WO2003074556A1

WO2003074556A1 - Tumor tag and the use thereof

Info

Publication number: WO2003074556A1
Application number: PCT/CN2002/000137
Authority: WO
Inventors: Jun Wu
Original assignee: Shanghai Genomics, Inc.
Priority date: 2002-03-04
Filing date: 2002-03-04
Publication date: 2003-09-12
Also published as: AU2002238359A1; CN1622955A; CN100358917C

Abstract

The present invention relates to new tumor tag-ULBP-4 proteins, the polynucleotides encoding the ULBP-4 proteins, and to the method of producing the ULBP-4 proteins by recombination technology. The present invention also discloses the use of the ULBP-4 proteins and the polynucleotides encoding them, e.g., diagnosing and treating for tumor. The present invention provides pharmaceutical compositions containing the ULBP-4 protein or the antibody against it.

Description

Tumor markers and uses thereof

Technical field

The present invention belongs to the field of biotechnology and medicine, and in particular, the present invention relates to a new tumor marker-ULBP-4 protein, a polynucleotide encoding a ULBP-4 protein, and a method for producing such a ULBP-4 protein by recombinant technology. method. The invention also discloses the use of the ULBP-4 protein and its coding sequence, such as for diagnosis and treatment of tumors, and a pharmaceutical composition containing the ULBP-4 protein or an antibody thereof. Background technique

Since the 1950s, medical science has made rapid progress. The progress of cancer treatment has also been very effective, and many oncogenes and tumor suppressor genes directly related to cancer have been discovered.

As early as 40 years ago, Lewis Thomas & Macfarlane Burnet proposed that the immune system can monitor and suppress the occurrence of cancer. The results of research on NK cells and T cells in the past two or three years have provided new supporting evidence for this hypothesis and revealed possible molecular mechanisms involved in this process.

NK cells (natural killer cells) are the first line of defense for immunity. NK cells secrete cytotoxins, which can quickly recognize and kill their target cells. Since the role of NK cells does not require antigen or mitogen stimulation, nor is it dependent on antibodies or complement, it should be able to distinguish between abnormal and normal tissues. It is currently believed that the activity of NK cells is regulated by the balance of inhibitory and activating receptors on the surface. MHC-I binds to receptors on the cell surface, including mouse Ly49 (recognizes H-2K, H-2D) and human KIR (immunoglobulin_like receptor, recognizes HU-A, -B, or -C), thereby inhibiting its active. In virus-infected cells and tumor cells, MHC-I expression is usually disrupted and cannot bind to inhibitory receptors on the surface of NK cells to activate and kill NK cells.

In addition to NK cells, T cells can prevent carcinogen-induced skin tumors. In humans and mice, Cellδ-T cell receptor (TCR) -positive T cells are located on the skin, digestive tract epithelial tissue, and mucosal surfaces, and participate in local immunity. Current experimental results show that these T cells also play an important role in destroying cells transformed by external carcinogens. Studies have also shown that Υδ-TCR + T cells can prevent the development of intestinal epithelial tumors. Two MHC-1 related molecules, MICA / MICB, have been found to act as signals of cellular abnormalities and cause immune responses.

Bauer et al. (Bauer S, et al., Science 1999 Jul 30; 285 (5428): 727-9) identified MICA / MICB receptor NKG2D by a representative difference analysis technique (RDA), a species Prior to this, its expression and function were unknown orphan C-type lectin-like NKcell receptor.

Several MHC-I and related molecular specific NK cell receptors have been found to be different from other NK cell receptors. NKG2D is located on the surface of all NK cells, γ δ -TCR + T cells, and CD8 + β -TCR + T cells. Is an activating receptor that interacts with the transmembrane adaptor protein DAP10 (Wu J, et al.,

Confirm this Science 1999 Jul 30; 285 (5428): 730-2). The cytoplasmic portion of the DAP10 protein contains a YxxM motif that activates the PI3 kinase pathway.

There is no human MICA / B homologous gene in mice. However, another glycoprotein family called RAE-1 was found in mice (Cerwenka A, et al., Immunity. 2000 Jun; 12 (6): 721-7), which also acts as a ligand for NKG2D (ligand ) And showed a weak homology with MHC-1. So far, it has been found that five profiled mouse RAE-1 proteins (RAE-1- α, - β , _ Υ, - δ, - ε) and a related protein, Η60. Expression analysis showed that the RAE-1 gene was not expressed or expressed very low in normal adult tissues, and was constitutively expressed in some tumor tissues, and its expression was increased by the effect of retinoic acid. There is no RAE-1 expression in normal skin, and it is expressed in tumors of mice after being induced by the carcinogen TPA. Recently Diefenbach et al. (Diefenbach A, et al., Nature 2001 Sep 13; 413 (6852): 165-71) and Cerwenka et al. (Cerwenka A, et al., Proc Natl Acad Sci USA 2001 Sep 25; 98 (20) : 11521-6) Studies have shown that in tumors expressing MHC Class I, if RAE-1 is also expressed, the tumors can also be eliminated by NK cells in vitro. Like NK cells, mouse γ δ -TCR + Τ cells can kill squamous cell carcinoma line cultured in vivo. Under certain circumstances, RAE-1 can also cause γ δ-TCR + Τ cells to transplant. Tumor memory.

Rae-1 homologs have also been found in humans, namely human ULBP-1, -2, -3. As a human cytomegalovirus (CMV) glycoprotein UL16 binding protein was first discovered, the ULBP gene is related to the C-1 family of bandits, but not closely related to the MICB that also binds to UL16. ULBP is also a ligand of NKG2D, which can stimulate NK cells to express cytokine and chemokine. The expression of ULBP in target cells against NK cells can make them attacked by NK cells. Cytomegalovirus-infected cells may escape the immune system's attack by blocking the surface of the cell's ULBP or MIC antigen with the UL16 protein.

In summary, these studies in humans and mice have shown that, whether targeting viruses or tumors, in NK cells, γ δ -TCR + T cells, CD8 + a β -TCR + T cell-mediated immune protection, NKG2D all played a key role. However, so far, the activation mechanism of NKG2D is not clear.

Since the 1990s, some breakthroughs have been made in the research of tumor immunology, and tumor immunotherapy has entered a new climax. Many immunotherapeutic methods have entered clinical trials, mainly through various methods of training in vivo or in vitro, activating patients' immune cells to identify malignant cells, and causing them to expand in large numbers, eventually removing or suppressing malignant cells.

In tumor immunology research, human tumor immune rejection antigen was first discovered in 1991. Many subsequent studies have shown that most tumor cells have components different from normal cells, which can cause the immune system to recognize and attack them, which is called tumor immune rejection antigen. The use of tumor immune rejection antigens can induce the generation of immune cells that can kill tumors in and outside patients. Therefore, tumor immune rejection antigen is the most critical component in tumor immunotherapy. So far, many tumor antigens have been found in melanoma and various tumor tissues including prostate cancer, thymic cancer, ovarian cancer, and gastrointestinal cancer. There are four types of human tumor immune rejection antigens that have been discovered so far. The first type of antigens are derived from somatic mutations of normal gene products, and the second type of antigens are derived from genetic mutations associated with carcinogenesis. Both types of antigens are patient-specific antigens and are not suitable for general treatment. The third type of antigen is the product of a gene that is also expressed in normal tissues but increased in tumors. If mutations are not involved, such antigens are universal in various cancer patients, but they are usually tissue-specific and not unique to tumors, and have little meaning in clinical applications. The fourth type of antigen has strict tumor specificity and is related to the general carcinogenesis process. Therefore, such antigens are widely expressed in human tumors and are most suitable as tumor markers and targets for anti-tumor immune attack. However, few of the antigens found so far fall into this category.

Therefore, there is an urgent need in the art to develop new Class 4 tumor immune rejection antigens as tumor markers for the diagnosis and treatment of tumors. Summary of invention

The object of the present invention is to provide a new human tumor marker ULBP-4 protein and fragments, analogs and derivatives thereof.

Another object of the invention is to provide polynucleotides encoding these polypeptides.

Another object of the present invention is to provide a method for producing these polypeptides and the use of the polypeptides and coding sequences. Through extensive and intensive research, the present invention has discovered and isolated a new antigen gene that can be used as a tumor marker: ULBP-4. This gene is not expressed or expressed in normal tissues at low levels and is widely expressed in tumor tissues. Its expression product is a membrane protein and may also be secreted extracellularly. The ULBP-4 membrane protein expressed on the cell surface can efficiently bind to the NKG2D receptor. The present invention has been completed on this basis.

In a first aspect of the invention, there is provided a novel isolated ULBP-4 polypeptide comprising:

NO: a polypeptide having a 2 or 4 amino acid sequence, or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof. Preferably, the polypeptide has the amino acid sequence of positions 1-263 or 24-263 in SEQ ID NO: 2 or 4.

In a second aspect of the present invention, there is provided a polynucleotide encoding these isolated polypeptides, the polynucleotide comprising a nucleotide sequence having at least 70 nucleotides with a nucleotide sequence selected from the group consisting of % Identity: (a) a polynucleotide encoding the aforementioned human ULBP-4 polypeptide; and (b) a polynucleotide complementary to the polynucleotide (a). Preferably, the polynucleotide encodes a polypeptide having the amino acid sequence of positions 1 to 263 or 24 to 263 in SEQ ID NO: 2 or 4. More preferably, the sequence of the polynucleotide is one selected from the group consisting of: (a) a sequence having positions 403-1194 in SEQ ID NO: 1; (b) a sequence having positions 1-1434 in SEQ ID NO: 1 (C) A sequence having positions 473-1194 in SEQ ID NO: 1; (d) A sequence having positions 1-792 in SEQ ID NO: 3; (e) A sequence having 70-792 in SEQ ID NO: 1 Sequence of bits. In a third aspect of the present invention, there are provided a vector containing the above polynucleotide, and a host cell transformed or transduced by the vector or a host cell directly transformed or transduced by the above polynucleotide.

In a fourth aspect of the present invention, a method for preparing a ULBP-4 protein is provided, the method comprising: (a) culturing the transformed or transduced host cell under conditions suitable for protein expression; (b) from a culture ULBP-4 protein was isolated.

In a fifth aspect of the present invention, there is provided an antibody that specifically binds to the aforementioned human ULBP-4 polypeptide. Nucleic acid molecules that can be used as primers or probes are also provided, which contain consecutive 15-1434 nucleotides in the polynucleotides described above.

In a sixth aspect of the present invention, there are provided compounds that mimic, promote, and antagonize the activity of human ULBP-4 polypeptide, and compounds that inhibit the expression of human ULBP-4 polypeptide. Methods of screening and / or preparing these compounds are also provided. Preferably, the compound is an antisense sequence of a human ULBP-4 polypeptide coding sequence or a fragment thereof.

In a seventh aspect of the present invention, a method for detecting the presence of ULBP-4 protein in a sample is provided, which includes: contacting a sample with a specific antibody to ULBP-4 protein, observing whether an antibody complex is formed, and an antibody complex is formed This indicates the presence of ULBP-4 protein in the sample. A method for detecting tumors is also provided, comprising the steps of: detecting the presence of ULBP-4 protein in a patient's sample (such as blood, urine, body fluids, saliva, etc.).

In an eighth aspect of the present invention, the present invention also provides a tumor detection kit, which contains a primer pair that specifically amplifies ULBP-4 and / or a ULBP-4 specific antibody. It may also contain specific probes and / or PCR buffers.

In a ninth aspect of the present invention, the use of the polypeptide and coding sequence of the present invention is provided. For example, the polypeptide of the present invention can be used to screen agonists that promote the activity of human ULBP-4 polypeptide, or antagonists that inhibit the activity of human ULBP-4 polypeptide, or can be used for identification of peptide fingerprints. The coding sequence or fragment of the human ULBP-4 protein of the present invention can be used as a primer for a PCR amplification reaction, or as a probe for a hybridization reaction, or used for manufacturing a gene chip or a microarray.

In a tenth aspect of the present invention, there is provided a pharmaceutical composition containing a safe and effective amount of the human ULBP-4 antagonist of the present invention and a pharmaceutically acceptable carrier. Preferred ULBP-4 antagonists are antibodies against ULBP-4 and ULBP-4 antisense sequences. These pharmaceutical compositions can treat tumors.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure of the techniques herein. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is an electropherogram of ULBP-4.

Figure 2 shows a human ULBP-4 cDNA coding sequence (SEQ ID NO: 3), and the differences compared with the sequence of positions 403-1194 in SEQ ID NO: 1 are underlined.

Figure 3 shows the amino acid sequence (SEQ ID NO: 4) of a human ULBP-4. Differences compared to the sequence of SEQ ID NO: 2 are underlined. Figure 4 shows the expression profile of ULBP-4 in various tumor cell lines. Each lane is: 1. U373 (astrocytoma); 2. JURKAT (acute T-cell leukemia); 3. LOVO (colon cancer); 4. HELA (cervical adenocarcinoma); 5. SW480 (colon cancer); 6. DU145 (prostate cancer); M: molecular weight standard.

Figure 5 shows the expression profile of ULBP-4 in various tissues. The lanes are: 1. liver; 2. skeletal muscle; 3. placenta; 4. spleen; 5. white blood cells; 6. pancreas; 7. lymph nodes; 8. heart; 9. lung; 10. brain; 11. thymus 12. Bone marrow; M: molecular weight standard.

Figure 6 shows that ULBP-4 specifically binds to NKG2D. Detailed description of the invention

In the present invention, the terms "ULBP-4 protein", "ULBP-4 polypeptide" or "tumor marker ULBP-

4 "is used interchangeably and refers to a protein or polypeptide with the human tumor marker ULBP-4 amino acid sequence (SEQ ID NO: 2 or 4). They also include mature ULBP- without a signal peptide (positions 1-23) 4.

As used herein, "isolated" refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment). For example, polynucleotides and polypeptides in a natural state in a living cell are not isolated and purified, but the same polynucleotides or polypeptides are separated and purified if they are separated from other substances existing in the natural state.

As used herein, "isolated ULBP-4 protein or polypeptide" means that the ULBP-4 polypeptide is substantially free of other proteins, lipids, carbohydrates, or other substances with which it is naturally associated. Those skilled in the art can purify the ULBP-4 protein using standard protein purification techniques. Substantially pure peptides can produce a single main band on a non-reducing polyacrylamide gel.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide, and preferably a recombinant polypeptide. The polypeptides of the present invention may be naturally purified products or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production protocol, the polypeptide of the invention may be glycosylated, or it may be non-glycosylated. Polypeptides of the invention may also include or exclude starting methionine residues.

The invention also includes fragments, derivatives and analogs of the human ULBP-4 protein. As used herein, the terms "fragment", "derivative" and "analog" refer to a polypeptide that substantially retains the same biological function or activity of the natural human ULBP-4 protein of the invention. A polypeptide fragment, derivative or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, and such substituted amino acid residues It may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a mature polypeptide and another compound (such as a compound that extends the half-life of a polypeptide, such as (Polyethylene glycol) fusion polypeptide, or (iv) a polypeptide formed by fusing additional amino acid sequences to this polypeptide sequence (such as a leader sequence or a secreted sequence or a sequence or protease sequence used to purify the polypeptide, or Antigen IgG fragment formed fusion protein). In accordance with the teachings herein, these fragments, derivatives, and analogs are within the scope of those skilled in the art.

In the present invention, the term "human ULBP-4 polypeptide" refers to SEQ ID NO having human ULBP-4 protein activity. A full-length or mature polypeptide of the 2 or 4 sequence. The term also includes variants of the SEQ ID NO. 2 or 4 sequence that have the same function as the human ULBP-4 protein. These variants include (but are not limited to): several (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10) amino acid deletions, insertions And / or substitutions, and adding one or several (usually 20 or less, preferably 10 or less, more preferably 5 or less) amino acids at the C-terminus and / or N-terminus. For example, in the art, the substitution of amino acids with similar or similar properties usually does not change the function of the protein. As another example, adding one or more amino acids to the C-terminus and / or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of human ULBP-4 protein.

The variants of the polypeptide include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, encoded by DNA that can hybridize to human ULBP-4 DNA under high or low stringency conditions Protein, and a polypeptide or protein obtained using an antiserum against human ULBP-4 polypeptide. The invention also provides other polypeptides, such as fusion proteins comprising human ULBP-4 polypeptide or fragments thereof. In addition to almost full-length peptides, the invention also includes soluble fragments of the human ULBP-4 polypeptide. Generally, the fragment has at least about 10 consecutive amino acids, usually at least about 30 consecutive amino acids, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 80 consecutive amino acids in a human ULBP-4 polypeptide sequence, most preferably At least about 100 consecutive amino acids.

The invention also provides analogs of human ULBP-4 protein or polypeptide. The differences between these analogs and natural human ULBP-4 peptides may be differences in amino acid sequences, differences in modified forms that do not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by a variety of techniques, such as random mutagenesis by radiation or exposure to mutagens, or by site-directed mutagenesis or other known molecular biology techniques. Analogs also include analogs with residues other than naturally occurring L-amino acids, such as D-amino acids, and analogs with non-naturally occurring or synthetic amino acids. It should be understood that the polypeptide of the present invention is not limited to the representative polypeptides exemplified above.

Modified (usually unchanged primary structure) forms include: Chemically derived forms of polypeptides in vivo or in vitro such as acetylated or carboxylated. Modifications also include glycosylation, such as those produced by glycosylation modification in the synthesis and processing of polypeptides or in further processing steps. This modification can be accomplished by exposing the polypeptide to an enzyme that undergoes glycosylation, such as mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (such as phosphotyrosine, phosphoserine, and phosphothreonine). Also included are peptides that have been modified to increase their resistance to proteolysis or to optimize their solubility.

In the present invention, the "human ULBP-4 protein conservative variant polypeptide" means that there are at most 10, preferably at most 8 and more preferably at most 5 compared to the amino acid sequence of SEQ ID NO: 2 or 4. Optimally up to 3 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. These conservative variant polypeptides are preferably produced by amino acid substitutions according to Table 1. Table 1

The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be coding or non-coding. The coding region sequence encoding the mature polypeptide may be the same as the coding region sequence shown in SEQ ID NO: 1 or 3 or a degenerate variant. As used herein, "degenerate variant" refers in the present invention to a nucleic acid sequence encoding a protein having SEQ ID NO: 2 or 4 but different from the coding region sequence shown in SEQ ID NO: 1 or 3.

The polynucleotide encoding a mature polypeptide of ULBP-4 includes: a coding sequence that encodes only a mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences); and non-coding sequence. The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or a polynucleotide that also includes additional coding and / or non-coding sequences.

The present invention also relates to a variant of the above-mentioned polynucleotide, which encodes a polypeptide having the same amino acid sequence as the present invention or fragments, analogs and derivatives of the polypeptide. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants. As known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially change its encoding. The function of the peptide.

The present invention also relates to a polynucleotide that hybridizes to the above sequence and has at least 50%, preferably at least 70%, more preferably at least 80%, and most preferably at least 90% or 95% identity between the two sequences. The invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the invention under stringent conditions. In the present invention, "strict conditions" means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 ° C; or ( 2) A denaturant is added during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42 ° C, etc .; or (3) only between two sequences Crosses occur only when the identity is at least 90%, and more preferably 95%. Also, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide shown in SEQ ID NO: 2 or 4.

The invention also relates to a nucleic acid fragment that hybridizes to the sequence described above. As used herein, a "nucleic acid fragment" is at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides in length. Nucleic acid fragments can be used in nucleic acid amplification techniques, such as PCR, to identify and / or isolate polynucleotides encoding ULBP-4 protein.

The human ULBP-4 nucleotide full-length sequence or a fragment thereof of the present invention can usually be obtained by a PCR amplification method, a recombinant method, or a synthetic method. For the PCR amplification method, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and a commercially available cDNA library or a cDNA prepared according to a conventional method known to those skilled in the art The library is used as a template and the relevant sequences are amplified. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then stitch the amplified fragments together in the correct order.

Once the relevant sequences are obtained, the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.

In addition, relevant methods can also be synthesized by artificial synthesis, especially when the fragment length is short. Generally, long fragments can be obtained by synthesizing multiple small fragments first and then joining them.

At present, a DNA sequence encoding a protein (or a fragment, a derivative thereof) of the present invention can be obtained completely through chemical synthesis. This DNA sequence can then be introduced into a variety of existing DNA molecules (or such as vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

A method of amplifying DNA / RNA using a PCR technique is preferably used to obtain the gene of the present invention. Primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by conventional methods. The amplified DNA / RNA fragments can be isolated and purified by conventional methods such as by gel electrophoresis.

The present invention also relates to a vector comprising a polynucleotide of the present invention, a host cell genetically engineered using the vector of the present invention or a ULBP-4 protein coding sequence, and a method for producing a polypeptide of the present invention by recombinant technology.

The polynucleotide sequence of the present invention can be used to express or produce recombinant ULBP-4 polypeptide by conventional recombinant DNA technology. Generally there are the following steps:

(1) Use the polynucleotide (or variant) encoding the human ULBP-4 polypeptide of the present invention, or use the polynucleotide (or variant) containing the polypeptide A recombinant expression vector of nucleotides transforms or transduces a suitable host cell;

(2) host cells cultured in a suitable medium;

(3) Isolate and purify protein from culture medium or cells.

In the present invention, the human ULBP-4 polynucleotide sequence can be inserted into a recombinant expression vector. In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host. An important feature of expression vectors is that they usually contain origins of replication, promoters, marker genes, and translation control elements.

Methods known to those skilled in the art can be used to construct expression vectors containing human ULBP-4 coding DNA sequences and appropriate transcription / translation control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, and in vivo recombinant technology. The DNA sequence can be operably linked to an appropriate promoter in an expression vector to guide mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.

In addition, the expression vector preferably contains one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, and green for eukaryotic cell culture. Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.

A vector containing the above-mentioned appropriate DNA sequence and an appropriate promoter or control sequence can be used to transform an appropriate host cell so that it can express a protein.

The host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples are: Escherichia coli, Streptomyces bacterial cells; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CH0, COS. Or 293 cells, etc.

Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as E. coli, competent cells capable of DNA uptake can be harvested after exponential growth phase, ₀₁₂ ^ Treatment with steps used are well known in the art. Another method is to use MgCl ₂ . If necessary, transformation can also be performed by electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, and liposome packaging.

The obtained transformants can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the medium used in the culture may be selected from various conventional mediums. Culture is performed under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

The recombinant polypeptide in the above method can be expressed in a cell, or on a cell membrane, or secreted into a cell. If desired, recombinant proteins can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional renaturation, treatment with a protein precipitant (salting out method), centrifugation, osmotic bacteria, ultra-treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.

Recombinant human ULBP-4 protein or polypeptide has many uses. These uses include, but are not limited to: screening antibodies, peptides or other substances that function against ULBP-4 protein function. In another aspect, the present invention also includes polyclonal antibodies and monoclonal antibodies, especially monoclonal antibodies, which are specific for human ULBP-4 DNA or a polypeptide encoded by a fragment thereof. Here, "specificity" means that the antibody is capable of binding to a human ULBP-4 gene product or fragment. Preferably, it refers to those antibodies that can bind to the human ULBP-4 gene product or fragment but do not recognize and bind to other unrelated antigen molecules. The antibodies of the present invention can be prepared by various techniques known to those skilled in the art.

The invention includes not only intact monoclonal or polyclonal antibodies, but also antibody fragments with immunological activity, such as Fab 'or (Fab) ₂ fragments; antibody heavy chains; antibody light chains; genetically engineered single-chain Fv molecules; Or chimeric antibodies.

Antibodies against human ULBP-4 protein can be used in immunohistochemistry to detect human ULBP-4 protein in biopsy specimens.

The antibodies of the present invention can be used to treat or prevent human ULBP-4 protein related diseases such as tumors. One method is to attack the amino group of the antibody with a thiol cross-linking agent such as SPDP and bind the toxin to the antibody through the exchange of disulfide bonds. This hybrid antibody can be used to kill human ULBP-4 protein-positive cells, such as tumors. cell.

By using the protein of the present invention, substances that interact with the ULBP-4 protein, such as receptors, inhibitors, agonists or antagonists, can be screened through various conventional screening methods.

The protein, the antibody, the inhibitor, the agonist, the antagonist or the receptor of the present invention, when administered therapeutically (administration), can provide different effects. Generally, these materials can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, where the pH is usually about 5-8, and preferably about 6-8, although the pH can vary with the material being formulated. And the nature of the condition to be treated. The formulated pharmaceutical composition may be administered by conventional routes, including (but not limited to): intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, or topical administration.

ULBP-4 antagonists (such as antibodies and antisense sequences) can be used directly in the treatment of diseases, for example, in the treatment of tumors. In addition, other therapeutic agents can be used in combination, such as TNF-a, TNF-β, and the like.

The present invention also provides a pharmaceutical composition containing a safe and effective amount of the ULBP-4 antagonist of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods. Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions. The active ingredient is administered in a therapeutically effective amount, for example, about 1 microgram to 10 milligrams per kilogram of body weight per day.

The invention also relates to a diagnostic test method for quantitative and localized detection of human ULBP-4 protein levels. These tests are well known in the art and include FISH assays and radioimmunoassays. The level of human ULBP-4 protein detected in the test can be used to diagnose tumors.

A method for detecting the presence of ULBP-4 protein in a sample is to use a specific antibody of ULBP-4 protein for detection, which includes: contacting the sample with a ULBP-4 protein-specific antibody; observing whether an antibody complex is formed, and The antibody complex indicates the presence of ULBP-4 protein in the sample. The polynucleotide of ULBP-4 protein can be used for the diagnosis and treatment of diseases related to ULBP-4 protein. A part or all of the polynucleotide of the present invention can be used as a probe to be fixed on a microarray or a DNA chip, and used for analyzing differential expression analysis and gene diagnosis of genes in tissue. ULBP-4 protein-specific primers for RNA-polymerase chain reaction (RT-PCR) in vitro amplification can also detect ULBP-4 protein transcription products.

The present invention also provides a tumor detection kit, which contains a primer pair that specifically amplifies ULBP-4 and / or a ULBP-4 specific antibody. It may also contain specific probes and / or PCR buffers.

The sequencing results of ULBP-4 showed that it had poor homology with the three genes of ULBP-1, -2 and -3, but all could bind to NKG2D and be located at the same position on the chromosome. The expression analysis results show that ULBP-4 gene is only expressed in tumor tissues, not expressed in normal cells or the expression level is very low. A comparison of the ULBP-4 gene with the RAE-1 family and H60 genes in mice shows that ULBP-4 may be the H60 homologous gene in humans. It has been reported (O 'Callaghan CA, et al., Immunity 2001 Aug; 15 (2): 201-11) that murine H60 and RAE-1 families competitively bind to NKG2D receptors, while H60 and NKG2D The affinity is about 10 times higher than that of the RAE-1 family. Since human ULBP-1, -2, and -3 are homologous genes of the RAE-1 gene, the binding capacity of ULBP-4 to the NKG2D receptor may also be higher than ULBP_1, -2, _3.

In addition, it is interesting that the ULBP gene, like the RAE-1 gene family, is a GPI-anchored membrane protein. Can be secreted into body fluids under the action of phospholipase C. Therefore, ULBP-4 may be an effective tumor marker and play a role in the diagnosis and treatment of numerous cancers.

Given that ULBP-4 may be a tumor cell-specific immune rejection antigen, it is secreted into body fluids. Therefore, the direct determination of ULBP-4 in blood or urine can be used not only as an aid for tumor diagnosis and post-healing observation, but also as a basis for early diagnosis of tumors.

In addition to being a diagnostic tool, ULBP-4 also has great application value in tumor surgery, which helps determine the depth of tumor invasion, specific edges and hidden parts. Radioimmuno-guided surgery (RIGS) is to inject antibodies labeled with radioisotope-labeled tumor-associated antigens into the body for imaging analysis. It can accurately locate the extent of tumor invasion.

In terms of tumor immunotherapy, as an immunorejection antigen, the fusion protein of ULBP-4 or ULBP4 can bind to NKG2D receptors on the surface of NK cells, γ δ -TCR + T cells, CD8 + α β -TCR + T cells, and activate these cells Activity, releases cytotoxins and kills tumor cells, while expanding the number of such anti-tumor cells in the body. Because ULBP-4 is only located on the surface of tumor cells, it can also be used as a guidance system for biological missiles. When treating tumors, it can focus the therapeutic drug on the tumor area to improve the cure rate, while reducing the damage of the drug to the whole body.

For tumor vaccines, ULBP-4 can be used to make molecular vaccines. Tumor vaccines are the main content of active immunity, and are divided into four types: cell tumor vaccines, subcellular tumor vaccines, molecular tumor vaccines and genetic tumor vaccines. The former two are early tumor vaccines, which have mixed clinical effects, and most of the long-term effects are not ideal. Molecular tumor vaccines are based on the relationship between antibodies and antigens to prepare anti-tumor idiotype anti-antibodies. As unique tumor vaccines, certain clinical effects have also been seen. Tumor antigen peptides can be produced industrially without the risk of tumor implantation, and there is no inhibitory component of tumor cells. Using ULBP-4 as tumor antigen peptide, the obtained molecular vaccine is not restricted by MHC System and can be applied to a variety of tumors. In addition to molecular tumor vaccines, tandem ULBP-4 genes are inserted into viral vectors as gene tumor vaccines.

In addition, the outer membrane portion of ULBP-4 can be used as an immune stimulating factor and binds to NKG2D, thereby activating immune cells such as NK cells and T cells to achieve the effect of enhancing immunity. The present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods without specific conditions in the following examples are generally performed according to conventional conditions such as those described in Sambrook et al., Molecular Cloning Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions. Suggested conditions. Example 1: Acquisition and localization of ULBP-4 gene

The protein sequences of the three genes of the ULBP family, ULBPl, ULBP2, and ULBP3, were tblastn to the HUMAN-EST library to find relevant ESTs. As a result, three ESTs were found upstream of the segment of chromosome contig (NT007285. 7) located at ULBP123: BI258059, BE545401, AL602601. Splice with assembly software and analyze with contig (NT007285. 6) to obtain the ULBP-4 coding region. Prediction of the coding protein for the spliced sequence revealed that there is a large reading frame starting from 403 bases, but without a termination code, it is estimated that the 3 'end is incomplete. Using the ensembl genescan tool to analyze the exon prediction of this chromosome segment, it was found that the chromosome segment has an exon-dense region and a continuous exon, showing that the 3 'end of the gene can be extended on the chromosome. As a result, the complete coding region of the gene was obtained. The specific sequence is shown in SEQ ID NO: 1. The 0RF is located at positions 403-1194 and encodes a 263 amino acid protein (SEQ ID NO: 2), in which amino acids 1-23 are signal peptides. According to the EST information of ULBP-4, it was located on chromosome 6, q25.1. Example 2: Polymorphism of ULBP-4

The following primers were synthesized and a human lymphocyte strain Jurkat cDNA library constructed by a conventional method was used as a template, and the gene ULBP_4 was cloned by a conventional PCR method.

5'-cgggatccatgcgaagaatatccctgacttctag -3 '(SEQ ID NO: 5)

5'-gccaagcttagacgtcctcaagggccagagac -3 '(SEQ ID NO: 6)

From the results shown in Figure 1, the size of the amplified fragment is about 800bp, which is consistent with the predicted 792bp.

The PCR amplified fragment was cloned into a vector and then sequenced to obtain the sequence shown in SEQ ID NO: 3, which was related to SEQ ID

Compared with NO: 1, there are 7 different nucleotides (Figure 2). The encoded ULBP-4 protein differs by 6 amino acids (Figure 3). Consistent with MICA / B, the ULPB family is highly polymorphic, which confirms that ULBP-4 is also polymorphic. Example 3: Sequence analysis of ULBP-4

The homologous alignment of ULBP-4 with the RAE-1 family and H60 revealed that ULBP-4 has no homology with RAE-1, but has 23.8% homology with H60. This suggests that human ULBP-4 may be a homologous gene of H60. 7 Example 4: Expression of ULBP-4

A monoclonal antibody recognition sequence (FLAG) was added to the signal peptide sequence of ULBP-, and the modified cDNA was inserted into a retroviral expression vector, and then transfected into mouse BaF / 3 cells, which was confirmed by immunoprecipitation and protein electrophoresis The expression of ULBP-4 cDNA in mammalian cells was obtained, and the molecular weight obtained in the experiment was consistent with the prediction, about 32 kDa. Example 5: Expression profile of ULBP-4

Using internal primers of the ULBP-4 coding region, the expression of ULBP-4 in various tumor cells and normal tissues was analyzed by RT-PCR:

Forward: 5 '-atgcgaagaatatccctgacttctagc-3' (SEQ ID NO: 7)

Reverse: 5 '-attttgccaccagacacagatgagaa-3' (SEQ ID NO: 8)

It can be seen from Fig. 4 that with the housekeeping gene GAPDH (right) as a positive control, obvious positive fragments can be obtained in each cancer cell line. When PCR was performed with primers in the coding region of the ULBP-4 gene, libraries of five cell lines were used: U373 (astrocytic tumor), JURKAT, HELA (cervical squamous cell carcinoma), SW480 (colon cancer), and DU145 (prostate cancer). Positive fragments can be amplified, indicating that the ULBP-4 gene is expressed in these cancer cells.

It can be seen from Figure 5 that ULBP-4 is not expressed in normal tissues. Because ULBP-4 is specifically expressed in tumor tissues, it can be used as a tumor marker. Example 6: Combination of ULBP-4 and NKG2D

Mouse Ba / F3 cell lines were transfected with retroviral expression vectors containing ULBP-1, -2, -3, and ULBP-4 cDNA, respectively. Transfected cells were labeled with soluble NKG2D-Ig fusion protein (indicated by a thick line in the figure) or human Ig (as a control, indicated by a thin line in the figure), and then labeled by flow cytometry and immunofluorescence.

It can be seen from Fig. 6 that the untransfected Ba / F3 cell line does not bind to NKG2D-Ig fusion protein and human Ig, while the cell lines transfected with ULBP-1, -2, -3 can bind to NKG2D_Ig fusion protein. It is shown that the products are all ligands of NKG2D receptors, which is consistent with previous reports. At the same time, the cell line transfected with the ULBP-4 gene can also specifically bind to MG2D, which not only confirms the expression of ULBP-4 cDNA in mammalian cells, but also indicates that the gene product is also a ligand of NKG2D. Example 7 ULBP-4 protein recombinant expression and purification

In this example, the PCR amplification product in Example 2 is used as a template, and PCR oligonucleotide primers at the 5 ′ and 3 ′ ends of the sequence are used for amplification to obtain human ULBP-4 DNA as an insert.

The 5 'terminal oligonucleotide primer sequence is: 5, -ccgggatccTTGGAGATCATGGTTG GTGGTCACT-3' (SEQ ID NO: 9). The primer contains a restriction site of a BamHI restriction enzyme, and a partial coding sequence without a signal peptide sequence is followed by the restriction site; The 3 'end primer sequence is: 5'-ggccaagcttCCATCTATCTGGTAGACTAGAAGAA-3' (SEQ ID NO: 10). The primer contains a restriction site for Hindlll restriction enzyme, a translation terminator, and a partial coding sequence of human ULBP-4.

The human ULBP-4 protein cDNA PCR product was purified and double digested with BamHI and Hindlll, and then recombined with plasmid pProEXHTa (GIBCOBRL) according to conventional methods to form the vector pProEXHTa_ULBP_4 and transformed into competent E. coli DH5 α. The positive clones were picked for identification, purified and sequenced (Type 377 sequencer of ΑΒΙ, BigDye Terminator kit, PE company). Sequencing confirmed the insertion of the complete ULBP-4 coding sequence.

Positive DH5a clones expressing ULBP-4 were inoculated in 10ml LB medium, cultured at 37 ° C with shaking at 300rpm overnight, 1: 100 diluted in LB medium inoculated and shaken for 2.5 hours, and added 100mM IPTG to 0.1mM after 37 Induced for 2 to 3 hrs at 5,000 g, centrifuged at 5,000 g for 10 min at 4 ° C to remove the supernatant, and placed on ice with 50 ml of loading buffer (0.5 M NaCl, 20 mM imidazole 2. 7 mM KC1, 10. 1 mM Na ₂ HP0 ₄ , 1. 8 mM KH ₂ P0 ₄ , pH 8. 0) Resuspend, break with ultrasound (B. Braun Labsonic U), and then 12, OOOg 4. After centrifugation for 10 min, the supernatant was filtered through a 0.8 μm filter membrane, and passed through a 1 ml Ni ²⁺ metal chelate Sepharose 4B chromatography column. After the loading buffer was sufficiently washed, 500 ul of imidazole elution buffer (0.5 M NaCl, 500mM imidazole 2. 7 mM KC1, 10. 1 mM Na ₂ HP0 ₄ , 1. 8 mM KH ₂ P0 ₄ , pH 8. 0) After standing at room temperature for 30 minutes, collect the eluate and repeat the elution 2-3 times to obtain human ULBP -4 proteins. Example 8 Production of anti-ULBP-4 protein antibodies

The recombinant human ULBP-4 protein obtained in Example 7 was used to immunize animals to produce antibodies. The specific method is as follows. Recombinant molecules are separated by chromatography before use. It can also be separated by SDS-PAGE gel electrophoresis. The electrophoretic band is cut from the gel and emulsified with an equal volume of complete Freund's adjuvant. Mice were injected intraperitoneally with 50-100 μ _§ /0.2 ml of emulsified protein. After 14 days, mice were injected intraperitoneally with the same antigen emulsified with incomplete Freund's adjuvant at a dose of 50-100 μ _§ /0.2 ml to boost immunity. Booster immunizations are performed every 14 days, at least three times. The specific reactivity of the obtained antisera was evaluated by its ability to precipitate human ULBP-4 protein gene translation products in vitro. As a result, it was found that the antibody can specifically bind to the protein of the present invention. All documents mentioned in the present invention are incorporated by reference in this application, as if each document was individually incorporated by reference. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

Claim

An isolated human ULBP-4 polypeptide, characterized in that it comprises: a polypeptide having the amino acid sequence of SEQ ID NO: 2 or 4, or a conservative variant polypeptide thereof, or an active fragment thereof, or an active derivative thereof.

The polypeptide according to claim 1, characterized in that the polypeptide has an amino acid sequence selected from the group consisting of amino acid sequences 1-263 or 24-263 in SEQ ID NO: 2 or 4.

3. An isolated polynucleotide, characterized in that it comprises a nucleotide sequence that is at least 70% identical to a nucleotide sequence selected from the group consisting of:

(a) a polynucleotide encoding the polypeptide of claims 1 and 2;

(b) A polynucleotide complementary to the polynucleotide (a).

The polynucleotide according to claim 3, wherein the polynucleotide encodes a sequence having SEQ ID NO:

Polypeptide with amino acid sequence 1-263 or 24-263 in 2 or 4.

The polynucleotide according to claim 3, wherein the sequence of the polynucleotide is one selected from the group consisting of:

(a) a sequence having positions 403-1194 in SEQ ID NO: 1;

(b) a sequence having positions 1-1434 in SEQ ID NO: 1;

(c) a sequence having positions 473-1194 in SEQ ID NO: 1;

(d) a sequence having positions 1-792 in SEQ ID NO: 3;

(e) a sequence having positions 70-792 in SEQ ID NO: 1.

6. A vector comprising the polynucleotide according to claim 3.

7. A genetically engineered host cell, characterized in that it contains the vector according to claim 6.

8. A method for preparing ULBP-4 protein, characterized in that the method includes:

(a) culturing the host cell of claim 7 under conditions suitable for expression;

(b) ULBP-4 protein is isolated from the culture.

An antibody capable of specifically binding to the ULBP-4 protein according to claim 1.

10. A method for detecting the presence of ULBP-4 protein in a sample, comprising: contacting the sample with an antibody that specifically binds to the ULBP-4 protein, and observing whether an antibody complex is formed, which is indicated by the formation of an antibody complex The ULBP-4 protein was present in the sample.