EP1177288A1 - Antigene specifique de tumeurs - Google Patents

Antigene specifique de tumeurs

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Publication number
EP1177288A1
EP1177288A1 EP00926997A EP00926997A EP1177288A1 EP 1177288 A1 EP1177288 A1 EP 1177288A1 EP 00926997 A EP00926997 A EP 00926997A EP 00926997 A EP00926997 A EP 00926997A EP 1177288 A1 EP1177288 A1 EP 1177288A1
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EP
European Patent Office
Prior art keywords
peptides
seq
tumor
peptide
immunogenic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00926997A
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German (de)
English (en)
Inventor
Günther Adolf
Karl-Heinz Heider
Wolfgang Sommergruber
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Boehringer Ingelheim International GmbH
Original Assignee
Boehringer Ingelheim International GmbH
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Application filed by Boehringer Ingelheim International GmbH filed Critical Boehringer Ingelheim International GmbH
Publication of EP1177288A1 publication Critical patent/EP1177288A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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 an immunogenic tumor antigen.
  • 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.
  • CD8-expressing cytotoxic T-lymphocytes (CTLs) play a major role (Coulie, 1997).
  • CTLs cytotoxic T-lymphocytes
  • a spontaneous remission rate showed a correlation between clinical course and the increased occurrence of CD8 + and CD4 + T cells (Schendel et al., 1993; Mackensen et al., 1993; Halliday et al., 1995; Kawakami et al., 1995; Kawakami et al., 1996; Wang, 1997; Celluzzi and Falo, 1998).
  • Specific CTL clones were obtained either from tumor infiltrating lymphocytes (TIL) or peripheral mononuclear blood cells (PBMC) after cocultivation with mostly autologous tumor cells and cytokine stimulation in vitro.
  • 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 melanoma 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 molecules Major histocompatibility 1 complex
  • HLA human leukocyte antigen
  • MHC I Molecules occur on most cells with a nucleus and present peptides (usually 8-10-mers) which are formed by 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, but not all 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 vaccines; Tighe et al., 1998) , or viral vectors (Restifo, 1997) are applied.
  • DNA vaccines 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 standard 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).
  • the methods for identifying and characterizing TAAs 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.
  • CTLs cellular immune response
  • antibodies humoral immune response
  • 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 .
  • tumor-specific tumor-associated
  • the object of the present invention was to provide a new tumor-associated antigen (TAA).
  • the human B99 CDNA was clomerized, 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 levels, 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 has a length of 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 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 subsequently 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 B99 to be amplified 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 not to the desired target
  • genomic libraries can be examined in genomic libraries by, for example, as in the screening of cDNA libraries, isolation by cloning by hybridization with DNA probes specific for B99, which clones upstream from the 5 'end obtained 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 NO: 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 NO: 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 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 (cf. 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 such as e.g. the
  • tumor-associated antigens can have tumor-specific mutations that contribute to an immunological differentiation between tumor and normal tissue (Mandruzzato et al., 1997; Hogan et al., 1998; Gaudi et al., 1999; W ⁇ lfel et al., 1995 ).
  • 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.
  • the invention thus relates to B99 peptides derived from regions of a tumor-expressed B99 which have tumor-specific mutations.
  • 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 MHC subtype of the patient thus represents one of the essential prerequisites for the effective application of a peptide to this patient with regard to the drawing of 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 Tie motif.
  • Methods have recently been developed, starting from the X-ray structure analysis of MHC-II molecules, which allow the exact 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 there ).
  • 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 in the sequence by, for example
  • Cytokm secretion and enhancing the efficiency of antigen presentation by 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
  • IS are carried out, provided that the effective immunogenicity of the peptide, which is composed of its binding affinity for the MHC molecule and its ability to stimulate T-cell receptors, is not only not impaired, but preferably enhanced, by these variations.
  • artificial peptides or peptide equivalents are used, which are designed according to the requirements of the binding ability to an MHC molecule.
  • 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 CTL induction
  • MHC binding and immunogenicity MHC binding and immunogenicity
  • Another method suitable for the purposes of the present invention for finding peptides with a greater immunogenicity than that of natural B99 peptides consists in screening 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 “peptidomimetics” 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 creo 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, particularly in the case of kidney, lung, colon, pancreas, breast and stomach carcinoma.
  • the immune response in the form of induction of CTLs can be caused in vivo or ex vivo.
  • a pharmaceutical composition containing as active component TAA B99 or fragments or peptide (s) derived therefrom is administered to a patient suffering from a tumor disease associated with 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, carrier.
  • 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. Charging 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 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).
  • a strategy has been around proven several times, for example the detection of the estrogen receptor as a basis for decision-making for endocrine therapy in breast cancer; c-erbB-2 as a relevant marker in the prognosis and course of therapy for breast cancer (Ravaioli et al., 1998; Revillion et al., 1998); PSMA ("prostate specific membrane antigen") as a marker for epithelial cells of prostate cancer in serum or by using a ni In-labeled monoclonal antibody against PSMA in immunoscintigraphy for prostate cancer (Murphy et al., 1998 and included references); CEA (“carcinoembryonic antigen”) as a serological marker for the prognosis and course in patients with colorectal cancer (Jessup and Lo
  • the present invention relates to isolated DNA molecules coding for a protein with the immunogenic properties of B9S 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 the in SEQ ID NO: 2, SEQ ID NO: 4 or SEQ ID NO: 6 shown amino acid sequence 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 that 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 which code for a protein (fragment) or peptides with a higher immunogenicity, the same considerations applying to the modifications at the DNA level as for the peptides described above. Another type of modification is the juxtaposition 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 m 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,
  • Microsphere, microsphere or liposome 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 which 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 Kohler 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 (Bruggemann 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 for the visualization of tumors which express B99 can be used. This is useful for the diagnosis and evaluation of the course of therapy. Therapeutic and diagnostic uses of antibodies which are suitable for anti-B99 antibodies are described in WO 95/33771 for.
  • the TAA of the name 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 determinants. 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 a proliferation-inhibiting effect can be used for the treatment of tumors with strong B99 expression, in particular 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
  • RDA Representative Difference Analysis
  • the human lung adenocarcinoma cell line A549 (CCL 185) obtained from ATCC was in T150
  • the 4 ml were transferred into 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 , 140mM NaCl, 1.5 mM MgCl 2 , 0.5% NP40), shaken vigorously and centrifuged in a 2 ml Eppendorf tube at 12,000 rpm and 4 ° C. for 5 min in a Sig a table centrifuge (Sigma 202 MK). The supernatant was transferred to a new Eppendorf tube and, after adding 55 ⁇ l of 20% SDS solution, twice with the double
  • tester the poly-A (+) RNA of the lung adenocarcinoma cell line A549 was called "tester", that of normal lung tissue
  • the newly generated primer / adapter sequences enable the presence of three new restriction enzyme interfaces (Kpn I, Sac I and Xho I) in the sequence of the nested PCR primer-2-alt after cloning the subtracted cDNA fragments in the pPCRII- Vector a subsequent cutting out of the respective cDNA fragments.
  • the design of a primer / adapter sequence with several available restriction enzyme interfaces was necessary because point mutations could often be observed especially in the primer sequences due to the PCR amplification steps.
  • 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 "tester” -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 approach in competent E. coli (OneShot TM, Invitrogen) transfected.
  • 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 total volume per PCR mixture contained Ix TaqPol buffer (50 mM KC1, 10 mM TrisHCl pH9, 0.1% Triton X-100), 1.5 mM MgCl 2 , 0.2 mM dNTPs (Promega), 0.025 U / ⁇ l Taq DNA polymerase (Promega), each 5 pM at 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 were used.
  • the primer pairs were also tested in parallel for the isolated plasmid.
  • the detectability of fragments of the expected length in one of the critical normal tissues (heart, liver, lung, kidney and leukocytes), but not in the cDNA libraries from immune-privileged tissues (brain, fetal brain and testis) under these PCR conditions (1 Cycle: 3 '94 ° C; 35 cycles: 1' 94 ° C - 1 '55 ° C - 1' 72 ° C; 1 cycle: 7 '72 ° C) was defined as the elimination criterion.
  • the number of candidates was reduced to 56. In game 3
  • 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 amplified 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.
  • 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, while no or only a few transcriptions were 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.
  • PCR amplification of cDNA from tumor cell lines (786-0, A549) and tissue samples from colon carcinomas with the primers according to SEQ ID NO: 16 to 18 gave the fragments expected after the cloning with the original primers.
  • cDNA fragment which now consists of 2216 bp
  • No further reading frames could be identified further in the 5 'region of the sequence, from which it can be concluded that the region from 0 to 427 already belongs to the 5' untranslated region of the B99 mRNA.
  • 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
  • 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. 20: 343-353.

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Abstract

L'invention concerne un antigène spécifique de tumeurs, des peptides immunogènes qui en sont dérivés et des molécules d'ADN les codant, ainsi que leur utilisation en immunothérapie d'affections cancéreuses.
EP00926997A 1999-04-28 2000-04-19 Antigene specifique de tumeurs Withdrawn EP1177288A1 (fr)

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