DE10148236A1 - Tumor peptide antigens from human PRDI-BF1 protein - Google Patents

Abstract

The invention relates to tumor-associated oligopeptides which are recognized by CD8-positive cytotoxic T-lymphocytes (ZTL) as peptide antigens and which cause ZTL-induced lysis and / or apoptosis of tumor cells, in particular cells of multiple myeloma. The oligopeptides have amino acid sequences which correspond to partial sequences of the human PRDI-BF1 protein. Each oligopeptide represents an epitope for CD8-positive ZTL and is suitable for a (restricted) immune response of CD8-positive ZTL against multiple- to human leukocyte antigen of the molecular group "MHC class I", allelic variant A2 To induce myeloma cells and other tumor cells.

Description

The invention relates to a tumor-associated oligopeptide that CD8-positive cytotoxic T lymphocytes (ZTL) is recognized as a peptide antigen, and that a ZTL- induced lysis and / or apoptosis of tumor cells, in particular cells of the multiple Causes myeloma.

CD8-positive ZTL represent effector cells of the cellular immune system. Their function consists in the specific elimination of infected or degenerate body's own Lines. The ZTL recognize, among other things, tumor-specific or tumor-associated Peptide antigens (TAA) attached to major histocompatibility complex (MHG) molecules of the Class I bound and presented on the surface of the degenerate cells. The The peptide antigens in the context of MHC class I molecules are recognized by 1 specific membrane-bound T cell receptors (TZR) of the ZTL. After detection the cell in question is killed by the ZTL lysing the target cells and / or the programmed cell death (apoptosis) of these target cells or induce cytokines release.

The detection of target cells by ZTL is supported by the expression of the CD8 Koreceptors relieved on ZTL. The CD8 coreceptor binds to conserved regions of the α2 and α3 domains of the MHC class I molecule and thus contributes to stabilization of the TZR-peptide-MHC complex.

The identification of human, tumor-associated peptide antigens and the generation Tumor-reactive T lymphocytes with specificity for these antigens are essential Requirements for the establishment of immunological strategies in therapy malignant, human tumors, especially of multiple myeloma. So far Treatment methods used on patients with multiple myeloma (plasmacytoma) are not very successful. However, there are increasing clinical and experimental ones Studies suggesting that multiple myeloma is caused by a ZTL-based Immunotherapy can be influenced. As for multiple myeloma immunotherapy only a few universal multiple myeloma-associated peptide antigens are currently described there is still a need to identify peptide antigens that are derived from a protein that in multiple myeloma cells (= malignant plasma cells) in Compared to normal cells is overexpressed.

The PRDI-BF1 protein is involved in the terminal differentiation of B cells that plasma cells and their neoplasms, namely cells of multiple myeloma, a Show overexpression of PRDI-BF 1. For the murine homolog of PRDI-BF1 (Blimp-1) different normal tissues were examined for RNA expression. there was poor expression in bone marrow, spleen and lymphocytes proven, also in the brain, lungs, heart, kidneys, skin and testis. The results Recent research suggests that PRDI-BF 1 also differentiates of myeloid cells is involved.

The oligopeptides resulting from the cellular processing of the PRDI-BF1 protein can in the context of MHC class I molecules of the allele variant A2, subtype A2.1 (short: A2.1; the most common MHC class I allele in the Caucasian population) are presented on the cell surface and represent attractive target structures for CD8 positive ZTL. You can therefore use it as a vaccine under certain conditions for the induction of T cells in general and of tumor-reactive T cells in be used with the aim that these T cells and the remission Cause eradication of multiple myeloma cells. In the case of melanoma Already known some peptide antigens that are used for immunotherapy within clinical trials can be used.

The object of the present invention is tumor-associated, in particular Provide multiple myeloma-associated peptide antigens derived from CD8-positive ZTL are recognized and a ZTL-induced lysis and / or apoptosis of tumor cells, in particular induce multiple myeloma cells.

One solution to this problem is to provide an oligopeptide that (a1) one of the amino acid sequences shown in the sequence listing 1 to 30 has that specified in the relevant sequence listing Corresponds to amino acid positions of the human PRDI-BF1 protein, or the (a2) one by amino acid substitution, deletion, insertion, addition, inversion and / or by chemical or physical modification of one or more amino acids of one of these amino acid sequences has a derivable amino acid sequence, and (b) represents an epitope for CD8-positive ZTL and (c) is suitable for converting one to human Leukocyte antigen of the molecular group "MHC class I", allele variant A2 (short: A2) restricted (restricted) immune response of CD8-positive ZTL against multiple To induce myeloma cells and other tumor cells.

An equivalent solution is to provide one for this oligopeptide analog retro-inverse peptide or pseudopeptide according to the invention, the instead of the -CO-NH peptide bonds non-peptide bonds such. B. -NH-CO bonds (see Meziere et al. 1997).

With the oligopeptides according to sequence protocols 1 to 30 are the first Peptide antigens are provided, the amino acid sequence of which is derived from the human PRDI-BF1 Protein. The PRDI-BF1 oligopeptides according to sequence protocols 1 to 30 and their derivatives represent quantitatively tumor-associated, especially multiple-myeloma Cells associated ZTL epitopes and thus provide the molecular basis for one PRDI-BF1-specific immunotherapy for malignant diseases, especially the Multiple myeloma.

The oligopeptides according to the invention (i.e. those in the sequence protocols 1 to 30 represented oligopeptides and their derivatives) can in particular in the active and passive immunization of multiple myeloma patients in whom this Epitopes corresponding to oligopeptides are presented in the context of A2.1, used to induce, generate and expand cytotoxic To induce T lymphocytes that are relevant for the PRDI-BF1- Amino acid sequences are specific, and are capable of multiple myeloma Specifically kill cells of the patient concerned and thereby cause healing convey.

In the course of the present invention it was found that PRDI-BF1 in resting B- Cells, T cells and in mononuclear cells of the peripheral blood only slightly is expressed and therefore not for killing by PRDI-BF1-specific cells susceptible, d. H. is not a suitable target. For those in the sequence listing 1 up to 30 oligopeptides and their derivatives shown, this gives the advantage of broad indication area with negligible risk of an unwanted Attack on normal cells.

The derivatives of the oligopeptides and / or shown in the sequence listing 1 to 30 the derived retro-inverse peptides or pseudopeptides also have the original oligopeptide itself the advantage that a potential functional self-tolerance (towards the corresponding original Oligopeptides) can be bypassed at the T cell level. While the PRDI-BF1- Oligopeptides according to sequence listing 1 to 30 due to the (low) expression in some normal tissues u. U. in the organism in question (patient's body) represent so-called tolerogen and for the organism's own (patient's own) ZTL are not immunogenic, the derivatives of these oligopeptides are usually used as antigens recognized and induce the activation and expansion of ZTL. This Derivative-induced ZTL usually have a high cross-reactivity towards the / the relevant PRDI-BF1 oligopeptide wild type sequence (s) and induce consequently also the lysis and / or apoptosis of such (tumor) cells that PRDI- BF1 oligopeptides according to sequence listing 1 to 30 (in the context of A2, present in particular of A2.1) on their surface.

Particularly preferred derivatives of those shown in sequence listing 1 to 30 Oligopeptides are those that are naturally found in other mammals or vertebrates occur, e.g. B. corresponding homologues from the mouse. This PRDI-BF1- Oligopeptide homologs and the nucleic acids encoding them can be relatively easily, namely directly and with common isolation procedures, from the respective organism be won.

The PRDI-BF1 oligopeptides shown in the sequence protocols 1 to 30 and their Derivatives as well as the retro-inverse peptides or pseudopeptides can be used by means of common Peptide synthesis methods are prepared, and the coding for these oligopeptides Nucleotide sequences can be with known chemical or with molecular biological Procedures can be obtained.

There is also the possibility of the invention described above Oligopeptides, a flexible linker and a heavy chain of the HLA molecule To construct fusion protein in such a way that the oligopeptide enables (in capable or suitable) to occupy the peptide binding groove of the HLA molecule. These fusion proteins and polynucleotides encoding them are particularly suitable as (Active ingredient of a) diagnostic or therapeutic or prophylactic or generally for the detection and / or manipulation of T cells that are one of those in the Sequence listings 1 to 30, in particular 1-4 PRDI-BF1 oligopeptides shown detect. The invention therefore also relates to a fusion protein which consists of one of the Oligopeptides described above, from a heavy chain of the HLA molecule and consists of a flexible linker and is constructed such that the oligopeptide suitable (capable), the peptide binding groove of the HLA molecule to occupy, and which for use or for use as a diagnostic or Therapeutic or prophylactic or generally for detection and / or Manipulation of T cells that one of the in the sequence protocols 1 to 30, recognize in particular 1-4 PRDI-BF1 oligopetides shown is suitable. The for polynucleotides encoding this fusion protein are also the subject of the present invention Invention.

The oligopeptides and their derivatives shown in sequence listing 1 to 30 as well as the retro-inverse peptides or pseudopeptides and the fusion proteins are suitable for in vivo induction of T lymphocytes in patients as well as for in vitro Induction and expansion according to reactive patient's own or non-patient's T-lymphocytes.

Come for an in vivo induction and expansion of T lymphocytes in the patient various methods are considered, for example (a) the injection of one of the in the Sequence listing 1 to 30 PRDI-BF1 oligopeptides and / or one or several derivatives of these oligopeptides and / or a retro-inverse peptide or Pseudopeptide and / or a fusion protein as a pure peptide or together with Adjuvantieri or with cytokines or in a suitable release system such. B. Liposomes, (b) the injection of a nucleic acid necessary for one of those in the Sequence listing 1 to 30 PRDI-BF1 oligopeptides shown or their derivatives and / or for one of the retro-inverse peptides or pseudopeptides and / or for one of the Fusion proteins encoded - in "naked" or complexed form or in the form of viral or non-viral vectors or together with release systems such as cationic Lipids or cationic polymers, (c) autologous cell loading; allogeneic, xenogenic or microbiological origin with one or more of those in the Sequence listing 1 to 30 PRDI-BF1 oligopeptides shown or their derivatives or retro-inverse peptides or pseudopeptides analogous thereto, (d) the loading of Cells with autologous, allogeneic, xenogeneic or microbiological origin at least one of the PRDI-BF1- shown in the sequence listing 1 to 30 Oligopeptides or homologues or retro-inverse peptides or pseudopeptides other species, so that as a result the relevant PRDI-BFl oligopeptide (s) and / or its derivative (s) and / or or the retro-inverse peptide (s) or Pseudopeptide (s) is / are presented on the respective cells, or (e) the Transfection or infection of autologous, allogeneic, xenogeneic or cells microbiological origin with at least one of the in the Sequence listing 1 to 30 PRDI-BF1 oligopeptides shown or their derivatives or retro-inverse peptide or pseudopeptide or fusion protein derived therefrom coding nucleic acids (again either in "naked" or complex form or in the form of viral or non-viral vectors).

In the case of in vitro induction and expansion, the in vitro obtained T- Lymphocytes are then given to the patient by infusion or injection or the like fed.

The invention therefore also relates to the use of the in the sequence listing 1 to 30 shown PRDI-BF1 oligopeptides and / or their derivatives and / or to it analog retro-inverse peptides or pseudopeptides and / or fusion proteins and / or of a polynucleotide which is used for at least one of the sequence protocols 1 to 30 PRDI-BF1-oligopeptides encoded, for the production of diagnostics - in particular MHC tetramers or MHC dimers or other structures to which at least one such oligopeptide or retro-inverse peptide according to the invention or Pseudopeptide is associated by covalent or non-covalent binding - and / or Prophylactic and / or therapeutic agents (especially vaccines) for detection and / or influencing and / or generation and / or expansion and / or Control of the activation and functional status of T cells, in particular CD8 positive ZTL.

Vaccines or in particular come as therapeutic agents and / or prophylactic agents Injections or infusion solutions that are considered as active ingredient (a) one of the in the Sequence listing 1 to 30 PRDI-BF1 oligopeptides shown and / or at least a derivative of one of these oligopeptides and / or at least one of these Oligopeptides or their inverse analogue retro-inverse peptide or pseudopeptide and / or contain at least one of the fusion proteins described above, and / or which (b) contain a nucleic acid which is at least for one of the in the Sequence listing 1 to 30 PRDI-BF1 oligopeptides shown or at least for encodes a derivative of one of these oligopeptides, and / or the (c) T- generated in vitro Lymphocytes that are specific against one of the sequence protocols 1 to 30 shown PRDI-BF1 oligopeptides and / or their derivative (s) and / or against one one of these oligopeptides or a derivative of these oligopeptides analog retroinverse peptide or pseudopeptide are included.

For the production of diagnostics or therapeutic agents or Prophylactic drugs are also particularly suitable for recombinant DNA or RNA vector molecules containing one or more polynucleotide (s) which are suitable for at least one of the PRDI-BFI- shown in sequence listing 1 to 30 Code oligopeptides and / or for at least one derivative of one of these oligopeptides, and those of cells of autologous, allogeneic, xenogeneic or microbiological origin are transcribable or expressible. The invention therefore also includes such recombinant DNA or RNA vector molecules and host cells carrying them Contain vector molecules.

As a diagnostic or therapeutic or prophylactic or in general for one Detection and / or manipulation of cells that one of the in the sequence listing 1 to overexpress the PRDI-BF1 oligopeptides shown can be according to the invention polyclonal, monoclonal or recombinant antibodies can also be used against at least one of the PRDI-BF1- shown in the sequence listing 1 to 30 Oligopeptides and / or against at least one derivative of this oligopeptide and / or against at least one retro-inverse peptide analogous to this oligopeptide or its derivative or pseudopeptide or fusion protein, or which are complexed one of the oligopeptides in question or its derivatives or retro-inverse to it Peptide (s) and / or pseudopeptide (s) and HLA-A2 react.

The use of one of the PRDI-BF1- shown in the sequence protocols 1 to 30 Oligopeptides and / or a derivative of these oligopeptides and / or one to one of these oligopeptides or a retro-inverse analogue to a derivative of these oligopeptides Peptide or pseudopeptide or fusion protein for the production of polyclonal, monoclonal or recombinant antibody against such an inventive Oligopeptide or retro-inverse peptide or pseudopeptide and the relevant one (s) Antibodies per se are therefore also part of the present invention.

• - die Verwendung wenigstens eines der in den Sequenzprotokollen 1 bis 30 dargestellten PRDI-BF1-Oligopeptide und/oder Derivate eines dieser Oligopeptide und/oder dazu analoger retro-inverser Peptide oder Pseudopeptide oder die Verwendung von Polynukleotiden mit einer Nukleotidsequenz, die mindestens für eines der in den Sequenzprotokollen 1 bis 30 dargestellten PRDI-BF1-Oligopeptide und/oder ein Derivat dieser Oligopeptide kodiert, zur Herstellung polyklonaler, monoklonaler oder rekombinanter A2-restringierter T-Zellrezeptoren oder dazu funktionell äquivalenter Moleküle mit Spezifität für ein solches erfindungsgemäßes Oligopeptid bzw. retro-inverses Peptid oder Pseudopeptid,
• - der/die betreffende(n) T-Zellrezeptor(en) an sich und dazu funktionell äquivalente Moleküle,
• - sowie Polynukleotide, die für diese T-Zellrezeptoren oder dazu funktionell äquivalenten Moleküle kodieren,
• - Expressionsvektoren mit der Fähigkeit zur Expression dieser T-Zellrezeptoren oder dazu funktionell äquivalenten Moleküle.

According to the invention, polyclonal, monoclonal or recombinant A2-restricted T-cell receptors can also be used as a diagnostic or therapeutic or prophylactic or in general for the detection and / or manipulation of cells which overexpress at least one of the PRDI-BF1 oligopeptides shown in sequence protocols 1 to 30 or for this purpose functionally equivalent molecules are used which are specific for one of the PRDI-BF1 oligopeptides shown in sequence protocols 1 to 30 and / or for a derivative of one of these oligopeptides and / or for analogous retro-inverse peptides or pseudopeptides or for one are described fusion protein. The T cell receptors or molecules functionally equivalent to them can be of autologous, allogeneic or xenogeneic origin. The subject matter of the present invention therefore also primarily includes:

• - The use of at least one of the PRDI-BF1 oligopeptides and / or derivatives of one of these oligopeptides and / or derivatives of these oligopeptides and / or retro-inverse peptides or pseudopeptides analogous to that shown in the sequence listing 1 to 30 or the use of polynucleotides with a nucleotide sequence which is at least for one of the encoded in sequence protocols 1 to 30 PRDI-BF1 oligopeptides and / or a derivative of these oligopeptides, for the production of polyclonal, monoclonal or recombinant A2-restricted T cell receptors or functionally equivalent molecules with specificity for such an inventive oligopeptide or retro- inverse peptide or pseudopeptide,
• - the relevant T cell receptor (s) per se and functionally equivalent molecules,
• polynucleotides which code for these T cell receptors or molecules which are functionally equivalent thereto,
• - Expression vectors with the ability to express these T cell receptors or their functionally equivalent molecules.

The invention also includes reagents for in vivo or in vitro activation of T cells, in particular CD8-positive ZTL, which are characterized in that they using at least one of those shown in Sequence Listing 1 to 30 PRDI-BF1 oligopeptides and / or at least one derivative of one of these oligopetides and / or at least one retro-inverse peptide or pseudopeptide analogous thereto or fusion protein and / or using at least one polynucleotide which at least for one of the oligopeptides shown in the sequence listing 1 to 30 or their derivative (s) and / or retro-inverse peptides analogous thereto or Pseudopeptides or fusion proteins encoded and / or using at least one of the PRDI-BF1 oligopeptides shown in sequence protocols 1 to 30 or homologues of other species are produced. These reagents can especially therapeutic agents, and above all vaccines.

In the following the invention is based on manufacturing and application examples explained in more detail with figures.

The abbreviations used mean

A2: human leukocyte antigen of the molecular group "MHC class I", allele variant "A2"
A2.1: human leukocyte antigen of the molecular group "MHC class I", allele variant "A2", subtype "A2.1"
A2K ^b : A2.1 / K ^b = MHC class I molecule from α ₁ and α ₂ domains of A2 and α ₃ domains of K ^b
APZ: antigen-presenting cell
ATCC: American Type Culture Collection
ATP: adenosine 5'-triphosphate
bkgd: unspecific fluorescence intensity
Blimp-1: B lymphocyte induced maturation protein 1
bp: base pairs
PRDI-BF1: positive regulatory domain I binding factor 1
BSA: bovine serum albumin
C-terminal: carboxyl-terminal
CD: Differentiation Cluster
CD8: human CDBα / β coreceptor
CDR: complementarity determining region
cDNA: complementary DNA
CMV: Cytomegalovirus
Con A: Concanavalin A
DMSO: dimethyl sulfoxide
DNA: deoxyribonucleic acid
dNTP: deoxynucleoside triphosphate
DSMZ: German collection of microorganisms and cell cultures
DTT: dithiothreitol
DZ: dendritic cells
E: T: effector to target cell ratio
EBV: Epstein-Barr virus
EDTA: ethylenediaminetetraacetate
ER: endoplasmic reticulum
EtBr: ethidium bromide
FACS: fluorescence-activated cell sorter
FCS: fetal calf serum
FITC: fluorescein isothiocyanate
Flu M1: A / PR / 8/34 influenza virus matrix protein M1
G-418: Geneticin (neomycin antibiotic)
GM-CSF: Granulocyte-macrophage-colony stimulating factor
HBV pol: hepatitis B virus polymerase
HEPES: N- (2-hydroxyethyl) piperizan N'-ethanesulfonic acid
HLA: human leukocyte antigen
HLA-A2.1 human leukocyte antigen of the molecular group "MHC class I", allele variant "A2", subtype "A2.1"
IFA: incomplete Freund's adjuvant
IFN: interferon
Ig: immunoglobulin
IL: Interleukin
kb: kilobase pairs
K ^b : H-2K ^b
kDa: kilo Dalton
LCL: lymphoblastoid cell line
LPS: lipopolysaccharide
MHC: major histocompatibility complex
Mio: million
MM: Multiple myeloma
courage: mutated
N-terminal: amino terminal
OD: optical density
PBMC: peripheral blood mononuclear cells
PBS: phosphate buffered saline
PCR: polymerase chain reaction
PG-E ₂ : prostaglandin E ₂
Rad: radiation absorbed dose
RNA: ribonucleic acid
RT: reverse transcriptase
SL: specific lysis
TAA: tumor-associated antigen (s)
TAP: Transporter associated with antigen processing
Tg: transgene
TIL: Tumor-infiltrating lymphocytes
TNF-α: tumor necrosis factor-α
Tris: tris (hydroxymethyl) aminomethane
TZR: T cell receptor
u: international units
Rpm: revolutions per minute
VSV-N: Vesicular stomatitis virus nucleoprotein
v / v: volume per volume
wt: wild type
w / v: mass per volume
ZTL: cytotoxic T lymphocytes

Abbreviations for amino acids

A: Alanine
C: cysteine
D: aspartate
E: glutamate
F: phenylalanine
G: glycine
H: histidine
I: isoleucine
K: Lysine
L: leucine
M: methionine
N: asparagine
P: Proline
Q: Glutamine
R: arginine
S: serine
T: Threonine
V: Valine
W: tryptophan
Y: tyrosine

The figures show

Fig. 1 binding selected synthetic PRDI-BF1 peptides. The relative A2.1 binding affinity (expressed as% inhibition) was determined by the ability of the respective peptide to inhibit the A2.1 binding of peptide p53 264-272. This was measured based on the inhibition of p53-specific CTL lysis of p53 264-272-loaded T2 target cells by PRDI-BF 1 peptides of different concentrations. The inhibition values for the peptides FluM1 and VSV-N were averaged from 6 independent experiments.

Fig. 2 A2.1-restricted immunogenicity of synthetic PRDI-BFI peptides in A2K ^b or CD8 × A2 (K ^b ) -tg mice. The immunogenicity was checked on the basis of the lytic activity of the CTL-induced in these mice by peptide immunization in a 4-hour cytotoxicity test. The target cells used were 0.2Ag peptide-loaded or unloaded T2A2K ^b cells or T2 cells (for checking the immunogenicity of PRDI-BFI 377-386). Representative specific lyses of individual ZTL cultures from an average of 2 immunized mice are shown.

Fig. 3 H-2 ^b -restricted immunogenicity of synthetic peptides BF1 ^PRDI-b in A2K or CD8 × A2 (K ^b) TG mice. The immunogenicity was checked on the basis of the lytic activity of the CTL-induced in these mice by peptide immunization in a 4-hour cytotoxicity test. EL-4 cells loaded or unloaded with 0.2 μg of peptide were used as target cells. Representative specific lyses of individual ZTL cultures from an average of 2 immunized mice are shown.

Fig. 4 peptide specificity and A2.1 restriction of the ZTL lines CD8 × A2K ^b 377 (A), CD8 × A2K ^b 402p (B) and CD8 × A2K ^b 401 (C) by repeated in vitro stimulation with the respective relevant peptide were established. To check the peptide specificity, T2 cells with FluM1 58-66 peptide (♦), the relevant peptide: PRDI-BFI 377-386 in (A), PRDI-BF1 402-410 in (B) and PRDI-BFI 401- 410 in (C) (●), as well as PRDI-BF I 402-410A in (B) or PRDI-BF1 402-410 in (C) (▪) or no peptide (○) and loaded in a 6-hour ⁵¹ Cr release test used as target cells at the specified E: T ratios. To check the restriction, peptide-loaded (▴) and unloaded (▵) EL-4 cells were used.

Fig. 5 CTL lines CD8 × A2K ^b 377 (A), CD8 × A2K ^b 402p (B) and CD8 × A2K ^b 401 (C): efficiency of peptide recognition. For this purpose, T2 cells with the specified peptide concentrations of PRDI-BF1 377-386 in (A), PRDI-BF1 402-410 in (B) and PRDI-BF1 401-410 in (C) (●), as well as PRDI-BF1 402 -410A in (8) or PRDI-BF1 402-410 in (C) (▪) and as target cells in a 4 (B, C) and 6 (A) hourly cytotoxicity test with an E: T ratio of 10: 1 tested.

Fig. 6 PRDI-BF1 RNA expression from multiple myeloma cell lines. Cytoplasmic RNA from the multiple myeloma cell lines L363, MC-CAR, U266, IM9, OPM-2 and NCI-H929 was reverse transcribed in cDNA and amplified with PRDI-BF1 or β-actin-specific primers in a subsequent PCR reaction. The PCR products were separated electrophoretically in a 1.6% agarose gel containing EtBr. EL-4 acted as a negative control. The arrows mark the 390 bp (β-actin PCR amplificate) and the 540 bp gel band (PRDI-BF1 PCR amplificate).

Fig. 7 PRDI-BF1 RNA expression from the osteosarcoma cell lines Saos-2 and U2OS. Cytoplasmic RNA from Saos-2 and U2OS was reverse transcribed in cDNA and amplified with PRDI-BF1 or β-actin-specific primers in a subsequent PCR reaction. The PCR products were separated electrophoretically in a 1.6% agarose gel containing EtBr. EL-4 acted as a negative control, L363 as a positive control. The arrows mark the 390 bp (β-actin PCR amplificate) and the 540 bp gel band (PRDI-BF1 PCR amplificate).

Fig. 8 ZTL detection of PRDI-BF1 overexpressing plasmacytoma cell lines. ZTL CD8 × A2K ^b 402 p, CD8 × A2K ^b 401 and CD8 × A2K ^b 377 were used as effector cells under the specified E: T ratios in a 6-hour cytotoxicity test against the A2.1-positive target cells Saos-2 (○), U266 (●), OPM-2 (▪), MC-CAR (▴) and the A2.1 negative target cell NCI-H929 (▵) tested. The ZTL lines CD8 allo A2 and CD8 × A2K ^b FluM1 served as controls.

Fig. 9 PRDI-BF1 RNA expression from normal cells. Cytoplasmic RNA from monocytes, T cells and B cells was reverse transcribed in cDNA and amplified with PRDI, BFI or β-actin-specific primers in a subsequent PCR reaction. The PCR products were separated electrophoretically in a 1.6% agarose gel containing EtBr. EL-4 acted as a negative control, L363 and NCI-H929 as a positive control. The arrows mark the 390 bp (β-actin PCR amplificate) and the 540 bp gel band (PRDI-BF1 PCR amplificate).

Fig. 10 Lack of substantial recognition of resting lymphohemopoietic cells. ZTL CD8 × A2K ^b 402p (A), CD8 × A2K ^b 401 (B), CD8 × A2K ^b 377 (C) were used as effector cells in a 6-hour ⁵¹ Cr release test at the given E: T ratios against resting B- Cells (○) and the same cells loaded with PRDI-BFI 402-410 (●) in (A, B, D, E) and PRDI-BF1 377-386 (▪) tested in (C, D, E). The ZTL lines CD8 × A2K ^b FluM1 (D) and CD8 allo A2 (E) served as controls.

Fig. 11 Lack of substantial recognition of resting lymphohemopoietic cells. ZTL CD8 × A2K ^b 402p (A), CD8 × A2K ^b 401 (B), CD8 × A2K ^b 377 (C) were used as effector cells in a 6-hour ⁵¹ Cr release test at the given E: T ratios against resting T- Cells (○) and the same cells loaded with PRDI-BFI 402-410 (●) in (A, B, D, E) and PRDI-BF1 377-386 (▪) tested in (C, D, E). The ZTL lines CD8 × A2K ^b FIuM1 (D) and CD8 allo A2 (E) served as controls.

Fig. 12 Lack of substantial recognition of activated mature dendritic cells (DZ). ZTL CD8 × A2K ^b 402p (A), CD8 × A2K ^b 377 (B) were loaded as effector cells in a 6-hour ⁵¹ Cr release test at the indicated E: T ratios against activated mature DC (○) and the same cells PRDI-BF1 402-410 (●) tested in (A, C and D) and PRDI-BF1 377-386 (▪) in (B, C and D). The ZTL lines CD8 × A2K ^b FluM1 (C) and CD8 from A2 (D) served as controls.

A) Materials mentioned in the examples (1) mice

• 1. A2.1/K^b (A2K^b)-Tg Mäuse - sie sind homozygot für ein chimäres MHC Klasse I- Tg, das sich aus den humanen α₁- und α₂-Domänen von A2.1 und aus der α₃- Domäne von H-2K^b der Maus zusammensetzt, sowie für das H-2^b-Gen.
• 2. huCD8α/β (CD8)-Tg Mäuse - sie sind homozygot für die α- und β-Kette des humanen CD8-Korezeptors.
• 3. [huCD8α/β × A2.1/K^b]_F1 (CD8 × A2K^b)-Tg Mäuse - sie exprimieren heterozygot das chimäre A2K^b-Molekül und zusätzlich die α- und β-Kette des humanen CD8. Außerdem sind sie homozygot für H-2^b.
• 4. A2.1-Tg Mäuse (([A2.1 × C57BL/6] × C57BL/6)_F1-Tg) - sie exprimieren die α₁-, α₂- und α₃-Domänen des humanen A2.1-Moleküls heterozygot und sind homozygot für H-2^b.
• 5. C57BL/6-Mäuse - sie besitzen den H-2^b-Phänotyp.

Transgenic (Tg) mice expressing the human MHC class I Tg HLA-A2.1 (A2.1) were crossed into the C57BL / 6 background using standard methods (Irwin et al., 1989). The following strains were used for this:

• 1. A2.1 / K ^b (A2K ^b ) -Tg mice - they are homozygous for a chimeric MHC class I Tg, which is derived from the human α ₁ and α ₂ domains of A2.1 and from the α ₃ - Domain of H-2K ^{b composed of} the mouse, as well as for the H-2 ^b gene.
• 2. huCD8α / β (CD8) -Tg mice - they are homozygous for the α and β chain of the human CD8 coreceptor.
• 3. [huCD8α / β × A2.1 / K ^b ] _F1 (CD8 × A2K ^b ) -Tg mice - they heterozygously express the chimeric A2K ^b molecule and additionally the α and β chain of human CD8. They are also homozygous for H-2 ^b .
• 4. A2.1-Tg mice (([A2.1 × C57BL / 6] × C57BL / 6) _F1 -Tg) - they express the α ₁ -, α ₂ - and α ₃ domains of human A2.1- Molecule heterozygous and are homozygous for H-2 ^b .
• 5. C57BL / 6 mice - they have the H-2 ^b phenotype.

(2) Synthetic peptides

Synthetic peptides were obtained from SNPE (Neosystem laboratoire, Strasbourg, France). The purity of the synthesized peptides was at least 75%, that of the peptides PRDI-BF1 401 and PRDI-BF1 402 at least 97%. The purity and correct amino acid composition of all peptides was checked by HPLC analysis and mass spectrometry. The peptides were dissolved in DMSO or H ₂ O at 10 mg / ml. The storage took place in aliquots at -20 to -80 ° C. In addition, a peptide representing residues 128-140 of the hepatitis B virus core protein (TPPAYRPPNAPIL) was synthesized.

(3) Antibodies

For the HLA typing of tumor cell lines and of A2-Tg mice, that of the Mouse hybridoma line BB7.2 (ATCC HB-82) produced monoclonal antibodies used.

For the detection of mouse monoclonal antibodies in flow cytometry, a FITC-conjugated polyclonal secondary antibody (goat anti-mouse IgG F (ab) ₂ fragment; 1:30 dilution; Jackson [Dianova], Hamburg) was used.

(4) cells, cell lines and transfectants

All cells and cell lines were in RPMI 1640 (Biowhittaker, Verviers, Belgium) in the presence of 10% heat inactivated (30 min. 56 ° C) FCS (PAA Laboratories, Linz, Austria), 1% 0.2 M L-glutamine (Biowhittaker ) and 50 µg / ml gentamycin (Gibco BRL, Eggenstein). For the propagation of cells and ZTL lines from the mouse, β-mercaptoethanol was additionally added to the medium in a final concentration of 5 × 10 ^-5 M. For the cultivation of neomycin-transfected cells, the medium Geneticin (G-418) (Gibco BRL) was added in an effective concentration of 280-560 µg / ml. All cells were cultivated at 37 ° C. and 5% CO ₂ in a water vapor-saturated atmosphere in cell culture bottles or 24-hole plates (ZTL) (Corning Costar, Bodenheim).

(4.1) cells

To obtain peripheral blood mononuclear cells (PBMZ) the blood of a healthy A2-positive donor with PBS (Biowhittaker, Walkersville, MA) diluted 1: 3 and with the same volume of Ficoll (Seromed Biochrom, Berlin) underlayed. After centrifugation (1400 rpm, 21 ° C, 20 min) the PBMZ were isolated from the interphase and washed.

The resting T and B cells were obtained after negative selection of A2-positive PBMZ with antibody-coated "beads" (Dynal, Hamburg). For the isolation of T cells, the PBMZ were incubated according to the manufacturer's instructions with anti-CD19 and anti-CD14 "beads", for the isolation of B cells with anti-CD2 and anti-CD14 "beads" and for isolation of monocytes with anti-CD2 and anti-CD19 beads. Dendritic cells (DC) were generated using standard methods from PBMC from an A2 positive donor. After incubating the PBMZ for 45 min at 37 ° C. in a 6-well plate (Corning Costar, Bodenheim), non-adherent cells were rinsed off and the adherent PBMZ in X-Vivo 15 (Biowhittaker, Verviers, Belgium), which was taken up with 1.5% autologous heat-inactivated plasma, 1000 U / ml IL-4 (PBH Strathmann Biotech, Hanover) and 800 U / ml GM-CSF ("Leucomax"; Sandoz, Nuremberg) was supplemented (Jonuleit et al., 1997). On days 3 and 5 there was a partial change of medium with the addition of 1000 U / ml IL-4 and 1600 U / ml GM-CSF, but without autologous plasma. The adherent PBMZ differentiated to non-adherent "Dendriphagen". On day 7, these immature DCs were sown in X-Vivo 15 with 1.5% autologous plasma and with 500 U / ml IL-4, 800 U / ml GM-CSF, 10 ng / ml TNF-α (Genzyme, Cambridge, MA), 10 ng / ml IL-1β (PBH Strathmann Biotech), 1000 U / ml 1L-6 (PBH Strathmann Biotech) and 1 µg / ml PG-E ₂ ("Minprostin E2"; Pharmacia Biotech, Freiburg) treated ( Jonuleit et al., 1997). The mature DCs expressed HLA-DR, CD58, CD80, CD83 and CD86 on days 9 and 10.

All of the cells listed served as target cells in the cytotoxicity test ("ZTL- Recognition").

(4.2) Cell lines and transfectants

• - die humane A2.1-positive T2-Zellinie ist ein BIT-Zellhybridom der Fusionspartner 721.147 und CEM (Salter und Cresswell, 1986),
• - T2-Zellen, die gemäß Theobald et al., 1995, mit dem A2K^b-Gen transfiziert wurden (T2A2^b),
• - die Thymom-Zellinie EL-4 aus der C57BL/6-Maus (Theobald et al., 1995),
• - die humane T-Zell Leukämie-Linie Jurkat (Theobald et al., 1995),
• - Jurkat-Zellen, die mit A2.1 transfiziert waren (JA2) (Theobald et al., 1995),
• - die konstitutiv A2.1-positive und p53-defektmutante Osteosarkom-Zellinie Saos-2 (Dittmer et al., 1993)
• - die Osteosarkom-Zellinie U2OS (HTB-96, ATCC, VA, USA)
• - die A2-positive Zellinie Plasmozytom OPM-2 (DSM ACC 50; DSMZ, Braunschweig, Deutschland)
• - die A2-positive Zellinie Plasmozytom U266 (TIB-196; ATCC, VA, USA)
• - die A2-positive Zellinie Plasmozytom MC-CAR (CRL-8083; ATCC, VA, USA)
• - die A2-positive Zellinie Plasmozytom L363 (DSM ACC 49; DSMZ, Braunschweig)
• - die A2-positive Zellinie Plasmozytom IM9 (CCL-159; ATCC, VA, USA)
• - die A2-negative Zellinie Plasmozytom NCI-H929 (CRL-9068, ATCC, VA, USA)

For the investigations described here, the cell lines listed below, manufactured according to (4.1) or known in the prior art and available at any time:

• the human A2.1 positive T2 cell line is a BIT cell hybridoma of the fusion partners 721.147 and CEM (Salter and Cresswell, 1986),
• T2 cells transfected with the A2K ^b gene according to Theobald et al., 1995 (T2A2 ^b ),
• the thymoma cell line EL-4 from the C57BL / 6 mouse (Theobald et al., 1995),
• the Jurkat human T-cell leukemia line (Theobald et al., 1995),
• Jurkat cells transfected with A2.1 (JA2) (Theobald et al., 1995),
• - the constitutive A2.1-positive and p53-defective mutant osteosarcoma cell line Saos-2 (Dittmer et al., 1993)
• - the osteosarcoma cell line U2OS (HTB-96, ATCC, VA, USA)
• - the A2-positive cell line plasmacytoma OPM-2 (DSM ACC 50; DSMZ, Braunschweig, Germany)
• - the A2-positive cell line plasmacytoma U266 (TIB-196; ATCC, VA, USA)
• - the A2-positive cell line plasmacytoma MC-CAR (CRL-8083; ATCC, VA, USA)
• - the A2-positive cell line plasmacytoma L363 (DSM ACC 49; DSMZ, Braunschweig)
• - the A2-positive cell line plasmacytoma IM9 (CCL-159; ATCC, VA, USA)
• - the A2-negative cell line plasmacytoma NCI-H929 (CRL-9068, ATCC, VA, USA)

All of the cells listed served as target cells in the cytotoxicity test.

B) Methods used in the examples (1) Flow cytometry

A2.1 expression of cells and cell lines was measured in the fluorescence-activated cell sorter (FACS) (Becton Dickinson, San Jose, CA). In each case 0.5 million cells were centrifuged off and labeled with the anti-A2.1 monoclonal antibody BB7.2 (or RPMI 1640, 10% FCS, see 2.4) in a volume of 50 μl (Lustgarten et al., 1997). After incubation on ice for one hour, the batches were washed twice with PBS (Biowhittaker, Walkersville, MA) and the cells were then washed with a FITC-conjugated secondary antibody (goat anti-mouse IgG F _ab fragment; 50 μl of a 1:30 dilution counterstained in PBS). After 25 min incubation on ice, the samples were washed twice with PBS and finally fixed in PBS and 1% formalin. The fluorescence activity of the cell populations selected in the forward scattered light was determined in the FACS.

(2) Determination of the peptide binding affinity for HLA-A2.1

A competition test was used to determine the binding of the PRDI-BF1 peptides to A2.1. T2 cells were loaded with 0.01 or 0.003 µg of the A2.1-binding peptide p53 264-272 (Theobald et al., 1995) and 3 or 10 µg PRDI-BF1 peptide. The A2.1-binding peptide 58-66 of the A / PR / 8/34 influenza virus matrix protein M1 (Flu M1 58-66) (Theobald et al, 1995) served as a positive control, the H-2K ^b -binding peptide 52-59 of Vesicular Stomatitis Virus nucleoprotein (VSV-N 52-59) (Theobald et al., 1995) as a negative control. The A2.1-restricted and p53 264-272-specific ZTL A2 264 clone 46 were tested at various effector to target cell (E: T) ratios for their lytic activity against peptide-loaded and unloaded T2 target cells in a 4-hour cytotoxicity test (see Chapter B 6) (Theobald et al., 1995). The percentage inhibition of the ZTL A2 264-mediated specific lysis (SL) of p53 264-272-loaded T2 cells by the test peptides was calculated at an E: T ratio of 3: 1 using the following formula:

(3) Immunization of A2.1-Tg mice and induction of peptide-specific as well alloreactive ZTL

To generate A2.1-restricted peptide-specific ZTL, 8 µg-old A2.1-Tg mice were given 100 µg of the respective test peptide and 120 µg HBV core 128-140 (an IA ^b -binding synthetic T-helper peptide) ( Theobald et al., 1995), emulsified in 100 ul incomplete Freund's adjuvant (IFA Difco Laboratories, Detroit, USA), injected subcutaneously into the tail (Theobald et al., 1995). After about 10 days, the spleen was removed, ground and the spleen cell suspension washed twice (1500 rpm, 5 ° C., 7 min). The spleen cells were sown at 7 million / ml / well in a 24-well plate. LPS-activated B-cell blasts irradiated with 3000 Rad ( ¹³² cesium), loaded with 5 µg / ml of the respective test peptide and 10 µg / ml human β ₂ -microglobulin, were washed as stimulator cells after washing twice at 3 million / ml / well added (Theobald et al., 1995). The LPS blasts were obtained by stimulating spleen cells (1 million / ml) from A2.1-Tg mice with 25 µg / ml LPS (Salmonella typhosa) and 7 µg / ml dextran sulfate (Pharmacia Biotech, Freiburg) for three days. The effector and stimulator cell batches were incubated for 6 days (I ° cultures) and subjected to a cytotoxicity test.

(4) Establishment of ZTL lines

Polyclonal peptide-specific ZTL lines with specificity for PRDI-BF1 377-386; 402-410 and 401-410 (ZTL CD8 × A2K ^b 377, CD8 × A2K ^b 402p, CD8 × A2K ^b 401) and for Flu M1 58-66 (ZTL CD8 × A2K ^b Flu M1) were determined by weekly restimulation of the effector cells with peptide-loaded stimulator cells established. JA2 cells, which were irradiated with 20,000 rads, were used as stimulator cells, then loaded in RPMI 1640 (Biowhittaker, Verviers, Belgium) with 5 μg / ml of the respective peptide and 10 μg / ml human β ₂ -microglobulin for about 30 min and finally twice were washed. The effector cells were sown together with 0.5 million JA2 cells and 6 million C57BL / 6 spleen cells irradiated with 3000 Rad in a total volume of 2 ml / hole in a 24-hole plate. 2-5% (v / v) supernatant from the culture medium Con A-activated spleen cells (TCGF) from Lewis rats was added to the batches (Theobald et al., 1995).

Allo-A2.1-reactive CTL lines were determined by intraperitoneal immunization of CD8- Tg mice induced with 20 million JA2 cells / mouse. After three weeks, the Spleen cells isolated and in vitro (7 million / ml / well) with irradiated JA2 cells (0.5 million / ml / hole) or spleen cells (6 million / ml hole) A2.1-Tg mice stimulated. By repeated weekly in vitro restimulation with JA2 cells in the presence of irradiated C57BL / 6 spleen cells (6 million / ml / well) and 2-5% TCGF were eventually allo-A2.1-reactive ZTL lines generated.

(5) RT-PCR a) Isolation of cytoplasmic RNA

Cytoplasmic RNA has been identified Instructions of the manufacturer with the RNeasy Mini Kit ("RNeasy Mini Protocol for the Isolation of RNA from the Cytoplasm of Animal Cells ", Quiagen, Hilden) Cells and cell lines isolated.

b) RNA determination

The RNA concentration was measured photometrically the absorption (OD) at a wavelength of 260 nm.

c) reverse transcription

In the reverse transcription (RT) reaction, 2 µg RNA with an oligo-dT-V primer (5'-TTT TTT TTT TTT TTT TTT (AGC) -3 ') and 20U AMV reverse transcriptase (Roche) in the presence of 2U RNase Inhibitor (Roche) and 1 mM deoxynucleotide triphosphates (dNTPs) (Sigma,) in 1 × cDNA synthesis buffer (Roche) rewritten into single-strand cDNA. The RT Reaction took place in a thermal cycler (Master Cycler gradient 5331, Eppendorf) at 55 ° C for 60 min and then 10 min at 65 ° C. The purification of the cDNA products were then processed using the High Pure PCR Product Purification Kit (Roche) according to the manufacturer's instructions.

d) PCR reaction

One tenth of the purified cDNA was treated with PRDI-BF1 specific primers (forward primer: 5'-CAC ACG GGA GAA AAG CCA CAT- 3 'and reverse primer: 5'-GAG GCA ACT TCA TGA GGG GTA GAT-3') and in parallel with β-actin-specific primers (forward primer: 5'-CTG GCA CCA CAC CTT CTA CAA T-3 'and reverse primer: 5'-CTT CTC CTT AAT GTC ACG CAC GAT-3 ') and 2.5U Taq polymerase (Sigma) in 1st PCR buffer (Sigma) 0.25 mM amplified dNTPs. The PCR reaction included a hot start (2 min 30 sec 94 ° C), 40 cycles (denaturation: 30 sec 94 ° C, annealing: 1 min 65 ° C and Elongation: 1 min 30 sec 72 ° C + 5 sec / cycle from cycle 11) and final Elongation of 7 min at 72 ° C. The PCR products were then in one ETBr-containing 1.6% (w / v) agarose gel analyzed. Served as the length standard a 100 bp marker.

(6) Cytotoxicity test

The lytic reactivity of effector cells towards different target cells was checked in a ⁵¹ Cr release test (Theobald et al., 1995). T2 cells were used as target cells for peptide titration tests. 1-5 million target cells were labeled with 150 μCi Na ( ⁵¹ Cr) O ₄ (1 mCi / ml) (NEN Life Science, Belgium) for 60-90 min. Before this labeling, 2 μl of peptide solution of different concentrations and 15 μl of FCS (PAA Laboratories, Linz, Austria) or FCS without peptide were added to the cells in peptide titration tests. The marked target cells were washed four times and the cell number was set to 0.1 million / ml. The effector cells were serially diluted 1: 3 with cell culture medium and seeded at 0.1 ml / well in 96-well plates. A total of five different E: T ratios were tested. Then 0.1 ml / well of the target cell suspension was added to the effector cells and the batches were incubated for 4-6 hours. The cells were then centrifuged off (1300 rpm, 5 ° C., 9 min), the supernatant (0.1 ml / hole) was removed and the ⁵¹ Cr release was measured using a gamma counter (Canberra Packard, Dreieich). The percentage specific lysis (SL) was calculated using the following formula:

The maximum ⁵¹ Cr release corresponded to the total ⁵¹ Cr incorporation by the target cells, the spontaneous ⁵¹ Cr release corresponded to the target cell lysis in the absence of effector cells and was generally less than 10% of the maximum ⁵¹ Cr release. The values for spontaneous and maximum lysis were averaged from four approaches, and those for experimental lysis from two approaches.

C) Examples example 1 Experimental extraction of the PRDI-BF1 oligopeptides with the in Sequence listing 1 to 30 amino acid sequences shown (1.1) Selection of potentially A2.1-binding PRDI-BF1 peptides

• 1. Sie weisen als sogenannte primäre Ankeraminosäuren, das sind Aminosäuren innerhalb des Peptids, die mit Resten der Bindungstasche des MHC Klasse I- Moleküls interagieren und die sich bei endogen prozessierten und im Kontext von MHC Klasse I-Molekülen präsentierten Peptide an Position 2 und am C-Terminus des Epitops befinden, an Position 2 klassischerweise die Aminosäuren L, M, I, V oder T und nichtklassischerweise die Aminosäuren A, Q oder K auf und am C- Terminus klassischerweise die Aminosäuren V, L oder I und nichtklassischerweise die Aminosäuren A, M oder T (Theobald et al., 1995).
• 2. Die betreffenden PRDI-BF1 Peptide sind bis auf die Peptide mit den SEQ ID NO 13- 16, 19, 20, 22, 23 und 29 nicht homolog zu den korrespondierenden Blimp-1 Peptiden der Maus.

Based on the known amino acid sequence of the PRDI-BF1 protein, 8mers, 9mers, 10mers and 11mers were determined, which represent partial sequences of this PRDI-BF 1 polypeptide and which meet the following criteria:

• 1. They have so-called primary anchor amino acids, which are amino acids within the peptide that interact with residues of the binding pocket of the MHC class I molecule and that are present at position 2 and am in endogenously processed and presented in the context of MHC class I molecules The C-terminus of the epitope is classically located at position 2, the amino acids L, M, I, V or T and not classically the amino acids A, Q or K, and at the C-terminus classically the amino acids V, L or I and not classically the amino acids A , M or T (Theobald et al., 1995).
• 2. The relevant PRDI-BF1 peptides, with the exception of the peptides with SEQ ID NO 13- 16, 19, 20, 22, 23 and 29, are not homologous to the corresponding Blimp-1 peptides of the mouse.

With this method, the PRDI-BF1 peptides shown in the sequence protocols 1 to 30 were selected (see FIG. 1), which show a strong or intermediate binding affinity to A2.1 (see example 1.2). The partial sequences of the PRDI-BF1 protein with the Gen Bank Accession Number: A39564 corresponding to these oligopeptides are listed in Table 1.

(1.2) Binding of selected synthetic PRDI-BF1 peptides to A2.1

The PRDI-BF1 peptides selected according to (1.1) on the basis of their theoretical binding strength were examined for their actual binding affinity for A2.1. For this purpose, a competitive binding test, which was published in Theobald et al. (1995) is functionally tested, the ability of the PRDI-BF1 peptides to inhibit the A2.1 binding of the competing synthetic peptide p53 264-272. This inhibition was measured from the decrease in lysis of T2 cells loaded with p53 264-272 peptide and the individual PRDI-BF1 test peptide, mediated by an A2.1-restricted p53 264-272-specific CTL clone. The binding results are summarized in Fig. 1. The known A2.1-binding peptide Flu M1 58-66 (cf. Theobald et al., 1995) served as a positive control and showed more than 80% inhibition at 3 µg and also at 10 µg, while the H-2K ^b -binding Peptide VSV-N 52-59 (Theobald et al., 1995) as a negative control showed no A2.1 binding activity. The PRDI-BF1 peptides were divided into 4 groups according to their binding strength. Of a total of 61 peptides tested, 16 had a high binding activity (at least 85% inhibition with 10 µg test peptide), 28 a medium (40-84% inhibition), 10 a weak (14-39%) and 7 no binding activity (<13% or no dose dependence of inhibition). The most binding PRDI-BF1 peptides were peptide 402-410 (SEQ ID NO. 1) and the two homologous peptides 747-755 (SEQ ID NO. 13) and 681-690 (SEQ ID NO. 19), both of which showed a 100% inhibition of the binding of the competing peptide p53 264-272 at 10 µg and at 3 µg. Dose dependency for these peptides only became apparent at an E: T of 1: 1. The 13 other strongly binding peptides including the peptides with the sequences according to SEQ ID NO. 2-4, 14, 16 and 18 showed a clear dose-dependent inhibition. The peptides with the sequences according to SEQ ID NO. 5-12, 15, 17 and 20-30 showed intermediate binding activity.

Overall, 72% of all selected peptides showed a strong or intermediate A2.1- Binding, only 11% could not bind to A2.1.

Example 2 Experimental proof of the suitability of the PRDI-BF1 oligopeptides 402-410, 401-410, 377-386 and 406-415 (SEQ ID NO. 1-4), for Generation of a specific, ZTL-mediated immunogenicity (2.1) Immunogenicity of A2.1-binding synthetic PRDI-BF1 peptides in A2.1-Tg mice

An obstacle to the detection of human MHC class I molecules by mouse T cells is the inability of mouse CD8 to interact with HLA molecules such as A2.1. Two strategies have been used to circumvent or remove this obstacle. One strategy consisted in the construction of the chimeric molecule A2.1 / K ^b (A2K ^b ), which is composed of the human α1 and α2 domains of A2.1 and the α3 domain of mouse K ^b , which is responsible for the interaction with CD8 is essential. In A2K ^b -Tg mice induced ZTL with restriction for the A2K ^b -Tg recognize the same peptide antigens that are also immunogenic in A2.1 positive humans.

The other strategy to enhance the A2.1 restricted response was to create a double-Tg mouse "CD8 × A2.1 / K ^b " by crossing an A2K ^b -Tg with a huCD8α / β-Tg mouse. The expression of the α and β chain of the huCD8 molecule enables the generated CTL to interact with the α3 domain of the A2.1 molecule of human cells.

A2K ^b - and CD8 × A2K ^b -Tg mice were immunized with the strongly or intermediate-binding peptides obtained according to Example 1 (see FIG. 1) in order to obtain PRDI-BF1 peptide-reactive ZTL. 9 to 11 days after the immunization, spleen cells of the mice in question were stimulated in vitro with peptide-loaded syngeneic LPS blasts and examined for an A2.1-restricted peptide-specific CTL response in a cytotoxicity test 6 days later. The results are summarized in Fig. 2. The induction of A2.1-restricted ZTL was already known for the positive control Flu M1 58-66 (Theobald et al., 1995). An A2.1-restricted and peptide-specific ZTL response has so far been used for the strongly binding peptides 401-410 (SEQ ID NO. 2), 402-410 (SEQ ID NO. 1), 377-386 (SEQ ID NO. 3) and 406-415 (SEQ ID NO. 4). The level of lysis was dependent on the E: T ratio. The ZTL were peptide-specific, since they lysed cells loaded with the corresponding peptide, but not cells that were loaded with irrelevant A2.1-binding peptides.

ZTL induced by PRDI-BF1 401-410, 402-410, 377-386 were A2.1-restricted, since the corresponding peptide-loaded A2.1-negative EL-4 cells (H-2 ^b ) of the mouse were not were recognized ( Fig. 3). For the 406-415 oligopeptide, an H-2 ^b- restricted ZTL response was primarily induced in addition to an A2.1-restricted, since in addition to PRDI-BF1 406-415-loaded T2 cells, EL-4 cells loaded with this peptide were also induced were specifically recognized ( Fig. 3).

(2.2) PRDI-BF1 401-410-, 402-410- and 377-386-specific ZTL: A2.1- Restriction, peptide specificity and efficiency of peptide recognition

A2T-restricted and for PRDI-BF1 401-410-, 402-410- and 377-386-specific ZTL were examined in more detail below. As there were only PRDI-BF1 401-410 and 402-410-specific ZTL lines generated from A2K ^b -Tg mice, CD8 × A2K ^b -Tg mice with PRDI-BF1 401-410 and 402 -410 immunized with the intention of obtaining ZTL with higher avidity. CD8 × A2K ^b -Tg mice have already been used to demonstrate the immunogenicity of PRDI-BF1 377-386 (FIGS . 2 and 3).

After immunization of CD8 × A2K ^b -Tg mice with PRDI-BF1 401-410 and 402-410, the spleen cells were stimulated with peptide-loaded syngeneic LPS blasts (I ° culture) and incubated 6 days later in the cytotoxicity test against T2 target cells with PRDI-BF1 401-410 or 402-410, tested. Peptide-specific CTLs were induced for both peptides (data not shown).

By repeated restimulation of I ° ZTL from CD8 × A2K ^b -Tg mice with peptide-loaded stimulator cells, stable ZTL lines with specificity for PRDI-BF1 401-410, 402-410 and 377-386 were generated. The peptide specificity and A2.1 restriction of the established ZTL lines CD8 × A2K ^b 377, CD8 × A2K ^b 402p and CD8 × A2K ^b 401 are shown in FIG. 4 AC. These ZTL did not recognize unloaded T2-cells or the irrelevant peptide Flu M1 58-66. However, T2 cells presenting Flu M1 58-66 were lysed from a CD8 × A2K ^b T cell population specific for Flu M1 58-66 (without figure). The ZTL were A2.1-restricted, since the corresponding peptide-loaded A2.1-negative EL-4 cells (H-2 ^b ) of the mouse were not recognized ( FIG. 4 AC).

In addition to the peptide 402-410, the ZTL CD8 × A2K ^b 402p also recognized the peptide variant 402-410A, which has an amino acid substitution of cysteine for alanine at position 408 ( FIG. 4B). If T2 cells were incubated at different concentrations of synthetic peptide 402-410 and 402-410A and used as target cells for CD8 × A2K ^b 402p in the cytotoxicity test, the ZTL line showed a more efficient logging of the peptide variant 402-410A ( Fig. 5B). The ZTL line CD8 × A2K ^b 401 also showed cross-reactivity towards the peptide PRDI-BF1 402-410 shortened by an amino acid ( FIG. 4C). The peptide 402-410 was recognized by this ZTL line by one log level more efficiently than the peptide PRDI-BF1 401-410 used to propagate the ZTL (detection of PRDI-BF1 402-410 even at a peptide concentration of 1nM compared to 10 nM for PRDI-BF1 401-410) ( Fig. 5C). For the ZTL CD8 × A2K ^b 377, the efficiency of peptide recognition with an E: T ratio of 10: 1 is also shown in FIG. 5A. The ZTL recognized the specific peptide PRDI-BF1 377-386 up to a peptide concentration of 1-10 nM.

The end result was A2.1-restricted ZTL populations with specificity for PRDI- BF1 401-410, 402-410 and 377-386 generated.

Example 3 Use of PRDI-BF1 401-410-, 402-410- and 377-386- specific ZTL for the specific detection of multiple myeloma cell (3.1) PRDI-BF1 expression in MM cell lines

In order to determine whether the PRDI-BF1 protein is actually overexpressed in malignant plasma cells and thus endogenously processed and A2.1-presented PRDI-BF1 peptides represent MM-associated peptide antigens, various MM cell lines were first tested for the expression of PRDI-BF1 RNA examined. For this purpose, the cytoplasmic RNA was isolated, the polyA-RNA (mRNA) was transcribed into cDNA (reverse transcription) with an oligo-dT primer and amplified with PRDI-BF1-specific primers in a subsequent PCR reaction. As a control, the cDNA was also amplified with β-actin-specific primers. Since β-actin is constitutively expressed in all cells, a comparable β-actin signal can be expected in all cells. Both the β-actin-specific primers and the PRDI-BF1-specific primers were chosen to be intron-spanning, so that due to the size of the PCR product, an RNA amplificate (PRDI-BF1: 540bp fragment; β-actin: 390bp fragment ) and a possibly contaminating DNA-derived amplificate (PRDI-BF1: 950bp fragment; β-actin: 830bp fragment). FIG. 6 shows the PRDI-BF1 RNA expression of the mm cell lines L363, MC-CAR, U266, IM9, OPM-2 and NCI-H929. In contrast to the PRDI-BF1-negative cell line EL-4, the mm cell lines show a strong PRDI-BF1 expression (540 bp amplificate). All examined cell lines show a comparable expression of β-actin (390 bp amplificate). PRDI-BF1 RNA expression was also examined for the two osteosarcoma cell lines Saos-2 and U2OS ( FIG. 7). For the U2OS cell line, the expression of PRDI-BF1 RNA was already proven by Keller and Maniatis (1991). U2OS express significantly more PRDI-BF1 RNA than Saos-2. Both cell lines show a lower expression compared to the mm cell line L363, which served here as a positive control ( FIG. 7). EL-4 acted as a negative control ( Fig. 7)

(3.2) Recognition of PRDI-BF1 Overexpressing MM Cells by PRDI BF1 401-410-, 402-410- and 377-386-specific ZTL

In order to check whether the MM cell lines are susceptible to recognition by PRDI-BF1 401-410-, 402-410- and 377-386- specific ZTL due to their strong expression of PRDI-BF1, the A2-positive mm- Cell lines U266, OPM-2 and MC-CAR and the A2-negative MM cell line NCI-H929 (data on A2.1 expression not shown in flow cytometry) in comparison with the A2-positive osteosarcoma cell line Saos-2 as target cells used for the ZTL CD8 × A2K ^b 377, CD8 × A2K ^b 402p and CD8 × A2K ^b 401 in the cytotoxicity test ( FIG. 8). The ZTL lines showed a selective and efficient lysis of the A2 ⁺ MM cell lines, the A2 ^- NCI-H929 cells were not lysed by any ZTL line ( FIG. 8). A2 ⁺ Saos-2 cells which showed a significantly lower PRDI-BF1 expression in the RT-PCR ( FIG. 7) were accordingly not recognized by the PRDI-BF1-specific CTL ( FIG. 8). The ZTL line CD8 allo A2 served as a positive control. Both the cell lines U266 mm, OPM-2 and MC-CAR as well as Saos-2 cells were lysed by the allo-A2.1-reactive effector cells, but not the A2 ^- Cell Line NCI-H929 (Fig. 8). The A2.1-restricted CDT line CD8 × A2K ^b FluM1, which did not lyse any of the target cells tested, acted as a negative control ( FIG. 8). The results indicate the endogenous processing and A2.1 presentation of the PRDI-BF1 peptide antigens 401-410, 402-410 and 377-386. The specific lysis of mm cell lines by PRDI-BF1 401-410-, 402-410- and 377-386-specific ZTL and the lack of susceptibility of tested tumor entities of different origins such as e.g. B. Breast cancer, colorectal cancer and hepatoblastoma compared to these CTLs (data not shown) indicate that PRDI-BF1 peptide antigens can serve as MM-associated tumor antigens.

Example 4 Use of PRDI-BF1 401-410-, 402-410- and 377-386- specific ZTL for the selective detection of MM cells (4.1) PRDI-BF1 expression from non-transformed cells lymphohematopoietic origin

For potential, PRDI-BF1-specific CTL-mediated immunotherapy, it is desirable that normal cells not be lysed. PRDI-BF1 RNA is overexpressed in MM cells (see Example 3, (3.1)) and is known to be also expressed by some normal cells, including lymphohematopoietic cells. FIG. 9 shows the PRDI-BF1 RNA expression for resting monocytes, T cells and B cells. In comparison to the two mm cell lines L363 and NCI-H929, these lymphohemopoetic cells have a significantly lower, but still substantial amount of PRDI-BF1 RNA. EL-4 cells acted as a negative control.

(4.2) Cytolytic reactivity PRDI-BF1 401-410-, 402-410- and 377-386- specific ZTL compared to non-transformed cells lymphohematopoietic origin

Non-transformed lymphohemopoietic cells were subsequently used as target cells for A2.1-restricted CTLs with specificity for PRDI-BF1 401-410, 402-410 and 377-386. Dormant B cells and T cells were not recognized by any of the PRDI-BF1-specific CTLs ( Figures 10 and 11A-C). ZTL lysis could be reconstituted by adding peptide PRDI-BF1 402-410 for CD8 × A2K ^b 402p and CD8 × A2K ^b 401 and PRDI-BF1 377-386 for CD8 × A2K ^b 377 (FIGS . 10 and 11A- C), which confirmed sufficient A2.1 expression of the target cells. A2.1 expression was also confirmed with the allo-A2.1-reactive ZTL line CD8 allo A2, which showed an efficient lysis of B cells and T cells ( FIGS. 10 and 11 E). No detection was carried out by FluM1-specific ZTL ( FIGS. 10 and 11D).

Since recent studies have shown that PRDI-BF1 is also involved in the differentiation of myeloid cells (Chang et al., 2000), monocytes were differentiated into dendritic cells (DZ) in vitro and were also used as target cells for PRDI-BF1 402-410- and 377-386-specific ZTL used. No lytic activity against DZ was observed for the ZTL CD8 × A2K ^b 402p and CD8 × A2K ^b 377 ( FIGS. 12A and B). Loading the DC with the corresponding peptide led to the reconstitution of the lysis, which indicated sufficient expression of A2.1 molecules (FIGS . 12A and B). ZTL CD8 allo A2 also served as a positive control and showed efficient lysis of the DC ( Fig. 12D). No lytic activity was found on the part of the ZTL CD8 × A2K ^b Flu M1 ( FIG. 12C). In summary, the results indicate that, despite substantial expression of PRDI-BF1-RNA, normal cells are not susceptible to PRDI-BF1-specific CTL-mediated lysis.

Example 5 Production of A2.1-restricted T cell receptors that are specific for a PRDI-BF1 oligopeptide according to the invention with a in the Sequence listing 1 to 30 amino acid sequences are shown

A2.1-Tg mice are treated with a PRDI-BF1 oligopeptide according to the invention, the one of the amino acid sequences shown in the sequence listing 1-30, immunized. The spleen is removed after 10 days. The spleen cells are covered with previously prepared, A2.1-positive antigen-presenting cells, which with the oligopeptide according to the invention are loaded, stimulated in vitro. The making of this A2.1-positive antigen-presenting cells were made using the ones in the prior art known techniques known to those skilled in the art. After several weeks of culture the T cells for their peptide and tumor recognition, peptide specificity and A2.1- Restriction checked. After successful testing, the T cell line is cloned. The resulting T cell clones are again used for peptide and tumor detection, Peptide specificity and A2.1 restriction tested.

The total mRNA is prepared from a T cell clone with a positive test result. The T cell receptor α and β chains are amplified by means of RT-PCR. The respective Chains are first cloned into bacterial plasmids and sequenced. The chains are partially humanized by the constant mouse regions by the homologous human regions. The cloning is then carried out resulting constructs into suitable retroviral vectors.

Peripheral blood lymphocytes of an A2.1-positive cancer patient, whose tumor cells Overexpressing PRDI-BF1 protein are removed using the vectors for α- and β chain of the T cell receptor transduced in vitro and gene expression at the protein level examined. T cell receptor expressing T lymphocytes are assessed for their ability to Lysis of tumor cells analyzed. After successful testing, the gene-modified lymphocytes are transfused in the patient and are said to be there Killing the degenerated cells and thus causing healing.

Alternatively, PRDI-BF1 specific α / β T cell receptor chains are cloned into vectors that have the ability to infect patient T cells in vivo. These infected T cells express PRDI-BF1-specific T cell receptors and are therefore able to recognize and kill PRDI-BF1-overexpressing tumor cells of the patients. Bibliography Chang DH, Angelin-Duclos C., Calame K .: Blimp-1: trigger for differentiation of myeloid lineage. Nat. Immunol. 1: 169; 2000
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Parker KC, Bednarek MA, Coligan JE (1994). Scheme for ranking potential HLA-A2 binding peptides based on independent binding of individual peptide side-chains. J. Immunol. 152, 163-175.
Satter RD, Cresswell P. (1986). Impaired assembly and transport of HLA-A and -B antigens in a mutant TxB cell hybrid. EMBO JS 943-949.
Theobald M., Biggs J., Dittmer D., Levine AJ, Sherman LA (1995). Targeting p53 as a general tumor antigen. Proc. Natl. Acad Sci. USA 92, 11993-11997.
Theobald M., Ruppert T., Kuckelkorn U., Hernandez J., Häußler A., Antunes Ferreira E., Liewer U., Biggs J., Levine AJ, Huber C., Koszinowski UH, Kloetzel P.-M., Sherman LA (1998). The sequence alteration associated with a mutational hotspot in p53 protects cells from lysis by cytotoxic T lymphocytes specific for a flanking peptide epitope. J. Exp. Med 188, 1017-1028.
Tuner CA, Mack DH, Davis MM: Blimp-1, a novel zinc finger-containing protein that can drive the maturation of B lymphocytes into immunoglobulin-secreting cells. Cell 77: 297, 1994
Wölfel T., Van Pel A., Brichard V., Schneider J., Seliger B., Meyer zum Büschenfelde KH, Boon T. (1994). Two tyrosinase nonapeptides recognized on HLA-A2 melanomas by autologous cytolytic T lymphocytes. Eur. J. Immunol. 24, 759-764. Table 1 Correlation between the PRDI-BF 1 oligopeptides shown in sequence protocols 1 to 30 and partial sequences of the PRDI-BF1 protein with the Gen Bank Accession number A39564

SEQUENCE LISTING

Claims (24)

1. tumor-associated oligopeptide which is recognized as a peptide antigen by CD8-positive cytotoxic T-lymphocytes (ZTL) and which causes ZTL-induced lysis and / or apoptosis of tumor cells, in particular multiple myeloma cells,
characterized in that the oligopeptide 1. has one of the amino acid sequences shown in the sequence listing 1 to 30, which corresponds to a partial sequence of the human PRDI-BF1 protein, or 2. has an amino acid sequence which can be derived from one of these amino acid sequences by amino acid substitution, deletion, insertion, addition, inversion and / or by chemical or physical modification of one or more amino acids and which has a functional equivalent to one of those in the sequence listing 1 to 30 amino acid sequences listed, and that it a) represents an epitope for CD8-positive ZTL
and b) is suitable for this; to induce a restricted (restricted) immune response of CD8-positive CTL against tumor cells, in particular multiple myeloma cells, to human leukocyte antigen of the molecular group "MHC class I", allele variant A2 (short: A2). 2. tumor-associated oligopeptide according to claim 1, characterized in that it is the amino acid sequence shown in the sequence listing SEQ ID NO 1 having. 3. tumor-associated oligopeptide according to claim 1, characterized in that that it is the amino acid sequence shown in the sequence listing SEQ ID NO 2 having. 4. tumor-associated oligopeptide according to claim 1, characterized in that that it is the amino acid sequence shown in the sequence listing SEQ ID NO 3 having. 5. tumor-associated oligopeptide according to claim 1, characterized in that it is the amino acid sequence shown in the sequence listing SEQ ID NO 4 having. 6. Retro-inverse peptide or pseudopeptide, characterized in that it is one Oligopeptide according to one of claims 1 to 5, in which instead of -CO-NH peptide bonds Nile-CO bonds or other non-peptide bonds are trained. 7. fusion protein which consists of an oligopeptide according to any one of claims 1 to 6, a heavy chain of the HLA molecule and a flexible linker and is constructed such that the oligopeptide is capable and capable of Occupy the furrow of the HLA molecule. 8. Polynucleotide with a nucleotide sequence which is at least for one oligopeptide encoded one of claims 1 to 7. 9. Use of an oligopeptide according to one of claims 1 to 5 and / or one retro-inverse peptide or pseudopeptide according to claim 6 and / or one Fusion protein according to claim 7 and / or a polynucleotide according to claim 8 for Manufacture of diagnostic and / or therapeutic and / or prophylactic for the Evidence and / or influencing and / or generation and / or expansion and / or control of the activation and functional state of T cells, especially CD8 positive cytotoxic T lymphocytes. 10. Reagent for the in vivo or in vitro activation of T cells, in particular CD8 positive cytotoxic T lymphocytes, characterized in that the reagent using at least one oligopeptide according to one of the Claims 1 to 5 and / or a retro-inverse peptide or pseudopeptide according to Claim 6 and / or a fusion protein according to Claim 7 and / or one Polynucleotide according to claim 8 is produced. 11. Recombinant DNA or RNA vector molecule that has at least one or more Contains polynucleotide (s) according to claim 8 and that in cells autologous, allogeneic, xenogenic or microbiological origin is expressible. 12. Host cell which according to a polynucleotide according to claim 8 or a vector molecule Claim 11 contains. 13. Use of at least one oligopeptide according to one of claims 1 to 5 and / or a retro-inverse peptide or pseudopeptide according to claim 6 and / or a fusion protein according to claim 7 for the production of polyclonal, monoclonal or recombinant antibody against the relevant (s) Oligopeptide (s) or against a complex of the concerned Oligopeptide (s) and HLA-A2. 14. Antibody specific with at least one oligopeptide according to one of the Claims 1 to 5 and / or a retro-inverse peptide or pseudopeptide according to Claim 6 and / or a fusion protein according to claim 7 or with one Complex of the relevant oligopeptide (s) and HLA-A2 reacts. 15. Oligopeptide according to one of claims 1 to 5 and / or fusion protein Claim 7, characterized in that it is covalent or non-covalent to MHC Class I tetramers or MHC class I dimers or pharmaceutically acceptable ones Carrier or other structures are bound. 16. Retro-inverse peptide or pseudopeptide according to claim 6, characterized characterized in that it is covalent or non-covalent to MHC class I tetramers or MHC class I dimers or pharmaceutically acceptable carriers or others Structures are bound. 17. Use of at least one oligopeptide according to one of claims 1 to 5 and / or a retro-inverse peptide or pseudopeptide according to claim 6 and / or a fusion protein according to claim 7 or a polynucleotide according to Claim 8 for the production of polyclonal or monoclonal or recombinant A2 restricted T cell receptors or molecules that are functionally equivalent to them the oligopeptide (s) in question. 18. T cell receptor or molecule that is functionally equivalent to it, that specifically with at least one oligopeptide according to one of claims 1 to 5 and / or one retro-inverse peptide or pseudopeptide according to claim 6 and / or one Fusion protein according to claim 7 reacts. 19. A polynucleotide encoding a T cell receptor according to claim 18. 20. Expression vector which has the ability to a T cell receptor according to claim 18 to express. 21. A host cell which is a polynucleotide according to claim 19 or an expression vector according to claim 20. 22. Process for the manufacture of a medicament for the treatment of PRDI-BF1- Protein-associated diseases, characterized in that at least one Oligopeptide and / or at least one retro-inverse peptide or pseudopeptide and / or at least one fusion protein and / or at least one polynucleotide and / or at least one T cell receptor and / or at least one vector molecule and / or at least one host cell and / or at least one antibody after one the aforementioned claims together with suitable additives and auxiliary substances is combined. 23. Medicines for the treatment of diseases associated with PRDI-BF1 protein. characterized in that it contains at least one oligopeptide and / or at least one retro-inverse peptide or pseudopeptide and / or at least one fusion protein and / or at least one polynucleotide and / or at least one T cell receptor and / or at least one vector molecule and / or at least one host cell and / or at least one antibody according to one of the preceding claims, optionally together with suitable additives and auxiliaries. 24. Use of a medicament according to claim 23 for the treatment of PRDI-BF1- Protein Associated Diseases.