WO2000066727A9 - Tumorassoziiertes antigen - Google Patents
Tumorassoziiertes antigenInfo
- Publication number
- WO2000066727A9 WO2000066727A9 PCT/EP2000/003552 EP0003552W WO0066727A9 WO 2000066727 A9 WO2000066727 A9 WO 2000066727A9 EP 0003552 W EP0003552 W EP 0003552W WO 0066727 A9 WO0066727 A9 WO 0066727A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- peptides
- seq
- tumor
- peptide
- immunogenic
- Prior art date
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Classifications
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- 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
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5156—Animal cells expressing foreign proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
Definitions
- the invention relates to the immunotherapy of tumor diseases.
- the immune system's task is to protect the organism from a variety of different microorganisms or to actively combat them.
- the importance of an intact immune system is particularly evident in inherited or acquired immunodeficiencies.
- the use of prophylactic vaccine programs has proven to be an extremely effective and successful immunological intervention in the fight against viral or bacterial infectious diseases.
- the immune system is also significantly involved in the elimination of tumor cells.
- TAAs tumor-associated antigens
- Stimulation of an immunological response leads to act as a tumor antigen in the unogenous.
- those tumor antigens that not only cause an immunological reaction, but also cause rejection of the tumor.
- the identification of defined antigens that can cause such an immunological reaction is an important step in the development of a molecularly defined tumor vaccine.
- T-lymphocytes (CTLs) play a major role (Coulie,
- TIL tumor infiltrating lymphocytes
- PBMC peripheral mononuclear blood cells
- TAA tumor associated antigens
- CD8-positive CTLs are recognized, a stated main goal on the way to the development of a tumor vaccine (Pardoll, 1998; Robbins and Kawakami, 1996). It is still unclear whether other cell types of the immune system such as CD4 + T helper cells also play an important role; some studies with MAGE-3 / HLA-A1 peptides in melano patients suggest this (Marchand et al., 1995; Boon et al., 1998). A number of TAAs recognized by CTLs have been identified in recent years (Boon et al., 1994; van den Eynde and van der Bruggen, 1997).
- MHC-I molecules MHC-I molecules occur on most cells with a nucleus and present peptides (usually 8-10 mers) , which result from proteolytic degradation of endogenous proteins (so-called antigen processing, "antigen processing").
- Peptide MHC-I complexes are recognized by CD8-positive CTLs. MHC-II molecules only come on so-called
- MHC-II complexes are recognized by CD4 helper T cells
- MHC complex can trigger various effector mechanisms that lead to apoptosis of the target cell in the case of CTLs, either when the MHC (eg in the case of transplant rejection) or the peptide (eg in the case of intracellular) Pathogens) is recognized as foreign, however, not all of the peptides presented meet the structural and functional requirements for effective interaction with T cells (as described by Rammenee et al., 1995 and below).
- the antigen can either be used as a recombinant protein with suitable adjuvants or carrier systems, or as a cDNA coding for the antigen in plasmid (DNA vaccine; Tighe et al., 1998) , or viral vectors (Restifo, 1997) are applied.
- DNA vaccine Tighe et al., 1998)
- viral vectors Restifo, 1997) are applied.
- Another possibility is the use of recombinant bacteria (eg Listeria, Salmonella), which recombinantly express the human antigen and which have an adjuvative effect due to their additional components (Paterson, 1996; Pardoll, 1998). In all of these cases, processing and presentation of the antigen by so-called "professional antigen-presenting cells" (APC) is necessary.
- APC professional antigen-presenting cells
- Antigens or their epitopes include molecules that can originate from all protein classes (eg transcription factors, receptors, enzymes; for an overview see Rammenee et al., 1995; Robbins and Kawakami, 1996). These proteins do not necessarily have to be located on the cell surface, as is the case with detection by antibodies is required. In order to function as a tumor-specific antigen for recognition by CTLs or to be used for therapy, the proteins must meet certain conditions: firstly, the antigen should be expressed mainly by tumor cells and not or only to a lesser extent in so-called "critical" normal tissues Concentration than in tumors. Critical normal tissues are essential tissues; an immune reaction directed against them could have serious, sometimes lethal consequences.
- the antigen is said to be present not only in the primary tumor, but also in the metastases. Furthermore, in view of a wide clinical use of the antigen, it is desirable if it is present in high concentration in several types of tumor.
- Another prerequisite for the suitability of a TAA as an effective component of a vaccine is the presence of T cell epitopes in the amino acid sequence of the antigen; Peptides derived from TAA are said to lead to an in vitro / in vivo T cell response ("immunogenic" peptide).
- Another selection criterion for a clinically widely applicable immunogenic peptide is the frequency with which the antigen is found in a given patient population.
- TAAs tumor-associated antigens
- viral proteins viral proteins
- mutated proteins overexpressed proteins
- fusion proteins formed by chromosomal translocation fusion proteins formed by chromosomal translocation
- differentiation antigens oncofetal antigens (Van den Eynde and Brichard, 1995; van den Eynde and van der Bruggen, 1997).
- TAAs which represent the starting point for the development of a tumor vaccine
- CTLs cellular immune response
- antibodies humor immune response
- differential transcription profiles between tumors and normal tissues are based on the one hand on the use of CTLs (cellular immune response) or antibodies (humoral immune response) that have already been induced in patients, or are based on the creation of differential transcription profiles between tumors and normal tissues.
- patient CTLs are used for screening eukaryotic tumor cDNA expression libraries which present the CTL epitopes via MHC-I molecules (Boon et al., 1994), while using high-affinity patient antisera prokaryotic cDNA expression libraries can be examined directly for the presence of TAAs via an immunoblot analysis of the individual plaques (Sahin et al., 1995).
- a combination of CTL reactivity and protein chemical methods represents the isolation of peptides isolated from MHC-I from tumor cells which have been preselected for reactivity with patient CTLs.
- the peptides are washed out of the MHC-I complex and identified using mass spectrometry (Falk et al., 1991; Woelfel et al., 1994; Cox et al., 1994).
- the approaches that use CTLs to characterize antigens are associated with considerable effort or not always successful due to the required cultivation and activation of CTLs.
- TAAs which are based on the comparison of the transcription profile of normal with tumor tissue
- these include differential hybridization, the creation of subtraction cDNA banks ("representational difference analysis”; Hubank and Schatz, 1994; Diatchenko et al., 1996) and the use of DNA chip technology or the SAGE method (Velculescu et al., 1995).
- the use of molecular biological methods must show that the potential antigen candidates found with them are tumor-specific (tumor-associated) and actually have T-cell epitopes that can trigger a cytotoxic T-cell response , In at least one case
- the object of the present invention was to provide a new tumor-associated antigen (TAA).
- RDA representational difference analysis
- the human B99 CDNA was cloned, the sequence obtained is shown in SEQ ID NO: 1. Sequence analysis of the cloned human B99 cDNA showed that from position 427 to position 1743 there is a continuous open reading frame which, at the nucleotide and protein level, has a high identity with the open reading frame of beta-1,3-galactosyl-o-glycosyl -Glycoprotein beta-1, 6-n-
- Acetyglucosaminyltransferase possesses. It can be concluded from the data obtained from Northern blot experiments that the B99 transcript is approx.
- the cloned region of the B99 cDNA is 2216 bp, with the presence of a polyA tail at the 3 'end of the sequence speaks for the completeness of the cDNA in this area.
- the difference in the size of the cloned B99 cDNA compared to the size derived from the Northern blot analysis can be explained by the presence of a polyA tail of unknown length and an additional sequence in the 5 'untranslated region of B99. Due to the fact that there is no continuous reading frame in the 5 'region of the cloned cDNA from position 0 to 427, it can be concluded that the ATG at position 427 is the start codon of B99.
- RNA preferably mRNA
- RNA is reverse transcribed from cells or tissues in which B99 is transcribed (eg colon carcinoma tissue or cell lines derived from lung adenocarcinoma such as A549) and then ligated with an adapter of known sequence, PCR with an adapter primer (binds specifically to the adapter at the 5 'end of the cDNA) and a B99-specific primer (eg SEQ ID NO: 8, 10, 11) allows the corresponding amplification B99 fragments: As described in Example 1, these PCR products can be cloned by standard methods and characterized, in particular by DNA sequencing.
- 5 ' end is the screening of cDNA libraries Hybridization with DNA probes or antisera specific for B99.
- RNA can be examined in genomic libraries by, for example, as in the screening of cDNA libraries, by cloning by cloning with DNA probes specific for B99 to isolate the clones upstream from the obtained 5 'end of the cDNA lying sequence information eg contain the promoter region of B99.
- the isolated cDNA codes for the tumor-associated antigen (TAA) of the designation B99 with the amino acid sequence given in SEQ ID N0: 2 (B99-1).
- TAA tumor-associated antigen
- the sequence of B99-1 is defined by the start codon at position 427 of the isolated B99 CDNA.
- the cDNA isolated from A549 cells a nucleotide exchange at position 622 compared to sequence SEQ ID N0: 1.
- This nucleotide exchange requires replacement of arginine (SEQ ID NO: 2, B99-1) by tryptophan (SEQ ID NO: 4, B99-2) at position No. 66.
- the amino acid sequence from B99-2 up to and including position 166 is identical to B99-1.
- a protein expressed by a cDNA with this reading frame has the amino acid sequence shown in SEQ ID NO: 6 (B99-3).
- the sequence of B99-3 differs from items 1 to 27 to B99-1 and from item 28 is identical to B99-1.
- the invention thus relates in a first aspect to a tumor-associated antigen of the designation B99, selected from the group of polypeptides with the amino acid sequence given in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6.
- the amino acid sequences shown in SEQ ID NO: 2 (B99-1), SEQ ID NO: 4 (B99-2) and SEQ ID NO: 6 (B99-3) can have deviations, for example those which are caused by the exchange of amino acids if the B99 derivative has the immunogenic properties desired for use in a tumor vaccine.
- SEQ ID NO: 2 B99-1
- SEQ ID NO: 4 B99-2
- SEQ ID NO: 6 B99-3
- the natural amino acid sequence of B99 (or correspondingly the sequences of the B99 cDNA) can optionally be modified by exchanging individual amino acids in a B99 CTL epitope in order to increase the affinity of B99 compared to the natural B99 CTL epitope -Peptides to MHC-I molecules and thus an increased immunogenicity and ultimately an increased reactivity to tumors.
- Modifications in the area of the B99 epitopes can be carried out on the total B99 protein (this is processed by the APCs into the corresponding peptides) or on larger B99 protein fragments or on B99 peptides (see below).
- the present invention relates to immunogenic fragments and peptides derived from B99.
- B99 peptides The latter are referred to below as "B99 peptides”.
- a first group are
- B99 peptides that trigger a humoral immune response are selected sections of B99 (at least 12 to 15 amino acids), which by means of so-called prediction algorithms ("prediction algorithms") such as the surface probability blot “(Emini et al., 1985), the” hydrophobicity blot “(Kyte and Doolittle, 1982) and the "antigenic index” (Jameson and Wolf, 1988).
- prediction algorithms such as the surface probability blot "(Emini et al., 1985), the” hydrophobicity blot “(Kyte and Doolittle, 1982) and the "antigenic index” (Jameson and Wolf, 1988).
- prediction algorithms such as the surface probability blot "(Emini et al., 1985), the” hydrophobicity blot “(Kyte and Doolittle, 1982) and the "antigenic index” (Jameson and Wolf, 1988).
- tumor-associated antigens can have tumor-specific mutations that contribute to an immunological differentiation between tumor and normal
- the B99 cDNA is expediently cloned from one or more different tumors with the aid of probes from the isolated cDNA according to the invention and the sequences obtained are compared with normal tissue B99 cDNA. It is to be expected that tumor B99 peptides from a sequence section mutated compared to normal tissue B99 have an increased immunogenicity compared to normal tissue B99 peptides from the corresponding section.
- Invention thus relates in a further aspect B99- peptides derived from regions of a tumor discourse appearing in a further aspect.
- the regions of B99-2 and B99-3 that differ from B99-1 deserve special interest. Provided that the insertion of the B99 DNA, which leads to these differences in the amino acid sequence, is a tumor-specific one
- peptides from this area have an increased immunogenicity compared to peptides from B99-1.
- antibodies against this area can be generated and tumor cells can be examined for expression of B99-2 and B99-3.
- B99- ⁇ Peptides are those that are presented by MHC molecules and cause a cellular immune response.
- MHC molecules There are two classes of MHC molecules, namely MHC-I molecules that are recognized by CD8-positive CTLs and MHC-II molecules that are recognized by CD4-positive T helper cells.
- a peptide In order for a peptide to trigger a cellular immune response, it must bind to an MHC molecule, and the patient to be treated must have the MHC molecule in its repertoire.
- the determination of the patient's MHC subtype thus represents one of the essential prerequisites for the effective application of a peptide to this patient with regard to triggering a cellular immune response.
- the sequence of a B99 peptide to be used therapeutically is predetermined by the respective MHC molecule with regard to anchor amino acids and length. Defined anchor positions and lengths ensure that a peptide fits into the peptide binding groove of the patient's respective MHC molecule. As a result, the immune system is stimulated and a cellular immune response is generated which, in the case of using a peptide derived from a tumor antigen, is directed against the patient's tumor cells.
- Immunogenic B99 peptides can be identified by known methods, one of the bases for this is the relationship between MHC binding and CTL induction.
- B99 peptides that represent CTL epitopes are identified and synthesized based on the B99 protein sequence.
- Various methods are suitable for this, which have been used to identify CTL epitopes of known protein antigens; e.g. the method described by Stauss et al., 1992, for the identification of T cell epitopes in human papillomavirus.
- the peptide candidates can also be examined for non-anchor residues which have a negative or positive effect on the binding or which make this possible (Ruppert et al., 1993). With this approach, however, it should be considered that the peptide binding motif is not the only decisive factor in the search for natural ligands; other aspects, e.g. the enzyme specificity during antigen processing, in addition to the specificity of the MHC binding, contribute to the identity of the ligand.
- the peptides can also be selected for their ability to bind to MHC-II molecules.
- Amino acids has a higher degree of degeneration in the anchor positions than the MHC-I Binding motif.
- Methods have recently been developed, based on the X-ray structure analysis of MHC-II molecules, which allow the precise analysis of the MHC-II binding motifs, and on the basis thereof, variations of the peptide sequence (Rammenee et al., 1995, and the original literature cited therein ).
- Peptides that bind to MHC-II molecules are typically presented to CD4-T cells by dendritic cells, macrophages or B cells. The CD4-T cells in turn then directly activate CTLs by, for example
- APC dendritic cells, macrophages and B cells
- Binding properties determined (stability of the peptide-MHC interaction correlates in most cases with immunogenicity; van der Burg et al., 1996). To determine the immunogenicity of the selected peptide or peptide
- Heteroclitic peptides They can be obtained by the following methods:
- the length of the peptide in the case of its adaptation to MHC-I molecules preferably corresponds to a minimum sequence of 8 to 10 amino acids with the required anchor amino acids.
- the peptide can also be extended at the C- and / or at the N-terminus, provided that this extension does not impair the ability to bind to the MHC molecule or the extended peptide can be processed cellularly for the minimal sequence.
- TILs tumor-infiltrating lymphocytes
- CTL induction for CTL induction
- MHC binding and immunogenicity as described by Parkhurst et al., 1996, and Becker et al., 1997, described.
- Another method suitable for the purposes of the present invention for finding peptides with greater immunogenicity than that of the natural B99 peptides is to screen peptide libraries with CTLs which recognize the naturally occurring B99 peptides on tumors, as described by Blake et al. , 1996, described; in this context, the use of combinatorial peptide libraries is proposed to design molecules that mimic tumor epitopes recognized by MHC-I restricted CTLs.
- the B99 polypeptides of the present invention or immunogenic fragments or peptides derived therefrom can be produced recombinantly or by means of peptide synthesis, as described in WO 96/10413, the disclosure of which is hereby incorporated by reference.
- the corresponding DNA molecule is inserted into an expression vector according to standard methods, transfected into a suitable host cell, the host is cultivated under suitable expression conditions and the protein is purified.
- Conventional methods can be used for the chemical synthesis of B99 peptides, e.g. commercially available automatic peptide synthesizers.
- B99 peptides or heteroclitic peptides substances which simulate such peptides, for example "peptido imetica” or “retro-inverse peptides", can be used. The same methods are used to test these molecules with regard to their therapeutic utility in a tumor vaccine as for the natural B99 peptides or B99 peptide equivalents.
- the TAA of the designation B99 according to the present invention and the protein fragments, peptides or peptide equivalents or peptidomimetics derived therefrom can be used in cancer therapy, for example to induce an immune response against tumor cells which express the corresponding antigen determinants. They are preferably used for the therapy of B99-positive tumors, in particular in renal cell, lung, colon, pancreas, breast and stomach carcinoma.
- the immune response in the form of induction of CTLs can be brought about in vivo or ex vivo.
- a pharmaceutical composition containing the active component TAA B99 or fragments or peptide (s) derived therefrom is administered to a patient suffering from a tumor disease associated with the TAA, the amount of TAA ( Peptide) must be sufficient to achieve an effective CTL response to the antigen-bearing tumor.
- the invention thus relates to a pharmaceutical composition for parenteral, topical, oral or local administration.
- the composition is preferably used for parenteral administration, for example for subcutaneous, intradermal or intramuscular use.
- the B99 TAAs / peptides are dissolved or suspended in a pharmaceutically acceptable, preferably aqueous, vehicle.
- the composition can also contain customary auxiliaries, such as buffers, etc.
- the TAAs / peptides can be used alone or in combination with adjuvants, for example incomplete Freund's adjuvant, saponins, aluminum salts or, in a preferred embodiment, polycations such as polyarginine or polylysine.
- the peptides can also attach to components that make up the
- Support CTL induction or activation be bound, e.g. on T helper peptides, lipids or liposomes, or they are used together with these substances and / or together with immunostimulating substances, e.g. Cytokines (IL-2, IFN- ⁇ ) administered.
- immunostimulating substances e.g. Cytokines (IL-2, IFN- ⁇ ) administered.
- B99 (fragments) or B99 peptides can also be used to trigger a CTL response ex vivo.
- An ex vivo CTL response to a tumor expressing B99 is induced by incubating the CTL progenitor cells together with APCs and B99 peptides or B99 protein.
- the activated CTLs are then allowed to expand, after which they are re-administered to the patient.
- APCs can be loaded with B99 peptides, which can lead to an efficient activation of cellular immune responses against B99 positive tumors (Mayordomo et al., 1995; Zitvogel et al., 1996).
- a suitable method for peptides on cells e.g. Loading dendritic cells is disclosed in WO 97/19169.
- B99 peptides are combined with peptides derived from other TAAs.
- the selection of peptides for such combinations is made with a view to the detection of different MHC types in order to cover the widest possible patient population and / or it is based on the widest possible range of indications by combining peptides from several different tumor antigens.
- Composition can vary over a wide range, typically a clinically applicable vaccine contains 1 to 15, preferably 3 to 10 different peptides.
- the peptides according to the invention can also be used as diagnostic reagents.
- the peptides can be used to test a patient's response to the humoral or cellular immune response elicited by the immunogenic peptide. This makes it possible to improve a treatment protocol.
- the dosage form (peptide, total protein or DNA vaccine) of the TAA for example, the increase in precursor T cells in the PBLs that are reactive to the defined peptide epitope can be investigated (Robbins and Kawakami, 1996 and references cited therein) .
- the peptides or the total protein or antibodies directed against the TAA can be used to characterize the course of the disease of a B99-positive tumor (for example by immunohistochemical analyzes of the primary tumor and metastases).
- the present invention relates to isolated DNA molecules coding for a protein with the immunogenic properties of B9-9 or for fragments thereof.
- the present invention relates to an isolated DNA molecule which contains a polynucleotide with the sequence shown in SEQ ID NO: 1 or which contains a polynucleotide which hybridizes with a polynucleotide of the sequence shown in SEQ ID NO: 1 under stringent conditions ,
- the present invention relates to an isolated DNA molecule which comprises a
- the DNA molecules or fragments thereof according to the invention code for (poly) peptides of the designation B99 (B99-1, B99-2 or B99-3) with that in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 amino acid sequence shown or for protein fragments or peptides derived therefrom; this also includes DNA molecules which, due to the degeneration of the genetic code, deviate from the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 or SEQ ID NO: 5.
- the invention also relates to DNA molecules which, due to the conservative exchange of amino acids, have deviations from the sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 (or SEQ ID NO: 5), provided they are for a B99 derivative or encode fragments or peptides with the immunogenic properties desired for use as tumor vaccines.
- the B99 DNA molecules of the present invention or the corresponding RNAs, which are also the subject of the present invention, are used, like the (poly) peptides encoded therein, for the immunotherapy of cancerous diseases.
- DNA molecules coding for natural B99 polypeptides are used.
- B99-CDNA DNA molecules coding for natural B99 polypeptides
- Fragments thereof can be used in modified derivatives. These include sequences with modifications that code for a protein (fragment) or peptides with greater immunogenicity, the same considerations for the modifications at the DNA level as for the peptides described above. Another type of modification is the purification of numerous sequences, coding for immunologically relevant peptides, in the manner of a string of pearls ("string-of-beads"; Toes et al., 1997). The sequences can also be modified by adding auxiliary elements, for example functions which ensure more efficient delivery and processing of the immunogen (Wu et al., 1995).
- the present invention relates to a recombinant DNA molecule which contains B99 DNA.
- the B99 DNA molecules of the present invention can be administered, preferably in recombinant form as plasmids, directly or as part of a recombinant virus or bacterium.
- any gene therapy method for the immunotherapy of cancer based on DNA (“DNA vaccine”) on B99-DNA can be used, both in vivo and ex vivo.
- Examples of in vivo administration are the direct injection of "naked" DNA, either intramuscularly or by means of a gene gun, which has been shown to lead to the formation of CTLs against tumor antigens.
- Examples of recombinant organisms are vaccinia virus, adenovirus or Listeria monocytogenes (an overview was given by Coulie, 1997).
- synthetic carriers for nucleic acids such as cationic lipids,
- Microspheres, microspheres or liposomes for the in vivo administration of nucleic acid molecules coding for B99 peptide can be used. Similar to peptides, various adjuvants that enhance the immune response can be co-administered, for example cytokines, either in the form of proteins or plasmids encoding them.
- the application can optionally be combined with physical methods, eg electroporation.
- An example of ex vivo administration is the transfection of dendritic cells, as described by Tuting, 1997, or other APCs that are used as cellular cancer vaccines.
- the present invention thus relates to the use of cells which express B99, either on their own or, in optionally modified form, after transfection with the corresponding coding sequence, for the production of a cancer vaccine.
- the invention relates to antibodies against B99 or fragments thereof.
- Polyclonal antibodies can be obtained in a conventional manner by immunizing animals, in particular rabbits, by injecting the antigen or fragments thereof, and then purifying the immunoglobulin.
- Monoclonal anti-B99 antibodies can be obtained according to standard protocols according to the principle described by Köhler and Milstein, 1975, by
- these animal antibodies can optionally be chimerized in a conventional manner (Neuberger et al., 1984, Boulianne et al., 1984) or humanized (Riechmann et al., 1988, Graziano et al., 1995).
- Human monoclonal anti-B99 antibodies fragments can also be obtained from so-called “phage display libraries” (Winter et al., 1994, Griffiths et al., 1994, Kruif et al., 1995, Mc Guiness et al., 1996) and by means of transgenic animals (Brüggemann et al., 1996, Jakobovits et al., 1995).
- the anti-B99 antibodies according to the invention can be used in immunohistochemical analyzes for diagnostic purposes.
- the invention relates to the use of B99-specific antibodies in order to selectively bring any substances into or into a tumor which expresses B99. Examples of such
- Substances are cytotoxic agents or radioactive nuclides, the effect of which is to damage the tumor on site. Due to the tumor-specific expression of B99, no or only minor side effects are expected.
- substances can be used to visualize tumors that express B99 using B99 antibodies. This is useful for the diagnosis and evaluation of the course of therapy. Therapeutic and diagnostic applications of antibodies which are suitable for anti-B99 antibodies are described in WO 95/33771 for.
- the TAA of the designation B99 according to the present invention and the protein fragments, peptides or peptide equivalents or peptidomimetics derived therefrom can be used in cancer therapy, e.g. B. to induce an immune response against tumor cells that express the corresponding antigen detectors inanten. They are preferably used for the therapy of B99-positive tumors, especially in the
- B99 DNA
- B99 (DNA) can therefore be used in screening assays to identify substances that modulate, in particular inhibit, the activity of this protein.
- such an assay can consist of introducing the B99 protein, or an active fragment thereof, into cells which react to the activity of B99 with proliferation or to bring the corresponding B99 DNA into expression in the cell, and the Determine cell proliferation in the presence and absence of a test substance.
- Substances with an anti-proliferative effect can be used for the treatment of tumors with strong B99 expression, especially in the kidney
- Fig. 1 RT-PCR analysis of cDNA pools of various human tumor and normal tissues using B99-specific primers
- Fig. 4 Immunohistochemical analysis of four different cases of adenocarcinoma with B99 serum
- Fig. 5 MHC stabilization on T2 cells using different concentrations of B9-9 peptides
- RDA Representative Difference Analysis
- the human lung adenocarcinoma cell line A549 (CCL 185) obtained from ATCC was in T150
- the 4 ml were transferred to a 15 ml falcon tube, mixed with 8 ml PBS, centrifuged at 1200 rpm in a Haereus table centrifuge (Megafuge 2.0R) for 5 min at 4 ° C, the cell pellet with 1 ml lysis buffer (10 mm TrisHCl pH8, 140 mM NaCl, 1.5 mM MgCl 2 , 0.5% NP40) were added, shaken vigorously and centrifuged in a 2 ml Eppendorf tube at 12,000 rpm and 4 ° C. for 5 min in a Sigma table centrifuge (Sigma 202 MK).
- 1 ml lysis buffer (10 mm TrisHCl pH8, 140 mM NaCl, 1.5 mM MgCl 2 , 0.5% NP40
- the supernatant was transferred to a new Eppendorf tube and, after addition of 55 ⁇ l 20% SDS solution, extracted twice with twice the volume of a CHCl 3 / phenol (1: 1 v / v) mixture and once with the single volume of CHC1 3 .
- the aqueous RNA-containing phase was mixed with 1/10 volume of 3M NaAc (pH5) and twice the volume of 96% EtOH and the RNA was precipitated overnight at -20 ° C.
- the isolation of poly-A (+) RNA using the PolyATtract Kit (Promega) was carried out in accordance with the manufacturer's protocol.
- the A549 poly-A (+) RNA with a concentration of 1 mg / ml in DEPC-treated H 2 0 was stored in aliquots at -80 ° C.
- Restriction enzyme interfaces were necessary because point mutations were often observed, particularly in the primer sequences due to the PCR amplification steps.
- the cDNA obtained was digested by "fixed” and “driver” with Rsal (Rsal is a 4-base-recognizing restriction enzyme and provides a statistical average of 256 bp long cDNA fragments).
- tester cDNA Identical parts of "tester cDNA” were ligated either with adapters 1 or 2 and then hybridized separately with an excess of "driver cDNA” at 65 ° C. The two approaches were then combined and subjected to a second hybridization with freshly denatured "driver cDNA”. The enriched "solid" -specific cDNAs were then amplified exponentially by PCR with primers specific for adapters 1 or 2. For further enrichment, an aliquot of this reaction was subjected to a second PCR with specific nested primers.
- the exponentially amplified cDNA fragments resulting from this reaction were directly inserted into the pCRII vector (Invitrogen; carefulTA cloning vector ”) and then a third of the ligation mixture was transfected into competent E. coli (OneShot TM, Invitrogen).
- Obtained subtraction library which was available both in the form of E. coli glycerol stock cultures and in the form of purified plasmids.
- the isolated plasmid DNA of all 712 clones was sequenced according to the Sanger method on an ABI-Prism device. The sequences obtained were annotated using the BioScout software (LION, Heidelberg) and subjected to database comparisons (Genbank). From 712 clones, 678 could be sequenced and annotated. The rest (34) had either only poly (A) sequences as an insert or corresponded to a religious vector or could not be sequenced. Of the 678 annotable sequences, 357 proved to be genes with a known function. The remaining 321 represented clones encoding genes with unknown function; 59 of them did not even have entries in the human EST database. Known genes were not further treated. The expression profile was estimated for those unknown genes for which an EST entry was available: all those ESTs were> 95%
- BLAST Altschul et al. (1994), which belonged to the experimentally determined sequence of the subtraction libraries, was checked. The annotation was divided into i) critical normal tissues, ii) fetal, "dispensable” and immune-privileged tissues and iii) tumors and tumor cell lines. On the basis of this "virtual mRNA profile", 200 clones for which no ESTs were used in group i) were selected for further experimental analyzes (including the 59 clones for which there was no EST entry). To further narrow the candidate clones, oligonucleotide primer pairs were designed and synthesized from the sequences determined from the 200 selected clones.
- cDNA libraries 8 different human tissue-derived cDNA libraries (GibcoBRL “SUPERSCRIPT TM”), which are directionally cloned into pCMV-SPORT, were initially analyzed by means of qualitative PCR tested for the presence of each candidate.
- the cDNA libraries used came from tissue from the heart (# 10419-018), liver (# 10422-012), leukocytes (# 10421-022), kidney (# 10420-016), lung (# 10424-018), Testis (# 10426-013), brain (# 10418-010) and fetal brain (# 10662-013).
- the PCR conditions were as follows: 20 ⁇ l i total volume per PCR mixture contained 1 ⁇ TaqPol
- Buffer 50 mM KC1, 10 mM Tris-HCl pH 9, 0.1% Triton X-100), 1.5 mM MgCl 2, 0.2 mM dNTPs (Promega), 0.025 U / ul Taq DNA polymerase (Promega), respectively 5 pM of specific oligonucleotide primers SEQ ID NO: 7 and SEQ ID NO: 8 and 100 ng of the plasmid DNA to be examined in each case.
- specific primers for GAPDH SEQ ID NO: 14 and 15
- the primer pairs were also tested in parallel for the isolated plasmid.
- cDNA pools were used which were produced from 3 ⁇ g total RNA from 3 different tissues of the same type.
- the 9 ⁇ g total RNA per tissue pool from tumor or normal tissues was reverse transcribed using AMV-RT (Promega) according to the manufacturer's recommendation.
- AMV-RT Promega
- the RNA was previously incubated with DNAse I (Boehringer Manheim).
- the quality and amount of the cDNAs were checked by PCR with GAPDH-specific primers (SEQ ID NO: 14 and 15) after 20 cycles (30 "95 ° C, 90" 60 ° C).
- B99 cDNA was plicated by 25, 30 and 35 cycles of the program 1 '95 ° C, 1' 55 ° C, 1 '72 ° C with the B99-specific primers according to SEQ ID NO: 7 and 8 a.
- the other 55 candidate clones, each with specific primers, were examined analogously.
- the PCR products were by means of agarose gel electrophoresis and
- FIG. 1 An example for candidate B99 is shown in FIG. 1: The RT-PCR analysis of cDNA pools of various human tumor and normal tissues using B99-specific primers gave a strong signal in colon carcinoma and in lung adenoma carcinoma line A549 as well as a weak signal in Breast carcinoma and renal cell carcinoma. A weak signal was only of all examined normal tissues noticeable in colon tissue. In the further course, the candidate B99 was evaluated in more detail.
- Colon tissue Using RT-PCR analysis of individual cDNAs from various human tumor and normal tissues using B99-specific primers, B99-CDNA was detected in 6 of 7 tumor samples, whereas only one of the examined normal tissues (1/6) showed a weak expression of B99 , Table 1
- Example 4 shows that B99 is clearly transcribed in a high percentage of tumors of various indications, whereas no or only isolated transcription was found in all examined normal tissues.
- B99-specific antibodies were generated in rabbits.
- the bacterial fusion protein pGEX-ORF2-1 / 1 (position 1278 to 1740 SEQ ID No: 1) was used for the immunization, and the serum obtained was affinity-purified using peptide B99-KML (SEQ ID NO: 61).
- SEQ ID NO: 61 peptide B99-KML
- Clone B99 has a 271 bp insert of an unknown human gene between the adapters introduced by the RDA.
- the complete cloning of the human sequence was carried out as follows: a UniGene analysis (National Center for Biotechnology Information) revealed the following ESTs homologous to B99: AA315469, AA345780, AA295520. With the help of these ESTs, the B99 sequence could be extended to 439 nucleotides. New primers within this sequence were synthesized (SEQ ID NO: 9 to 12). The respective theoretical fragment lengths from A549 cDNA could be amplified by PCR with various combinations of these primers.
- one of the clones isolated from A549 cells showed a nucleotide exchange at position 622 in comparison to sequence SEQ ID NO: 1.
- This nucleotide exchange requires a replacement of arginine (SEQ ID NO: 2, B99-1) with tryptophan (SEQ ID NO: 4, B99-2) at position No. 66 of the amino acid sequence.
- the amino acid sequence from B99-2 to position 166 is identical to B99-1.
- the aforementioned insertion requires a second potential reading frame from items 845 to 1744 the sequence shown in SEQ ID NO: 3 (or SEQ ID NO: 5).
- a protein expressed by a cDNA with this reading frame has the amino acid sequence shown in SEQ ID NO: 6 (B99-3).
- the sequence of B99-3 differs from items 1 to 27 to B99-1 and from item 28 is identical to B99-1.
- B99-MHC binding peptides were tested in a T2 peptide loading test for their ability to stabilize HLA-A2 molecules on the surface of T2 cells, which is an indication of their MHC binding ability.
- the experiment was carried out as described by Böhm et al. , 1998. Stabilization was measured by FACS analysis with an HLA-A2 specific antibody (BB7.2). Five peptides showed a stabilizing effect when used in a concentration of 100 ⁇ g / ml, represented by an increase in the mean fluorescence intensity compared to the control without peptide or to a MAGE-3 Al control peptide which shows no binding. (Table 4):
- these peptides were examined in a dilution series in the same test system in order to show a possible concentration dependence of the binding.
- 5 shows the MHC stabilization on T2 cells by means of different concentrations of B99 peptides.
- the peptides B99-19, B99-187 and B99-209 in particular show a clear one
- Paterson Y Ikonomidis G (1996), Curr. Opin. Immunol. 5: 664-9
- Rapellino M, Pecchio, F, Baldi, S, Scappaticci, E, and
- van Elsas A., van der Minne, CE, Borghi, M., van der Spek, CW, Braakman, E., Osanto, S., and Schrier, PI (1996), CTL Recognition of an IL-2 Producing Melanoma Vaccine.
- van Elsas A., van der Minne, CE, Borghi, M., van der Spek, CW, Braakman, E., Osanto, S., and Schrier, PI (1996), CTL Recognition of an IL-2 Producing Melanoma Vaccine.
- van Elsas A., van der Minne, CE, van der Spek, CW, Brouwenstijn, N., Osanto, S., and Schrier, PI (1997), J. Immunother. 2TJ: 343-353.
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MXPA01010740A MXPA01010740A (es) | 1999-04-28 | 2000-04-19 | Antigeno asociado con tumores. |
AU45531/00A AU4553100A (en) | 1999-04-28 | 2000-04-19 | Tumour-associated antigen |
EP00926997A EP1177288A1 (de) | 1999-04-28 | 2000-04-19 | Tumorassoziiertes antigen |
CA002365278A CA2365278A1 (en) | 1999-04-28 | 2000-04-19 | Tumour-associated antigen |
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WO2003063770A2 (en) | 2001-11-07 | 2003-08-07 | Mannkind Corporation | Expression vectors encoding epitopes of target-associated antigens and methods for their design |
JP2006238757A (ja) * | 2005-03-02 | 2006-09-14 | Eiken Chem Co Ltd | 癌を検出するためのマーカー |
DE102005041616B4 (de) * | 2005-09-01 | 2011-03-17 | Johannes-Gutenberg-Universität Mainz | Melanom-assoziierte MHC Klasse I assoziierte Oligopeptide und für diese kodierende Polynukleotide und deren Verwendungen |
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WO2000034449A2 (en) * | 1998-12-04 | 2000-06-15 | Henrik Clausen | UDP-N- ACETYL GLUCOSAMINE: GALACTOSE-β1, 3-N- ACETYL GALACTOSAMINE- α-R/ N- ACETYL GLUCOSAMINE -β1, 3-N- ACETYL GALACTOSAMINE- α-R (GlcNAc TO GalNAc) β1,6-N- ACETYL GLUCOSAMINYL TRANSFERASE, C2/4GnT |
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