials (e.g. adjuvants, immunostimulatory components and/or carriers). Adjuvants are included in vaccine compositions to enhance specific immune responses. It is therefore of particular importance to identify adjuvants which, when combined with one or more antigens, result in vaccine compositions capable of inducing a strong specific cytotoxic T cell response, and most importantly a clinical response against target injury.

Adjuvant

A number of adjuvants have been described and used to produce antibodies in laboratory animals such as mice, rats and rabbits. In this context, the tolerance to side effects may be higher, since its main purpose is to obtain a strong antibody response.

For use in medicine and for being approved for use in medicine, especially for use in humans, it is desirable to have well characterized components of the vaccine composition, including adjuvants. It is further desirable that the composition have a minimal risk of any adverse reactions, such as granulomas, abscesses or fever.

In a preferred embodiment, the vaccine composition is suitable for administration to a human subject, and thus a preferred adjuvant is suitable for administration to a human subject.

The adjuvants selected may further be selected according to the type of immune response, B cell and/or T cell activation that they are required to stimulate, and may be formulated into vaccine compositions to optimize distribution and presentation to relevant lymphoid tissues.

Recently, methods involving loading appropriate antigen presenting cells with antigenic peptides have been considered as an efficient way to generate immune responses against cancerous diseases. The method comprises isolating APC (pbl) or APC precursor cells from a patient and loading these with antigenic peptides, optionally using dendritic cells and differentiating in vitro into APCs and loading with antigenic peptides prior to injection into the patient. This method is very complicated and time consuming. The use of cell cultures makes them very inflexible vaccine compositions, which require special preparation and storage facilities. It is an object of the present invention to identify vaccine compositions that are easy to prepare and/or store.

Adjuvants for use in therapeutic vaccines may be mineral salts (such AS aluminium hydroxide and aluminium phosphate or calcium phosphate colloids), oil emulsion and surfactant based formulations (such AS MF59 (microfluidised detergent stabilised oil-in-water emulsion), QS21 (purified saponin), AS02(SBAS2, oil-in-water emulsion + monophosphoryl lipid a (MPL) + QS21), Montanide ISA51 and ISA-720 (stabilised water-in-oil emulsion), adjuvants 65 (peanut oil, mannide monooleate and aluminium monostearate) (rubiimunochem Research inc., Hamilton, Utah)), specific adjuvants (such AS viral particles (single thin layer liposome carriers incorporating influenza hemagglutinin), AS04 (aluminium salt with MPL), ISCOMS (structured complexes of saponin and lipids (such AS cholesterol monophosphate), polyglycolide (polyglycolide) (PLG)), (natural and synthetic) microbial lipid derivatives (such AS MPL a) derivatives (such AS mplyl lipid (e) Detox (MPL + m. phlei cell wall skeleton), AGP (RC-529 (synthetic acetylated monosaccharide)), DC chol (lipoidal immunostimulant capable of self-assembling into liposomes), OM-174 (lipid a derivatives), CpG motifs (synthetic oligonucleotides containing immunostimulatory CpG motifs), modified bacterial toxins, LT and CT (with non-toxic adjuvant effect), endogenous human immunomodulators (e.g. hGM-CSF or hll-12 or immudatin (C3d tandem array)), inert carriers (e.g. gold particles).

QS-21 and Montanide ISA-51 adjuvants can be provided in the form of sterile, single-use bottles.

In a specific embodiment, the vaccine composition comprises an adjuvant and a survivin peptide or survivin peptide variant as described below. In a preferred embodiment, the Adjuvant can be Montanide Incomplex Seppic Adjuvant (ISA) (Seppic, Belgium) which includes Montanide eISA-51, Montanide ISA50, Montanide ISA70, Montanide ISA206, Montanide eISA708, Montanide ISA-720, Montanide ISA763A, Montanide ISA207, Montanide ISA264, Montanide ISA27, Montanide ISA35, Montanide ISA740, Montanide ISA773, Montanide ISA266, Montanide ISA267, Montanide ISA28, Montanide ISA51F, Montanide ISA016D, and Montanide IMS. The last three mentioned have not been approved for use in humans, but are potentially useful in vaccine compositions according to the invention.

Montanide ISA-51 and Montanide ISA-720 are particularly useful in humans, and therefore they are preferred, and they are oil-based adjuvants that must be administered as an emulsion (see below).

In some embodiments, the vaccine composition may further comprise one or more additional immunostimulatory components. These include, without limitation, Muramyl Dipeptide (MDP); such as N-acetyl-muramyl-L-alanyl-D-isoglutamine (ala-MDP), N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-orthopolymuramyl-L-alanyl-D-isoglutamine (CGP11637, N-MDP) and N-acetyl-muramyl-L-alanyl-D-isoglutamyl-L-alanine-2- (1 '-2' -dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (CGP19835A, MTP-PE), dimethylglycine, ghrelin, and trehalose dimycolate, monophosphoryl lipid A (MPL), And formyl-methionine containing tripeptides (e.g., N-formyl-Met-Leu-Phe). These compounds are commercially available from sources such as Sigma Chemical Co, (st. louis, MO) and RIBI ImmunoChem Research, Inc (Hamilton, MT).

The carrier may be present independently of the adjuvant. For example, the carrier function may be to increase the molecular weight of particular survivin fragments, thereby enhancing their activity or immunogenicity, bringing about stability, increasing biological activity, or increasing serum half-life. The carrier may be any suitable carrier known to those skilled in the art. The carrier protein may be, but is not limited to, keyhole limpet hemocyanin, serum proteins (such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins), or hormones (such as insulin or palmitic acid). In order to immunize a human, the carrier must be a physiologically acceptable carrier, which is acceptable and safe to humans. In one embodiment of the invention, however, tetanus toxoid and/or diphtheria toxoid are also suitable carriers. Alternatively, the carrier may be dextran, such as sepharose.

Vaccines based on oil emulsions and surfactants can be divided into water-in-oil, oil-in-water and water-in-oil-in-water formulations.

Adjuvants for oil-in-water formulations can be mineral oils or/and non-mineral oils and surfactants/emulsifiers. Adjuvants are mixed with water-soluble antigen compositions to provide vaccine formulations.

According to the invention, the oil may be metabolizable or non-metabolizable, or a mixture of metabolizable and non-metabolizable oils. Non-mineral oils are rapidly metabolized and removed from the injection site, so they have few side effects, but the immune response is correspondingly small, while mineral oils are only partially metabolized, with a higher risk of inducing undesired effects and a good immune response.

Preferably, the oil is premixed with an emulsifier (such as mannide monooleate) and emulsified with a colloid mill or continuous mechanical or fluid ultrasonic emulsifier prior to addition to the aqueous vaccine phase.

More complex double emulsions (water/oil/water) can be prepared by once more emulsifying in an aqueous phase containing a small amount of tween 80.

Significant advances that have been achieved in recent years are: optional 'ready-to-use' oil adjuvants were introduced. For example, oils containing esters of octadecenoic acid and mannitol anhydrous can conveniently form double or mixed emulsions (water/oil/water) which are stable and of low viscosity, without the need for elaborate emulsification equipment.

In particular embodiments, it is preferred that the adjuvant is for a water-in-oil vaccine formulation.

In particular embodiments, the preferred oil component may be a non-mineral oil. In a preferred embodiment, the adjuvant is selected from the group of non-mineral oil based Montanide ISA (incomplete lateral justant), such as Montanide ISA708, Montanide ISA-720, Montanide ISA763A, Montanide ISA207, Montanide ISA264, Montanide ISA27 and Montanide ISA 35.

Optionally, the adjuvant may comprise a non-mineral oil or/and a mineral oil. Thus in a preferred embodiment, the adjuvant is selected from the group of non-mineral oil or/and mineral oil based Montanide ISA (uncomplexed adjuvant) such as Montanide ISA740, Montanide ISA773, Montanide ISA266, Montanide ISA267, Montanide ISA28, and Montanide IMS, the last of which has not been approved for human use.

In a most preferred embodiment, the oil component is a mineral oil. In a preferred embodiment, the adjuvant is selected from the group of mineral oil based Montanide ISA (incomplete lateral add patent (Seppic, belgium)), such as Montanide ISA50, Montanide ISA-51, Montanide ISA70, Montanide ISA206, and Montanide ISA51F and Montanide ISA016D, the last two of which have not been approved for human use.

The composition of the mineral oil (e.g. carbon chain length) affects the efficacy of the adjuvant, the short chain induces a strong immune response but carries local side effects, whereas the response with the long chain is less but without significant side effects, so the mineral oil should be well characterized, balancing the composition of the long and short carbon chains. Preferably the mineral oil has less than 8%, or such as less than 7%, or such as less than 6% and most preferably less than 5% hydrocarbons with a chain length of less than C14.

Preferably, the mineral oil is free of unsaturated or aromatic hydrocarbons.

Preferably the oil component has an a.p.i. gravity of from 32 to 40, or such as from 35 to 37 and especially such as from 36.2 to 36.8.

In addition, it is preferred that the mineral oil has a specific gravity at 25 ℃ of 0.82 to 0.84 or, for example, 0.83 to 0.84. More preferably, the mineral oil has a specific gravity of 0.834-0.838 at 25 ℃.

Mineral oils having a viscosity of 55 to 65SSU at 37.8 deg.C (100F) are preferred, with mineral oils having a viscosity of 27 to 61 being more preferred, and mineral oils having a viscosity of 59 to 61SSU at 37.8 deg.C (100F) being especially preferred.

Preferably the mineral oil has a refractive index at 25 ℃ of from 1.2 to 1.6, or such as from 1.3 to 1.6 or such as from 1.4 to 1.5 and most preferably from 1.458-.463,

further preferred mineral oils have one or more of the following characteristics;

a) the acid test is superior to the lowest value,

b) the fluorescence at 360nm is negative,

c) the suspended matter can be seen to be negative,

d) having a minimum ASTM flash point of 295F,

e) all RN requirements for light mineral oil and uv absorption are met.

The mineral oil should be of pharmaceutical grade.

Drakeol6VR (Penreco, Texas) is a pharmaceutical grade mineral oil. Drakeol6VR, which is free of unsaturated or aromatic hydrocarbons, has an A.PI. gravity of 36.2-36.8, a specific gravity of 0.834-0.838 at 25 ℃, a viscosity of 59-61SSU or 10.0-10.6 centistokes at 37.8 ℃ (100F), a refractive index of 1.458-463 at 25 ℃, is superior to the lowest value in acid testing, is negative in fluorescence at 360nm, is negative in visible suspensions, has an ASTM pour test value of 0-15F, has a minimum ASTM flash point of 295F, and meets all RN requirements for light mineral oil and ultraviolet absorption. Less than 5% of the mineral oil consists of hydrocarbons with short chains.

In a most preferred embodiment, the mineral oil is Drakeol6 VR.

Surface active agent

The surface tension of the adjuvant can be adjusted by the surfactant, whereby it can influence the viscosity of the adjuvant and the vaccine formulation. The toxic effects of surfactants are related to the residual level of fatty acids, and therefore high quality surfactants with low levels of fatty acids are needed. The surfactant should be of pharmaceutical grade.

Adjuvants according to the present invention may comprise a surfactant.

In a specific embodiment, the adjuvant is formulated as a water-in-oil emulsion comprising a mineral oil and a surfactant, wherein the adjuvant comprises up to 14.5 volume percent of said surfactant.

One aspect of the present invention relates to a vaccine composition for use as a medicament, comprising:

i. one or more survivin peptides or survivin peptide variants, wherein the sequence of the peptide variant is identical over the entire length to the sequence of SEQ ID NO: 23 is at least 85% identical in consecutive amino acid sequences, and

an adjuvant formulated for a water-in-oil emulsion comprising a mineral oil and a surfactant, wherein the adjuvant contains up to 14.5 volume percent of said surfactant.

In another embodiment, the adjuvant comprises 2 to 14 volume percent of said surfactant, such as 5 to 14 volume percent of said surfactant, such as 6 to 13 volume percent of said surfactant, such as 7 to 12 volume percent of said surfactant or such as 8 to 12 volume percent of said surfactant.

Preferably the major fatty acid species of the surfactant is C18', which constitutes 65 to 88% of the composition.

It is further preferred that the surfactant is an oil, such as a lipid liquid with a maximum acid number of 1.

It is also preferred that the surfactant have a saponification value of 150-.

It is also preferred that the surfactant has a hydroxyl number of from 70 to 120, or such as from 80 to 110, 85 to 105, and most preferably a hydroxyl number of from 89 to 100.

It is also preferred that the surfactant has an iodine value of from 40 to 100 or such as from 50 to 90, or such as from 60 to 80, or such as from 65 to 78, and most preferably an iodine value of from 67 to 75.

It is also preferred that the surfactant has a heavy metal value of less than 40ppm or such as 30ppm or such as 25ppm or such as 22ppm, and most preferably a heavy metal value of less than 20 ppm.

It is also preferred that the surfactant has a maximum water content of 50%, or such as 45%, or such as 40%, or such as 37%, and most preferably a maximum water content of 0.35%.

It is also preferred that the surfactant has a viscosity at 25 ℃ of, for example, 200-400mPaS, or for example 225-375mPaS, or for example 250-350mPaS, or for example 275-325mPaS, and most preferably a viscosity at 25 ℃ of about 300 mPaS.

In a preferred embodiment, the surfactant is mannide oleate, also known as anhydrous mannitol octadecanol.

Montanide80 is based on oleic acid (with various fatty acids distributed), the predominant fatty acid species being C18', making up 65 to 88% of the composition. The oil is a lipid liquid having a maximum acid value of 1, a saponification value of 164-172, a hydroxyl value of 89-100, an iodine value of 67-75, a maximum peroxide value of 2, a heavy metal value of less than 20ppm, a maximum water content of 0.35%, a maximum color value of 9 and a viscosity of about 300mPaS at 25 ℃.

In a most preferred embodiment, the adjuvant comprises the surfactant mannide oleate (Montanide80(Seppic, belgium)).

In another preferred embodiment, the adjuvant comprises mineral oil Drakeol6VR and surfactant diglycolic oleate (Montanide 80).

In a preferred embodiment, the adjuvant comprises mineral oil Drakeol6VR and surfactant mannide oleate, wherein the adjuvant contains up to 14.5% of the surfactant anhydrous mannitol octadecanol ester (Montanide 80).

In a more preferred embodiment, the adjuvant comprises from 2 to 14 volume percent of said surfactant anhydrous mannitol octadecane, such as from 5 to 14 volume percent of said surfactant anhydrous mannitol octadecane, such as from 6 to 13 volume percent of said surfactant anhydrous mannitol octadecane, such as from 7 to 12 volume percent of said surfactant anhydrous mannitol octadecane or such as from 8 to 12 volume percent of said surfactant anhydrous mannitol octadecane.

In particular embodiments, the adjuvant is a clear yellow liquid having a density of about 0.7 to 1.0 or 0.8 to 0.9 or preferably about 0.85 at 20 ℃.

In particular embodiments, the adjuvant has a viscosity of less than 500mPaS, such as less than 250mPaS or such as 25-150mPaS or preferably about 150mPaS at 20 ℃.

In a particular embodiment, the adjuvant has a maximum acid number of 0.5,

in particular embodiments, the adjuvant has a saponification number of 10 to 40, such as 12 to 30, such as 14 to 25 or preferably 16 to 20.

In particular embodiments, the adjuvant has a hydroxyl number of from 5 to 20, or such as from 6 to 18 or such as from 7 to 15 or preferably from 9 to 13.

In particular embodiments, the adjuvant has a maximum peroxide value of 5 or 4 or 3 or preferably 2.

In particular embodiments, the adjuvant has an iodine value of 1 to 20, such as2 to 15, such as 3 to 12 or preferably such as 5 to 9.

In particular embodiments, the adjuvant has a maximum water content of 2 percent, such as 1.5 percent, such as 1 percent or preferably such as 0.5 percent.

In a particular embodiment, the adjuvant has a refractive index between 1.450 and 1.470 or between 1.455 and 1.465 or preferably between 1.461-1.463 at 25 ℃.

In a specific embodiment, 50: 50 conductivity of adjuvant and saline mixed less than 20 μ Scm^-1、15μScm^-1、12μScm^-1Or preferably less than 10 μ Scm^-1。

Montanide ISA51 contains mannide oleate (Montanide80) in mineral oil solution (Drakeol 6 VR). Montanide ISA51 contains about 8 to 12 percent anhydrous mannitol octadecanol and about 88 to 92 percent mineral oil. Montanide ISA51 is a clear yellow liquid having a density of about 0.85 at 20 ℃ and a viscosity of about 5 at 20 ℃0 mPaS. MontanideiSA51 is characterized by a maximum acid number of 0.5, a saponification number of 16-20, a hydroxyl number of 9-13, a maximum peroxide number of 2, an iodine number of 5-9, a maximum water content of 0.5 percent, and a refractive index at 25 ℃ of between 1.455 and 1.465 or preferably between 1.461 and 1.463. 50: conductivity of 50 mixed MontanideeISA 51 and saline was less than 10 μ Scm^-1。

In a more preferred embodiment, the adjuvant is a Montanide complete adjuvant.

In a most preferred embodiment, the vaccine composition comprises:

i. one or more survivin peptides or survivin peptide variants, wherein the sequence of the peptide variant is identical over the entire length to the sequence of SEQ ID NO: 23, and a contiguous amino acid sequence of at least 85%, and

an adjuvant formulated for a water-in-oil emulsion comprising a mineral oil and a surfactant, wherein the adjuvant comprises up to 14.5 volume percent of the surfactant, wherein the adjuvant is Montanide ISA 51.

Survivin peptides or peptide variants

The ideal immunotherapeutic target is a gene product that is silenced in normal tissues, overexpressed in cancer cells, and directly involved in tumor cell survival and development. Survivin potentially achieves these features because it inhibits apoptosis and is involved in the regulation of cell division. Therefore, survivin prevents physiological cell death, thereby prolonging cell survival.

Survivin is a 16.5kDa cytoplasmic protein containing a single BIR and a multiply charged carboxy-terminal coiled region replacing the RING finger structure. The coding sequence was 429 nucleotides long (SEQ ID NO: 22) including a stop codon, while the encoded survivin protein was 142 amino acids long (SEQ ID NO: 23).

The present invention relates to vaccine compositions comprising one or more survivin peptides or survivin peptide variants.

In a specific embodiment, one or more survivin peptide or survivin peptide variant comprises a full length survivin polypeptide consisting of 142 amino acid residues (SEQ ID NO: 23).

Preferably, the one or more survivin peptides or survivin peptide variants comprise at least 5 amino acid residues, and more preferably it comprises at least 7 amino acid residues, at least 8 amino acid residues, at least 9 amino acid residues, at least 10 amino acid residues, at least 11 amino acid residues, at least 12 amino acid residues, at least 14 amino acid residues, at least 16 amino acid residues, at least 18 amino acid residues or at least 20 amino acid residues.

In a specific embodiment it is further preferred that the one or more survivin peptides or survivin peptide variants consist of at most 142 amino acid residues or such as at most 120 amino acid residues, or such as at most 100 amino acid residues, and preferably at most 80 amino acid residues or such as at most 50 amino acid residues, and more preferably it consists of at most 20 amino acid residues, such as at most 18, such as at most 16, such as at most 14, such as at most 12, such as at most 11, such as at most 10 amino acid residues.

In a specific embodiment, the vaccine composition comprises one or more peptides or peptide variants consisting of seq id NO: 23 and at most 20 consecutive amino acid residues.

In a specific embodiment, further preferred is one or more survivin peptide or survivin peptide variants consisting of up to 142 consecutive amino acid residues or such as up to 120 consecutive amino acid residues, or such as up to 100 consecutive amino acid residues, and preferably up to 80 consecutive amino acid residues or such as up to 50 consecutive amino acid residues of SEQ ID NO23, and more preferred it consists of up to 20 consecutive amino acid residues, such as up to 18 consecutive amino acids, such as up to 16 consecutive amino acids, such as up to 14 consecutive amino acids, such as up to 12 consecutive amino acids, such as up to 11 consecutive amino acids, such as up to 10 consecutive amino acid residues of SEQ ID NO 23.

In particular embodiments, the peptide consists of a heptapeptide, an octapeptide, a nonapeptide, a decapeptide, or an undecapeptide, which consists of SEQ ID NO: 23, 7, 8, 9, 10, 11 consecutive amino acid residues.

The present invention also encompasses survivin peptide variants and functional equivalents disclosed herein. "functional equivalents" as used herein are defined by reference to the corresponding function of the predetermined fragment of the sequence in question. For example, functional equivalence can be determined by similar binding affinity, or similar potency, as demonstrated by ELISPOT assays with molecules of the HLAI class.

Functional equivalents or variants of the survivin-derived peptides described herein will be understood to display progressively different amino acid sequences from the preferred, pre-determined sequences over the number and extent of insertions, deletions and substitutions (including conservative substitutions). This difference can be measured by a decrease in identity between the preferred, predetermined sequence and the survivin-derived variant or survivin-derived functional equivalent.

The identity between amino acid sequences can be calculated using algorithms known in the art. Fragments homologous to fragments comprising or consisting of contiguous survivin-derived amino acid residues are to be considered as falling within the scope of the present invention when they are preferably such as at least 75% identical, such as at least 80% identical, such as at least 85% identical, such as at least 88% identical, at least 90% identical, such as at least 94% identical, including 95%, 96%, 97%, 98% or 99% identical over their entire length to the predetermined survivin-derived peptide.

The vaccine composition according to the invention comprises one or more survivin peptides or survivin peptide variants, wherein the sequence of the peptide variant is identical over the entire length to the sequence of SEQ ID NO: 23 are at least 85% identical.

An example of a peptide variant according to the invention is the peptide LLLGEFLKL (Presence of peptide LLLGEFLKL)Live protein_96-104L2) (SEQ ID NO: 4) wherein the peptide variant is 100% (89%) identical to the survivin sequence "LTLGEFLKL" 8/9.

The names used above to describe peptide variants are used throughout the application: the pre-numeric letter (e.g., L2) represents a variant amino acid (L) that replaces the other naturally occurring amino acid at position 2 of a given peptide. A given peptide is either represented by a number indicating the number of first amino acids of the peptide relative to the full-length survivin sequence (survivin)₉₆Or Sur96 starting at residue 96 of survivin), or by two numbers, the second number giving the terminal residue of the peptide corresponding to survivin.

Peptide variants may be derived from known survivin sequences, such as the sequence disclosed in US6.245.523 (SEQ ID NO: 23 herein). Peptides are selected that potentially have the ability to bind to a particular HLA molecule by utilizing a pairing arrangement of known sequences that bind to a given particular HLA molecule to show the dominance of some of the relevant amino acids at a particular position of the peptide. This dominant amino acid residue is also referred to herein as an "anchor residue" or "anchor residue motif. By this relatively simple process based on known sequence data that can be found in accessible databases, peptides can be derived from survivin protein molecules, which are likely to bind to specific HLA molecules. Representative examples of such analyses of some HLA molecules are given in table 2 below:

TABLE 2 major anchor residue motifs used.

HLA alleles

1 st position

Position 2

Position 3

Position 5

Position 6

Position 7

C-terminal end

HLA-A1HLA-A2HLA-A3HLA-A11HLA-A23HLA-A24HLA-A25HLA-A26HLA-A28HLA-A29HLA-A30HLA-A31HLA-A32HLA-A33HLA-A34HLA-A66HLA-A68HLA-A69HLA-A74HLA-B5HLA-B7HLA-B8HLA-B14HLA-B15(B62)HLA-B17HLA-B27HLA-B35HLA-B37HLA-B38HLA-B39HLA-B40

E，DE，DE，DE，D

T，SL，ML，V，MV，I，F，YI，YYM，A，TV，T，I，L，FV，A，LEY，L，F，VI，LY，I，L，VV，LT，VT，VV，T，ATA，PPR，KQ，L，K，P，H，V，I，M，S，TRPD，EHR，HE

D，EF，YM，L，F，Y，IL，M，F，YF，YKD，EF，I，V

I，VK，R

VFI，L，V

L

YL，VK，Y，FK，RW，II，L，FWY，FA，RY，LYRWRRR，KR，KV，LV，LI，LL，FLL，VF，Y，WL，VY，K，F，LI，L，M，YI，L，MF，LL，FL，V，

(B60，61)HLA-B42HLA-B44HLA-B46HLA-B48HLA-B51HLA-B52HLA-B53HLA-B54HLA-B55HLA-B56HLA-B57HLA-B58HLA-B67HLA-B73HLA-Cw1HLA-Cw2HLA-Cw3HLA-Cw4HLA-Cw6HLA-Cw6HLA-Cw8HLA-Cw16

L，PEM，I，L，VQ，KA，P，GQPPPPA，T，SA，T，SPRA，LA，LA，LY，P，FYYA，L

F，Y

A，W，M，T，RY，LF，Y，WY，FLF，Y，I，VI，VW，F，LA，VA，VF，W，YF，W，YLPLF，YL，ML，M，F，YL，I，V，YL，Y，FL，I，L，V

Thus, as an example, a nonapeptide potentially having the ability to bind HLA-A1 has one of the following sequences: Xaa-T-D-Xaa-Xaa-Xaa-L-Xaa-Y, Xaa-T-E-Xaa-Xaa-Xaa-L-Xaa-Y, Xaa-S-D-Xaa-Xaa-Xaa-L-Xaa-Y or Xaa-S-E-Xaa-Xaa-L-Xaa-Y (Xaa represents any amino acid residue). In a similar manner, sequences can be designed that potentially have the ability to bind any other HLA molecule.

It will be appreciated that one of ordinary skill in the art will be able to identify other "anchor residue motifs" for a given HLA molecule.

Thus, peptide variants according to the invention include peptides comprising a sequence including any of the amino acid residues listed in table 2 for each particular HLA.

Alternatively, the difference can be measured by directly comparing the number of substitutions in the peptide compared to the contiguous amino acid sequence of survivin. Thus in a specific embodiment, the vaccine comprises a survivin peptide variant consisting of 7-20 consecutive amino acids that are identical to the amino acid sequence of SEQ id no: 23 comprises one or two amino acid substitutions. In a more preferred embodiment, the vaccine composition comprises a survivin peptide variant consisting of 7-12 consecutive amino acids that differ from the amino acid sequence of SEQ ID NO: 23 comprises one amino acid substitution compared to the contiguous amino acid sequence.

The binding groove of MHC class 1 molecules fits perfectly well for peptides of 9 or 10 amino acids. In a more preferred embodiment, the vaccine composition comprises one or more survivin peptide or survivin peptide variants consisting of 9 or 10 amino acid residues.

Based on the sequence of the survivin protein selected, one or more survivin peptides or survivin peptide variants may be derived by any suitable chemical or enzymatic treatment of the survivin starting material forming the above-mentioned suitably sized peptide, or may be synthesized by conventional peptide synthesis procedures familiar to those of ordinary skill in the art.

The peptides of the invention have the sequence of the native sequence of the survivin protein from which they are derived. However, peptides with higher affinity for any given HLA molecule can be derived from this native sequence by substitution, deletion or addition of at least one amino acid residue, such as based on the process of identifying anchor residue motifs for a given HLA molecule as described above (see table 2).

Thus, in a specific embodiment, the vaccine composition comprises one or more survivin peptides or peptide variants, wherein the sequence of the one or more survivin peptide variants may be derived from the native sequence by substitution, deletion or addition of at least one amino acid residue, thereby obtaining a peptide having the anchor residue motif for a given HLA molecule.

Thus, to enhance the immunogenicity of survivin-derived peptides, amino acid substitutions may be introduced at the anchor positions, rather than at the TCR contact residues, to enhance the binding of the peptide to the hla class molecules. This results in more immunogenic epitopes, for example, which enhance the ability to induce cancer-reactive CTLs, and has proven to be more suitable for inducing clinically meaningful CTL responses. It may be important that the target cancer cell not only express and present the native survivin-derived peptide on the cell surface. Thus, it is very important that therapeutic CTLs specific for the modified survivin-derived peptides are induced to cross-react with the natural analogs.

In a specific embodiment, the one or more survivin peptide or survivin peptide variants are restricted to at least one of the MHC class I molecules selected from the group consisting of: HLA-A1, HLA-A2, HLA-A3, HLA-A11, HLA-A23, HLA-A24, HLA-A25, HLA-A26, HLA-A28, HLA-A29, HLA-A30, HLA-A31, HLA-A32, HLA-A33, HLA-A33, HLA-A33, HLA-A33, HLA-B33, HLA-B33, HLA-B33, HLA-B33, HLA-B33, HLA-B33, HLA-B33, HLA-B33, HLA-B58, HLA-B67, HLA-B73, HLA-Cw1, HLA-Cw2, HLA-Cw3, HLA-Cw4, HLA-Cw6, and HLA-Cw 6.

Thus, in a further embodiment, the one or more peptides are restricted to at least one of the MHC class I molecules selected from the group consisting of: HLA-A1, HLA-A2, HLA-A3, HLA-A11, HLA-A24, HLA-B7, HLA-B35, HLA-B44, HLA-B8, HLA-B15, HLA-B27 and HLA-B51, HLA-Cw1, HLA-Cw2, HLA-Cw3, HLA-Cw4, HLA-Cw5, HLA-Cw6, HLA-Cw7 and HLA-Cw 16.

In a particularly preferred embodiment, the one or more peptides are restricted to at least one of the MHC class I molecules selected from the group consisting of: HLA-A1, HLA-A2, HLA-B7 and HLA-B35.

The vaccine according to the invention may comprise two or more peptides with the same tissue specificity, as the efficacy of each peptide may vary from individual to individual. Thus in a specific embodiment, the two or more survivin peptides or survivin peptide variants are restricted to MHC class I HLA molecules selected from the group consisting of: HLA-A1, HLA-A2, HLA-A3, HLA-A11, HLA-A23, HLA-A24, HLA-A25, HLA-A26, HLA-A28, HLA-A29, HLA-A30, HLA-A31, HLA-A32, HLA-A33, HLA-A33, HLA-A33, HLA-A33, HLA-B33, HLA-B33, HLA-B33, HLA-B33, HLA-B33, HLA-B33, HLA-B33, HLA-B33, HLA-B58, HLA-B67, HLA-B73, HLA-Cw1HLA-Cw2, HLA-Cw3, HLA-Cw4, HLA-Cw6, and HLA-Cw 6.

In another embodiment, one or more survivin peptide or survivin peptide variants is restricted to at least one of the MHC class I HLA-a molecules selected from the group consisting of: HLA-A1, HLA-A2, HLA-A3, HLA-A9, HLA-A10, HLA-A11, HLR-Aw19, HLA-A23(9), HLA-A24(9), HLA-A25(10), HLA-A26(10), HLA-A28, HLA-A29(w19), HLA-A30(w19), HLA-A31(w19), HLA-A32(w19), HLA-Aw33(w19), HLA-Aw34(10), HLA-Aw36, HLA-Aw43, HLA-Aw66(10), HLA-Aw68(28), HLA-A69 (28). Simpler names are also used in the literature, where only major numerical names are used, such as HLA-A19 or HLA-A24 instead of HLA-Aw19 and HLA-A24, respectively (9). In particular embodiments, one or more survivin peptide or survivin peptide variants are restricted to MHC class I HLA species selected from the group consisting of; HLA-A1, HLA-A2, HLA-A3, HLA-A11 and HLA-A24.

In a particular embodiment, one or more survivin peptide or survivin peptide variants are restricted to HLA-a 1.

In particular embodiments, one or more survivin peptide or survivin peptide variants is an HLA-a1 restricted peptide having a sequence selected from the group; MAEAGFIHY (SEQ ID NO: 17) (survivin)_38-46Survivin of Y9, replacement of "C" with "Y" at position 9_38-46) PTENEPDLAY (SEQ ID NO: 18) (survivin)_47-56Y10 survivin with "Y" in place of "Q" at position 10_47-56) QFEELTLGEF (SEQ ID NO: 15) (survivin)_92-101) And FTELTLGEF (SEQ ID NO: 16) (survivin)_93-101T2 survivin with "T" instead of "E" at position 2_93-101). The names in parentheses show the residue positions in the survivin protein disclosed in US6,245,523 and the amino acid changes in the peptide.

In a particular embodiment, one or more survivin peptide or survivin peptide variants are restricted to HLA-a 2.

In other particular embodiments, one or more survivin peptide or survivin peptide variants are HLA-a 2-restricted survivin-derived peptides having a sequence selected from the group consisting of: FLKLDRERA (survivin)_101-109) (SEQ ID NO: 1) TLPPAWQPFL (survivin)_5-14) (SEQ ID NO: 2) ELTLGEFLKL (survivin)_95-104) (SEQ ID NO: 3) LLLGEFLKL (stock)Live protein_96-104L2 survivin with "L" instead of "T" at position 2_96-104) (SEQ ID NO: 4) and LMLGEFLKLL survivin_96-104M2 (survivin with "M" in place of "T" at position 2)_96-104(SEQ ID NO：5)。

One or more survivin peptide or survivin peptide variants may also be an HLA-A3 restricted peptide (e.g. RISTFKNWPK (Sur18K10) survivin with "K" replacing "F" at position 10_18-27(SEQ ID NO: 20)) and/or HLA-A11-restricted peptides (e.g., DLAQCFFCFK (survivin 53-62) (SEQ ID NO: 19), DVAQCFFCFK (Sur53/V2) (SEQ ID NO: 45), DFAQCFFCFK (Sur53/F2) (SEQ ID NO: 46), DIAQCFFCFK (Sur53/I2) (SEQ ID NO: 47)) and/or HLA-A2-restricted peptides (e.g., RISTFKNWPFL (survivin 18-28) (SEQ ID NO: 21)) and/or HLA-A24-restricted peptides (e.g., STFKNWPFL (Sur20-28) (SEQ ID NO: 41), or SYFKNWPFL (Sur20-28/Y2) (SEQ ID NO: 48) with "Y" in position 2 in place of "T").

In a further embodiment, one or more survivin peptide or survivin peptide variants is restricted to a MHC class I HLA-B molecule comprising any one of: HLA-B5, HLA-B7, HLA-B8, HLA-B12, HLA-B13, HLA-B14, HLA-B15, HLA-B16, HLA-B17, HLA-B118, HLA-B21, HLA-Bw22, HLA-B27, HLA-B35, HLA-B37, HLA-B38, HLA-B39, HLA-B40, HLA-Bw41, HLA-Bw42, HLA-B44, HLA-B45, HLA-Bw46 and HLA-Bw 47. In a particular embodiment, the MHC class I HLA-B class to which one or more survivin peptide or survivin peptide variant binds is selected from the group consisting of: HLA-B7, HLA-B8, HLA-B15, HLA-B27, HLA-B35, HLA-B44, HLA-B51 and HLA-B58.

In a more preferred embodiment, one or more survivin peptide or survivin peptide variants is/are restricted to HLA-B7 or HLA-B35.

In a particular embodiment, one or more survivin peptide or survivin peptide variants are restricted to HLA-B7.

In other specific embodiments, one or more survivin proteins are present in the compositionThe peptide or survivin peptide variant is a survivin-derived peptide that binds HLA-B7 having a sequence selected from the group consisting of: LPPAWQPFL (survivin)_6-14) (SEQ ID NO: 10) QPFLKDHRI (survivin)_11-19) (SEQ ID NO: 11) CPTENEPDL (survivin)_51-59) (SEQ ID NO: 6) TPERMAEAGF (survivin)_34-43) (SEQ ID NO: 12) APPAWQPFL (survivin)_6-14A1) (SEQ ID NO: 13) RPPAWQPFL (survivin)_6-14R1) (SEQ ID NO: 14) RAIEQLAAM (Sur133-141) (SEQ ID NO: 44) TAKKVRRAI (Sur127-135) (SEQ ID NO: 49) RPIEQLAAM (Sur133P2) (SEQ ID NO: 50) or TPKKVRRAI (Sur127P2) (SEQ ID NO: 51). APPAWQPFL (SEQ ID NO: 13) is derived from survivin by replacing "L" with "A" at position 1 of the peptide_6-14And RPPAWQPFL (SEQ ID NO: 14) is derived from survivin by replacing "L" with "R" at position 1 of the peptide_6-14Derived from (a).

In a particular embodiment, one or more survivin peptide or survivin peptide variants are restricted to HLA-B35.

In other particular embodiments, one or more survivin peptide or survivin peptide variants are HLA-B35-restricted survivin-derived peptides having a sequence selected from the group consisting of: CPTENEPDL (survivin)_46-54) (SEQ ID NO: 6) EPDLAQCFF (survivin)_51-59) (SEQ ID NO: 7) CPTENEPDY (survivin)_46-54Y9) (SEQ ID NO: 8) and EPDLAQCFY (survivin)_51-59Y9) (SEQ ID NO: 9). The names in parentheses indicate the residue positions of the survivin proteins disclosed in US6,245,523. CPTENEPDY (SEQ ID NO: 8) is derived from survivin by replacing "L" with "Y" at the C-terminus of the peptide_46-54And EPDLAQCFY (SEQ ID NO: 9) is derived from survivin by replacing the "F" residue with a "Y" at the C-terminus_51-59Derived from (a).

In a particular embodiment, one or more survivin peptide or survivin peptide variants are restricted to HLA-B51. Survivin peptides are in particular HLA-B51-restricted survivin derived peptides having the sequence: RAIEQLAAM (Sur133-141) (SEQ ID NO: 44). This peptide is also HLA-B7 restricted.

In a particular embodiment, one or more survivin peptide or survivin peptide variants are restricted to HLA-B27.

In other particular embodiments, one or more survivin peptide or survivin peptide variants are HLA-B27-restricted survivin-derived peptides having a sequence selected from the group consisting of: ERMAEAGFI (Sur36-44) (SEQ ID NO: 43), ERAKNKIAK (Sur107-115) (SEQ ID NO: 52), DRERAKNKI (Sur105-113) (SEQ ID NO: 53), KEFEETAKK (Sur122-130) (SEQ ID NO: 54), ERMAEAGFL (Sur36/L9) (SEQ ID NO: 55), ERMAEAGFF (Sur36/F9) (SEQ ID NO: 56), ERMAEAGFR (Sur36/R9) (SEQ ID NO: 57), ERMAEAGFK (Sur36/K9) (SEQ ID NO: 58) or KRFEETAKK (Sur122/R2) (SEQ ID NO: 59).

In a particular embodiment, one or more survivin peptide or survivin peptide variants are restricted to HLA-B44.

In other particular embodiments, one or more survivin peptide or survivin peptide variants are HLA-B44-restricted survivin-derived peptides having a sequence selected from the group consisting of: KETNNKKKEY (Sur115Y10) (SEQ ID NO: 42), KETNNKKKEF (Sur115-124) (SEQ ID NO: 60), EELTLGEFL (Sur94-102) (SEQ ID NO: 61) or EELTLGEFY (Sur94Y9) (SEQ ID NO: 62).

In a particular embodiment, one or more survivin peptide or survivin peptide variants are restricted to HLA-B8.

In other particular embodiments, the one or more survivin peptide or survivin peptide variants are HLA-B8-restricted survivin-derived peptides having a sequence selected from the group consisting of: ISTFKNWPFL (Sur19-28) (SEQ ID NO: 63), ISKFKNWPFL (Sur19/K3) (SEQ ID NO: 64), LSVKKQFEEL (Sur87-96) (SEQ ID NO: 65), LSKKKQFEEL (Sur87/K3) (SEQ ID NO: 66), RAKNKIAKET (Sur108-117) (SEQ ID NO: 67), RAKNKIAKEL (Sur108/L10) (SEQ ID NO: 68), NNKKKEFEET (Sur118) (SEQ ID NO: 69), NNKKKEFEEL (Sur118/L10) (SEQ ID NO: 68670), QPKLKDHRI (Sur11/K3) (SEQ ID NO: 71), FLKDHRIST (Sur13) (SEQ ID NO: 72), FLKDKRIST (Sur13/K5) (SEQ ID NO: 73), 5 (Sur 5/L5) (SEQ ID NO: 3674), 5(SEQ ID NO: 5) (SEQ ID NO: 3675), Sur 5/K5) (SEQ ID NO: 5) (SEQ ID 5/5), and 5 (Sur 5/5) (SEQ ID NO: 5) (SEQ ID 5/5) (SEQ ID NO: 5), FLSVKKQFE (Sur86) (SEQ ID NO: 79), FLSVKKQFL (Sur86/L9) (SEQ ID NO: 80), FLKVKKQFE (Sur86/K3) (SEQ ID NO: 81), SVKKQFEEL (Sur88) (SEQ ID NO: 82), SVKKKFEEL (Sur88/K5) (SEQ ID NO: 83), FLKLKRERA (Sur101/K5) (SEQ ID NO: 84), FLKLDRERL (Sur101/L9) (SEQ ID NO: 85), TAKKKRRAI (Sur127/K5) (SEQ ID NO: 86) or TAKKVRRAL (Sur127/L9) (SEQ ID NO: 87), or TAKKVRRAI (Sur127) (SEQ ID NO: 49), FLKLDRERA (Sur101) (SEQ ID NO: 1) or QPFLKDHRI (Sur11) (SEQ ID NO: 11).

In a particular embodiment, one or more survivin peptide or survivin peptide variants are restricted to HLA-B15.

In other particular embodiments, the one or more survivin peptide or survivin peptide variants are HLA-B15-restricted survivin-derived peptides having a sequence selected from the group consisting of: TLPPAWQPF (Sur5) (SEQ ID NO: 88), TLPPAWQPY (Sur5/Y9) (SEQ ID NO: 89), WQPFLKDHRI (Sur10) (SEQ ID NO: 90), WQPFLKDHRY (Sur10/Y9) (SEQ ID NO: 91), FLKDHRISTF (Sur13) (SEQ ID NO: 92), FLKDHRISTY (Sur13/Y9) (SEQ ID NO: 93), RISTFKNWPF (Sur RISTFKNWPF) (SEQ ID NO: 94), RISTFKNWPF (Sur RISTFKNWPF/L RISTFKNWPF) (SEQ ID NO: 95), RISTFKNWPF (Sur RISTFKNWPF) (SEQ ID NO: 96), RISTFKNWPF (Sur RISTFKNWPF/Y RISTFKNWPF) (SEQ ID NO: 97), RISTFKNWPF (Sur RISTFKNWPF) (SEQ ID NO: 98), RISTFKNWPF (Sur RISTFKNWPF/Q RISTFKNWPF) (SEQ ID NO: 3699), RISTFKNWPF (Sur RISTFKNWPF/L RISTFKNWPF) (SEQ ID NO: 100), Sur RISTFKNWPF (Su RISTFKNWPF/Y RISTFKNWPF) (SEQ ID NO: RISTFKNWPF), Sur RISTFKNWPF (Sur RISTFKNWPF/Y RISTFKNWPF) (SEQ ID NO: RISTFKNWPF/RISTFKNWPF) (SEQ ID NO: RISTFKNWPF) and SEQ ID NO: RISTFKNWPF/RISTFKNWPF (SEQ ID NO: RISTFKNWPF), KKHSSGCAF (Sur78) (SEQ ID NO: 104), KQHSSGCAF (Sur78/Q2) (SEQ ID NO: 105), KLHSSGCAF (Sur78/L2) (SEQ ID NO: 106), RAIEQLAAY (Sur133/Y9) (SEQ ID NO: 107) or RLIEQLAAM (Sur133/L2) (SEQ ID NO: 108) or RAIEQLAAM (Sur133) (SEQ ID NO: 44).

In a particular embodiment, one or more survivin peptide or survivin peptide variants are restricted to HLA-B58.

In other particular embodiments, the one or more survivin peptide or survivin peptide variants are HLA-B58-restricted survivin-derived peptides having a sequence selected from the group consisting of: PTLPPAWQPF Sur5(SEQ ID NO: 109), CTPERMAEAGF (Sur33) (SEQ ID NO: 110), ETNNKKKEF (Sur116) (SEQ ID NO: 111), ISTFKNWPF (Sur19) (SEQ ID NO: 98), GAPTLPPAW (Sur2) (SEQ ID NO: 112) or CAFLSVKKQF (Sur84) (SEQ ID NO: 113).

In other useful embodiments, one or more survivin peptide or survivin peptide variants are peptides that are restricted to MHC class I HLA-C molecules selected from the group consisting of: HLA-Cw1, HLA-Cw2, HLA-Cw3, HLA-Cw4, HLA-Cw5, HLA-Cw6, HLA-Cw7 and HLA-Cw 16.

In addition, post-translational modifications of the peptides of the invention are beneficial.

Thus in a specific embodiment, the one or more survivin peptide or survivin peptide variant is a peptide comprising one or more post-translational modifications.

The peptide may comprise any kind of modification. Thus, nearly 200 structurally diverse covalent modifications have been identified, ranging in size and complexity from amide to carboxylic acid to the incorporation of multiple complex oligosaccharides. These modifications include phosphorylation, acetylation, ubiquitination, lipidation (acetylation, prenylation, farnesylation, geranylation, palmitoylation, myristoylation), methylation, carboxylation, sulfonation, and O-or N-glycosylation.

It has been shown that exposure of breast cancer MCF-7 or cervical cancer HeLa cells to anticancer agents (including doxorubicin, paclitaxel, or UVB) results in a 4-5 fold increase in survivin expression. Survival protein water after anticancer therapyThe flat changes are not involved in regulating survivin mRNA expression, nor are they dependent on re-transcription of the gene. In contrast, inhibition of survivin phosphorylation on Thr34 by the cyclin-dependent kinase inhibitor flavopiridol resulted in loss of survivin expression, whereas survivin Thr, which was not phosphorylated, resulted in loss of survivin expression₃₄Mutants to Ala show an increased clearance compared to wild type survivin. The sustained elimination of survivin phosphorylation on Thr34 enhanced anticancer agent-induced apoptosis independent of p53 and inhibited tumor growth without toxicity in an in vivo breast cancer xenograft model. These data suggest that Thr34 phosphorylates critically regulated survivin levels in tumor cells, and that continued elimination of p34 kinase activity removes survivability checkpoints of survivin and enhances apoptosis in tumor cells.

It is therefore contemplated that survivin and survivin-derived peptides of the invention encompass phosphorylated peptides. Native survivin phosphopeptide antigens may be identified by scanning for the presence of MHC peptide binding motifs around the phosphorylation site Thr 34. Thus, possible survivin-derived phosphopeptide sequences include TPERMAEAGF (SEQ ID NO: 114) -a putative HLA-B35-and/or HLA-B7-and/or HLA-B51-restricted peptide antigen. Other natural phosphopeptides included herein include: HLA-A2: CACTPERMA (SEQ ID NO: 115), and CTPERMAEA (SEQ ID NO: 116); HLA-A3: FLEGCACTP (SEQ ID NO: 117); HLA-B7/HLA-B35/HLA-B51: WPFLEGCACT (SEQ ID NO: 118) (phosphorylated Thr residues are indicated in bold).

It is well known that different HLA molecules have different distributions in the main population. Therefore, there is a need to identify peptide epitopes restricted by several HLA class I molecules in order to expand the patient population that can be treated with the method according to the invention. Characterization of multiple survivin epitopes with different HLA restriction elements broadens the clinical potential of the target antigen in two important ways: (i) it increases the number of patients eligible for survivin-derived peptide-based immunotherapy. About 50% of the caucasian and Asian populations express HLA-A2 antigen, about 25% of the caucasians and 5% of the Asians express HLA-A1 and HLA-A3 antigens, and about 15% of the caucasians and 30% of the Asians express HLA-A11 antigen. Although these numbers cannot be accumulated due to co-expression, the combination of peptides that would be restricted to multiple of these would indeed cover most cancer patients, (ii) collective targeting of several restriction elements in each patient could reduce the risk of immune escape through HLA allele deletion. Deletion of a single HLA allele is a significant component of MHC alterations performed by cancer cells, while complete deletion of class I expression is a rather rare event. Thus, it is now possible to identify survivin epitopes restricted by different HLA alleles, targeting more than one HLA molecule simultaneously in patients with overlapping alleles.

Although many potential peptides for use in vaccine compositions can be predicted, the actual identification of peptides and vaccine compositions that achieve useful responses still requires testing of multiple parameters. The present invention describes particularly effective vaccine compositions. The vaccine composition according to the invention comprises a survivin peptide and a survivin peptide variant.

Examples of currently preferred multi-epitope vaccines include "tailored" combinations of epitopes for survivin-derived peptides depending on the tissue type of a given patient, e.g. subjects with HLA-1, HLA-a2, HLA-A3, and HLA-B35 phenotypes may be immunized with a vaccine comprising the following peptides: ELTLGEFLKL (survivin)_95-104) (SEQ ID NO: 3) LMLGEFLKLL survivin_96-104M2((SEQ ID NO: 5), CPTENEPDY (survivin)_46-54Y9) (SEQ ID NO: 8) and EPDLAQCFY (survivin)_51-59Y9) (SEQ ID NO: 9) and/or RISTFKNWPK (Sur18K10) (SEQ ID NO: 20)

alternatively, epitopes can be selected based on the prevalence of various HLA phenotypes in a given population. As an example, HLA-A2 is the most widely distributed phenotype in the caucasian population, so peptides that bind HLA-A2 will be active in a large portion of this population.

However, the vaccine composition according to the invention may also comprise a combination of two or more survivin peptides or survivin peptide variants, each of which specifically interacts with a different HLA molecule, thereby covering a larger proportion of the target population. Thus, by way of example, the vaccine composition may comprise a combination of peptides restricted by HLA-A molecules and peptides restricted by HLA-B molecules, such as including HLA-A and HLA-B molecules corresponding to the prevalence of HLA phenotypes in the target population, e.g., HLA-A2 and HLA-B35. In addition, the vaccine combination may comprise peptides restricted by HLA-C molecules. Other combinations according to the invention include peptides restricted by HLA-A1 and HLA-A2, or peptides restricted by HLA-A1 and HLA-B35. In addition, a combination of 3 peptides with different specificities, such as peptides restricted by HLA-A1, HLA-A2 and HLA-B35, can be used.

It may be advantageous to include in the vaccine combination an HLA-B7 restricted peptide or two peptides, such as HLA-A1 and HLAA2 binding peptides or such as HLA-A1 and HLA-B35 binding peptides.

One aspect of the invention relates to HLA-B7-restricted peptides, such as LPPAWQPFL (survivin)_6-14) (SEQ ID NO: 10) QPFLKDHRI (survivin)_11-19) (SEQ ID NO: 11) CPTENEPDL (survivin)_51-59) (SEQ ID NO: 6) TPERMAEAGF (survivin 34-43) (SEQ ID NO: 12) APPAWQPFL (survivin)_6-14A1) (SEQ ID NO: 13) or RPPAWQPFL (survivin)_6-14R1)(SEQ ID NO：14)。

In one embodiment, the HLA-B7-restricted peptide is APPAWQPFL (survivin)_6-14A1) (SEQ ID NO: 13) or RPPAWQPFL (survivin)_6-14R1) (SEQ ID NO: 14). In a particular embodiment, the HLA-B7-restricted peptide is APPAWQPFL (survivin)_6-14A1) (SEQ ID NO: 13) in various specific embodiments, the HLA-B7-restricted peptide is RPPAWQPFL (survivin)_6-14R1)(SEQ ID NO：14)。

One aspect of the invention relates to a vaccine composition comprising one or more survivin peptides or peptide variants, wherein the sequence of the peptide variant is identical over the entire length to the sequence of SEQ ID NO: 23 and wherein the composition comprises

HLA-B7 binding peptides and/or

HLA-A1 and HLA-A2 restricted peptides and/or

HLA-A1 and HLA-B35 restricted peptides

And any of the above adjuvants, such as Montanide ISA 51.

In a particularly preferred embodiment, the vaccine composition comprises:

i. a peptide comprising APPAWQPFL (SEQ ID NO: 13) and consisting of up to 15, preferably 10 amino acids,

and/or

A peptide comprising RPPAWQPFL (SEQ ID NO: 14 and consisting of up to 15, preferably 10 amino acids,

and/or

A peptide comprising FTELTLGEF (SEQ ID NO: 16) and consisting of at most 15, preferably 10 amino acids, and a peptide comprising LMLGEFLKL (SEQ ID NO: 5) and consisting of at most 15, preferably 10 amino acids,

and/or the presence of a gas in the gas,

a peptide comprising FTELTLGEF (SEQ ID NO: 16) and consisting of at most 15, preferably 10 amino acids, and a peptide comprising EPDLAQCFY (SEQ ID NO: 9) and consisting of at most 15, preferably 10 amino acids,

and/or

A peptide comprising LMLGEFLKL (SEQ ID NO: 5) and consisting of up to 15, preferably 10 amino acids, and a peptide comprising EPDLAQCFY (SEQ ID NO: 9) and consisting of up to 15, preferably 10 amino acids,

any of the above adjuvants, such as Montanide ISA 51.

Three or more survivin peptides or survivin peptide variants are included and the efficacy of the vaccine composition can be further improved, especially if the peptides are restricted to different HLA molecules.

In a particular embodiment, the vaccine composition comprises,

a) three or more survivin peptides or survivin peptide variants, wherein the sequence of the peptide variants is identical over the entire length to the sequence of SEQ ID NO: 23 are at least 85% identical,

b) And any of the above adjuvants, such as Montanide ISA 51.

In particular embodiments, the HLA-a1 binding peptide, HLA-a2 binding peptide and/or HLA-B35 binding peptide preferably consists of at most 15, such as at most 14, 13, 12, 11, and most preferably at most 10 amino acids.

In a particular embodiment, the vaccine composition comprises HLA-A1-restricted peptide FTELTLGEF (SEQ ID NO: 16). In a second particular embodiment, the vaccine composition comprises the HLA-A2 binding peptide LMLGEFLKL (SEQ ID NO: 5), while in a third particular embodiment, the vaccine composition comprises the HLA-B35 binding peptide EPDLAQCFY (SEQ ID NO: 9).

In a most particular embodiment, the vaccine composition comprises HLA-A1-restricted peptide FTELTLGEF (SEQ ID NO: 16), HLA-A2-binding peptide LMLGEFLKL (SEQ ID NO: 5), and HLA-B35-binding peptide EPDLAQCFY (SEQ ID NO: 9).

In particular embodiments, the HLA-a1 binding peptide, HLA-a2 binding peptide and/or HLA-B35 binding peptide described above preferably consists of at most 15, such as at most 14, 13, 12, 11, and most preferably at most 10 amino acids.

Seven or more survivin peptides or survivin peptide variants are included and the efficacy of the vaccine composition can be further improved, especially if the peptides are restricted to different HLA molecules.

In a particular embodiment, the vaccine composition comprises,

a) seven or more survivin peptides or survivin peptide variants, wherein the sequence of the peptide variants is identical over the entire length to the sequence of SEQ ID NO: 23 is at least 85% identical,

And wherein at least one peptide or peptide variant is selected from the group of HLA-B7 binding peptides

b) And any of the above adjuvants, such as Montanide ISA 51.

In a particular embodiment, the HLA-A1, HLA-A2, HLA-A3, HLA-A24, HLA-A11, HLA-B35 and/or HLA-B7 binding peptides described above preferably consist of at most 15, such as at most 14, 13, 12, 11, and most preferably at most 10 amino acids.

In a preferred embodiment, the vaccine composition comprises HLA-A1 binding peptide FTELTLGEF (SEQ ID NO: 16), HLA-A2 binding peptide LMLGEFLKL (SEQ ID NO: 5), HLA-A3 binding peptide RISTFKNWPK (SEQ ID NO: 20), HLA-A24 binding peptide STFKNWPFL (SEQ ID NO: 41), HLA-A11 binding peptide DLAQCFFCFK (SEQ ID NO: 19), HLA-B35 binding peptide EPDLAQCFY (SEQ ID NO: 9) and HLA-B7 binding peptide LPPAWQPFL (SEQ ID NO: 10) and an adjuvant, such as Montanide ISA 51.

In another preferred embodiment, the vaccine composition further comprises

i. At least one peptide or peptide variant selected from the group of HLA-B44 binding peptides and/or,

at least one peptide or peptide variant selected from the group of HLA-B27 binding peptides and/or,

at least one peptide or peptide variant selected from the group of HLA-B51 binding peptides.

In a particularly preferred embodiment, the vaccine composition comprises HLA-A1 binding peptide FTELTLGEF (SEQ ID NO: 16), HLA-A2 binding peptide LMLGEFLKL (SEQ ID NO: 5), HLA-A3 binding peptide RISTFKNWPK (SEQ ID NO: 20), HLA-A24 binding peptide STFKNWPFL (SEQ ID NO: 41), HLA-A11 binding peptide DLAQCFFCFK (SEQ ID NO: 19), HLA-B35 binding peptide EPDLAQCFY (SEQ ID NO: 9) and HLA-B7 binding peptide LPPAWQPFL (SEQ ID NO: 10), and any one or more of HLA-B44 binding peptide is KETNNKKKEY (SEQ ID NO: 42), HLA-B27 binding peptide is ERMAEAGFI (SEQ ID NO: 43), and HLA-B51 binding peptide is RAIEQLAAM (SEQ ID NO: 44), and an adjuvant, such as Montanide ISA 51.

In any of the above embodiments, the HLA-a11 binding peptide may be selected from the group consisting of: DLAQCFFCFK (SEQ ID NO: 19), DVAQCFFCFK (SEQ ID NO: 45), DFAQCFFCFK (SEQ ID NO: 46) or DIAQCFFCFK (SEQ ID NO: 47).

In any of the above embodiments, it is preferred that the number of peptides multiplied by up to 13, such as 12, such as 11 or preferably 10 or 9, is the maximum number of amino acids which make up the vaccine. For example, if the vaccine comprises 5 peptides, it is therefore preferred that the vaccine consists of up to 65, such as 60, such as 55, such as 50 or 45 amino acids.

To select survivin peptides or survivin peptide variants for use in the vaccine compositions according to the invention, the ability of survivin peptides and peptide variants to bind HLA class I molecules may be assessed. In addition, the ability of the survivin peptide or survivin peptide variant to elicit INF- γ producing cells in the PBL population of cancer patients can be assessed.

These measurements may give an indication of the usefulness of the survivin peptide or survivin peptide variant for use in a vaccine composition, but preferably a vaccine composition comprising the survivin peptide or survivin peptide variant is capable of inducing a strong specific T cell response in cancer patients after administration. It is also preferred to administer vaccine compositions to induce a clinical response characterized as described in the section relating to assessment of target lesions. The clinical response may be characterized by at least stable disease (at most a 20% increase in the sum of the longest diameters of the target lesions), more preferably a decrease in the sum of the longest diameters of the target lesions, such as a partial response or, most preferably, a complete regression.

The vaccine composition comprises one or more survivin peptides or peptide variants, wherein the one or more survivin peptides or one or more peptide variants are restricted to HLA class I molecules, wherein the restricted peptides or peptide variants are characterized by at least one of the following features;

(i) peptides capable of binding to HLA class I molecules, e.g. by the highest number of HLA class I molecules that can be recovered (C)₅₀Value), the affinity of which, as determined by the integrated binding assay described in WO2004/067023, is at most 50 μ M.

(ii) Capable of eliciting INF-gamma-producing cells in a PBL population of cancer patients, as determined by an ELISPOT assay (as described in WO 2004/067023) at a frequency of at least every 10⁴Each PBL has 1.

Integrated binding assays provide a simple method of screening candidate peptides that bind to a given HLA allele molecule with the above affinities. In a preferred embodiment, one or more survivin peptide or survivin peptide variant has a C of at most 30. mu.M₅₀Values, such as at most 20. mu.M, include, for example, at most 10. mu.M, at most 5. mu.M and at most 2. mu.M or at most 1. mu.M. C of selected peptide₅₀The values are shown inIn Table 4 of WO 2004/067023.

One or more survivin peptide or survivin peptide variants for use in the vaccine composition according to the invention is characterized by being able to recognize or elicit INF-gamma responder T cells, i.e. cytotoxic T Cells (CTLs) that specifically recognize a specific peptide in the PBL population or tumor cells (target cells) of a cancer patient. This activity can be readily determined by subjecting PBLs or tumor cells from the patient to an ELISPOT assay as described in WO2004/067023 and the references herein. Before the assay, it is beneficial to stimulate the PBL population or tumor cells to be tested by contacting the cells with the peptide to be tested. Preferably the peptide can be present at every 10⁴A frequency of at least 1 for each PBL elicits or recognizes INF- γ -producing T cells, as determined by the ELISPOT assay used herein. More preferably the frequency is every 10⁴At least 5 PBLs, most preferably every 10 PBLs⁴At least 10 PBLs, e.g. every 10⁴There are at least 50 or 100 PBLs. In a particularly preferred embodiment, the frequency is every 10⁴At least 200 PBLs or, e.g., every 10 PBLs⁴There are at least 250 PBLs.

IFN- γ production in response to melanoma-specific antigens (e.g., MART-1 and gp100 peptides) was demonstrated by immunization of cancer patients with the indicated peptides. But no significant correlation with clinical response was observed (Hersey, p. et al, Cancer immunol. immunother.2004, sep.21).

Thus, once a putative immunogen is identified, it is preferable to assess in vivo function. It is highly preferred that the vaccine composition comprises one or more survivin peptides or one or more peptide variants which are capable of eliciting a very strong immune response, as measured before and after immunization, as an ELISPOT assay against INF- γ, inducing a very strong specific cytotoxic T-cell response. The assay involves testing a cytotoxic T-cell response in a patient by analyzing the responsiveness of PBMCs obtained before and after administration of an immune composition to an immunogen used in a vaccine composition, for example by the ELISPOT assay described in example 1.

The use of dendritic cells in vaccine compositions has resulted in higher efficacy in the prior art compared to oil-based adjuvants (Schreurs MW et al, Cancer Res.2000 Dec15; 60 (24): 6995-. Dendritic cells are currently the preferred adjuvant.

The unexpected results of clinical trials described in example 1 show that administration of a vaccine composition according to the invention induces a surprisingly large number of cells specifically releasing INF- γ.

Preferably administration of a vaccine composition according to the invention induces a strong specific T-cell response in a subject, measured by the number of INF-gamma releasing cells, every 10⁴More than 50 PBMC cells, or e.g. every 10⁴More than 100 PBMC cells, or e.g. every 10⁴More than 150 PBMC cells, or e.g. every 10⁴More than 200 PBMC cells. Most preferably, the specific T-cell response measured by the number of INF-gamma releasing cells is every 10⁴More than 250 PBMCs.

The specific T-cell response measured by ELISPOT may depend on the administration regimen used, e.g. the number of immunizations and the time of administration of the vaccine composition (see instructions relating to treatment). In particular embodiments, a strong specific cytotoxic T cell response may be detected after 12 months, or such as after 10 months (e.g. after 8 months). In a preferred embodiment, the specific cytotoxic T cell response may be detected after 6 months. In a further most preferred embodiment, the specific cytotoxic T cell response may be detected after 4 or 3 months.

The anti-angiogenic effect of the peptides can be further assessed accordingly, since inhibition of angiogenesis has a profound effect on the development of solid tumors.

An indicator of a potential anti-angiogenic effect is the infiltration of tumor stroma by antigen-specific T cells. Antigen-specific T cells on tumor lesions in cancer patients can be detected in situ using the polypeptide/HLA complex by testing the tumor stroma for the presence of antigen-specific T cells using the tissue staining method described in example 2. Antigen-specific T cells may recognize survivin peptide or survivin peptide variants according to the invention, preferably complexes with HLA 1-class molecules.

Preferably, administration of a vaccine composition according to the invention induces infiltration of antigen-specific T-cells in the tumor stroma, such as survivin-specific T-cells in the tumor stroma.

The evaluation of the vaccine composition further comprises examining the ability of the administered vaccine composition to elicit a clinical response. For the treatment of cancer, the clinical response is determined using the solid tumor response assessment criteria (RECIST) described in the section below relating to the assessment of target lesions.

In a specific embodiment, the vaccine composition according to the invention is capable of eliciting a clinical response, which is referred to as stable disease, partial response or complete regression, characterized by an increase of at most 20% in the sum of the longest diameters of the target lesions.

Thus, a simple method of identifying potential uses of peptide variants in a vaccine composition according to the invention comprises the steps of: selecting particular HLA molecules, e.g., molecules that occur at high rates in a given population, performing the above alignment analysis to identify "anchor residue motifs" in survivin protein, isolating or constructing a peptide of suitable size that comprises one or more of the identified anchor residues and (i) testing the ability of the produced peptide to bind to a particular HLA molecule using the integration assay described herein, and/or (ii) testing the produced peptide in the PBL population of cancer patients every 10 as determined by the ELISPOT assay described in WO2004/067023⁴The PBLs have a frequency of at least 1 to elicit the ability of INF-gamma producing cells.

To identify whether a peptide or peptide variant can be used in a vaccine composition according to the invention, the ability of a vaccine composition comprising a peptide to elicit an immune response, such as the ability to elicit a strong specific cytotoxic T-cell response, can be determined by the ELISPOT assay against INF- γ before and after immunization (as described in example 1 herein).

WO2004/067023 shows that survivin reactive cells isolated by HLA/peptide complexes possess the functional ability to lyse target cells. In addition, it was further demonstrated that dendritic cell vaccines using survivin peptide and survivin peptide variants can elicit weak immune responses in cancer cell lines and in PBL populations of cancer patients, but the clinical response in cancer patients is not great because there is a report that the disease is still progressing.

Relative to the total number of PBMCs in the population or CD8⁺The number of cells, specific immune responses can be assessed. The latter evaluation reference is used in WO2004/067023, and the results described herein use the total number of PBM cells as a reference. If the same reference is used for the results described in FIG. 17 of WO2004/067023, the number of reactive cells will be every 10⁴There are 8-30 PBMC cells.

Since the intensity of the immune response is a key parameter for the evaluation of potential immunogens, this assay is a powerful tool for the evaluation of vaccine compositions. According to the present invention, the ability to stimulate a strong specific T cell response after administration to a cancer patient is an important feature. Before and after immunization with the vaccine composition, the response can be measured by the number of INF-gamma producing cells in the PBMC population. In a particular embodiment of the invention, the vaccine composition is capable of stimulating a strong specific T cell response in cancer patients, wherein the strong T cell response measured by the ELISPOT assay after administration is every 10⁴The PBL cells are greater than 50, such as greater than 100, such as greater than 150, such as greater than 200, such as greater than 225, or such as greater than 250 peptide-specific spots.

The results of the evaluation of the anti-angiogenic effect can be used to select useful peptides, since it is believed (without being limited to theory) that a vaccine composition capable of eliciting an anti-angiogenic effect will prove to be very effective in inhibiting tumor growth. If combined with one or more peptides that elicit a strong specific T cell response against survivin, it is expected that a vaccine composition comprising the one or more peptides will have a very high probability of inducing an excellent clinical response.

One aspect of the invention relates to a vaccine composition comprising one or more survivin peptides or one or more variants of a peptide (wherein the sequence of the peptide variant is at least 85% identical over the entire length to the contiguous amino acid sequence of SEQ ID NO: 23) and an adjuvant, which is capable of inducing infiltration of antigen-specific T cells in the tumor stroma of a subject.

In a preferred embodiment, the vaccine composition is capable of inhibiting angiogenesis in a subject.

It is also necessary to assess the effect on target lesions and therefore the clinical response after administration of the vaccine composition must be assessed as described in the following section relating to assessment of target lesions. Preferably the vaccine composition is capable of eliciting a clinical response, which is referred to as stable disease, partial response or complete regression, characterized by an increase of at most 20% in the sum of the longest diameters of the target lesions as seen in example 1. More preferably, the sum of the longest diameters of the target lesions is reduced (partial response), and the most preferred response is complete regression.

Survivin peptide or survivin peptide variants are expected to form complexes of HLA and peptide on the cell surface in addition to binding to HLA molecules, which complexes in turn serve as epitopes or targets for cytolytic T cells. It is possible that the survivin peptide or survivin peptide variant will elicit other types of immune responses, such as B cell responses that can generate antibodies against the complex and/or Delayed Type Hypersensitivity (DTH) responses.

The latter type of immune response is characterized by congestion and significant sclerosis at the site of injection of the vaccine composition of the invention.

Possible side effects of immunization may be systemic or local toxicity. Changes in blood vessels (e.g., vasculitis) or impaired wound healing are possible side effects. Changes in hemoglobin, leukocytes and platelets, as well as lactate dehydrogenase, creatinine and cholinesterase are other undesirable effects.

In a particular embodiment of the invention, administration of the vaccine composition does not produce a vascular change. In a second embodiment, administration of the vaccine composition does not induce impaired wound healing.

Thus in the most preferred embodiment of the invention, the vaccine composition is administered without substantial adverse side effects. In particular, the relevance of side effects should be assessed in relation to the severity of the disease.

As also mentioned above, increasing attention has been given to eliciting tumor-specific T helper cell immunity, i.e., immunity with class II-MHC restricted epitopes, despite the fact that tumors do not normally express class II MHC. This is based on the recent finding that in many cases the induction and efficacy of vaccine-induced anti-tumor responses requires the coordination of tumor-specific CD 4-positive Th cells.

Thus, an important factor driving the development of vaccines with more complex compositions is the desire to target multiple tumor antigens, as can be achieved by designing vaccines that contain or encode a carefully selected set of CTL and Th cell epitopes.

Multi-epitope vaccine

Clearly, polyepitope vaccines constitute an effective way to generate immunity against epitopes derived from several different antigens without the need to introduce potentially harmful proteins, such as proto-oncoproteins, (genes encoding these proteins). The vaccine also allows selective induction of immunity against subdominant and cryptic T cell epitopes, which is particularly important in the case of tumour-associated autoantigens, for which tolerance to epitopes that appear predominantly in normal tissues may exist.

Some of the challenges associated with epitope vaccines include the inability of antigen-presenting cells to present certain epitopes. In particular, antigens expressed on tumor cells can be differentially presented due to functional differences between the immunoproteasome of the antigen-presenting cell and the 'constitutive' proteasome present in most tumor cells.

Thus, identifying peptides suitable for vaccine compositions includes testing and selecting based on experimental studies to evaluate the efficacy of various compounds included in the vaccine composition, including the antigen and adjuvant components of the vaccine composition.

Thus, in a further aspect, the invention provides a vaccine composition comprising one or more survivin peptide or survivin peptide variants alone or in combination with other proteins or peptide fragments, as appropriate. In particular embodiments, these other proteins or peptide fragments include, but are not limited to, proteins or peptide fragments thereof involved in regulating apoptosis. Suitable examples of such proteins may be selected from the Bcl-2 protein family, such as the Bcl-2 protein, the Bcl-XL protein, the Bcl-w protein, the Mcl-1 protein, the TRAG-3 protein, and peptide fragments derived from any protein. Other known apoptosis inhibitors include members of the Inhibitor of Apoptosis Proteins (IAP) family, such as X-IAP, C-IAP1 and C-IAP 2. These proteins are relatively ubiquitously expressed, whereas the apoptosis polypeptide inhibitor ML-IAP is expressed quite selectively and is detected mainly in melanoma. Thus, ML-IAP fragments that elicit a specific T cell response (i.e., a cytotoxic T cell response or a helper T cell response) may optionally be included in the vaccine compositions of the present invention.

Useful peptide fragments of ML-IAP include ML-IAP₂₄₅(RLQEERTCKV)(SEQ ID NO：24)，ML-IAP₂₈₀(QLCPICRAPV)(SEQ ID NO：25)，ML-IAP₉₀(RLASFYDWPL)(SEQ ID NO：26)，ML-IAP₁₅₄(LLRSKGRDFV)(SEQ ID NO：27)，ML-IAP₂₃₀(VLEPPGARDV)(SEQ ID NO：28)，ML-IAP₉₈(PLTAEVPPEL)(SEQ ID NO：29)，ML-IAP₃₄(SLGSPVLGL)(SEQ ID NO：30)，ML-IAP₅₄(QILGQLRPL)(SEQ ID NO：31)，ML-IAP₉₉(LTAEVPPEL)(SEQ ID NO：32)，ML-IAP₈₃(GMGSEELRL) (SEQ ID NO: 33) and ML-IAP₂₀₀(ELPTPRREV)(SEQ ID NO：34)。

Advantageously, the pharmaceutical composition of the invention may further comprise at least one further immunogenic protein or a peptide fragment thereof selected from proteins or peptide fragments not belonging to or derived from a survivin protein. In particular embodiments, the immunogenic protein or peptide fragment thereof is derived fromThe Bcl-2 protein family described in PCT/DK 2004/000799. Another Bcl-2 derived immunogenic peptide is an HLA-a2 restricted peptide having a sequence selected from the group consisting of: bcl₁₇₂(NIALWMTEYL)(SEQID NO：35)，Bcl₁₈₀(YLNRHLHTWI)(SEQ ID NO：36)，Bcl₂₀₈(PLFDFSWLSL) (SEQ ID NO: 37) and Bcl₂₁₄(WLSLKTLLSL)(SEQ ID NO：38)，Bcl₂₁₈(KTLLSLALV) (SEQ ID NO: 39) and Bcl₈₀(AAAGPALSPV)(SEQ ID NO：40)。

A particular embodiment of the invention relates to a vaccine composition according to the invention, further comprising one or more peptides or peptide variants selected from the group consisting of: ML-IAP, BCL-2, BCL-X, MCL-1 or TRAG-3 peptide (as described in PCT/DK 2004/000798) or a peptide variant thereof which binds to HLA 1-like molecules.

In addition, the composition according to the invention may be provided in the form of a multi-epitope vaccine comprising class I and/or class II restriction epitopes as defined above.

Dosage form

Vaccine compositions contemplated to be useful in the present invention comprise an immunologically effective amount of a survivin peptide or survivin peptide variant.

The amount of survivin peptide or survivin peptide variant in the vaccine composition may vary depending on the particular application. However, wherever a single dose of immunogen is preferred, it is from about 10. mu.g to about 5000. mu.g, more preferably from about 25. mu.g to about 2500. mu.g, or from about 50. mu.g to about 1000. mu.g, or from about 50. mu.g to about 500. mu.g, or from about 50. mu.g to about 250. mu.g, or as from about 50. mu.g to about 200. mu.g, or from about 75. mu.g to about 150. mu.g. In a preferred embodiment, the dose of immunogen is from about 75 μ g to about 150 μ g. In a most preferred embodiment, the dose is about 100 μ g.

Administration of

Modes of administration include intradermal, subcutaneous and intravenous administration, implantation in dosage forms of controlled release timing, and the like. Any and all forms of administration known in the art are included herein. Subcutaneous administration, especially deep subcutaneous administration, is preferred. It is further preferred that the vaccine composition according to the invention is administered in a replacement hand or foot in the vicinity of the draining lymph node.

Also included are any and all conventional dosage forms known in the art suitable for formulation into injectable immunogenic peptide compositions, such as lyophilized dosage forms and solutions, suspensions or emulsion dosage forms, including conventional pharmaceutically acceptable carriers, diluents, preservatives, adjuvants, buffer components, and the like, if desired.

The immunological effect of the composition of the invention can be determined by several methods known to the person skilled in the art and as described in the examples of WO 2004067023. Examples of how to determine the CTL response elicited by a vaccine composition are provided in WO 97/28816. Successful immune responses may also be determined by Delayed Type Hypersensitivity (DTH) reactions occurring after immunization and/or detection of antibodies specifically recognizing one or more peptides of the vaccine composition.

In a preferred embodiment, the pharmaceutical composition of the invention is an immunogenic composition or vaccine capable of eliciting an immune response against a cancerous disease.

The expression "immunogenic composition or vaccine" as used herein refers to a composition that elicits at least one type of immune response against cancer cells. Thus, the immune response may be of any of the types described above: CTL responses in which CTLs are produced that recognize HLA/peptide complexes present on the cell surface and cause cell lysis, i.e. vaccines elicit effector T cells that produce cytotoxic effects against cancer cells in the vaccinated subject; b cell responses to produce anti-cancer antibodies; and/or DTH-type immune responses.

Nucleic acid vaccines

The vaccine composition according to the invention may comprise a nucleic acid encoding a survivin polypeptide (SEQ ID NO: 23), a peptide fragment thereof or a survivin peptide variant thereof. The nucleic acid may thus encode any of the above proteins and peptide fragments. For example, the nucleic acid may be DNA, RNA, LNA, HNA, PNA, preferably the nucleic acid is DNA or RNA.

In a specific embodiment the invention relates to a vaccine composition comprising:

i. a nucleic acid encoding:

a) survivin polypeptide (SEQ ID NO: 23),

b) survivin peptide or

c) Survivin peptide variants.

And

any of the above adjuvants.

The nucleic acids of the invention may be contained in any suitable vector, such as an expression vector. A large number of vectors are available and the skilled person will be able to select a vector for a particular purpose. For example, the vector may be in the form of a plasmid, cosmid, viral particle, or artificial chromosome. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures, for example, the DNA may be inserted into an appropriate restriction endonuclease site using techniques well known in the art. In addition to the nucleic acid sequences according to the invention, the vector may further comprise one or more signal sequences, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. The vector may also comprise additional sequences. Suitable vectors containing one or more of these components are constructed using standard ligation techniques known to those skilled in the art. The vector is preferably an expression vector comprising a nucleic acid operably linked to a regulatory nucleic acid sequence which directs expression in a suitable cell. Within the scope of the present invention, the regulatory nucleic acid sequence should generally be capable of directing expression in a mammalian cell, preferably a human cell, more preferably an antigen-presenting cell.

In a preferred embodiment, the vector is a viral vector. The viral vector comprises, in addition to the nucleic acid encoding survivin or a peptide fragment thereof, a second nucleic acid sequence encoding a T cell stimulating polypeptide. The T cell stimulating polypeptide is preferably selected from the group consisting of B7.1, ICAM-1 and LFA-3.

The vector may also be a bacterial vector, such as an attenuated bacterial vector. To induce a sustained mucosal immune response at the site of infection and persistence, attenuated bacterial vectors may be used. Different recombinant bacteria can be used as vectors, for example a bacterial vector can be selected from the group consisting of salmonella, lactococcus, and listeria. In general, it could be shown to induce immunity against the heterologous antigen HPV16L1 or E7, showing strong CTL response induction and tumor regression in mice.

Pharmaceutical agent

One aspect of the invention relates to a vaccine composition according to the invention for use in the preparation of a medicament. In a particular embodiment, the medicament is for the treatment of cancer.

In a specific embodiment, the medicament according to the invention is for subcutaneous administration, and thus the medicament may be formulated as a solution or suspension, or as a lyophilized product which may be formulated as a suspension prior to administration.

The invention further relates to a medicament for the treatment of cancer comprising a vaccine composition comprising one or more survivin peptides or survivin peptide variants as active ingredients and an adjuvant.

Treatment of

Survivin molecules were found to be down-regulated in a large number of cancer disease populations. Vaccine compositions comprising survivin peptide or survivin peptide variants are useful for treating clinical conditions, such as cancer diseases. Treatment according to the present invention includes alleviation of disease symptoms and inhibition of disease progression, thereby producing the clinical response described below.

The vaccine composition according to the invention may be administered more than once, such as two, three, four, five, or such as more than five, such as more than 7, such as more than 10, such as more than 15. The disease treated with the vaccine composition of the present invention may be recurrent or chronic, and thus treatment may be continued for a long period at regular intervals in order to minimize symptoms and inhibit disease development or recurrence. For example, the disease to be treated may be a cancerous disease, and treatment may continue until or until a clinical response of complete resolution or stable disease is achieved, or for the lifetime of the patient.

For example, the vaccine composition may be administered once every 14 days for at least 1 month, such as at least 2 months, such as at least 3 months, such as at least 5 months, such as at least 8 months, such as at least 12 months, such as at least 20 months. The vaccine composition may be administered at regular intervals throughout the life of the subject. The vaccine composition may be administered when disease recurrence or disease progression is detected.

In a specific embodiment, the present invention relates to a method of stimulating a strong specific T cell response against survivin in a subject, the method comprising:

a) there is provided a vaccine composition according to the invention,

c) thereby stimulating a strong specific T cell response in the subject, wherein the specific T cell response, as measured by the ELISPOT assay, is every 10 before and after administration of the vaccine composition⁴There were more than 50 peptide-specific spots for each PBMC cell.

In a particular embodiment, the strong specific T cell response, as measured by the ELISPOT assay before and after administration of the vaccine composition, is every 10⁴There were more than 200 peptide-specific spots for each PBMC cell.

In another specific embodiment, the method according to the present invention may comprise administering the vaccine composition once per month.

In various preferred embodiments, the vaccine composition is administered every 2 months.

In a specific embodiment, the present invention relates to a method of treating or preventing a disease comprising;

a) providing a vaccine composition comprising any of the peptides described above and optionally any of the adjuvants described above,

c) thereby stimulating a strong specific T cell response in the subject, wherein the strong specific T cell response, as measured by the ELISPOT assay, is every 10 th of the prior and subsequent administration of the vaccine composition⁴There were more than 50 peptide-specific spots for each PBMC cell.

d) A clinical response is obtained in the subject.

Clinical response was assessed as described in the section below relating to assessment of target lesions.

In a preferred embodiment, the vaccine composition is for use in the treatment of a clinical condition.

In a preferred embodiment of the invention, the clinical condition is cancer. The term "cancer" as used herein is meant to encompass any cancer, neoplasm, and pre-neoplastic disease. For example, the cancer may be selected from the group consisting of: colon cancer, breast cancer, pancreatic cancer, ovarian cancer, prostate cancer, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver carcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioblastoma, neuroma, craniopharyngioma, neuroma, glioma, astrocytoma, medulloblastoma, craniopharyngioblastoma, ependymoma, thecal tumor, adenocarcinomas, adeno, Pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, retinoblastoma, leukemia and lymphoma, acute lymphocytic leukemia and polycythemia vera, multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease, acute non-lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, rectal cancer, urinary organ cancer, uterine cancer, oral cancer, skin cancer, gastric cancer, brain tumor, liver cancer, laryngeal cancer, esophageal cancer, breast tumor, Acute Lymphoid Leukemia (ALL) not present in childhood, thymic ALL, B-cell ALL, acute myeloid leukemia, myeloblastoid (myeloblastoid) leukemia, acute megakaryocytic (megakaryocytic) leukemia, Burkitt's lymphoma, acute myelogenous leukemia, chronic myelogenous leukemia, and T-cell leukemia, small and large non-small cell lung cancer, acute myelogenous leukemia, germ cell tumors, endometrial cancer, gastric cancer, head and neck cancer, chronic lymphoid leukemia, hairy cell leukemia, and thyroid cancer.

In a specific embodiment, the vaccine composition according to the invention is for use in the treatment of a cancer selected from the group consisting of; malignant melanoma, pancreatic cancer, cervical cancer or colon cancer.

In a preferred embodiment, the vaccine composition according to the invention is used for the treatment of malignant melanoma and pancreatic cancer.

One aspect of the present invention relates to a method of inhibiting angiogenesis, comprising;

a) providing a vaccine composition according to the invention

b) Administering the vaccine composition to a subject.

The subject in need of treatment is any subject, preferably a human. The peptides will typically have different affinities for different HLA molecules. Thus, in embodiments of the invention wherein the vaccine composition or pharmaceutical composition comprises a survivin peptide, it is preferred that the vaccine composition or pharmaceutical composition administered to a given individual will comprise at least one peptide capable of associating with an HLA molecule of that particular individual.

Combination therapy

The invention also relates to pharmaceutical compositions for combination therapy and kits comprising the components.

Combination therapy as used herein means treatment of a subject in need thereof with more than one different drug. Thus in one aspect, the combination therapy may comprise administering a pharmaceutical composition comprising the vaccine composition described above and a second drug or a kit comprising the components. The second agent may be any agent described below, such as a chemotherapeutic agent or an angiogenesis inhibitor.

Combination therapy may comprise, inter alia, administration of a chemotherapeutic and/or immunotherapeutic agent to an individual, in combination with one or more of i) a survivin peptide or survivin peptide variant according to the invention, ii) a vaccine composition according to the invention. However, combination therapy may also include radiation therapy, gene therapy, and/or surgery.

One aspect of the invention relates to a combination therapy method comprising the simultaneous, sequential or separate administration in any order:

i) vaccine composition according to the invention

ii) and a second drug.

In a preferred embodiment, the second agent is a chemotherapeutic agent.

For example, the chemotherapeutic agent may be methotrexate, vincristine, adriamycin, cisplatin, sugarless chloroethylnitrosourea, 5-fluorouracil, mitomycin C, bleomycin, doxorubicin, dacarbazine, taxol, fragylamine, meglumine GLA, valrubicin, carmustine and polifeprosan, MM1270, BAY12-9566, RAS farnesyltransferase inhibitors, MMP, MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470, Hycamtin/topotecan, PKC412, sevelavada/PSC 833, nosalin/Mitroxantrone, Metaret/suramin, batistat, E7070, BCH-4556, CS-682, 9-AC 3340, AG3433, Incmonel/VX-853, VX-693, VXK-6975, VX-9, DX-AA 3340, AG3433, Inc/VX-2518, CDPT 2516, CDPD 2518/25151, CDPD 25151, CDPT 2518/25151, CDPT 2515, L25151, CDPT 2515, CDPT 2518, and L2512, FK317, Picibanil/OK 432, AD 32/valrubicin, strontium chloride/strontium derivatives, Temodal/temozolomide, Evacet/liposomal doxorubicin, Yewtaxan/paclitaxel, taxol/paclitaxel, Hirodada/Capecitabine, Fluorofenolone/doxifluridine, Cyclopax/oral paclitaxel, oral taxane, SPU-077/cisplatin, HMR 1275/Flaveridol, CP-358(774)/EGFR, CP-609(754)/RAS proto oncogene inhibitor, BMS-182751/oral platinum, 776T (tegafur/uracil), levorotation UF/levo mi oxazole hydrochloride, eniluracil/C85/FU 5 synergist, irinotecan/levo oxazole, Camptosar/irinotecan, modTUex/Ralitxe, Claritpine/paclitaxel, Paxex/paclitaxel, Doxil/liposomal doxorubicin, Caelyx/liposomal doxorubicin, Fludara/fludarabine, Pharmarubicin/epirubicin, Depocyt, ZD1839, LU79553/Bis-Naphtalimide, LU 103793/dolastatin, Caetyx/liposomal doxorubicin, R.opti/2, 2-difluorodeoxycytidine, ZD0473/Anormed, YM116, iododine seeds, CDK4 and CDK2 inhibitors, PARP inhibitors, D4809/Dexifosamide, Ifes/Mesnex/isophosphoramide, Weigemeng/teniposide, Palaplatin/carboplatin, Plantol/isophosphide, Vesperidine/etoposide, ZD9331, Texostat/docetaxel, prodrugs of guanine arabinoside, taxanes, nitrosoureas, actinoelphine (e.g. melphel and aminophosphonic acid), aminophosphonic acid D, Chlamydamine, cytosine D, cytosine hydrochloride, cytosine B-D, cysteine, and D, Daunomycin hydrochloride, estramustine sodium phosphate, etoposide (VP16-213), 5-fluorodeoxyuridine, fluorouracil (5-FU), flutamide, hydroxyurea (hydroxyurea), ifosfamide, interferon alpha-2 a, alpha-2 b, leuprolide acetate (LHRH-releasing factor analogue), lomustine (CCNU), dichloromethyldiethylamine hydrochloride (mechlorethamine), mercaptopurine, thioethanesulfonic acid, mitoxantrone (o.p '-DDD), mitoxantrone hydrochloride, octreotide, plicamycin, procarbazine hydrochloride, streptozotocin, tamoxifen citrate, thioguanine, thiotepa, vinblastine sulfate, amacridine (m-AMSA), azacitidine, Erthropoietin, Hexamethylmelamine (HMM), interleukin 2, propylhydrazone (methyl-GAG; methylglyoxalin; MGdeoxyBG), penoxstatin (2's, Mechlorethamine (methyl-CCNU), teniposide (VM-26), and vindesine sulfate. Additionally, the chemotherapeutic agent may be a chemotherapeutic agent as described in table 3 of US6,482,843, columns 13 to 18.

The therapeutic or vaccine compositions of the present invention may also be used in combination with other anti-cancer strategies, which combination therapies are effective in inhibiting and/or eliminating tumor growth and metastasis. The methods of the present invention may be beneficially used with other forms of treatment, including, but not limited to, radiation, surgery, gene therapy, and chemotherapy.

Survivin is highly expressed in endothelial cells during angiogenesis, suggesting that it is involved in the cytoprotective effects of Vascular Endothelial Growth Factor (VEGF), and thus targeting cells expressing survivin may directly target cancer cells and further prevent tumor growth by inhibiting angiogenesis.

The anti-angiogenic effect can be enhanced by a combination of treatment with a vaccine according to the invention and treatment with an angiogenesis inhibitor. The anti-angiogenic therapy targets tumor vasculature and prevents tumor growth beyond a certain size, and thus in a second preferred embodiment, the second agent is an angiogenesis inhibitor.

For example, the angiogenesis inhibitor can be, but is not limited to, BMS-275291, Dalteparin: (B)) Suramin, 2-methoxyestradiol (2-ME), Salipramine, CC-5013 (Salipramine analogue), combretastatin A4 phosphate, LY317615 (protein kinase C beta inhibitor)) Soy isoflavones (genistein; soy protein isolate), AE-941 (Neovastat)^TM(ii) a GW786034), anti-VEGF antibodies (bevacizumab; avastin^TM) Interferon-alpha, PTK787/ZK222584, VEGF-Trap, ZD6474, EMD121974, Carboxyamidrazole (CAI), celecoxib (C.E.)) Halofuginone hydrobromide (Tempostatin)^TM) AdPEDF, Macugen, tryptophanyl-tRNA synthetase (TrpRS), rhufab V2(aka lucentis), squalamine, Retanane 15mg (a hydroxyprogesterone caproate acetate anecortave suspension) and interleukin-12.

"combination therapy" may include the introduction of a heterologous nucleic acid into an appropriate cell, which is generally referred to as gene therapy. For example, gene therapy may involve the introduction of a tumor suppressor gene or an apoptosis-promoting gene into a tumor cell. Alternatively, a nucleic acid sequence that inhibits the expression of a protooncogene or an apoptosis-inhibiting gene may be introduced into a tumor cell. In addition, genes can be introduced which encode enzymes that confer sensitivity to chemotherapeutic agents on tumor cells. Thus, in one embodiment, the invention provides a method comprising the step of treating cancer by introducing a genetic vector encoding a protein capable of enzymatically converting a prodrug (i.e., a non-toxic compound) to a toxic compound. In the methods of the invention, the therapeutic nucleic acid sequence is a nucleic acid encoding a product that by itself or in the presence of other drugs causes cell death. Representative examples of such therapeutic nucleic acids are nucleic acids encoding herpes simplex virus thymidine kinase. Other examples are the thymidine kinase of varicella zoster virus and the bacterial gene cytosine deaminase, which converts 5-fluorocytosine to the highly toxic compound 5-fluorouracil.

Assessing target damage

Therapeutic response was determined using RECIST (response assessment criteria in solid tumors) criteria reported in the original WHO manual (world health organization printed publication No. 48; 1979) which examined the determination of the longest diameter of target lesions (therase P et al J.Natl.cancer Inst.2000, 2.2/d; 92 (3): 205-16). Responses are divided into; complete response, partial response, disease progression, and stable disease. A complete response is the disappearance of all target lesions, while a partial response means that the sum of the longest diameters of the target lesions is reduced by at least 30%. Disease progression means an increase of at least 20% in the sum of the longest diameters of the target lesions. The stable disease condition refers to the condition without the above condition. The duration of a complete or partial response should be measured from the time the assay criteria are met, until the first day that disease recurrence or progression is recorded. Preferably, the effect is observed in at least one patient.

Partial response refers to a response in which at least a 30% reduction in the sum of the longest diameters of the target lesions was observed. The response may be further subdivided, where a partial response of 40%, or a partial response of 50%, or a partial response of 60%, or a partial response of 70%, or a partial response of 80%, or a partial response of 90% is observed, which relates to treatments in which the sum of the longest diameters of the target lesions is reduced by at least 40%, 50%, 60%, 70%, 80%, or 90%, respectively.

In a specific embodiment, the vaccine composition according to the invention is used for the treatment of cancer, thereby achieving a partial response of at least 30%.

In a specific embodiment, the method comprising administering a vaccine composition according to the invention is used for treating cancer and thereby achieving a partial response of at least 40%, such as at least 50%, such as at least 60%, such as at least 70%, such as at least 80% or such as at least 90%.

Stable disease refers to a response in which the sum of the longest diameters of the target lesions is reduced by 30% or less, and also includes a response in which the sum of the longest diameters of the target lesions is increased by 20% or less. Such responses may be subdivided into responses in which the sum of the longest diameters of the target lesions is reduced by 30% or less, such as 25% or less, such as 20% or less, such as 15% or less, such as 10% or such as 5% or less, and into subgroups in which the sum of the longest diameters of the target lesions is increased by 20% or less, such as 15% or less, such as 10% or such as 5% or less. Also included are subgroups where the sum of the longest diameters of the target lesions is reduced or increased by at most 1%, such as 3%, or such as at most 5%.

Disease progression means an increase of at least 20% in the sum of the longest diameters of the target lesions. Such responses may be subdivided into responses in which the sum of the longest diameters of the target lesions increases by at most 25%, such as at most 30%, such as at most 35%, such as at most 40%, such as at most 45%, or such as at most 50%. According to the present invention, a treatment that would cause a disease-progressive response in which the sum of the longest diameters of the target lesions is increased by at most 30% is considered a positive result if the diagnosis predicts a 50% increase. Thus, particular embodiments include treatments wherein the response is characterized by a disease-progressive response, and wherein the sum of the longest diameters of the target lesions is increased by at most 25%, such as at most 30%, such as at most 35%, such as at most 40%, such as at most 45%, or such as at most 50%.

Particular embodiments of the present invention relate to methods of treating or preventing a disease, comprising;

a) there is provided a vaccine composition according to the invention,

b) administering the vaccine composition to a subject

c) For treating cancer, and

d) wherein administration of the vaccine composition results in stable disease, partial response, or complete regression characterized by an increase in the sum of the longest diameters of the target lesions of up to 20%.

The data described in example 1 demonstrate that even in already heavily treated patients with very advanced disease, a very strong survivin-specific T cell response was obtained in the circulating lymphocyte population in all patients examined. As seen in table 3 of example 1, both subjects (JUSC and OTSC) experienced complete regression, while in some cases stable disease was observed and partial responses were seen in one subject.

Kit comprising a component

Combination therapy includes the separate, sequential or simultaneous administration of two or more active ingredients formulated in one or more medicaments. For ease of use, the medicaments may be included in a single combined product or kit of parts.

One aspect of the invention relates to a kit comprising components comprising:

a) a vaccine composition comprising

i. One or more survivin peptides or survivin peptide variants, wherein the sequence of the peptide variant is identical over the entire length to the sequence of SEQ ID NO: 23 are at least 85% identical,

and an adjuvant as described herein, and wherein,

b) and a second drug.

For the treatment of cancer, the medicament in the kit of parts according to the invention may be a chemotherapeutic agent. For treating immune disorders, the medicament may be an immunotherapeutic.

In a specific embodiment, the medicament in the kit according to the invention comprises a chemotherapeutic or immunotherapeutic agent. In a preferred embodiment, the medicament comprises a chemotherapeutic agent. According to the invention, the kit of parts comprises a vaccine composition and a medicament for separate, sequential or simultaneous administration. The kit may further comprise and be labeled with appropriate usage/administration methods/dosage information for the included pharmaceutical and vaccine compositions.

Drawings

FIG. 1 kinetic analysis of immunity of pancreatic cancer patients PBL to the survivin peptide Sur1M2 assessed by IFN- γ ELISPOT. PBMCs of the patient OTSC were obtained before and one, three and six months after the first immunization of sur1M 2/Montanide.Stimulating T-lymphocytes once with the peptide, then subjecting 10⁵Individual cells were plated in each well, and three wells were repeated with or without peptide. By usingThe series 2.0 analyzer (CTL analyzer, LLC, Cleveland, usa) calculates the average number of peptide-specific spots for each patient (after subtraction of the spots without added peptide).

FIG. 2 kinetic analysis of melanoma patient PBL immunity to the survivin peptide Sur1M2 assessed by IFN-. gamma.ELISPOT. PBMCs of patient JUSC were obtained before and one, three and six months after the first immunization of sur1M 2/Montanide. Stimulating T-lymphocytes once with the peptide, then subjecting 10⁵Individual cells were plated in each well, and three wells were repeated with or without peptide. By usingThe series 2.0 analyzer (CTL analyzer, LLC, Cleveland, usa) calculates the average number of peptide-specific spots for each patient (after subtraction of the spots without added peptide).

FIG. 3 kinetic analysis of melanoma patient PBL immunity to the survivin peptide Sur1M2 assessed by IFN-. gamma.ELISPOT. PBMCs from SIST of patients were obtained before, and one and four months after the first immunization with sur1M 2/Montanide. Stimulating T-lymphocytes once with the peptide, then subjecting 10⁵Individual cells were plated in each well, and three wells were repeated with or without peptide. By usingThe series 2.0 analyzer (CTL analyzer, LLC, Cleveland, usa) calculates the average number of peptide-specific spots for each patient (after subtraction of the spots without added peptide).

Examples

Example 1

Clinical results using a vaccine composition comprising a survivin-derived epitope and montanide ISA51 as adjuvant. A series of patients with advanced cancer as described below were treated.

All clinical trials were performed in compliance with the declaration of helsinki and all patients gave informed consent prior to treatment. The clinical studies were approved by the ethical evaluation committees of the university of murzburg (study No. 7/03) in germany and the Paul-Ehrlich institute (draft No. 0899/01) in Langen, germany.

Patient's health

Eligible patients participating in the study must meet the following criteria:

detectable metastatic melanoma, pancreatic, colon or cervical cancer

The confirmed slow progression of the disease

At least one standard treatment failure

The expected life is still at least 3 months

No treatment over the last 4 weeks

Absence of significant organ failure

Class I tissue types HLA-A1, -A2 or-B35

Peptides

The peptides included in this study are survivin peptide variants, modified from the survivin peptide by substitution of one amino acid. Thereby obtaining better anchoring residues and improved binding affinity of a given peptide to MHC molecules. The peptides used included:

HLA-A1-restricted epitope FTELTLGEF (SEQ ID NO: 16) (survivin)_93-1012T, wherein the native glutamine at position 2 is replaced by threonine).

HLA-A2-restricted epitope LMLGEFLKL (SEQ ID NO: 5) (survivin)_96-1042M, wherein the natural threonine at position 2 is replaced with methionine).

HLA-B35 restriction epitope EPDLAQCFY (SEQ ID NO: 9) (survivin)_51-599Y, wherein the leucine at position 9 is replaced by tyrosine).

100 μ g of HLA-A1, HLA-A2 or HLA-B35 restricted survivin peptide were mixed with 1ml of Montanide ISA51 according to the manufacturer's instructions (Seppic, Brussels, Belgium). The mixture is administered by deep subcutaneous injection into the hand or foot by administration adjacent to the draining lymph node. Patients were immunized twice at 7 day intervals, followed by other immunizations at 28 day intervals.

Toxicity, clinical efficacy and immune response were evaluated.

ToxicityAssessed by physical examination/history, hematological tests, and serum chemistry. These checks were performed before each immunization.

Clinical efficacyEvaluation is performed by physical examination and appropriate imaging studies (e.g., CT scan, NMR scan, chest X-ray, ultrasound or bone scintigraphy) before and every 3 months after treatment initiation.

Immunological responseMonitored by the ELISPOT assay, which detects survivin 96-104 specific IFN- γ release using PBMC. To increase the sensitivity of the ELISPOT assay, 1X 10 per ml of X-vivo medium (Bio Whittaker, Walkersville, Maryland) in 24-well plates (Nunc, Denmark) in the presence of 10. mu.M peptide⁶PBMC were stimulated once in vitro at individual cell concentrations, and the medium was supplemented with 5% heat-inactivated human serum and 2mM L-glutamine. After 2 days, 40IU/ml recombinant interleukin-2 (IL-2) (Chiron, Ratingen, Germany) was added. After 10 days, the cells were tested for reactivity. Briefly, nitrocellulose-based 96-well plates (MultiScreen MAIP N45, Millipore, Hedehuene, Denmark) were coated with anti-IFN-. gamma.antibodies (1-D1K, Mabtech, Nacka, Sweden). Washing the wells, blocking with X-vivo medium, and adding 10⁴Individual stimulatory T2 cells (with or without 10 μ M peptide) and different concentrations of effector cells. The plates were incubated overnight. The following day the medium was discarded and the plate washed, followed by addition of biotinA secondary antibody (7-B6-1-biotin, Mabtech). Plates were incubated for 2 hours, washed and avidin-enzyme conjugate (AP-avidin, Calbiochem, Life Technologies) was added to each well. The plates were incubated at room temperature for 1 hour and the enzyme substrate NBT/BCIP (Gibco, Life technologies) was added to each well and incubated at room temperature for 5-10 minutes. Once a dark purple point appeared, the reaction was stopped by washing with tap water. By usingThe number of spots was counted by a series 2.0 analyzer (CTL analyzer, LLC, Cleveland, USA), and the peptide-specific CTL frequency was calculated from the number of cells forming the spots. All experiments against each peptide antigen were performed in triplicate.

Results

A summary of clinical results obtained using survivin peptide includes; patient abbreviation, number of immunizations and clinical response, which are summarized in table 3.

Patient's health

Date of birth

Diagnosis result

HLA type

Number of immunizations

Are being studied

End day

Quit

Clinical response

AGSC

28.05.26

MM

A2

2

X

ALKA

19.04.36

MM

A2

5

22.10.03

X

BRHI

27.08.34

MM

A2

5

X

SD

CHPF

18.12.34

MM

A2

12

X

PR

CHPF

18.12.34

MM

B35

1

X

EREI

04.05.55

MM

A2

5

08.10.03

X

HAKO

27.02.63

MM

A1

2

X

JUSC

03.01.57

MM

A2

16

X

CR

MAKR

20.08.40

MM

B35

3

X

OSRO

11.07.35

MM

A2

4

05.08.04

OTSC

10.05.29

PC

A2

15

X

CR

REPA

27.09.45

MM

A2

4

19.04.04

SIST

08.02.34

MM

A2

7

30.03.04

SD

WESE

23.07.42

MM

A2

5

X

SD

Table 3. results of ongoing clinical trials.

Metastatic Melanoma (MM), Pancreatic Cancer (PC). Complete Response (CR), Partial Response (PR), Stable Disease (SD), and Progressive Disease (PD).

Toxicity:no treatment-induced side effects were observed. No signs of systemic or local toxicity were observed at the injection site. Of particular interest to vascular changesSymptoms of chemotherapy, such as vasculitis or impaired wound healing. Hemoglobin, leukocytes and platelets, as well as lactate dehydrogenase, creatinine and cholinesterase were not affected by immunization (data not shown). Thus, no clinical or histological signs of vascular changes were detected. In addition, no signs of impaired wound healing, hemorrhage, cardiac disorders, vasculitis, or inflammatory bowel disease have occurred. Thus, vaccination with survivin is tolerable and safe for cancer patients who maintain normal hematopoiesis.

Clinical efficacy: objective clinical responses appeared in this group of patients with fairly unfavorable prognosis. Responses included complete regression of tumor visceral metastases in some patients, but most were disease stable. Notably, despite previous chemotherapy (with separate treatments)Or) The response failed, but resulted in complete tumor regression in both patients diagnosed with cutaneous melanoma (JuSc) and pancreatic adenocarcinoma (OtSc), respectively (table 3). Thus, clinical results demonstrate that it is a very successful complete treatment with unusually high response rates and clinical efficacy.

Survivin-specific CD8+ T cell responses.

The kinetics of the cytotoxic T cell response in the patient is followed. PBMCs obtained before and after immunization were tested for reactivity to the modified survivin 96-104 epitope by ELISPOT against IFN- γ. In all patients tested, survivin-reactive T cells were clearly induced. For two patients with complete responses OtSc (with pancreatic cancer) and JuSc (with melanoma), PBL were analyzed before immunization and one, three and six months after initiation of immunization. In patients OtSc, a strong response was already present one month after the start of the immunization trial. The response was stronger after three and six months. Thus, every 10 can be detected⁴More than 600 of individuals survivedProtein-specific cells (fig. 1). In patient JuSc, no response was observed until six months later (fig. 2), at which point it was as strong as in patient OtSc. In addition, we analyzed the PBLs of the SiSt of melanoma patients one and four months prior to immunization and after the start of the immunization trial. In patients with SiSt, a strong response was observed four months after immunization (fig. 3). Finally, two very advanced melanoma patients (AlKa and ErEi) were analyzed for PBL and received only four immunizations before they died due to disease. In both patients, an anti-survivin peptide response was also induced (data not shown). The data confirm that even in these heavily treated patients with very advanced disease, a very strong survivin-specific T cell response was obtained within the circulating lymphocyte population in all patients examined. Thus, every 10 th in vitro stimulation of PBL from immunized patients was observed⁴There are greater than 250 cells that release INF- γ, and in particular embodiments, greater than 600 cells that release INF- γ.

Example 2

Immunohistochemical staining

Biotinylated peptide/HLA complexes were multimerized with streptavidin-FITC-conjugated dextran molecules (DAKO, Glostrup, denmark) to produce multivalent HLA-dextran compounds for immunohistochemical analysis. The tissue sections were dried overnight and then fixed in cold acetone for 5 minutes. All incubation steps were performed at room temperature in the dark: (a) primary antibody (1: 100 dilution) for 45 minutes (b) Cy 3-conjugated goat anti-mouse antibody (1: 500 dilution; code 115-; and finally (c) 75 minutes with polymer. Between each step, the slides were washed 2 times with PBS/0.1% BSA for 10 min each. Sections were covered with coverslips and stored in a refrigerator until examined with a confocal microscope (Leica).

Sequence listing

<110> Sawack company (Survac ApS)

<120> survivin peptide vaccine

<130>P984PC00

<160>118

<170>PatentIn version 3.3

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<211>9

<212>PRT

<213> synthetic peptide

<220>

<221>misc_feature

<222>(1)..(9)

<223> survivin 101-

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Phe Leu Lys Leu Asp Arg Glu Arg Ala

1 5

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<211>10

<212>PRT

<213> synthetic peptide

<220>

<221>MISC_FEATURE

<222>(1)..(10)

<223> survivin 5-14

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Thr Leu Pro Pro Ala Trp Gln Pro Phe Leu

1 5 10

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<212>PRT

<213> synthetic

<220>

<221>misc_feature

<222>(1)..(10)

<223> survivin 95-104

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Glu Leu Thr Leu Gly Glu Phe Leu Lys Leu

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<211>9

<212>PRT

<213> synthetic peptide

<220>

<221>misc_feature

<222>(1)..(9)

<223> survivin 96-104L2

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Leu Leu Leu Gly Glu Phe Leu Lys Leu

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<220>

<221>misc_feature

<222>(1)..(9)

<223> survivin 96-104M2

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Leu Met Leu Gly Glu Phe Leu Lys Leu

1 5

<210>6

<211>9

<212>PRT

<213> synthetic peptide

<220>

<221>misc_feature

<222>(1)..(9)

<223> survivin 46-54

<400>6

Cys Pro Thr Glu Asn Glu Pro Asp Leu

1 5

<210>7

<211>9

<212>PRT

<213> synthetic peptide

<220>

<221>misc_feature

<222>(1)..(9)

<223> survivin 51-59

<400>7

Glu Pro Asp Leu Ala Gln Cys Phe Phe

1 5

<210>8

<211>9

<212>PRT

<213> synthetic peptide

<220>

<221>misc_feature

<222>(1)..(9)

<223> survivin 46-54Y9

<400>8

Cys Pro Thr Glu Asn Glu Pro Asp Tyr

1 5

<210>9

<211>9

<212>PRT

<213> synthetic peptide

<220>

<221>misc_feature

<222>(1)..(9)

<223> survivin 51-59Y9

<400>9

Glu Pro Asp Leu Ala Gln Cys Phe Tyr

1 5

<210>10

<211>9

<212>PRT

<213> synthetic peptide

<220>

<221>misc_feature

<222>(1)..(9)

<223> survivin 6-14

<400>10

Leu Pro Pro Ala Trp Gln Pro Phe Leu

1 5

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<211>9

<212>PRT

<213> synthetic peptide

<220>

<221>misc_feature

<222>(1)..(9)

<223> survivin 11-19

<400>11

Gln Pro Phe Leu Lys Asp His Arg Ile

1 5

<210>12

<211>10

<212>PRT

<213> synthetic peptide

<220>

<221>misc_feature

<222>(1)..(10)

<223> survivin 34-43

<400>12

Thr Pro Glu Arg Met Ala Glu Ala Gly Phe

1 5 10

<210>13

<211>9

<212>PRT

<213> synthetic peptide

<220>

<221>misc_feature

<222>(1)..(9)

<223> survivin 6-14A1

<400>13

Ala Pro Pro Ala Trp Gln Pro Phe Leu

1 5

<210>14

<211>9

<212>PRT

<213> synthetic peptide

<220>

<221>misc_feature

<222>(1)..(9)

<223> survivin 6-14R1

<400>14

Arg Pro Pro Ala Trp Gln Pro Phe Leu

1 5

<210>15

<211>10

<212>PRT

<213> synthetic peptide

<220>

<221>misc_feature

<222>(1)..(10)

<223> survivin 92-101

<400>15

Gln Phe Glu Glu Leu Thr Leu Gly Glu Phe

1 5 10

<210>16

<211>9

<212>PRT

<213> synthetic peptide

<220>

<221>misc_feature

<222>(1)..(9)

<223> survivin 93-101T2

<400>16

Phe Thr Glu Leu Thr Leu Gly Glu Phe

1 5

<210>17

<211>9

<212>PRT

<213> synthetic peptide

<220>

<221>misc_feature

<222>(1)..(9)

<223> survivin 38-46Y9

<400>17

Met Ala Glu Ala Gly Phe Ile His Tyr

1 5

<210>18

<211>10

<212>PRT

<213> synthetic peptide

<220>

<221>misc_feature

<222>(1)..(10)

<223> survivin 47-56Y10

<400>18

Pro Thr Glu Asn Glu Pro Asp Leu Ala Tyr

1 5 10

<210>19

<211>10

<212>PRT

<213> synthetic peptide

<220>

<221>misc_feature

<222>(1)..(10)

<223> survivin 53-62

<400>19

Asp Leu Ala Gln Cys Phe Phe Cys Phe Lys

1 5 10

<210>20

<211>10

<212>PRT

<213> synthetic peptide

<220>

<221>misc_feature

<222>(1)..(10)

<223> survivin 18-27K10

<400>20

Arg Ile Ser Thr Phe Lys Asn Trp Pro Lys

1 5 10

<210>21

<211>11

<212>PRT

<213> synthetic peptide

<220>

<221>misc_feature

<222>(1)..(11)

<223> survivin 18-28

<400>21

Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu

1 5 10

<210>22

<211>429

<212>DNA

<213> human (homo sapiens)

<220>

<221>CDS

<222>(1)..(426)

<400>22

atg ggt gcc ccg acg ttg ccc cct gcc tgg cag ccc ttt ctc aag gac 48

Met Gly Ala Pro Thr Leu Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp

1 5 10 15

cac cgc atc tct aca ttc aag aac tgg ccc ttc ttg gag ggc tgc gcc 96

His Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Cys Ala

20 25 30

tgc acc ccg gag cgg atg gcc gag gct ggc ttc atc cac tgc ccc act 144

Cys Thr Pro Glu Arg Met Ala Glu Ala Gly Phe Ile His Cys Pro Thr

35 40 45

gag aac gag cca gac ttg gcc cag tgt ttc ttc tgc ttc aag gag ctg 192

Glu Asn Glu Pro Asp Leu Ala Gln Cys Phe Phe Cys Phe Lys Glu Leu

50 55 60

gaa ggc tgg gag cca gat gac gac ccc ata gag gaa cat aaa aag cat 240

Glu Gly Trp Glu Pro Asp Asp Asp Pro Ile Glu Glu His Lys Lys His

65 70 75 80

tcg tcc ggt tgc gct ttc ctt tct gtc aag aag cag ttt gaa gaa tta 288

Ser Ser Gly Cys Ala Phe Leu Ser Val Lys Lys Gln Phe Glu Glu Leu

85 90 95

acc ctt ggt gaa ttt ttg aaa ctg gac aga gaa aga gcc aag aac aaa 336

Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Lys Asn Lys

100 105 110

att gca aag gaa acc aac aat aag aag aaa gaa ttt gag gaa act gcg 384

Ile Ala Lys Glu Thr Asn Asn Lys Lys Lys Glu Phe Glu Glu Thr Ala

115 120 125

aag aaa gtg cgc cgt gcc atc gag cag ctg gct gcc atg gat tga 429

Lys Lys Val Arg Arg Ala Ile Glu Gln Leu Ala Ala Met Asp

130 135 140

<210>23

<211>142

<212>PRT

<213> human (homo sapiens)

<400>23

Met Gly Ala Pro Thr Leu Pro Pro Ala Trp Gln Pro Phe Leu Lys Asp

1 5 10 15

His Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu Glu Gly Cys Ala

20 25 30

Cys Thr Pro Glu Arg Met Ala Glu Ala Gly Phe Ile His Cys Pro Thr

35 40 45

Glu Asn Glu Pro Asp Leu Ala Gln Cys Phe Phe Cys Phe Lys Glu Leu

50 55 60

Glu Gly Trp Glu Pro Asp Asp Asp Pro Ile Glu Glu His Lys Lys His

65 70 75 80

Ser Ser Gly Cys Ala Phe Leu Ser Val Lys Lys Gln Phe Glu Glu Leu

85 90 95

Thr Leu Gly Glu Phe Leu Lys Leu Asp Arg Glu Arg Ala Lys Asn Lys

100 105 110

Ile Ala Lys Glu Thr Asn Asn Lys Lys Lys Glu Phe Glu Glu Thr Ala

115 120 125

Lys Lys Val Arg Arg Ala Ile Glu Gln Leu Ala Ala Met Asp

130 135 140

<210>24

<211>10

<212>PRT

<213> human (homo sapiens)

<400>24

Arg Leu Gln Glu Glu Arg Thr Cys Lys Val

1 5 10

<210>25

<211>10

<212>PRT

<213> human (homo sapiens)

<400>25

Gln Leu Cys Pro Ile Cys Arg Ala Pro Val

1 5 10

<210>26

<211>10

<212>PRT

<213> human (homo sapiens)

<400>26

Arg Leu Ala Ser Phe Tyr Asp Trp Pro Leu

1 5 10

<210>27

<211>10

<212>PRT

<213> human (homo sapiens)

<400>27

Leu Leu Arg Ser Lys Gly Arg Asp Phe Val

1 5 10

<210>28

<211>10

<212>PRT

<213> human (homo sapiens)

<400>28

Val Leu Glu Pro Pro Gly Ala Arg Asp Val

1 5 10

<210>29

<211>10

<212>PRT

<213> human (homo sapiens)

<400>29

Pro Leu Thr Ala Glu Val Pro Pro Glu Leu

1 5 10

<210>30

<211>9

<212>PRT

<213> human (homo sapiens)

<400>30

Ser Leu Gly Ser Pro Val Leu Gly Leu

1 5

<210>31

<211>9

<212>PRT

<213> human (homo sapiens)

<400>31

Gln Ile Leu Gly Gln Leu Arg Pro Leu

1 5

<210>32

<211>9

<212>PRT

<213> human (homo sapiens)

<400>32

Leu Thr Ala Glu Val Pro Pro Glu Leu

1 5

<210>33

<211>9

<212>PRT

<213> human (homo sapiens)

<400>33

Gly Met Gly Ser Glu Glu Leu Arg Leu

1 5

<210>34

<211>9

<212>PRT

<213> human (homo sapiens)

<400>34

Glu Leu Pro Thr Pro Arg Arg Glu Val

1 5

<210>35

<211>10

<212>PRT

<213> synthetic sequence

<400>35

Asn Ile Ala Leu Trp Met Thr Glu Tyr Leu

1 5 10

<210>36

<211>10

<212>PRT

<213> synthetic sequence

<400>36

Tyr Leu Asn Arg His Leu His Thr Trp Ile

1 5 10

<210>37

<211>10

<212>PRT

<213> synthetic sequence

<400>37

Pro Leu Phe Asp Phe Ser Trp Leu Ser Leu

1 5 10

<210>38

<211>10

<212>PRT

<213> synthetic sequence

<400>38

Trp Leu Ser Leu Lys Thr Leu Leu Ser Leu

1 5 10

<210>39

<211>9

<212>PRT

<213> synthetic sequence

<400>39

Lys Thr Leu Leu Ser Leu Ala Leu Val

1 5

<210>40

<211>10

<212>PRT

<213> synthetic sequence

<400>40

Ala Ala Ala Gly Pro Ala Leu Ser Pro Val

1 5 10

<210>41

<211>9

<212>PRT

<213> synthetic peptide

<220>

<221>MISC_FEATURE

<222>(1)..(9)

<223>Sur 20-28

<400>41

Ser Thr Phe Lys Asn Trp Pro Phe Leu

1 5

<210>42

<211>10

<212>PRT

<213> synthetic peptide

<220>

<221>MISC_FEATURE

<222>(1)..(10)

<223>Sur115Y10

<400>42

Lys Glu Thr Asn Asn Lys Lys Lys Glu Tyr

1 5 10

<210>43

<211>9

<212>PRT

<213> synthetic peptide

<220>

<221>MISC_FEATURE

<222>(1)..(9)

<223>Sur36-44

<400>43

Glu Arg Met Ala Glu Ala Gly Phe Ile

1 5

<210>44

<211>9

<212>PRT

<213> synthetic peptide

<220>

<221>MISC_FEATURE

<222>(1)..(9)

<223>Sur133-141

<400>44

Arg Ala Ile Glu Gln Leu Ala Ala Met

1 5

<210>45

<211>10

<212>PRT

<213> synthetic peptide

<400>45

Asp Val Ala Gln Cys Phe Phe Cys Phe Lys

1 5 10

<210>46

<211>10

<212>PRT

<213> synthetic peptide

<400>46

Asp Phe Ala Gln Cys Phe Phe Cys Phe Lys

1 5 10

<210>47

<211>10

<212>PRT

<213> synthetic peptide

<400>47

Asp Ile Ala Gln Cys Phe Phe Cys Phe Lys

1 5 10

<210>48

<211>9

<212>PRT

<213> synthetic peptide

<400>48

Ser Tyr Phe Lys Asn Trp Pro Phe Leu

1 5

<210>49

<211>9

<212>PRT

<213> synthetic peptide

<400>49

Thr Ala Lys Lys Val Arg Arg Ala Ile

1 5

<210>50

<211>9

<212>PRT

<213> synthetic peptide

<400>50

Arg Pro Ile Glu Gln Leu Ala Ala Met

1 5

<210>51

<211>9

<212>PRT

<213> synthetic peptide

<400>51

Thr Pro Lys Lys Val Arg Arg Ala Ile

1 5

<210>52

<211>9

<212>PRT

<213> synthetic peptide

<400>52

Glu Arg Ala Lys Asn Lys Ile Ala Lys

1 5

<210>53

<211>9

<212>PRT

<213> synthetic peptide

<400>53

Asp Arg Glu Arg Ala Lys Asn Lys Ile

1 5

<210>54

<211>9

<212>PRT

<213> synthetic peptide

<400>54

Lys Glu Phe Glu Glu Thr Ala Lys Lys

1 5

<210>55

<211>9

<212>PRT

<213> synthetic peptide

<400>55

Glu Arg Met Ala Glu Ala Gly Phe Leu

1 5

<210>56

<211>8

<212>PRT

<213> synthetic peptide

<400>56

Glu Arg Ala Glu Ala Gly Phe Phe

1 5

<210>57

<211>9

<212>PRT

<213> synthetic peptide

<400>57

Glu Arg Met Ala Glu Ala Gly Phe Arg

1 5

<210>58

<211>9

<212>PRT

<213> synthetic peptide

<400>58

Glu Arg Met Ala Glu Ala Gly Phe Lys

1 5

<210>59

<211>9

<212>PRT

<213> synthetic peptide

<400>59

Lys Arg Phe Glu Glu Thr Ala Lys Lys

1 5

<210>60

<211>10

<212>PRT

<213> synthetic peptide

<400>60

Lys Glu Thr Asn Asn Lys Lys Lys Glu Phe

1 5 10

<210>61

<211>9

<212>PRT

<213> synthetic peptide

<400>61

Glu Glu Leu Thr Leu Gly Glu Phe Leu

1 5

<210>62

<211>9

<212>PRT

<213> synthetic peptide

<400>62

Glu Glu Leu Thr Leu Gly Glu Phe Tyr

1 5

<210>63

<211>10

<212>PRT

<213> synthetic peptide

<400>63

Ile Ser Thr Phe Lys Asn Trp Pro Phe Leu

1 5 10

<210>64

<211>10

<212>PRT

<213> synthetic peptide

<400>64

Ile Ser Lys Phe Lys Asn Trp Pro Phe Leu

1 5 10

<210>65

<211>10

<212>PRT

<213> synthetic peptide

<400>65

Leu Ser Val Lys Lys Gln Phe Glu Glu Leu

1 5 10

<210>66

<211>10

<212>PRT

<213> synthetic peptide

<400>66

Leu Ser Lys Lys Lys Gln Phe Glu Glu Leu

1 5 10

<210>67

<211>10

<212>PRT

<213> synthetic peptide

<400>67

Arg Ala Lys Asn Lys Ile Ala Lys Glu Thr

1 5 10

<210>68

<211>10

<212>PRT

<213> synthetic peptide

<400>68

Arg Ala Lys Asn Lys Ile Ala Lys Glu Leu

1 5 10

<210>69

<211>10

<212>PRT

<213> synthetic peptide

<400>69

Asn Asn Lys Lys Lys Glu Phe Glu Glu Thr

1 5 10

<210>70

<211>10

<212>PRT

<213> synthetic peptide

<400>70

Asn Asn Lys Lys Lys Glu Phe Glu Glu Leu

1 5 10

<210>71

<211>9

<212>PRT

<213> synthetic peptide

<400>71

Gln Pro Lys Leu Lys Asp His Arg Ile

1 5

<210>72

<211>9

<212>PRT

<213> synthetic peptide

<400>72

Phe Leu Lys Asp His Arg Ile Ser Thr

1 5

<210>73

<211>9

<212>PRT

<213> synthetic peptide

<400>73

Phe Leu Lys Asp Lys Arg Ile Ser Thr

1 5

<210>74

<211>9

<212>PRT

<213> synthetic peptide

<400>74

Phe Leu Lys Asp His Arg Ile Ser Leu

1 5

<210>75

<211>9

<212>PRT

<213> synthetic peptide

<400>75

Ala Phe Leu Ser Val Lys Lys Gln Phe

1 5

<210>76

<211>9

<212>PRT

<213> synthetic peptide

<400>76

Ala Phe Lys Ser Val Lys Lys Gln Phe

1 5

<210>77

<211>9

<212>PRT

<213> synthetic peptide

<400>77

Al a Phe Leu Ser Lys Lys Lys Gln Phe

1 5

<210>78

<211>9

<212>PRT

<213> synthetic peptide

<400>78

Ala Phe Leu Ser Val Lys Lys Gln Leu

1 5

<210>79

<211>9

<212>PRT

<213> synthetic peptide

<400>79

Phe Leu Ser Val Lys Lys Gln Phe Glu

1 5

<210>80

<211>9

<212>PRT

<213> synthetic peptide

<400>80

Phe Leu Ser Val Lys Lys Gln Phe Leu

1 5

<210>81

<211>9

<212>PRT

<213> synthetic peptide

<400>81

Phe Leu Lys Val Lys Lys Gln Phe Glu

1 5

<210>82

<211>9

<212>PRT

<213> synthetic peptide

<400>82

Ser Val Lys Lys Gln Phe Glu Glu Leu

1 5

<210>83

<211>9

<212>PRT

<213> synthetic peptide

<400>83

Ser Val Lys Lys Lys Phe Glu Glu Leu

1 5

<210>84

<211>9

<212>PRT

<213> synthetic peptide

<400>84

Phe Leu Lys Leu Lys Arg Glu Arg Ala

1 5

<210>85

<211>9

<212>PRT

<213> synthetic peptide

<400>85

Phe Leu Lys Leu Asp Arg Glu Arg Leu

1 5

<210>86

<211>9

<212>PRT

<213> synthetic peptide

<400>86

Thr Ala Lys Lys Lys Arg Arg Ala Ile

1 5

<210>87

<211>9

<212>PRT

<213> synthetic peptide

<400>87

Thr Ala Lys Lys Val Arg Arg Ala Leu

1 5

<210>88

<211>9

<212>PRT

<213> synthetic peptide

<400>88

Thr Leu Pro Pro Ala Trp Gln Pro Phe

1 5

<210>89

<211>9

<212>PRT

<213> synthetic peptide

<400>89

Thr Leu Pro Pro Ala Trp Gln Pro Tyr

1 5

<210>90

<211>10

<212>PRT

<213> synthetic peptide

<400>90

Trp Gln Pro Phe Leu Lys Asp His Arg Ile

1 5 10

<210>91

<211>10

<212>PRT

<213> synthetic peptide

<400>91

Trp Gln Pro Phe Leu Lys Asp His Arg Tyr

1 5 10

<210>92

<211>10

<212>PRT

<213> synthetic peptide

<400>92

Phe Leu Lys Asp His Arg Ile Ser Thr Phe

1 5 10

<210>93

<211>10

<212>PRT

<213> synthetic peptide

<400>93

Phe Leu Lys Asp His Arg Ile Ser Thr Tyr

1 5 10

<210>94

<211>10

<212>PRT

<213> synthetic peptide

<400>94

Arg Ile Ser Thr Phe Lys Asn Trp Pro Phe

1 5 10

<210>95

<211>10

<212>PRT

<213> synthetic peptide

<400>95

Arg Leu Ser Thr Phe Lys Asn Trp Pro Phe

1 5 10

<210>96

<211>9

<212>PRT

<213> synthetic peptide

<400>96

Asp Leu Ala Gln Cys Phe Phe Cys Phe

1 5

<210>97

<211>9

<212>PRT

<213> synthetic peptide

<400>97

Asp Leu Ala Gln Cys Phe Phe Cys Tyr

1 5

<210>98

<211>9

<212>PRT

<213> synthetic peptide

<400>98

Ile Ser Thr Phe Lys Asn Trp Pro Phe

1 5

<210>99

<211>9

<212>PRT

<213> synthetic peptide

<400>99

Ile Gln Thr Phe Lys Asn Trp Pro Phe

1 5

<210>100

<211>9

<212>PRT

<213> synthetic peptide

<400>100

Ile Leu Thr Phe Lys Asn Trp Pro Phe

1 5

<210>101

<211>9

<212>PRT

<213> synthetic peptide

<400>101

Arg Met Ala Glu Ala Gly Phe Ile Tyr

1 5

<210>102

<211>9

<212>PRT

<213> synthetic peptide

<400>102

Arg Met Ala Glu Ala Gly Phe Ile Phe

1 5

<210>103

<211>9

<212>PRT

<213> synthetic peptide

<400>103

Arg Leu Ala Glu Ala Gly Phe Ile Tyr

1 5

<210>104

<211>9

<212>PRT

<213> synthetic peptide

<400>104

Lys Lys His Ser Ser Gly Cys Ala Phe

1 5

<210>105

<211>9

<212>PRT

<213> synthetic peptide

<400>105

Lys Gln His Ser Ser Gly Cys Ala Phe

1 5

<210>106

<211>9

<212>PRT

<213> synthetic peptide

<400>106

Lys Leu His Ser Ser Gly Cys Ala Phe

1 5

<210>107

<211>9

<212>PRT

<213> synthetic peptide

<400>107

Arg Ala Ile Glu Gln Leu Ala Ala Tyr

1 5

<210>108

<211>9

<212>PRT

<213> synthetic peptide

<400>108

Arg Leu Ile Glu Gln Leu Ala Ala Met

1 5

<210>109

<211>10

<212>PRT

<213> synthetic peptide

<400>109

Pro Thr Leu Pro Pro Ala Trp Gln Pro Phe

1 5 10

<210>110

<211>11

<212>PRT

<213> synthetic peptide

<400>110

Cys Thr Pro Glu Arg Met Ala Glu Ala Gly Phe

1 5 10

<210>111

<211>9

<212>PRT

<213> synthetic peptide

<400>111

Glu Thr Asn Asn Lys Lys Lys Glu Phe

1 5

<210>112

<211>9

<212>PRT

<213> synthetic peptide

<400>112

Gly Ala Pro Thr Leu Pro Pro Ala Trp

1 5

<210>113

<211>10

<212>PRT

<213> synthetic peptide

<400>113

Cys Ala Phe Leu Ser Val Lys Lys Gln Phe

1 5 10

<210>114

<211>10

<212>PRT

<213> synthetic peptide

<400>114

Thr Pro Glu Arg Met Ala Glu Ala Gly Phe

1 5 10

<210>115

<211>9

<212>PRT

<213> synthetic peptide

<400>115

Cys Ala Cys Thr Pro Glu Arg Met Ala

1 5

<210>116

<211>9

<212>PRT

<213> synthetic peptide

<400>116

Cys Thr Pro Glu Arg Met Ala Glu Ala

1 5

<210>117

<211>9

<212>PRT

<213> synthetic peptide

<400>117

Phe Leu Glu Gly Cys Ala Cys Thr Pro

1 5

<210>118

<211>10

<212>PRT

<213> synthetic peptide

<400>118

Trp Pro Phe Leu Glu Gly Cys Ala Cys Thr

1 5 10

Claims

1. A vaccine composition for use as a medicament, comprising:

i. a survivin peptide consisting of SEQ ID NO: 5 is composed of, and

montanide ISA51 adjuvant.

2. The vaccine composition of claim 1, comprising:

i. a nucleic acid encoding SEQ ID NO: 5,

and

montanide ISA51 adjuvant.

3. Use of a vaccine composition as characterized in any one of claims 1-2 for the manufacture of a medicament for the treatment of metastatic melanoma.

4. Use of a vaccine composition as characterized in any one of claims 1-2 for the manufacture of a medicament for the treatment of pancreatic cancer.