Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

• the present invention relates to a novel modified human oncofoetal antigen immature laminin receptor (OFA/iLR)- derived peptide, its stereoisomers, its peptide analogues and mixtures thereof for use in diagnostic and therapeutic applications, specifically for use in the treatment of cancer.
• the present invention relates to immunotherapeutic methods, and molecules and cells for use in
• the present invention relates to the immunotherapy of cancer, in particular several tumor entities including hematological malignancies such as leukemia, multiple myeloma and T cell lymphoma, solid cancers such as breast cancer, colon cancer, lung cancer, kidney cancer, gastric cancer, ovarian cancer, prostate cancer, renal cancer, as well as any cancer that expresses the human oncofoetal antigen immature laminin receptor protein (OFA/iLRP).
• the present invention furthermore relates to a tumor-associated T-helper cell peptide epitope, its stereoisomers, its peptide analogues and mixtures thereof, alone or in combination with other tumor-associated peptides that serve as active
• the present invention relates to one novel modified peptide sequence derived from HLA class I of the human oncofoetal antigen immature laminin receptor protein (OFA/iLRP), its stereoisomers, its peptide analogues and mixtures thereof which can be used in vaccine or other pharmaceutical compositions for eliciting anti-tumor immune responses.
• OFA/iLRP human oncofoetal antigen immature laminin receptor protein
• B cells bone marrow- derived lymphocytes
• T cells T cells
• An important facet of the system is the T cell response. Stimulation of an immune response is dependent upon the presence of antigens recognized as foreign by the host immune system. T cells react with foreign antigens via receptors on their surfaces.
• the T cell receptor is capable of recognizing a particular antigen only when it is associated with a surface marker on an antigen-presenting cell (APC) .
• APC antigen-presenting cell
• MHC histocompatibility complex
• MHC-I-molecules that can be found on most cells having a nucleus that present peptides which result from proteolytic cleavage of endogenous proteins and larger peptides.
• MHC- II-molecules can be found on professional antigen
• APC presenting cells
• Macrophages such as Macrophages, Dendritic Cells, on B cells, on endothelial cells and on altered cells of tumors and tumor stroma which do, under normal circumstances, not express MHC class II-molecules on their cell surfaces, and present either peptides stemming
• MHC-I-presented peptides are recognized by CD8 + -positive cytotoxic T lymphocytes
• MHC-II-presented peptides are recognized by CD4 + -helper-T-cells (generally described in Immunobiology by Charles A., Jr. Janeway, Paul Travers, Mark Walport, Mark J. Shlomchik) .
• the APC processes the antigenic protein into shorter peptides called epitopes.
• the epitopes are usually 8 to 12 residues in length and contain two conserved residues ("anchor") in their sequence that interact with the corresponding binding groove of the MHC- molecule (see 2nd listing as published in Immunogenetics (Rammensee H, Bachmann J, Emmerich NP, Bachor OA,
• a first signal results from the binding
• Cytokines include the group of
• cytotoxic T cells that recognize and are specific to the tumor rejection antigen are stimulated and destroy tumor cells that express the antigen (Cheever et al . , Annals N.Y. Acad. Sci. 1993 690:101-112; Rosenberg SA. Shedding light on immunotherapy for cancer. N Engl J Med. 2004 Apr 1; 350(14) : 1461-3) .
• the antigens that are recognized by the tumor specific cytotoxic T lymphocytes can be molecules derived from all protein classes, such as enzymes, receptors, transcription factors, etc. A comprehensive listing of peptides binding to or eluted from MHC class I or class II molecules can be found on www.syfpeithi.org.
• tumor associated antigens for example, can also be present in tumor cells as products of mutated genes. Good examples are MHC class I ligands, which function as T cell epitopes, from K-ras, BCR-abl and mutated p53.
• Another important class of tumor associated antigens are tissue-specific structures, such as CT ("cancer testis ") -antigens that are expressed in
• tumor associated peptides binding to MHC molecules stem from genes, which are expressed in higher copy numbers in cancer cells compared to healthy cells of the same organ or tissue, as well as compared to healthy cells from other tissues.
• genes which are expressed in higher copy numbers in cancer cells compared to healthy cells of the same organ or tissue, as well as compared to healthy cells from other tissues.
• C-met oncogene as a broadly expressed tumor-associated antigen recognized by cytotoxic T- lymphocytes. Clin Cancer Res. 2004 Jun 1; 10 (1) : 3658-66.
• Other tumor-associated peptides stem from antigens which are in tumor cells retained and not secreted (e.g. proteins from the mucin gene family) .
• Other sources can be aberrant transcripts (frameshift) , peptides from junction sites of post-translational protein-protein fusions.
• tumor associated antigens are tissue specific. Examples include, but are not limited to, tyrosinase for melanoma, PSA and PSMA for prostate cancer and chromosomal cross-overs such as bcr/abl in lymphoma.
• tumor associated antigens identified occur in multiple tumor types, and some, such as oncogenic proteins and/or tumor suppressor genes (tumor suppressor genes are, for example reviewed for renal cancer in Linehan WM,
• tumor antigens can be the target of a tumor specific immune response in multiple types of cancer.
• the ideal tumor antigen for use in a vaccine or at which to direct immunotherapy would be one which is present on all tumor types, absent or masked in normal tissues, evolutionarily conserved, and its function required for the malignancy of the tumor cells.
• Such an immunogen would be less likely to be able to be down regulated or mutated and still have the tumor cells grow and metastasize optimally.
• the discovery of the existence of tumor associated antigens has now raised the possibility of using a host's immune system to intervene in tumor growth.
• T-helper cells play an important role in orchestrating the effector function of CTLs in antitumor immunity.
• T-helper cell epitopes that trigger a T-helper cell response of the Thl type support effector functions of CD8 + Killer T cells, which include cytotoxic functions directed against tumor cells displaying tumor-associated peptide/MHC complexes on their cell surfaces. In this way tumor-associated T-helper cell peptide epitopes, alone or in combination with other tumor-associated peptides, can serve as active
• 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.
• Various mechanisms of harnessing both the humoral and cellular arms of the immune system are currently being explored for cancer
• the proteins In order for the proteins to be recognized by the cytotoxic T-lymphocytes as tumor-specific antigen, and in order to be used in a therapy, particular prerequisites must be fulfilled.
• the antigen should be expressed mainly by tumor cells and not by normal healthy tissues or in rather small amounts. It is furthermore desirable, that the respective antigen is not only present in one type of tumor, but also in high concentrations (e.g. copy numbers per cell) .
• Essential is the presence of epitopes in the amino acid sequence of the antigen, since such peptide (“immunogenic peptide") that is derived from a tumor associated antigen should lead to an in vitro or in vivo T cell response. It is therefore an object of the present invention, to provide a novel amino acid sequence for such peptide that have the ability to bind to a molecule of the human major histocompatibility complex (MHC) class-I and trigger T cell responses against cells bearing the peptide in conjunction with MHC molecules on their cell surfaces.
• MHC human major histocompatibility complex
• this object is solved by providing a tumor associated modified peptide that is selected from the amino acid sequence of the human
• HLA/iLRP human major histocompatibility complex
• MHC human major histocompatibility complex
• OFA/iLRP oncofoetal antigen-immature laminin receptor protein
• OFA-iLRP can be specifically recognized by both T and B lymphocytes making it an attractive target molecule for vaccination approaches in several cancer entities.
• DC dendritic cells
• hematopoietic target cells could be generated both in vitro and in vivo (Siegel S, Wagner A, Lucasitz D et al . Coggin, JJr . , Barsoum, A., Rohrer, J., Schmitz, N., Zeis, M.
• US 4,861,710 discloses a clone comprising a recombinant cDNA clone for encoding cell surface receptor for laminin as well as respective probes.
• WO 2006/114307 discloses two naturally processed peptide epitopes from the OFA/iLR protein and their medical use fighting malignant cells.
• first polypeptide comprising a first polypeptide and a second polypeptide, wherein said complex comprises the amino acid sequences of a first polypeptide (SMI1, SEQ ID NO: 359), and a second polypeptide (BAS1, SEQ ID NO: 518), denoted as ProPair 267a-267b.
• SMI1 first polypeptide
• BAS1 second polypeptide
• 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 is a 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 modified peptide of the
• the inventors have identified a novel modified HLA-A*0201- specific peptide epitope derived from the OFA/iLR protein. This peptide represents a useful tool for both conducting tumor immunological studies and vaccination strategies in OFA/iLRP-expressing malignancies .
• the present invention is defined in the claims and provides a tumor associated modified peptide comprising the sequence according to SEQ ID NO. 1, its stereoisomers, its peptide analogues and mixtures thereof.
• the novel modified peptide of the present invention has an increased ability to bind to a molecule of the human major histocompatibility complex (MHC) class-I, in particular to HLA-A*0201, compared to its naturally processed analogue.
• MHC human major histocompatibility complex
• the peptide of the present invention when bound to HLA-A*0201, is capable of eliciting the production of a cytotoxic T lymphocyte (CTL) , which then recognizes a cell that expresses a polypeptide comprising the given amino acid sequence.
• CTL cytotoxic T lymphocyte
• the modified peptides of the present invention may include non-peptide bonds or may be fusion peptides.
• compositions comprising a modified peptide of the
• the invention or its stereoisomers or its peptide analogues or mixtures thereof and a pharmaceutically acceptable carrier.
• the invention further provides a tumor associated peptide of the invention, its stereoisomers, its peptide analogues and mixtures thereof for use in medicine.
• Another embodiment of the present invention provides a cancer vaccine comprising a novel modified peptide of the invention, or its stereoisomers or its peptide analogues or mixtures thereof.
• the invention further provides the use of a modified peptide of the invention, its stereoisomers, its peptide analogues and mixtures thereof in the manufacture of a medicament for killing target cells in a patient, which targets cells expressing a polypeptide comprising an amino acid sequence of the peptide of the present invention, as provided in SEQ ID NO. 1, or its peptide analogues.
• the present invention further provides activated cytotoxic T lymphocyte (CTL) , produced by methods discussed above, which selectively recognize a cell that expresses a poly ⁇ peptide comprising a peptide of the present invention or its peptide analogues.
• CTL cytotoxic T lymphocyte
• the present invention also provides a T-cell receptor (TCR) that recognizes a cell that expresses a polypeptide com ⁇ prising a peptide of the present invention or its peptide analogues, the TCR being obtainable from the cytotoxic T lymphocyte (CTL) described above, or a functionally equiva ⁇ lent molecule to the TCR.
• TTL cytotoxic T lymphocyte
• the present invention also provides use of cytotoxic T lym ⁇ phocytes in the manufacture of a medicament for killing target cells in a patient.
• the cytotoxic T lymphocytes tar ⁇ get cells expressing a polypeptide comprising the peptide of the present invention or its peptide analogues.
• the invention in a further aspect relates to a method of killing target cells in a patient, which targets cells expressing a polypeptide comprising an amino acid sequence as given herein or an analogous amino acid sequence with conservative substitutions, the method comprising
• administering to the patient an effective amount of a peptide or peptides according to the present invention wherein the amount of said peptide or said peptides is effective to provoke an anti-target cell immune response in said patient.
• the invention in a further aspect relates to a method of killing target cells in a patient which targets cells ex ⁇ pressing a polypeptide comprising an amino acid sequence as given according to the present invention or an analogous amino acid sequence with conservative substitutions, the method comprising the step of obtaining cytotoxic T lym ⁇ phocytes (CTL) from the patient.
• the target cells are cancer cells. More pref ⁇ erably, said cancer is leukemia or lymphoma which expresses the polypeptide which comprises an amino acid sequence as given according to the present invention or an analogous amino acid sequence with conservative substitutions.
• the HLA system represents the human major histocompatibility (MHC) system.
• MHC human major histocompatibility
• MHC systems control a range of characteristics: transplantation antigens, thymus dependent immune responses, certain complement factors and predisposition for certain diseases.
• 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.
• Class I molecules are so-called HLA-A- molecules that are presented on the surface of most
• the present invention further relates to one novel modified peptide sequence derived from HLA class I molecules of the oncofoetal antigen-immature laminin receptor protein, its stereoisomers, its peptide analogues and mixtures thereof, which can be used for diagnostic or in vaccine compositions for eliciting anti-tumor immune responses.
• HLA class I molecules of the oncofoetal antigen-immature laminin receptor protein its stereoisomers, its peptide analogues and mixtures thereof, which can be used for diagnostic or in vaccine compositions for eliciting anti-tumor immune responses.
• amino acid refers to an organic compound having an amino group (-N3 ⁇ 4) , a carboxyl (or carboxy) group (-COOH) in the same molecule, and as used in the art also includes imino acids having an imino group, e.g. proline and hydroxyproline .
• the amino acid may be -amino acid, ⁇ -amino acid or the like. In preferred embodiments, an amino acid is an a-amino acid.
• amino acids used herein may be naturally-occurring, non- naturally occurring or synthetic amino acids.
• non-naturally occurring amino acid refers to an amino acid which is not found in a naturally-occurring protein.
• naturally occurring amino acid refers to an amino acid which is found in a naturally-occurring protein.
• amino acids and amino acid residues disclosed herein may be denominated by either a three letter code or a single letter code as indicated in the following: Alanine (Ala, A), Arginine (Arg, R) , Aspar- agine (Asn, N) , Aspartic Acid (Asp, D) , Cysteine (Cys, C) , Glutamine (Gin, Q) , Glutamic Acid (Glu, E) , Glycine (Gly, G) , Histidine (His, H) , Isoleucine (lie, I), Leucine (Leu, L) , Lysine (Lys, K) , Methionine (Met, M) , Phenylalanine (Phe, F) , Proline (Pro, P) , Serine (Ser, S) , Threonine (Thr, T) , Tryptophan (Trp, W) , Tyrosine (T
• peptides disclosed herein include all isomeric (e.g., enantiomeric and diastereomeric) forms of amino acid residues in their sequences.
• L or D as in the L/D nomenclature, may be omitted where the amino acid is stated to be or is obviously derived from a protein source and is therefore assumed to be L.
• Peptides disclosed herein may either contain one kind of stereoisomers of amino acid residues or mixtures of various stereoisomers of amino acid residues, e.g.
• peptides disclosed herein may contain L-amino acids, D-amino acids, or both (L- and D-amino acids) . It is known in the art that peptides containing D-amino acids may ad ⁇ dress certain limitations associated with peptides contain ⁇ ing L-amino acids, e.g. they are more resistant to prote- olytic degradation, which may increase serum and saliva half-life of respective peptides.
• the modified peptide of the invention is derived from the known (wild-type) sequence of OFA/iLRP, e. g. the sequence disclosed in WO-A-8802407.
• wild-type refers to a gene or gene product which has the characteristics of that gene or gene product when isolated from a naturally
• a wild-type gene is what is most of the genes
• modified or mutant refers to a gene or gene product which displays modifications in sequence and/or functional properties (i.e. altered characteristics) when compared to the wild-type gene or gene product.
• the modified peptide of the present invention is characterized by its ability to bind to (being restricted to) a
• novel peptide sequence have been identified by a new and generally applicable combined approach for the identification of processed HLA class I ligands of defined - e.g. tumor associated - antigens. It is preferred if the peptide of the invention is able to bind to HLA- A2. It is preferred if the peptide of the invention is able to bind to HLA- A2. It is preferred if the peptide of the invention is able to bind to HLA- A2. It is a new and generally applicable combined approach for the identification of processed HLA class I ligands of defined - e.g. tumor associated - antigens. It is preferred if the peptide of the invention is able to bind to HLA- A2. It is
• the peptide bind selectively to HLA-A*0201.
• the modified peptide has the sequence VLIENPADV (SEQ ID No. 1) .
• the respective position of the modified peptide in the respective protein is ⁇ - AA 74 .
• the Accession-numbers of human-OFA/iLR in the Genbank of the "National Centre for Biotechnology Information" of the National Institute of Health are, for example, AAC50652 or AAP35883.
• the invention provides a modified peptide, comprising an modified amino acid sequence according to SEQ ID No .1 or a variant thereof provided that the peptide is not the intact human polypeptide from which the amino acid sequence is derived (i.e. oncofoetal antigen-immature laminin receptor protein (OFA/iLRP) .
• OFA/iLRP oncofoetal antigen-immature laminin receptor protein
• peptide analogues or “peptide variants” may be used interchangeably and may refer to the modified peptide of the present invention (SEQ ID No. 1) in which
• conservative amino acid substitutions replace amino acid residues of SEQ ID No. 1 with other analogous amino acids having similar Hydrophobicity Indexes.
• hydrophobic properties of amino acids contribute strongly to the secondary structure of the respective peptides. It is therefore generally understood in the art that Hydrophobicity Indexes of amino acids play an important role in providing a peptide with its function. Therefore, conservative substitutions with analogous amino acids having similar Hydrophobicity Indexes as used herein means that the peptides with such substitutions will most likely still be able to bind to an HLA molecule in
• Hydrophobicity Indexes relative to glycine are provided in brackets following respective amino acid' s three- and sin ⁇ gle letter codes for all groups of amino acids) : Phe F (100), He I (99), Trp W (97), Leu L (97), Val V (76), Met M (74), hydrophobic group of amino acids: Tyr Y (63), Cys C (49), Ala A (41) , neutral group of amino acids: Thr T (13), His H (8), Gly G (0) , Ser S (-5) , Gin Q (-10) , hydrophilic group of amino acids: Arg R (-14), Lys K (-23), Asn N (-28), Glu E (-31), Pro P (-46), Asp D (-55). Examples of conservative substitutions with analogous amino acids having similar Hydrophobicity Indexes include
• substitutions of amino acids within each of the groups mentioned above with another amino acid from the same group e.g. V with F, or I or W or L or M; I with F or W or L or V or M; E with R or K or N or P or D; N with R or K or E or P or D; P with R or K or N or E or D; A with Y or C; D with R or K or N or E or P.
• analogues of the novel modified peptide with SEQ ID No. 1 all possible combinations of conservative substitutions with analogous amino acids from the same group according to Hydrophobicity Index as disclosed above are explicitly disclosed therein, including, e.g., "ILIENPADV",
• VLIEKPADV and "ILIEKPADV” which are based on SEQ ID No .1 (VLIENPADV) .
• the modified peptide that forms the basis of the present invention has been identified as being presented by MHC class I bearing cells.
• this particular peptide as well as other peptides containing the sequence of said peptide i.e. derived peptides
• the persons of skill in the present art are well aware of methods that can be applied in order to determine the extent to which a response is induced by an individual peptide, in particular with reference to the examples herein and the respective literature .
• the inventors identified one HLA-A*0201-specific T cell epitope derived from the OFA/iLR-protein able to induce specific T cell reactivity against human tumor cells, including but not limited to various hematological malignancies .
• a peptide according to the present invention consists essentially of an amino acid sequence according to SEQ ID No. 1 or a variant thereof.
• Consisting essentially of shall mean that a peptide according to the present invention, in addition to the sequence according to SEQ ID No. 1 or a variant thereof, could contain additional N- and/or C-terminally located stretches of amino acids that are not necessarily forming part of the peptide that functions as core sequence of the peptide comprising the binding motif and as an immunogenic T-helper epitope.
• a peptide may be modified so that it at least maintains, if not improves, the ability to
• HLA-A MHC molecule
• HLA-A suitable MHC molecule
• certain positions of HLA-A binding peptides are typically anchor residues forming a core sequence fitting to the binding motif of the HLA binding groove .
• the peptide of the invention may be any peptide (by which term the inventors include oligopeptide or polypeptide) which includes the amino acid sequences or a portion or variant thereof as given.
• the peptide of the invention is one which, if expressed in an antigen presenting cell, may be processed so that a fragment is produced which is able to bind to an appropriate MHC molecule and may be presented by a suitable cell and elicit a suitable T cell response. It will be appreciated that a fragment produced from the peptide may also be a peptide of the invention.
• the peptide of the invention contains a portion which includes the given amino acid sequence or a portion or variant thereof and a further portion which confers some desirable property.
• the further portion may include a further T cell epitope (whether or not derived from the same polypeptide as the first T cell epitope-containing portion) or it may include a carrier protein or peptide.
• the peptide of the invention is a truncated human protein or a fusion protein of a protein fragment and another polypeptide portion provided that the human portion includes one or more inventive amino acid sequences .
• the peptide of the invention includes the modified amino acid sequence of the invention and at least one further T cell epitope wherein the further T cell epitope is able to facilitate the production of a T cell response directed at the type of tumor that expresses a tumor-associated antigen.
• the peptides of the invention include so-called "beads on a string" polypeptides which can also be used as vaccines.
• the inventors include the meaning that the polypeptide is overexpressed compared to normal levels of expression or that the gene is silent in the tissue from which the tumor is derived but in the tumor it is expressed.
• overexpressed the inventors mean that the polypeptide is present at a level at least 1.2 x that present in normal tissue; preferably at least 2 x and more preferably at least 5 x or 10 x the level present in normal tissue .
• a further aspect of the invention provides a method of killing target cells in a patient which targets cells expressing a polypeptide comprising an amino acid sequence of the invention, or its peptide analogues, the method comprising administering to the patient an effective amount of a peptide or peptides according to the invention, or an effective amount of a polynucleotide or an expression vector encoding a said peptide or said peptides, wherein the amount of said peptide or said peptides or amount of said polynucleotide or expression vector is effective to provoke an anti-target cell immune response in said
• the target cell is typically a tumor or cancer cell, in particular a leukemia or lymphoma cell.
• expression vector refers to DNA sequences containing a desired coding sequence and appropriate DNA sequences necessary for the expression of the operably linked coding sequence in a particular host organism or host cell.
• DNA sequences necessary for expression in pro- caryotes include a promoter, optionally an operator se ⁇ quence, a ribosome binding site and possibly other se- quences.
• Eukaryotic cells are known to utilize promoters, polyadenylation signals and enhancers.
• operably linked refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule ca ⁇ pable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.
• the term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
• regulatory element refers to a genetic element which controls some aspect of the expres ⁇ sion of nucleic acid sequences.
• a promoter is a regulatory element which facilitates the initiation of transcription of an operably linked coding region.
• Promoters and enhancers consist of short arrays of DNA sequences that interact specificallv with cellular proteins involved in transcription [Maniatis T. et al . , Science 236:1237 (1987)]. Promoter and enhancer elements have been isolated from a variety of eukaryotic sources in ⁇ cluding genes in yeast, insect and mammalian cells and vi- ruses (analogous control elements, i.e., promoters, are also found in procaryotes) .
• a particular promoter and enhancer depends on what cell type is to be used to express the protein of interest. Some eukaryotic promoters and enhancers have a broad host range while oth- ers are functional in a limited subset of cell types [for review see Voss S.D. et al . , Trends Biochem. Sci., 11:287 (1986) and Maniatis. T. et al . , supra (1987)].
• a still further aspect of the present invention provides the use of a peptide, its stereoisomers, its peptide analogues and mixtures thereof according to the invention in the manufacture of a medicament for killing target cells in a patient which targets cells expressing a polypeptide comprising an amino acid sequence of the invention or its peptide analogues.
• the target cells are cancer cells, more
• leukemia or lymphoma cancer cells preferably leukemia or lymphoma cancer cells.
• the patients who are treated by the methods of the invention have the HLA-A2 type.
• the HLA haplotype of the patient is determined prior to treatment. HLA haplotyping may be carried out using any suitable method; such methods are well known in the art .
• the invention includes the use of the modified peptide of the invention, its stereoisomers, its peptide analogues and mixtures thereof for active in vivo vaccination.
• the vaccine of the present invention is administered to a host either alone or in combination with another cancer therapy to inhibit or suppress the
• modified peptide of the invention may be used directly (i.e. they are not produced by
• the peptide or peptides has fewer than 100 or 50 residues.
• present invention exhibit an overall length of between 8 and 12 consecutive amino acid (AA) residues, further
• the peptide for use in a cancer vaccine may be any suitable peptide.
• it may be a suitable 9-mer peptide or a suitable 7-mer or 8-mer or 10-mer or 11-mer peptide or 12-mer. Longer peptides may also be suitable, but 9-mer or 10-mer peptides are preferred.
• a combination with other peptides for example MHC class II specific peptides can be used.
• the person of skill will be able to select preferred combinations of immunogenic peptides by testing, for example, the generation of T cells in vitro as well as their efficiency and overall presence, the
• the most efficient peptides are then combined as a vaccine for the purposes as described above.
• a further aspect of the invention therefore provides a vaccine effective against cancer, or cancer or tumour cells, comprising an effective amount of a peptide or peptides according to the invention.
• a vaccine comprising a (synthetic) peptide or peptides (i.e. either alone or in combinations of 1, 2, 3, 4, 5 or 6 or even more peptides) .
• the invention in a further aspect relates to a
• composition that contains said modified peptide, or its stereoisomers, or its peptide analogues or mixtures thereof according to the invention.
• composition is used for parenteral administration, such as subcutaneous, intradermal, intramuscular or oral
• peptide or its stereoisomers, or its peptide analogues or mixtures thereof are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous carrier.
• a pharmaceutically acceptable, preferably aqueous carrier preferably aqueous carrier.
• the peptide or peptides may be
• the composition can contain excipients, such as buffers, binding agents, blasting agents, diluents, flavors, lubricants, etc.
• excipients such as buffers, binding agents, blasting agents, diluents, flavors, lubricants, etc.
• the peptide, or its stereoisomers, or its peptide analogues or mixtures thereof can also be administered together with immune stimulating substances, such as cytokines.
• An extensive listing of excipients that can be used in such a composition can be, for example, taken from A. Kibbe, Handbook of Pharmaceutical Excipients, 3. Ed., 2000,
• an adjuvant such as, for example, IL-2, IL-12, GM-CSF, incomplete Freund's adjuvant, complete Freund's adjuvant or liposomal formulations.
• an adjuvant such as, for example, IL-2, IL-12, GM-CSF, incomplete Freund's adjuvant, complete Freund's adjuvant or liposomal formulations.
• the most preferred adjuvants can be found in, for example, Brinkman JA, Fausch SC, Weber JS, Kast WM. Peptide-based vaccines for cancer immunotherapy. Expert Opin Biol Ther. 2004 Feb;4(2): 181-98.
• the peptide, or its stereoisomers, or its peptide analogues or mixtures thereof may also be conjugated to a suitable carrier such as keyhole limpet hemocyanin (KLH) or mannan (see WO
• the peptide or peptides vaccine may be administered without adjuvant.
• the peptide or peptides may also be tagged, or be a fusion peptide or fusion peptides, or be a hybrid molecule.
• the peptide or peptides which sequence is or are given in the present invention is or are expected to stimulate CD8 + CTL. However, stimulation is more efficient in the presence of help provided by CD4 + T cells.
• the fusion partner or sections of a hybrid molecule suitably provide epitopes which stimulate CD4 + T cells.
• CD4 + stimulating epitopes are well known in the art and include those identified in tetanus toxoid.
• composition can be used for a prevention, prophylaxis and/or therapy of tumoros diseases.
• the activated CTL express a T cell receptor (TCR) which is involved in recognizing cells which express the polypeptide or polypeptides. It is useful if the cDNA encoding the TCR is cloned from the activated CTL and transferred into a further CTL for expression.
• TCR T cell receptor
• the target cells for the CD8 + CTL can be cells of the tumor, leukemia or lymphoma (which express MHC class I) and/or stromal cells surrounding the tumor (tumor cells) (which sometimes also express MHC class I) .
• a still further aspect of the invention provides a method of killing target cells in a patient which targets cells expressing a polypeptide or polypeptides comprising an amino acid sequence of the invention or its peptide
• analogues the method comprising the steps of (1) obtaining CTL from the patient; (2) introducing into said cells a polynucleotide encoding a TCR, or a functionally equivalent molecule, as defined above; and (3) introducing the cells produced in step (2) into the patient.
• the pharmaceutical preparation containing the peptide or peptides of the present invention is or are administered to a patient that suffers from a tumorous disease that is associated with the respective peptide or antigen.
• OFA/iLRP such as leukemias (e.g. CLL) myelomas (e.g. MM) or T cell lymphomas, solid cancers (e.g. breast cancer, colon cancer, lung cancer, kidney cancer, gastric cancer, ovarian cancer, prostate cancer, renal cancer) as well as any cancer that expresses the human oncofoetal antigen immature laminin receptor protein (OFA/iLRP) .
• the peptide or peptides according to the invention can also be used as diagnostic reagent. Using the peptide or
• CTL-population CTLs it can be analyzed, whether in a CTL-population CTLs are present that are specifically directed against a peptide or peptides or are induced by a therapy.
• the increase of precursor T cells can be tested with this peptide or these peptides that have reactivity against the defined peptide or peptides.
• the peptide or peptides can be used as marker or markers in order to monitor the progression of the disease of a tumor that expresses said antigen of which the peptide is derived from.
• the peptide is used or peptides are used for the production of an antibody.
• Polyclonal antibodies can be obtained in a standard fashion by immunization of animals via injection of the peptide or peptides and subsequent purification of the immune globulin.
• Monoclonal antibodies can be produced according to standard protocols such as described, for example, in Methods Enzymol. (1986), 121, Hybridoma
• the vaccine can be dependent from the specific type of cancer that the patient to be treated is suffering from as well as the status of the disease, earlier
• treatment regimens the immune status of the patient, and, of course, the HLA-haplotype of the patient.
• a peptide was synthesized which was predicted by the computer programs PAProC (http://www.uni- tuebingen.de/uni/kxi/) and SYFPEITHI
• the modified peptide (referred to as "iLR-ASK-A2"; SEQ ID No. 1) having an amino acid sequence in which the second amino acid "Ala” of the known amino acid sequence Val-Ala- Ile-Glu-Asn-Pro-Ala-Asp-Val (SEQ ID No. 2) is changed to "Leu”, namely Val-Leu-Ile-Glu-Asn-Pro-Ala-Asp-Val, has a higher binding affinity for the HLA-A*0201-molecule .
• cytotoxic T lymphocytes CTL
• 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.
• the preferred embodiment of the present invention provides a modified peptide
• This peptide is preferably a polypeptide consisting of 9-12 amino acids and comprising the amino acid sequence indicated in Sequence ID No . 1 and, more preferably, a peptide consisting of the amino acid sequence indicated in Sequence ID No. 1.
• the peptide showed strong binding affinity to HLA-A* 0201 if compared with the naturally processed peptide (Sequence ID No. 2) .
• a significant feature of the modified peptide of the invention is its capability to elicit INF-y-/GrB-producing responder T cells, i. e. cytotoxic T cells (CTLs) that specifically recognize the particular peptide on tumor cells of a cancer patient (target cells) .
• CTLs cytotoxic T cells
• This activity is readily determined by subjecting PBMC or tumor cells from a patient to an ELISPOT assay (as
• the peptide Prior to the assay, it would be advantageous to stimulate the PBMC population to be assayed by contacting the cells with the peptide to be tested.
• the peptide is capable of eliciting INF-y-/GrB-producing T cells at a frequency of at least 1 per 10 4 PBMC as determined by an ELISPOT assay as used herein. More preferably the frequency is at least 5 per 10 4 PBMC, most preferably at least 10 per 10 4 PBMC, such as at least 50 or 100 per 10 4 PBMC.
• the ELISPOT assay represents a strong tool to monitor
• OFA/iLR peptide specific T cell responses Such direct evidence is provided herein, as it was that OFA/iLRP reactive cells isolated by means of HLA/peptide complexes possess the functional capacity of lysing target cells. Accordingly the modified peptide of the invention is capable of eliciting INF-y-/GrB-producing cells in a PBMC population of a patient having a cancer disease where
• OFA/iLRP is expressed including a haematopoietic malignancy such as chronic lymphocytic leukemia, multiple myeloma and malignant lymphoma.
• the modified peptide of the invention is able to elicit an immune response in the form of T cell having cytotoxic effect against OFA/iLRP expressing cells of a cancer and the TAP-deficient T2 cell line loaded with the appropriate iLR-ASK-A2 peptide.
• Specific CTL lines elicited strong cytolytic activity against allogenic OFA/iLRP expressing HLA-A*0201 + CLL cells but spared HLA-A2-negative / OFA/iLRP-positive targets and HLA-A2-positive / OFA/iLRP- negative targets.
• Antibody blocking experiments revealed an MHC-class I-restricted killing induced by peptide specific CD8 + T lymphocytes.
• the inventors identified a novel modified HLA- A*0201-specific peptide epitope derived from the OFA/iLR protein. This peptide represents a useful tool for both conducting tumor immunological studies and vaccination strategies in OFA/iLRP- expressing malignancies.
• the invention in a further aspect relates to a method of killing target cells in a patient which targets cells expressing a polypeptide comprising an amino acid sequence as given herein or amino acid sequence of its peptide analogues, the method comprising administering to the patient an effective amount of a peptide or peptides according to the present invention wherein the amount of said peptide or peptides is effective to provoke an anti- target cell immune response in said patient.
• the target cells are cancer cells. More particularly, the target cells are cancer cells. More particularly, the target cells are cancer cells. More particularly, the target cells are cancer cells. More
• said cancer is leukemia or lymphoma which expresses the polypeptide which comprises an amino acid sequence as given according to the present invention or amino acid sequence of its peptide analogues.
• Table 1 shows T-cell epitope containing modified peptide SEQ ID No 1 (iLR-ASK-A2) and naturally processed SEQ ID No 2 that are presented by MHC class I according to the present invention.
• Figure 1A shows the binding of the Influenza matrix protein FluMl to the HLA-A2-molecule of the TAP-deficient T2 cell line as a positive control in a T2 binding assay.
• Figure IB shows the strenghtened binding ability of the modified peptide iLR-ASK-A2 to bind to the HLA-A*0201 compared to the binding ability of the naturally processed peptide (SEQ ID No 2) determined by an T2 binding assay.
• Figure 2 shows the capability of the modified peptide iLR- ASK-A2 of elicting INF- ⁇ - and GrB-producing T cells compared to the native peptide determined by an ELISPOT assay .
• Figure 3 shows the lysis of peptide-pulsed TAP (transporter associated with antigen processing) -deficient T2 target cells by iLR-ASK-A2-specific CTL.
• Figure 4 shows that CTL specific for HLA-A* 02 /iLR-ASK-A2 kill tumor cells from HLA-A2-positive CLL patients but spared HLA-A2-negative CLL cells.
• Figure 5 shows the blocking of target cell lysis by antibodies recognizing MHC class I or TCR for iLR-ASK-A2.
• APC antigen presenting cell
• CD Cluster of Differentiation cpm: counts per minute
• DC Dendritic Cell
• HLA Human Leukocyte Antigen
• IF Interferon IL: Interleukin
• PBMC Peripheral Blood Mononuclear Cells
• PBMC peripheral blood mononuclear cells
• TAP-deficient T2 cell line used in this study were obtained from American Type Culture Collection (Manassas, VA, USA) .
• PBMC and tumor samples were collected from patients with chronic lymphocytic leukemia (CLL) , multiple myeloma (MM) and T cell lymphoma respectively, after informed consent and approval by the institutional review board.
• CLL chronic lymphocytic leukemia
• MM multiple myeloma
• T cell lymphoma T cell lymphoma
• the peptides FIuMl 58 _ 66 (GILGFVFTL, positive control in T2 binding assay), HIV-P0I476-84 (ILKEPVHGV, negative control in ELISPOT assay) msurv33 (LYLKNYRIA, murine survivin peptide epitope specific for H2 d , negative control in T2 binding assays and 51 Chromium release assay) ; native 1LR 66 -74 (VAIENPADV, naturally processed OFA/iLR peptide) , iLR-ASK- A2 66 _ 74 (VLIENPADV, modified OFA/iLR peptide) were purchased from peptides & elephants (Berlin, Germany) provided with more than 90% purity and were analyzed by high-performance liquid chromatography and mass spectrometry.
• the T2 whole cell-binding assay was performed using a protocol adopted from Casati et al . (Casati C, Dalerba P, Rivoltini L et al .
• the apoptosis inhibitor protein survivin induces tumor- specific CD8 + and CD4 + T cells in colorectal cancer patients. Cancer Res. 2003; 63, 4507- 4515).
• the influenza matrix protein served as a positive control.
• PBMC form patients with CLL, MM and T- NHL were stimulated with the iLR-ASK-A2 peptide in the presence of IL-2 and IL-7 in the same concentration used for the stimulation of CTL as mentioned above for at least 10 days with one restimulation at day 7.
• ELISPOT-assays were performed as described elsewhere (Siegel, S., Wagner, A., Friedrichs, B., Wendeler, A., Wendel, L., Lucasitz, D., Steinman, J., Barsoum, A., Coggin, J., Rohrer, J., Dreger, P., Schmitz, N. & Zeis, M.
• PBMC from patients with CLL, MM and T-NHL were stimulated with the native iLR66-74 peptide in the presence of IL-2 and IL-7 in the same concentration used for the stimulation of CTL as mentioned above for at least 10 days with one
• Table 1 Peptides and tumor-associated T-helper cell peptide epitopes as identified in the present invention
• iLR-ASK-A2 66 _74 VLIENPADV SEQ ID No. 1 2. native iLR 66 _ 7 4 VAIENPADV SEQ ID No. 2
• msurv33 LYLKNYRIA SEQ ID No. 5 murine survivin peptide epitope specific for H2 d , negative control in T2 binding assays
• T2-binding assay revealed a strenghtened binding of the iLR-ASK-A2 peptide (SEQ ID No.l) to the HLA-A2-molecule compared to the naturally processed iLR peptide (SEQ ID 2) by loading the TAP-deficient T2 cell line with different amonts of peptide determined by flow cytometry (Fig. 1A/B) .
• iLR-ASK-A2 is able to bind the HLA-A*02-molecule 2.
• iLR-ASK-A2 has an increased ability to bind the HLA- A*02-molecule compared to the naturally processes iLR peptide .
• T2 cells are deficient for expression of TAP, the
• T2 cells expressing empty HLA-A*02 molecules on the cell surface are optimal targets for peptide-specif ⁇ c killing by HLA-A* 02- restricted T cells.
• T cells specific for iLR-ASK-A2 were tested on T2 cells pulsed with the appropriate peptide VLIENPADV, with no peptide or the irrelevant murSurv peptide (SEQ ID No. 5) .
• T cells recognized only T2 cells pulsed with the iLR-ASK-A2 peptide. T2 cells pulsed with no peptide or the irrelevant murSurv peptide were not recognized (Fig. 3) .
• Fig. 3 T2 cells pulsed with no peptide or the irrelevant murSurv peptide were not recognized (Fig. 3) .
• iLR-ASK-a2 is capable of elicting OFA/iLRP-specific cytotoxic T-lymphocytes in A*02-positive CLL patients
• T cells specific for HLA-A* 02-restricted iLR-ASK-a2 peptide were tested in a standard 51 Chromium release assay on tumor cells from CLL patients.
• Cells from A*02- positive/OFA/iLRP-positive patient were recognized by the iLR-ASK-A2-specific CTL while A*02-negative/OFA/iLRP- positive as well as A*02-positive/OFA/iLRP-negative
• iLR-ASK-A2 is capable of elicting OFA/iLRP-specific cytotoxic T-lymphocytes in A*02-positive CLL patients
• iLR-ASK-A2 peptide is recognized in an HLA-restricted fashion .
• Fig.5 shows antibody blocking experiments to further characterize the specificity of the T cell response. Prior to 51 Cr-release experiments at different E:T ratio,
• blocking mAbs were incubated with HLA-A*0201- positive/OFA/iLRP-positive CLL cells at mAb concentrations as indicated by the manufacturer.
• the blocking experiments indicate that the recognition of iLR-ASK-A2 is mediated by cells bearing T-cell receptors (TCR) , recognizing their target in the context of MHC class I (HLA class I) .
• TCR T-cell receptors
• Control mAbs specific for irrelevant, HMFG-I did not have an effect on the recognition of CLL cells by the effector T cells.
• PBS was used as a negative control in these blocking experiments .
• iLR-ASK-A2 is capable of elicting OFA/iLRP-specific cytotoxic T-lymphocytes in A*02-positive CLL patients
• iLR-ASK-A2 peptide is recognized in an HLA-restricted fashion .
• the restriction is allele-specific (specific for A*02).
• OFA/iLRP-specific cytotoxic T lymphocytes are able to kill OFA/iLRP expressing allogenic target cells.
• targets and effector cells can be specifically inhibited with mAbs against MHC class I or TCR .
• the inventive modified peptide iLR-ASK-A2 from the OFA/iLR protein is a candidate for developing peptide- based therapeutic vaccines for cancer patients in general.

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