Patent 6,511,830

The present invention relates to a polypeptide which is a constituent of a killer T cell receptor and is capable of injuring human immunodeficiency virus-infected cells; a DNA encoding this polypeptide; a recombinant vector comprising this DNA and a vector; a transformant obtained by transferring this recombinant vector into a host cell; a process for producing the above polypeptide characterized by culturing the above transformant in a medium, thus forming and accumulating the polypeptide in the culture and then recovering the polypeptide from the culture; an antibody reacting specifically with the above polypeptide; human-mouse type killer T cell receptor .alpha.chain and .beta.chain in each of which the variable region site of the above polypeptide is sustained while the constant region site thereof has been replaced by that of the human type; transgenic animals having the above polypeptide expressed therein; and anti-HIV agents containing the above polypeptide.

DISCLOSURE OF THE INVENTION

The present invention relates to (1) to (17) as shown below.

(1) A polypeptide which is a constituent of a killer T cell receptor and is capable of injuring specifically human immunodeficiency virus-infected cells;

(2) The polypeptide according to the above (1) wherein the human immunodeficiency virus is HIV-1;

(3) The polypeptide according to the above (2) wherein the HIV-1 is HIV-1 IIIB;

(4) The polypeptide according to any one of the above (1) to (3), wherein a polypeptide constitutes a killer T cell receptor which recognizes specifically human immunodeficiency virus envelope protein gp160.

(5) The polypeptide according to the above (4) wherein the recognition region of the killer T cell receptor which recognizes specifically human immunodeficiency virus envelope protein gp160 is a V3 region of the gp160;

(6) The polypeptide according to the above (5) wherein the recognition region is a region comprising the amino acid sequence 315 to 329 in the human immunodeficiency virus envelope protein gp160 V3 region;

(7) A polypeptide which comprises an amino acid sequence shown in SEQ ID NO: 7 or 9, or a polypeptide, which comprises an amino acid sequence wherein one or more of amino acids in the amino acid sequence are substituted, deleted or added, and is capable of injuring specifically human immunodeficiency virus infected-cells.

The above-mentioned substitutions, deletions or additions of one or more of amino acids can be performed by means of a well-known art before the filing of the present applicaiton, the site-directed mutagenesis method. In addition, the term "one or more of amino acids" used herein means the number of amino acids which can be substituted, deleted, or added by the site-directed mutagenesis method.

The polypeptide which comprises an amino acid sequence wherein one or more amino acids are substituted, deleted, or added, and is capable of injuring specifically human immunodeficiency virus-infected cells can be prepared according to the methods described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) (hereinafter abbreviated as Molecular Cloning ² nd ed., ), Current Protocols in Molecular Biology, Supplement 1 to 38, John Wiley & Sons (1987-1997) (hereinafter abbreviated as Current Protocols in Molecular Biology), Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Nucleic Acids Research, 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488(1985), Proc. Natl. Acad. Sci. USA, 81, 5662 (1984), Science, 224, 1431(1984), PCT WO85/00817(1985), Nature, 316, 601(1985) and the like.

(8) A DNA encoding the polypeptide according to any one of the above (1) to (7).

(9) The DNA having the nucleotide sequence shown in SEQ ID NO:6 or 8.

(10) A DNA which encodes the polypeptide, capable of injuring specifically human immunodeficiency virus-infected cells, which can hybridize with the DNA according to the above (8) or (9) under stringent conditions.

As used herein, the term "the DNA which encodes the polypeptide, capable of injuring specifically human immunodeficiency virus-infected cells, which can hybridize under stringent conditions" means a DNA which can be obtained by using the DNA of the above (8) or (9) as a probe according to the colony hybridization technique, the plaque hybridization technique or the southern blot hybridization technique or the like. For example the DNA can be identified by performing hybridization using a filter, to which DNA derived from a colony or a plaque is immobilized, under the presence of 0.7 to 1.0M NaCl at 65^oC. and then by washing the filter using 0.1-2xSSC (saline-sodium citrate) solution (where the composition of 1xSSC solution is 150 mM sodium chloride, 15 mM sodium citrate) at 65^oC.

Hybridization can be performed according to the methods shown in protocols including Molecular Cloning 2^nd ed., Current Protocols in Molecular Biology, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995), and the like.

The DNA which can be hybridized is, for example, a DNA having homology of 80% or more, preferably 95% or more, to the nucleotide sequence shown in SEQ ID NO: 6 or 8.

(11) A recombinant vector comprising the DNA according to any one of the above (8) to (10) and a vector.

(12) A transformant obtained by introducing the recombinant vector according to the above (11) into a host cell.

(13) A process of producing the polypeptide according to any one of the above (1) to (7), which comprises culturing the transformant of the above (12) in a medium, forming and accumulating the polypeptide of any one of the above (1) to (7) in the culture, and recovering the polypeptide from the culture.

(14) An antibody which specifically reacts with the polypeptide according to any one of the above (1) to (7).

(15) The polypeptide according to any one of the above (1) to (7), having a human type constant region site.

(16) Transgenic animals, having the polypeptide according to any one of the above (1) to (7) expressed therein.

(17) Anti-HIV agents containing the polypeptide according to any one of the above (1) to (7).

The killer T cell clone injuring HIV-infected cells can be established by preparing antigens, administering the antigens to animals for immunization, removing sensitized lymphocytes from the cells of the immunized animals and stimulating the sensitized lymphocytes.

As the HIV strain, which is used for producing the killer T cell clone injuring HIV infected cells, includes HIV-1 III3 strain or the like can be mentioned. As the epitope, Env-K1 containing the amino acid sequence 315 to 329 presented in V3 region within HIV-1 envelope protein gp160 [amino acid sequence; RIQRGPGRAFVTIGK (Takahashi, H. et al., Proc. Natl. Acad. Sci. USA, 85, 3105 (1988), hereinafter referred to as P18) can be mentioned.

The methods for administering the antigen include the following: a method using ISCOM (Immunostimulating complex) which is a special immunopotentiating substance (adjuvant) [Takahashi, H. et al., Nature, 344, 873 (1990)]; a method using a complex of QS-21, one of constituent of ISCOM, and HIV envelope protein gp160 [Wu, J. et al., J. Immunol., 148, 1438(1992)]; a method using a recombinant vaccinia virus wherein the gp160 gene is introduced [Takahashi, H. et al., Proc. Natl. Acad. Sci. USA, 85, 3105 (1988)] and a method using self-dendritic cell formed by binding, a self-cell, into which the gp160 gene is introduced, and Env-K1 [Takahashi, H. et al., Int. Immuno., 5, 849 (1993)] since it is known to be difficult for general purified protein antigens and the like to induce the killer T cells.

Example of animals for immunization includes mice, rats, rabbits, monkeys and the like. For example, the mice for immunization have various genetic characters such as B10.PL(H-2^u), B10.P(H-2^p), B10.Q(H-2^q), and B10.A(H-2^a). In particular, a BALB/c(H-2^d) mouse which shows a high reactivity with P18 is preferred.

Sensitized lymphocytes are obtained by removing the spleen from the immunized animal, and performing a treatment such as the removal of erythrocytes. To stimulate the sensitized lymphocytes, antigen-presenting cells, fibroblasts and the like, which express antigens or to which antigens are bound, are irradiated with radiation or treated with mitomycin-C are used. These cells are preferably the same type of cell line as the immunized cells. P18 specific killer T cell clone can be established by stimulating continuously with the cells. The killer T cell clones injuring HIV infected-cells according to the present invention include RT-1, RT-2, RT-3 and the like. A method for confirming T cells is, e.g., FACScan using an antibody to a molecular marker expressed on the cell.

A T cell .alpha..beta.receptor is a heterodimer protein formed by disulfide bonds of .alpha.chain and .beta.chain polypeptides. The receptor forms a complex with CD3 and is expressed on the surface layer of a T cell. The specific T cella .alpha..beta.receptor comprises many different V-(D)-J-C regions. The type of the receptor itself is considered to be defined according to the amino acid sequence of V region and the specificity to a foreign matter according mainly to the amino acid sequences of D and J regions. Accordingly, the T cell receptor gene is identified from P18-specific killer T cell clone by determining V regions for T cell receptor .alpha.chain and .beta.chain, and by identifying the entire gene sequence.

The V regions of the T cell receptor .alpha.chain and .beta.chain are identified by polymerase chain reaction (hereinafter referred to as PCR) with primers produced based on the sequences of the obtained mRNA and of each V region. Then the reverse transcription-PCR (RT-PCR) is performed for the obtained mRNA to produce cDNA. Thus the sequence can be determined.

The full-length DNA having a junctional region specific to p18 is produced by the recombinant PCR technique to determine the whole gene sequence.

The total RNA is prepared from the T cell clone by the guanidine thiocyanate--cesium trifluoroacetate method [Methods in Enzymology, 154, 3 (1987)], acidic guanidine thiocyanate-phenol-chloroform (AGPC) method [Analytical Biochemistry, 162, 156 (1987), Experimental Medicine 9, 1937 (1991)] and the like.

From the total RNA mRNA is prepared as poly (A)⁺ RNA according to the method using the oligo(dT) immobilized cellulose column technique (Molecular Cloning 2^nd ed., ), the method using an oligo dT latex, and the like.

Alternatively, mRNA can be prepared directly from tissues or cells by using Fast Track mRNA Isolation Kit (manufactured by Invitrogen), Quick Prep mRNA Purification Kit (manufactured by Pharmacia), and the like.

From the total RNA or mRNA obtained, cDNA libraries are obtained by using conventional method.

For example the cDNA library can be prepared according to the method described in Molecular Cloning 2^nd ed., Current Protocols in Molecular Biology, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995) and the like, or by using commercially available kits, such as, SuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (manufactured by Gibco BRL) and ZAP-cDNA Synthesis Kit (manufactured by STRATAGENE).

As the cloning vectors for preparing the cDNA library, any of phage vectors and plasmid vector can be used so long as it is capable of autonomously replicating in Escherichia coli K12.

Examples of suitable vectors are ZAP Express [manufactured by STRATAGENE, Strategies, 5, 58 (1992)], pBluescript II SK(+) [Nucleic Acids Research, 17, 9494 (1989)], Lambda ZAP II (manufactured by STRATAGENE), .lambda. gt10, .lambda. gt11 [DNA Cloning, A Practical Approach, 1, 49 (1985)], .lambda. TriplEx (manufactured by CLONTECH), .lambda. ExCell (manufactured by Pharmacia), pT7T318U (manufactured by Pharmacia), pcD2 [Mol. Cell. Biol., 3, 280 (1983)], pUC18 [Gene, 33, 103 (1985)], and pAMo[J. Biol. Chem., 268, 22782-22787 (1993), another name, pAMoPRC3Sc (JP-A-05-336963)].

Any microorganism belonging to Escherichia coli can be used as a host microorganism. Examples of the host microorganisms are Escherichia coli XL1-Blue MRF' [manufactured by STRATAGENE, Strategies, 5, 81 (1992)], Escherichia coli C600 [Genetics, 39, 440 (1954)], Escherichia coli Y1088 [Science, 222, 778 (1983)], Escherichia coliY1090 [Science, 222, 778 (1983)], Escherichia coli NM522 [J. Mol. Biol., 166, 1 (1983)], Escherichia coli K802 [J. Mol. Biol., 16, 118 (1966)], Escherichia coli JM105 [Gene, 38, 275 (1985)], Escherichia coli SOLR.TM. Strain (manufactured by STRATAGENE), and Escherichia coli LE392 (Molecular Cloning 2^nd ed.,).

In addition to the cDNA library constructed by the above-mentioned methods, commercially available cDNA library can be used.

From the cDNA library constructed by the above-mentioned methods, the cDNA clone containing the DNA of the present invention can be selected the colony hybridization, or the plaque hybridization [Molecular Cloning 2^nd ed.,] using probes labeled with isotope or fluorescence.

The probes can include a fragment obtained by amplifying a part of cDNA using PCR [PCR Protocols, Academic Press (1990)] with primers based on a partially known nucleotide sequence, and an oligonucleotide based on a partially known nucleotide sequence.

The primer prepared based on such sequences can be employed when both nucleotide sequences of the full-length cDNA on the 5'-end side and 3'-end side are known in sequences such as ESTs,.

cDNA is synthesized from the mRNA using the cDNA clone having the DNA of the present invention selected as described above, according to the above techniques.

By the use of 5'-RACE (rapid amplification of cDNA ends) and 3'-RACE [Proc. Natl. Acad. Sci. USA, 85, 8998 (1988)] wherein PCR is conducted with primers based on a nucleotide sequence of an adapter which is added to both ends of the cDNA and with those based on a partially known nucleotide sequence, cDNA which is upstream (5'-end side) and downstream (3'- end side) from the amplified fragment can be obtained.

The full-length DNA of the present invention can be obtained by ligating the obtained cDNA fragments.

To determine the nucleotide sequence of the DNA obtained by the above methods, the DNA fragments or those cleaved by an appropriate restriction enzyme(s) are introduced into a vector by standard techniques, then the product is analyzed by a standard nucleotide sequence analysis technique, e.g., the dideoxy technique by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)] or using nucleotide sequence analyzers of Perkin Elmer (373A.DNA sequencer), those of Pharmacia, and of LI-COR.

The DNA of interest can be prepared by chemical synthesis using a DNA synthesizer based on the nucleotide sequence information obtained by the above-mentioned methods. The DNA synthesizers include the one manufactured by Shimazu Corp. using the thiophosfite technique, the one (model 392) by Perkin Elmer using the phosphoamidite technique, and the like.

The novelty of the obtained nucleotide sequence can be confirmed by searching a nucleotide sequence database of GenBank, EMBL, DDBJ and the like, using a homology search program i.e., BLAST.

For a novel nucleotide sequence, after converting it to an amino acid sequence, an amino acid sequence database, e.g., GenPept, PIR, or Swiss-Prot, is searched using a homology search program e.g., FASTA, and Frame Search, thereby searching the existing genes having homologies.

The DNA of the present invention obtained by the above emthod can be expressed in a host cell to produce the polypeptide of the present invention, according to the methods described in Molecular Cloning 2^nd ed., Current protocols in Molecular Biology and the like.

That is, the polypeptide of the present invention can be produced by constructing a recombinant vector wherein the DNA of the present invention is inserted an appropriate expression vector at an insertion site located downstream of the promoter therein, transferring this vector to a host cell to obtain a transformant expressing the polypeptide of the present invention, and culturing this transformant.

As the host cells, any bacterial cells, yeast cells, animal cells, insect cells, plant cells and the like can be used, so long as the desired gene can be expressed therein. Particularly, a transformant obtained by transferring the recombinant vector, in which the DNA of the present invention is inserted to introduce into a peripheral blood cell of a healthy individual, can be employed for the gene therapy of HIV-infected individuals.

As the expression vectors, which are capable of autonomously replicating in the host cell or being integrated into a chromosome and contain a promoter at a site appropriate for the transcription of the DNA of the present invention are used.

When a prokaryote cell such as a bacterial cell is used as the host cell, the preferable recombinant vector expressing the polypeptide gene which is a constituent of a T cell receptor of the present invention can autonomously replicate in the prokaryotes and is a recombinant vector consisting of a promotor, ribosome binding sequence, the DNA of the present invention, and a transcription termination sequence. The vector may further comprise a gene requlating the promoter.

Examples of suitable expression vectors are pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega), pQE-8 (manufactured by QIAGEN), pKYP10 (JP-A-58-110600), pKYP200[Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci., USA, 82, 4306 (1985)], pBluescript II SK(-) (STRATAGENE), pTrs30 (FERM BP-5407), pTrs32 (FERM BP-5408), pGHA2 (FERM BP-400), pGKA2 (FERM B-6798), pTerm2 (JP-A-3-22979, US4686191, US4939094, US5160735), pKK233-3 (manufactured by Amersham Pharmacia Biotech), pGEX (manufactured by Pharmacia), pET system (manufactured by Novagen), pSupex, pTrxFus (Invitrogen), and pMAL-c2 (New England Biolabs).

As the promoters, any promoters capable of being expressed in host cells can be used. When Escherichia coli is used as a host, promoters derived from such as Escherichia coli or phages include trp promotor (Ptrp), lac promotor (Plac), P_L promoter, T7 promoter, P_R promoter and the like. In addition, promoters, artificially designed and modified e.g., Ptrpx2 formed by joining two Ptrp in series, tac promoter, T7lac promoter, and let I promoter can be used. When Bacillus subtilis is used as a host, the promoters include SP01 and SP02 that are phages of Bacillus subtilis, penP promoters, and the like.

As the ribosome binding sequence, a plasmid in which the distance between Shine-Dalgarno sequence and a starting codon is appropriately adjusted (e.g., 6 to 18 bases) can be used preferably.

A transcription termination sequence is not always necessary for the expression of the DNA according to the present invention. Preferably, the transcription termination sequence is arranged directly after the structural gene.

Examples of suitable host cells are cells of microorganisms belonging to genus Escherichia, genus Serratia, genus Bacillus, genus Brevibacterium, genus Coryneabacterium, genus Microbacterium, genus Pseudomonas, for example, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No. 49, Escherichia coli W3110, Escherichia coli NY49, Serratia ficaria, Serratia fonticola, Serratia liquefaciens, Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens, Brevibacterium ammoniagenes, Brevibacterium immariophilum ATCC14068, Brevibacterium saccharolyticum ATCC 14066, Corynebacterium glutamicum ATCC13032, Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869, Corynebacterium acetoacidophilum ATCC13870, Microbacterium ammoniaphilum ATCC15354, Pseudomonas sp. D-0110 and the like.

Introduction of the recombinant vector can be carried out by any of the method for introducing DNA into the above host cell, for example, the method using calcium ion [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast method (JP-A-63-248394), and the electroporation method[Gene, 17, 107 (1982), Molecular & General Genetics, 168, 111 (1979)].

As the plasmid containing the DNA encoding the polypeptide, which is a constituent of the killer T cell receptor of the present invention, for example, pH-RT1.alpha. containing the DNA encoding the killer T cell receptor .alpha.chain or pH-RT1.beta. containing the DNA encoding the killer T cell receptor .beta.chain, or the like can be mentioend. Escherichia coli TG1/pH-RT1.alpha. containing the plasmid pH-RT1.alpha. and Escherichia coli TG1/pH-RT1.beta. containing the plasmid pH-RT1.beta. were deposited with National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology (1-3, Higashi-1-chome, Tsukuba-shi, Ibaraki-ken, Japan) as FERM BP-6078 and FERM BP-6079, respectively.

When a yeast cell is used as the host cell, YEp13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, pHS15 and the like can be used as the expression vectors.

As the promoter, any promoters capable of expressing in a yeast cell can be used. Examples of suitable promoters are PH05 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MF.alpha. 1 promoter, and CUP 1 promoter.

The host cells can include yeast cells belonging to a genus Saccharomyces, genus Schizosaccharomyces, genus Kluyveromyces, genus Trichosporon, genus Schwanniomyces, genus Pichia, for example Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichia pastoris, and the like.

Introduction of the recombinant vector can be carried out by any of the methods for introducing DNA into yeast cells, for example the electroporation [Methods in Enzymology, 194, 182 (1990)], the spheroplast method[Proc. Natl. Acad. Sci. USA, 81, 4889 (1984)], and the lithium acetate method [Journal of Bacteriology, 153, 163 (1983)].

When an animal cell is used as a host cell, pcDNAI/Amp (manufactured by Invitrogen), pcDNAI, pAMoERC3Sc, pCDM8 [Nature, 329, 840 (1987)], pAGE107 [JP-A-3-22979, Cytotechnology, 3, 133 (1990)], pREP4 (manufactured by Invitrogen), pAGE103 [Journal of Biochemistry, 101, 1307 (1987)], pAMo, pAMoA, pAS3-3 (JP-A-2-227075) and the like can be used as the expression vector.

As the promotor, any promoters capable of expressing in animal cells can be used. Example of suitable promoters are cytomegalovirus (CMV) IE (immediate early) gene promoter, SV40 initial promoter or metallothionein promoter, retrovirus promoter, heat shock promoter, SR a promoter and the like. In addition, human CMV IE gene enhancer can be used with the promoter.

Examples of animal cells are mouse myeloma cells, rat myeloma cells, mouse hybridomas, human Namalwa cells, or Namalwa KJM-1 cells, human fetal kidney cells, human leukocytes, African green monkey kidney cells, Chinese hamster CHO cells, HBT5637 (JP-A-63-299) and the like.

The mouse myeloma cells include SP2/0, NS0 and the like, the rat myeloma cells include YB2/0 and the like, the human fetal kidney cells include HEK293 (ATCC: CRL-1573) and the like, the human leukocytes include BALL-1 and the like, and the African green monkey kidney cells include COS-1, COS-7 and the like.

Introduction of the recombinant vector can be carried out by any of the methods of introducing DNA into animal cells, for example the electroporation [Cytotechnology, 3, 133 (1990)], the calcium phosphate transfection (JP-A-2-227075), and the lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] and methods shown in Virology, 52, 456 (1973) and the like.

When an insect cell is used as a host cell, the polypeptide can be expressed by the methods described in Baculovirus Expression Vectors, A Laboratory Manual (W. H. Freeman and Company, New York (1992)), Molecular Biology, A Laboratory Manual, Current protocols in Molecular Biology, Bio/Technology, 6, 47 (1988) and the like.

That is, a recombinant vector for transferring a recombinant gene and baculovirus are co-introduced into an insect cell to obtain a recombinant virus in the insect cell culture supernatant, then the insect cell is infected with the recombinant virus, therefore the polypeptide can be expressed.

Examples of the gene transfer vector suitable for use in this method are pVL1392, pVL1393, and pBlueBacIII (both manufactured by Invitrogen).

An example of the Baculoviruses is Autographa californica nuclear polyhedrosis virus, which is a virus infecting insects belonging to family Barathra.

Examples of the insect cells are the ovarian cells of Spodoptera frugiperda and of Trichoplusia ni, culture cells derived from a silk worm ovarium.

The ovarian cells of Spodoptera frugiperda include Sf9, Sf21 (Baculovirus Expression Vectors, A Laboratory Manual) and the like, those of Trichoplusia ni include High 5, BTI-TN-5B1-4 (manufactured by Invitrogen) and the like, the culture cells from a silk worm ovarium include Bombyx mori N4 and the like.

Methods of transferring both said vector for transferring the recombinant gene and said baculovirus into an insect cell to prepare a recombinant virus include calcium phosphate transfection (JP-A-2-227075), lipofection [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)] and the like.

Methods of expressing genes include secretory production, fusion protein expression and the like according to the techniques shown in Molecular Cloning 2^nd ed in addition to direct expression.

When the gene is expressed in yeast cell, an animal cell, or insect cells, a sugar or sugar chain-attached protein can be obtained.

The polypeptide that is a constituent of a T cell receptor of the present invention can be produced by culturing the transformant obtained as described above to form the polypeptide that is a constituent of a killer T cell receptor of the present invention is formed, accumulated in the culture, and recovering the polypeptide accumulated in the culture.

Further, the polypeptide, which is a constituent of a T cell receptor of the present invention, can be expressed in vivo by transferring the expression vector to express the appropriate polypeptide, which is a constituent of a T cell receptor of the present invention, into a cell collected from a patient, and then by returning the cell into the body.

Culturing of the transformant of the present invention can be carried out by conventional methods for culturing the host cell of the transformant.

As the media for culturing of the transformant prepared by using microorganisms such as Escherichia coli or yeasts as a host cell, any of natural media and synthetic media can be used insofar as it contains a carbon source, a nitrogen source, and inorganic salts, and the like which can be assimilated by the microorganism used, and the transformant is efficiently cultured therein.

As the carbon sources, any glucose, fructose, sucrose, molasses, starch, carbonhydrates such as hydrolysates of starch, organic acids e.g., acetic acids and propionic acids, and alcohols e.g., ethanol and propanol can be used.

As the nitrogen sources any ammonia, salts of inorganic acids or organic acids, such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other substances nitrogen containing compounds, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysates, soybean meal and soybean meal hydrolysate, various fermentation microorganic cells or their digests, and the like can be used.

The inorganic substances used in the present invention include potassium dihydrogenphosphate, dipotassium hydrogenphosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like.

Culturing is usually carried out under aerobic conditions, for example, by shaking cultures or submerged aeration stirring culture, at 15 to 40^oC. for 16 to 96 hours. The pH is maintained at 3.0 to 9.0 during the culturing. The pH adjustment is carried out by using an inorganic or organic acid, alkaline solution, urea, calcium carbonate, ammonia, and the like.

If necessary, antibiotics such as ampicillin and tetracycline may be added to the medium.

When a microorganism transformed with the expression vector comprising an inducible promoter is cultured, an inducer may be added to the medium if necessary. For example isopropyl-.beta.-D-thiogalactopyranoside (IPTG) or the like may be added in the case of microorganisms transformed with an expression vector comprising lac promoter, and in the case of microorganisms transformed with an expression vector comprising trp promoter, indoleacetic acid (IAA) or the like may be added.

For the culturing of the transformants prepared by using an animal cell as host cells include a generally used RPMI1640 media, Eagle MEM media or those to which fetal calf serum or the like is added may be used. Culturing is usually carried out in the presence of 5% CO₂ at 35 to 37^oC. for 3 to 7 days. If necessary, antibiotics such as kanamycin and penicillin may be added to the medium while culturing.

For the culturing of the transformant prepared by using an insect cell as the host cell, TNM-FH medium (manufactured by Pharmingen), Sf900 II SFM (manufactured by Life Technologies), ExCell1400 and ExCell405 (both manufactured by JRH Biosciences) and the like may be used.

Culturing is usually carried out at 25 to 30^oC., at a pH ranging from 6 to 7 and normally for 1 to 5 days. If necessary, antibiotics such as gentamicin may be added to the medium while culturing.

The polypeptide expressed in the above-described manner can be purified from the culture of the transformant by conventional methods for isolating and purifying enzymes to obtain the polypeptide which is a constituent of T cell receptor of the present invention.

For example, when the polypeptide of the present invention is expressed in a soluble form within the cell, after the completion of culturing and the cells are recovered by centrifugation, suspended in an aqueous buffer, followed by disruption using an ultrasonic disruption, a French press, a Manton Gaulin homogenizer, a Dyno Mill, and the like to obtain a cell-free extract.

The cell-free extract is centrifuged, and from the obtained supernatant, a purified sample can be produced from the supernatant obtained by centrifugation of the cell-free extract by conventional methods for isolating and purifying enzymes including a solvent extracting, salting-out with ammonium sulfate, desalting, precipitation with organic solvents, anion exchange chromatography using resins such as diethylaminoethyl (DEAE)--Sepharose and DIAION HPA-75 (manufactured by Mitsubishi Chemical Corp.), cation exchange chromatography using resins e.g., S-Sepharose FF (manufactured by Pharmacia) and the like, hydrophobic chromatography using resins such as butyl sepharose, phenyl sepharose and the like, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing, alone or in combination.

When the polypeptide is expressed in cells in an insoluble form, the cells are similarly disrupted, and separated by centrifugation, and fractions are precipitated, fraction. The polypeptide is recovered from the precipitate fraction by conventional method and the insoluble polypeptide is solubilized with a protein denaturing agent.

The solubilized solution is diluted or dialyzed to give a solution containing no protein-denaturing agent or containing protein-denaturing agent at a low concentration so that proteins are not denatured and the normal protein structure is restored, followed, by the same isolation and purification step as mentioned above to obtain a purified protein preparation.

When the polypeptide of the present invention or its derivatives such as a sugar-modified proteins are extracellularly secreted, the polypeptide or its derivatives such as the sugar chain-added from can be recovered from the culture supernatant.

That is, the culture is treated by the above-described means such as centrifugation, and the obtained soluble fractions is subjected to the same isolation and purification methods as described above to obtain a purified sample.

Further, the polypeptide of the present invention can be produced as a fusion protein with another protein and purified by affinity chromatography using substances having affinity for the fusion protein. For example according to the technique by Row et al., [Proc. Natl. Acad. Sci. USA, 86, 8227(1989), Genes Develop., 4, 1288 (1990)] or to methods described in JP-A-05-336963 and in JP-A-06-823021, the polypeptide of the present invention can be produced as a fusion protein with protein A, and purified by affinity chromatography using immunoglobulin G. Moreover, the polypeptide of the present invention can be produced as a fusion protein with a Flag peptide, and purified by affinity chromatography using an anti Flag antibody [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)]. Furthermore, the polypeptide of the present invention can be purified by affinity chromatography using an antibody specific to the polypeptide itself.

Moreover, the polypeptide of the present invention can be produced by chemical synthetic methods such as the Fmoc method (the fluorenylmethyloxycarbonyl method) and tBoc method (t-butyloxycarbonyl method) based on the amino acid sequence information contained in the polypeptide.

Further, the peptide of the present invention can be chemically synthesized by using peptide synthesizers manufactured by Advanced ChemTech, Perkin Elmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimazu Corp., and the like.

The structural analysis for the purified polypeptide of the present invention can be carried out by methods conventionally used in Protein Chemistry, for example by techniques shown in Protein Structure Analysis for Gene Cloning (Hisashi Hirano, Tokyo Kagaku Dojin, 1993).

The transgenic animals used herein means animals into which foreign genes are introduced at their initial developmental stage. The transgenic animals include mice, rats, or livestock such as cattle and sheep. The transgenic mouse is prepared as described below.

The transgenic mouse of the present invention can be prepared according to the methods of Hogan, B. et al., [Manipulating the mouse embryo. A laboratory manual. 2^nd ed. 1994. Cold Spring Harbor Laboratory Press, New York.] and Yamamura, K. et al., [J. Biochem., 9, 357-363 (1984)]. That is, a female C57BL/6 mouse treated with a hormone is allowed to cross, and the fertilized ovum is taken out, a fragment of a gene to be transferred but having no part of a vector, which is prepared in advance, is micro-injected using a micro-glass pipette into the male pronucleus of the fertilized ovum. Of the ova obtained to which the genes are introduced, several hundreds of surviving ova are transplanted into the uterine tubes of pseudo-pregnant mice, thereby generating transgenic mice. can be prepared as follows.

Animals are immunized using the proteins obtained by the above-mentioned method as antigens. For immunization the intact antigens may be administered subcutatenously, intravenously, or intraperitoneally to the animals. It is preferred to administer, the antigen in combination with a carrier protein with high antigenicity or an appropriate adjuvant.

The carrier proteins include Macroschisma sinense hemocyanin, Keyhole limpet hemocyanin, bovine serum albumin, bovine thyroglobulin and the like. The adjuvants include complete Freund's adjuvant, alminium hydroxide gel, pertussis vaccine and the like.

The animals for immunization include non-human mammals, including rats, goats, 3 to 20 weeks old mice, rats, hamsters and the like.

The antigen is administered 3 to 10 times every 1 to 2 weeks after the first administration. The dose of the antigen is preferably 50 to 100 .mu.g per animal. On 3rd to 7th days after each administration, a blood sample is collected from fundus oculi veniplex, and the obtained serum is examined for reactivity to the antigen according to enzyme-linked immunosorbent assay [ELISA: IGAKU-SHOIN Ltd. (1976)] and the like.

Then non-human mammals, the serum of which shows a sufficient antibody titer, are employed as a source for serum- or antibody-producing cells.

The polyclonal antibodies can be prepared by subjecting the serum to separation and purification procedure.

The monoclonal antibody can be prepared by fusing the antibody-producing cells and a myeloma cells derived from a non-human mammal to obtain hybridoma, and culturing the obtained hydridoma or administering the obtained hybridoma to an animal to cause ascites tumor, and subjecting the culture or the ascites to isolation and purification steps.

The antibody-producing cells are collected from splenic cells, the lymph node, peripheral blood of a non-human mammal administered with the antigen.

As the myeloma cells, any myeloma cells capable of proliferating in vitro can be used. Examples of suitable cells lines are 8-azaguanine resistant mouse (derived from BALB/c) myeloma cell line P3-X63Ag8-U1 (P3-Ul) [G.Kohler et al.,; Europ. J. Immunol., 6, 511 (1976)], SP2/0-Ag14(SP-2) [M. Shulman et al., ; Nature, 276, 269 (1978)], P3-X63-Ag8653(653) [J. F. Kearney et al.,; J. Immunol., 123, 1548 (1979)], and P3-X63-Ag8(X63) [G.Kohler et al.,; Nature, 256, 495 (1975)] which is derived from a mouse. For culture or subculture of these cells, 2x10⁷ or more of cells are secured before cell fusion according to Antibodies--A Laboratory Manual, Cold Spring Harbor Laboratory, 1988 (herein after abbreviated as A Laboratory Manual).

After the antibody producing cells obtained as described above and the myeloma cells are washed, a cell agglutination medium such as polyethylene glycol-1000 (PEG-1000) is added to fuse these cells, and then suspended in the medium. As the cell washing solution, examples of the solutions are an MEM medium, and a PBS (1.83 g of disodium hydrogenphosphate, 0.21 g of potassium dihydrogenphosphate, 7.65 g of sodium chloride, 1 l of distilled water, pH 7.2). As the medium used to suspend fusion cells, examples of the media are a HAT medium, which is an normal medium (RPMI-1640 medium to which 1.5 mM of glutamine, 5x10^-5 M 2-mercaptoethanol, 10 .mu.g/ml of gentamicin and 10% fetal calf serum (FCS) (manufactured by CSL) are added) supplemented with 10^-4 M hypoxantine, 1.5x10^-5 M thymidine and 4x10^-7 M aminopterin, so that only the fusion cells of interest can be selectively obtained.

After the culturing, a portion of the culture supernatant is subjected to enzyme immunoassay, to select cells which react with an antigenic protein and do not react with an non-antigenic protein. Then cloning is carried out by limiting dilution, and cells showing a high and stable antibody titer according to enzyme immunoassay are selected as monoclonal antibody producing hybridoma cell lines.

Enzyme Immunoassay

Antigenic proteins or cells expressing antigenic proteins is coated on a 96-well plate and allowed to react with a primary antibody, namely a hybridoma culture supernatant or a purified antibody.

After the primary antibody reaction, the plate is washed and a secondary antibody are added.

The secondary antibody is an antibody obtained by labeling an antibody, which can recognize the immunoglobuline of the primary antibody with a biotin, an enzyme, a chemiluminescent substance, a radioactive compound or the like. For example when a mouse is used to prepared hybriodmas, an antibody capable of recognizing the mouse immunoglobulin is used as the secondary antibody.

After the above-mentioned reaction is finished, a reaction suitable for a substance labeling the secondary antibody is performed, thereby selecting hybriodmas that produce monoclonal antibodies specifically reacting with the antigens.

The monoclonal antibodies can be prepared by separating and purifying from the culture fluid obtained by culturing the hymbridomas; or from the ascites of the 8 to 10 week mice or nude mice, which are treated with 0.5 ml Pristane (2,6,10,14-tetramethylpentadecane) by administering it intraperitoneally to the mice and are kept for 2 weeks, and to which the monoclonal antibody-producing hybridomas are administered so as to cause ascites tumor.

Monoclonal antibodies can be separated or purified by one or more of the methods including centrifugation, salting out using 40 to 50% saturated ammonium sulfate, caprylic acid precipitation method, chromatographies using DEAE-Sepharose column, anion exchange column, protein-A or -G column, or gel filtration column, and the like. The method allows to recover IgG or IgM fractions and obtain purified monoclonal antibodies.

Claim 1 of 25 Claims

What is claimed is:

1. A polypeptide which is a constituent of a killer T cell receptor and is capable of specifically recognizing human immunodeficiency virus envelope protein gp 160.

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