WO2004067023A2

WO2004067023A2 - Survivin-derived peptides and use thereof

Info

Publication number: WO2004067023A2
Application number: PCT/DK2004/000062
Authority: WO
Inventors: Eivind Per Thor Straten; Mads Hald Andersen
Original assignee: Survac Aps
Priority date: 2003-01-30
Filing date: 2004-01-30
Publication date: 2004-08-12
Also published as: CN1767848A; EA008026B1; ATE406906T1; EP1587532A2; EA200501206A1; CN1767848B; AU2004208469B2; EP2359841A2; EP1587532B1; ES2339193T3; EP2857034A1; US20070148184A1; EP2857034B1; PT2005966E; MXPA05008128A; EP2092938B1; NO20053999L; NO20053999D0; CN101906148A; JP2006517529A

Abstract

MHC Class I-restricted peptides derived from the tumor associated antigen, survivin, which peptides are capable of binding to Class I HLA molecules at a high affinity, capable of eliciting INF-Ϝ-producing cells in a PBL population of a cancer patient and capable of in situ detection of cytotoxic T cells in a tumor tissue, therapeutic and diagnostic composition comprising the peptide and uses thereof.

Description

SURVIVIN-DERIVED PEPTIDES AND USE THEREOF

FIELD OF INVENTION

The present invention relates to novel survivin-derived peptides and their use for diagnostic and therapeutic purposes, specifically in cancer. In particular, the novel peptides are MHC Class I-restricted T-cell epitopes that are capable of eliciting cytotoxic T-cell responses in cancer patients including in situ and ex vivo responses. Specifically, such novel peptides are derived from the apoptosis inhibitor protein survivin, a recognized tumor associated antigen (TAA).

TECHNICAL BACKGROUND AND PRIOR ART

The process by which the mammalian immune system recognizes and reacts to foreign or alien materials is a complex one. An important facet of the system is the T-cell response. This response requires that T cells recognize and interact with complexes of cell surface molecules referred to as human leukocyte antigens (HLA) constituting the human major histocompatibility complex (MHC), and peptides. The peptides are derived from larger molecules, which are processed by the cells, which also present the HLA/MHC molecule. The interaction of T cells and complexes of HLA/peptide is restricted, requiring a T cell that is specific for a particular combination of an HLA molecule and a peptide. If a specific T cell is not present, there is no T-cell response even if its partner complex is present. Similarly, there is no response if the specific complex is absent, but the T cell is present.

The mechanism by which T cells recognize cellular abnormalities has also been implicated in cancer. E.g. in WO92/20356, a family of genes is disclosed which are processed into peptides which, in turn, are expressed on cells surfaces, and can lead to lysis of the tu- mour cells by specific CTLs. These genes are referred to as the MAGE family and are said to code for "tumour rejection antigen precursors" or "TRAP" molecules, and the peptides derived therefrom are referred to as "tumour rejection antigens" or "TRAs".

In WO 94/05304, nonapeptides are disclosed which bind to the HLA-A1 molecule. The re- ference discloses that given the known specificity of particular peptides for particular HLA molecules, one should expect a particular peptide to bind one HLA molecule, but not others. This is significant, because different individuals possess different HLA phenotypes. As a result, while identification of a particular peptide as being a partner for a specific HLA molecule has diagnostic and therapeutic ramifications, these are only relevant for individu- als with that particular HLA phenotype.

Several peptides presented by MHC molecules have been characterized and it has been found that some of these may carry posttranslational modifications possibly having an impact on the functionality of the HLA-peptide complex. Thus, a number of studies have associated alterations in the pattern of phosphorylation with malignant transformation. Furthermore, it has been shown that phosphorylation could have a neutral, negative or even a positive effect on peptide binding to class I MHC and that phosphopeptide-specific CTL, which discriminated between the phosphorylated and the non-phosphorylated versions of the peptide, could be generated, showing that such CTL most likely are part of the class I MHC-restricted CTL repertoire. Recently, it has been shown that phosphorylated peptides indeed are processed naturally and presented by MHC class I molecules in vivo. Additionally, the presence of phosphorylated peptides in extracts from isolated class I molecules from several different EBV-transformed B-cells has been established.

Thus, it is well established that peptide epitopes derived from tumor associated antigens (TAAs) can be recognized as antigens by cytotoxic T lymphocytes (CTLs) in the context of MHC molecules (1). However, although it is generally accepted that most if not all, tumours are antigenic, only a few are indeed immunogenic in the sense that tumour pro- gression is readily controlled by the immune system.

To overcome this limitation, several immunotherapeutic trials have been initiated, e.g. vaccinations with TAA-derived peptides. For melanoma, the tumour for which the largest number of CTL-defined TAAs has been characterized, powerful CTL responses against anti- gens have been induced by vaccination and some patients experienced a complete remission of their disease (2,3). However, most of the peptide epitopes used in these vaccination trials are melanocyte specific, and these peptides cannot be applied for tumours of non-melanocyte origin. Furthermore, expression of these TAAs is heterogeneous among tumours from different patients and can even vary among metastases obtained from one patient. However, during the last couple of years a number of tumour specific peptide antigens, which are expressed in a number of different cancers, have been identified, i.e. HER-2 (4), Muc-1 (5) and telomerase (6).

It has also been shown that by proper manipulation tumor antigens present in tumors can be exposed to the immune system. Studies have shown that the CD8+ CTL arm of the immune response, alone or in combination with CD4+ T_h cells, constitutes the primary anti-tumor effector arm of the adaptive immune response. Up till now the focus has mainly been on the CTL arm of the immune response. However, it is becoming more and more clear that the CD4 T cell response plays an essential role in tumor rejection, especially in the induction phase or in the extension of a CTL response in vivo. Consequently, the incorporation of class II-restricted tumor antigens into effective tumor vaccination protocols might increase the effectiveness of the vaccines.

Apoptosis is a genetic program of cellular suicide, and inhibition of apoptosis has been suggested to be an important mechanism involved in cancer formation by extending the life span of cells favouring the accumulation of transforming mutations (7). Survivin is a recently identified member of the family of inhibitors of apoptosis proteins (IAPs). In a global gene expression analysis of about 4 million transcripts, survivin was identified as one of the top genes invariably up-regulated in many types of cancer but not in normal tissue (8). Solid malignancies overexpressing survivin include lung, colon, breast, pancreas, and prostate cancer as well as hematopoietic malignancies (9). Additionally, series of melanoma and non-melanoma skin cancers have been reported to be invariably survivin positive (10,11). The overexpression of survivin in most human cancers suggests a general role of apoptosis inhibition in tumor progression, a notion substantiated by the observation that in the case of colorectal and bladder cancer, as well as neuroblastoma, expression of survivin was associated with an unfavourable prognosis. In contrast, survivin is undetect- able in normal adult tissues. These characteristics qualify survivin as a suitable TAA for both diagnostic and therapeutic purposes.

Thus, during the last decade a large number of TAAs have been identified which are recognized by CTLs in a major histocompatibility complex (MHC)-restricted fashion. As survivin is overexpressed in most human cancers and inhibition of its function results in increased apoptosis, this protein may serve as a target for therapeutic CTL responses. The survivin protein and the potential diagnostic and therapeutic use hereof are disclosed in (8) and US 6.245.523, which are incorporated herein by reference. Survivin is a 16.5 kDa cytoplasmic protein containing a single BIR and a highly charged carboxy-terminal coiled region instead of a RING finger, which inhibits apoptosis induced by growth factor (IL-3) withdrawal when transferred in B cell precursors. The gene coding for survivin is nearly identical to the sequence of Effector Cell Protease Receptor-1 (EPR-1), but oriented in the opposite direction, thus suggesting the existence of two separate genes duplicated in a head-to- head configuration. Accordingly, survivin can be described as an antisense EPR-1 product. Functionally, inhibition of survivin expression by up-regulating its natural antisense EPR-1 transcript results in massive apoptosis and decreased cell growth.

US 6.245.523 discloses the isolation of purified survivin and it provides nucleic acid molecules that encode the survivin protein, and antibodies and other molecules that bind to survivin. US 6.245.523 also discloses anti-apoptotically active fragments of the survivin protein and variants hereof wherein an amino acid residue has been inserted N- or C-ter- minal to, or within, the disclosed survivin sequence. It is specifically disclosed that such peptides should contain key functional residues required for apoptosis, i.e. Trp at position 67, Pro at position 73 and Cys at position 84.

The present invention is based on the discovery that MHC Class I restricted peptides can be derived from the survivin protein, which are capable of binding to MHC Class I HLA molecules and thereby eliciting both eλ vivo and in situ CTL immune responses in patients suffering from a wide range of cancer diseases. These findings strongly suggest that survivin acts as a TRAP molecule, which is processed by cells into peptides having TRA functionality. Evidently, these findings open the way for novel therapeutic and diagnostic ap- proaches which, due to the fact that survivin appears to be expressed universally by tumour cells, are generally applicable in the control of cancer diseases. SUMMARY OF THE INVENTION

Accordingly, the invention pertains in a first aspect to a MHC Class I-restricted epitope peptide derived from survivin, said epitope having at least one of the following characte- ristics:

(i) capable of binding to the Class I HLA molecule to which it is restricted at an affinity as measured by the amount of the peptide that is capable of half maximal recovery of the Class I HLA molecule (C₅₀ value) which is at the most 50 μM as determined by the assem- bly binding assay as described herein,

(ii) capable of eliciting INF-γ -producing cells in a PBL population of a cancer patient at a frequency of at least 1 per 10⁴ PBLs as determined by an ELISPOT assay, and/or

(iii) capable of in situ detection in a tumour tissue of CTLs that are reactive with the epitope peptide.

Preferably, the peptide of the invention has at least two, most preferably all of these three features.

In further aspects the invention provides a pharmaceutical composition and a composition for ex vivo or in situ diagnosis of the presence in a cancer patient of survivin reactive T- cells among PBLs or in tumour tissue, which composition comprises a peptide as defined above.

In yet further aspects the invention relates to a diagnostic kit for ex vivo or in situ diagnosis of the presence in a cancer patient of survivin reactive T-cells among PBLs or in tumor tissue, which kit comprises a peptide according of the invention, and a complex of such a peptide and a Class I HLA molecule or a fragment of such molecule.

In another aspect there is also provided a method of detecting in a cancer patient the presence of survivin reactive T-cells, the method comprising contacting a tumour tissue or a blood sample with a complex as defined above and detecting binding of the complex to the tissue or the blood cells.

In still further aspects the invention pertains to a molecule that is capable of binding specifically to a peptide of the invention such as an antibody or a fragment hereof, and to a molecule that is capable of blocking the binding of such a molecule.

An important aspect of the invention relates to the use of the peptides of the invention for the preparation of a medicament for the treatment of cancer. A further aspect relates to the use of the composition or the molecule as mentioned above for the preparation of a medicament for the treatment of cancer. Still further aspects relate independently to a method for treating cancer in a mammal, such as a human, comprising the administration to a patient suffering from the disease an effective amount of the peptide, composition or a molecule of the invention.

DETAILED DISCLOSURE OF THE INVENTION

The novel MHC Class I-restricted peptide of the invention is characterised by having at least one of several features, one of which is the ability to bind to the Class I HLA molecule to which it is restricted at an affinity, which, when it is measured by the amount of the peptide that is capable of half maximal recovery of the Class I HLA molecule (C₅₀ value) in an assembly assay as described herein, is at the most 50 μM. This assembly assay is carried out as described previously (12,13), and it is based on stabilisation of the HLA molecule after loading of peptide to the peptide transporter deficient cell line T2. Subsequently, correctly folded stable HLA heavy chains are immunoprecipitated using conformation dependent antibodies and the peptide binding is quantitated.

This assay provides a simple means of screening candidate peptides for their ability to bind to a given HLA allele molecule at the above affinity. In preferred embodiments, the peptide of the invention in one having a C₅₀ value, which is at the most 30 μM, such as a C₅₀ value, which is at the most 20 μM including C₅₀ values of at the most 10 μM, at the most 5 μM and at the most 2 μM.

As mentioned above, the HLA system represents the human major histocompatibility (MHC) system. Generally, MHC systems control a range of characteristics: transplantation antigens, thymus dependent immune responses, certain complement factors and predisposition for certain diseases. More specifically, the MHC codes for three different types of molecules, i.e. Class I, II and III molecules, which determine the more general characteristics of the MHC. Of these molecules, the Class I molecules are so-called HLA-A, HLA-B and HLA-C molecules that are presented on the surface of most nucleated cells and throm- bocytes.

The peptides of the present invention are characterised by their ability to bind to (being restricted to) a particular MHC Class I HLA molecule. Thus, in one embodiment the peptide is one which is restricted to a MHC Class I HLA-A molecule including HLA-Al, HLA-A2, HLA- A3, HLA-A9, HLA-A10, HLA-A11, HLA-Awl9, HLA-A23(9), HLA-A24(9), HLA-A25(10), HLA- A26(10)„ HLA-A28, HLA-A29(wl9), HLA-A30(wl9), HLA-A31(wl9), HLA-A32(wl9), HLA- Aw33(wl9), HLA-Aw34(10), HLA-Aw36, HLA-Aw43, HLA-Aw66(10), HLA-Aw68(28), HLA- A69(28). More simple designations are also used throughout the literature, where only the primary numeric designation is used, e.g. HLA-A19 or HLA-A24 instead of HLA-Awl9 and HLA-A24(9), respectively. In specific embodiments, the peptide of the invention is restricted to a MHC Class I HLA species selected from the group consisting of HLA-Al, HLA- A2, HLA-A3, HLA-A11 and HLA-A24. The peptides of the invention are derived from the known sequence of survivin, e.g. the sequence disclosed in US 6.245.523. The selection of peptides potentially having the ability to bind to a particular HLA molecule can be made by the alignment of known sequences that bind to a given particular HLA molecule to thereby reveal the predominance of a few related amino acids at particular positions in the peptides. Such predominant amino acid residues are also referred to herein as "anchor residues" or "anchor residue motifs". By following such a relatively simple procedure based on known sequence data that can be found in accessible databases, peptides can be derived from the survivin protein molecule which are likely to bind to the particular HLA molecule. Representative examples of such analyses for a range of HLA molecules are given in the below table:

,

Thus, as an example, nonapeptides potentially having the ability to bind to HLA-Al would have 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-Xaa-L-Xaa-Y (Xaa indi- eating any amino acid residue). In a similar manner, sequences potentially having the ability to bind to any other HLA molecule can be designed. It will be appreciated that the person of ordinary skill in the art will be able to identify further "anchor residue motifs" for a given HLA molecule.

Thus, in useful embodiments, the peptides of the invention include peptides, the se- quences of which comprise, for each of the specific HLA alleles listed in the table, any of the amino acid residues as indicated in the table.

Thus, a simple approach to identifying peptides o the invention includes the following steps: selecting a particular HLA molecule, e.g. one occurring at a high rate in a given population, carrying out an alignment analysis as described above to identify "anchor residue motifs" in the survivin protein, isolating or constructing peptides of a suitable size that comprise one or more of the identified anchor residues and testing the resulting peptides for (i) capability to bind to the particular HLA molecule using the assembly assay as described herein, (ii) the capability of the peptides to elicit INF-γ -producing cells in a PBL population of a cancer patient at a frequency of at least 1 per 10⁴ PBLs as determined by an ELISPOT assay as described herein, and/or (iii) the capability of the peptides to detect in situ in a tumour tissue CTLs that are reactive with the epitope peptides being tested.

In specific embodiments, the peptide of the invention is an HLA-A2 restricted survivin-de- rived peptide having a sequence selected from the following: FLKLDRERA (survivin₁₀ι-ιo₉) (SEQ ID NO: l), TLPPAWQPFL (survivin₅_₁₄) (SEQ ID NO:2), ELTLGEFLKL (survivin_95-ιo₄) (SEQ ID NO:3), LLLGEFLKL (SEQ ID NO:4) and LMLGEFLKL (SEQ ID NO: 5). (The designations in brackets indicate the positions of the residues in the survivin protein as disclosed in US 6.245.523). LLLGEFLKL (SEQ ID NO:4) is a sequence derived from survivin_96-1o₄ by substituting "T" in position 2 of the peptide with an "L" and LMLGEFLKL (SEQ ID NO:5) is derived from survivin_96-ιo₄ by substituting "T" in position 2 with "M".

In further useful embodiments, the peptide of the invention is a peptide, which is restricted by a MHC Class I HLA-B molecule including any of the following: HLA-B5, HLA-B7, HLA-B8, HLA-B12, HLA-B13, HLA-B14, HLA-B15, HLA-B16, HLA-B17, HLA-B18, 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-Bw47. In specific embodiments, the MHC Class I HLA-B species to which the peptide of the invention is capable of binding is selected from HLA-B7, HLA-B35, HLA-B44, HLA-B8, HLA-B15, HLA-B27 and HLA-B51.

In specific embodiments, the peptide of the invention is an HLA-B35-restricted survivin- derived peptide having a sequence selected from the following: CPTENEPDL (survivin₄₆-₅₄) (SEQ ID NO:6), EPDLAQCFF (survivin₅₁_₅₉) (SEQ ID NO:7), CPTENEPDY (SEQ ID NO:8) and EPDLAQCFY (SEQ ID NO:9). (The designations in brackets indicate the positions of the residues in the survivin protein as disclosed in US 6.245.523). CPTENEPDY (SEQ ID NO:8) is a sequence derived from survivin₄₆-₅₄ by substituting "L" in the C-terminal of the peptide with a "Y" and EPDLAQCFY (SEQ ID NO:9) is derived from survivin₅₁.₅₉ by substituting an "F" residue in the C-terminal 2 with a "Y". In further specific embodiments, the peptide of the invention is a HLA-Al restricted peptide having a sequence selected from the following: Survivin₃₈.₄₆ (Sur38Y9) (a C changed to a Y at P9, MAEAGFIHY)(SEQ ID NO:38), Survivin₄₇.₅₆ (Sur47Y10) (a Q changed to a Y at P10, PTENEPDLAY(SEQ ID NO:39)), Survivin_92-10ι (Sur92-101) (QFEELTLGEF) (SEQ ID NO: 27), and Survivin_93-10ι (Sur93T2 (a E changed to a T at P2, FTELTLGEF (SEQ ID NO:36)). The peptide of the invention may also be a HLA-A3 restricted peptide such as Survivin_18-24 (Surl8K10) (a F changed to a K at P10, RISTFKNWPK (SEQ ID NO:57) and/or a HLA-Al 1 restricted peptide such as Survivin₅₃.₆₂ (Sur53-62)(DLAQCFFCFK)(SEQ ID NO:45) and/or a HLA-A2 restricted peptide such as Survivin₁₈.₂₈ (Surl8-28) (RISTFKNWPFL)(SEQ ID NO:66).

In further useful embodiments, the peptide of the invention is a peptide, which is restricted to a MHC Class I HLA-C molecule including any of the following: HLA-Cwl, HLA-Cw2, HLA- Cw3, HLA-Cw4, HLA-CW5, HLA-Cw6, HLA-Cw7 and HLA-Cwl6.

Preferably, the peptide of the invention comprises less than 50 amino acid residues, and more preferably it comprises at the most 20 amino acid residues, such as at the most 10 amino acid residues. In specific embodiments, the peptide is a heptapeptide, an octopep- tide, a nonapeptide, a decapeptide or an undecapeptide.

The peptide of the invention is, as mentioned above, derived from a survivin protein or a fragment hereof. The survivin protein from which the peptide can be derived is survivin protein from any animal species in which the protein is expressed. In preferred embodiments, the survivin starting protein is from a mammal species including a rodent species, rabbit and a primate species such as humans. Based on the sequence of the selected survivin protein, the peptide of the invention is derived by any appropriate chemical or enzymatic treatment of the survivin starting material that results in a peptide of a suitable size as indicated above, or it can be synthesised by any conventional peptide synthesis procedures with which the person of ordinary skills in the art is familiar.

The peptide of the invention may have a sequence which is a native sequence of the survivin protein from which is derived. However, peptides having a higher affinity to any given HLA molecule may be derived from such a native sequence by modifying the sequence by substituting, deleting or adding at least one amino acid residue, e.g. on the basis of the procedure described above whereby anchor residue motifs in respect of the given HLA molecule are identified.

Accordingly, to increase the immuogenicity of survivin-derived peptides, amino acid substitutions can be introduced at anchor positions, but not at TCR contact residues, to increase peptide binding to the HLA class I molecule. This has resulted in more immunogenic epitopes, e.g., this has enhanced the capacity to induce cancer-reactive CTL and it has been demonstrated to be more suitable for the induction of clinically meaningful CTL responses. Importantly, however, the target cancer cells do only express and present the native survivin-derived peptide on the cell-surface. In that respect, it is of crucial importance that therapy-induced CTL specific for the modified survivin-derived peptides cross-react with the native analogues.

The present invention also encompasses variants and functional equivalents of the survivin-derived peptides as disclosed herein. "Functional equivalents" as used in the present context is established by means of reference to the corresponding functionality of a predetermined fragment of the sequence in question. Functional equivalence can be established by e.g. similar binding affinities to HLA class I molecules, or similar potency demonstrated by the ELISPOT assay.

Functional equivalents or variants of a survivin-derived peptide as described herein will be understood to exhibit amino acid sequences gradually differing from the preferred, predetermined sequences, as the number and scope of insertions, deletions and substitutions including conservative substitutions, increases. This difference is measured as a reduction in homology between a preferred, predetermined sequence and the survivin- derived variant or survivin-derived functional equivalent.

The homology between amino acid sequences may be calculated using algorithms well known in the art. Fragments sharing homology with fragments comprising or consisting of consecutive survivin-derived amino acid residues are to be considered as falling within the scope of the present invention when they are preferably at least about 90% homologous, such as at least 94% homologous, including 95%, 96%, 97%, 98% or 99% homologous with a predetermined survivin-derived peptide.

Furthermore, it may be advantageous to carry out post-translational modifications of the peptides of the invention. It has been shown that exposure of breast carcinoma MCF-7 or cervical carcinoma HeLa cells to anticancer agents including Adriamycin, Taxol, or UVB resulted in a 4-5-fold increased survivin expression. Changes in survivin levels after anticancer treatment did not involve modulation of survivin mRNA expression and were independent of de novo gene transcription. Conversely, inhibition of survivin phosphorylation on Thr³⁴ by the cyclin-dependent kinase inhibitor flavopiridol resulted in loss of survivin expression, and nonphosphorylatable survivin Thr³⁴— *Ala exhibited accelerated clearance as compared with wild-type survivin. Sequential ablation of survivin phosphorylation on Thr³⁴ enhanced tumor cell apoptosis induced by anticancer agents independently of p53 and suppressed tumor growth without toxicity n a breast cancer xenograft model in vivo. These data suggest that Thr³⁴ phosphorylati on critically regulates survivin levels in tumor cells and that sequential ablation of p34^cdc2 k nase activity may remove the survivin viability checkpoint and enhance apoptosis in tumor cells.

Accordingly, it is contemplated that the 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 Thr34. Thus, possible survivin-derived phosphopeptide sequences include T P E R M A E A G F, a putative HLA-B35- and/or HLA-B7- and/or a HLA-B51- restricted peptide antigen. Additional native phosphopeptides encompassed herein include: HLA-A2: C A C T P E R M A and C T P E R M A E A; HLA-A3: F L E G C A C T P; HLA- B7/HLA-B35/HLA-B51 : W P F L E G C A C T (Phoshorylated Thr residue marked in bold).

A significant feature of the peptide of the invention is its capability to recognise or elicit INF-γ -producing responder T cells, i.e. cytotoxic T cells (CTLs) that specifically recognise the particular peptide in a PBL population or tumour cells of a cancer patient (target cells). This activity is readily determined by subjecting PBLs or tumour cells from a patient to an ELISPOT assay as described in reference (16) and in the following examples. Prior to the assay, it may be advantageous to stimulate the PBL population or the tumour cells to be assayed by contacting the cells with the peptide to be tested. Preferably, the peptide is capable of eliciting or recognising INF-y -producing T cells at a frequency of at least 1 per 10⁴ PBLs as determined by an ELISPOT assay as used herein. More preferably the frequency is at least 5 per 10⁴ PBLs, most preferably at least 10 per 10⁴ PBLs, such as at least 50 or 100 per 10⁴ PBLs.

The ELISPOT assay represents a strong tool to monitor survivin peptide specific T-cell responses. However, although it has been shown that ELISPOT reactivity in most cases correlates with the capacity of the CLLs to lyse target cells, the conclusive evidence for this notion can only be given directly. Such direct evidence is provided herein, as it was de- monstrated (see Example 2) that survivin reactive cells isolated by means of HLA/peptide complexes possess the functional capacity of lysing target cells. Additionally, it was demonstrated that the isolated CTLs specifically recognising a peptide of the invention were capable of lysing HLA-matched tumour cells of different origin, e.g. melanomas and breast cancer. This finding strongly suggests that cancer cells in general process and present the same endogenous survivin peptide. Therefore, a major implication of the findings herein is that the peptides of the invention are expressed and complexed with HLA molecules on a variety of cancer cells of different histological origins. This renders these cancer cells susceptible to destruction by CTLs and emphasizes the potential usefulness of survivin immunization to control the growth of different neoplasms. The presence of spontaneous CTL- responses in PBLs and tumour cells to HLA-restricted survivin-derived peptide epitopes from patients suffering from three unrelated cancer types, i.e., breast cancer, melanoma and CLL, further substantiates the universal immunotherapeutic potential of this tumour antigen.

Accordingly, in another preferred embodiment the peptide of the invention is capable of eliciting INF-γ -producing cells in a PBL population of a patient having a cancer disease where survivin is expressed including a haematopoietic malignancy including chronic lymphatic leukemia and chronic myeloid leukemia, melanoma, breast cancer, cervix cancer, ovary cancer, lung cancer, colon cancer, pancreas cancer and prostate cancer. Specifically, the peptide of the invention is able to elicit an immune response in the form of T cell having cytotoxic effect against survivin expressing cells of a cancer cell line, including a cell line selected from the breast cancer cell line MCF-7 and the melanoma cell line FM3. In addition to their capacity to elicit immune responses in PBL populations and cancer cell lines, it was demonstrated that the peptides of the invention are also capable of eliciting cytolytic immune responses in situ, i.e. in solid tumour tissues. This was demonstrated by providing HLA-peptide complexes, e.g. being multimerised and being provided with a de- tectable label, and using such complexes for immunohistochemistry stainings to detect in a tumour tissue CTLs that are reactive with the epitope peptide of the invention. Accordingly, a further significant feature of the peptide of the invention is that it is capable of in situ detection in a tumour tissue of CTLs that are reactive with the epitope peptide.

It is contemplated that the peptides of the invention, in addition to their capacity to bind to HLA molecules resulting in the presentation of complexes of HLA and peptides on cell surfaces, which complexes in turn act as epitopes or targets for cytolytic T cells, may elicit other types of immune responses, such as B-cell responses resulting in the production of antibodies against the complexes and/or a Delayed Type Hypersensitivity (DTH) reaction. The latter type of immune response is defined as a redness and palpable induration at the site of injection of the peptide of the invention.

It is well known, that the different HLA molecules are of different prevalence in the major human populations. Accordingly, there is a requirement for identifying peptide epitopes restricted to several HLA class I molecules to extend the patient cohort that can be treated according to the methods of the present invention. The characterisation of multiple survivin epitopes with different HLA restriction elements broadens the clinical potential of this target antigen in two important ways: (i) It increases the number of patients eligible for immunotherapy based on survivin-derived peptides. The HLA-A2 antigen is expressed by around 50 % of the Caucasian and Asian populations, the HLA-Al and HLA- A3 antigens are both expressed by around 25 % of Caucasians and 5 % of Asians, whereas the HLA- All antigen is expressed by around 15 % of Caucasians and 30 % of Asians. Even though these numbers cannot be summed up due to co-expression, a combination of peptides restricted by a multiplicity of these would certainly encompass most cancer patients, (ii) The collective targeting of several restriction elements in each patient is likely to decrease the risk of immune escape by HLA-allele loss. Loss of a single HLA allele is a significant component of MHC alterations described for cancer cells, whereas total loss of Class I expression is a rather infrequent event . Thus, with the identification of survivin epitopes restricted to different HLA alleles, it is now possible to target more than one HLA-molecule simultaneously in patients with allelic overlap.

Accordingly, based on the disclosure of the present invention the person of skill in the art would be able to develop highly immunogenic multi-epitope vaccines. Preferably, such vaccines should be designed so as to facilitate a simultaneous delivery of the best-suited survivin-derived peptides optionally in combination with other suitable peptides and/or adjuvants as described hereinafter.

Furthermore, as previously described, there has been an increased focus on eliciting tumor-specific T helper cell immunity, i.e., vaccinating with class II-MHC restricted epitopes despite the fact that tumors generally do not express class II MHC. This is based on the recent finding that the induction and efficacy of the vaccine-induced anti-tumor response in many cases requires the cooperation of tumor-specific CD4 positive T_h cells. Thus, an important factor driving the development of vaccines having a more complex composition is the desire to target multiple tumor antigens e.g. by designing vaccines comprising or encoding a collection of carefully selected CTL and T_h cell epitopes.

Obviously, multi-epitope vaccines constitute an efficient way to raise immunity against epitopes derived from several different antigens without the need for introducing (genes encoding) potentially hazardous proteins such as oncoproteins. Such vaccines also permit selective induction of immunity against subdominant and cryptic T cell epitopes, which can be especially important in the case of tumor-associated autoantigens for which tolerance may exist for the epitopes that are prominently presented in normal tissues. Furthermore, antigen-presenting cells may fail to present certain epitopes that are expressed on tumor cells because of functional differences between the immunoproteasomes of antigen- presenting cells and the 'constitutive' proteasomes present in most tumor cells. In the case of peptide-based vaccines, such epitopes can be administered in an 'MHC-ready' form, which enables presentation through exogenous loading independently of antigen uptake and processing by host antigen-presenting cells.

It is evident that the findings of the present invention provide the basis for therapeutic as well as diagnostic applications of the survivin-derived peptides.

Thus, an important aspect of the present invention relates to a composition comprising:

Accordingly, in a further aspect the present invention provides a pharmaceutical composition comprising one or more of the peptides of the invention alone or in suitable combination with other proteins or peptide fragments. In specific embodiments such other proteins or peptide fragments include but are not limited to proteins involved in regulation of cell apoptosis or peptide fragments hereof. Suitable examples of such proteins can be selected from the Bcl-2 protein family, e.g., the Bcl-2 protein, the Bcl-w protein, the Mcl-1 protein, the Bcl-X_L protein, and peptide fragments derived from any of the proteins. Other known apoptosis inhibitors include members of the inhibitor of apoptosis protein (IAP) family such as X-IAP, C-IAP1 and C-IAP2 these proteins are all relatively ubiquitously expressed whereas the inhibitor of apoptosis polypeptide ML-IAP has a rather selective expression, and is predominantly detected in melanomas. Thus, fragments of ML-IAP capable of eliciting a specific T-cel! response i.e a cytotoxic T-cell response or a helper T- cell response may optionally be included in the composition of the present invention. Useful peptide fragments of ML-IAP include ML-IAP₂₄₅ (RLQEERTCKV)(SEQ ID NO:75), ML- IAP₂₈₀ (QLCPICRAPV)(SEQ ID NO:76), ML-IAP₉₀ (RLASFYDWPL)(SEQ ID NO:77), ML-IAP₁₅₄ (LLRSKGRDFV)(SEQ ID NO:78), ML-IAP₂₃₀ (VLEPPGARDV)(SEQ ID NO:79), ML-IAP₉₈ (PLTAEVPPEL)(SEQ ID NO:80), ML-IAP₃₄ (SLGSPVLGL)(SEQ ID NO:81), ML-IAP₅₄ (QILGQLRPL)(SEQ ID NO:82), ML-IAP₉₉ (LTAEVPPEL)(SEQ ID NO:83), ML-IAP₈₃ (GMGSEELRL)(SEQ ID NO:84) and ML-IAP₂₀₀ (ELPTPRREV)(SEQ ID NO:85). Additionally, the composition according to the present invention may be provided as a multiepitope vaccine comprising class I restricted epitope and/or class II restricted epitopes as defined hereinbefore.

Example of a presently preferred multiepitope vaccines include "tailor made" combinations of survivin-derived peptide eptiopes depending of the tissue type of the given patient, e.g., a subject carrying HLA-A2, HLA-A3, and HLA-B35 phenotypes could be vaccinated with a vaccine comprising surlM2, sur9, surl8K10, sur46Y9, sur51Y9. Additionally, the pharmaceutical composition of the invention may advantageously comprise at least one further immunogenic protein or peptide fragment hereof selected from a protein or peptide fragment not belonging to or derived from the survivin protein. In specific embodiments, the immunogenic protein or peptide fragment thereof is derived from the Bcl-2 protein family as described hereinbefore. A further immunogenic Bcl-2-derived peptide is an HLA- A2 restricted peptide having a sequence selected from the following: Bcl₁₇₂, Bcl₁₈₀, Bcl₂₀s, and Bcl₂ι₄

As the peptides of the invention are relatively small molecules it may be required in such compositions to combine the peptides with various materials such as adjuvants, to produce vaccines, immunogenic compositions, etc. Adjuvants, broadly defined, are substances which promote immune responses. Frequently, the adjuvant of choice is Freund's complete or incomplete adjuvant, or killed B. pertussis organisms, used e.g. in combination with alum precipitated antigen. A general discussion of adjuvants is provided in Goding, Monoclonal Antibodies: Principles & Practice (2nd edition, 1986) at pages 61-63. Goding notes, however, that when the antigen of interest is of low molecular weight, or is poorly immunogenic, coupling to an immunogenic carrier is recommended. Examples of such carrier molecules include keyhole limpet haemocyanin, bovine serum albumin, ovalbumin and fowl immunoglobulin. Various saponin extracts have also been suggested to be useful as adjuvants in immunogenic compositions. Recently, it has been proposed to use granulocyte-macrophage colony stimulating factor (GM-CSF), a well known cytokine, as an adjuvant (WO 97/28816).

Accordingly, the invention encompasses a therapeutic composition further comprising any adjuvant substance including any of the above or combinations thereof. It is also contem- plated that the antigen, i.e. the peptide of the invention and the adjuvant can be administered separately in any appropriate sequence.

The choice of antigen in the pharmaceutical composition of the invention will depend on parameters determinable by the person of skill in the art. As it has been mentioned, each of the different peptides of the invention is presented on the cell surfaces by a particular HLA molecule. As such, if a subject to be treated is typed with respect to HLA phenotype, a peptide/peptides are selected that is/are known to bind to that particular HLA molecule. Alternatively, the antigen of interest is selected based on the prevalence of the various HLA phenotypes in a given population. As an example, HLA-A2 is the most prevalent phe- notype in the Caucasian population, and therefore, a composition containing a survivin-derived peptide binding to HLA-A2 will be active in a large proportion of that population. However, the composition of the invention may also contain a combination of two or more survivin-derived peptides, each interacting specifically with a different HLA molecule so as to cover a larger proportion of the target population. Thus, as examples, the pharmaceutical composition may contain a combination of a peptide restricted to a HLA-A molecule and a peptide restricted to a HLA-B molecule, e.g. including those HLA-A and HLA-B molecules that correspond to the prevalence of HLA phenotypes in the target population, such as e.g. HLA-A2 and HLA-B35. Additionally, the composition may comprise a peptide restricted to an HLA-C molecule.

It is comtemplated that useful immunogenic compositions of the inventions in addition to a survivin-derived peptide as defined herein may comprise an immunologically effective amount of the survivin protein as such as it is defined herein or an immunogenic fragment hereof.

The amount of the immunogenic peptide of he invention in the pharmaceutical composi- tion may vary, depending on the particular application. However, a single dose of the im- munogen is preferably anywhere from about 10 μg to about 5000 μg, more preferably from about 50 μg to about 2500 μg such as about 100 μg to about 1000 μg. Modes of administration include intradermal, subcutaneous and intravenous administration, implantation in the form of a time release formulation, etc. Any and all forms of administration known to the art are encompassed herein. Also any and all conventional dosage forms that are known in the art to be appropriate for formulating injectable immunogenic peptide composition are encompassed, such as lyophilised forms and solutions, suspensions or emulsion forms containing, if required, conventional pharmaceutically acceptable carriers, diluents, preservatives, adjuvants, buffer components, etc.

The immunoprotective effect of the composition of the invention can be determined using several approaches. Examples hereof are provided in the following examples. A further example on how to determine a CTL response provoked by the immunogenic composition is provided in WO 97/28816, supra. A successful immune response may also be determined by the occurrence of DTH reactions after immunisation and/or the detection of antibodies specifically recognising the peptide(s) of the vaccine composition.

In preferred embodiments, the pharmaceutical composition of the invention is an immunogenic composition or vaccine capable of eliciting an immune response to a cancer disease. As used herein, the expression " immunogenic composition or vaccine" refers to a composition eliciting at least one type of immune response directed against cancer cells. Thus, such an immune response may be any of the types mentioned above: A CTL response where CTLs are generated that are capable of recognising the HLA/peptide complex presented on cell surfaces resulting in cell lysis, i.e. the vaccine elicits the production in the vaccinated subject of effector T-cells having a cytotoxic effect against the cancer cells; a B-cell response giving rise to the production of anti-cancer antibodies; and/or a DTH type of immune response.

In useful embodiments an immunogenic response directed against a cancer disease is elicited by administering the peptide of the invention either by loading MHC class I molecules on antigen presenting cells (APCs) from the patient, by isolating PBLs from the patient and incubating the cells with the peptide prior to injecting the cells back into the patient or by isolating precursor APCs from the patient and differentiating the cells into professional APCs using cytokines and antigen before injecting the cells back into the patient. Thus, in one embodiment of the present invention, a method for treating cancer patients is one wherein the peptide is administered by presenting the peptide to the patient's antigen presenting cells (APCs) ex vivo followed by injecting the thus treated APCs back into the patient. There are at least two alternative ways of performing this. One alternative is to iso- late APCs from the cancer patient and incubate (load) the MHC class I molecules with the peptide. Loading the MHC class I molecules means incubating the APCs with the peptide so that the APCs with MHC class I molecules specific for the peptide will bind the peptide and therfore be able to present it to T cells. Subsequently, the APCs are re-injected into the patient. Another alternative way relies on the recent discoveries made in the field of den- dritic cell biology. In this case, monocytes (being dendritic cell precursors) are isolated from the patient and differentiated in vitro into professional APC (or dendritic cells) by use of cytokines and antigen. This is described in Examples 3 and 5, where adherent PBLs (being mainly monocytes) are cultured in vitro together with GM-CSF, IL-4 and TNF-α. Subsequently, the in vitro generated DCs are pulsed with the peptide and injected into the patient.

Due to the fact that survivin appears to be expressed in most cancer forms, it is very likely that vaccines of the invention can be provided to control any type of cancer disease where survivin is expressed. Thus, as examples, the vaccine composition of the invention is im- munologically active against a haematopoietic malignancy including chronic lymphatic leukemia and chronic myeloid leukemia, melanoma, breast cancer, cervix cancer, ovary cancer, lung cancer, colon cancer, pancreas cancer and prostate cancer.

From the above description, the skilled person will readily realise that the peptides of the invention are useful as cancer diagnostic tools, particularly so, as the peptides are derived from survivin expressed in all cancer types. Therefore, the peptides of the invention provide the basis for developing universally applicable diagnostic and prognostic procedures in respect of cancer diseases. Thus, in other useful embodiments the composition of the invention is a composition for ex vivo or in situ diagnosis of the presence in a cancer patient, e.g. based on the detection of survivin reactive T-cells among PBLs or in tumour tissue.

Accordingly, there is, in still further aspects, provided a diagnostic kit for ex vivo or in situ diagnosis of the presence of survivin reactive T-cells among PBLs or in tumor tissue comprising one or more peptides of the invention, and a method of detecting in a cancer patient the presence of survivin reactive T-cells, the method comprising contacting a tumor tissue or a blood sample with a complex of a peptide of the invention and a Class I HLA molecule or a fragment of such molecule and detecting binding of the complex to the tissue or the blood cells.

Another useful diagnostic or prognostic approach is based on generating antibodies in a heterologous animal species, e.g. murine antibodies directed against a human survivin-derived peptide of the invention, which can then be used, e.g. to diagnose for the presence of cancer cells presenting the peptide. For such immunisation purposes, the amount of peptide may be less than that used in the course of in vivo therapy, such as that mentioned above. In general, a preferred dose can range from about 1 μg to about 750 μg of peptide. It is also possible to produce monoclonal antibodies based on immunisation with a peptide of the invention. Accordingly, the present invention also relates to a molecule, in particular a monoclonal or polyclonal antibody including a fragment hereof, that is capable of binding specifically to a peptide of the invention and to a molecule that is capable of blocking such a binding, e.g. an antibody raised against the monoclonal or polyclonal antibody directed against a peptide of the invention.

In one aspect, the invention provides a complex of a peptide of the invention and a Class I HLA molecule or a fragment of such molecule, which is useful as a diagnostic reagent such as it is described supra. The complex is made by any conventional means including those described in the following examples. Such a complex may be monomeric or multimeric.

The present invention provides the means for alleviating or curing a cancer disease. Ac- cordingly, it is a further aspect of the invention to use the peptides as defined hereinbefore for the preparation of a medicament for the treatment of cancer. A still further aspect of the present invention relates to the use of a molecule or a composition as defined hereinbefore for the preparation of a medicament for the treatment of cancer. Preferably, a cancer disease associated with the expression of survivin, including as examples: a haematopoietic malignancy including chronic lymphatic leukemia and chronic myeloid leukemia, melanoma, breast cancer, cervix cancer, ovary cancer, lung cancer, colon cancer, pancreas cancer and prostate cancer. The use comprises administering to a patient suffering from the disease an effective amount of the pharmaceutical composition according to the invention, a molecule that is capable of binding specifically to a peptide of the invention and/or a molecule that is capable of blocking the binding of such a molecule.

In some cases it will be appropriate to combine the use of the invention with a conventional cancer treatment such as radiotherapy or chemotherapy.

The invention will now be described in further details in the below, non-limiting examples and the figures, wherein Fig. 1 illustrates T-cell response as measured in an ELISPOT in patient CLL1 to no peptide, Surl (LTLGEFLKL, SEQ ID NO: 10) peptide and Sur9 (ELTLGEFLKL, SEQ ID NO:3) peptide. PBLs were stimulated once with peptide before plated at 6χl0⁵ cells per well in duplicate. The average number of spots per peptide was calculated using a CCD scanning device and a computer system,

Fig. 2 illustrates T-cell response as measured in an ELISPOT in patient CLL1 to no peptide, the peptide analogue SurlL2 (LLLGEFLKL, SEQ ID NO:4), and the peptide analogue SurlM2 (LMLGEFLKL, SEQ ID NO:5). PBLs were stimulated once with peptide before plated at 10⁴ cells per well in duplicate. The average number of spots per peptide was calculated using a CCD scanning device and a computer system,

Fig. 3 shows responses as measured in an ELISPOT in patient CLL2 and CLL3 to no peptide (black bar), the Surl (LTLGEFLKL, SEQ ID NO: 10) peptide (grey bar), the Sur9 (ELTLGEFLKL, SEQ ID NO:3) peptide (white bar), the analogue peptide SurlL2 (LLLGEFLKL, SEQ ID NO:4) (light grey bar), and the analogue peptide SurlM2 (LMLGEFLKL, SEQ ID NO:5) (dark grey bar). Each experiment was performed with 10⁵ cells per well in duplicate, and the average number of spots was calculated,

Fig. 4 represents T cells that were isolated from tumour infiltrated lymph nodes from patient Mell, Mel2, and Mel3, stimulated once in vitro and analyzed in an ELISPOT assay for response to no peptide (black bar) the peptides Surl (LTLGEFLKL, SEQ ID NO: 10) (grey bar) and Sur9 (ELTLGEFLKL, SEQ ID NO:3) (white bar). Each experiment was performed in duplicate with 10⁵ cells per well. In each experiment two wells without addition of peptide was also included. The average number of spots per peptide was calculated for each patient,

Fig. 5 shows functional activity of survivin specific CTLs. CTLs were isolated from a melanoma infiltrated lymph node using survivin coated magnetic beads. (A) Specific lysis of melanoma cell lines; the HLA-A2 positive FM3 (triangle) and the HLA-A2 negative FM45 (square). (B) Specific lysis of breast cancer cell lines; the HLA-A2 positive MCF-7 (triangle) and the HLA-A2 negative BT-20 (square),

Fig. 6 shows frequency of survivin reactive CTLs in PBL from breast cancer patients. Reac- tivity was examined in three breast cancer patients (top, middle, and bottom, respectively) by the ELISPOT. For each patient the assays were performed in the absence of peptide, in the presence of surl peptide, in the presence of sur9, and in the presence of the modified surlM2 peptide. 1 x 10⁴ effector cells per well were used. The graph depicts the quantification of reactive cells; grey columns represent the average number of IFN-γ producing cells,

Fig. 7 illustrates HLA-35 binding of survivin-derived peptides and analysis of the peptide- mediated recovery of HLA-B35 molecules by survivin-derived peptides. Lysates of meta- bolically labeled T2-B35 cells were incubated at 4°C in the presence of 50, 5, 0.5, 0.05 and 0.005 mM of peptide. The recovery of HLA-B35 was analyzed in an assembly assay and quantified subsequent to IEF-gel electrophoresis, using ImageGauge phosphorimager software (FUJI photo film Co., LTD., Japan). The C₅₀ value is the concentration of the peptide required for half-maximal binding to HLA-B35,

Fig. 8 shows spontaneous T-cell responses observed in PBLs from cancer patients. A) The number of IFNγ spot forming cells measured in ELISPOT assay without peptide (white bars), with sur51-59 (black bars) or sur46-54 (gray bars), among in vitro stimulated PBLs from patient CLL5 (10⁵ cells/well), HEM12 (10⁵ cells/well), and HEM8 (5xl0⁴ cells/well). B) The number of spot forming cells among 1.7xl0⁵ PBLs from HEM 12, cultured for 10 days with peptide-pulsed matured autologous dendritic cells. The columns represent the average of two measurements,

Fig. 9 demonstrates spontaneous T-cell responses against native and modified survivin peptides in melanoma patients. A) The number of spot forming cells measured in ELISPOT assay against sur51-59 and sur51Y9 from patient FM25 in PBLs (4xl0³cells/well) and TILs (7xl0⁴ cells/well) as well as TILs from FM45 (10⁴ cells/well). B) The number of spot forming cells measured in ELISPOT assay against sur46 and sur46Y9 measured in TILs from FM74 (5xl0³ cells/well). The columns represent the average of two measurements with the non-specific IFNγ release subtracted,

Fig. 10 illustrates binding affinity of survivin-derived peptides to HLA-Al. Class I MHC heavy chain bands were quantified on a Phosphorimager. The mount of stabilized HLA-Al heavy chain is directly related to the binding affinity of the added peptide. The peptide- mediated recovery of HLA-Al (arbitrary units) induced by 40, 4, 0.4, 0.04 μM of Sur93- 101 (line), Sur93T2 (square), Sur49-58 (circle) or Influenza A, PB1 591-599 (triangle),

Fig. 11 shows spontaneous responses against HLA-Al restricted peptides. Spontaneous T- cell responses against survivin-derived peptides as measured by ELISPOT assay. The average number of peptide specific IFNγ spots formed in response to Sur92-101, Sur38Y9, Sur47Y10, and Sur93T2 among 5xl0⁴ in vitro stimulated PBL or TIL from melanoma patients. The peptide specific responses showed were observed among analyses of 6 PBL samples and 3 TIL samples from melanoma (Mel) patients. Non-specific IFNy spots are subtracted. Bars: range of duplicates,

Fig. 12 shows spontaneous responses against HLA-Al 1 restricted peptides. Spontaneous T- cell responses against survivin-derived peptides as measured by the ELISPOT assay. The average number of peptide specific IFNγ spots formed in response to Sur53-62 among 5xl0⁴ in vitro stimulated PBL or TIL from cancer patients. The peptide specific responses showed were observed among analyses of 5 melanoma (Mel) patients (5 PBL, 1 TIL) and 2 CLL (CLL) patients (PBL). Non-specific IFNy spots are subtracted. Bars: range of duplicates,

Fig. 13 illustrates spontaneous responses against HLA-A3 restricted peptides. Spontaneous T-cell responses against survivin-derived peptides as measured by the ELISPOT assay. The average number of peptide specific IFNγ spots formed in response to SurlδKlO among 5xl0⁴ in vitro stimulated PBL or TIL from melanoma patients. The peptide specific responses showed were observed among analyses of 23 PBL samples and 4 TIL samples from melanoma (Mel) patients. Non-specific IFNy spots are subtracted. Bars: range of duplicates,

Fig. 14 illustrates spontaneous responses against HLA-A2 restricted peptides. Spontaneous T cell responses against survivin-derived peptides as measured by the ELISPOT assay. The average number of peptide specific IFNγ spots formed in response to the llmer peptide, Surl8-28 among 5xl0⁴ in vitro stimulated PBL from cancer patients. The peptide specific responses showed were observed among analyses of 10 PBL samples from 2 melanoma (Mel), 6 CLL (CLL), and 2 mamma carcinoma (MC) patients. Non-specific IFNy spots are subtracted. Bars: range of duplicates,

Fig. 15 illustrates spontaneous T cell responses against survivin-derived peptides as measured by the ELISPOT assay. The average number of peptide specific IFNγ spots formed in response to sur6-14 (LPPAWQPFL) among 10⁵ in vitro stimulated PBL from five melanoma patients (mel25, mel26, mel3, me!6, mel39), two CLL patients (CLL1, CLL54) and 2 breast cancer patients (breastll, breast 15). Non-specific IFNy spots are subtracted,

Fig. 16 illustrates the laboratory values of stable detection of LDH, cholinesterase, creatinine, hemoglobin, leucocytes and thrombocytes following vaccination therapy of four patients (ARW, • KN, - WWE, ■ GB), and

Fig. 17 demonstrates kinetic analysis of immunity to survivin peptides assessed by IFNγ ELISPOT. PBMCs were obtained before the first DC vaccination and three months thereafter. The numbers of IFNγ spot-forming cells above background are depicted.

In the following table, amino acid sequences for peptides used herein and their respective SEQ ID NOs are listed:

EXAMPLE 1

Identification of a cytotoxic T-lymphocyte response to the apoptosis inhibitor protein survivin in cancer patients

Summary

Using CTL epitopes derived from survivin, specific T-cell reactivity against such antigens in peripheral blood from chronic lymphatic leukemia (CLL) patients and in tumor-infiltrated lymph nodes from melanoma patients by ELISPOT analysis have been studied. CTL re- sponses to survivin-derived peptide epitopes were detected in three out of six melanoma patients and in three out of four CLL patients. No T-cell reactivity was detected in PBL from six healthy controls. Thus, survivin-derived peptides may serve as important and widely applicable targets for anti-cancer immunotherapeutic strategies.

Introduction

The survivin protein was scanned for the presence of HLA-A*0201 (HLA-A2) binding peptide motifs and after successful identification, the peptides were used to test for specific T- cell reactivity in leukemia and melanoma patients by ELISPOT assay. In both patient co- horts CTL responses to two survivin-derived peptide epitopes were detected, whereas no T-cell reactivity could be detected in the healthy controls. These data suggest that survivin represent a widely expressed tumor antigen recognized by autologous T cells.

Materials and Methods

Patients and normal controls

Peripheral vein blood samples from 4 patients diagnosed with CLL (designated CLLl-4) and blood samples from 6 normal individuals were collected into heparinised tubes. PBLs were isolated using Lymphoprep separation and frozen in fetal calf serum (FCS) with 10% di- methylsulphoxide. Additionally, T lymphocytes from tumor-infiltrated lymph nodes were obtained from 6 melanoma patients (designated mell-6). Freshly resected lymph nodes were minced into small fragments, crushed to release cells into culture and cryopreserved. PBLs were available from 4 of the melanoma patients. All individuals included were HLA-A2 positive as determined by FACS analysis using the HLA-A2 specific antibody BB7.2. The antibody was purified from hybridoma supernatant. Patient samples were obtained from the State University Hospital, Herlev, Denmark. Informed consent was obtained from the patients prior to any of theses measures.

Survivin-derived peptides

All peptides were obtained from Research Genetics (Huntsville, AL, USA) and provided at >90% purity as verified by HPLC and MS analysis. The peptides used are listed in Table 1.

Table 1. Peptides examined in this study and their binding affinity to HLA-A2

Name Protein⁹ Sequence SEQ ID NO: C₅o (μM)⁶

CI HIV-1 pol₄₇₆.₄₈₄ ILKEPVHGV 11 0.7

Surl Survivin_96-ιo4 LTLGEFLKL 10 >100

Sur2 Survivin_133-14ι RAIEQLAAM 12 Not binding

Sur3 Survivinι₃₀-ι₃₈ KVRRAIEQL 13 >100

Sur4 Survivin_20-28 STFKNWPFL 14 Not binding

Sur5 Survivin_88-96 SVKKQFEEL 15 Not binding

Sur6 Survivin₁₀ι-ιo₉ FLKLDRERA 1 30

Sur7 Survi ini27-i35 TAKKVRRAI 16 Not binding

Sur8 Survivin_5-14 TLPPAWQPFL 2 30

Sur9 Survivin₉₅.₁₀ ELTLGEFLKL 3 10

SurlO Survivin_126-135 ETAKKVRRAI 17 Not binding

SurlL2 LLLGEFLKL 4 1

SurlM2 LMLGEFLKL 5 1

^aThe value range listed in subscript indicates the position of the peptide in the survivin se- quence as disclosed in US 6.245.523 ^ft The C₅₀ value is the concentration of the peptide required for half maximal binding to HLA-A2 determined as described below

Assembly assay for peptide binding to class I MHC molecules

Assembly assays for binding of the synthetic peptides to class I MHC molecules metaboli- cally labeled with [35S]-methionine were carried out as described (12,13). The assembly assay is based on stabilization of the class I molecules after loading of peptide to the peptide transporter deficient cell line T2. Subsequently, correctly folded stable MHC heavy chains are immunoprecipitated using conformation-dependent antibodies. After IEF elec- trophoresis, gels were exposed to phosphorimager screens, and peptide binding was quantified using the Imagequant Phosphorimager program (Molecular Dynamics, Sunnyvale, CA).

Antigen stimulation of PBLs

To extend the sensitivity of the ELISPOT assay, PBLs were stimulated once in vitro prior to analysis (14,15). Fresh and previously frozen PBLs gave similar results in the ELISPOT assay. On day 0, PBLs or crushed lymph node were thawed and plated in 2 ml/well at a con- centration of 2x106 cells in 24-well plates (Nunc, Denmark) in AIM V medium (Life Technologies, Roskilde, Denmark), 5% heat-inactivated human serum and 2 mM of L-glutamine in the presence of 10 μM of peptide. In each experiment a well without peptide was included. Two days later 300 IU/ml recombinant interleukin-2 (IL-2) (Chiron, Ratingen, Germany) was added to the cultures. The cultured cells were tested for reactivity in the ELISPOT assay on day 12.

ELISPOT assay

The ELISPOT assay used to quantify peptide epitope-specific interferon-γ-releasing effector cells was performed as in (16). Briefly, nitrocellulose bottomed 96-well plates (Multiscreen MAIP N45, Millipore, Hedehusene, Denmark) were coated with anti-IFN-γ antibody (1-D1K, Mabtech, Nacka, Sweden). The wells were washed, blocked by AIM V medium, and cells were added in duplicates at different cell concentrations. Peptides were then added to each well and the plates were incubated overnight. On the following day, medium was discarded and the wells were washed prior to addition of biotinylated secondary antibody (7-B6-1- Biotin, Mabtech). The plates were incubated for 2 hours, washed and Avidin-enzyme conjugate (AP-Avidin, Calbiochem, Life Technologies) was added to each well. Plates were incubated at RT 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 min. The reaction was terminated by washing with tap water upon the emergence of dark purple spots. The spots were counted using the Alphalmager System (Alpha Innotech, San Leandro, CA. USA) and the peptide specific CTL frequency could be calculated from the numbers of spot-forming cells. The assays were all performed in duplicate for each peptide antigen. Results

Binding of survivin derived peptides to HLA-A2

The amino acid sequence of the survivin protein was screened for the most probable HLA- A2 nona- and decamer peptide epitopes, using the main HLA-A2 specific anchor residues (17). Ten survivin-derived peptides were synthesized and examined for binding to HLA-A2. An epitope from HIV-1 pol476-484 (ILKEPVHGV, SEQ ID NO: ll) (Table 1) was used as a positive control. The peptide concentration required for half maximal recovering of class I MHC (C₅₀ value) was 0.7 μM for the positive control. In comparison, the peptide designated Sur9 (ELTLGEFLKL, SEQ ID NO:3) bound at an affinity of C₅₀ = 10 μM. The peptides designated Sur6 (FLKLDRERA, SEQ ID NO: l) and Surδ (TLPPAWQPFL, SEQ ID NO:2), respectively bound to HLA-A2 at C₅₀ = 30 μM, whereas Surl (LTLGEFLKL, SEQ ID NO: 10) and Sur3 (KVRRAIEQL, SEQ ID NO: 13) bound weaker (C₅₀ >100 μM). Five of the peptides examined (Sur2, Sur4, Sur5, Sur7, and SurlO) did not bind to HLA-A2.

Since Surl is a weak HLA-A2 binder, two analogue peptides designated SurlL2 and SurlM2, respectively in which a better anchor residue (leucine or methionine) replaced the native threonine at position 2 were synthesized. Both of these peptides bind with almost similar high affinity to HLA-A2 as the positive control (C₅₀ = 1 μM).

CTL response to survivin in CLL patients

PBLs from four HLA-A2 positive CLL patients were stimulated once in vitro before exami- nation in the ELISPOT assay. This procedure was chosen to extend the sensitivity of the ELISPOT. All of the above 10 survivin-derived peptides were included in the first line of experiments. Responses were detected to Surl and Sur9 and only data for these peptides are given in the figures. Fig. 1 shows CTL reactivity to Surl and Sur9 as determined in patient CLL1. Each spot represents a peptide reactive, INF-γ-producing cell. The average number of spots per peptide was calculated using a CCD scanning device and a computer system. Fifty-two Sur9 peptide specific spots (after subtraction of spots without added peptide) per 6χl0⁵ were detected in the CLL1 patient (Fig. 1). No response was detected to the weak HLA-A2 binding peptide Surl, however the patient responded strongly to the strong HLA-A2 binding peptide analogue SurlM2 (35 peptide specific spots per 10⁴ cells) (Fig. 2). No response was detected to the other strong HLA-A2 binding peptide analogue SurlL2 in this patient (Fig. 2). Patient CLL2 responded strongly to Sur9 (128 peptide specific spots per 10⁵ cells) and weakly to Surl (22 peptide specific spots per 10⁵ cells) (Fig. 3). The response to the SurlL2 analogue was only slightly increased relative to the natural epitope, whereas the patient responded similarly strongly to the SurlM2 peptide as to the decamer peptide Sur9. In patient CLL3 a weak response to Sur9 was observed (Fig. 3). No response to Surl or the modified Surl peptides were observed in the patient. No survivin responses were detected in the last patient CLL4 (data not shown). PBLs from 6 healthy HLA-A2 positive controls were analyzed to investigate whether a response to survivin could be detected in healthy individuals. No response was observed in any of the controls to any of the survivin-derived peptides.

CTL response to survivin in melanoma patients

T lymphocytes isolated from tumour infiltrated lymph nodes from HLA-A2 positive melanoma patients were examined. The freshly resected lymph node was minced into small fragments and crushed to release cells into culture. Cells were stimulated once with peptide in vitro before examination in the ELISPOT assay. Survivin specific T cells were de- tected in three of the six patients analyzed. A strong Sur9 response was detected in patient Mel2 and Mel3. A weaker response to the Surl peptide was also detected in these patients (Fig. 4). In Mell the response to the weakly binding peptide Surl was stronger than the response to the stronger HLA-A2 binder Sur9 (Fig. 4). No response was detected in the tumor-infiltrated lymph nodes from the last three melanoma patients (Mel4-6). PBLs from two of the survivin reacting patients, Mell and Mel2, and from two of the non-reacting patients, Mel4 and Mel5, were examined. No response could be detected to either Sur9 or Surl in PBLs from any of these patients (data not shown).

EXAMPLE 2

Spontaneous cytotoxic T-cell responses to survivin-derived MHC class I- restricted T-cell epitopes in situ and ex vivo in cancer patients

Summary

Spontaneous cytotoxic T-cell responses to survivin-derived MHC class I restricted T-cell epitopes were demonstrated in situ as well as ex vivo in breast cancer, leukemia, and melanoma patients. Moreover, survivin reactive T cells isolated by magnetic beads coated with MHC/peptide complexes were cytotoxic to HLA-matched tumours of different tissue types. Being a universal tumor antigen, survivin may serve as a widely applicable target for anti-cancer immunotherapy.

Materials and Methods

Construction of HLA-peptide complexes for T-cell staining and T-cell sorting

A recognition site for enzymatic biotinylation using biotin protein ligase (BirA) in fusion with the 5'-end of the extracellular domains of HLA A*0201 (residues 1-275) was ex- pressed in E. coli BL21 (DE3). The recombinant protein was purified by size- (Sephadex G25, Pharmacia) and ion exchange (mono-Q, Pharmacia) chromatography from inclusion bodies solubilised in δ M urea. The HLA A*0201 was folded in vitro by dilution in the presence of the modified survivin peptide SurlM2 (LMLGEFLKL, SEQ ID NO:5) or the MAA peptide gpl00154-163, and subsequently biotinylated as described previously (35, 36). After gel filtration on a Pharmacia Sephadex G25 column to remove unbound biotin, the protein was multimerised with streptavidin-FITC conjugated dextran molecules (kindly provided by L. Winther, DAKO, Denmark) to generate multivalent HLA-dextran compounds for immunohistochemistry. The HLA A*0201 construct was a kind gift of Dr. Mark M. Davis (Dept. of Microbiology and Immunology, Stanford University, Palo Alto, CA). Cell separation was performed as previously described (37). Briefly, 5 x 10⁶ streptavid in-conjugated magnetic beads (Dynal, Oslo, Norway) were washed twice in 200 μl cold PBS, 0.5μg pep- tide/A*0201 monomers were added and the mixture incubated for 15 min. at room temperature. After two washes these beads were mixed with PBLs at a ratio of 1: 10 and sub- sequently incubated for 1 h followed by a precipitation of bead-bound cells in a magnetic field. The precipitation step was repeated once.

Immunohistochemistry stainings

For staining with FITC-conjugated multimeric peptide/MHC complexes, tissue sections were dried overnight and subsequently fixed in cold acetone for 5 min. All incubation steps were performed at room temperature and in the dark: (i) 45 min. of the primary antibody (1: 100 diluted), (ii) Cy 3-conjugated goat anti-mouse (1 : 500 diluted; code 115-165-100, Jackson ImmunoResearch, obtained from Dianova, Hamburg, Germany) for 45 min. and finally (iii) the multimers for 75 min. Between each step the slides were washed two times for 10 min. in PBS/BSA 0.1%. The slides were mounted in vectashield and kept in the refrigerator until observed under the confocal microscope.

Cytotoxicity assay

Conventional [51Cr]-release assays for CTL-mediated cytotoxicity were carried out as described in (13). Target cells were autologous EBV-transformed B-cell lines, the HLA-A2 positive breast cancer cell line MCF-7 (available at ATCC), the HLA-A2 positive melanoma cell line FM3 (38), the HLA-A2 negative breast cancer cell line BT-20 (available from ATCC) and the HLA-A2 negative melanoma cell line FM45 (38). All cancer cell lines expressed survivin as examined by RT-PCR (data not shown).

ELISPOT assay

The ELISPOT assay was used to quantify peptide epitope-specific IFN-γ releasing effector cells and has been described previously (39). Briefly, nitrocellulose bottomed 96-well plates (Multiscreen MAIP N45, Millipore) were coated with an anti-IFN-γ antibody (1-D1K, Mabtech, Sweden) and non-specific binding was blocked using AIM V (GibcoBRL, Life Technologies Inc., Gaithersburg, MD, USA). Lymphocytes were added at different cell con- centrations together with the specific peptides and T2 cells and incubated overnight at 37°C. Following two washes the biotinylated detection antibody (7-B6-l-Biotin, Mabtech) was added. Specific binding was visualised using alkaline phosphatase-avidin together with the respective substrate (GibcoBRL). The reaction was terminated upon the appearance of dark purple spots, which were quantitated using the Alphalmager System (Alpha Innotech, San Leandro, CA, USA). The peptides used for the ELISPOT were Surl, Sur9 and the Surl analogue peptide SurlM2 as described in Example 1.

Results

In situ staining of HLA-A2/survivin reactive T cells

In Example 1 two survivin-derived peptide epitopes recognized by T cells in leukemia and melanoma, i.e., Surl were identified. The weak binding affinity of Surl to HLA-A2 was im- proved substantially by replacing threonine at position 2 with a better anchor residue (methionine; SurlM2). This measure enabled the construction of stable HLA-A2/peptide complexes. These complexes were multimerised using dextran molecules, which were conjugated with streptavidin and FITC. Multimerised MHC-complexes were used to stain acetone-fixed, frozen material. Using a confocal laser microscope, SurlM2/HLA-A*0201 reactive CTLs could readily be detected in situ in the tumor microenvironment. We depicted such cells in the primary tumor and the sentinel lymph node of a stage III melanoma patient as well as in a primary breast cancer lesion. To ensure the specificity of the staining, a series of negative controls was carried out. Neither the use of peptide/HLA- dextran multimers with peptides derived from the melanoma differentiation antigen gplOO on the same tumour, nor SurlM2/HLA-dextran multimers in case of a tumour sample obtained from an HLA-A2 negative donor resulted in a positive staining.

Isolated survivin reactive CTLs lyse tumour cell lines of different origin

To characterise the functional capacity of survivin-reactive CTLs, these cells were isolated by means of magnetic beads coated with HLA-A2/SurlM2-complexes (36). A freshly resected melanoma infiltrated lymph node was minced into small fragments and crushed to release cells into culture. Cells were stimulated once with peptide in vitro prior to isolation. One day after isolation IL-2 was added, and on day 5 the capacity of these cells to kill tu- mour cells was tested either by ELISPOT or in standard 51Cr release assays. First, by means of ELISPOT analysis it was possible to establish that CTLs isolated using the modified SurlM2/HLA-A2-complex also responded to the native Surl peptide (data not shown). Second, the cytotoxicity of the survivin reactive CTLs against the HLA-A2 positive melanoma cell-line FM3 (Fig. 5 A) and the HLA-A2 positive breast-cancer cell line MCF-7 (Fig. 5 B) was tested. The isolated T cells effectively lysed both HLA-A*0201 cell lines. In contrast, no cytotoxicity was observed against the HLA-A2 negative melanoma cell line FM45 (Fig. 5 A) or the HLA-A2 negative breast cancer cell line BT-20 (Fig. 5 B).

Survivin reactivity measured in PBL by ELISPOT

The presence of survivin reactive T cells in PBLs from ten HLA-A2 positive breast cancer patients was examined by the ELISPOT. Before analysis, PBLs were stimulated once in vitro to extend the sensitivity of the assay. Reactivity to the following survivin peptides was examined: Surl, Sur9 and SurlM2. Survivin specific T cells were detected in six out of the ten HLA-A2 positive breast cancer patients. Representative examples are given in Fig. 6. In PBLs from two patients a response against Surl and the modified analogue SurlM2, but not against Sur9 (Fig. 6, top, middle) was detected, in three patients a response to Sur9 was detected, but not to Surl or SurlM2 (Fig. 6 bottom), and one patient responded only to SurlM2. In contrast, no survivin responses were detected in PBLs from 20 healthy HLA- A2 positive donors. Similarly, PBLs from fourteen HLA-A2 positive melanoma patients were examined. Survivin responses were present in seven of these patients (Table 2). Two patients responded to the Sur9 peptide, three to the SurlM2 peptide, one to both Surl and SurM2, and one to all three peptides. In Example 1, T-cell response to survivin in 3 chronic lymphatic leukemia (CLL) patients was tested (Table 2; CLLl, CLL2, CLL3). These studies were extended using PBLs from three additional CLL patients. Notably, all patients produced a T-cell response to at least one survivin epitope (Table 2; CLL5, CLL6, CLL7). In addition, PBLs from one patient suffering from chronic myeloid leukemia (CML) was examined. In this patient, a response to all three peptides was identified (data not shown). The data are summarized in Table 2.

Table 2. Patients with survivin peptide-specific T lymphocytes in PBLs as measured by ELISPOT

Melanoma a)

Patient Surl Sur9 SurlM2

P4 - - 97

Pll - - 112

P13 - - 71

P15 61 - 101

P17 - 172 -

P39 - 127 -

P64 112 70 128

Breast cancer b)

Patient Surl Sur9 SurlM2

Bl 122 - 208

B2 67 - 72

B3 - 54 -

B4 - 45 -

B5 - 19 -

B6 - - 24

CLL c)

Patient Surl Sur9 SurlM2

CLLl - 27 320

CLL2 - 39 -

CLL3 23 127 122

CLL5 - 100 124

CLL6 - 121 360

CLL7 68 132 174 a) Frequency of reactive cells per 10⁴; 14 patients examined. b) Frequency of reactive cells per 10⁴; 10 patients examined. c) Frequency of reactive cells per 10⁵; 7 patients examined.

EXAMPLE 3

HLA-B35-restricted immune responses to survivin-derived peptides in cancer pa- tients

Summary

In this study, two survivin-derived epitopes, which are restricted to HLA-B35 were identi- fied and characterized. Specific T-cell reactivity against both of these epitopes was present in the peripheral blood from patients with different haematopoietic malignancies and melanoma. Substitutions of the C-terminal anchor residue improved the recognition by tumor infiltrating lymphocytes from melanoma patients. Furthermore, spontaneous cytotoxic T- cell responses to survivin in situ in a primary melanoma lesion was demonstrated. These epitopes extends the applicability of future vaccine strategies based on survivin peptides in relation to malignancies as well as the HLA profile of the patients involved.

In Examples 1 and 2, HLA-A2 restricted survivin-derived T-cell epitopes were studied. Since HLA-A2 is only expressed in about 30% of the Caucasian population (63), peptide epitopes restricted to other HLA class I molecules need to be identified to extend the fraction of patients that could be treated. In this study, two novel T-cell epitopes from survivin restricted to HLA-B35, which is expressed in 9% of the Caucasian population (63), were identified, and spontaneous immune responses to these survivin peptides were detected in patients with different haematopoietic malignancies and melanoma.

Materials and Methods

Patients

Peripheral vein blood samples from cancer patients were collected, PBLs were isolated using Lymphoprep separation, HLA-typed (Department of Clinical Immunology, University Hospital, Copenhagen) and frozen in FCS with 10% DMSO. Ten HLA-B35 positive patients were selected for further analysis. These patients suffered from melanoma, CLL, follicular lymphoma (FL), diffuse large B-cell lymphomas (DLBCL) and Multiple Myeloma (MM), re- spectively. At the time blood samples were collected patients had not been medically treated within the previous four months. Additionally, tumor-infiltrating lymphocytes (TIL) isolated from lymph nodes were collected from three of the melanoma patients and frozen in FCS with 10% DMSO. Peptides

Seven synthetic survivin-derived peptides were used in this study: Sur6-14, Surll-19, Sur34-43, Sur46-54, Sur51-59, Sur46Y9, Sur51Y9, and one EBV-derived peptide, EBNA3A 457-466 (63). All peptides were obtained from Research Genetics (Huntsville, AL) and provided at >90% purity, as verified by HPLC and MC analyses. The peptides are listed in Table 3 below.

Table 3. HLA-B35 binding of survivin-derived peptides

Name Protein and position Sequence SEQ ID NO: C₅₀ (μM)

Sur6-14 Survivin_6-14 LPPAWQPFL 18 >100

Surll-19 Survivinn_-19 QPFLKDHRI 19 Not binding

Sur34-43 Survivin_{34- 3} TPERMAEAGF 20 >100

Sur46-54 Survivin_46-54 CPTENEPDL 6 20

Sur51-59 Survivin₅₁.₅₉ EPDLAQCFF 7 13

Sur46Y9 Modified peptide CPTENEPDY 8 4

Sur51Y9 Modified peptide EPDLAQCFY 9 1.5

C24 EBNA3A_458-466 YPLHEQHQM 21 0.8

Assembly assay for peptide binding to MHC class I molecules

The assembly assay described in Examples 1 and 2 was used to measure binding affinity of the synthetic peptides to HLA-B35 molecules metabolically labeled with [S35]methionine. Briefly, the assay is based on peptide-mediated stabilization of empty HLA molecules released, upon cell lysis, from the TAP deficient cell line T2, stably transfected with HLA-B35 (kindly provided by Dr J. Haurum, Symphogen ApS, Lyngby, Denmark). Stably folded HLA- molecules were immunoprecipitated using the conformation-dependent mAb W6/32. The HLA molecules were separated by IEF electrophoresis, gels were exposed to phosphorimager screens (Imaging plate, FUJI photo film Co., LTD., Japan), analyzed and the amount of correctly folded HLA molecules were quantified using ImageGauge phosphorimager software (FUJI photo film Co., LTD., Japan).

Antigen stimulation of PBLs

To extend the sensitivity of the ELISPOT assay, lymphocytes were stimulated once in vitro with peptide prior to analysis (14, 15). PBLs or TILs were thawed and stimulated with 50μM of the individual peptide epitopes in 96-well plates for 2 h at 26°C (5xl0⁵-10⁶ cells per peptide), and pooled for further 10 days of culture at 37°C in x-vivo with 5% human serum (HS), in 24 well plates (Nunc, Roskilde, Denmark), with 2xl0⁶ cells per well. At the second day of incubation 40 μg/ml IL-2 (Apodan A/S, Denmark) were added. At day 10, the cultured cells were tested for reactivity in the ELISPOT assay.

The ELISPOT assay The ELISPOT assay used to quantify peptide specific, IFN-γ releasing effector cells in PBLs or TILs collected from cancer patients was performed as described in Example 1. Briefly, nitrocellulose-bottomed 96-well plates (Multiscreen MAIP N45; Millipore, Hedehusene, Denmark) were coated with mAb against human IFN-γ, 7.5 μg/ml (1-D1K; Mabtech, Nacka, Sweden). Wells were washed and blocked in x-vivo (x-vivo 15TM BioWhittacker, Molecular Applications Aps, Denmark) and cells were added in duplicates at different concentrations. For antigen presentation, 10⁴ T2-B35 cells, with and without 10 μM peptide, were added per well. Plates were incubated overnight, the cells discarded, and wells washed prior to addition of biotinylated secondary antibody (7-B6-l-Biotin; Mabtech). Plates were incubated 2 h at room temperature, washed and avidin-alkaline phoεphatase conjugate was added (AP-Avidin; Calbiochem, Life Technologies, Inc.). After 1 h of incubation at room temperature, the enzyme substrate nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (Code No.K0598, DakoCytomation Norden A/S) was added, and dark purple spots emerged in 3-7 min. The reaction was terminated by washing with tap water. Spots were counted using the Alpha Imager System (Alpha Innotech, San Leandro, CA), and the frequency of peptide specific T cells were calculated from the number of spot forming cells.

All assays were performed in duplicates for each peptide antigen, and lymphocytes cul- tured in the same well, were tested in equal cell numbers with and without peptide, to measure the number of peptide specific cells in the culture.

Maturation of dendritic cells (DCs)

Adherent cells were isolated from PBLs after 2 h of culture. These were cultured for 10 additional days in RPMI 1640 (GibcoTM Invitrogen corporation, UK) with 10% FCS. 800 ng/ml GM-CSF (PreproTech, London, UK) and 40 ng/ml IL-4 (PreproTech) were added every third day. At day 10, DCs were matured for 24 h by adding 50 ng/ml TNF-α (PreproTech). After maturation, DCs were released and pulsed with 20μM peptide in the presence of 3μg/ml β2-microglobulin for 2 h at 26°C.

Isolation of peptide specific T cells

Antigen specific cells were isolated using sur51Y9/HLA-B35-coated magnetic beads as de- scribed in Example 2. Biotinylated monomers of HLA-B35 with sur51Y9 (obtained from Prolmmune, Oxford, UK) were coupled to streptavidin coated magnetic beads (Dynabeads M-280, Dynal A/S, Oslo, Norway) by incubating 2.5 μg monomers with 5xl0⁶ beads in 40 μl PBS for 20 min. at room temperature. The magnetic complexes were washed three times in PBS, using a magnetic device (Dynal A/S, Oslo, Norway) and subsequently mixed with PBLs at a ratio of 1: 10 in PBS with 5% BSA, and rotated very gently for 1 h. Antigen specific CD8^÷ T cells associating with the magnetic complexes were gently washed two or three times. Isolated cells were resuspended several times in x-vivo supplemented with 5% human serum and incubated for 2 h before the magnetic beads were released and re- moved from the cell suspension. The isolated antigen specific CDδ⁺ T cells were used in ELISPOT assay to analyze the cross-reactivity between the native and modified peptide.

TCR clonotype mapping by denaturing gradient gel electrophoresis (DGGE)

DGGE clonotype mapping of he human TCR BV regions 1-24 has been described in details (66). Briefly, RNA was isolated using the Purescript Isolation Kit (Gentra Systems Inc. MN) and transcribed cDNA was amplified by PCR using primers for the variable regions of the TCR beta chains in conjunction with a common constant region primer. The computer pro- gram MELTδ7 was used to ensure that the amplified DNA molecules were suited for DGGE analysis provided a 50 bp GC-rich sequence (GC-clamp) was attached to the 5'-end of the constant region primer. DGGE analysis was done in 6% polyacrylamide gels containing a gradient of urea and formamide from 20% to δ0%. Electrophoresis was performed at 160 V for 4.5 hours in lx TAE buffer at a constant temperature of 54°C.

Immunohistochemistry stainings

Multimerised peptide/HLA complexes were used to identify antigen specific T cells in situ in tumor lesions of cancer patients using the procedure described in Example 2. Biotinylated sur51Y9/HLA-B35 monomer was supplied by Proimmune limited, Oxford, UK. The biotinylated monomers of sur51Y9/HLA-B35 were multimerised with streptavidin-FITC-con- jugated dextran molecules (kindly provided by L. Winther, DAKO, Glostrup, Denmark) to generate multivalent HLA-dextran compounds for immunohistochemistry. Tissue sections were dried overnight and subsequently fixed in cold acetone for 5 min. All the incubation steps were performed in the dark at room temperature: (a) 45 min of the primary antibody (1: 100 diluted) (b) Cy 3-conjugated goat-anti-mouse antibody (1 :500 diluted; code 115-165-100; Jackson ImmunoResearch, obtained from Dianova, Hamburg, Germany) for 45 min; and finally (c) the multimers for 75 min. Between each step, the slides were washed two times for 10 min in PBS/BSA 0.1%. The slides were mounted in vectashield and kept in the refrigerator until observed under the confocal microscope (Leica).

Results

Identification of HLA-B35 binding survivin-derived peptides

The amino acid sequence of survivin was screened for nonameric and decameric peptides with anchor residues, according to the peptide-binding motif of HLA-B35 (67). Five peptides were selected containing proline as the N-terminal anchor in position 2 and phenyl- alanine, leucine, isoleucine or tyrosine as C-terminal anchor residues (Table 3). Assembly assay revealed two peptides, sur51-59 (EPDLAQCFF, SEQ ID NO:7) and sur46-54

(CPTENEPDL, SEQ ID NO:6) that were able to stabilise HLA-B35 efficiently. Additionally, two peptides, sur34-43 (TPERMAEAGF, SEQ ID NO:20) and sur6-14 (LPPAWQPFL, SEQ ID NO: lδ) showed a weak stabilization, whereas the remaining peptide did not stabilize HLA- B35 at all. The peptide concentration required for half maximal recovery of HLA-B35 (C50) was estimated at 13μM for sur51-59 and 20μM for sur46-54. In comparison, the positive control-epitope C24 from EBNA3A45δ-466 (YPLHEQHQM, SEQ ID NO:21) had an estimated C₅₀ value of 0,δμM.

To enhance the binding affinity of sur46-54 and sur51-59 the C-terminai amino acid was replaced with tyrosine, a better anchor residue (67). The recovery of HLA-B35 mediated by the modified peptides was analyzed in the assembly assay, and C₅₀ values were estimated at 1.5μM for sur51Y9 and 4μM for sur46Y9 (Fig. 7).

Spontaneous immune responses against native peptide epitopes

Initially, five patients were analyzed for spontaneous immune responses to the four native HLA-B35 binding peptides sur51-59, sur46-54, sur34-43 and sur6-14. These five patients had different haematopoietic malignancies: HEMδ and HEMlδ suffered from MM, HEM12 from FL, HEM9 had DLBCL, and CLL5 had CLL.

INF-γ ELISPOT assays were performed on PBLs after 10 days of in vitro stimulation to detect peptide precursor CTLs. Spontaneous immune responses were detected against two of the native HLA-B35 binding peptides, sur51-59 and sur46-54. Two patients, HEM12 and CLL5 showed a response to both sur51-59 and sur46-54, whereas HEM8 only showed a response to sur51-59 (figure 8A and B). No response could be detected in the two remaining patients, HEM9 and HEMlδ, and no response could be detected to the poorly binding peptides sur34-46 and sur6-14 in any patients.

An alternative approach to in vitro stimulation was used in patient HEM12, i.e. PBLs were co-cultured with matured autologous dendritic cells pulsed with sur51-59 to stimulate a CTL response in vitro. PBLs from this culture showed strong reactivity towards sur51-59 in ELISPOT (figure δB).

Increased recognition of modified peptides

As described above, peptide modifications to enhance the HLA-B35 affinity resulted in a 5- 10-fold higher affinity for HLA-B35 relative to the native peptides. A group of five melanoma patients were analyzed for spontaneous immune responses to both the native and modified peptides by means of ELISPOT assay. PBL samples were analyzed after in vitro stimulation, whereas TIL samples were analyzed directly. Spontaneous immune responses were observed in either PBLs or TILs from three of the five patients. FM25 showed reactivity against sur51-59 and sur51Y9 in both PBL and TIL samples (Fig. 9A). FM45 responded only to the modified peptide sur51Y9, with a strong response detectable in TILs. No PBLs were available from this patient (figure 9A). FM74 showed a strong response to sur46Y9 in TIL, but no response to the native peptide was detectable (figure 9B). A weak response to sur46Y9 was also observed in PBLs from FM74 (data not shown).

Cross-reactivity between the native and modified peptide The high affinity of sur51Y9 to HLA-B35 enables the production of stable monomers of HLA-B35 with sur51Y9. Having established the presence of survivin reactive T lymphocytes in tumor infiltrated lymph nodes and PBLs from different cancer patients, magnetic beads were coated with such HLA-B35/Sur51Y9-complexes and these were used to isolate survivin peptide reactive T lymphocytes from PBL from patient CLL5. This patient showed a strong response to sur51-59. Beads were tightly bound to the cell surface of the specific cells, as visualized by microscopy (data not shown), permitting precipitation of antigen specific cells by a magnetic field. The isolated sur51Y9 specific cells responded strongly to sur51-59, (figure 9), whereas no response could be detected in the remaining PBLs (data not shown). The isolation was analyzed by the RT-PCR/DGGE based TCR clonotype mapping. This technique allows the analysis for T-cell clonality in complex cell populations, even if only small numbers of cells are available. These analyses showed that δ distinct clones were isolated (data not shown).

Antigen specific T cells present in situ in a melanoma lesions

Sur51Y9/HLA-B35 monomers were multimerised using dextran molecules conjugated with streptavidin and FITC. Multimerised MHC-complexes were used to stain acetone-fixed, fro- zen material using the procedure described in Example 2. Antigen specific cells were visualized using a confocal laser microscope. Sections of primary melanoma from three patients were analyzed, and Sur51Y9/HLA-B35-reactive CTLs could readily be detected in situ in the tumor microenvironment in one of the patients. Co-staining with a mAb against granzyme B showed that these survivin specific CTLs released granzyme B, exerting cytotoxic activity, HLA-B35 negative melanoma patients were used as controls (data not shown).

EXAMPLE 4

Identification of novel survivin-derived CTL epitopes with different HLA-A- restriction profiles

Summary

Novel HLA-A1-, HLA-A2-,HLA-A3- and HLA-Al 1-restricted survivin epitopes were characterised on the basis of CTL responses in cancer patients. These epitopes significantly increase the number of patients eligible for immunotherapy based on survivin-derived peptides. Additionally, the collective targeting of several restriction elements is likely to decrease the risk of immune escape by HLA-allele loss.

Materials and Methods Patients

Patient samples were received from the University of Wϋrzburg, Germany and the University Hospital in Herlev, Denmark. Informed consent was obtained from the patients prior to any of these measures. Tissue typing was conducted at Department of Clinical Immunology, University Hospital, Copenhagen, Denmark. Peripheral blood lymphocytes (PBL) from cancer patients with melanoma, mamma carcinoma, and chronic lymphocytic leukemia (CLL) were isolated using Lymphoprep separation and frozen in fetal calf serum (FCS) with 10% dimethylsulphoxide. Furthermore, T lymphocytes from primary lesions and from tumor infiltrated lymph nodes from melanoma patients were obtained. Freshly resected tumor tissue was minced into small fragments, and crushed to release tumor- infiltrating lymphocytes (TIL) for cryopreservation.

Peptides All peptides were purchased from Invitrogen (Carlsbad, CA, USA) and provided at >80% purity as verified by HPLC and MS analysis. All peptides used are listed in Table 4, Example 5 below.

Cell lines

The human T2 cell line is a TAP1 and TAP2 defective hybrid of the B-LCL.174 and the T- LCL CEM cells and thus only express low levels of HLA class I molecules (HLA-A*0201 and HLA-B*5101) at the cell surface. T2 cells transfected with HLA-A*0301 were kindly provided by Dr A McMicheael, IMM, John Radcliffe Hospital, Oxford. T2 cells transfected with HLA-A*1101 were kindly provided by Dr M Masucci, MTC, Karolinska Institute, Stockholm, Sweden. The BM36.1 cell line is also defective in TAP function and has a similar phenotype as T2 with low expression of HLA class I (HLA-A*0101, HLA-B*3501) at the surface. The BM36.1 cells were kindly provided by Dr A Ziegler, Humboldt University, Berlin, Germany.

Assembly assay for peptide binding to MHC class I molecules

The binding affinity of synthetic peptides (Invitrogen, Carlsbad, CA, USA) to HLA-Al, -A2, A3, or -All molecules metabolically labeled with [³⁵S]-methionine was measured in the assembly assay, as described previously (12). The assay is based on peptide-mediated stabilization of empty HLA molecules released upon cell lysis, from the TAP-deficient cell lines. Stably folded HLA-molβcules were immune-precipitated using the HLA class I- specific, conformation-dependent mAb W6/32, and separated by isoelectric focusing (IEF) gel electrophoresis. MHC heavy chain bands were quantified using the ImageGauge Phosphorimager program (FUJI photo film Co., Carrollton, TX, USA). The intensity of the band is proportional to the amount of peptide-bound class I MHC complex recovered during the assay. Subsequently, the extent of stabilization of the HLA-molecule is directly related to the binding affinity of the added peptide. The peptide concentration used to analyze the recovery of the HLA-molecules was 40, 4, 0.4, 0.04 μM for HLA-Al and HLA- All, and 100, 10, 1, 0.1, 0.01 μM for HLA-A2 and HLA-A3. The C_so value was subsequently calculated for each peptide as the peptide concentration sufficient for half maximal stabilization.

Antigen stimulation of PBL

To extend the sensitivity of the ELISPOT assay, PBL were stimulated once in vitro prior to analysis. At day 0, PBL or crushed lymph nodes were thawed and plated as 2 x 10⁶ cells in 2 ml/well in 24-well plates (Nunc, Roskilde, Denmark) in x-vivo medium (Bio Whittaker, Walkersville, Maryland), 5% heat-inactivated human serum, and 2 mM of L-glutamine in the presence of 10 μM of peptide. Two days later 20 IU/ml recombinant interleukin-2 (IL- 2) (Chiron, Ratingen, Germany) was added to the cultures. The cultured cells were tested for reactivity in the ELISPOT on day 10.

ELISPOT assay

The ELISPOT assay was used to quantify peptide epitope-specific interferon-γ releasing effector cells as previously described (16). Briefly, nitrocellulose bottomed 96-well plates (Multiscreen MAIP N45, Millipore, Hedehusene, Denmark) were coated with anti-IFN-γ antibody (1-D1K, Mabtech, Nacka, Sweden). The wells were washed, blocked by X-vivo medium, and the cells were added in duplicates at different cell concentrations. The peptides were then added to each well and the plates were incubated overnight. The following day, media was discarded and the wells were washed prior to addition of biotinylated secondary antibody (7-B6-l-Biotin, Mabtech). The plates were incubated for 2 hours, washed, and avidin-alkaline phosphatase conjugate (Calbiochem, Life Technologies, Inc. San Diego, CA, USA ) was added to each well. The plates were incubated at room temperature for one hour, washed, and the enzyme substrate NBT/BCIP (DakoCytomation Norden A/S, Glostrup, Denmark) was added to each well and incubated at RT for 5-10 min. Upon the emergence of dark purple spots, the reaction was terminated by washing with tap-water. The spots were counted using the ImmunoSpot® Series 2.0 Analyzer (CTL Analyzers, LLC, Cleveland, US) and the peptide specific CTL frequency could be calculated from the numbers of spot-forming cells. All assays were performed in duplicates for each peptide antigen.

Results

Identification of HLA-Al restricted survivin epitopes

Binding of survivin-derived peptides to HLA-Al

The amino acid sequence of the survivin protein was screened for the most probable HLA- Al nonamer or deca-mer peptide epitopes, using the main HLA-Al anchor residues, aspartic acid (D), glutamic acid (E) at position 3 and tyrosine (Y), phenylalanine (F) at the 3δ

C-terminus. Accordingly, six survivin-derived peptides were synthesized and examined for binding to HLA-Al (table 4). Additionally, the two peptides Sur3δ-46 (MAEAGFIHC)(SEQ ID NO:23) and Sur47-56 (PTENEPDLAQ) (SEQ ID NO:25) was included, in spite they only contain one of the main anchors, since both were identified as possible good binders by the predictive algorithm by Rammensee et al. available at http://syfpeithi.bmi- heidelberg.com/. C₅₀ values were estimated for each peptide as the peptide concentration needed for half maximal stabilization of HLA-Al (table 4). However, only one of these peptides Sur92-101 (QFEELTLGEF) (SEQ ID NO: 27) bound with almost similar high affinity as a known positive control epitope from the Influenza A protein, basic polymerase 1 (PB1) (VSDGGPNLY) as exemplified in figure 10. Sur93-101 (FEELTLGEF) (SEQ ID NO:24) had a low binding affinity for HLA-Al, whereas none of the other peptides analyzed bound to HLA-Al (Table 4). Consequently, we synthesized a number of analogue peptides in which better anchor residues replaced the natural amino acids. We modified the two peptides Sur3δ-46 (MAEAGFIHC) (SEQ ID NO:23) and Sur47-56 (PTENEPDLAQ) (SEQ ID NO:25) introducing tyrosine (Y) instead of cysteine (C) or glutamine (Q) respectively at the C- terminus. Both of the modified peptides bound strongly to HLA-Al (table A). Additionally, we substituted the amino acids at position 2 with the auxiliary anchors threonine (T) or serine (S) in the two peptides Sur92-101 and Sur93-101. These modifications did not have a positive effect of the binding of Sur92-101 to HLA-Al. In contrast, the Sur93T2 (FTELTLGEF) (SEQ ID NO:36) bound with high affinity to HLA-Al (Table 4). Figure 10 illustrates the binding of the native low affinity peptide Sur93-101, the high affinity modified peptide Sur93T2 and the non-binding peptide Sur49-5δ as compared to the positive control epitope from influenza. Finally, we modified Surl4-22, Sur34-43, Sur49- 5δ, Sur51-59, Sur92-101, and Sur93-101 with tyrosine (Y) at the C-terminus, however this did not improve binding affinity to HLA-Al for any of these peptides (data not shown).

HLA-Al restricted CTL responses against survivin-derived peptides in cancer patients

PBL from six melanoma patients and TIL from three melanoma patients were analyzed for the presence of CTL specific against any of the four high affinity survivin deduced peptides Sur38Y9, Sur47Y10, Sur92-101, and Sur93T2 by means of ELISPOT. T-cell reactivity against at least one of the survivin-derived peptides was observed in three PBL samples and one TIL sample from the total of nine patients analyzed. As seen in figure 11, PBL from one patient, Mel.Al-3 hosted a T-cell response against all four peptides, Sur38Y9, Sur47Y10, Sur92-101, and Sur93T2. Mel.Al-2 showed responses against Sur47Y10, Sur92-101 and Sur93T2, whereas in Mel.Al-1/TIL and Mel.Al-4/PBL responses were observed against Sur47Y10 and Sur93T2, respectively (figure 11).

In addition, ten melanoma patients were tested for immune reactivity against the native peptides Sur93-101, Sur38-46 and Sur47-56 by means of ELISPOT; however, no peptide- specific responses were detected in any of these patients (data not shown).

Identification of HLA-A11 restricted survivin epitopes

Binding of survivin-derived peptides to HLA-Al 1 The amino acid sequence of the survivin protein was screened for nonamer or deca-mer peptides with binding motifs corresponding to that of the HLA-A3 super-family, including HLA-A3 and HLA-A11. Peptide sequences with the main anchor residues, leucine (L) in position 2 and lysine (K) at the C-terminus, were chosen together with peptide sequences having related amino acids at these positions according to the predictive algorithm by Rammensee et al. (table 4).

Thirteen peptides were predicted from the protein sequence of survivin and analyzed for binding to HLA-A11 and HLA-A3. Three of these peptides, Sur53-62 (DLAQCFFCFK) (SEQ ID NO:47), Sur54-62 (LAQCFFCFK) (SEQ ID NO:42) and Surll2-120 (KIAKETNNK) (SEQ ID NO:44) bound HLA-A11 with high affinity, comparable to the viral epitope from EBV nuclear antigen 4 (AVFDRKSDAK) (SEQ ID NO:63). In addition, one peptide, Surll2-121 (KIAKETNNKK) (SEQ ID NO:5l) bound weakly to HLA-A11 (Table 4).

HLA-Al 1 restricted CTL responses against survivin-derived peptides in cancer patients

PBL from five melanoma patients and two CLL patients were tested for T-cell reactivity against the four HLA-A11 binding peptides Sur53-62; Sur54-62, Surll2-120, and Surll2- 121. We were able to detect responses against the survivin-derived peptide Sur53-62 in PBL from two of the melanoma patients, Mel.All-1, Mel.All-2, by means of ELISPOT (figure 12). Additionally, we were able to detect Sur53-62 specific T-cells among tumor infiltrating lymphocytes (TIL) from a tumor infiltrated lymph node in patient Mel .Al 1-2 (figure 12). In the patient Mel.All-1 a strong immune response against the survivin peptide Sur53-62 was observed in five different blood samples taken over a period of two years (data not shown).

Identification of HLA-A3 restricted survivin epitopes

Binding of survivin-derived peptides to HLA- A3

The survivin-derived peptides predicted for binding to the HLA-A3 super-family were additionally analyzed for the binding to HLA-A3. Only two of the peptides Surll2-120 (KIAKETNNK) (SEQ ID NO:44) and Surll2-121 (KIAKETNNKK) (SEQ ID NO:57) bound HLA-A3 with high affinity, similar to the viral epitope, Influenza A nucleoprotein 265-273 (ILRGSVAHK) (SEQ ID NO:74) (Table 4). Furthermore, two peptides Sur53-62

(DLAQCFFCFK) (SEQ ID NO:47) and Sur95-103 (ELTLGEFLK) (SEQ ID NO:43) bound weakly to HLA-A3.

Some of the peptides with no detectable binding were modified in an attempt to increase the binding affinity for HLA-A3. Thus, we synthesized two analogue peptides of Sur54-62 and Surll3-122 in which a better anchor residue leucine (L) replaced the natural alanine (A) at position 2. Sur54L2 (LLQCFFCFK) (SEQ ID NO:56) bound HLA-A3 with high affinity, whereas Surll3L2 (ILKETNNKKK) (SEQ ID NO: 59) only bound weakly (Table 4). In addition, we synthesized four analogue peptides of Sur5-13, Surl3-22, Surlδ-27, and Sur53-61 in which the better anchor residue lysine (K) replaced the natural phenylalanine (F) at the C-terminus. Sur5K9 (TLPPAWQPK) (SEQ ID NO: 54) and SurlδKlO (RISTFKNWPK) (SEQ ID NO: 5δ) bound to HLA-A3 with high affinity, whereas the substitutions had no detectable effect on the binding to HLA-A3 of Surl3K9 (FLKDHRISTK) (SEQ ID NO:57) and Sur53K9 (DLAQCFFCK) (SEQ ID NO:55) compared to the native analogues.

HLA- A3 restricted CTL responses against survivin-derived peptides in cancer patients

Nine samples from melanoma patients (five PBL and four ΗL) were analyzed for immune reactivity against the two native high affinity HLA-A3 binding peptides Surll2-120 and Surll2-121, as well as the two native, weak binding peptides Sur53-62 and Sur95-103. However, no immune responses against these peptides could be detected by ELISPOT in any of the patients. Subsequently, the same patients were analyzed for spontaneous immune reactivity against the three high affinity, modified survivin-derived peptides, Sur5K9, SurlδKlO, and Sur54L2. CTL reactivity was detected against SurlδKlO in TIL samples from three patients, Mel.A3-l, Mel.A3-2, Mel.A3-3 (figure 13). No responses were detected against the two other peptides, Sur5K9 and Sur54L2. To further verify these responses, PBL from additional eighteen melanoma patients were analyzed for CTL reactivity against SurlδKlO. Three responding patients, Mel.A3-4, Mel.A3-5, and Mel.A3-6, were found among these, resulting in a total of six responding patients among the twenty- seven patients analyzed (figure 13).

Identification of a novel HLA-A2 restricted survivin epitope

Binding of 11-mer survivin-derived peptides to HLA-A2

The amino acid sequence of the survivin protein was screened for the most probable HLA- A2 11-mer peptide epitopes, using the main HLA-A2 specific anchor residues. Six survivin deduced peptides were synthesized and examined for binding to HLA-A2. None of the peptides examined bound with similar high affinity as a known positive control epitope from Epstein-Barr virus BMLF_280-288 peptide (GLCTLVAML) (SEQ ID NO:72) (Table 4). The peptide concentration required for half maximal recovery of HLA-A2 (C₅₀ value) was 0.9 μM for the positive control. In comparison, the peptides Surlδ-2δ (RISTFKNWPFL) (SEQ ID NO:67) and Sur86-96 (FLSVKKQFEEL) (SEQ ID NO:69) bound weakly to HLA-A2 (C₅₀ = 69 μM and 72 μM respectively). However, the two known HLA-A2- restricted survivin epitopes bound in a similar way weakly to HLA-A2; Sur95-104 (ELTLGEFLKL) (SEQ ID NO:43) bound with intermediate affinity (C₅₀ = 10 μM) whereas Sur96-104 (LTLGEFLKL) (SEQ ID NO: 10) bound only weakly (C₅₀ >100 μM). The remaining four 11-mer peptides examined (Sur4-14 (PTLPPAWQPFL) (SEQ ID NO: 66), Sur54-64 (LAQCFFCFKEL) (SEQ ID NO: 68), Sur88-9δ (SVKKQFEELTL) (SEQ ID NO:70), and Surl03-113 (KLDRERAKNKI) (SEQ ID NO:74)) did not bind to HLA-A2.

HLA-A2 restricted CTL responses against survivin-derived peptides in cancer patients PBL from ten cancer patients (two melanoma (Mel), six CLL (CLL), and two mamma carcinoma (MC) patients) was initially analyzed to investigate whether the two weak binding llmer peptides, Surlδ-2δ and Surδ6-96 were presented by HLA-A2 and recognized by the immune system of cancer patients. CTL responses against Surlδ-28 were found in PBL from two of the ten patients analyzed (CLL-1, CLL-2, figure 14), whereas no responses could be detected against Sur86-96 (data not shown). To further verify these Surlδ-2δ specific responses, PBL from additional twelve patients (seven melanoma, one CLL, and four mamma carcinoma patients) were analyzed for CTL reactivity against this peptide. Among these, four patients (CLL-3, MC-1, MC-2, Mel.A2-l) had Surlδ-2δ specific immune activity detectable by ELISPOT (Figure 14). Thus, altogether PBL from six out of twenty-two patients analyzed hosted a CTL response against Surlδ-2δ.

Identification of HLA-B7 restricted survivin epitopes

Binding of survivin derived peptides to HLA-B7

The amino acid sequence of the survivin protein was screened for peptides of nine to ten amino acids, with anchor residues according to the peptide binding motif of HLA-B7. Five peptides were selected and analyzed for their ability to stabilize HLA-B7 in the assembly assay. C₅₀ values were estimated for each peptide as the peptide concentration needed for half maximal stabilization of HLA-B7 (table 4). Two survivin-derived peptides, sur6-14 (LPPAWQPFL) (SEQ ID NO: 18) and surll-19 (QPFLKDHRI) (SEQ ID NO: 19) stabilized HLA- B7 weakly, with C₅₀ values above 100 μM; whereas sur46-54 (CPTENEPDL) (SEQ ID NO:6), sur51-59 (EPDLAQCFF) (SEQ ID NO:7), and sur34-43 (TPERMAEAGF) (SEQ ID NO:20) did not bind to HLA-B7 (table 4).

HLA-B7 restricted CTL responses against survivin derived peptides in cancer patients

HLA-B7 positive PBL from five melanoma patients (mel25, mel26, mel3, mel6, me!39), two CLL patients (CLLl, CLL54) and 2 breast cancer patients (breastll, breast 15) were tested for T-cell reactivity against the weak HLA-B7 binding peptides sur6-14 (LPPAWQPFL) (SEQ ID NO: lδ)and surll-19 (QPFLKDHRI) (SEQ ID NO: 19). We were able to detect a strong spontaneous CTL response against the survivin derived peptide sur6-14 in PBL in a CLL patient and in a breast cancer patient (figure 15). Additionally, we were able to detect a weak response against this peptide in the melanoma patient mel3 (figure 15).

Summarv of HLA allele-restricted immune responses to survivin-derived peptides in cancer patients

A range of survivin-derived peptides comprising 9-11 amino acid residues were tested for binding to the following HLA alleles: HLA-Al, HLA-A3, HLA-A11 and HLA-B7 using the as- sembly assay for peptide binding to MHC class I molecules described in the preceding examples. In addition, several of the peptides were tested for their capacity to elicit a CTL immune response using the ELISPOT assay as also described above.

A summary of the results, including results obtained in the previous examples, are given in the below Table 4:

Table 4. Cm and ELISPOT data for selected survivin-derived peptides

'An response was observed against the peptide Surl 12-120 in one lymphoma patient (HEM34) by means of ELISPOT. "Responses were detected against the peptide Sur53-62 in 3 lymphoma patients (HEM9, 11, 34) by means of ELISPOT. ^IVA weak response was observed in a melanoma patient (FM-TIL95) by means of ELISPOT "A response was observed against Surll2-121 in a melanoma patient (PM6), most evident in metastatic lymph-node suspension, and weaker in the ΗL from primary tumor and PBL by means of ELISPOT.

™'An response against the peptide Sur6-14 was observed in a CLL patient (CLL9), and a weaker response was observed in a lymphoma patient by means of elispot (HEM 21) (data not shown.

EXAMPLE 5

Therapeutic trial procedures using survivin-derived peptides as immunogens

Summary

Five heavily pretreated stage IV melanoma patients were vaccinated with the modified HLA-A2-restπcted survivin epitope, namely the surlM2 peptide, presented by autologous dendritic cells in a compassionate use setting. Four of the patients mounted strong T-cell response to this epitope as measured by ELISPOT assay. Furthermore, in situ peptide/HLA- A2 multimer staining revealed the infiltration of survivin reactive cells into both visceral and soft tissue metastases Notably, vaccination associated toxicity was not observed. The data demonstrate that it is feasible to induce T-cell response against survivin, even in late stage melanoma patients, and that these vaccinations are well tolerated.

Materials and Methods

Patient eligibility criteria and treatment regimen All clinical procedures were in accordance with the Declaration of Helsinki and all patients provided informed consent prior to therapy. Stage IV cutaneous or uveal melanoma patients were eligible when their disease was progressive despite at least two different chemo-, immuno, or chemoimmunotherapies. In addition, a patients had to be 18 years or older, express HLAA*0201, and suffer from measurable disease validated by cranial, thoracic and abdominal computed tomography scans. Patients' Karnofsky index had to be 60% or better. No systemic chemo-, and/or immunotherapy was allowed within 4 week prior to vaccination. Important exclusion criteria were evidence of CNS metastases, active autoimmune or infectious diseases, pregnancy and lactation, as well as significant psychiatric abnormality. Peptide pulsed dendritic cells were generated as previously described (82). Briefly, PBMCs from leukapheresis were isolated on LymphoprepTM (Nycomed Pharma), frozen in aliquots and stored in liquid nitrogen. One week prior to vaccination, PBMCs were thawed, washed and cultured in medium containing gentamycin, glutamine and heat inactivated autologous plasma. On day 1 and 5, IL-4 and GM-CSF were added. To differentiate mature DCs, TNF-γ and prostaglandin E2 were added on day 6. On day 7, cells displaying phenotypical and morphological characteristics of mature DCs, i.e. a veiled appearance and = 75% CD83 expression, were pulsed with a modified survivin- derived HLA-A2 restricted survivin₉₆.₁₀₄ epitope, LMLGEFLKL (SEQ ID NO 10)(Clinalfa, Switzerland) 14. Cells were only used for vaccination if microbial tests of samples taken from cultures on days 1 and 5 proved to be sterile.

Patients were vaccinated at 7-day intervals for the first two vaccinations followed by 26- day intervals for further vaccinations. A total of 10-20 x 106 mature, survivin₉₆.₁₀₄ pulsed DCs were resuspended in PBS, containing 1% human serum albumin, and injected intradermally in aliquots of 1.5 x 106 DCs per injection site in the ventromedial regions of the thighs close to the regional lymph nodes. Limbs where draining lymph nodes had been removed and/or irradiated were excluded. Leukapheresis was repeated after 5 vaccinations in absence of severe deterioration of patient's state of health or occurrence of CNS metastases.

Measurement of clinical and immunological responses

CT scans were performed prior to vaccination and every three months thereafter or in case of severe clinical signs of disease progression. Immunological responses were monitored by the ELISPOT assay, using PBMCs obtained every three months, to detect survivin₉₆.₁₀₄ specific IFN-γ release. To extend the sensitivity of the ELISPOT assay, PBMCs were stimulated once in vitro at a concentration of 1 x 10⁶ cells per ml in 24-well plates (Nunc, Denmark) in X-vivo medium (Bio Whittaker, Walkersville, Maryland), supplemented with 5% heat-inactivated human serum and 2 mM of L-glutamine in the presence of 10 μM of peptide. Two days later, 40 IU/ml recombinant interleukin-2 (IL-2) (Chiron, Ratingen, Germany) were added. After 10 days the cells were tested for reactivity. To this end, nitrocellulose bottomed 96-well plates (Multiscreen MAIP N45, Millipore, Glostrup, Denmark) were coated with an anti-IFN-γ antibody (1-D1K, Mabtech, Sweden). Lymphocytes were added at 10⁴ - 10^s cells in 200 μl X-vivo medium per well together with 10⁴ T2-cells and the relevant peptides at a final concentration of 2 μM. After an overnight incubation at 37°C and two washes, the biotinylated detection antibody (7-B6-l-Biotin, Mabtech, Sweden) was added; its specific binding was visualised using alkaline phosphatase-avidin together with the respective substrate (GibcoBRL). The reaction was terminated upon the appearance of dark purple spots, which were quantitated using the Alphalmager System (Alpha Innotech, San Leandro, CA, USA).

Survivin_96-104/HLA-A*0201 reactive CDδ+ T lymphocytes were also tracked in situ both at the vaccination sites as well as in visceral, soft tissue, or cutaneous metastases by means of multimeric survivin₉₆_₁₀₄/HLA-A*0201 complexes. Vaccination sites were excised 24 h after intradermal injection in all patients, whereas metastatic lesions were only removed in selected patients, if easily accessible (patients KN and GB), or removed during a curative intent (patient WW). The staining procedure for multimeric peptide/MHC complexes has been described recently (6δ). The multimeric survivin_96-104/HLA-A*0201 complexes were generated by introduction of a recognition site for enzymatic biotinylation at the 5' end of the extracellular domains of HLA-A*0201 (residues 1-275). The recombinant protein was purified by size-exclusion (Sephadex G25, Pharmacia, Erlangen, Germany) and ionexchange (mono-Q, Pharmacia) chromatography and folded in vitro by dilution in presence of the respective peptides and β2-microglobulin. After gel filtration on a Sephadex G25 column, the protein was multimerized with streptavidin-FITC conjugated to dextran molecules (kindly provided by L. Winther, DAKO, Copenhagen, Denmark) to generate multivalent HLA-dextran complexes. Cryopreserved sections of the respective samples were dried over-night and subsequently fixed in cold acetone for 5 min. All incubation steps were performed in the dark at room temperature as follows: (i) 45 min of an anti-CDδ antibody (1: 100, clone HITδa, Pharmingen, San Diego, CA), (ii) Cy3- conjugated goat antimouse (1:500 diluted; code 115-165-100, Dianova, Hamburg, Germany) for 45 min and finally (iii) the multimers for 75 min. Between each step the slides were washed twice for 10 min in PBS/BSA 0.1%. Finally, slides were mounted in vectashield and observed under a Leica Confocal Microscope (TCS 4D, Leica, Mannheim, Germany).

Results

Patient characteristics, toxicity and clinical course

Five far-advanced stage IV melanoma patients were enrolled, two suffering from uveal melanoma, one from soft tissue melanoma and the remaining two from cutaneous melanoma. Due to the manifestation of symptomatic brain metastases, one patient was taken off therapy after only two vaccinations. The other four patients received up to 15 vaccinations. One patient died from cardiac arrest in tumor free status after surgical resection of remaining metastases. Another patient was taken off therapy after 10 vaccinations because of appearance of visceral metastases (RW). One patient remained on study after 15 vaccinations. Detailed patient characteristics, previous therapy, number of vaccinations and survival status are summarized in table 5. No major toxicities occurred. Thus, hemoglobin, leucocytes and thrombocytes, as well as lactate dehydrogenase, creatinine and cholinesterase were not influenced by the vaccination therapy (Fig. 16). No signs of systemic or local toxicity were observed at the injection sites. Furthermore, there was no detection of impaired wound healing, hemorrhagic disorders, cardiac dysfunction, vasculitis or inflammatory bowel disease. In one patient (WW), pre-existing liver metastases could be stabilized under vaccination therapy, but a new adrenal metastasis still occurred. Unfortunately, this patient died due to cardiac arrest, even though tumor-free after curative surgery. A brain metastasis was detected in patient PB only 4 weeks after initiation of vaccination. Therefore, this patient had to be excluded from further vaccinations after only two DC injections. The other three patients demonstrated slow progression of metastatic disease without substantial impairment in their general state of health. Remarkably, for patient KN, an overall survival of 13 months (from vaccination start to death) could be achieved despite a heavy metastatic load and fast disease progression at the start of vaccination. Patient GB remained on protocol 14 months after initiation of vaccination with survivin-peptide pulsed DCs. It should be noted, however, that both patients (RW and GB) received additional localized treatment for tumor control, either radiation of subcutaneous tumors (RW) or local chemotherapy (GB).

Survivin-specific CD8+ T cell responses

To monitor the kinetics of cytotoxic T cell responses, PBMCs obtained prior to and three months after vaccination were tested for reactivity to the modified survivin_96-104 epitope by ELISPOT for IFN-γ. Before analysis, PBMCs were stimulated once in vitro to extend the sensitivity of this assay. In all four patients tested, an induction of survivin₉₆-₁₀₄ reactive T cells was evident (Fig. 17). Analysis for reactivity to other HLA-A*0201 restricted survivin peptides, i.e. the non-modified survivin₉₆.₁₀₄ and the adjacent Sur9 epitope, demonstrated a T cell response against these peptides in two of the patients (KN and RW)(data not shown).

The prognostic and clinical value of measurements of tumor-specific T-cell responses in peripheral blood has been questioned repeatedly; thus, we also tested for the presence of survivin_96-104/HLA-A*0201 reactive CDδ+ T lymphocytes among tumor infiltrating lymphocytes in situ by peptide/MHC multimer staining. To validate the method, we first analyzed tissue samples from delayed type hypersensitivity reactions occurring at the vaccination site within 24 hrs. This analysis confirmed earlier observations that intradermal injections of peptide-pulsed DC induce a strong peptide-specific inflammatory T-cell infiltrate. Subsequently, the peptide/MHC multimer staining procedure was applied on soft tissue and visceral metastases, which revealed the presence of survivin_96-ι₀₄/HLA-A*0201 reactive cells among the CDδ+ infiltrate. This observation suggests that the vaccination does not only induce T cell with the desired specificity, but also endows them with the necessary homing capacity. Table 5. Summary of vaccination trials: patient characteristics

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Claims

5δCLAIMS

1. A MHC Class I-restricted epitope peptide derived from survivin, said epitope having at least one of the following characteristics:

(iii) capable of in situ detection in a tumor tissue of CTLs that are reactive with the epitope peptide.

2. A peptide according to claim 1 having a C₅₀ value, which is at the most 30 μM.

3. A peptide according to any of claims 1-2 having a C₅₀ value, which is at the most 20 μM.

4. A peptide according to any of claims 1-3, which is restricted by a MHC Class I HLA-A molecule.

5. A peptide according to claim 4, which is restricted by a MHC Class I HLA species selected from the group consisting of HLA-Al, HLA-A2, HLA-A3, HLA-Al 1 and HLA-A24.

6. A peptide according to any one of claims 4 or 5, which is restricted by HLA-A2.

7. A peptide according to any one of claims 4-6, which is selected from the group consisting of FLKLDRERA (SEQ ID NO: l), TLPPAWQPFL (SEQ ID NO:2), ELTLGEFLKL (SEQ ID NO:3), LLLGEFLKL (SEQ ID NO:4) and LMLGEFLKL (SEQ ID NO:5).

δ. A peptide according to any one of claims 1-3, which is restricted by a MHC Class I HLA-B molecule.

9. A peptide according to claim 8, which is restricted by a MHC Class I HLA-B species selected from the group consisting of HLA-B7, HLA -B35, HLA -B44, HLA-Bδ, HLA-B15, HLA- B27 and HLA-B51.

10. A peptide according to any one of claims δ or 9, which is restricted by HLA-B35 or HLA-B7.

11. A peptide according to any one of claims δ-10, which is selected from the group consisting of CPTENEPDL (SEQ ID NO:6), EPDLAQCFF (SEQ ID NO:7), CPTENEPDY (SEQ ID NO:8), EPDLAQCFY (SEQ ID NO:9), LPPAWQPFL (SEQ ID NO: 18) and QPFLKDHRI (SEQ ID NO: 19).

12. A peptide according to any of the preceding claims comprising at the most 20 amino acid residues.

13. A peptide according to claim 12 that comprises at the most 10 amino acid residues.

14. A peptide according to any one of the preceding claims, which is a nonapeptide or a deca peptide.

15. A peptide according to any one of the preceding claims, which is a native sequence of survivin of a mammal species.

16. A peptide according to claim 15 that is derived from human survivin.

17. A peptide according to any one of the preceding claims, which is derived from a native sequence of survivin by substituting, deleting or adding at least one amino acid residue.

lδ. A peptide according to any of the preceding claims, which is phosphorylated.

19. A peptide according to claim lδ, which comprises Thr34 of the native survivin disclosed in US 6.245.523.

20. A peptide according to any one of the preceding claims comprising, for each specific HLA allele, any of the amino acid residues as indicated in the following table:

Cw6

HLA- Y L,Y,F

Cw6

HLA- ,I,

Cwδ

HLA- A,L L,V

Cwl6

21. A peptide according to any one of the preceding claims that is capable of eliciting INF-γ -producing cells in a PBL population of a cancer patient at a frequency of at least 10 per 10⁴ PBLs.

5

22. A peptide according to any one of the preceding claims, which is capable of eliciting INF-γ -producing cells in a PBL population of a patient having a cancer disease where survivin is expressed.

10 23. A peptide according to claim 22 where the cancer disease is selected from the group consisting of a haematopoietic malignancy including chronic lymphatic leukemia and chronic myeloid leukemia, melanoma, breast cancer, cervix cancer, ovary cancer, lung cancer, colon cancer, pancreas cancer and prostate cancer.

15 24. A peptide according to any one of the preceding claims, which is capable of eliciting INF-γ -producing cells in a PBL population of a patient having a cancer disease, said INF-γ - producing cells having cytotoxic effect against survivin expressing cells of a cancer cell line, including a cell line selected from the group consisting of the breast cancer cell line MCF-7 and the melanoma cell line FM3.

20

25. A pharmaceutical composition comprising the peptide according to any one of claims 1- 24.

26. A composition according to claim 25 that comprises a peptide according to claim 4 in 25 combination with a peptide according to claim δ.

27. A composition according to claim 26 comprising a peptide according to claim 6 in combination with a peptide according to claim 10.

30 28. A composition according to any one of claims 25-27, which is a vaccine capable of eliciting an immune response against a cancer disease.

29. A composition according to any one of claims 25-2δ, further comprising an immunogenic protein or peptide fragment selected from a protein or peptide fragment not 35 belonging to or derived from the survivin protein family.

30. A composition according to claim 29, where the protein or peptide fragment not belonging to or derived from the survivin protein family is a protein, or peptide fragment hereof, involved in regulation of cell apoptosis.

5 31. A composition according to claim 29 where the immunogenic protein or peptide fragment selected from a protein or peptide fragment hereof not belonging to or derived from the survivin protein family is Bcl-2 or a peptide fragment hereof.

32. A composition according to any one of claims 25-31, which is a multiepitope vaccine. 10

33. A composition according to claim 32 where the vaccine is capable of eliciting an immune response against a cancer disease where survivin is expressed.

34. A composition according to claim 33 where the cancer disease is selected from the

15 group consisting of a haematopoietic malignancy including chronic lymphatic leukemia and chronic myeloid leukemia, melanoma, breast cancer, cervix cancer, ovary cancer, lung cancer, colon cancer, pancreas cancer and prostate cancer.

35. A composition according to claims 33 or 34 where the vaccine elicits the production in 20 the vaccinated subject of effector T-cells having a cytotoxic effect against the cancer cells.

36. A composition for ex vivo or in situ diagnosis of the presence of survivin reactive T- cells among PBLs or in tumor tissue, the composition comprising a peptide according to claim 1.

25

37. A diagnostic kit for ex vivo or in situ diagnosis of the presence of survivin reactive T- cells among PBLs or in tumour tissue, the kit comprising a peptide according to any one of claims 1-24.

30 3δ. A complex of a peptide according to claims 1 and a Class I HLA molecule or a fragment of such molecule.

39. A complex according to claim 3δ which is monomeric.

35 40. A complex according to claim 3δ which is multimeric.

41. A method of detecting the presence of survivin reactive T-cells, the method comprising contacting a tumour tissue or a blood sample with a complex according to claim 38 and detecting binding of the complex to the tissue or the blood cells.

40

42. A molecule that is capable of binding specifically to a peptide according to any of claims 1-24.

43. A molecule according to claim 42 which is an antibody or a fragment hereof.

44. A molecule that is capable of blocking the binding of a molecule according to claim 42 or 43.

5 45. Use of a peptide as defined in an any of claims 1-24 for the preparation of a medicament for the treatment of cancer.

46. Use of a composition according to claim 25, a molecule according to claim 42 or a molecule according to claim 44 for the preparation of a medicament for the treatment of

10 cancer.

47. The use according to any one of claims 45 or 46, wherein the disease to be treated is a cancer disease where survivin is expressed.

15 48. The use according to claim 46 wherein the cancer disease is selected from the group consisting of a haematopoietic malignancy including chronic lymphatic leukemia and chronic myeloid leukemia, melanoma, breast cancer, cervix cancer, ovary cancer, lung cancer, colon cancer, pancreas cancer and prostate cancer.

20 49. The use according to any one of claims 45-48, which is combined with a further treatment.

50. The use according to claim 49 wherein the further treatment is radiotherapy or chemotherapy.