KR100229114B1 - Cd27 cell surface antigen and a recombinant dna encoding the same - Google Patents

Abstract

The present invention is directed to a simple and highly efficient method for cloning cDNAs from mammalian expression libraries based on transient expression in mammalian host cells.

A novel expression vector is also described that allows for the highly efficient construction of mammalian cDNA libraries. The cloning method of the present invention, which has been used to clone genes for cell surface antigens of human lymphocytes, is generally applied to cloning genes. The cell surface antigen cloned according to the present invention was purified and its nucleotide sequence and amino acid sequence determined. These antigens have utility in the diagnosis and treatment of immune mediated infectious diseases in mammals, including humans.

Description

CD27 cell surface antigen and a recombinant DNA encoding the same}

The present invention is part of the pending US Patent Application No. 160,416 of February 25, 1988, and part of the pending US Patent Application No. 379,076 of July 13, 1989, pending US 23 March 1990 Part of the ongoing application of patent application number 498,809.

[Background]

The primary means in the field of recombinant genetics is to convert poly (A) + mRNA into double stranded (hereinafter referred to as ds) cDNA, insert it into a cloning vector and express it in the appropriate host cell. Molecular cloning methods for ds cDNA are described, for example, in Williams, 'The Preparation and Screening of a cDNA Clone Bank' in Willaimson,

ed. Genetic Engineering, Vol. 1, p. 2, Academic Press, New York (1981); Maniatis, 'Recombinant DNA', in Prescott, ed., Cell Biology, Academic Press, New York (1980); And Efstratiadis et al., 'Cloning of Double-Stranded DNA,' in Stelo et al., Genetic Engineering, Vol. 1, p. 15, Plenum Press, New York (1979).

Significant variables that influence the successful cloning of specific genes and cDNA cloning methods should be carefully selected.

For many cDNA cloning methods, a common method involves the construction of a 'cDNA library', which is a collection of cDNA clones derived from total poly (A) + mRNA derived from organism cells of interest.

Mammalian cells contain up to 30,000 different mRNA sequences and, for example, the number of clones needed to obtain non-abundant mRNA will have to be much higher. Methods of constructing eukaryotic genomic DNA libraries in different transgenic expression vectors, including lambda bacteriophage, cosmid and viral vectors, are known. Some commonly used methods are described, for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, publisher, Cold Spring Harbor, New York (1982).

Once the genomic cDNA library is constructed, it is necessary to isolate cells containing thousands of specific human genes of interest from thousands of host cells. Many different methods of separating target genes from cDNA libraries have been used with ever changing changes.

Such methods include, for example, using nucleic acid probes which are labeled mRNA fragments having nucleic acid sequences complementary to the DNA sequence of the target gene. When this method is applied to cDNA clones of abundant mRNA in transformed bacterial hosts, the colonies that strongly hybridize to this probe are likely to contain the target DNA sequence. Identification of the clones is described, for example, by in situ hybridization / selection (see Methods Enzymol., 68: 206 (1979), Goldberg et al.), Hybrid inhibition translation methods [Paterson et al., Proceedings of the National Academy of Sciences, 74: 4370 (1977)] or direct DNA sequencing [see Maxam and Gilbert, Proceedings of the National Academy of Sciences, 74: 560 (1977) and Maat and Smith, Nucleic Acids Res., 5: 4537 (1978)] Can be made by.

However, such methods have a major drawback when their purpose is to replicate mRNA that is relatively abundant from the cDNA library. For example, using direct in situ hybridization, it is very difficult to detect clones containing cDNA complementary to mRNA species present in the initial library population of less than one part of 200. As a result, various methods of enriching mRNA in the entire population (e.g., size fractionation, methods using synthetic oligodeoxynucleotides, differential hybridization or immunopurification) have been developed and cloned mRNA that is not abundant Often used when making Such methods are described, for example, in Molecular Cloning: A Laboratory Manual by Maniatis et al., Supra.

Many functional eukaryotic proteins initially exist in the form of precursor molecules containing a leader sequence or signal sequence at their N-terminus. These leader sequences bind to the cell membrane and the signal sequence is cleaved from the protein by a signal peptidase enzyme and then pulls the remaining protein through the lipid bilayer. In this way it functions only after the protein is secreted from the cell (eg insulin, serum albumin, antibodies and gut enzymes) or after the protein is bound to the outer surface of the cell membrane (eg histocompatibility antigen).

Mammalian T lymphocytes with cell surface antigenic properties are additional examples of proteins that bind to the cell surface. In mammals, all cells induced by lymphocytes in mature bone marrow are present in lymphoid organs, including the thymus, spleen, lymph nodes, and lymphoid aggregates, and also actively circulate through the blood and lymphatic system. Mature lymphocyte cells can be divided into two groups: thymic-dependent (T) lymphocytes and thymic-independent (B) lymphocytes. T lymphocytes migrate inside the thymus and proliferate there.

During differentiation, T lymphocytes express characteristic cell surface membrane homologous antigens including Thy-1, TLA, gv-1, Ly-1, Ly-2, Ly-3 and Ly-5. As T lymphocytes mature, T lymphocytes lose some of the TLA antigen and Thy-1 antigen and acquire histocompatibility antigens, taking the typical membrane conformation of recycled T lymphocytes. See, for example, Mota, 'Activity of Immune Cells,' in Bier et al., Eds., Fundamentals of Immunology, 2d Ed., Springer-Verlag, Berlin, pp. 35-62 (1986).

Since T lymphocytes are indirectly involved in antibody formation and activity, complex assays of cell function were required rather than simple antibody titration measurements. Because of this, in part their importance in the development of immunoadaptation has not been recognized until relatively recently. Mature T lymphocytes synthesize and express a unique type of glycoprotein surface antigen that serves as a marker for identifying other T lymphocyte subpopulations, including T helper cells, T suppressor cells, and T cytotoxic cells. Each of these subgroups plays a very important role in regulating the immune system (see Mota, supra).

Functional and phenotypic heterotypic T lymphocytes in the human body are well accepted. Two major subgroups are known, regulatory T cells containing helper T lymphocytes and suppressor T lymphocytes, and mediated immune T cells, which are directed to cell surface antigens, autologous T cells that mediate cell immunity. Antibodies and monoclonal antibodies. For example, at the beginning of formation, human lymphoid cells in the thymus express an antigen called T11 that strongly responds to a monoclonal antibody called the strain group of differentiation 2 (CD2) and monoclonal to the cell surface antigen T1. Reacts weakly with ronal antibody CD5. During maturation, these cells lose T11 (CD2) and obtain three new antigens defined by monoclonal antibodies CD4, CD8 and CD1. As it matures, thymic cells finish expressing cell surface antigens that react with monoclonal antibody CD1 and finish expressing T3 antigens that react with monoclonal antibody CD3, followed by T4 (CD4) or T8 (CD8) antigens. It is divided into two subgroups that are expressed. Immunoadaptation is obtained at this stage but does not fully develop until thymic lymphocytes migrate out of the thymus (see Mota, supra). In contrast to most thymus, circulating T lymphocytes express T1 (CD5) and T3 (CD3) antigens. T4 (CD4) antigen is present on about 55-65% of peripheral T lymphocytes, while T8 (CD8) antigen is on 20-30% of peripheral T lymphocytes. These two subgroups correspond to helper T cells, suppressor T cells and cytotoxic T cells, respectively.

In addition to providing a convenient way to distinguish T lymphocyte subpopulations, these cell surface antigens are important for mature T cell activation and function. T cell activation involves a complex series of cell surface interactions between T cells and target cells or stimulating cells, in addition to binding of T cell receptors to T cell specific antigens.

For example, CD2, the human T cell erythrocyte receptor, allows thymic cells and T-lymphocytes to adhere to target cells (eg erythrocytes) and thymic epithelium. It occurs through a specific molecular ligand of CD2, called LFA-3, which is a widely distributed surface antigen in the human body. This phenomenon has long been used to detect, analyze, and purify human cells producing antibodies against sheep erythrocytes, and serves as the basis for the E-rosette experiments described for the first time by Zaalberg, Nature 202: 1231 (1964). do. CD2 / LFA-3 interactions were described for cell soluble target conjugation (see Nature et al., 323: 262-264 (1986) by Shaw et al.) And mixed lymphocyte responses [J. Immunol. 131: 180-185 (1983). Anti-CD2 monoclonal antibodies directly activate peripheral T-lymphocytes via antigen-independent pathways (see Meuer et al. Cell 36: 897-906 (1984)) that exhibit broader immune regulatory roles for CD2. Can be.

The recognition that T lymphocytes are the major effector of cell-mediated immunity and may also be involved as helper T cells or suppressor T cells in regulating the immune response has resulted in a significant contribution to increasing the practical application of clinical immunology to medicine.

The scope of the present application includes the defense against infectious diseases, immunotherapy, organ transplantation, prevention of diseases by blood storage and treatment of defects of the immune system and treatment of various diseases mediated by immunological mechanisms. Moreover, immunological techniques are commonly used in clinical laboratories for hormone and drug measurements. For example, clinical immunology is described in Weir, ed., Handbook of Experimental Immunology in Four Volumes: Volume 4: Applications of Immunological Methods in Biomedical Sciences. 4th Ed. Blackwell Scientific Publications, Oxford (1986); Boguslaski et al., Eds., Clinical Immunochemistry: Principles of Methods and Applications, Little, Brown & Co., Boston (1984); Holborow et al., Eds., Immunology in Medicine: A Comprehensive Guide to Clinical Immunology, 2d Ed., Grune & Stratton, London (1983); And Petersdorf et al., Eds., Harrison's Principles of Internal Medicine, 10th ed. McGraw-Hill, New York, Publisher, pp. 344-391 (1983). Clearly, a more complete understanding of the proteins that regulate the immune system is of great value for clinical immunology.

The use of mammalian transgenic libraries to isolate cDNAs encoding mammalian proteins as described above provided several advantages. For example, proteins expressed in mammalian host cells must be functional and will undergo all normal post-translational modifications. Proteins that are transported through the intracellular membrane system usually must go through a complete transport process. Mammalian expression systems have also enabled us to study intracellular transport mechanisms and the mechanisms by which cell surface proteins are attached and inserted into membranes.

A common host cell in mammals called 'COS' cells is a mutant viral vector named Simian Virus Strain 40 (SV 40), which has early and late functional genes but lacks the origin of functional replication, which leads to monkey kidney cells. It is formed by infection. Since the SV 40 T antigen is present in COS cells, all foreign DNA cloned on the vector containing the SV 40 replication origin will replicate in COS cells. The foreign DNA will be independent of the cellular DNA and temporarily replicated.

In recent years, except for some lymphokines, cDNA has been isolated by COS intracellular expression [Wong, G.G et al. Science 228: 810-815 (1985); Lee, F. et al. Proceedings of the National Academy of Sciences, USA 83: 2061-2065 (1986); Yokota, T. et al. Proceedings of the National Academy of Sciences, USA 83: 5894-5898 (1986); See Cell 47: 3-10 (1986) by Yang, Y. et al.], In general, few cDNAs are isolated from mammalian transgenic libraries. There are two main reasons for this. Firstly, existing techniques for constructing large plasmid libraries (see Mol. Cell, Biol. 2: 161-170 (1982), Okayama, H. et al.) Are difficult to master and are obtained by phage cloning techniques. The possible library sizes are rarely studied [Huynh, T. et al. In: DNA Cloning Vol. I, A Practical Approach, Glover, DM (ed.), IRL Press, Oxford (1985), pp. 49-78 Secondly, existing vectors are exceptionally inadequately adapted for high levels of expression, particularly in COS cells (see Wong, GG et al., Science 228: 810-815 (1985)). The success reported with lymphokin cDNAs does not suggest that the methods used are generally suitable because cDNAs are particularly prone to isolation from the expression library, and lymphokin bioassays are very sensitive [Wong, GG et al. 228: 810-815 (1985); by Lee, F. et al. Proceedings of the National Academy of Sciences, USA 83: 2061-2065 (1986); Yokota, T. et al. Proceedings of the National Academy of Sciences, USA 83: 5894-5898 (1986); Yang, Y. et al. Cell 47 : 3-10 (1986), mRNA is typically abundant and short [Song, GG et al. Science 228: 810-815 (1985); Lee, F. et al. Proceedings of the National Academy of Sciences, USA 83 2061-2065 (1986); Proceedings of the National Academy of Sciences, USA 83: 5894-5898 (1986); See, Yang, Y. et al., Cell 47: 3-10 (1986).

Therefore, expression in mammalian hosts has previously been most commonly used only as a variety of methods for identifying proteins encoded by genes isolated by more traditional cloning methods. See, eg, J. Virol, Stuve et al. 61 (2): 327-335 (1987) Herpes simplex Type II strain 333 by plaque hybridization of a recombinant phage vector based on M13 used to transform E. coli JM101 sensitized cells. The gene for glycoprotein gB2 of was cloned. The homogeneity of the protein encoded by the isolated clones was thus demonstrated by transfecting mammalian COS cells and Chinese hamster ovary (CHO) cells. Expression was demonstrated by immunofluorescence and radioimmunoprecipitation.

Oshima et al. Used plaque hybridization to screen phage lambda gt11 cDNA libraries for genes encoding beta-glucuronidase in the human placenta [Proceedings of the National Academy of Sciences, U.S.A. 84: 685-689 (1987). Homogeneity of the isolated cDNA clones was demonstrated by immunoprecipitation of proteins expressed by COS-7 cells transfected with cloned inserts using an effector after SV 40.

Transient expression in mammalian cells has been used as a means of confirming homogeneity of genes previously isolated by other screening methods. See Gerald et al., Journal of General Virology 67: 2695-2703 (1986). Mackenzie, Journal of Biological Chemistry 261: 14112-14117 (1986); Gene 43: 1111-1121 (1986) by Seif et al .; See also Orkin et al., Molecular and Cellular Biology 5 (4): 762-767 (1985).

These methods are often ineffective, tedious, and require multiple screening processes to identify clones of sufficient length or duplicates. Prior screening methods based on the expression of fusion proteins are ineffective and require large amounts of monoclonal antibodies. Such weakness is aggravated by the use of inefficient expression vectors, resulting in inadequate protein expression levels for effective selection.

Summary of the Invention

The present invention relates to cDNAs encoding cell surface antigens, isolated nucleotide sequences and encoded products thereof. The present invention also relates to new and robust methods for cloning cDNAs encoding cell surface antigens and to construct cDNA libraries for high efficiency expression vectors that are particularly suitable for high expression in eukaryotic host cells.

The highly efficient cloning methods of the present invention are based on the transient selection of antigens in eukaryotic cells and the physical selection of cells expressing antigens by adhering to antigen coatings such as culture dishes. The method of the present invention is useful for the isolation and molecular cloning of all proteins that can be transported and expressed on the cell surface membranes of eukaryotic cells.

The method for cloning cDNA encoding cell surface antigens of the present invention produces a cDNA library, introduces this cDNA library into eukaryotic mammalian cells, preferably tissue culture cells, and can express this cell surface antigen. The cells are cultured under conditions and exposed to the antibody or first antibody directed against the cell surface antigen to form a cell surface antigen-first antibody complex, which is subsequently directed to the first antibody. 2 Cells expressing cell surface antigens by exposure to a substrate coated with an antibody are allowed to adhere to the substrate through the formation of a cell surface antigen-first antibody-second antibody complex, and the adhesion is separated from the unbonded cells. It involves doing.

Using the cloning method of the present invention, genes encoding cell surface antigens can be isolated and molecularly cloned as follows: CD1a, CD1b, CD1c, CD2, CD6, CD7, CD13, CD14, CD16, CD19, CD20, CD22, CD26, CD27, CD28, CD31, CDw32a, CDw32b, CD33, CD34, CD36, CD37, CD38, CD39, CD40, CD43, CD44, CD53, ICAM, LFA-3, FcRia, FcRib, T1isa and Leu8 antigens.

The nucleotide sequence of the cloned gene was determined by the method of the invention and the amino acid sequence of the encoded protein was identified.

CD1a, CD1b, CD1c, CD2, CD6, CD7, CD13, CD14, CD16, CD19, CD20, CD22, CD26, CD27, CD28, CD31, CDw32a, CDw32b, CD33, CD34, CD36, CD37, CD38, CD39, CD40, Cloned genes encoding CD43, CD44, CD53, ICAM, LFA-3, FcR1a, FcRIb, TLiSa, and LEU8 are also subjects of the present invention.

Once the gene encoding the antigen is cloned according to the method of the invention, the gene can be expressed in prokaryotic or eukaryotic host cells to produce a portion or encoded protein in substantial pure form that does not exist in nature. . Aspects of the invention are substantially pure cell surface antigens, in particular CD1a, CD1b, CD1c, CD2, CD6, CD7, CD13, CD14, CD16, CD19, CD20, CD22, CD26, CD27, CD28, CD31, CDw32a, CDw32b, CD33, CD34, CD36, CD37, CD38, CD39, CD40, CD43, CD44, CD53, ICAM, LFA-3, FcRIa, FcRIb, TLiSa, and Leu8 antigens and functional analogs and equivalents thereof.

CD1a, CD1b, CD2, CD7, CD14, CD16, CD19, CD20, CD22, CD27, CD28, CDw32a, CDw32b, CD33, CD34, CD40, CD44, CD53, ICAM, LFA-3, FcRIa, FcRIb, TLiSa and Leu8 antigens The primary amino acid sequence of was determined. The invention thus also relates to the amino acid sequences of these antigens and their functional equivalents and the nucleotide sequences encoding these antigens.

The present invention also relates to a highly efficient cDNA expression vector that forms a very large mammalian expression library and yields a large amount of protein in the mammalian host cell for efficient selection. In a particular type of the invention, the cDNA expression vector is an inhibitory tRNA gene; SV 40 origin; A synthetic transcription unit comprising a chimeric promoter consisting of human cytomegalovirus AD 169, a direct early enhancer sequencer fused to HIV LTR-60 to +80 sequences, interposed between the inhibitory tRNA gene and the SV 40 origin; A polylinker comprising two Bst XI sites separated by a replicable DNA sequence and flanked by XbaI sites; And the SV 40 small t antigen splice polyadenylation signal initial site.

Another aspect of the invention includes a chimeric promoter composed of human cytomegalovirus AD 169, a direct initial enhancer sequencer fused to HIV LTR -60 to +80 sequences and comprises a synthetic transcription unit for use in a cDNA expression vector. . The small size and specific arrangement of the cDNA expression vector sequences of the present invention allows for efficient replication in host mammalian tissue culture cells such as COS cells. Moreover, this vector allows the use of a highly effective oligonucleotide based cDNA insertion method using a polylinker containing two converted Bst XI sites separated by short replicable DNA fragments.

Another aspect of the invention includes a vector comprising two identical Bst XI sites that are redirected with respect to each other, separated by short replicable DNA fragments. Another aspect of the invention includes a polylinker as mentioned above.

Another aspect of the invention incorporates a synthetic DNA oligonucleotide that provides a terminal sequence similar to the short replicable DNA fragment of the polylinker of the invention to a cDNA fragment to be inserted into a vector, and the resulting cDNA fragment and the synthetic DNA oligonucleotide A method of cDNA insertion based on oligonucleotides comprising inserting a terminal sequence into a polylinker of the vector from which a short replicable DNA fragment has already been removed.

In preparing a cDNA library according to the method of the present invention, a large number of tumor cells can be infiltrated by macrophages and lymphocytes and used as a raw material of macrophages or lymphocyte transcripts to produce a significant effect instead of the commonly used tumor cell lines. I found it. Therefore, another aspect of the present invention involves the use of tumor cells, in particular human tumor cells, to prepare cDNA libraries to be used by the methods of the present invention.

Another advantage of the robust selection system of the present invention is that there is no need to insert cDNA directly. The method of the present invention results in library construction efficiencies such as those described for phage vectors such as lambda gt10 and lambda gt11 and has the additional advantage that clones generated according to the method of the present invention are easy to manipulate.

The immunological selection techniques of the present invention allow for the efficient use of antibodies which can be monoclonal or polyclonal in relatively small absolute amounts. The method of the invention is also very fast. In general, three or several cycles of immunological selection and rescue are required to isolate target cDNA clones. Thus, the method of the present invention also results in the efficient use of labor and materials in cloning genes encoding cell surface antigens. As noted above, the methods of the present invention provide cell surface antigens that bind to mammalian T lymphocytes (eg, antigens CD1a, CD1b, CD1c, CD2, CD6, CD7, CD13, CD14, CD16, CD19, CD20, CD22, Genes encoding CD26, CD27, CD28, CD31, CDw32a, CDw32b, CD33, CD34, CD36, CD37, CD38, CD39, CD40, CD43, CD44, CD53, ICAM, LFA-3, FcRIa, FcRIb, TLiSa and Leu8) Was used to clone successfully.

Purified genes and purified proteins of the present invention are useful in the application of immunodiagnostics and immunotherapy comprising diagnosing and treating immune mediated infections, diseases and diseases of animals including humans. Purified genes and purified proteins of the invention can also be used to identify, isolate and purify other antibodies and antigens. Such diagnostic and therapeutic uses are not yet included in other aspects of the present invention. Moreover, substantially pure proteins of the present invention can be prepared as medicaments or pharmaceutical compositions for therapeutic administration. The present invention further relates to such pharmaceutical and pharmaceutical compositions.

Detailed description of the invention

The present invention relates to novel methods for cloning cDNAs encoding cell surface antigens and to construct cDNA libraries. In particular, the present invention relates to substantially pure cell surface antigens encoded by specific cDNA expression vectors and components thereof, nucleotide sequences or genes, cDNA fragments isolated by the methods of the present invention. The invention also relates to methods of using the isolated nucleotide sequences and encoded products.

The following detailed description will refer to various methodologies known to those skilled in the art of recombinant genetics. Publications and other materials in which known methodologies are incorporated by reference are incorporated herein by reference in their entirety. Standard references established in accordance with the general principles of recombinant DNA technology include Darnell, J.E. Molecular Cell Biology, Scientific American Books, Inc., publisher, New York, N.Y. (1986);

Lewin, B.M., Genes II, John Wiley & Sons, publisher, New York, N.Y. (1985); Old, R.W. Principles of Gene Manipulation: An Introduction to Genetic Engineering, 2d edition, University of California Press, Berkeley, CA (1981); And Molecular Cloning: A Laboratroy Manual, Cold Spring Harbor, NY (1982) by Maniatis, T. et al.

Cloning refers to the insertion of a specific gene or other DNA sequence into a vector molecule using in vitro recombinant techniques. To successfully clone the desired gene, a DNA fragment is formed and the fragment is combined with a vector molecule to introduce a complex DNA molecule into a host cell that the molecule can replicate and to select a clone with the target gene from the receptor host cells. You need to use the method.

'cDNA' means complementary or copy DNA produced from an RNA template by the action of an RNA dependent DNA polymerase (reverse transcriptase). Thus 'cDNA clone' refers to a double DNA sequence complementary to the RNA molecule of interest carried in a cloning vector.

By 'cDNA library' is meant a collection of recombinant DNA molecules containing cDNA inserts that together contain the entire genome of an organism. Such cDNA libraries can be prepared by methods known in the art that have already been described, for example described in Maniatis et al., Molecular Cloning: A Laboratory Manual, supra.

In general, RNA is first isolated from the genome of the organism cell in which the particular gene is to be cloned. For the purposes of the present invention, mammalian cell lines, in particular human cell lines, are preferred. More preferred are human tumor cell line HPB-ALL and human lymphoblast cell line JY.

Alternatively, the RNA may be isolated from tumor cells derived from tumors of the animal, preferably from tumor cells of the human body. Thus, the library can be prepared, for example, from adrenal tumors of the human body, but any other tumor can be used.

The immunoselective cloning method of the present invention extracts total RNA comprising a specific gene from a cell, synthesizes a series of complementary double stranded cDNA fragments from the RNA, and introduces the cDNA fragment into mammalian cells in tissue culture. Methods of making the library. Mammalian cells are conserved under conditions that allow for the expression of a protein (ie, cell surface antigen). The resulting cells are exposed to a first antibody or group of antibodies directed against cell surface antigens. As a result, a cell surface antigen-first antibody complex is formed.

This complex is exposed to a substrate coated or bound to a second antibody directed towards the first antibody. Cells expressing cell surface antigens are attached to a substrate (because complexes are formed in the cell surface antigen-the first antibody-the second term).

Isolation of Total RNA

As a method for separating total RNA, a guanidium thiocyanate / cesium chloride (hereinafter referred to as GuSCN / CsCl) method is preferable. More preferred are GuSCN / LiCl variants of the GuSCN / CsCl method with additional capacity and rate. Briefly, 0.5 g of GuSCN was dissolved in 0.58 ml of 25% LiCl (stock filtered through a 0.45 micron filter) for each ml of the desired mixture and 20 μl of mercapto ethanol was added. The cells are taken out little by little and the pellet is lightly dispersed to the wall and 1 ml of solution is added to 5 x 10 ⁷ cells. The resulting mixture is cut out with polytron until it is inviscid.

For small scale preparation (less than 10 ⁸ cells), 2 ml of layer of the cut mixture is added to 1.5 ml of 5.7 M CsCl (without RNase; 1.26 g of CsCl is added to each ml of 10 mM EDTA pH 8) and RNase Covered with free water and spun for 2 hours at SW 55 50 K rpm. For large scale preparation, 25 ml of layer was added to 12 ml CsCl in SW 28 test tube, covered with RNase free water and spun at SW 55 24 K rpm for 8 hours. Carefully aspirate the contents with a sterile Pasteur pipette connected to a vacuum flask. Once past the CsCl interface, scrape the band around the test tube with the tip of the pipette to prevent the layer on the test tube wall from flowing down. Inhale remaining CsCl solution. The pellet is absorbed (but not re-dissolved) in water. 1/10 volume of NaOAc and 3 volumes of EtOH were added and the final mixture was spun. If necessary, the pellet is resuspended in water (eg 70 ° C.).

The concentration is adjusted to 1 mg / ml and frozen. Small RNAs (eg 5S) do not sink. For small cells, reduce the volume and cover GuSCN in gradient form with RNase-free water (precipitation is not effective when RNA is dilute).

Preparation of Poly A ⁺ RNA

Poly A ⁺ RNA can be preferably prepared by the oligo dT selection method. Briefly, disposable polypropylene columns are prepared by washing with 5M NaOH and then washing with RNase free water. About 0.3 ml (final packing bed) of oligo dT cellulose is used for each milligram of total RNA. Oligo dT cellulose is prepared by resuspending about 0.5 ml of dry powder in 1 ml of 0.1 M NaOH and transferring it to a column or filtering 0.1 M NaOH through a previously used column (the column can be reused multiple times). .

It is washed with several column volume of RNase-free water until the pH is neutral and washed with 2-3 ml dropping buffer.

The column bed is discarded into 15 ml sterile test tubes using 4-6 ml dropping buffer. The total RNA was placed at 70 ° C. for 2-3 minutes, LiCl of RNase free stock was added (to 0.5 M) and mixed with oligo dT cellulose in 15 ml test tubes. Then vortex or stir for 10 minutes. The resulting product was poured onto a column, washed with 3 ml of dropping buffer and washed with 3 ml of medium flush buffer. mRNA was eluted directly into SW 55 in vitro with 1.5 mL of 2 mM EDTA, 0.1% SDS; Discard the first two or three drops.

Eluted mRNA was precipitated by adding 1/10 volume of 3M NaOAc and filling the test tube with EtOH. It was then mixed and cooled for 30 minutes at -20 ° C and spun at 50 K rpm for 30 minutes at 5 ° C. EtOH was poured out and the test tubes were dried in air. mRNA pellets were resuspended in 50-100 μl of RNase-free water.

About 5 μl is melted at 70 ° C. in MOPS / EDTA / formaldehyde and run on a 1% agarose gel without RNase to check vagina.

cDNA synthesis

CDNA is synthesized from the following. Preferred methods for the synthesis of cDNA are various methods described by Gubler and Hoffman (see Gene, 25: 263-269 (1982)). This method is carried out as follows:

a. First strand manufacturing

4 μg of mRNA was prepared and heated to about 100 ° C. in a microfuse in vitro for 30 seconds and cooled on ice. The volume was adjusted to 70 μl with no RNAse water.

Then 20 μl of RT1 buffer, 2 μl of RNAse inhibitor (36 u / μl from Boehringer), 5 μg / μl of oligo dT (1 μl, from Collaborative Research) and 20 mM dXTP's ( Purchased from UltraPure, 2.5 μl), 1 μl of 1 M DTT, 4 μl of RT-LX (purchased from LifeScience, 24 u / μl) were added. The resulting mixture was incubated at 42 ° C. for 40 minutes. Then heat was added to make it inert (10 min at 70 ° C).

b. Second strand manufacturing

Prepare 320 μl of RNAse free water, 80 μl of RT2 buffer, 5 μl of DNA polymerase I (Billinger, 5u / μl), and 2 μl of RNAse H (obtained from BRL, 2u / μl). . This was incubated at 15 ° C for 1 hour and at 22 ° C for 1 hour. 20 μl of 0.5 M EDTA at pH 8.0 is added, phenol is extracted and NaCl is added to 20 μg / ml of 0.5 M straight polyacrylamide (carrier) to precipitate EtOH and fill the test tube with EtOH. Rotate for 2-3 minutes in the microfuge test tube, then vortex to repel deposits high on the test tube wall and re-rotate for 1 minute.

c. Adapter manufacturer

Precipitated cDNA was resuspended in 240 μl of TE (10/1). 30 μl of 10 × low salt buffer, 30 μl of 10 × binding additive, 12-mer adapter with 3 μl (2.4 μg) kinase, 8-mer adapter with 2 μl (1.6 μg) kinase and 1 μl T4 DNA ligase (purchased from BioLabs, 400 u / μl or from Schöllinger, 1 vice unit / ml) was added. It was then incubated overnight at 15 ° C. Phenol extraction and EtOH precipitation (extra carriers are no longer needed) as mentioned above and resuspended in 100 μl of TE.

Use of cDNA Fragments for Expression Vectors

For use with a cDNA expression vector based on Bst XI of the present invention (see below), an oligonucleotide fragment comprising the terminal sequence corresponding to the Bst XI site on the vector was bound to the cDNA fragment to be inserted. The pool was prepared by fractionating the resulting fragments. The preferred method is as follows:

20% KOA, 2 mM EDTA, 1 μg / ml EthBr solution and 5% KOAc, 2 mM EDTA, 1 μg / ml EthBr solution are prepared.

2.6 ml of 20% KOAc solution is added to the bag chamber with low slope marker. Air bubbles are removed from the test tube connecting the two chambers by flowing the solution through the front chamber and then tilting backwards. Block the passage between the chambers and add 2.5 ml of 5% solution to the front chamber. If there was liquid in the test tube from the previous run, run a 5% solution at the end of the test tube and return it to the chamber. Place the device on the stir plate, secure the stir bar moving as soon as possible, open the stopper connecting the two chambers and open the front stopper. Fill the KOAc solution from the polyalomer SW 55 test tube bottom. Cover the slope with 100 μl of cDNA solution. Equilibrium test tubes are prepared and the slope is rotated for 3 hours at 22 ° C. at 50 K rpm. To collect fractions from that slope, penetrate the SW 55 test tubes with the butterfly infusion set (with the cut luer herb) close to the bottom of the SW 55 test tubes and collect three 0.5 ml fractions and then 0.25 ml fractions. 6 are placed in a microfuse test tube (22 drops and 11 drops, respectively). Fractions are EtOH precipitated by the addition of 20 μg / ml of straight polyacrylamide and EtOH is filled to the end of the tube. After cooling the test tube, it is spun in a microfuse for 3 minutes.

Then vortex and re-rotate for 1 minute. The pellet is washed with 70% EtOH (return). Do not dry completely. Resuspend each 0.25 ml fraction in 10 μl of TE.

Run 1 μl on 1% agarose minigel. The first three fractions are collected and the last six fractions contain no material smaller than 1 kb.

Inhibitory tRNA plasmids can be propagated by known methods. In a preferred method of the present invention the supF plasmid can be selected from non-inhibiting hosts comprising p3, the second plasmid, and comprises elements of amber mutated ampicillin (amp) and tetracycline (tet) drug resistance (Seed, See 1983). The p3 plasmid derived from PR 1 is 57 kb in length and is a single radiation episome that remains stable. Ampicillin resistance of this plasmid returns to high speed, so usually ampr plasmid cannot be used for p ³ -containing strains. Choosing a plasmid that is only resistant to tet is usually as good as choosing a plasmid that is amp + tet resistant. However, the spontaneous appearance of chromosomal inhibitory tRNA mutations represents the inevitable side (about 10 ^-9 frequency) in this system. Colonies resulting from naturally occurring inhibitory mutations are usually larger than colonies resulting from plasmid transformation. Typically inhibitory plasmids are selected from LB medium containing 12.5 μg / ml amp and 7.5 μg / ml tet. For large scale plasmid production, M9 casamino acid medium containing glycerol (0.8%) can be used as the carbon source and the bacteria grow saturated.

Vector DNA can be isolated by known methods.

The following method is the preferred method of separating plasmid from 1 liter of saturated cells:

Rotate the cells in one liter of J6 bottle at 4.2 K rpm for 25 minutes. Resuspend in 40 ml of 10 mM EDTA at pH 8. (Muddy on a soft surface). Add 80 ml 0.2 M NaOH, 1% SDS and return until clear and viscous.

40 ml of 5 M KOAc (2.5 M KOAc, 2.5 M HOAc) at pH 4.7 is added and shaken to a point of viscous (until the mass is 2-3 mm). Rotate for 5 minutes at 4.2K rpm in a 1 liter J6 bottle. The supernatant is poured into a 250 ml bottle through a thin, thin cotton ball. Fill the bottle with isopropyl alcohol. Then spin in a 1 liter J6 bottle at 4.2 K rpm for 5 minutes. Pour the liquid from the bottle and rinse it slowly with 70% EtOH (do not break the pellet). Turn the bottle upside down to remove traces of EtOH with Kimwipe.

Resuspend in 3.5 ml Tris base / EDTA 20 mM / 10 mM.

3.75 mL of resuspended pellet is added to 4.5 g of CsCl.

0.75 mL of 10 mg / mL EtBr is added and mixed. The solution is filled into a VTi 80 test tube. Run at 80 rpm for 2.5 hours or more. The band indicated by visible light is extracted with a 1 ml syringe and a needle with a gauge of 20 or less. Cut off the top of the test tube, insert (ie as thin as possible) the needle above the test tube at an angle of about 30 ° C. with respect to the test tube and diagonal the needle about 3 mm below the band. After removing the band, the tube contents are poured into the bleach. Place the extracted band in 13 ml Sarstedt test tube. Extract n-butanol saturated with 1 M NaCl in a test tube. When large amounts of DNA are obtained, they are reextracted. Inject butanol into a trap containing 5 M NaOH (to destroy ethidium). Almost equal volume of 1 M ammonium acetate is added to the DNA (squirt bottle). Add about 2 volumes of 95% ethanol. Rotate for 5 minutes at 10 K rpm. J2-21. The pellet is carefully washed with 70% ethanol. Dry with a cotton swab or lyophilize.

Vectors can be produced by known methods for cloning.

A preferred method is to start cutting 20 μg of vector in 200 μl of reaction with 100 units of Bst XI (purchased from Biolabs, New York, USA) to 50 ° C. in a thermostated bath (ie circulating structure). By cutting all night. Prepare 2 KOAc at 5-20% slope in SW 55 test tube as described above. 100 μl of the cleaved vector is added to each test tube and run at 50 ° C. at 22 ° C. for 3 hours. The tube is irradiated at 300 nm ultraviolet. The desired band will travel about two-thirds of the tube length. Overload the slope toward the front of the band means. Remove the band with a 1 ml syringe and a 20 gauge needle. Straight polyacrylamide is added and 3 volumes of EtOH are added to precipitate the plasmid. Resuspend in 50 μl of TE. Combine by adding a certain amount of vector and cDNA. Based on this binding attempt, binding on a large scale, which can be done by known methods. Usually, 1-2 μg of cleavage vector is required for the whole cDNA preparation.

The adapter can be prepared by known methods but the crude adapter is resuspended at a concentration of 1 μg / μl, MgSO ₄ is added 10 mM and 5 volumes of EtOH are added to precipitate. Wash with 70% EtOH and resuspend in TE at a concentration of 1 μg / μl. To the kinase with 25 μl of resuspended adapter was added 3 μl of 10 × kinase buffer and 20 units of kinase and incubated overnight at 37 ° C.

Among the preferred methods of the present invention, methods for preparing the buffers mentioned in the above detailed description will be apparent to those skilled in the art. For convenience, preferred buffer compositions are as follows:

Dropping buffer: 0.5 M LiCl, 10 mM Tris pH 7.5, 1 mM EDTA 0.1% SDS.

Medium wash buffer: 0.15 M LiCl, 10 mM Tris pH 7.5, 1 mM EDTA 0.1% SDS.

Rt 1 buffer: 0.25 M Tris pH 8.8 (pH 8.2 at 42 ° C.), 0.25 M KCl, 30 mM MgCl ₂ .

RT 2 buffer: 0.1 M Tris pH 7.5, 25 mM MgCl 2, 0.5 M KCl, 0.25 mg / ml BSA, 50 mM DTT.

10 × Low Salt Buffer: 60 mM Tris pH 7.5, 60 mM MgCl ₂ , 50 mM NaCl, 2.5 mg / ml BSA, 70 mM Me.

10 X binding additive: 1 mM ATP, 20 mM DTT, 1 mg / ml BSA, 10 mM spermidine.

10 X Kinase Buffer: 0.5 M Tris pH 7.5, 10 mM ATP, 20 mM DTT, 10 mM spermidine, 1 mg / ml BSA 100 mM MgCl ₂ .

"Vector" refers to a DNA molecule derived from plasmid or bacteriophage and into which DNA fragments can be inserted or cloned. The vector may be autonomously replicated in a host or carrier organism that contains one or more unique restriction sites and that allows the cloned sequence to replicate. Thus, a 'DNA expression vector' refers to any autologous element that can be replicated independently in a host with the chromosome of that host after the DNA of the additional sequence has been inserted into the genome of the autologous element. Such DNA expression vectors include bacterial plasmids and phages.

Preferred for the purposes of the present invention are viral vectors such as those derived from Simian virus strain 40 (SV40). SV40 is a papovavirus containing a double stranded cyclic DNA molecule having a molecular weight of 28 Mdal and having a molecular weight of 3 Mdal covering the entire genome of the virus. The total nucleotide sequence of this single small, covalent pulmonary cyclic DNA molecule was determined. Nature 273: 113-120 (1978) by Fiers et al .; See Reddy et al., Science 200: 494-502 (1978). Viral DNA of the SV 40 can be obtained in large quantities and the genomic sites responsible for the various viral functions are precisely located in the detailed physical map of the DNA. Fiers et al., Supra; See Reddy et al., Supra. The viral genome of SV 40 is able to proliferate silently or as a complete part of the cell chromosome, with a wealth of information present upon replication and expression of this genome.

Also preferred for the purposes of the present invention is a single stranded bacteriophage cloning vehicle named M13 having a pulmonary cyclic DNA genome of about 6.5 kb. The advantage of using M13 as a cloning carrier is that phage particles released from infected cells can be used as templates for DNA sequencing analysis because they contain single stranded DNA homologous to one of two complementary strands of cloned DNA. to be.

Even more preferred for the purposes of the present invention are the expression vectors piH3, piH3M and CDM8 deposited in the American Type Culture Collection (ATCC), Rockville Parkrow Drive 12301, Maryland, USA. piH3 is deposited as ATCC 67634, dated February 24, 1988. piH3M has been deposited as ATCC 67633, February 24, 1988.

CDM8 is deposited as ATCC 67635, dated February 24, 1988.

By 'tissue culture' is meant to persist or grow animal tissue cells in vitro to further differentiate and preserve cell structures or functional groups or both. Primary tissue cells are those obtained directly from a population of homogeneous cells that perform the same function in an organism. For example, treatment of such tissue cells with trypsin, a proteolytic enzyme, is dissociated into each primary tissue cell that grows well when planted at high density on the culture plate. Cell cultures produced by proliferating primary tissue cells in tissue culture are referred to as 'secondary cell culture'. Most secondary tissue cells divide a limited number of times and then die. However, some secondary cells can proliferate indefinitely to form continuous 'cell lines' beyond the 'separation time'.

Often cell lines contain extra chromosomes and are usually abnormal in other respects. The persistence of these cells is a characteristic that cancer cells have in common.

Preferred cell lines for use as tissue culture cells according to the present invention include monkey kidney cell lines named 'COS'. COS cells are cells that have been transformed with SV40 DNA that contain a functional initial gene site except for the incomplete origin of viral DNA replication. COS cell clone M6 is particularly preferred for use in the methods of the present invention. Also preferred for the purposes of the present invention are 'WOP' cells of mice in which NIH 3T3 cells have been transfected with polyoma originating deletion DNA.

cDNA can be introduced into the host tissue culture cells of the present invention by methods known in the art. Transfection is described, for example, in plasma fusion, spherical fusion or DEAE dextran method [Sussman et al., Cell. Biol. 4: 1641-1643 (1984).

If the spherical fusion method is used, Mol. Cell Bio. The following modification method based on 1: 743-752 (1981) is a preferable method. For example, in short, a set of six fusions requires 100 ml of cells in gravy. Cells containing proliferative plasmids are grown in LB such that OD 600 = 0.5.

100 μg / mL spectinomycin (or 150 μg / mL chloramphenicol) is added. Continue to incubate at 37 ° C. with shaking for 10-16 hours (cells start to lyse with extended incubation in spectinomycin or chloramphenicol medium).

100 ml of culture (JA 14 / GSA rotor, 250 ml bottle) is spun at 10,000 rpm for 5 minutes. Beat well and resuspend the pellet in the bottle with 5 ml of cold 20% sucrose, 50 mM Tris-HCl in pH 8.0. Incubate for 5 minutes on ice. 2 ml of cold 0.25 M EDTA at pH 8.0 is added and incubated at 37 ° C. (water bath) for 5 min. Place on ice and examine the percent conversion of the spheres under the microscope.

In a flow hood, slowly add 20 ml of cold DME / 10% sucrose / 10 mM MgCl ₂ (approx. 2 drops per second). Remove medium (50% fusion) from cells plated in 60 cm dishes the day before. 5 ml of globular suspension is added to each dish. Place the dish on top of the test tube carrier in a centrifuge with a winged bucket. Six plates can be conveniently prepared at one time. Dishes can be stacked on top of other dishes, but stacking three is undesirable because the spherical layer on the top dish is often torn or separated after centrifugation. Rotate for 10 minutes at 1000 xg. Calculate the force based on the radius on the plate bottom. Carefully inhale the fluid from the dish. Pipette 1.5-2 mL of 50% (w / w) PEG 1450 (or PEG 1000) / 50% DME (serum free) into the center of the dish. If necessary, clean the area around the pipette end so that the PEG extends radially flat on the plate. After adding PEG to the last dish, support all dishes with a rod so that the PEG solution collects on the floor. Then inhale PEG. The remaining PEG thin layer on the cell surface is sufficient to promote fusion, and the remaining PEG thin layer is easy to wash and better cell viability can be obtained than if the PEG stock was left behind. In 90-120 seconds (PEG 1000) or 120-150 seconds (PEG 1450) after contact with the PEG solution, 1.5 ml of DME (serum-free) is pipetted into the center of the dish. The PEG layer is washed radially with DME. Tilt the dish at an angle and inhale. Repeat wash with DME. 3 ml of DME / 10% serum containing 15 μg / ml of gentamicin sulfate are added. Incubate for 4-6 hours in the incubator. The medium and residual bacterial suspension are removed and further medium is incubated for 2-3 days.

Excess washing of the cell layer to remove PEG removes only a large number of cells with no substantial benefit. If cells are allowed to be washed for a few minutes with a second DME, most of the globular layer will form naturally. However, it is preferred to simply wash and terminate to 37 ° C. in complete medium.

PEG solutions can be conveniently prepared by melting fresh bottles with PEG to 60 ° C. and pouring about 50 ml aliquots into pre-weighed bottles using 50 ml centrifuge tubes. Aliquoted PEG is stored at 5 ° C. in the dark. Aliquots are weighed and remelted to prepare fresh bottles and the same volume of DME (serum free) is added. If necessary, adjust the pH with 7.5% sodium bicarbonate solution and filter sterilize. The resulting PEG solution can be stored up to 3 months at room temperature with no appreciable adverse effect.

Transfected host cells will be cultured according to the present invention to express the protein encoded by the cDNA clone and increase the absolute number of cells available for subsequent immunoselection. Those skilled in the art will be able to recognize methods and media suitable for the purposes of the present invention and to take into account cell types and other variables generally considered. For example, COS cells can be cultured in Dulbecco's Modified Eagle's Medium (DME) supplemented with 10% bovine serum and gentamicin sulfate. Transient expression of normally transfected cells can predict posterior transfection within 48 to 72 hours. However, this period will vary depending on the type or strain of host cell used and the cell culture conditions, which will be apparent to the skilled artisan.

Immunoprecipitation, blotting, and cDNA sequencing of genes cloned according to the methods of the present invention can be performed by any convenient method known to those skilled in the art. For example, Clark et al., Leukocyte Typing II, Vol. II, pp. Preference is given to the immunoprecipitation protocols described in 155-167 (1986). Southern, Northern or other blot analysis methods known in the art are used, using hybrid probes prepared by known methods such as Hu et al. (See Gene 18: 271-277 (1982)). . CDNA sequencing is also described in Sanger et al., Proc. Natl. Acad. Sci. (USA) 74: 5463-5467 (1977) by the known method, including the dideoxynucleotide method of literature.

The antibodies used according to the invention may be polyclonal antibodies or monoclonal antibodies. Such antibodies can be used alone or in combination with other polyclonal or monoclonal antibodies to affect the immunoselection of the antigen or cells expressing the antigen by the methods of the invention.

Methods of preparing antibodies or fragments thereof for use in accordance with the present invention are known to those skilled in the art.

Standard references that set general guidelines for immunology include Klein, J., Immunology: The Science of Self-Nonself Discrimination, John Wiley & Sons, publisher, New York (1982); Eds., Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses, Plenum Press, publisher, New York (1980); Campbell, A., 'Monoclonal Antibody Technology' in Burden, R. et al., Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 13, Elsevere, publisher, Amsterdam (1984).

'Antibody' means fragments in addition to the original molecule capable of binding to antigens such as, for example, Fab and F (ab) ' ₂ fragments. Polyclonal antibody preparations can be derived directly from the blood of the appropriate animal species after immunization with the desired antigen or fragment thereof using all standard protocols known to those skilled in the art. Similarly, monoclonal antibodies can be prepared using known methods [Kohler et al. Eur. J. Immunol. 6: 292 (1976). Monoclonal antibodies are preferably used for the purposes of the present invention.

For the purpose of immune selection according to the present invention, tissue culture host cells exposed to an antibody against a target cell surface antigen express the target antigen by distributing the cells on a substrate coated with the antibody against the antibody to the antigen. Are separated from host cells. The technique 'panning' is known to those skilled in the art and is described, for example, in J. Immunol. Meth. 15: 47-56 (1977) and Proc. Of Wysocki and Sato. Natl. Acad. Sci (USA) 75: 2844-2848 (1978).

Panning according to the method of the invention may be carried out as follows:

a. Antibody-coated dish. Bacteriologic 60 mm plates, Falcon 1007 or equivalent, or 10 cm plates such as Fuchsia 8-757-12 can be used. Positive anti-mouse affinity purified antibodies (eg purchased from Cooper Biomedical (Cappel)) are diluted to 10 μg / ml in 50 mM Tris HCl at pH 9.5.

Add 3 ml per 6 cm dish or 10 ml per 10 cm dish. Leave carefully for an hour and a half, move to the next dish, leave for an hour and a half, and move to the third dish. Wash the plate 3 × with 0.15 M NaCl (in this case the wash bottle is convenient) and incubate overnight in 3 ml of 1 mg / ml BSA in PBS, inhale and freeze.

b. Panning. The cells will be in a 60 mm dish.

The medium is aspirated from the dish and 2 ml of PBS / 0.5 mM EDTA / 0.02% azide is added to incubate the dish at 37 ° C. for 30 minutes to remove cells from the dish. Break three cells with a short Pasteur pipette and collect the cells from each dish into centrifuge tubes. Fix at 2.5 (200 xg) (5 minutes) and spin for 4 minutes. Resuspend the cells in 0.5-1.0 ml of PBS / EDTA / azide / 5% FBS and add antibody. Incubate on ice for at least 30 minutes. Add equal volume of PBS / EDTA / azide and carefully layer on 3 ml PBS / EDTA / azide / 2% picol, fix at 2.5 and spin for 4 minutes. Carefully inhale the supernatant. Antibody-coated dishes containing 3 ml of PBS / EDTA / azide / 5% FBS by absorbing cells in 0.5 ml of PBS / EDTA / azide and pipetting through a 100 micron nylon net (purchased from Teteco) An aliquot is added to the.

Add cells from up to two 60 mm dishes to one 60 mm plate with antibody-coated. Leave at room temperature for 1-3 hours. Excess cells that have not adhered to the plate are removed by washing gently with PBS / 5% serum or medium. Usually it is enough to wash 2 or 3 times with 3 ml.

c. Hirt supernatant. Hirt, J. Molec. Biol. 26: 365-369 (1967) The preferred method of modification of the literature is as follows: 0.4 ml of 0.6% SDS, 10 mM EDTA is added to the panned plate. Leave for 20 minutes (or 1 minute if there are no cells on the plate). The viscous mixture is pipetted into a microfuse test tube.

Add 0.1 ml of 5 M NaCl, mix and leave on ice for at least 5 hours. Keeping the mixture as cold as possible seems to improve the quality of the hilt supernatant. Rotate for 4 minutes and carefully remove the supernatant, extract phenol (perform twice if the first interface is not clean), add 10 μg straight polyacrylamide (or other carrier), fill the test tube with EtOH and settle to 0.1 ml Resuspend in. Three volumes of EtOH / NaOAc are added to reprecipitate and suspended in 0.1 ml. Reprecipitate by adding 3 volumes of EtOH / NaOAc and resuspend in 0.1 ml. MC 1061 / p3 is preferably transformed using a high rate protocol to be described later. If the DNA volume exceeds the 2% sensitive cell aliquot, the transformation efficiency will be poor. 5% represents the same number of cells as 2.5% (efficiency is halved).

For aspects of the invention it is preferred to use 'blockers' in the culture medium. Blockers that are certain that nonspecific proteins, proteolytic enzymes or antibodies are present do not destroy or cross-link with antibodies present on the substrate or host cell surface and result in false positives or false negatives. The choice of blocking agent can substantially improve the specificity of the immunoselection step of the present invention. For example, many nonspecific monoclonal antibodies of the same type or subtype (isotype) (eg, IgG ₁ , IgG ₂ A, IgGm, etc.) as used in the immunoselection step can be used as blocking agents. Blocker concentrations (usually 1-100 μg / μl) are important to maintain adequate sensitivity to suppress unwanted interference. Those skilled in the art will also recognize that the buffer system used in the culture may be selected to optimize the blocking action and reduce nonspecific binding.

First, the panned cell populations for cells expressing the target cell surface antigen are removed from the trypsin-free cell culture dish (cultured). The cells were then exposed to the first antibody, which is a polyclonal or monoclonal antibody directed against the relevant antigenic family or antigen of interest. At this early stage, one antibody or population of antibodies can be used and selection issues, expected frequencies and other variables depending on the nature of the target antigen will be apparent to those skilled in the art.

The target antigen expressed on the surface of the host cell will form an antigen-antibody complex.

Subsequently, the cells are juxtaposed near substrates such as culture dishes, filter discs, or the like previously coated with a second antibody or group of antibodies. This second antibody will be directed towards the first antibody, for example the problem of selecting the animal from which the first antibody is produced will be self-defined by one of ordinary skill. For example, if the first antibody is produced in a mouse, the second antibody will be directed against the immunoglobulin of the mouse from goat or sheep. Cells that express the target antigen will adhere to the substrate via a complex formed between the antigen, the first antibody and the second antibody. Adherent cells can then be washed to separate from non-adhered cells. DNA encoding the target antigen is described in Hirt, J. Molec. Biol. 26: 365-369 (1967) prepared from adherent cells by known methods, such as those in the literature. This DNA is obtained in abundance to the desired degree by transformation into E. coli or other suitable host cell for subsequent fusion and selection.

In the normal case, the first one immunoselection will use the first panel of antibodies directed towards the epitope or epitope group common to the antigen family to which the target antigen belongs. This will be enough to significantly reduce the number of clones for the next immunoselection. Usually these two immunoselections have been shown to be appropriate but the number of immunoselections may vary as described above. Thereafter, one selection can be made using either the first group of antibodies or one first antibody that recognizes only the target antigen.

"Substrate" means a solid surface to which an antibody may bind upon immunoselection according to the invention. Suitable suitable substrates include glass, polystyrene, polypropylene, dextran, nylon and other materials. Test tubes, beads, microtiter plates, bacterial plates, and the like coated with or formed from such materials can be used. Antibodies can be covalently or physically bound by known methods by covalent or ester bonds via amides, or by absorption. One skilled in the art will know many other suitable substrates and methods of immobilizing antibodies on the substrates, or will be able to identify substrates and methods using only typical experiments.

Host tissue culture cells for preferably use in accordance with the present invention should be selected to avoid a condition in which the antibody used for panning recognizes a determinant on uninfected cells. COS cells are therefore preferred for transient expression of certain surface antigens, while more preferred are WOP cells of mice.

Among the rat WOP cells, WOP 3027 cells are more preferred.

Since WOP cells rarely cross-react between murine antibodies and murine cell surface determinants, virtually all antibodies can be used.

The insertion size of the recombinant DNA molecule should be chosen to maximize the likelihood of obtaining the entire coding sequence. Those skilled in the art will know a variety of ways to determine the predetermined optimal insertion size of a given gene.

Vector construction and cDNA insertion

Vectors suitable for expressing cDNA in mammalian tissue culture cells can be constructed by known methods.

Preferred for the purposes of the present invention are expression vectors comprising SV 40 origin. Vectors can be naturally derived or include synthetic transcription origin and SV 40 early site promoters. More preferred are chimeric promoters composed of direct early enhancer sequences of the human cytomegalovirus. In addition, various 'enhancer sequences' can be used with the SV 40 vector. Such content is described, for example, in Cell 27: 299-308 (1981) to Banerji et al .; Nature 295: 568-572 (1982) by Levinson et al .; And Mol., Conrad et al. Cell. Biol. 2: 949-965 (1982).

CDNA insertion into a vector of the present invention can occur, for example, by homopolymer end sites with terminal transferases. However, homopolymer tubes positioned 5 'to the cDNA insert can inhibit in vitro and in vivo expression. Therefore, for the purposes of the present invention, it is preferred to use inverted identical cleavage sites separated by short replicable DNA fragments. Such reversed cleavage sites preferably use Bst XI restriction endonucleases and can be used in parallel with cDNA synthetic oligonucleotides that provide similar ends to the clotable fragment of the vector. In this way, the cDNA cannot bind to itself but to the vector. This allows the most efficient use of both cDNA and vectors.

Another type of the invention is the above method based on the above oligonucleotides sufficient to facilitate the insertion of cDNA into the vector. The piH3M of the present invention uses preferred and inverted endonuclease sites. This vector may include the SV 40 origin of replication but a more preferred type includes the M13 origin. Vectors containing the M13 origin allow for high levels of expression of the coding sequence under control in COS cells. The small sized and sequenced sequences in the plasmid also allow for high levels of replication in COS cells.

'Cell surface antigen' refers to any protein transported to the cell surface through an intracellular membrane system. Such antigens are usually attached to cell surface membranes through carboxy terminal domains containing hydrophobic amino acids lying in the lipid layer of the membrane, where they exert biological antigenic effects.

Antigens similar to T-lymphocytes are particularly suitable for gene cloning by the methods of the invention. However, cell surface antigens of all cells can be cloned according to the methods of the present invention.

Moreover, proteins which are not usually expressed on the cell surface can be cloned according to the method of the present invention using, for example, a fluorescence cytometry sorter (FACS) to enrich for fixed cells expressing intracellular antigens.

By "substantially pure" is meant that all antigens of the present invention or all genes encoding such antigens are present in a type that is substantially free of other contaminants or other antigens or genes normally found together in nature and not found in nature. . 'Functional derivative' means 'fragment', 'modifier', 'analog' or 'chemical derivative' of a molecule. 'Fragment' of a molecule, such as all antigens of the invention, means all polypeptide subsets of the molecule. 'Modified' of such molecule means a naturally occurring molecule that is substantially similar to the entire molecule or fragment thereof. By 'analog' of a molecule is meant a non-natural molecule that is substantially similar to the entire molecule or fragment thereof.

Fragments, variants, analogs and / or chemical derivatives of antigens are fragments, variants, analogs, chemical derivatives and the like when the amino acid sequences of the two molecules are substantially similar, ie at least about 50% homology and the two molecules have similar biological activity, and / Or 'substantially similar' to the antigen.

If the two sequences are substantially similar, that is, the first nucleotide sequence encodes fragments, variants, analogs, chemical derivatives having an amino acid sequence that is at least about 50% homologous to the amino acid sequence encoded by the second nucleotide sequence And the biological activity of the fragments, variants, analogs, chemical derivatives and / or antigens encoded by the first nucleotide sequence with the fragments, variants, analogs, chemical derivatives and / or antigens encoded by the first nucleotide sequence. In the case of similar biological activity, one nucleotide sequence is said to be "substantially similar" to the other. Thus, if two molecules have similar biological activity, they are considered variants if the term is used, even if the molecule contains additional amino acid residues not found in the other molecule or if the sequences of amino acid residues are not identical. . As used herein, when a molecule contains additional chemical moieties that are not usually part of a molecule, that molecule is said to be a 'chemical derivative' of another molecule. Such chemical moieties can improve the solubility, absorption, biological half-life, etc. of the molecule. The chemical moiety can reduce the toxicity of the molecule or eliminate or attenuate all unwanted side effects of the molecule. Chemical moieties that may mediate such actions are described, for example, in Remington's Pharmaceutical Sciences, 16th ed., Mack Publishing Co., Easton, Penn. (1980).

Similarly, a 'functional derivative', which is a gene of all antigens of the present invention, may be said to be "substantially similar" in its nucleotide sequence and is a "fragment", "variant", or It includes 'analog'.

Antigens, fragments, variants, analogs, and / or chemical derivatives may have a 'similar biological activity' for other antigens, fragments, variants, analogs and / or chemical derivatives if the former has at least about 25% of the latter's biological activity. Say you have it. Biological activity is an action, function or process that is characteristic of life.

In addition, biological activity is the regeneration, extension of biological processes in vitro or non-naturally occurring such that biological activity occurs when artificially inserting antigens, fragments, variants, analogs and / or chemical derivatives into an experimental animal to induce antibody production. Or adaptation. Antigens, fragments, variants, analogs and / or chemical derivatives may have one or more biological activities. Biological activity can be detected or measured by methods or assays characteristic of that activity. Functional derivatives having a biological activity similar to that of an antigen should have a biological activity of at least about 25% of the antigenic activity as determined by a characteristic assay for that activity and known to those skilled in the art.

Substantially pure antigens expressed by the methods of the invention include immunodiagnostic assays known to those of skill in the art, including radioimmunoassays (RIAs), enzyme immunoassays (EIAs) and enzyme-linked immunosorbent assays (ELISAs). Can be used for Soluble, substantially pure proteins of the present invention may be used alone or in combination with other agents or other antigens of the present invention, including lymphokines and monocaine or drugs to treat immune related diseases and disorders in animals, including humans. May be administered. Examples of disorders that may benefit from treatment with substantially pure proteins of the invention include immunodeficiency and acute hypersensitivity, asthma, irritable pneumonia, immunocomplex disease, vasculitis, systemic lupus erythematosus, rheumatoid arthritis, immunopathological kidney disorders, Acute inflammation, hemolytic anemia, platelet disorders, plasma and other cell tumors, amyloidosis, parasites, multiple sclerosis, murine-Barre syndrome, acute and subacute myopathy, myasthenia gravis, endocrine diseases and tissue and organ transplantation Rejection, all of which are described in Petersdorf et al., Eds., Harrison's Principles of Internal Medicine, supra. See also Weir, ed., Supra; Boguslaski et al., Eds., Supra; And Holborow et al., Eds., Supra.

When used in immunotherapy, the antigens of the invention may or may not be labeled with a therapeutic agent. Examples of therapeutic agents that can be combined with the antigens of the present invention for immunotherapy are drugs, radioisotopes, lectins and toxins.

The range of dose for administering the antigen of the present invention is large enough to produce the desired immunotherapeutic effect, but not large enough to cause side effects such as unwanted cross reactions and hypersensitivity.

In general, the dose used will depend on the age, condition, sex and severity of the patient. In addition, reverse signs (if any), immune tolerance and other variables will also affect the titer dose. Administration may be parenterally by injection or by gradual perfusion over time. It may also be administered intravenously, subcutaneously, intramuscularly, intraperitoneally or transdermally.

Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-water soluble solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol solutions, aqueous solutions, emulsions or suspensions containing saline and buffered medium. Parenteral excipients include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or nonvolatile oils. Intravenous excipients include fluid and nutritional supplements, electrolyte supplements and the like based on Ringer's dextrose. There are also antiseptics and other additives such as antimicrobial agents, antioxidants, chelating agents, inert gases and the like. Such formulations and methods for their preparation are described, for example, in Remington's Pharmaceutical Science, 16th ed., Supra.

In addition, the antigen of the present invention may be used alone or in combination with other drugs such as lymphokine, monocaine and drugs, drugs used in the treatment of animals including humans, immunological indicators, or other antigens. It can be made into a composition.

The antigen of the invention may be administered alone, but is preferably administered in a pharmaceutical composition. The pharmaceutical composition of the present invention comprises at least one antigen or a pharmaceutically acceptable salt thereof, one or more carriers and any other therapeutic agent. 'Acceptable' means that the medicament or carrier is compatible with the other ingredients of the pharmaceutical composition and does not harm the patient. The composition includes those suitable for oral, rectal, nasal, topical (including cheek and tongue), vaginal or parenteral administration. The compositions are conveniently in unit dosage form and may be prepared by methods known in the art of pharmacy. Such methods include combining the active ingredient with the carrier which constitutes one or more accessory ingredients. Generally, the composition is prepared uniformly by combining the active component with a liquid carrier or a finely ground solid carrier or both, and molding the product formed thereby, if necessary.

Pharmaceutical compositions administered orally in accordance with the present invention may be of any convenient type, including capsules, cachets or tablets, each containing a predetermined amount of the active ingredient. Also possible are powders or granules other than solutions or suspensions in liquid emulsions of water- or water-insoluble liquids or oil-in-water, or liquid emulsions of water-in-oil. The active ingredient may also be presented as a bullion, a paper or an ointment.

The invention described so far will be more fully understood by reference to the following examples which do not in any way limit the scope of the invention.

EXAMPLE

Example I Isolation, Molecular Cloning and Structure of Human CD2 Antigen

cDNA expression vector piH3

COS cell expression vectors are constructed from piSV by inserting a synthetic transcription unit between the inhibitor tRNA gene and the SV 40 origin [Luttle et al. Mol. Biol. Med. 1: 473-488 (1983).

The transcription unit consists of a chimeric promoter consisting of human cytomegalovirus AD169 direct early enhancer sequences fused to HIV LTR-67 to +80 sequences. Directly downstream from the HIV LTR +80 sequence is a polylinker containing two Bst X1 sites separated by a 350 bp stuffer: on the side of the Bst X1 site is an Xba1 site that may be used to delete the insert. have. Downstream of the polylinker is the small t antigen splice of SV 40 and the early site polyadenylation signal derived from pSV2. The nucleotide sequence of the transfection vector is shown in FIG.

cDNA Library Configuration

RNA can be prepared from HPB-ALL cells by the guanidium thiocyanate / CSCl method described above. Poly A ⁺ RNA is prepared from total RNA by oligo dt selection. See Maniatis et al. Molecular Cloning: A Laboratory Manual, supra. cDNA is synthesized by the method of Gubler and Hoffman (Gene 25: 263-269 (1982)). The Bst XI adapter binds to cDNA and the reaction product is fractionated by centrifugation through a 5 ml 20% potassium acetate gradient containing 1 mM EDTA for 3 hours at 50 k rpm in SW 55 rotor. Collection was manually via a syringe needle or butterfly inserted just above the bend of the test tube. Individual fractions are added to straight polyacrylamide followed by ethanol precipitation to 20 μg / ml [see Cell 37: 889-901 (1984) by Strauss and Varshavsky). Fractions containing cDNA greater than 700 bp are pooled and bound to gradient purified Bst X1 cleaved piH3 vectors.

The bound DNA is transformed into E. coli MC1061 / p3 suitably prepared by the following protocol:

The desired strains are lined up on the LB plate. The next day inoculate one colony into 20 ml TYM broth (preparation method described below) in 250 ml flask. The cells are grown in midlog steps (OD ₆₀₀ is about 0.2-0.8), poured into 21 flasks containing 100 ml of TYM broth and shaken vigorously until the cells grow to 0.5-0.9 OD and then 500 in the same vessel. Dilute again with ml. When cells grow to OD ₆₀₀ 0.6, place the flask in ice water and shake slowly to cool rapidly. When the cultures are cooled, rotate for 15 minutes (J6) at 4.2 K rpm. The supernatant is poured out and shaken slowly on ice to resuspend the pellet in about 100 ml of cold TfB I (see below). Then re-rotate for 8 minutes (J6) at 4.2 K rpm in the same bottle. The supernatant is poured out and the pellet is shaken slowly on ice and resuspended in 20 ml of cold TfB II. Aliquots of 0.1-0.5 ml are placed in pre-chilled microfuse test tubes, cooled in liquefied nitrogen and stored at -70 ° C. Aliquots are removed for transformation, thawed at room temperature until melted and placed on ice. Add DNA and leave on ice for 15-30 minutes and incubate at 37 ° C for 5 minutes (6 minutes for 0.5 ml aliquots). The suspension containing DNA is then diluted 1: 10 in LB and allowed to grow for 90 minutes before applying platelet or antibiotic selection. Alternatively, the heat-vibration transformation mixture is plated directly onto an antibiotic plate that is poured just prior to flattening thin layers (4-5 ml) of antibiotic-free LB agar.

Medium and buffers:

TYM: 2% bacto-trypton, 0.5% yeast extract, 0.1 M NaCl, 10 mM MgSO ₄ (can be added before autoclave).

TfB I: 30 mM KOAc, 50 mM MnCl ₂ , 100 mM KCl, 10 mM CaCl ₂ , 15% (v / v) glycerol.

TfB II: 10 mM Na-MOPS, pH 7.0, 75 mM CaCl ₂ , 10 mM KCl, 15% glycerol.

Recovery of cDNA Clone by Panning

A biological culture dish for panning (Falcon 1007) was prepared by washing the dish with 0.15 M NaCl in a wash bottle instead of PBS and blocking the unreacted site by incubating overnight in PBS containing 1 mg / ml BSA. Except for affinity purified amounts of anti-mouse IgG antibodies, Wysocki and Sato [Proc. Nat1. Acad. Sci. USA 75: 2844-2848 (1978), which was prepared by coating as described. Typically, dishes are prepared in bulk and frozen after inhalation of PBS / BSA. In one screening procedure, 50% fused COS cells in 24.6 cm plates were collected from Mol. Cell Biol. 1: 743-752 (1981) Transfection is carried out by plasma fusion according to the method described in the literature.

After 72 hours of post-fusion, cells are isolated by incubating for 30 minutes at 37 ° C. in PBS / 1 mM EDTA / 0.02% sodium azide. The isolated cells are pooled together and centrifuged and resuspended in cold PBS / EDTA / 5% fetal bovine serum containing the monoclonal antibody as a plurality diluted 1: 1000 as well as conventional reagents at the concentrations indicated by the manufacturer. . After 1 hour on ice cells are diluted 1: 1 with PBS / EDTA / azide and layered in 10 ml of PBS / EDTA / azide containing 2% Picol 400.

After centrifugation (400 × g, 5 min), carefully aspirate the supernatant and resuspend the pellet in a small amount of PBS / EDTA / 5% FBS and pan the cells with 3 ml of PBS / EDTA / 5% FBS. Distributed on the plate.

Then, the reputation must be in Wysocki and Sato's Proc. Natl. Acad. Sci. USA. 75: 2844-2848 (1978). Episomal DNA is recovered from adherent cells by the method of Hirt [J. Mol. Biol. 26: 365-269 (1967)] and transformed into MC 1061 / p3.

Cell Lines and Cell Cultures

COS cell clone M6 cells are grown in Dulbecco's modified Eagle's medium supplemented with 10% bovine serum and 15 μg / ml gentamicin sulfate (DME / 10% bovine serum). The cells are divided in a ratio of about 1: 8 from the storage plate so as compact as possible without damaging the cells the day before transfection in a 6 cm dish. T cell lines are grown in modified Dulbecco's medium (IMDM) and NuSerum (purchased from Collaborative Research) or 10% fetal calf serum of iscoves containing gentamycin as described above.

COS Cell Transfection for Immunofluorescence Experiments

50% fused COS cells in a 6 cm dish with 10% new serum (purchased from Collaborative Research). Transfection is carried out in a volume of 1.5 ml cocktail consisting of DME or IMDM medium comprising 400 μg / ml DEAE dextran, 10 μM diphosphate chloroquine and 1 μg / ml DNA.

After 4 hours (or faster if the cell is in poor condition) at 37 ° C., the transfection mixture is removed and the cells are treated with 10% DMSO in PBS for 2 minutes [Sussman and Milman's Cell Biol.4: 1641-1643 (1984). The cells are then allowed to express in DME / 10% bovine serum for 48-72 hours.

Immunoprecipitation, Northern and Southern Blot Analysis

T cells were labeled and lysed by treatment with lactoperoxidase, and Clark and Einfeld [Leukocyte Typing II, Vol. 2] were used with commercially available goat's anti-mouse IgG agarose beads (purchased from Cooper Biomedical). II, pp. 155-167 (1986), which was immunoprecipitated. COS cells were transfected with the DEAE dextran method, trypsinized and passed to new plates 24 hours after transfection without dilution. After 36 hours the cells were isolated by exposure to PBS / EDTA as described above, centrifuged and labeled by the lactoperoxidate method. For T cell immunoprecipitation, clear lysates were prepared. However, the lysis buffer contains 1 mM PMSF and the incubation with the first antibody is performed only at 4 ° C. for 2 hours.

Elution samples were fractionated on 11.25% discontinuous polyacrylamide gels using the buffer system of Laemmli [Nature 227: 680-685 (1970)].

Northern blotting analysis substantially eliminated DMSO from the dropping buffer, denatured at 70 ° C. for 5 minutes, and substantially all of Molecular Cloing, a Laboratory Manual by Maniatis et al. Except that the gel contained 0.6% formaldehyde rather than 6%. (1982) as described in the literature. Gels were stained and photographed in two volumes of water containing 1 μg / ml of ethidium bromide and transferred to nylon (Gene Screen, purchased from DuPont) in the dye solution. The transfer RNA is a five-minute Saran wrap (Saran Wrap) [Church and Gilbert at 0.22 mW / cm ² flow rate (measured at 254 nm) Proc. Natl. Acad. Sci. USA 8: 1991-1995 (1984).] Was irradiated by exposure to a bactericidal lamp. Southern blot analysis was performed using Reed and Mann [Nucl. Acids Res. 13: 7207-7221 (1986), which was performed by alkaline transfer to nylon (gin screen, purchased from DuPont). Hybridization probes were prepared by the methods of Hu and Messing [Gene 18: 271-277 (1992)] and the blots were prepared in SDS / phosphate buffer containing 10 DNA μg equivalent of M13 mp 19 phage [Church and Gilbert Proc. . Natl. Acad. Sci. USA 8: 1991-1995 (1984).

Erythrocyte Rosetting

Erythrocytes were prepared from total blood by centrifugation three times in PBS. COS cells were transfected into 6 cm dishes with CD2 or other surface antigen expressing clones by the DEAE method. The medium was aspirated and 2 ml of PBS / 5% FDS / azide was added to each plate for post-transfection for 48-72 hours, followed by 0.4 ml of a suitable red blood cell sample as 20% suspension in PBS. After 1 hour at room temperature, non-adherent erythrocytes were washed off slowly and the plates were tested.

CDNA encoding the CD2 antigenic determinant was isolated by the following method. cDNA was prepared from RNA extracted from human T cell tumor line HPB-ALL and inserted into the expression vector piH3 based on SV 40 origin as described above. About 3 × 10 ⁵ recombinant cDNA libraries were constructed and the library introduced into COS cells by plasma fusion. After 3 days the cells are exposed to EDTA to isolate and treated with a pool of three monoclonal antibodies directed to the CD2 antigen determinant (OKT 11, Leu 5b and Coulter T11). Cells treated with the antibody were distributed in a dish coated with an affinity purified amount of anti-mouse IgG antibody, allowed to adhere, and gently washed to separate from non-adhered cells. This method of enrichment is known from immunological literature [Mage et al. J. Immunol. Methods 15: 47-56 (1977).

The resulting colonies are pooled, fused to COS cells and subjected to two pannings as before. In three times, portions of the detached cells were treated with three monoclonal antibody mixtures specific for CD2, and Hirt supernatants were again generated and transformed into E. coli. DNA was prepared from 8 final colonies and transfected into COS cells. After 3 days, surface expression of the CD2 antigen was detected by immunofluorescence indirectly in 6 of 8 transfected dishes. Restriction digestion of the corresponding plasmid DNA revealed 1.5 kb of inserts in all six isolates.

One of the six clones was prepared in bulk for subsequent analysis. After transfection of COS cells, direct immunofluorescence analysis with a partial panel of antibodies provided by a third international research assembly for leukocyte differentiation antigens revealed that not all of the antibodies provided reacted with phytohemagglutinin activated T lymphocytes. Except for one sample, it showed a positive reaction. Of the 17 antibodies tested, at least eight distinguishable groups were defined by varying the type of reaction with various primate lymphocytes.

Jonker and Nooij, Leukocyte Typing II, Vol. I, pp. 373-387 (1986).

cDNA sequence analysis

CD2 cDNA inserts were subcloned in M 13 mp 19 in two directions (see Gene 19: 259-268 (1982) by Vieira and Messing) and the sequence was determined by the dideoxynucleotide method (see FIG. 2). Sanger et al., Proc. Natl. Acad. Sci. USA 74: 5463-5467 (1977). Transcription decipherment frame for Kozak [Microbiol. Rev. : 1-47: 45 (1983), 360 residues extending from ATG triplets that meet the consensus criteria were observed (see FIG. 1). The predicted amino acid sequence results in a membrane integration protein with a single membrane that spans hydrophobic moieties in much larger intracellular domains. N-terminal amino acid sequence and Heijne [Nucl. Acids Res. 14: 4683-4690 (1986), the comparison of the signal sequence residue frequency matrix constructed by, reveals that mature CD2 peptides are formed by cleaving precursor peptides between residues 19 and 20. there was.

A surprising and unexpected feature of this sequence is the presence of any N-linked glycosylation sites that are close to the proposed cleavage site. The final polypeptide backbone has an expected molecular weight of 38.9 kd divided into an external domain of mass 21.9 kd and a cytoplasmic domain of mass 14.6 kd. Three N-binding glycosylation sites are present in the extracellular domain.

The membrane spanning domain contains 26 constant residues with dominant hydrophobicity. Immediately following nine residues are seven basic residues of lysine or arginine. The prominent appearance of hydrophobic residues followed by basic residues is a common tissue feature of proteins inside and outside the membrane with carboxy termini.

Another surprising feature of the intra and endodomain is the beta turn motif in terms of hydrophobic residues (commonly found in flank beta turns) [see Chou and Fasman's Annual Review of Biochemistry 47: 251-276 (1978)] cys-gly-gly the appearance of -gly. Theoretically, only 20 residues arranged in the alpha helix are needed to traverse the 3 nm membrane bilayer [see Tanford's Science 200: 1012-1018 (1978)], and about 14 hydrophobic residues insert the membrane integrating protein. And release (see cell 41: 1007-1015 (1985), by Adams and Rose), intra and outer membrane fragments of the CD2 antigen may include flexion or entanglement.

A rather large cytoplasmic domain gives the possibility of CD2 having inherent enzymatic activity. The cytoplasmic domain contains three sites in proline that are very abundant and have a high potential for rotation.

Comparison of the amino acid sequence and the NBRF database did not show substantial similarity with other proteins. In particular, no similarity with the alpha or beta chains of the T cell receptor was observed and the possibility that CD2 was the first T cell receptor was excluded (see Science et al., Science 231: 1118-1122 (1986)).

Proteins directly within the cytoplasm are expressed in vivo (EGF) and in vitro (HLA) phosphorylation or cyclic AMP-dependent protein kinases by EGF receptors and class I histocompatibility genes and protein kinase C, respectively. Is a type of basic protein followed by a serine residue followed by a type found at the same point in the known site. Hunter et al. Nature 311: 480-482 (1984); Davis and Czech, Proc. Nat1. Acad. Sci. 82: 1974-1978 (1985); J. Biol in Guild and Strominger. Chem. 259: 9235-9240 and 13504-13510 (1984). Similar sites are found in the cytoplasmic domain of the interleukin 2 receptor and phosphorylated in vivo by protein kinase C. Nature 311: 626-631 (1984) by Leonard et al .; Nature 311: 631-635 (1984) from Nikaido et al .; J. Biol. Of Shackelford and Trowbridge. Chem. 259: 11706- (1984).

Immunoprecipitation of CD2 Antigens Expressed by Transfected Cells

COS cells were transfected with a CD2 transgenic plasmid and the surface was labeled ¹²⁵ I by the lactoperoxidase method 60 hours after transfection. Cell lysates were prepared and incubated with monoclonal anti-CD2 antibody (OKT 11) or foreign antibody (OKT 4; anti-CD4) at 4 ° C. for 2 hours. After addition of several anti-mouse antibodies bound to Sepharose and washing several times, the adsorbed proteins were eluted, T lymphocytes activated by phytohemagglutinin, cDNA donor HPB-ALL, or organ T produced in our laboratory. Similarly prepared immunoprecipitates from cell lines were subjected to 11.25% acrylamide gel electrophoresis. Autoradiography demonstrated that prominent bands of immune reactants precipitate from COS cells transfected with anti-CD2 antibodies but not by control.

The calculated average molecular weight of the COS cell material is 51 kd, which is compared with 54 kd which is the average molecular weight for T blast cells and T cell line material; Antigens from HPB-ALL cells were found to have a molecular weight of about 61 kd. The difference observed in size is believed to be due to the different types of glycosylation that occur in different cell types. Minor bands with an apparent molecular weight of 38 kd are present in the immunoprecipitated material from COS cells but not T cells or HPB-ALL cells. The size of this species is consistent with the predicted molecular weight of 39 kd of mature peptides that are not glycosylated within error.

CD2 Expressing COS Cells Forming Positive Red Blood Cell Rosettes

COS cells transfected with CD2 expressing clones were purified from a purified MT910 (IgG, kappa) anti-CD2 antibody at a concentration of 1 μg / ml by Leukocyte Typing II, Vol. I, pp. 233-242 (1986)] or purified MB40.5 [IgG1. Kappa; J Exp. Med. 160: 633-651 (1984)] for 1 hour with antibody. MB40.5 recognizes monomorphic HLA-ABC determinants and cross reacts with histocompatibility antigens in African green monkeys;

It is chosen because it represents an isotype-antibody that recognizes a surface antigen that is rich enough to be almost similar to the CD2 antigen expressed by the transfected cells. Positive red blood cell rosettes are observed in the presence of MB40.5 but not MT910. Rosette inhibition was observed with OKT 11 antibody and not with various other control antibodies.

Transfected COS Cells Forming Red Blood Cell Rosettes from Other Animals

In addition to sheep red blood cells, human T cells are known to form red blood cell rosettes in horses, pigs, dogs, goats, and rabbits that are not red blood cells in mice or rats. Johansen et al. J. Allergy Clin. Immunol. 54: 86-94 (1974); In Amiot et al., A. Bernard et al. Eds., Springer, publisher, New York, N.Y., pp. 281-293 (1984); Nalet and Fournier, Cell. Immunol. 96: 126-136 (1985). Autoroset between red blood cells and thymic cells of human body [Baxley et al. Clin. Exp. Immunol. 15: 385-393 (1973). COS cells transfected with CD2 expressing clones are treated with MT910 or control antibody MB40.5 and exposed to red blood cells from the species. Rosettes are observed with red blood cells in horses, pigs, dogs, goats, sheep, rabbits, and humans, but not with red blood cells in mice or rats. Rosette formation is blocked by pretreatment of transfected COS cells with MT910 but not MB40.5. In this experiment, it is noteworthy that horse red blood cells usually form dense rosettes and goat red blood cells form rather poor rosettes, perhaps because their small size makes them more sensitive to washing. Mouse erythrocytes bind weakly and spontaneously in culture dishes other than pretreated MT910 and MB40.5 cells, while rat erythrocytes do not show all sorts of detectable binding.

Binding of Human Red Blood Cells Blocked by LFA3 Antibodies

Since LFA3 has been proposed on the basis of antibody blocking studies that target structures against CD2 antibodies [see Shaw et al. Nature 323: 262-264 (1986)], the ability of anti-LFA 3 antibodies to block rosette formation Researched. The transfected cells are ascites diluted 1: 1000 and are as potent or more potent as anti-LFA3 (IgG1, kappa) or LFA3: G10 / B11 and D10, anti-K14 antigens, D6, anti-Wr ^b antigens. Exposure to human erythrocytes pretreated for 2 hours at a concentration of 10 μg / ml of each of the four isotype non-aggregated antibodies directed against human erythrocyte antigens (see Nichols et al. Vox Sang, in press). Erythrocytes are washed away from excess LFA3 antibody, but the rosette is allowed to form in the presence of the control antibody to protect by desorption a possible loss of antigen blocking ability. Rosette formation is observed in the presence of four control antibodies but not with erythrocytes pretreated with anti-LFA3.

Other T cell antigen expressing COS cells that do not form a rosette

Many clones are isolated by transfection techniques such as those used to clone CD2 and are characterized by varying degrees by antibody reactivity, nucleic acid restriction and sequencing and immunoprecipitation. Representative clones analyze the ability to transfect COS cells and maintain rosette formation. CD1a, CD1b, CD1c, CD4, CD5, CD6, CD7, CD8, and CD28 (Tp 44) clones did not form rosettes with human red blood cells.

RNA Blot Assay

The same amount of total PNA prepared from cell types that express or lack the CD2 antigen is electrophoresed through a modified agarose gel and transferred to nylon. Thymic cells, active T cells, and aged T due to hybridization of strand-selective probes prepared from M13 clones containing CD2 cDNA inserts with their migrated RNAs (see Gene 17: 271-277 (1982) by Hu and Messing) It was found that there are potent 1.65 kb and 1.3 kb transcripts present in RNA derived from the cell population. Smaller amounts are found in RNA extracted from cDNA donor HPB-ALL and also extracted from MOLT4; Only detectable levels have been recorded in RNA from HSB-2 strains. Reactive with RNA from Namalwa (bucket lymphoma), U937 (histocytic leukemia), HuT-78 (adult T cell leukemia), PEER (T cell leukemia) or Jurkat clone J3R7 (T cell leukemia) strains Not observed.

The type of responsiveness corresponds to the known or measured type of expression of the CD2 antigen, which is absent or undetectable on Namalwa, * 937, HuT-78, J3R7, PEER and HSB-2 cell lines, weakly present in MOLT4, EPB-ALL Is more strongly present and most strongly present in activated T cells. Thymic cells are also known to express high levels of CD2 antigen.

Testing of the cDNA clone sequence suggested that 1.3 kb of RNA resulted from the formation of an alternative 3 'end at the end for the canonical polyadenylation signal AATAAA at position 1085 of the cDNA sequence. To test this opinion, RNA from HPB-ALL and activated T cells is analyzed by Northern blot and hybridized with a complete cDNA probe or a probe derived from the 3 'portion of the nucleotide 1131 terminal cDNA. While the latter probe reacted with only 1.65 kb of species, the former probe appeared similar to the type of reaction that can be observed in FIG. The result is consistent with the proposed origin with 1.3 kb of transcript.

A weakly hybridizing transcript of about 0.75 kb was detected in activated and aged T cell RNA preparations. The origin of this RNA is currently unknown.

Genome Composition of the CD2 Gene

Southern blot analysis of genomic DNA from placental, peripheral blood lymphocytes, T cells, HeLa cells or tumor lines used for RNA analysis showed the same BamHI cleavage type, which was independent of normal transduction of the CD2 gene during growth. To indicate. Similarly, large genome modifications do not proceed to prevent the tested T cell tumor lines from expressing the CD2 antigen. In addition to preliminary results with an incomplete collection of one phage recombinant with a CD2 sequence, restriction analysis of whole genomic DNA with many different enzymes revealed that the gene was divided into at least four exons.

d

Example 2 Cloning, Sequence and Expression of CD36

To isolate cDNA clones encoding CD36, human placental cDNA (see Simmons and Seed (1988) Nature 333: 568-570) was transferred to COS cells using DEAE-dextran as a promoter. [Example 1, Reference Documents]. 48 hours after posterior transfection, cells were removed from the dish without trypsin and incubated with the monoclonal anti-CD36 antibody 5F1 [Bernstein et al. (1982) J. Immunol. 128: 876-881; Andrews et al. (1984) J. Immunol. 128: 398-404] Panning was performed on dishes coated with goat's anti-mouse immunoglobulin antibody. Unadhered cells are carefully washed to remove, adhered cells are lysed and episomal plasmids recovered from the cells are purified and transformed into E. Coli. After two similar additions following globular fusion, plasmid DNA recovered from 11 of 12 randomly selected colonies was found to indicate the appearance of CD36 determinants in transfected COS cells.

Two clones were randomly selected for subsequent analysis.

It showed the same restriction enzyme fragment type as the two hole insert of about 1.9 kb. COS cells are transfected with clones reacted with monoclonal antibodies 5F1 and F13 and OKM5 (purchased from Ort, Raritan, NJ). Cells transfected with the anti-CD36 antibody pool were immunoprecipitated to find that 83 kd of molecules were present on the transfected COS cells and C32 melanoma cells and lacking on the control (transfected with CD25) COS cells. High molecular weight species, such as dimer CD36, were immunoprecipitated from transfected COS cell lysate rather than from C32 melanoma cell lysate. The nucleotide sequence is shown in Table 1. In Table 1, the numbers of the nucleotide sequences are shown in the left margin at the beginning of each line. The deduced amino acid sequence is represented by a single letter code below the beginning of each encoding nucleotide triplet and the starting methionine is shown in numeral 1 above the starting codon. The N-linked glycosylated potent site in the derived amino acid sequence is shown by one dotted line. The putative intramembrane domain is shown by a double dotted line. Although two consensus polyadenylation (AATAAA) features were found in the cDNA, there was no poly (A) terminus and if the transcript had approximately 5 'terminus as observed in the clone, by blot hybridization None of the RNA species observed is short enough to correspond to polyadenylation at this site. The estimated initiation codon is not the first ATG found in the clone, but the previous two are closely followed by the intraskeletal termination codon.

The scheduled initiation methionine was followed by a short hydrophobic site resembling a secretory signal sequence, but the cleavage site could not be clearly identified. The recently determined 36 amino acid sequence of the amino terminus in Table 1 [Tandon et al. J. Biol. Chem. 264: 7570-7575 (1989), indicates that the mature polypeptide starts at an amino acid residue immediately following the starting methionine. It is not clear whether one Arg residue preceding the hydrophobic moiety is sufficient to attach the amino terminus to the membrane. The resulting polypeptide has 471 residues with an expected molecular weight of about 53 kd. The proposed extracellular domain is followed by 27 extremely hydrophobic residues corresponding to the endo- and extracellular domains and 6 residues (three of which are basic) corresponding to the thin intracellular domain.

TABLE 1

The presence of 10 potent N-binding glycosylation sites appeared to be sufficient to account for the discrepancy in molecular weight between the 83 kd species found by immunoprecipitation and the expected polypeptide. Subsequent comparison of the entire sequence against various databases of known proteins revealed no major homology.

However, some internal structure was revealed. All cysteine residues in the extracellular domain are limited to the domain defined by the residues 937-1209 of the nucleotide sequence. Placing cysteine as a guide allows the extracellular domain to be divided into three fragments, followed by two domains without cysteine followed by cysteine-rich fragments.

However, the comparison of the sequences of these fragments showed no significant association with other molecules in the existing database, especially thrombospondine.

CD36 protein was purified by immunoprecipitation. Since the rapid immunoselective cloning method depends on the expression of transfected COS cells, the same cell line cloned with CD36 cDNA could also be used as a source of the expressed protein.

C32 melanoma cells, COS cells transfected with CD36 and COS cells transfected with CD25 (control) Surface label is dissolved in phosphate buffered saline solution containing 1 mM phenylmethylsulfonyl fluoride, 0.5% NP-40 and 0.1% sodium dodecyl sulfate. Anti-CD36 monoclonal antibody was added and allowed to be absorbed into the lysate at 4 ° C. for 12 hours, then goat's anti-mouse immunoglobulin beads (capel) were added and mixed for 2 hours and mixed by J. Clark and Einfeld. Immunol. 135: 155-167 (1986) wash as described. Large amounts of protein may also be obtained in purified type from COS cell lysates transfected by immunoaffinity column purification. Other antibodies to CD36 can be obtained using the CD36 protein and are expressed and / or purified as immunogens as previously described.

CD36 is a binding site for cell adhesion of parasitic erythrocytes by Plasmodum falciparum and the inventors of Ockenhouse, C.D. (1989) Science 243: 1469-1741, et al. Cell adhesion of parasitic infected red blood cells was shown to be blocked by monoclonal antibodies to CD36. Clear cell adhesion was demonstrated when incubating infected red blood cells with COS cells transfected with CD36 cDNA.

The ability to avoid splenic clearance by attaching parasitic erythrocytes to peripheral blood vessels plays an important role in the pathogenicity of tropical fever malaria and is thought to be involved in cerebral malaria mortality by causing occlusion of small tubes in the brain. .

Therefore, the cDNA and purified protein of the present invention are useful when provided purified CD36 sufficient to make a therapeutic monoclonal antibody.

Example 3. Isolation and Cloning of cDNA Encoding T Lymphocyte TLiSA Antigen

CDNA clones encoding TLiSA1 were obtained from human T-cell cDNA libraries transferred to COS cells as described above and subjected to the rapid immunoselective cloning method of the present invention.

The monoclonal antibody ACT-T-SET TLiSA1 (purchased from Massachusetts Cambridge T-Cell Science Corporation) was used to depot transfected COS cells expressing cloned cDNA by positive indirect immunofluorescence. The positive plasmid is contained in a 1.7 kb insert.

TLiSA protein was isolated by immunoprecipitation method described in Example 2. The protein has a molecular weight of about 50 kd measured by gel electrophoresis.

The nucleotide sequence of the cDNA is determined by dideoxy nucleotide chain termination as described above. Table 2 shows the nucleotide sequences with 1714 residues and the estimated amino acid sequences represented by a single letter code under the first nucleotide of each coding triplet. The ATG encoding the putative starting methionine is followed by a short hydrophobic site that matches the secretory signal sequence, and the site that is necessarily cleaved is 19 residues in the transcription translation frame. The resulting polypeptide, if no longer processed, has 317 residues with an expected molecular weight of about 36 kd.

The proposed extracellular domain is followed by 25 polar hydrophobic residues corresponding to the intracellular domain (shown in double lines in Table 2). The presence of nine potent N-binding glycosylation sites (indicated by one dotted line in Table 2) appeared to be sufficient to account for the difference in molecular weight between the expected polypeptide and the 50 kd species found by immunoprecipitation.

TliSA is involved in mediating the differentiation of T-cells into cell fusion form by IL-2 induction. Antibodies against TLiSA are useful for preventing T cell differentiation stimulated by IL-2 and for modulating the adverse effects of IL-2 upon treatment.

TABLE 2

Example 4 Isolation and Molecular Cloning of cDNA Encoding T Lymphocyte-Specific CD27 Antigens

CDNA clones encoding CD27 were obtained from human T lymphocyte cDNA transferred to COS cells and immunoselected by the method of the present invention. RNA was extracted from mononuclear cells derived from 1 unit of blood after 4 days of culture in a medium containing 1 μg / ml of phytohemagglutinin (PHA) using guanidium thiocyanate. Total RNA was chosen as poly-A. cDNA was prepared, cloned into CDM 8 to transfect COS cells, and CD27 cDNA was prepared from Seed and Aruffo's Proc. Natl. Acad. Sci. 84: 8573-8577 (1987); And in Aruffo and Seed, Proc. Natl. Acad. Sci. USA 84: 3365-3369 (1987) were immunoselected with the provided monoclonal antibodies OKT 18a and CLB-9F4 as described.

The vector contained 1.2 kb of cDNA insert.

The nucleotide sequence of cDNA was determined by dideoxynucleotide chain termination as described above.

The sequence of 1203 residues and the estimated amino acid sequence are shown in Table 3. The starting methionine is shown by the number 1 above the starting codon. The putative CD27 polypeptide speaks of typical features of type 1 membrane integration proteins.

It begins with a hydrophobic site of 20 amino acids that matches the secretory signal sequence. This hydrophobic site is followed by an extracellular domain of 171 residues, a hydrophobic membrane spanning domain of 20 residues (represented by doublets), and a cytoplasmic domain of 49 amino acids starting with a positively charged stop transfer sequence. There is no poly (A) end.

The putative CD27 amino acid sequence is very homologous over the entire length to the B lymphocyte and cancer antigen CD40 described in FIG. CD27 is also highly homologous to receptors for nerve growth factor (NGFR) across the extracellular and transmembrane domains [Stamenkovie et al. EMBO J. 8: 1403-1410 (1989); See Johnson et al., Cell 47: 545-554 (1989).

TABLE 3

Most of the conserved structural features found in these three proteins are in the abundance of cysteine or histidine in the extracellular site. This is commonly found in pairs separated by two or four intervening amino acid residues similar in configuration to the protein used to bind this construct to zinc ions.

Sites rich in cysteine and histidine are followed by membrane proximity domains rich in serine, threonine and proline, which have been proposed as sites where biochemically identified 0-linked glycans are added to NGFR [Johnson et al. (1989), supra ; Grob et al. J. Biol. Chem. 260: 8044-8049 (1985).

COS cells transfected with anti-CD27 antibody were immunoprecipitated followed by gel electrophoresis to reveal the presence of a 110 kd species and a 55 kd single band in the presence of a reducing agent. This indicates that on transfected COS cells, CD27 is a disulfide bound homodimer containing 55 kd of monomers similar to the type precipitated from T lymphocytes [Bigler et al. J. Immunol. 141: 21-28 (1988); Stockinger et al. Leukocyte Typing II, Vol. I: 513-529 (1986); Van Lier et al. Eur. J. Immunol. 18: 811-816 (1987).

CD27 is a T lymphocyte active antigen. The structure suggests that it can function as a receptor for lymphokines or growth factors. Recognition of CD27 results in the proliferation of T cells and increases the expression of specific genes required for the helper and effector functions of those T cells. Expression of CD27 on T cells increases stimulation with phytohemagglutinin (PHA) or anti-CD3 monoclonal antibodies two to five times and adds at least one CD 27 monoclonal antibody to PHA. Increase the proliferation of stimulated T cells [Leukocyte Typing II, Vol. Bigler and Chiorazzi. I: 503-512 (1986); (1987) by Van Lier. T cells positive for CD27 have been shown to help B cells for IgM synthesis and to secrete IL-2 when moderately stimulated [Van Lier et al. Eur. J. Immunol. 18: 811-816 (1988).

The ability to interfere with the binding of antigen presenting cells to CD27 positive T cells, or to allow such binding to occur on surfaces other than lymphocyte cells, can be useful for diagnosis and treatment. For example, fusion proteins of CD27 and receptor ligands immobilized on a substrate will be useful for detecting the presence of specific antigens in body fluids. Soluble CD27 fusion proteins will be useful, for example, in preventing unwanted T cell proliferation in certain autoimmune diseases.

Example 5 Isolation and Molecular Cloning of cDNA Encoding CD53 Antigen

Antibody MEM-53 [Hadam, M.R. (1989) Leucocyte Typing IV. Knapp. B. et al. Oxford Univ. Press, p. 674], CD53, an antigen recognized by HD 77, HI 29 and HI 36 and 63-5A3, is a glycoprotein widely distributed among nuclear cells of the hematopoietic lineage, although extremely limited [Stevanova, I. et al. (1989) Leucocyte Typing IV. Knapp. B. et al. Oxford Univ. Press, p. 678]. CD53 is expressed by monocytes and macrophages, granulocytes, dendritic dendritic cells, osteoclasts and osteoblasts and T and B cells at all stages of differentiation.

To obtain cDNA clones encoding CD53, a cDNA library was constructed from peripheral blood lymphocytes and promyelocytic tumor cell line HL 60 was transfected into COS cells by the DEAE-dextran method as described above. After 48 hours of infection, the cells were pooled and incubated with the monoclonal antibody MEM-53, followed by Seed and Aruffo's Proc. Nat1. Acad. Sci. USA 84: 3365-3369 (1987) and Proc. Aruffo and Seed. Nat1. Acad. Sci. USA 84: 8573-8577 (1987) panned as described. Following two rounds of globular fusion subsequent, plasmid DNA recovered from one colony isolate was transfected into COS cells and the extent of CD53 expression was recorded by immunofluorescence.

Two of the eight transfections are positive, each with an insert of about 1.5 kb. COS cells transfected with one of the clones were reacted with the respective antibodies MEM-53, HI 29, HI 36 and 63-5A3.

To isolate CD53 from peripheral blood lymphocytes and transfected COS cells for comparison purposes, the lymphocytes and transfected COS cells were surface labeled with ¹²⁵ I using lactopperoxidase and H ₂ O ₂ , and 1% NP40, Dissolve in a pH 8.0, 50 mM Tris-HCl, lysis buffer containing 150 mM sodium chloride, 5 mM magnesium chloride, 5 mM potassium chloride, 20 mM iodineacetamide and 1 mM phenylmethyl sulfonyl fluoride. The cells are solubilized for 45 min at a concentration of 2.5 x 10 ⁷ cells / ml in lysis buffer and then centrifuged at 12,000 g. After confirming the stability with goat's anti-mouse immunoglobulin beads (purchased from Capel, Melbourne, PA), immunoprecipitation was performed by Schneider et al. Biol. Chem. 257-10766 (1982) with monoclonal antibody MEM 53 or 63-5A3 and protein A-Sepharose CL-4B (purchased from Sigma, St. Louis, Missouri).

Immunoprecipitates were analyzed on 12.5% acrylamide gels eluting in SDS sample buffer and containing sodium dodecyl sulfate.

Wide bands of radioactive iodide proteins in the mass range of 34 kd to 42 kd are obtained from peripheral blood lymphocytes with MEM-53 or 63-5A3 monoclonal antibodies, and by CD53 extending from 36 kd to 46 kd Comparable to the band obtained from the transfected COS cells. Cell surface proteins recovered from transfected COS cells are not typical because the cDNA clones usually have a molecular weight lower than or equal to the molecular weight found on the cell of origin [Aruffo and Seed's Proc. Natl. Acad. Sci. USA 84: 3365-3369 (1987). A population of about 15 kd is released from glycoproteins when cleaved with endoglycosidase F (N-glycanase), while treatment with neuraminidase and O-glycanase does not affect the apparent molecular weight [Hadam, MR, (1989) Leucocyte Typing IV, Knapp, B. et al. (Eds.) Oxford Univ. Press, p. 674; Steucova et al. Leucocyte Typing IV, Knapp, B. et al. (Eds.) Oxford Univ. Press, p. An additional blurry band of 20 kd, a non-glycosylated precursor, was detected in immunoprecipitates of transfected COS cells, but not in immunoprecipitates of peripheral blood lymphocytes.

Blot hybridization of genomic DNA from T cell line PEER cut with several enzymes revealed a type consistent with one replicating gene. RNA blot analysis revealed one mRNA of 1.8 kb derived from B, T and bone marrow cell lines and peripheral blood lymphocytes. Expression levels are comparable in cell lines other than THP1 cell line with little CD53 mRNA. CD53 transcripts are more abundant in peripheral blood lymphocytes than in cultured cell lines, consistent with the higher surface expression of CD53 in these cells.

The nucleotide sequence of the CD53 cDNA was determined by dideoxynucleotide chain termination as described above using synthetic oligonucleotide primers.

The sequence of the CD53 insert consists of 1452 nucleotides (Table 4) and terminates close to two overlapping AATAAA motifs (represented by a single line). The 3 'unencoding sequence includes three examples of the ATTTA sequence (shown in quotation marks), which indicates the instability of the mRNA (see Cell 46: 659 (1986) by Shaw and Kamen). The transcription translation frame starting at residue 74 encodes a protein consisting of 219 amino acids with an expected molecular weight of 24,340 kd. The expected polypeptide is unusual in that it has four major hydrophobic fragments (shown in doublets), three of which are close to the amino terminus of the molecule. The first hydrophobic fragment contains at least three cysteine residues and glycine, which are atypical for the signal sequence or simple transmembrane alpha helix. Cysteine and glycine were found to be located immediately before the signal cleavage site [Hijne's Nucleic Acids Res. 14: 4683 (1986), which suggests that the amino terminus of the mature protein begins in the middle of the first hydrophobic domain.

Supporting this view is provided by the finding that the size of the polypeptide backbone of ME491, a membrane-integrating protein of related type III discussed below, is smaller than expected from the cDNA sequence as a result of cleavage of the signal peptide. . Because there are only two potent sites for adding N-linked glycans, which are located between the third and fourth hydrophobic fragments, the carboxy terminus is not limited to the short hydrophilic portion between the second and third hydrophobic fragments. It must be placed inside the cell. If the amino terminus is not processed, it must also remain in the cell.

CD53 is a type III membrane-integrating protein associated with three other membrane proteins: the ME491 antigen [Hotta et al., Cancer Res. 48: 2955 (1988);

CD37, which is not specific for B cell binding but is mainly expressed on B cells and widely glycosylated antigen [Schwartz et al. J. Immun. 140: 905 (1988); And the non-glycosylated antigen S5.7 [Pressano et al., Cancer Res. 43: 4812 (1983).

CD53 is also a type III membrane integrating protein carried by lactose to bacterial cells. indirectly related to coli lac Y permease. CD53 transcripts in peripheral blood lymphocytes increase in terms of diffusion after mitosis stimulation by PHA, suggesting that the protein is involved in transporting factors essential for cell proliferation.

Of the molecules that have a wide range of reactivity in the hematopoietic system, CD53 currently has the widest reactivity as well as the most stringent restriction on hematopoietic cells. Anti-CD53 antibodies are useful tools for identifying hematopoietic neoplasms and can help identify morphologically incompletely defined cells, such as in primary cultures of the spleen or bone marrow.

TABLE 4a

TABLE 4b

Equivalent

Those skilled in the art will be able to recognize or evaluate many equivalents of the specific forms of the invention referred to herein using routine experiments only. Such equivalents are intended to be included within the scope of this invention.

Missing technical challenges for invention

1 is a diagram showing the nucleotide sequence of the piH3 expression vector.

Nucleotides 1-589 are derived from pMB 1 origin (from pBR 322); Nucleotides 590-597 are derived from SacII linker (ACCGCGT);

Nucleotides 598-799 are derived from synthetic tyrosine inhibitory tRNA gene (supF gene);

Nucleotides 800-947 are derived from the residues of ASV LTR fragments (Pvu II to MIu 1); Nucleotides 948-1500 are derived from the cytomegalovirus AD 169 enhancer in the human body; Nucleotides 1501-1650 are derived from HIV TATA and tat − response elements; Nucleotides 1651-1716 are derived from piLNXAN polylinkers (Hind III to Xba); Nucleotides 1717-2569 are derived from pSV for splice and poly-addition signals; Nucleotides 2570-2917 are derived from the origin of replication of SV40 (pvu II to Hind III); And nucleotides 2918-2922 are derived from piVX, the residue of the R1 site from the polylinker.

2 shows the nucleotide sequence of a CD2 cDNA insert. Nucleotide numbers are shown in parentheses on the right and amino acid numbers on the left. The locations of possible sites for adding asparagine-associated carbohydrate (CHO) are shown with the expected transmembrane (TM) sequence. Amino acid sequences were numbered from the planned cleavage sites of the secretory signal sequence.

3 is a diagram showing a restriction map of the CDM8 expression vector. CDM8 vectors contain deleted conversions of mutant polyomavirus initial sites selected for high efficiency expression in mouse and monkey cells.

Substantially the entire immunodeficiency promoter region of the human body was replaced by a sequence of the same origin as the direct initial promoter of the cytomegalovirus of the human body and by the inclusion of the bacteriophage T7 promoter between the eukaryotic promoter and the cDNA insertion site. Arrows indicate the direction of transfer.

4 shows a restriction map of the piH3M vector. The direction of transcription is indicated by the arrow. Restriction endonuclease sites flanking the Bst XI cloning site are shown.

5 shows the nucleotide sequence of a piH3M vector. There are seven fragments. Residues 1-587 are derived from the replication origin of pBR322 and residues 588-1182 are of M13 origin, residues 1183-1384 from the supF gene, 1385-2238 from the chimeric cytomegalovirus / human immunodeficiency virus promoter, 2239- 2647 is from a replicable fragment, 2648-3547 is from plasmid pSV2 (splice and polyadenylation signal), and 3548-3900 is derived from SV 40 virus origin.

6 shows the nucleotide sequence of CD40.

Missing configuration and action of the invention

Missing Effect of Invention

Claims (2)

Recombinant DNA encoding a CD27 cell surface antigen, containing the nucleotide sequences set forth in Table 3. Substantially pure CD27 protein having the amino acid sequence of Table 3.