EP0551301A1 - Antigene de surface de cellule cd53 et son utilisation - Google Patents

Antigene de surface de cellule cd53 et son utilisation

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Publication number
EP0551301A1
EP0551301A1 EP91916292A EP91916292A EP0551301A1 EP 0551301 A1 EP0551301 A1 EP 0551301A1 EP 91916292 A EP91916292 A EP 91916292A EP 91916292 A EP91916292 A EP 91916292A EP 0551301 A1 EP0551301 A1 EP 0551301A1
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EP
European Patent Office
Prior art keywords
cells
cell
cdna
antigen
dna
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EP91916292A
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German (de)
English (en)
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Brian Seed
Alejandro Aruffo
Martine Massachussetts General Hospital AMIOT
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General Hospital Corp
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General Hospital Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • cDNA library is a collection of cDNA clones derived from the total poly(A) + mRNA derived from a cell of the organism of interest.
  • a mammalian cell may contain up to 30,000 different mRNA sequences, and the number of clones required to obtain low-abundance mRNAs, for example, may be much greater.
  • Methods of constructing genomic eukaryotic DNA libraries in different expression vectors including bacteriophage lambda, cosmids, 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).
  • genomic cDNA library Once a genomic cDNA library has been constructed, it is necessary to isolate from the thousands of host cells the cell containing the particular human gene of interest.
  • Many different methods of isolating target genes from cDNA libraries have been utilized, with varying success. These include, for example, the use of nucleic acid probes, which are labeled mRNA fragments having nucleic acid sequences complementairy to the DNA sequence of the target gene. When this method is applied to cDNA clones of abundant mRNAs in transformed bacterial hosts, colonies hybridizing strongly to the probe are likely to contain the target DNA sequences.
  • the identity of the clone then may be proven, for example, by in situ hybridization/selection (Goldberg et al., Methods Enzymol., (68:206 (1979)) hybrid-arrested translation (Paterson et al., Proceedings of the National Academy of Sciences. 74:4370 (1977)), or direct DNA sequencing (Maxam and Gilbert, Proceedings of the National Academy of Sciences. 74:560 (1977); Maat and Smith, Nucleic Acids Res., 5:4537 (1978)).
  • leader sequences bind to the cell membrane and draw the remainder of the protein through the lipid bilayer, after which the signal sequence is cleaved from the protein by a signal peptidase enzyme.
  • the protein thus functions only after secretion from the cells (for example, insulin, serum albumin, antibodies, and digestive tract enzymes), or after the proteins have been anchored to the outer surface of a cell membrane (for example, histocompatibility antigens).
  • T lymphocytes The cell surface antigens characteristic of mammalian T lymphocytes are additional examples of proteins that anchor to the cell surface.
  • certain cells derived from bone marrow mature into lymphocytes, which are present in the lymphoid organs, including the thymus, spleen, lymph nodes, and lymphoid aggregates, and also circulate actively through the blood and lymph systems.
  • Mature lymphocyte cells may be divided into two populations: thymus-dependent (T) lymphocytes and thymusindependent (B) lymphocytes. T lymphocytes migrate to the interior of the thymus, where they undergo differentiative proliferation.
  • T lymphocytes During their differentiation process, they express characteristic cell surface membrane alloantigens, including Thy-1, TLA, gv-1, Ly-1, Ly-2, Ly-3, and Ly-5. As they mature, T lymphocytes lose the TLA antigens and some of the Thy-1 antigens, and gain histocompatibility antigens, acquiring the membrane conformation typical of the recirculating T lymphocytes. This is described, for example, by Mota, "Activity of Immune Cells," in Bier et al., eds., Fundamentals of Immunology, 2d Ed., SpringerVerlag, Berlin, pp. 35-62 (1986).
  • T lymphocytes are involved indirectly in the formation of antibodies and their activities thus have required complex analysis of cell function, rather than simple antibody titer measurement. Partly due to this, their importance in development of immunologic competence was not recognized until relatively recently. Mature T lymphocytes synthesize and express an unique pattern of surface glycoprotein antigens which serve as markers for identification of different T lymphocyte subpopulations, including T helper cells, T suppressor cells, and T cytotoxic cells. Each of these subpopulations plays a very important role in regulating the immune system. (Mota, supra).
  • T lymphocytes In humans, the functional and phenotypic heterogeneity of T lymphocytes is well accepted. Two major subpopulations are known: effector T cells mediating cellular immunity; and regulator T cells containing helper and suppressor T lymphocytes. These two subpopulations have been defined with heteroantisera, autoantibodies, and monoclonal antibodies directed at cell surface antigens.
  • effector T cells mediating cellular immunity
  • regulator T cells containing helper and suppressor T lymphocytes These two subpopulations have been defined with heteroantisera, autoantibodies, and monoclonal antibodies directed at cell surface antigens.
  • T11 which reacts strongly to a monoclonal antibody designated Cluster of Differentiation 2 (CD2), and react slightly with monoclonal antibody CD5 to cell surface antigen T1.
  • CD2 Cluster of Differentiation 2
  • T11 CD2
  • CD4 T4
  • CD8 T8 antigen
  • Immunologic competence is acquired at this stage, but is not completely developed until thymic lymphocytes migrate outside the thymus. (Mota, supra.) In contrast with the majority of thymocytes, circulating T lymphocytes express the T1 (CD5) and T3 (CD3) antigens.
  • T4 (CD4) antigen is present on approximately 55-65% of peripheral T lymphocytes, whereas the T8 (CD8) antigen is expressed on 20-30%. These two subpopulations correspond to helper and to suppressor and cytotoxic T cells, respectively. In addition to providing a convenient means of distinguishing T lymphocyte subpopulations, these cell surface antigens are important for mature T cell activation and effector function. T cell activation involves a complex series of cell surface interactions between the T cell and the target cell or stimulator cell in addition to binding of the T cell receptor to its specific antigen.
  • CD2 the human T cell erythrocyte receptor
  • target cells e.g., erythrocytes
  • LFA-3 a specific molecular ligand for CD2, designated LFA-3, in humans, which is a widely distributed surface antigen.
  • LFA-3 a specific molecular ligand for CD2, designated LFA-3, in humans, which is a widely distributed surface antigen.
  • This phenomenon has long been employed to detect, assay and purify human cells producing antibodies to sheep erythrocytes and serves as the basis for the E-rosette test, first described by Zaalberg, Nature 202:1231 (1964).
  • CD2/LFA-3 interactions also have been shown to mediate cytolytic target conjugation (Shaw et al., Nature 323:262-264 (1986), and the mixed lymphocyte reaction
  • Anti-CD2 monoclonal antibodies can directly activate peripheral T-lymphocytes via an antigen-independent pathway (Meuer et al., Cell 36:897-906 (1984)), indicating an even wider immunoregulatory role for CD2.
  • T lymphocytes are the main effectors of cell-mediated immunity and also are involved as helper or suppressor cells in modulating the immune response has resulted in a significant contribution to the increasing practical application of clinical immunology to medicine.
  • the scope of this application includes defense against infections, prevention of diseases by immunization, organ transplantation, blood banking, and treatment of deficiencies of the immune system and a variety of disorders that are mediated by immunologic mechanisms.
  • immunologic techniques frequently are used in the clinical laboratory, as in the measurement of hormones and drugs.
  • the protein expressed in a mammalian host cell should be functional and should undergo any normal posttranslational modification.
  • a protein ordinarily transported through the intracellular membrane system to the cell surface should undergo the complete transport process.
  • a mammalian expression system also would allow the study of intracellular transport mechanisms and of the mechanism that insert and anchor cell surface proteins to membranes.
  • COS mammalian host cell
  • SV40 simian virus strain 40
  • any foreign DNA cloned on a vector containing the SV40 origin of replication will replicate because SV40 T antigen is present in COS cells.
  • the foreign DNA will replicate transiently, independently of the cellular DNA.
  • Lymphokine bioassays are very sensitive ((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); Yang, Y. et al., Cell 47:3-10 (1986)) and the mRNAs are typically both abundant and short (Wong, G.G. et al., Science 228:810-815 (1985); Lee, F., et al., Proceedings of the National Academy of Sciences.
  • Oshima et al. used plaque hybridization to screen a phage lambda gt11 cDNA library for the gene encoding human placental beta-glucuronidase. Oshima et al., Proceedings of the National Academy of Sciences. U.S.A. 84:685-689 (1987). The identity of isolated cDNA clones was verified by immunoprecipitation of the protein expressed by COS-7 cells transfected with cloned inserts using the SV40 late promoter.
  • the present invention relates to a powerful new method for cloning cDNA encoding cell surface antigens, to a method of constructing cDNA libraries to high efficiency expression vectors particularly suited for high level expression in eukaryotic host cells, and to the isolated nucleotide sequences and their encoded products.
  • the highly efficient cloning technique of the present invention is based upon transient expression of antigen in eukaryotic cells and physical selection of cells expressing the antigen by adhesion to an antibody-coated substrate, such as a culture dish.
  • the methods of the present invention are useful for the isolation and molecular cloning of any protein which can be expressed and transported to the cell surface membrane of a eukaryotic cell.
  • the method for cloning cDNA encoding a cell surface antigen of the present invention comprises preparing a cDNA library; introducing this cDNA library into eukaryotic mammalian preferably tissue culture cells; culturing these cells under conditions allowing expression of the cell surface antigen; exposing the cells to a first antibody or antibodies directed against the cell surface antigen, thereby allowing the formation of a cell surface antigenfirst antibody complex; subsequently exposing the cells to a substrate coated with a second antibody directed against the first antibody, thereby causing cells expressing the cell surface antigen to adhere to the substrate via the formation of a cell surface antigen-first antibody-second antibody complex; and separating adherent from non-adherent cells.
  • isolation and molecular cloning of genes encoding such cell surface antigens as the following have been accomplished: the 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.
  • a cloned gene such as that encoding 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, FcR1a, FcR1b, TLiSa, and Leu8, is also the subject of the present invention.
  • the gene encoding an antigen has been cloned according to the method of the present invention, that gene can be expressed in a prokaryotic or a eukaryotic host cell to produce the encoded protein or portion thereof in substantially pure form such as it does not exist in nature.
  • Another aspect of the present invention relates to substantially pure cell surface antigens, particularly: 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 their functional analogues and equivalents.
  • the primary amino acid sequences of the 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 have been determined.
  • the invention thus also relates to the amino acid sequences of those antigens and their functional equivalents and to the nucleotide sequences encoding those antigens.
  • This invention also relates to high efficiency cDNA expression vectors which allow the generation of very large mammalian expression libraries and yield large amounts of protein in mammalian host cells, resulting in efficient selection.
  • a cDNA expression vector comprises a suppressor tRNA gene; an SV40 origin; a synthetic transcription unit, comprising a chimeric promoter composed of human cytomegalovirus AD169 immediate early enhancer sequencer fused to the HIV LTR -60 to +80 sequences, inserted between the suppressor tRNA gene and the SV40 origin; a polylinker comprising two BstXI sites separated by a replaceable DNA sequence and flanked by XbaI sites; and an SV40 small t antigen splice and early region polyadenylation signals.
  • a further aspect of the present invention comprises a synthetic transcription unit for use in a cDNA expression vector, comprising a chimeric promoter composed of human ⁇ ytomegalovirus AD169 immediate early enhancer sequences fused to HIV LTR -60 to +80 sequences.
  • a chimeric promoter composed of human ⁇ ytomegalovirus AD169 immediate early enhancer sequences fused to HIV LTR -60 to +80 sequences.
  • the small size and particular arrangement of the sequences of the cDNA expression vector of the present invention allow highly efficient replication in host mammalian tissue culture cells, such as COS cells.
  • this vector employs a polylinker containing two inverted BstXI sites separated by a short replaceable DNA segment, which allows the use of very efficient oligonucleotide-based cDNA insertion strategy.
  • the present invention comprises a vector comprising two identical BstXI sites in inverted orientation each with respect to the other, which BstXI sites are separated by a short replaceable DNA fragment.
  • Another aspect of the invention is a polylinker as described above.
  • a further aspect of the invention relates to an oligonucleotide-based cDNA insertion method, comprising ligating synthetic DNA oligonucleotides to the cDNA segment desired to be inserted into a vector, the synthetic DNA oligonucleotides giving the same terminal sequences as those of the short replaceable DNA fragment of the polylinker of the invention, and inserting the resulting cDNA segment plus synthetic DNA oligonucleotide terminal sequences into the polylinker of the vector, from which the short replaceable DNA fragment previously has been removed.
  • the present invention relates to the use of tumor cells, particularly human tumor cells, to prepare cDNA libraries for use according to the methods of the present invention.
  • Another advantage of the powerful selection system of the present invention is that directional insertion of the cDNA is not necessary.
  • the method of the present invention results in library construction efficiencies which are on a par with those described for phage vectors such as lambda gt10 and lambda gt11, with the additional advantage that clones generated according to the methods of the present invention are easier to manipulate.
  • the immunoselection technique of the present invention allows efficient use of antibodies, which may be monoclonal or polyclonal, in relatively small absolute amounts.
  • the method of the present invention also is quite rapid. Generally, three or fewer cycles of immunoselection and rescue are required to isolate a target cDNA clone.
  • the method of the present invention also results in the efficient use of labor and materials when cloning genes encoding cell surface antigens. As described above, this method has been employed to successfully clone genes encoding cell surface antigens associated with mammalian T lymphocytes (e.g.
  • the purified genes and proteins of the present invention are useful for immunodiagnostic and immunotherapeutic applications, including the diagnosis and treatment of immune-mediated infections, diseases, and disorders in animals, including humans. They can also be used to identify, isolate and purify other antibodies and antigens. Such diagnostic and therapeutic uses comprise yet another aspect of the present invention.
  • the substantially pure proteins of the present invention may be prepared as medicaments or pharmaceutical compositions for therapeutic administration. The present invention further relates to such medicaments and compositions.
  • Nucleotides 1-589 are derived from pMB1 origin (pBR322 ori); nucleotides 590-597 are derived from the SacII linker (ACCGCGT); nucleotides 598-799 are derived from the synthetic tyrosine suppressor tRNA gene (supF gene); nucleotides 800-947 are derived from a remnant of the ASV LTR fragment (PvuII to MIu1); nucleotides 948-1500 are derived from the human cytomegalovirus AD169 enhancer; nucleotides 1501-1650 are derived from HIV TATA and tatresponsive elements; nucleotides 1651-1716 are derived from the piLNXAN polylinker (HindIII to Xba); nucleotides 1717-2569 are derived from pSV to splice and poly-Addition signals; nucleotides 2570-2917 are derived from the SV40 origin of replication (pyuII to (HindIII
  • Nucleotide numbering is given in parentheses at right, amino acid numbering, left. Locations of the potential sites for addition of asparagine-linked carbohydrate (CHO) are shown, as well as the predicted transmembrane (TM) sequence. The amino acid sequence is numbered from the projected cleavage site of the secretory signal sequence.
  • Figure 3 Restriction map of the CDM8 expression vector
  • the CDM8 vector includes a deleted version of a mutant polyoma virus early region selected for high efficiency expression in both murine and monkey cells. Substantially all of the human immunodeficiency promoter region has been replaced with the cognate sequences of the human cytomegalovirus immediate early promoter, and by inclusion of a bacteriophage T7 promoter between the eukaryotic promoter and the site of cDNA insertion. Arrows indicate the direction of transcription.
  • WOP cells transfected with a clone encoding the LFA-3 antigen were detected by indirect immunofluorescence, amplified and sequenced.
  • (A) shows the 874 base pair insert containing an open reading frame of 237 residues originating at a methionine codon, and terminating in a series of hydrophobic residues. Hydrophobic and hydrophilic regions within this open reading frame are shown in (B).
  • the direction of transcription is indicated by an arrow. Restriction endonuclease sites flanking the BstXI cloning sites are shown.
  • Residues 1-587 are from the pBR322 origin of replication, 588-1182 from the M13 origin, 1183-1384 from the supF gene, 1385-2238 are from the chimeric cytomegalovirus/human immunodeficiency virus promoter, 2239-2647 are from the replaceable fragment, 2648-3547 from plasmid pSV2 (splice and polyadenylation signals), and 3548-3900 from the SV40 virus origin.
  • FIG. 7 Nucleotide sequence of the CD28 cDNA Nucleotide numbering is given in parentheses at right, amino acid numbering, center and left. Location of the potential sites for addition of asparagine-linked carbohydrate (CHO) are shown, as well as the predicted transmembrane (TM) sequence. The amino acid sequence is numbered from the projected cleavage site of the secretory signal sequence.
  • FIG. 8 Nucleotide seguence of the CD7 cDNA insert Nucleotide numbering is given in parentheses at right. Splice donor and acceptor sites indicated by (/). The location of the potential sites for addition of asparaginelinked carbohydrate (CHO) are shown, the potential fatty acid esterification site is denoted (*), and the predicted transmembrane domain (TM) is underlined. Nucleotide sequences potentially involved in hairpin formation are denoted by (.). The presumed polyadenylation signal is underlined.
  • Nucleotide number is given in the parenthesis at right, amino acid numbering, center and left. Locations of the potential sites for addition of asparagine-linked carbohydrate (CHO) are shown, as well as the predicted transmembrane (TM) sequence. The amino acid sequence is numbered from the projected cleavage site of the secretory signal sequence. Cysteine residues are underscored with asterisks.
  • FIG. 10 Sequence of the CD20.4 cDNA A.
  • the sites of potential N-linked glycosylation are denoted by the symbol -CHO-; the hydrophobic regions are underscored.
  • the site of the poly(A) + tail in clone CD20.6 is denoted by an asterisk.
  • This invention relates to a novel method for cloning cDNA encoding a cell surface antigen and to a method of constructing cDNA libraries. It also relates to particular cDNA expression vectors and components thereof, nucleotide sequences or genes isolated by the method, substantially pure cell surface antigens encoded by the cDNA segments, and methods of using the isolated nucleotide sequences and encoded products.
  • cDNA expression vectors and components thereof nucleotide sequences or genes isolated by the method, substantially pure cell surface antigens encoded by the cDNA segments, and methods of using the isolated nucleotide sequences and encoded products.
  • cDNA complementary or copy DNA produced from an RNA template by the action of RNA-dependent DNA polymerase (reverse transcriptase).
  • a "cDNA clone” means a duplex DNA sequence complementary to an RNA molecule of interest, carried in a cloning vector.
  • cDNA library is meant a collection of recombinant DNA molecules containing cDNA inserts which together comprise the entire genome of an organism.
  • a cDNA library may be prepared by art-recognized methods described, for example, in Maniatis et al.. Molecular Cloning: A Laboratory Manual, supra.
  • RNA is first isolated from the cells of an organism from whose genome it is desired to clone a particular gene.
  • Preferred for the purposes of the present invention are mammalian, and particularly human, cell lines. More preferred are the human tumor cell line HPB-ALL and the human lymphoblastoid cell line JY.
  • RNA can be isolated from a tumor cell, derived from an animal tumor, and preferably from a human tumor.
  • a library may be prepared from, for example, a human adrenal tumor, but any tumor may be used.
  • the immunoselection cloning method of the present invention comprises the preparation of a cDNA library by extracting total RNA including a particular gene from a cell, synthesizing a series of complementary doublestranded cDNA fragments from the RNA and introducing these cDNA fragments into mammalian cells in tissue culture.
  • the mammalian cells are maintained under conditions which allow them to express the protein (i.e. the cell surface antigen).
  • the resulting cells are exposed to a first antibody or pool (group) of antibodies directed against the cell surface antigen. This results in formation of a cell surface antigen-first antibody complex.
  • the complexes are exposed to a substrate to which is coated or bound a second antibody directed against the first antibody. Cells expressing the cell surface antigen adhere to the substrate (because of formation of a cell surface antigen-first antibody-second antibody complex).
  • Adherent cells are separated from non-adherent cells. Isolation of total RNA
  • the guanidium thiocyanate/CsCl method of isolating total RNA is preferred. More preferred is a guanidium thiocyanate/LiCl variant of the GuSCN/CsCl method, which has added capacity and speed. Briefly, for each ml of mix desired, 0.5g GuSCN are dissolved in 0.58 ml of 25% LiCl (stock filtered through 0.45 micron filter) and 20 ul of mercaptoethanol is added. Cells are spun out and the pellet is dispersed on walls by flicking, add 1 ml of solution to up to 5 ⁇ 10 7 cells. The resulting combination is sheared by polytron until nonviscous.
  • polyA + RNA may be prepared, preferably by the oligo dT selection method. Briefly, a disposable polypropylene column is prepared by washing with 5M NaOH and then rinsing with RNase-free water. For each milligram total RNA about 0.3 ml (final packed bed) oligo dT cellulose is used. Oligo dT cellulose is prepared by resuspending about 0.5 ml of dry powder in 1 ml of 0.1M NaOH and transferring it into the column, or by percolating 0.1 NaOH through a previously used column (columns can be reused many times). This is washed with several column volumes of RNase-free water, until pH is neutral, and rinsed with 2-3 ml of loading buffer.
  • the column bed is then removed into a sterile 15 ml tube using 4-6 ml of loading buffer.
  • the result is poured into a column and washed with 3 ml loading buffer and then 3 ml of middle wash buffer.
  • mRNA is eluted directly into an SW55 tube with 1.5 ml of 2 mM EDTA, 0.1% SDS; the first two or three drops are discarded.
  • Eluted mRNA is precipitated by adding 1/10 vol. 3M NaOAc and filling the tube with EtOH. This is then mixed, chilled for 30 minutes at -20oC, and spun at 50k rpm at 5°C for 30 min. The EtOH is poured off and the tube is air dried. The mRNA pellet is resuspended in 50-100 ul of RNase-free water. Approximately 5 ul is melted at 70° in MOPS/EDTA/formaldehyde and run on an RNase-free 1% agarose gel to check quality.
  • cDNA is synthesized.
  • a preferred method of cDNA synthesis is a variant of that described by Gubler and Hoffman (Gene, 25:263-269 (1982)). This is carried out as follows: a. First Strand. 4 ug of mRNA and heated to about 100°C in a microfuge tube for 30 seconds and quenched on ice. The volume is adjusted to 70 ul with RNase-free water.
  • RNAse inhibitor Boehringer 36 u/ul
  • 1 ul of 5 ug/ul of oligo dT Collaborative Research
  • 2.5 ul of 20 mM dXTP's ultrapure
  • 1 ul of 1 M DTT and 4 ul of RT-LX (Life Science, 24 u/ul).
  • the resulting combination is incubated at 42°C for 40 min. It is heated to inactivate (70°C 10 min).
  • Second Strand Second Strand.
  • RNAse free water 80 ul of RT2 buffer, 5 ul of DNA Polymerase I (Boehringer, 5 U/ul), 2 ul RNAse H (BRL 2 u/ul).
  • DNA Polymerase I Boehringer, 5 U/ul
  • 2 ul RNAse H BBL 2 u/ul
  • Adaptors 20 ul of 0.5M EDTA pH 8.0, phenol extract and EtOH precipitate by adding NaCl to 0.5M, linear polyacrylamide (carrier) to 20 ug/m
  • oligonucleotide segments containing terminal sequences corresponding to BstXI sites on the vectors are ligated to the cDNA fragment desired to be inserted.
  • the resulting fragments are pooled by fractionation.
  • a preferred method is as follows:
  • Suppressor tRNA plasmids may be propagated by known methods.
  • supF plasmids can be selected in nonsuppressing hosts containing a second plasmid, p3, which contains amber mutated ampicillin and tetracycline drug resistance elements (Seed, 1983).
  • the p3 plasmid is derived from PR1, is 57 kb in length, and is a stably maintained, single copy episome.
  • the ampicillin resistance of this plasmid reverts at a high rate, so that amp r plasmids usually cannot be used in p3-containing strains. Selection for tet resistance alone is almost as good as selection for amp+tet resistance.
  • Suppressor plasmids typically are selected for in LB medium containing amp at 12.5 ug/ml and tet at 7.5 ug/ml.
  • M9 casamino acids medium containing glycerol 0.8% may be used as a carbon source, and the bacteria grown to saturation.
  • Vector DNA may be isolated by known methods. The following method is preferred for plasmid from 1 liter of saturated cells:
  • the vector may be prepared for cloning by known methods.
  • a preferred method begins with cutting 20 ug of vector in a 200 ul reaction with 100 units of BstXI (New York Biolabs), cutting at 50oC overnight in a well-thermostatted water bath (i.e., circulating water bath).
  • the desired band will have migrated 2/3 of the length of the tube. Forward trailing of the band means the gradient is overloaded.
  • Adaptors may be prepared by known methods, but it is preferred to resuspend crude adaptors at a concentration of 1 ug/ul, add MgSO 4 to 10 mM, and precipitate by adding 5 volumes of EtOH. Rinse with 70% EtOH and resuspend in TE at a concentration of 1 ug/ul. To kinase take 25 ul of resuspended adaptors, add 3 ul of 10X kinasing buffer and 20 units of kinase; incubate 37oC overnight.
  • buffers mentioned in the above description of preferred methods according to the present invention will be evident to those of skill.
  • preferred buffer compositions are as follows: Loading Buffer: 0.5 M LiCl, 10 mM Tris pH 7.5, ImM
  • Middle Wash Buffer 0.15 M LiCl, 10 mM Tris pH 7.5, ImM
  • RT2 Buffer 0.1 M Tris pH 7.5, 25 mM MgCl 2 , 0.5
  • vector is meant a DNA molecule, derived from a plasmid or bacteriophage, into which fragments of DNA may be inserted or cloned.
  • a vector will contain one or more unique restriction sites, and may be capable of autonomous replication, in a defined host or vehicle organism such that the cloned sequence is reproducible.
  • DNA expression vector is meant any autonomous element capable of replicating in a host independently of the host's chromosome, after additional sequences of DNA have been incorporated into the autonomous element's genome.
  • DNA expression vectors include bacterial plasmids and phages.
  • viral vectors such as those derived from simian virus strain 40 (SV40).
  • SV40 is a papovavirus having a molecular weight of 28 Mdal, and containing a circular double-stranded DNA molecule having a molecular weight of 3 Mdal, which comprises the entire genome of the virus. The entire nucleotide sequence of this single, small, covalentiy closed circular DNA molecule has been determined. Fiers et al., Nature 273:113-120 (1978); Reddy et al., Science 200:494-502 (1978).
  • the viral DNA of SV40 may be obtained in large quantities, and the genomic regions responsible for various viral functions have been accurately located with respect to a detailed physical map of the DNA. Fiers et al., supra; Reddy et al., supra.
  • the viral genome of SV40 can multiply vegetatively or as an integral part of cellular chromosomes, and a wealth of information exists on the replication and expression of this genome.
  • M13 single-stranded bacteriophage cloning vehicle
  • An advantage of utilizing M13 as a cloning vehicle is that the phage particles released from infected cells contain single-stranded DNA homologous to only one of the two complementary strands of the cloned DNA, which therefore can be used as a template for DNA sequencing analysis.
  • Even more preferred for the purposes of the present invention are the expression vectors designated piH3, piH3M, and CDM8, deposited at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, MD 20652.
  • piH3 was deposited at the ATCC on February 24, 1988, and has accession number ATCC 67634.
  • piH3M was deposited at the ATCC on February 24, 1968, and has accession number ATCC 67633.
  • CDM8 was deposited at the ATCC on February 24, 1986, and has accession number ATCC 67635.
  • tissue culture is meant the maintenance or growth of animal tissue cells in vitro so as to allow further differentiation and preservation of cell architecture or function or both.
  • Primary tissue cells are those taken directly from a population consisting of cells of the same kind performing the same function in an organism. Treating such tissue cells with the proteolytic enzyme trypsin, for example, dissociates them into individual primary tissue cells that grow well when seeded onto culture plates at high densities. Cell cultures arising from multiplication of primary cells in tissue culture are called “secondary cell cultures.” Most secondary cells divide a finite number of times and then die.
  • COS cells are those that have been transformed by SV40 DNA containing a functional early gene region but a defective origin of viral DNA replication. COS cell clone M6 is particularly preferred for use according to the method of the invention.
  • murine "WOP" cells which are NIH 3T3 cells transfected with polyoma origin deletion DNA.
  • cDNA may be introduced into the host tissue culture cells of the present invention by any methods known to those of skill. Transfection may be accomplished by, for example, protoplast fusion, by spheroplast fusion, or by the DEAE dextran method (Sussman et al., Cell. Biol. 4:1641-1643 (1984)). If spheroplast fusion is employed, a preferred method is the following variant based on Sandri-Goldrin et al., Mol. Cell Bio. 1:743-752 (1981).
  • a set of six fusions requires 100 ml of cells in broth.
  • Grow cells containing amplifiable plasmid to OD 600 0.5 in LB.
  • spectinomycin to 100 ug/ml (or chloramphenicol to 150 ug/ml).
  • Cells begin to lyse with prolonged incubation in spectinomycin or chloramphenicol medium).
  • Dishes can be stacked on top of each other, but 3 in a stack is not advisable as the spheroplast layer on the top dish is often torn or detached after centrifugation.
  • the thin layer of PEG that remains on the cells is sufficient to promote fusion; the layer remaining is easier to wash off, and better cell viability can be obtained, than if the bulk of the PEG is left behind.
  • PEG 1000 120 seconds
  • PEG 1450 120 to 150 seconds
  • DME no serum
  • the PEG layer will be swept radially by the DME. Tilt the dishes and aspirate. Repeat the DME wash.
  • the PEG solution can be conveniently prepared by melting a fresh bottle of PEG at 60oC and pouring approximate 50 ml aliquots by means of a 50 ml centrifuge tube into preweighed bottles. The aliquoted PEG is stored at 5oC in the dark. To make up a fresh bottle, weigh the aliquot, remelt, and add an equal volume of DME (no serum). Adjust the pH with 7.5% Na bicarbonate solution if necessary, and filter sterilize. The resulting PEG solution may be stored up to 3 months at room temperature without detectable adverse consequence.
  • Transfected host cells will be cultured according to the invention in order to accomplish expression of the protein encoded by the cDNA clone, and to increase the absolute numbers of cells available for subsequent immunoselection.
  • COS cells for example, may be cultured in Dulbecco's modified Eagle's medium (DME) supplemented with 10% calf serum and gentamycin sulfate.
  • DME Dulbecco's modified Eagle's medium
  • Transient expression of transfected cells normally can be expected between 48 and 72 hours posttransfection. However, this time period may vary depending upon the type or strain of host cell used and the cell culture conditions, as will be apparent to those of ordinary skill.
  • Immunoprecipitation, blotting, and cDNA sequencing of genes cloned according to the methods of the present invention may be carried out by any convenient methods known to those of skill.
  • the immunoprecipitation protocol of Clark et al., Leukocyte Typing II, Vol. II, pp. 155-167 (1986) is preferred.
  • Southern, Northern, or other blot analysis methods known to those of skill may be employed, using hybridization probes prepared by known methods, such as that of Hu et al. (Gene 18.:271-277 (1982)).
  • cDNA sequencing also may be accomplished by known methods, including the dideoxynucleotide method of Sanger et al., Proc. Natl. Acad. Sci. (USA) 74:5463-5467 (1977).
  • the antibodies used according to the present invention may be polyclonal or monoclonal. These may be used singly, or in conjunction with other polyclonal or monoclonal antibodies to effect immunoselection of cells expressing the desired antigen or antigens by the methods of the present invention.
  • Methods of preparing antibodies or fragments thereof for use according to the present invention are known to those of skill. Standard reference works setting forth general principles of immunology include Klein, J., Immunology: The Science of Self-Nonself Discrimination.
  • antibody is meant to include the intact molecule as well as fragments thereof, such as, for example. Fab and F(ab)' 2 fragments, which also are able to bind to antigen.
  • Polyclonal antibody preparations may be derived directly from the blood of the desired animal species after immunization with the antigen of interest, or a fragment thereof, using any of the standard protocols known to those of ordinary skill.
  • monoclonal antibodies may be prepared using known methods (Kohler et al., Eur. J. Immunol. 6:292 (1976)). Use of monoclonal antibodies is preferred for the purposes of the present invention.
  • the tissue culture host cells which have been exposed to antibodies directed against the target cell surface antigen are separated from host cells which do not express the target antigen by distributing the cells onto a substrate coated with antibody directed against the antibody for the antigen.
  • This technique termed “panning,” will be known to those of skill, and is described, for example, by Mage et al., J. Immunol. Meth. 15:47-56 (1977), and Wysocki and Sato, Proc. Natl. Acad. Sci. (USA) 75:2844-2848 (1978).
  • Panning according to the methods of the present invention may be carried out as follows: a. Antibody-coated dishes.
  • Aspirate medium from dish add 2 ml PBS/0.5 mM EDTA/0.02% azide and incubate dishes at 37oC for 30 min. to detach cells from dish. Triturate cells vigorously with short pasteur pipet, and collect cells from each dish in a centrifuge tube. Spin 4 min. setting 2.5 (200 x g) (takes 5 min). Resuspend cells in 0.5-1.0 ml PBS/EDTA/azide/5% FBS and add antibodies. Incubate at least 30 min. on ice. Add an equal volume of PBS/EDTA/azide, layer carefully on 3 ml PBS/EDTA/azide/2% Ficoll, and spin 4 min. at setting 2.5.
  • Blockers assure that non-specific proteins, proteases, or antibodies present do not cross-link with or destroy the antibodies present on the substrate or on the host cell surface, to yield false positive or false negative results. Selection of blockers can substantially improve the specificity of the immunoselection step of the present invention.
  • a number of non-specific monoclonal antibodies, for example, of the same class or subclass (isotype) as those used in the immunoselection step e.g., IgG 1 , IgG 2 A, IgGm, etc.
  • Blocker concentration normally 1-100 ug/ul
  • the buffer system used for incubation may be selected to optimize blocking action and decrease non-specific binding.
  • a population of cells to be panned for those expressing the target cell surface antigen is first detached from its cell culture dish (harvested) without trypsin.
  • the cells then are exposed to a first antibody, which may be polyclonal or monoclonal, directed against the antigen of interest or against a family of related antigens.
  • a single antibody or a group of antibodies may be used, the choice depending upon the nature of the target antigen, its anticipated frequency, and other variables that will be apparent to those of skill.
  • Target antigens expressed on the surfaces of host cells will form an antigen-antibody complex.
  • the cells subsequently are placed in close apposition to a substrate, such as a culture dish, filter disc, or the like, which previously has been coated with a second antibody or group of antibodies.
  • a substrate such as a culture dish, filter disc, or the like
  • This second antibody will be directed against the first antibody, and its choice will be a matter of ordinary skill dictated by, for example, the animal in which the first antibody was raised. For example, if the first antibody was raised in mice, the second antibody might be directed against mouse immunoglobulins, raised in goats or sheep. Cells expressing the target antigen will adhere to the substrate via the complex formed between the antigen, the first antibody, and the second antibody. Adherent cells then may be separated from nonadherent cells by washing.
  • DNA encoding the target antigen is prepared from adherent cells by known methods, such as that of Hirt, J. Molec. Biol. 26:365-369 (1967). This DNA may be transformed into E. coli or other suitable host cells for further rounds of fusion and selection, to achieve the desired degree of enrichment.
  • the initial rounds of immunoselection will employ a panel of first antibodies directed against an epitope or group of epitopes common to the family of antigens to which the target antigen belongs. This will be sufficient to narrow the number of clones for future rounds quite significantly. Two such rounds usually will be found adequate, but the number of rounds may vary as mentioned above. Thereafter, a single round of selection may be performed employing a single first antibody or a group of first antibodies recognizing only the target antigen.
  • substrate is meant a solid surface to which antibodies may be bound for immunoselection according to the present invention.
  • suitable substrates include glass, polystyrene, polypropylene, dextran, nylon, and other materials. Tubes, beads, microtiter plates, bacteriological culture dishes, and the like formed from or coated with such materials may be used.
  • Antibodies may be covalentiy or physically bound to the substrate by known techniques, such as covalent bonding via an amide or ester linkage, or by absorption. Those skilled in the art will know many other suitable substrates and methods for immobilizing antibodies thereupon, or will be able to ascertain such substrates and methods using no more than routine experimentation.
  • the choice of host tissue culture cells for use according to the present invention preferably should be such as to avoid the situation in which the antibodies used for panning recognize determinants on untransfected cells.
  • COS cells are preferred for transient expression of certain surface antigens
  • WOP 3027 cells are even more preferred. WOP cells allow virtually all antibodies to be used, since cross-reactions between murine antibodies and murine cell surface determinants are rare.
  • the insert size of the recombinant DNA molecule should be chosen to maximize the likelihood of obtaining an entire coding seguence. Those of skill will know various methods by which a preliminary determination of optimal insert size for a given gene may be determined. Vector construction and cDNA insertion
  • Vectors suitable for expression of cDNA in mammalian tissue culture cells may be constructed by known methods.
  • Preferred for the purposes of the present invention is an expression vector containing the SV40 origin.
  • the vector may contain a naturally derived or synthetic transcription origin, and the SV40 early region promoter. Even more preferred is a chimeric promoter composed of human cytomegalovirus immediate early enhancer sequences.
  • Various "enhancer sequences" also may be used with SV40 vectors. These are described, for example, by Banerji et al., Cell 27:299-308 (1981); Levinson et al., Nature 295:568-572 (1982); and Conrad et al., Mol. Cell. Biol. 2:949-965 (1982).
  • Insertion of cDNA into the vectors of the present invention can occur, for example, by homopolymeric tailing with terminal transferase.
  • homopolymeric tracts located 5' to cDNA inserts may inhibit in vitro and in vivo expression.
  • preferred for purposes of the present invention is the use of inverted identical cleavage sites separated by a short replaceable DNA segment.
  • Such inverted identical cleavage sites preferably employing the BstXI restriction endonuclease, may be used in parallel with cDNA synthetic oligonucleotides, giving the same terminii as the replaceable segment of the vector. In this manner, the cDNA cannot ligate to itself, but can ligate to the vector. This allows the most efficient use of both cDNA and vector.
  • Another embodiment of the present invention is the above-described efficient oligonucleotide-based strategy to promote cDNA insertion into the vector.
  • the piH3M vector of the present invention is preferred, and employs the inverted endonuclease sites.
  • This vector may contain an SV40 origin of replication, but a more preferred form contains an M13 origin.
  • This vector, containing the M13 origin allows high level expression in COS cells of coding sequences placed under its control. Also, the small size and particular arrangement of sequences in the plasmid permit high level replication in COS cells.
  • cell surface antigen any protein that is transported through the intracellular membrane system to the cell surface. Such antigens normally are anchored to the cell surface membrane through a carboxyl terminal domain containing hydrophobic amino acids that lie in the lipid bilayer of the membrane, and there exert their biological and antigenic effects. Antigens such as those of T-lymphocytes are particularly suited for gene cloning by the method of the present invention. However, cell surface antigens of any cells may be cloned according to the present method. Moreover, proteins not normally expressed on the cell surface may admit of cloning according to the present method by, for example, using fluorescence activated cell sorting (FACS) to enrich for fixed cells expressing intracellular antigens.
  • FACS fluorescence activated cell sorting
  • substantially pure is meant any antigen of the present invention, or any gene encoding any such antigen, which is essentially free of other antigens or genes, respectively, or of other contaminants with which it might normally be found in nature, and as such exists in a form not found in nature.
  • functional derivative is meant the “fragments,” “variants,” “analogs,” or “chemical derivatives” of a molecule.
  • a “fragment” of a molecule, such as any of the antigens of the present invention is meant to refer to any polypeptide subset of the molecule.
  • a “variant” of such molecules is meant to refer to a naturally occurring molecule substantially similar to either the entire molecule, or a fragment thereof.
  • An “analog” of a molecule is meant to refer to a non-natural molecule substantially similar to either the entire molecule or a fragment thereof.
  • a fragment, variant, analog and/or chemical derivative of an antigen is said to be "substantially similar" to another fragment, variant, analog, chemical derivative and/or antigen if the sequence of amino acids in both molecules is substantially the same, i.e., have at least about 50% homology, and if both molecules possess a similar biological activity.
  • a nucleotide sequence is said to be "substantially similar" to another nucleotide sequence if both sequences are substantially the same, i.e., the first nucleotide sequence encodes a fragment, variant, analog, chemical derivative or antigen having an amino acid sequence of at least about 50% homology to that encoded by the second nucleotide sequence, and the fragment, variant, analog, chemical derivative and/or antigen encoded by the first nucleotide sequence possesses a biological activity similar to the biological activity of the fragment, variant, analog, chemical derivative and/or antigen encoded by the second nucleotide sequence.
  • a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, etc. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, etc. Moieties capable of mediating such effects are disclosed, for example, in Remington's Pharmaceutical Sciences.
  • a “functional derivative" of a gene of any of the antigens of the present invention is meant to include “fragments,” “variants,” or “analogues” of the gene, which may be “substantially similar” in nucleotide sequence, and which encode a molecule possessing similar activity.
  • An antigen, fragment, variant, analog and/or chemical derivative is said to have "similar biological activity" to another antigen, fragment, variant, analog and/or chemical derivative if the former has a biological activity of at least about 25% of that of the latter.
  • Biological activities are those operations, functions or processes which are characteristic of living organisms. Biological activities can also include the reproduction, extension or adaptation of living processes to in vitro or non-natural systems, such as the biological activity exhibited when an antigen, fragment, variant, analog and/or chemical derivative is artificially introduced into a test animal to induce the production of antibodies.
  • An antigen, fragment, variant, analog and/or chemical derivative can have one or more biological activities. Biological activities can be detected or measured by methods or assays that are characteristic for that activity. For a functional derivative to have a biological activity similar to that of an antigen, it must have a biological activity of at least about 25% of that of antigen as measured by an assay characteristic for that activity and known to those of skill in the art.
  • the substantially pure antigens that have been expressed by methods of the present invention may be used in immunodiagnostic assay methods well known to those of skill, including radio-immunoassays (RIAs), enzyme immunoassays (EIAs) and enzyme-linked immunosorbent assays (ELISAs).
  • RIAs radio-immunoassays
  • EIAs enzyme immunoassays
  • ELISAs enzyme-linked immunosorbent assays
  • the substantially pure proteins of the present invention, in soluble form may be administered alone or in combination with other antigens of the present invention, or with other agents, including lymphokines and monokines or drugs, for the treatment of immune-related diseases and disorders in animals, including humans.
  • immune deficiency diseases diseases of immediate type hypersensitivity, asthma, hypersensitivity pneumonitis, immune-complex disease, vasculitis, systemic lupus erythematosus, rheumatoid arthritis, immunopathogenic renal injury, acute and chronic inflammation, hemolytic anemias, platelet disorders, plasma and other cell neoplasms, amyloidosis, parasitic diseases, multiple sclerosis, Guillain-Barre syndrome, acute and subacute myopathic paralysis, myasthenia gravis, immune endocrinopathies, and tissue and organ transplant rejection, all as 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.
  • the antigens of the present invention may be unlabeled or labeled with a therapeutic agent.
  • therapeutic agents which can be coupled to the antigens of the invention for immunotherapy are drugs, radioisotopes, lectins, and toxins.
  • the dose ranges for the administration of the antigens of the present invention are those large enough to produce the desired immunotherapeutic effect, but not so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage employed will vary with the age, condition, sex, and extent of the disease in the patient. Counterindications (if any), immune tolerance and other variables also will affect the proper dosage.
  • Administration may be parenteral, by injection or by gradual perfusion over time. Administration also may be intravenous, intraparenteral, intramuscular, subcutaneous, or intradermal. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions.
  • non-aqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic and aqueous solutions, emulsions, or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like.
  • Preservatives and other additives also may be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and the like.
  • Such preparations, and the manner and method of making them, are known and described, for example, in Remington's Pharmaceutical Science. 16th ed., supra.
  • the antigens of the present invention also may be prepared as medicaments or pharmaceutical compositions comprising the antigens, either alone or in combination with other antigens or other agents such as lymphokines, monokines, and drugs, the medicaments being used for therapy of animal, including human, immune-related indications.
  • the antigens of the present invention may be administered alone, it is preferred that they be administered as a pharmaceutical composition.
  • the compositions of the present invention comprise at least one antigen or its pharmaceutically acceptable salt, together with one or more acceptable carriers and optionally other therapeutic agents.
  • acceptable is meant that the agent or carrier be compatible with other ingredients of the composition and not injurious to the patient.
  • compositions include those suitable for oral, rectal, nasal, topical (including buccal and sublingual), vaginal, or parenteral administration.
  • the compositions conveniently may be presented in unit dosage form, and may be prepared by methods well known in the pharmaceutical arts. Such methods include bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients.
  • compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers, or both, and shaping the product formed thereby, if required.
  • Orally administered pharmaceutical compositions according to the present invention may be in any convenient form, including capsules, cachets, or tablets, each containing a predetermined amount of the active ingredient. Powders or granules also are possible, as well as solution or suspension in aqueous or nonaqueous liquids, or oil-in-water liquid emulsions, or water-in-oil liquid emulsions.
  • the active ingredient also may be presented as a bolus, electuary or paste.
  • a COS cell expression vector was constructed from piSV (Little et al., Mol. Biol. Med. 1:473-488 (1983)) by inserting a synthetic transcription unit between the suppressor tRNA gene and the SV40 origin.
  • the transcription unit consisted of a chimeric promoter composed of human cytomegalovirus AD169 immediately early enhancer sequences fused to the HIV LTR -67 to +80 sequences.
  • RNA was prepared from HPB-ALL cells by the guanidinium thiocyanate/CsCl method, as described above.
  • PolyA + RNA was prepared from total RNA by oligo dT selection. Maniatis et al. Molecular Cloning: A Laboratory Manual, supra.
  • cDNA was synthesized by the method of Gubler and Hoffman (Gene 25:263-269 (1982)).
  • BstXI adaptors were ligated to the cDNA, and the reaction products fractionated by centrifugation through a 5 ml-20% potassium acetate gradient containing 1 mM EDTA for 3 hours at 50k rpm in a SW55 rotor. 0.5 ml fractions were collected manually through a syringe needle or butterfly inserted just above the curve of the tube. Individual fractions were ethanolprecipitated after addition of linear polyacrylamide (Strauss and Varshavsky, Cell 37:869-901 (1984)) to 20 ug/ml. Fractions containing cDNA larger than 700bp were pooled and ligated to gradient purified BstXI digested piH3 vector.
  • the ligated DNA was transformed into E. coli MC1061/p3 made competent by the following protocol: The desired strain was streaked out on an LB plate. The next day a single colony was inoculated into 20 ml TYM broth (recipes below) in a 250 ml flask. The cells were grown to midlog phase (OD 600 about 0.2-0.8), poured into a 21 flask containing 100 ml TYM, and vigorously agitated until cells grew to 0.5-0.9 OD, then diluted again to 500 ml in the same vessel. When the cells grew to OD 600 0.6, the flask was placed in ice-water, and shaken gently to assure rapid cooling.
  • DNA was added, let sit on ice 15-30 minutes, and incubated at 37°C for 5 minutes (6 minutes for 0.5 ml aliquots). Thereafter the DNA-containing suspensions were diluted 1:10 in LB and grown for 90 minutes before plating or applying antibiotic selection. Alternatively, the heat-pulsed transformation mix was plated directly on antibiotic plates onto which a thin (4-5 ml) layer of antibiotic-free LB agar was poured just before plating.
  • Yeast Extract 0.1M NaCl, 10 mM MgSO 4 (can be added before autoclaving).
  • 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.
  • Bacteriological culture dishes (Falcon 1007) were prepared for panning by coating with an affinity purified sheep anti-mouse IgG antibody as described by Wysocki and Sato (Proc. Natl. Acad. Sci. USA 75:2844-2848 (1978)), except that dishes were washed with 0.15M NaCl from a wash bottle instead of PBS, and unreacted sites were blocked by overnight incubation in PBS containing 1 mg/ml BSA. Dishes were typically prepared in large batches and stored frozen, after aspiration of the PBS/BSA. In the first round of screening, 24 6 cm dishes of 50% confluent COS cells were transfected by protoplast fusion according to the method of Sandri-Goldrin et al., Mol.
  • COS cell clone M6 cells were propagated in Dulbecco's modified Eagle's medium supplemented with 10% calf serum and gentamycin sulfate at 15 ug/ml (DME/10% calf serum). Cells were split the day before transfection in 6 cm dishes at approximately 1:8 ratio from stock plates kept as dense as possible without overtly affronting the cells. T cell lines were grown in Iscove's modification of Dulbecco's medium (IMDM) containing gentamycin as above, and either NuSerum (Collaborative Research) or fetal bovine serum at 10%.
  • IMDM Iscove's modification of Dulbecco's medium
  • COS cells at 50% confluence in 6 cm dishes were transfected in a volume of 1.5 ml with a cocktail consisting of DME or IMDM medium containing 10% NuSerum (Collaborative Research), 400 ug/ml DEAE Dextran, 10uM chloroquine diphosphate, and 1 ug/ml DNA. After 4 hours at 37oC (or earlier if the cells appeared ill), the transfection mix was removed and the cells were treated with 10% DMSO in PBS for 2 minutes. Sussman and Milman, Cell Biol. 4: 1641-1643 (1984). Cells were then returned to DME/10% calf serum for 48 to 72 hours to allow expression.
  • T cells were labeled by lactoperoxidase treatment, lysed, and immunoprecipitated by the procedure of Clark and Einfeld (Leukocyte Typing II, Vol. II, pp. 155-167 (1986)), using commercially available goat anti-mouse IgG agarose beads (Cooper Biomedical).
  • COS cells were transfected by DEAE Dextran method and trypsinized and passed without dilution into new plates 24 hours after transfection. 36 hours later, cells were detached by exposure to PBS/EDTA as above, centrifuged, and labeled by the lactoperoxidate method.
  • a cleared lysate was prepared as for the T cell immunoprecipitations, except that the lysis buffer contained 1 mM PMSF, and incubation with the primary antibody was carried out for only 2 hours at 4°C. Eluted samples were fractionated on discontinuous 11.25% polyacrylamide gels using the buffer system of Laemmli (Nature 227:680-685 (1970)).
  • Northern blot analysis was carried out essentially as described (Maniatis et al., Molecular Cloning, a Laboratory Manual (1982)), except that DMSO was omitted from the loading buffer, denaturation was at 70°C for 5 minutes, and the gel contained 0.6% formaldehyde rather than 6%.
  • the gel was stained in two volumes of water containing 1 ug/ml ethidium bromide, photographed, and transferred to nylon (GeneScreen, DuPont) in the staining liquor.
  • the transferred RNA was irradiated by exposure to a germicidal lamp through Saran Wrap (Church and Gilbert, Proc. Natl. Acad. Sci.
  • Erythrocytes were prepared from whole blood by three centrifugations in PBS. COS cells were transfected in 6 cm dishes with CD2 or other surface antigen expression clones by the DEAE method. 48 to 72 hours posttransfection, the medium was aspirated and 2 ml of PBS/5% FDS/azide was added to each plate, followed by 0.4 ml of the appropriate erythrocyte samples as 20% suspensions in PBS. After 1 hour at room temperature, the nonadherent erythrocytes were gently washed off, and the plates examined.
  • a cDNA encoding CD2 antigen determinants was isolated in the following manner: cDNA was prepared from RNA extracted from the human T Cell tumor line HPB-ALL and inserted into the SV40 origin-based expression vector piH3 as described above. A cDNA library of approximately 3 ⁇ 10 5 recombinants was constructed, and the library was introduced into COS cells by protoplast fusion. Three days later the cells were detached by exposure to EDTA and treated with a pool of monoclonal antibodies, including three (OKT11, Leu5b, and Coulter T11) directed against CD2 determinants.
  • the antibody-treated cells were distributed into dishes coated with an affinity purified sheep anti-mouse IgG antibody, allowed to attach, and separated from the nonadherent cells by gentle washing. This method of enrichment is known in the immunological literature (Mage et al., J. Immunol. Methods 15:47-56 (1977).
  • the resulting colonies were pooled, fused into COS cells, and subjected to a second round of panning as before.
  • a portion of the detached cells was treated with a mixture of three monoclonal antibodies specific for CD2, and a Hirt supernatant was again generated and transformed into E. coli.
  • DNA was prepared from eight of the resulting colonies and transfected into COS cells. After three days, surface expression of the CD2 antigen was detected by indirect immunofluorescence in six of eight transfected dishes. Restriction enzyme digestion of the corresponding plasmid DNAs revealed a 1.5 kb insert in all six isolates.
  • the CD2 cDNA insert was subcloned into M13 mp19 (Vieira and Messing, Gene 19:259-268 (1982)) in both orientations, and the sequence determined by the dideoxynucleotide method ( Figure 2). Sanger et al., Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977). An open reading frame was observed to extend 360 residues from an ATG triplet satisfying the consensus criteria of Kozak (Microbiol. Rev.: 1-47:45 (1963)) for translational initiation codons ( Figure 1). The predicted amino acid sequence evokes an integral membrane protein with a single membrane spanning hydrophobic anchor terminating in a rather large intracytoplasmic domain.
  • a surprising and unexpected feature of this sequence is the presence of a potential N-linked glycosylation site just proximal to the proposed cleavage site.
  • the resulting polypeptide backbone has a predicted molecular weight of 38.9 kd divided into an external domain of mass 21.9 kb and a cytoplasmic domain of mass 14.6 kd. Three N-linked glycosylation sites are present in the extracellular domain.
  • the membrane spanning domain comprises 26 unchanged residues of predominantly hydrophobic character. In the nine residues immediately following are seven basic residues, either lysines or arginines. The appearance of predominantly hydrophobic residues followed by basic residues is a common organizational feature of transmembrane proteins bearing carboxyl-terminal anchors. Another surprising feature of the transmembrane domain is the appearance of a cys-gly-gly-gly, a beta turn motif (Chou and Fasman, Annual Review of Biochemistry 47:251-276 (1978)), flanked by hydrophobic residues (which are frequently found flanking beta turns).
  • the transmembrane segment of the CD2 antigen may contain a bend or kink.
  • the rather large size of the cytoplasmic domain leaves open the possibility that CD2 possesses an intrinsic enzymatic activity.
  • the cytoplasmic domain is very rich in prolines and contains three sites with high turn probability.
  • COS cells were transfected with the CD2 expression plasmid and surface labeled with 125 I by the lactoperoxidase method 60 hours post-transfection.
  • a cell lysate was prepared, and portions were incubated either with monoclonal anti-CD2 antibody (OKT11) or with an extraneous (OKT4; anti-CD4) antibody for 2 hours at 4°C.
  • Sepharosebound anti-mouse antibody was added, and after several washing steps, the adsorbed proteins were eluted and electrophoresed through a 11.25% acrylamide gel together with similarly prepared immunoprecipitates from phytohemagglutinin-activated T lymphocytes, the cDNA donor line HPB-ALL, or a long-term T cell line generated in this laboratory.
  • COS cells transfected with the CD2 expression clone were treated for 1 hour with purified MT910 (IgG, kappa) anti-CD2 antibody (Rieber et al., Leukocyte Typing II, Vol. I, pp. 233-242 (1986)) at a concentration of 1 ug/ml, or with purified MB40.5 (IgG1, kappa; Kawata et al., J. Exp. Med. 160:633-651 (1984)) antibody at the same concentration.
  • purified MT910 IgG, kappa
  • MB40.5 IgG1, kappa
  • Kawata et al. J. Exp. Med. 160:633-651 (1984)
  • MB40.5 recognizes a monomorphic HLA-ABC determinant and cross-reacts with African Green Monkey histocompatibility antigens; it was chosen because it represents an isotype-matched antibody recognizing a surface antigen of approximately the same abundance as the CD2 antigen expressed by transfected cells. Sheep erythrocyte rosettes were observed in the presence of MB40.5, but not of MT910. Rosette inhibition was also observed with OKT11 antibody, and not with various other control antibodies.
  • Transfected COS cells form rosettes with other animal erythrocytes
  • human T cells are known to form rosettes with horse, pig, dog, goat, and rabbit, but not mouse or rat erythrocytes.
  • Johansen et al. J. Allergy Clin. Immunol. 54:86-94 (1974); Amiot et al., in, A. Bernard et al., eds., Leucocyte Typing, Springer, publisher. New York, N.Y., pp. 281-293 (1984); Nalet and Fournier, Cell. Immunol. 96:126-136 (1985).
  • Transfected cells were exposed to human erythrocytes pretreated for 2 hours with either anti-LFA3 (IgG1, kappa) as ascites at 1:1000 dilution, or with a 10 ug/ml concentration of each of four isotype-matched nonagglutinating antibodies directed against human erythrocyte antigens as prevalent or more prevalent than LFA3:G10/B11 and D10, anti-K14 antigen, D6, anti-Wr b antigen; and F7/B9, anti-k antigen.
  • the erythrocytes were washed free of excess LFA3 antibody, but were allowed to form rosettes in the presence of the control antibodies to guard against possible loss of antibody blocking power by desorption. Rosette formation was observed in the presence of all four control antibodies, but not with erythrocytes pretreated with anti-LFA3.
  • a number of clones were isolated by the same expression technique used to clone CD2 and characterized to varying degrees by antibody reactivity, nucleic acid restriction and sequence analysis, and immunoprecipitation. Representative clones were transfected into COS cells and analyzed for ability to sustain rosette formation.
  • the CD1a, CD1b, CD1c, CD4 , CD5, CD6, CD6, CD8, and CD28 (Tp44) clones did not form rosettes with human erythrocytes.
  • RNA prepared from cell types expressing or lacking CD2 antigen were electrophoresed through denaturing agarose gels and transferred to nylon.
  • Hybridization of the transferred RNA with a strand selective probe (Hu and Messing, Gene 17:271-277 (1982)) prepared from an M13 clone containing a CD2 cDNA insert revealed the presence of prominent 1.65 and 1.3 kb transcripts present in RNA derived from thymocyte, activated T cell, and senescent T cell populations.
  • Lesser amounts were found in RNA extracted from the cDNA donor line, HPB-ALL and less still from MOLT4; barely detectable levels were recorded in RNA from the HSB-2 line.
  • RNA from Namalwa Bokitt lymphoma
  • U937 human lymphoma
  • HuT-78 HuT-78
  • PEER T cell leukemia
  • Jurkat clone J3R7 T cell leukemia
  • the pattern of reactivity conformed well with the known or measured pattern of expression of CD2 antigen, which was absent or indetectable on the Namalwa, *937, HuT-78, J3R7, PEER, and HSB-2 cell lines, weakly present on MOLT4, more strongly present on EPB-ALL, and most strongly present on activated T cells.
  • Thymocytes are also known to express high levels of CD2 antigen.
  • RNA from HPB-ALL and activated T cells was subjected to Northern blot analysis and hybridized either with a complete cDNA probe, or with a probe derived from the 3' portion of the cDNA distal to nucleotide 1131.
  • the latter probe reacted only with the 1.65 kb species, while the former showed the same reactivity pattern observed in Figure 5. This result is consistent with the suggested origin of the 1.3 kb transcript.
  • cDNAs encoding surface antigens can be isolated by the transient expression system of the present invention, in which COS cells transfected with cDNA libraries are allowed to attach to ("panned" on) antibody-coated plates. Plasmid DNA is recovered from cells adhering to the plates, transformed into E. coli. and the process is repeated, usually twice, to isolate the desired clone.
  • this approach cannot be used when the monoclonal antibodies used for panning recognize determinants on the untransfected cells. This appears to be the case for antiLFA3 monoclonal TS2/9.
  • a similar transient expression system based on polyoma virus replicationcompetent cells should allow almost all monoclonals to be used, since the probability of cross reaction between murine antibodies and murine cell surface determinants should usually be small.
  • a new expression vector, CDM8 ( Figure 3) was created from the COS cell vector piH3M described previously.
  • the new vector differs by the inclusion of a deleted version of a mutant polyoma virus early region selected for high efficiency expression in both murine and monkey cells, by the replacement of substantially all of the human immunodeficiency promoter region with the cognate sequences of the human cytomegalovirus immediate early promoter, and by inclusion of a bacteriophage T7 promoter between the eukaryotic promoter and the site of cDNA insertion. Expression in COS cells of chloramphenicol acetyltransferase by all of the vectors was equivalent.
  • a library of 1.9 ⁇ 10 6 recombinants having inserts greater than 0.8 kb in size was prepared in the CDM8 vector from a microgram of poly A+ RNA isolated from the human lymphoblastoid cell line JY.
  • the library was introduced into WOP cells (NIH 3T3 cells transfected with polyoma origin deletion DNA) by spheroplast fusion, and subjected to three rounds of panning and reintroduction into E. coli as described in Example I.
  • the amino acid sequence predicted from the nucleotide sequence of the LFA-3 clone was compared to the NBRF database, and no significant homologies were uncovered; the most significant scores were to the HIV envelope protein.
  • cysteine residues within the 200 residues comprising the presumed mature protein are 6 N-linked glycosylation sites, and 5 tandem serine or tandem threonine residues that frequently appear in O-linked glycosylated proteins. Ten cysteine residues appear in the complete sequence, 6 of which are distributed in the latter half of the mature protein, and one of which falls in the carboxy-terminal hydrophobic domain. Although esterification of cysteine thiols to fatty acids is a common occurrence in integral membrane proteins, and may play an alternate role in membrane anchoring of LFA-3, two examples are known of cysteine residues within or at the margin of the hydrophobic region of phosphatidylinositol linked proteins.
  • RNA and DNA blot hybridization analysis showed that the LFA-3 gene shares no closely related sequences in the genome, and encodes a single RNA species of about 1 kb in length. Cell lines that express large amounts of surface LFA-3 have greater amounts of LFA-3 RNA than those that express small or nondetectable amounts.
  • Radioimmunoprecipitation of the antigen expressed in transfected COS and murine cells shows a broad band of approximately 50 kd mean molecular mass, similar to that found in JY cells.
  • CD28 cDNA Antigen The previous examples illustrate the monoclonal antibody-based technique of the present invention for enrichment of cDNAs encoding surface antigens.
  • a method of constructing plasmid expression libraries is described which allows the enrichment technique to be fully exploited.
  • the method of the present invention for making plasmid expression libraries is of general use for expression cloning.
  • the antibody selection technique of the present invention has also been applied to isolate a cDNA clone encoding the CD28 antigen.
  • the antigen shares substantial homology with members of the immunoglobulin superfamily and forms a dimer structure on the surface of transfected COS cells similar to the dimer structure found on T lymphocytes.
  • Poly(A)+ RNA was prepared from the human T-cell tumor line HPB-ALL by oligo(dT) cellulose chromatography of total RNA isolated by the guanidinium thiocyanate method (Chirgwin, J.M. et al., Biochemistry 18:5294-5299 (1979)).
  • cDNA was prepared by a protocol based on the method of Gubler and Hoffman (Gubler, U. et al.. Gene 25:263-269 (1982)). 4 ug of mRNA was heated to approximately 100oC in a 1.5 ml centrifuge tube for 30 seconds, quenched on ice, and the volume adjusted to 70 ul with RNAse-free water.
  • RNAse free water 80 ul of buffer (0.1 M Tris pH 7.5, 25 mM MgCl 2 , 0.5 M KC1, 0.25 mg/ml BSA, and 50 mM DTT), 25 units of DNA Polymerase I (Boehringer), and 4 units of RNAse H (BRL).
  • buffer 0.1 M Tris pH 7.5, 25 mM MgCl 2 , 0.5 M KC1, 0.25 mg/ml BSA, and 50 mM DTT
  • 25 units of DNA Polymerase I Boehringer
  • 4 units of RNAse H BRL
  • Unpurified oligonucleotides having the sequence CTCTAAAG and CTTTAGAGCACA were dissolved at a concentration of 1 mg/ml, MgSO 4 was added to 10 mM, and the DNA precipitated by adding 5 volumes of EtOH. The pellet was rinsed with 70% ETOH and resuspended in TE at a concentration of 1 mg/ml.
  • 25 ul of the resuspended oligonucleotides were phosphorylated by the addition of 3 ul of buffer (0.5 M Tris pH 7.5, 10 mM ATP, 20 mM DTT, mM spermidine, 1 mg/ml BSA, and 10 mM MgCl 2 ) and 20 units of polynucleotide kinase followed by incubation at 37oC overnight.
  • buffer 0.5 M Tris pH 7.5, 10 mM ATP, 20 mM DTT, mM spermidine, 1 mg/ml BSA, and 10 mM MgCl 2
  • 3 ul of the 12-mer and 2 ul of the 8-mer phosphorylated oligonucleotides were added to the cDNA prepared as above in a 300 ul reaction mixture containing 6 mM Tris pH 7.5, 6 mM MgCl 2 , 5 mM NaCl, 0.35 mg/ml BSA, 7 mM mercaptoethanol, 0.1 mM ATP, 2 mM DTT, 1 mM spermidine and 400 units T4 DNA ligase (New England BioLabs) at 15° overnight.
  • the precipitate was washed with 70% ethanol, dried, and resuspended in 10 ul. 1 ul of the last 6 fractions was run on a gel to determine which fractions to pool, and material less than 1 kb in size was typically discarded. Remaining fractions were pooled and ligated to the vector.
  • the complete seguence and derivation of the vector is shown in Figure 5.
  • the vector was prepared for cloning by digestion with BstXI and fractionation on 5-20% potassium acetate gradients as described for the cDNA. The appropriate band was collected by syringe under 300 nm UV light and ethanol precipitated as above.
  • cDNA and vector were titrated in test ligations. Usually 1-2 ug of purified vector were used for the cDNA from 4 ug of poly A+ RNA.
  • the ligation reactions were composed as described for the adaptor addition above.
  • the ligation reactions were transformed into MC1061/p3 cells made competent as described above. The transformation efficiency for supercoiled vector was 3-5 ⁇ 10 8 colonies/ug. Recovery and characterization of the CD28 clone
  • Panning of the library was carried out as described herein above, using purified antibody 9.3 (DuPont) at a concentration of 1 ug/ml in the antibody cocktail.
  • the methods used for COS cell transfection, radioimmunoprecipitation, RNA and DNA blot hybridization, and DNA sequencing were all as described herein above.
  • a large plasmid cDNA library was constructed in a high efficiency expression vector containing an SV40 origin of replication.
  • a preferred version of the vector, containing an M13 origin, is shown in Figure 6.
  • Three features of the vector make it particularly suitable for this use: (i) the eukaryotic transcription unit allows high level expression in COS cells of coding sequences placed under its control; (ii) The small size and particular arrangement of sequences in the plasmid permit high level replication in COS cells; and (iii) the presence of two identical BstXI sites in inverted orientation and separated by a short replaceable fragment allows the use of an efficient oligonucleotide-based strategy to promote cDNA insertion in the vector.
  • the BstXI cleavage site creates a four base 3' extension which varies from site to site.
  • a vector was created in which two identical sites were placed in inverted orientation with respect to each other, and separated by a short replaceable segment of DNA. Digestion with BstXI followed by removal of the replaceable segment yielded a vector molecule capable of ligating to fragments having the same ends as the replaceable segment, but not to itself.
  • cDNA synthetic oligonucleotides were employed that give the same termini as the replaceable segment. The cDNA then could not ligate to itself, but could ligate to the vector. In this way, both cDNA and vector were used as efficiently as possible.
  • the library construction efficiencies observed according to the present invention between 0.5 and 2 ⁇ 10 6 recombinants per ug of mRNA, with less than 1% background and an insert size greater than 1 kb, compared favorably with those described for phage vectors lambda gt10 (7.5 X 10 5 /ug of mRNA) and lambda gt11 (1.5 X 10 6 /ug of mRNA) (Huynh, T., et al.. In: DNA Cloning Vol. I, A Practical Approach. Glover, D.M. (ed.), IRL Press, Oxford (1985), pp. 49-78); but the resulting clones were more convenient to manipulate.
  • Surface antigen CDNAs can be isolated from these libraries using the antibody enrichment method of the present invention.
  • the library is introduced into COS cells (for example, by spheroplast or protoplast fusion), where it replicates and expresses its inserts.
  • the cells are harvested by detaching without trypsin, treated with monoclonal antibodies specific for the surface antigens desired, and distributed in dishes coated with affinity purified antibody to mouse immunoglobulins. Cells expressing surface antigen adhere, and the remaining cells can be washed away. From the adherent cells, a Hirt fraction is prepared (Hirt, B., J. Molec. Biol. 26:365-369 (1967)), and the resulting DNA transformed back into E.
  • the CD28 cDNA was isolated from a library of about 3 X 10 5 recombinants prepared from cDNA from 0.8 ug of poly A + RNA using an earlier version of the protocol described in the Materials and Methods.
  • the library was screened for CD28 (and other surface antigen) cDNA clones by the method outlined above.
  • COS cells were panned with the 9.3 antibody alone.
  • a Hirt supernatant was prepared from the adherent cells and transformed into E. coli.
  • Plasmid DNA was isolated from eight colonies and transfected individually into COS cell cultures. The presence of the DC28 antigen was detected in three of eight transfected cultures by indirect immunofluorescence. All three plasmid DNAs contained an insert of about 1.5 kb.
  • the CD28 cDNA encodes a long open reading frame of 220 residues having the typical features of an integral membrane protein ( Figure 17). Removal of a predicted (von Heijne, Nucl. Acids Res. 14:4683-4690 (1986)) N-terminal signal sequence gives a mature protein of 202 residues comprising an extracellular domain with five potential N-linked glycosylation sites (Asn-X-Ser/Thr), a 27-amino acid hydrophobic membrane spanning domain, and a 41-amino acid cytoplasmic domain.
  • CD28 cDNA directs the production of a homodimer in transfected COS cells Immunoprecipitation of CD28 antigen from transfected
  • COS cells was carried out using the monoclonal antibody 9.3
  • the material obtained from COS cells migrated with a molecular weight of 74 kd under nonreducing conditions and 39 kd under reducing conditions, a pattern consistent with homodimer formation. Under the same conditions activated T cells give bands with molecular weights of 87 and 44 kd, and HPB-ALL cells give bands of 92 and 50 kd, under nonreducing and reducing conditions respectively.
  • the variation in molecular weight of the material obtained from different cell types arises as a result of differing glycosylation patterns characteristic of each type. Similar results were observed with other leukocyte surface antigens (Seed et al., Proc. Natl. Acad. Sci USA 87 (1987)).
  • the nucleotide sequence of the CD28 cDNA predicts a mature protein with molecule weight of 23 kd, much smaller than observed in these experiments, and probably attributable to utilization of the 5 N-linked glycosylation sites predicted by the amino acid sequence.
  • RNA blot analysis The nucleotide sequence of the CD28 cDNA predicts a mature protein with molecule weight of 23 kd, much smaller than observed in these experiments, and probably attributable to utilization of the 5 N-linked glycosylation sites predicted by the amino acid sequence.
  • RNA blot analysis Equal amounts of total RNA prepared from cell types expressing or lacking CD28 were subjected to RNA blot analysis as described hereinabove.
  • Four bands with molecular weights of 3.7, 3.5, 1.5, and 1.3 kb were visible in lanes containing RNA thymocytes, T blasts, senescent T cells, and the T cell leukemia cell lines PEER and HPB-ALL.
  • No bands were detected in lanes containing RNA prepared from the cell lines U937 (histiocytic leukemia), HuT-78 (Adult T cell leukemia), Jurkat (T cell leukemia), Namalwa (Burkitt lymphoma), MOLT4, and HSB-2, all of which do not express CD28.
  • the 1.5 kb transcript presumably corresponds to the isolated cDNA, and the 3.7 and 3.5 kb species reflect incomplete splicing or alternative polyadenylation site utilization.
  • the 1.3 kb transcript may terminate at an unconventional polyadenylation signal, since there is no obvious candidate in the sequence.
  • the CD28 gene is not rearranged DNA blot analysis (Seed et al., Proc. Natl. Acad. Sci USA 87 (1987)) of genomic DNA from placenta, peripheral blood lymphocytes, T cells, HeLa cells, or the tumor lines used in the RNA blot analysis above showed identical Dra 1 digest patterns indicating that rearrangement is not involved in the normal expression of the CD28 gene during development. Similarly, no gross genomic rearrangement underlies the failure of the examined T-cell tumor lines to express CD28 antigen. It may be inferred from the Dra 1 fragment pattern that the CD28 gene contains at least two introns.
  • the CD7 cluster of antibodies (Palker, et al., Leukocyte Typing II, Springer-verlag, New York, 303-313 (1985)) recognized a 40 kd glycoprotein (gp40) on the surface of peripheral blood T cells and thymocytes.
  • CD7 + T cells enhance immunoglobulin (Ig) synthesis by B cells (Miroshima et al., J. Immunol. 129:1091-1098 1982)), suppress B cell Ig synthesis when stimulated with Concanavalin A (Haynes et al.. Proc. Natl. Acad. Sci. U.S.A.
  • CD7 has been found to be the most reliable marker for the identification of T cell acute lymphoblastic leukemia (Link et al., Blood 62:722-728 (1983)). As such, studies have been carried out, in which cytotoxins coupled to the anti-CD7 antibody 3A1 were used to purge bone marrow prior to reinfusion to avoid early relapse in autologous bone marrow transplants or as prophylaxis against graft vs.
  • anti-CD7 antibodies also show promise as immunosuppressive agents in the treatment of allograft rejections (Raftery et al .. Transpl. Proc. 17:2737-2739 (1985)) which is in accord with the recent observation that the anti-CD7 antibody 7G5 significantly inhibits the primary mixed lymphocyte reaction (Lazarovits et al., Leukocyte Typing III. Oxford Univ. Press, Oxford (1987)).
  • CD7 tetanus toxoid
  • CD7 may be, or be part of, the T cell IgM receptor (Sandrin et al., Leukocyte Typing III. Oxford Univ. Press, Oxford (1987)), the ability of COS cells expressing CD7 to bind IgM or IgM immune complexes was evaluated. The results do not support the simple notion that CD7 itself is an IgM receptor.
  • Preparation of cDNA library and recovery and characterization of CD7 clones Preparation of an HPB-ALL cDNA library in the expression vector piH3 was carried out as described herein. Panning of the library was carried out according to the method of the present invention, using purified anti-CD7 antibody Leu9 (Becton Dickinson) and antibody 7G5 as ascites fluid was diluted 1/1000. Methods for cell transfection, radioimmunoprecipitation, DNA and RNA blot hybridization and DNA sequencing were all as described herein.
  • Human IgM, IgG, and IgA antibodies affinity purified FITC conjugated goat anti-human immunoglobulins antibodies (anti-Ig(G+M+A)), washed and preserved bovine red blood cells, and IgG and IgM fractions of rabbit anti-bovine red blood cell antibodies were purchased from Cooper Biomedical (Malverne, PA).
  • COS cells were transfected by the DEAE Dextran method with cDNAs encoding the CD7, CDw32, and CD28 surface antigens. 48 hours after transfection the cells were washed with PBS/0.5% BSA and incubated with either human IgM, IgG or IgA antibodies at a concentration of 1 ug/ml, at 4°C for 2 hours.
  • a 2% suspension of bovine erythrocytes was washed with PBS/0.5% BSA and treated with subagglutmating amounts of either IgG or the IgM fraction of rabbit anti-bovine erythrocyte antibodies at 4°C for 1 hour. Erythrocytes were then washed twice with PBS/0.5% BSA and adjusted to a 2% solution. 2 ml of antibody-coated erythrocytes were layered on 60 mm dishes containing COS cells which had been transfected 48 hours earlier with either CD7, CD32 or CD28 by the DEAE Dextran method. The dishes were then centrifuged at 150 X g at 4°C for 15 minutes.
  • Peripheral blood lymphocytes were obtained from heparinized blood by centrifugation at 4°C over a Ficoll-Hypaque gradient at 400 x g for 30 minutes. Leukocytes at the interface were washed two times with PBS. The leukocytes were adjusted to 10Y7 cells/ml in IMDM/10% Fetal Bovine Serum (FBS) and incubated in tissue culture dishes at 37oC for 30 minutes. Nonadherent cells were transferred to new dishes, and PHA was added to stimulate proliferation of T lymphocytes. On the next day the cells were washed with PBS and placed in fresh IMDM/10%FBS.
  • FBS Fetal Bovine Serum
  • Rosette assays were performed three days later. Cells were washed with PBS/0.5% BSA, and a 10 ul suspension of 2% Ig-coated erythrocytes prepared as described above was added to 10 ul of PBS/0.5% BSA containing 5 X 10 6 cells/ml. The mixtures were placed in Falcon round bottom 96 well plates and centrifuged at 150 X g for 15 min at 4oC. After an additional incubation of 45 min at 4oC pellets were resuspended with 10 ul of PBS/0.5% BSA, and the rosettes scored by phase contrast microscopy. The experiments were carried out in both the presence and absence of 0.1% sodium azide with no detectable difference.
  • a large plasmid library was constructed in the expression vector ⁇ 3M as describe hereinabove.
  • the library was introduced into COS cells by spheroplast fusion, and allowed to replicate and express its inserts.
  • the COS cells were harvested by detaching without trypsin 48 to 72 hours after transfection, treated with monoclonal antibodies specific for surface antigens believed to be encoded in the library, and distributed in dishes coated with affinity purified anti-mouse antibody as described herein. Under these conditions, cells expressing surface antigen adhere and the remaining cells can be washed way.
  • a Hirt (Hirt, J. Mol. Biol. 26:365-369 (1967)) fraction was prepared from adherent cells, and the resulting DNA transformed back into E. coli for further rounds of fusion and selection.
  • the detached cells were treated with a mixture of monoclonal antibodies specific for CD7 (765 and Leu9), and a Hirt supernatant was again generated and transformed into E. coli.
  • transformation of the DNA into E. coli 8 colonies were picked, and the plasmid DNA prepared from them by an alkaline miniprep procedure (Maniatis, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York (1982)).
  • DNA was prepared from 8 resulting colonies and transfected into COS cells. After 3 days, surface expression of the CD7 antigen was detected by indirect immunofluorescence in 7 of 8 transfected dishes. Restriction enzyme digest of the corresponding plasmid DNAs revealed two species. One contained a 1.2 kb insert, and the other a 1.3 kb insert.
  • the length of the cytoplasmic domain 39 residues, is in good agreement with the 30-40 amino acids predicted by protease digestion of the CD7 precursor in rough microsomal membrane fractions (Sutherland et al., J. Immunol. 133:327-333 (1984)).
  • CD7 contains a disulfide bond linking Cys 23 and Cys 89.
  • a second disulfide bond, linking Cys 10 and Cys 117, has been proposed, based on the structural similarity between CD7 and Thy-1.
  • the extracellular domains of both Thy-1 and CD7 have 4 cysteine residues, in roughly homologous positions.
  • Thy-1 The 4 cysteine residues of Thy-1 are joined in two internal disulfide bridges between Cys 9-111 and Cys 19-85 (Williams et al.. Science 216:696-703 (1982)).
  • Cys 111 forms an amide bond with the ethanolamine moiety of a substituted phosphatidylinositol, and is thus the last residue of the mature molecule (Tse et al., Science 230:1003-1008 (985)).
  • Cys 117 is followed by four repeats of a sequence whose consensus is Xaa-Pro-Pro-Xaa-Ala-Ser-Ala-Leu-Pro, and which, it is proposed, plays the role of a stalk projecting the V-like domain away from the surface of the cell.
  • the extracellular domain of CD7 has significant homology with both chains of the rat CD8 heterodimer (Johnson et al., Nature 323:74-76 (1986)), and the myelin P 0 protein (Lemke et al., Cell 40:501-508 (1985)).
  • CD7 directs the production of a 40 kd protein in transfected COS cells
  • Immunoprecipitation of CD7 antigen from transfected COS cells was carried out as described herein using monoclonal antibody 7G5 (Lazarovits et al., Leukocyte Typing III, Oxford Univ. Press, publisher, Oxford, England (1987).
  • the material obtained from COS cells migrated with as a broad band with molecular weight of 40 kd under reducing conditions.
  • HPB-ALL cells the cDNA donor line
  • activated T cells gave bands with molecular widths of 41 and 39 kd respectively.
  • RNA prepared from cell types expressing or lacking CD7 were subjected to Northern blot analysis as described herein.
  • a single 1.3 kb species was visible in lanes containing RNA from thymocytes, activated T cells, resting T cells, and the T cell leukemia lines HuT-78, HPB-ALL, Jurkat J3R7, HSB-2 and PEER. With the exception of the PEER cell line, none of the T cell tumors showed significant overexpression of CD7 transcripts.
  • CD7 RNA was detected in all of the thymus-derived cells, but not in RNA from U937 (histiocytic leukemia) and Namalwa (Burkitt Lymphoma) cells. No band corresponding to the 1.7 kb cDNA could be detected, suggesting that this species is artificially enriched during the cloning or library amplification process.
  • the CD7 gene is not rearranged
  • IgM binding to CD7-positive cells can be blocked by the anti-CD7 monoclonal antibody Huly-m2 (Thurlow et al., Transplantation 18:143-147 (1984)), and IgM columns bind a 37 kd protein from radiolabeled lysates of peripheral blood T lymphocytes (Sandrin et al., Leukocyte Typing III, Oxford Univ. Press, publisher, Oxford, England (1987)).
  • COS cells expressing CD7 were tested for their ability to bind IgM.
  • IgM receptor activity was assayed either by direct binding (Hardin et al., Proc. Natl. Acad. Sci. USA 76:912-914 (1979)) or by a rosette assay with ox erythrocytes coated with an IgM fraction of rabbit anti-bovine red cell serum as described by Ercolaniey al., J. Immunol. 127:2044-2051 (1981)).
  • Cells expressing CD7 neither bound human IgM nor formed rosettes with IgM-coated erythrocytes.
  • COS cells transfected with a cDNA encoding the human IgG receptor CDw32 bound IgG directly and formed rosettes with IgG-coated erythrocytes. Erythrocytes coated with IgM or IgG antibodies also adhered to a fraction of peripheral blood lymphocytes as reported (Moretta et al., Eur. J. Immunol. 5:565-569 (1975)). These results do not support the notion that the CD7 antigen is by itself an IgM receptor, although they do not exclude the possibility that COS cells suppress IgM binding activity in some manner, or that CD7 is part of, or modified to become, an IgM receptor.
  • CD7 is not by itself an IgM receptor is supported by the observation that a number of CD7 + T cell lines are FcRu-(Sandrin et al., Leukocyte Typing III, Oxford Univ. Press, publisher, Oxford, England (1987)).
  • the sequence of the isolated clone is most closely related to the murine beta 2 Fc receptor, but has diverged completely in the portion encoding the cytoplasmic domain.
  • the receptor expressed in COS cells shows a preference for IgG 1 among IgG subtypes, and no affinity for IgM, IgA or IgE.
  • cDNA libraries were prepared from tumor cell lines or from a human tumor and transfected into COS cells. After 48 hours, the cells were treated with mouse or human IgG antibodies, and allowed to settle on dishes coated with affinity-purified sheep antimouse IgG or goat anti-human IgG antibodies. After lysis, DNA recovery, and transformation in E. coli, the cycle was repeated for two more rounds. Although no positive clones were isolated from the tumor line libraries, a cDNA clone encoding an Fc receptor was isolated from a library prepared from a human adrenal tumor. It has been discovered that many tumors are heavily infiltrated by macrophages and lymphocytes. Thus, tumor RNA may be a productive source in general for transcripts of human macrophages.
  • Radioimmunoprecipitation of transfected COS cells with CDw32 antibodies showed the presence of a single 40 kd species, comparable in size to the antigen recognized on the surface of the myeloid CDw32 + line HL-60, and to the less abundant antigen present on the histiocytic leukemia line U937. This result reinforces the notion that the isolated receptor is CDw32, as the CD16 receptor is reported to be substantially larger (60-70 kd).
  • the nucleotide sequence of the isolated receptor ( Figure 9) is highly homologous to that of members of the recently isolated murine receptor family, and most closely related to the murine beta 2 receptor by nucleic acid homology.
  • the murine beta 2 receptor is found on T and B lymphocytes and macrophages, while the alpha receptor is restricted to macrophages; in the human system, CDw32 (shown here to be beta 2 -like) is restricted to macrophages while another Fc receptor (CD16) is found on lymphocytes and macrophages.
  • CDw32 shown here to be beta 2 -like
  • CD16 Fc receptor
  • junctions of the insertion site do not show obvious relationships to splice donor and acceptor sequences. Comparison of the human and murine peptide sequences showed that the peptide sequence diverges at the end of the transmembrane domain, before the nucleotide sequence diverges, suggesting the existence of a selective pressure favoring the creation of a differenct cytoplasmic domain.
  • RNA blot analysis showed that myeloid but not lymphocytic cell lines expressed RNA homologous to the
  • CDw32 probe DNA blot analysis showed multiple bands consistent with the existence of a small multigene family.
  • pan B cell antigen CD20 (B1, Bp35) plays an important role in B cell activation.
  • Monoclonal antibodies (mAb) to CD20 induce different cellular responses depending on the antibody used and the stage of differentiation or activation of the target B cells.
  • the monoclonal antibody 1F5 activates resting B cells by initiating the transition from the G 0 to the G 1 phase of the cell cycle, and induces dense tonsillar B cells to proliferate (Clark et al., Proc. Natl. Acad. Sci USA 82:1766 (1985); Clark and Shu, J. Immunol. 138:720 (1987)).
  • 1F5 does not induce an increase in cytoplasmic free calcium and does not induce circulating B cells to proliferate (Rabinovitch et al., In: Leukocyte Typing III (McMichael, Ed.), p. 435, Oxford University Press (1987)).
  • Other anti-CD20 mAbs, such as Bl have been shown to block B cell activation (Tedder et al., J. Immunol. 135:973 (1985)) and both 1F5 and B1 can inhibit B cell differentiation (Golay et al., J. Immunol. 135:3795
  • Poly (A) + RNA was prepared from the human Burkitt cell line Daudi by oligo (dT) cellulose chromatography of total
  • Anti CD20 mAbs 1F5, 2H7, B1, L27, G28-2, 93-1B3, B-C1, and NU-B2 were obtained from the International Leukocyte Typing Workshop (Valentine et al., In: Leukocyte Typing III (McMichael, Ed.), p. 440, Oxford University Press (1987)). Purified mAbs were used at a concentration of 1 ug/ml and ascites were used at a dilution of 1:1000. Panning was done according to the present method. In the first round of screening, eight 10 cm dishes of 50% confluent COS cells were transfected by the DEAE-Dextran method. Subsequent screening cycles were performed by spheroplast fusion.
  • Both cDNA dlones have the same coding sequence, and differ only in the 3' untranslated region.
  • the insert in clone CD20.6 has a short polyA tail and lacks a consensus polyadenylation signal, while the insert in CD20.4 lacks a polyA tail and extends 431bp beyond the 3' terminus in CD20.6 (Fig. 10A).
  • RNA blot analysis showed that three transcripts of 3.8, 3.0 and 1.5 kb were present in B cells but absent from other cell types, in agreement with the known pattern of antibody reactivity (Clark et al., Proc. Natl. Acad. Sci. USA 82:1766 (1985); Clark et al, J. Immunol. 138:720 (1987); Tedder et al., J. Immunol. 135:973 (1985); Golay et al., J. Immunol. 135:3795 (1985)). It appears likely that the CD20.6 clone is derived from the 1.5 kb transcript or possibly from an even shorter, undetectable species. Because the CD20.4 clone lacks a poly(A) + tail, its source cannot be inferred at present.
  • DNA blot analysis showed that the CD20 genomic sequences are not rearranged during development and are not amplified in the cell lines examined. A restriction fragment length polymorphism was observed in a DNA sample obtained from placenta.
  • the amino acid sequence predicted by the cDNA contains 297 residues and has a molecular mass of 33,097 daltons.
  • the sequence contains three major hydrophobic stretches involving residues 51-103, 117-141 and 183-203 (Fig.10).
  • Two other notable characteristics are the absence of an amino-terminal signal peptide and the presence of a highly charged carboxy-terminal domain.
  • a polyclonal anti-CD20 antibody that recognized the last 18 residues of the carboxy-terminus reacts with lysates of cells expressing CD20 but not with intact cells, suggesting that the CD20 carboxy terminus is located within the cytoplasm.
  • the amino-terminus is also intracellular, and that the first hydrophobic region acts as an internal membrane insertion signal (Zerial et al., EMBO J. 5: 1543 (1986)).
  • the first hydrophobic region is composed of 53 residues and is therefore long enough to span the membrane twice if organized as an alpha helix. Because there are two remaining hydrophobic regions, the intracellular localization of the carboxy-terminus requires that the first hydrophobic domain exit the membrane on the side.
  • the carboxy-terminal antibody may only recognize epitopes exposed by detergent treatment allowing the carboxy-terminus to be extracellular and forcing the first hydrophobic domain to exit the membrane on the extracellular side.
  • the sequence contains 2 potential N-glycosylation sites (Asn-Xaa-Ser/Thr, where Xaa cannot be Pro (Bause, Biochem. J. 209:331 (1983)) at positions 9 and 293, but neither of these is expected to be used if located in intracellular domains of the molecule.
  • the difference in molecular mass between CD20 expressed on COS cells and on B cells is therefore presumably due to O-linked glycosylation, although other forms of post-translational modification are not excluded. If the carboxy-terminus is intracellular, the only extracellular domain would lie between residues 142 and 182. This region is rich in serine and threonine residues which might support O-glycosylation.
  • LFA-1 antigen a major receptor of T cells, B cells and granulocytes (Rothlein, R., et al., Exp. Med. 163:1132-1149 (1987)), is involved in cytolytic conjugate formation, antibody-dependent killing by NK cells and granulocytes, and helper T cell interactions.
  • LFA-1 has been placed in the integrin family of cell surface receptors by virtue of the high sequence similarity between the LFA-1 and integrin beta chains (Kishimoto, T.K., et al., Cell 48:681-690 (1987); Hynes, R.O. Cell 41:549-554 (1987)).
  • the adhesion ligands of the integrin family are glycoproteins bearing the Arg-Gly-Asp (RGD) seguence motif, e.g., fibronectin, fibrinogen, vitronectin and von Willebrand factor (Ruoslahti, E., et al., Cell 44:517-518 (1987)).
  • ICAM-1 Intercellular Adhesion Molecule-1
  • a ligand for LFA-l Rosin, R., et al., J. Immunol. 137:1270-1275 (1986); Dustin, M.L., et al., J. Immunol. 137:245-254 (1986)
  • ICAM contains no RGD motifs, and instead is homologous to the neural cell adhesion molecule NCAM (Cunningham, B.A., et al. Science 236:799-806 (1987); Barthels, D., et al., EMBO J. 6:907-914 (1987)).
  • COS cells transfected with the ICAM cDNA clone bind myeloid cells by a specific interaction which can be blocked by monoclonal antibodies directed against either LFA-l or ICAM-1.
  • a cDNA library was constructed using RNA prepared from HL60 cells induced with phorbol myristyl acetate (PMA). The library was transfected into COS cells and cells expressing surface antigens were recovered according to the methods of the present invention by panning with the antiICAM monoclonal antibodies (mAbs) 8F5 and 84H10 (McMichael, A.J., et al., eds., Leukocyte Typing III. White Cell Differentiation Antigens. Oxford University Press (1987)). Episomal DNA was recovered from the panned cells and the expression-panning cycle repeated a further 2 times to obtain a cDNA clone designated pICAM-1.
  • mAbs monoclonal antibodies
  • COS cells transfected with pICAM-1 gave positive surface immunofluorescence reactions with three anti-ICAM-1 antibodies: 8F5; 84H10; and RR-1.
  • Immunoprecipitation of pICAM-1-transfected COS cells with the mAb 84H10 gave a band of molecular mass 100 kd. 30).
  • a slightly larger protein of 110 kd was precipitated from HL60 cells induced for 48 hours with either phorbol myristyl acetate (PMA), gamma-interferon (gammalFN), tumour necrosis factor (TNF), or interleukin-1 beta (IL-1 beta), but was absent from uninduced cells.
  • PMA phorbol myristyl acetate
  • gammalFN gamma-interferon
  • TNF tumour necrosis factor
  • IL-1 beta interleukin-1 beta
  • ICAM-1 expressed in COS cells The smaller molecular mass of ICAM-1 expressed in COS cells is consistent with the lower molecular masses observed for other surface antigens expressed in COS cells.
  • RNA blot analysis showed 2 species of 3.2 kb and 1.9 kb present in HL60 cells stimulated with either PMA, gamma IFN, TNF or IL-1 gamma, but absent in uninduced cells.
  • the expression of ICAM-1 is regulated by a number of cytokines, apparently at the level of transcription. Similar species were present in B cells (JY and Raji), T cells (Peer and T blasts) and Lymphokine Activated Killer cells (LAK).
  • the structure of these ICAM-1 transcripts and their relationship to the pICAM-1 cDNA remains to be established. Blot hybridization of genomic DNA from placenta revealed a pattern consistent with a single copy gene.
  • pICAM-1 encodes a functional cell adhesion molecule
  • COS cells expressing ICAM-1 were tested for their ability to bind HL60 cells. After 30 minutes at 37°C in the presence of Mg 2+ , HL60 cells strongly adhered to the ICAM-expressing COS cells, but not to mock transfected cells. The specificity of this adhesion was demonstrated by preincubating the ICAM-1 expressing COS cells with mAb 84H10. All HL60 binding was abolished under these conditions.
  • An isotype matched monoclonal antibody, W6/32 which recognizes a monomorphic HLA-ABC related determinant of approximately equal abundance to ICAM-1 on transfected COS cells, had no effect on the adhesion. Similarly, preincubation of the HL60 cells with either 84H10 or W6/32 did not inhibit binding.
  • LFA-1 was acting as the receptor for ICAM-1 in this system
  • HL60 cells were pretreated with antibodies against the beta chain of LFA-l (CD18 (McMichael, A.J., et al., eds., Leukocyte Typing III. White Cell Differentiation Antigens, Oxford University Press (1987))) and then subjected to the binding assay. All adhesion to ICAM-expressing COS cells was blocked. Pretreatment of COS cells with the CD18 antibodies had no effect on the adhesion. This provides direct evidence that ICAM-1 is indeed acting as an adhesion ligand for LFA-1.
  • CD18 McMichael, A.J., et al., eds., Leukocyte Typing III. White Cell Differentiation Antigens, Oxford University Press (1987)
  • the sequence of the pICAM-1 cDNA insert consists of 1846 nucleotides (Fig. 11).
  • the predicted peptide sequence of 532 residues has the typical features of a transmembrane protein including a putative signal sequence, which may be cleaved between glycine-25 and asparagine-26 (von Heijne, G., Nucl. Acids Res. 14:4683-4690 (1986)), and a single 25 residue membrane-spanning domain terminating in a short, highly charged cytoplasmic domain.
  • the extracellular domain contains seven potential N-linked glycosylation sites which could adequately explain the difference in size between the deglycosylated precursor (55 kd) and the final product (90-115 kd) (Dustin, M.L., et al., J. Immunol. 137:245-254 (1986)). Differential use of these putative glycosylation sites could also explain the heterogeneous molecular mass of ICAM-1 observed in different cell types (Dustin, M.L., et al., J. Immunol. 137:245-254 (1986)).
  • LFA-1 is a member of the integrin family of cell surface receptors (Kishimoto, T.K., et al., Cell 48:681-690 (1987); Hynes, R.O., Cell 48:549-554 (1987)).
  • the tripeptide motif Arg-Gly-Asp (RGD) is a common feature of the ligands for this family, e.g., fibronectin, fibrinogen, vitronectin and von Willebrand factor, and is crucial for ligand-receptor interaction (Ruoslahti, E, et al., Cell 44:517-518 (1967)).
  • ICAM-1 contains no RGD motifs, bearing instead a single RGE sequence at position 152.
  • NBRF National Biomedical Research Foundation
  • ICAM-1 may be divided into five Ig domains (28-112, 115-206, 217-310, 312-391, and 399-477) each of which shows significant similarity with other members of the Ig superfamily (Williams, A.F., Immunol. Today 8:298-3-3 (1987)).
  • domain I is similar to CD3 whilst domains IV and V are similar to domains of myelin associated glycoprotein (Arguint, M., et al.,, Proc. Natl. Acad. Sci.
  • ICAM-1 cDNA The availability of a functional ICAM-1 cDNA will allow a better assessment of the role of ICAM-l/LFA-1 mediated adhesion in antigen-specific leukocyte function, including T-cell mediated killing, T-helper responses and antibody-dependent cell mediated killing.
  • Example VIII Isolation and Molecular Cloning of the Human
  • the rapid immunoselection cloning method of the present invention was applied to isolate and clone the CD19, CD20, CDw32a, CDw32b, and CD40 antigens.
  • the nucleotide seguence of CD19 is shown in Figure 12.
  • the nucleotide sequence of CD20 is shown in Figure 13.
  • the nucleotide sequence of CDw32a is shown in Figure 15.
  • the nucleotide sequence of CDw32b is shown in Figure 16.
  • the nucleotide sequence of CD40 is shown in Figure 17.
  • a human placenta cDNA (Simmons and Seed (1988) Nature 333:568-570) was transferred into COS cells using DEAE-Dextran as a facilitator (Example I, supra). 48 hours posttransfeetion the cells were detached from the dishes without trypsin, incubated with monoclonal anti-CD36 antibodies 5F1 (Bernstein et al. (1982) J. Immunol. 128:876-881) (Andrews et al. (1984) J. Immunol. 121:398-404) and panned on dishes coated with goat anti-mouse immunoglobulin antibodies.
  • Nonadherent cells were removed by gentle washing, the adherent cells were lysed, and episomal plasmids recovered from the cells were purified and transformed into E. coli. After two similar rounds of enrichment following spheroplast fusion, plasmid DNAs recovered from 11 out of 12 randomly chosen colonies were found to direct the appearance of CD36 determinants in transfected COS cells.
  • the nucleotide sequence is given in Table 1.
  • Table 1 the nucleotide sequence numbering is shown in the left margin at the beginning of each line.
  • the deduced amino acid sequence is shown as single letter code underneath the beginning of each coding nucleotide triplet, with the initiation methionine indicated by the number 1 above the initiator codon.
  • the potential sites of N-linked glycosylaton in the derived amino acid sequence are underlined by a single dashed line.
  • the putative transmembrane domain is double-underlined.
  • CD36 protein was purified by immunoprecipitation. Since the rapid immunoselection cloning method depended upon expression of transfected COS cells, it follows that the same cell lines from which CD36 cDNA was cloned could also be used as a source of the expressed protein.
  • C32 melanoma cells, CD36 transfected cells COS cells, and CD25 (control) transfected COS cells were surface labelled with Na 125 I and lysed in a phosphate buffered saline solution containing 1 mM phenylmethylsulfonyl fluoride, 0.5% NP-40 and 0.1% sodium dodecyl sulfate.
  • Anti-CD36 monoclonal antibodies were added, and allowed to absorb to the lysate for 12 hours at 4°C, after which goat anti-mouse Immunoglobulin beads (Cappel) were added, mixed for two hours, and washed as described (Clark and Einfeld J. Immunol. 135:155-167 (1986)).
  • CD36 has been identified as a binding site for cytoadherence of Plasmodum falciparum parasitized erythrocytes, by the inventors herein and by Ockenhouse, C.D. et al. (1989) Science 243:1469-1741. Cytoadhesion of parasitized erythrocytes has been shown to be blocked by monoclonal antibodies to CD36.
  • cDNA libraries Three independent cDNA clones (designated p135, p90 and p98/X2) encoding human FcRI were isolated by the rapid immunoselection cloning method of the present invention from a cDNA library expressed in COS cells. (See also Allen, J.M. and Seed B., Science 243:378-381 (1989)).
  • the cDNA library was constructed from polyadenylated RNA obtained from cells of a single patient undergoing extracorporeal interleukin-2 induction therapy. Expression of the three cDNAs in COS cells gave rise to IgG binding of the appropriate affinity and subtype specificity. DNA sequence analysis revealed that the cDNAs encode similar type I integral membrane proteins with 3 extracellular immunoglobulin domains.
  • the intracellular domain of p98/X2 diverges from that of the other two cDNAs.
  • a composite sequence of the three cDNAs is shown in Table 2 with the nucleotide differences of the p89/X2 or p90 clones shown respectively below or above the pl35 sequence. Dashes denote gaps and no residues are shown above or below where the sequences are identical.
  • the p90 cDNA has the shortest 5' untranslated region, 7 additional residues between the polyadenylation motif and the poly A tract, and 2 polymorphisms in the coding region.
  • the p98/X2 cDNA has the longest 5' untranslated region, 1 polymorphism in the coding seguence, and diverges from the other two cDNAs at residue 1051, becoming a complex pattern of repeats of upstream sequences.
  • the p98/X2 clone lacks a polyadenylation site.
  • the FcRI protein from each of the three clones was purified from the respective COS cell lines which expressed them, by immuno-adsorption to IgG-agarose. (See Stengelin S. et al., EMBO J. 7:1053 (1988)). Gel electrophoresis of purified proteins showed a single species from pl35 and p90 COS cells, relative molecular size 70 kd. Cells transfected with p98/X2 expressed a protein of 67 kd. A slightly larger protein of 75 kd was adsorbed from untreated and interferon-gamma-treated U937 promonocyte cells.
  • the smaller mass observed in COS cells is consistent with the reduced masses observed from Table 2 other surface antigens expressed in COS cells, see e.g., Example IX.
  • the predicted polypeptide sequences show the typical features of a type I integral membrane protein, and include a short hydrophobic signal sequence, a single 21-residue hydrophobic membrane-spanning domain, and a short, highly charged cytoplasmic domain (Fig. 4).
  • the extracellular portion contains six potential N-linked glycosylation sites and six Cys residues distributed among three C2 set Igrelated domains.
  • FcRI is a high-affinity receptor for the Fc portion of IgG, normally located on the cell surfaces of macrophages.
  • the ability to interfere with such bonding, or to cause it to occur on surfaces other than macrophages, is useful in therapy.
  • a fusion protein of FcRI and a receptor ligand will be helpful to increase the potency of antibodies in therapy.
  • a cDNA clone encoding TLiSAl was obtained from a human T-cell cDNA library transferred into COS cell as described and subjected to the rapid immunoselection cloning method of the invention.
  • a monoclonal antibody ACT-T-SET TLiSA1 (T-Cell Sciences Corp., Cambridge, Massachusetts) was used to detect transfected COS cells expressing the cloned cDNA, by positive indirect immunofluorescence.
  • the positive plasmid contained in a 1.7 kb insert.
  • TLiSA protein was isolated by immunoprecipitation, as described supra, Example IX. The protein had a molecular weight of about 50 kd, as measured by gel electrophoresis.
  • the nucleotide sequence of the cDNA was determined by dideoxynucleotide chain termination as described, supra.
  • the sequence of 1714 residues is given in Table 3, together with the deduced amino acid sequence shown in single letter code under the first nucleotide of each coding triplet.
  • the ATG encoding the presumed initiator methionine is followed by a short hydrophobic region consistent with a secretory signal sequences, the most likely excision site being 19 residues into the open reading frame.
  • the resulting polypeptide if not further processed,would
  • TLiSA is involved in mediating IL-2 induced differentiation of T-cells into cytolytic forms. Antibodies to TLiSA are useful to prevent IL-2 stimulated T cell differentiation, and to modulate adverse effects of IL-2 in therapy.
  • an expression library was constructed from the Burkitt lymphoma cell line Daudi, introduced into COS cells by the DEAE-Dextran method described supra, and subjected to three rounds of panning and re-introduction into E. coli as described in Seed and Aruffo, Proc. Natl. Acad. Sci. USA 84: 3365-3369 (1987) and Aruffo and Seed, Proc Natl. Acad. Sci. USA 84: 8573-8577 (1987). Of 16 plasmids picked after the third round, two tested positive for CD22 expression by indirect immunofluorescence in COS cells. Of the five carbohydraterelated epitopes, A, B, C, D and E, recognized by anti-CD22 monoclonal antibodies, only epitopes A and D were expressed in COS cells.
  • RNA blot hybridization analysis has revealed the presence of a major 3 kb RNA species and 4 minor species of 2.6, 2.3, 2.0 and 1.5 kb in several B cell lines. RNA encoding CD22 has not been found in several T cell lines, including peripheral blood T cells, the T cell leukemia Jurkat, the myeloid leukemia lines HL60 and U937 and the hepatoma HepG2.
  • DNA blot hybridization of placental DNA gave a simple pattern consistent with a single copy gene.
  • DNA sequence analysis by the dideoxy method described supra showed that the 2107 bp insert encoded a polypeptide of 647 amino acids.
  • the nucleotide and amino acid sequences appear in Table 4.
  • the initial methionine is followed by 18 predominantly hydrophobic amino acids resembling a secretory signal sequence.
  • the mature protein, having a relative molecular weight of 71.1 kd consists of an extracellular portion of 491 residues, followed by a 19 residue membrane-spanning domain (doubly underlined), and an intracellular domain of 118 amino acids.
  • N-X-S/T Ten potential N-linked glycosylation sites (N-X-S/T, X not equal to P) are found in the predicted extracellular domain, as well as a large number of serine and threonine residues which may be sites of O-linked glycan addition.
  • the extracellular portion of CD22 consists of five segments having Ig-like domain organization.
  • the short intercysteine spacing (63 and 64 residues in domains 1 and 2, and 42 residues in domains 3-5) suggests that they fold into the 7 strand two layer beta-sheet structure characteristic of immunoglobulin constant regions rather than the 9 strand structure of variable regions.
  • CD22 has been found to be highly homologous to myelin associated glycoprotein (MAG), a neuronal cell surface protein which mediates cell-cell contacts during myelogenesis, it was postulated that CD22 has a role in B cell adhesion.
  • COS cells transfected with CD22 cDNA were contacted with erythrocytes or peripheral blood mononuclear cells and incubated under conditions which minimized nonspecific interaction. Erythrocyte and mononuclear cell resetting was observed with CD22-positive COS cells but not with COS cells transfected with an unrelated cDNA clone.
  • MAG myelin associated glycoprotein
  • B cell adhesion studies involving anti-epitope monoclonal antibodies have indicated that different epitopes of CD22 may participate in erythrocyte and monocyte adhesion and that different ligands may be recognized on each cell type.
  • B cell adhesion studies also suggest that CD22, in a manner analogous to T cell CD2, CD4 and CD8 adhesion to target cells, may promote recognition by the B cell antigen receptor by intensifying B cell-presenting cell contacts.
  • CD22 has been previously implicated in the transmission of signals synergizing with the antigen receptor (Pezutto et al., J. Immunol.
  • CD22 positive B cells The ability to interfere with the binding of CD22 positive B cells with accessory cells, or the ability to cause such binding to occur on surfaces other than lymphocyte cells can be useful in diagnostics and therapy.
  • a fusion protein of CD22 and a receptor ligand fixed to a substrate will be useful in detecting the presence of a particular antigen in body fluids.
  • a soluble form of CD22 can have immunomodulatory activity.
  • Example XIII The Isolation and Molecular Cloning of cDNA Encoding for T Lymphocyte-specific CD27 Antigen
  • the vector contained a 1.2 kb cDNA insert.
  • the nucleotide sequence of the cDNA was determined by dideoxynucleotide chain termination as described, supra.
  • the sequence of 1203 residues and the deduced amino acid sequence appear in Table 5.
  • the initiation methionine is indicated by the number 1 above the initiator codon.
  • the deduced CD27 polypeptide demonstrates the typical features of a type I integral membrane protein. It begins with a twenty amino acid hydrophobic region consistent with a secretory signal sequence. This hydrophobic region is followed by a 171 residue extracellular domain, a 20 residue hydrophobic membrane spanning domain (doubly underlined) and a 49 amino acid cytoplasmic domain beginning with a positively charged stop transfer sequence. There is no poly (A) tail.
  • CD27 amino acid sequence is highly homologous to the B lymphocyte and carcinoma antigen CD40, described supra, over its entire length. CD27 is also highly homologous to the receptor for nerve growth factor (NGFR) over the extracellular and transmembrane domains
  • CD27 is a T lymphocyte activation antigen. Its structure suggests that it may function as the receptor for a lymphokine or growth factor. The recognition of CD27 causes T cell proliferation and increased expression of certain genes needed for the helper and effector functions of the T cell. The expression of CD27 on T cells increases two to five fold with stimulation by phytohemagglutinin (PHA) or anti-CD3 monoclonal antibodies and the addition of at least one CD27 monoclonal antibody can augment PHA stimulated proliferation of T cells (Bigler and Chiorazzi, Leukocyte Typing II, Vol. I:503-512 (1986); Van Lier, (1987)).
  • PHA phytohemagglutinin
  • T cells positive for CD27 have been found to provide help to B cells for IgM synthesis and secrete 11-2 when appropriately stimulated (Van Lier et al., Eur. J. Immunol. 18:811-816 (1988)).
  • the ability to interfere with the binding of CD27 positive T cells with antigen presenting cells, or the ability to cause such binding to occur on surfaces other than lymphocyte cells, can be useful in diagnostics and therapy.
  • a fusion protein of CD27 and a receptor ligand fixed to a substrate will be useful in detecting the presence of a particular antigen in body fluids.
  • a soluble CD27 fusion protein will be useful to prevent undesired T cell proliferation, for example, in certain autoimmune diseases.
  • Example XIV The Isolation and Molecular Cloning of the
  • Two cDNA clones encoding Leu8 determinants were isolated from a human T cell library by the method of the present invention.
  • the nucleotide sequence of the cDNA was determined by dideoxynucleotide chain termination as described, supra.
  • the DNA sequence analyses (Table 6) shows that the longer insert of the two contains 2,350 residues, whereas the shorter lacks 436 internal residues but is otherwise identical.
  • the entire sequence of the longer clone is shown, with the portion deleted from the shorter clone overlined.
  • the predicted amino acid sequence is shown below the nucleotide sequence. Sites of potential N-linked glycosylation are designated --CHO-- and the proposed transmembrane region for the longer form is doubly underlined.
  • RNA blot hybridization revealed a major transcript of 2.4 kb in peripheral blood mononuclear cells, tonsillar B cells, and several lymphocytic cell lines; and a minor transcript of 2.0 kb, present in peripheral blood mononuclear cells, and the Jurkat and HSB-2 leukaemic T cell lines.
  • the deduced protein encoded by the larger insert bears a strongly hydrophobic putative membrane spanning domain near its C terminus, followed by several positively charged residues resembling a cytoplasmic anchor sequence.
  • the protein is closely related to the recently described murine Mel-14 homing receptor (Lasky et al., Cell 56:1045-1055 (1989); Siegelman et al .. Science 243:1165-1172 (1989)).
  • the protein encoded by the shorter insert bears a weakly hydrophobic C-terminal domain characteristic of surface proteins that are attached to the cell membrane by covalent linkage to a phosphatidylinositol-substituted glycan.
  • Leu8 is a homing receptor, allowing T cells to adhere to the specialized post-capillary endothelium of peripheral lymph nodes.
  • the presence or absence of Leu8 classifies the T cell in terms of homing potential and tissue distribution. Serological studies have indicated that Leu8 is a marker of resting lymphocytes in peripheral lymph nodes (Poletti et al., Hum. Pathol. 19:1001-1007 (1986)).
  • CD4 + Leu8 ⁇ cells have been found to directly inhibit pokeweed mitogen-induced IgG synthesis (Kanof et al., J. Immun. 139:49-54 (1967)). It therefore appears that CD4 + Leu8 ⁇ cells, activated to provide help for B cell Ig synthesis, exit the nodes and circulate peripherally to encounter antigen-presenting cells.
  • the ability to interfere with the binding of Leu8 ⁇ T cells to antigen presenting cells, or the ability to cause such binding to occur on surfaces other than lymphocyte cells can be useful in diagnostics and therapy.
  • the level of activated Leu8 ⁇ T cells relative to resting Leu8 + cells could serve as a measure of immune response to a particular antigen.
  • Leu8 transmembrane protein which mediates adhesion to specialized endothelial cells of lymph nodes, has been observed to be quite specific in its recognition of the lectin ligand, sulfated galactosyl ceramide (sulfatide). Modification of the specificity of this binding could serve to regulate the homing potential of resting T cells. Soluble forms of Leu8 can act as anti-inflammatory agents by reducing lymphocyte migration.
  • Example XV The Isolation and Molecular Cloning of cDNAs
  • CD44 is a polymorphic integral membrane protein. Immunochemical and RNA blot data have supported the existence of two forms of CD44: a mesenchymal form expressed by hematopoietic cells and an epithelial form weakly expressed by normal epithelium but highly expressed by carcinomas.
  • the nucleotide sequence of the hematopoietic CD44.5 cDNA (Table 7) consists of 1354 residues terminating in a short poly(A) tail 19 base pairs downstream from a CATAAA sequence.
  • the ATG encoding the first methionine is embedded in a consensus initiation sequence and followed by 19 predominantly hydrophobic residues resembling a secretory signal peptide sequence. Cleavage of this peptide would yield a mature protein of 341 residues with a predicted relative molecular mass of 37.2 kd.
  • the extracellular amino terminal domain of 248 residues is followed by 21 predominantly hydrophobic amino acids corresponding to the predicted transmembrane domain (doubly underlined) and a 72 residue hydrophilic (cytoplasmic) domain.
  • the discrepancy between the predicted mass of the protein backbone and the deglycosylated forms observed in immunoprecipitates suggest that extensive O-linked glycosylation is present.
  • the extracellular domain has six potential N-linked glycosylation sites, indicated in Table 7 by a --CHO-- designation, and is rich in serine and threonine residues (22% in aggregate).
  • the dipeptide SG that forms the minimal attachment site of serine-linked chondroitin sulfate in proteoglycan proteins appears at residues 160, 170, 211 and 238 in the predicted extracellular domain; these potential glycosylation sites are underlined.
  • RNA blot hybridization revealed three major messages of 1.6, 2.2 and 5.0 kb in a variety of hematopoietic cell lines, including the B lymphoblastoid line CESS, the T cell leukemias HUT-102 and HPB-ALL, lymphokine activated T cells, tonsillar B cells and the histiocytic lymphoma U937.
  • hematopoietic CD44 may play a role in lymphocyte homing. It has been shown that hematopoietic CD44 is an extracellular matrix receptor with affinity for collagens type I and VI (Stamenkovic et al., Cell 56: 1057-1062 (1989)). Hematopoietic CD44 may also have a lymphocyte activation role. The ability to interfere with the binding of hematopoietic CD44 to lymph node cells, or the ability to cause such binding to occur on other surfaces, can be useful in diagnostics and therapy. For example, modification of this binding can serve to regulate the homing potential of lymphocytes. Soluble forms of CD44 can have immunomodulatory activity.
  • a cDNA library prepared from the colon carcinoma line HT29 was transfected into COS cells by the DEAE-Dextran method described supra.
  • the cells were pooled 48 hours after transfection, incubated with anti-CD44 monoclonal antibody F-10-44-2 (Dalchau et al., Eur. J. Immunol. 10:745-749 (1980)) and panned on dishes coated with goat-anti-mouse affinity purified antibody. After several washes, the adherent cells were lysed, and episomal DNA purified and transformed into E. coli.
  • plasmid DNA recovered from seven out of ten randomly picked colonies was found to direct the appearance of epithelial CD44 determinants on transfected COS cells. All seven of the positive clones bore cDNA inserts of about 2.4 kb.
  • RNA blot analysis has revealed that the epithelial CD44 transcripts comprise 2.2, 2.7 and 5.5 kb species.
  • Epithelial CD44 isolated by immunoprecipitation has revealed that the glycoprotein is about 160 kd.
  • Transfected B cells expressing epithelial CD44 do not adhere to rat lymph node stromal cells in primary culture as do hematopoietic CD44 transfected lymphocytes.
  • the epithelial CD44 is weakly expressed by normal epithelium but highly expressed by carcinomas. It is possible that an extracellular matrix receptor function of epithelial CD44 may promote tumor invasiveness.
  • the ability to interfere with the binding of epithelial CD44 with extracellular matrices can be useful in therapy or diagnostics. For example, interference of the epithelial CD44 binding to extracellular matrices can diminish the likelihood of metastasis in cancer patients. Soluble forms of CD44 can act to prevent metastatic cells from "homing" to lymph nodes.
  • CD53 the antigen recognized by antibodies MEM-53
  • HI36 is a glycoprotein widely distributed among, but strictly restricted to, nucleated cells of the hematopoietic lineages (Stevanova, I., et al., (1969) in
  • CD53 is expressed by monocytes and macrophages, by granulocytes, dendritic cells, osteoclasts and osteoblasts, and by T and B cells from every stage of differentiation.
  • cDNA libraries constructed from peripheral blood lymphocytes and the promyelocytic tumor cell line HL60 were transfected into
  • COS cells by the DEAE-Dextran method, described supra.
  • the cells were pooled 48 hours after transfection, incubated with monoclonal antibodies MEM-53, and panned as described in Seed and Aruffo, Proc. Natl. Acad. Sci. USA 84: 3365-3369
  • lymphocytes and transfected COS cells were surface labeled with 125 I using lactoperoxidase and H 2 O 2 , and then lysed in a lysis buffer of 50 mM Tris-HCl pH 8.0 containing 1% NP40, 150 mM NaCl, 5 mM MgCl 2 , 5 mM KCl, 20 mM iodoacetamide and 1 mM phenylmethylsulfonyl- fluoride. Cells were solubilized at a concentration of 2.5 ⁇ 10 7 cells/ml in lysis buffer for 45 minutes then centrifuged at 12,000 g.
  • Immunoprecipitations were performed with monoclonal antibodies MEM53 or 63-5A3 and protein A-Sepharose CL-4B (Sigma, St. Louis, MO) as described by Schneider et al. (J. Biol. Chem. 257:10766 (1982)). Immunoprecipitates were eluted in SDS- sample buffer and analyzed on 12.5% acrylamide gels containing sodium dodecyl sulfate.
  • a broad band of radioiodinated protein ranging in mass from 34 kd to 42 kd was obtained from peripheral blood lymphocytes either with MEM-53 or 63-5A3 monoclonal antibodies, comparable to the band obtained from CD53-transfected COS cells, which extended from 36 kd to 46 kd.
  • the higher molecular mass in COS cells is atypical, as cell surface proteins recovered from transfected COS cells usually display unchanged or lower molecular mass than those found on the cell from which the cDNA clone originated (Aruffo and Seed, Proc. Natl. Acad. Sci. USA84:3365-3369 (1987)).
  • RNA blot analysis revealed a single 1.8 kb mRNA derived from B, T, and myeloid cell lines and from peripheral blood lymphocytes. The level of expression was comparable in the different cell lines except in THP1 cell line, which had little CD53 mRNA. CD53 transcripts are more abundant in peripheral blood lymphocytes than in cultivated cell lines, consistent with the higher surface expression of CD53 among these cells.
  • the nucleotide sequence of CD53 cDNA was determined by dideoxynucleotide chain termination as described, supra. using synthetic oligonucleotide primers.
  • the sequence of the CD53 insert consists of 1452 nucleotides (Table 9), and terminates close to two overlapping AATAAA motifs (singly underlined).
  • the 3' noncoding sequence contains three examples of the ATTTA sequence (indicated by quotation marks), which has been shown to mediate mRNA instability (Shaw and Kamen, Cell 46:659 (1986)).
  • An open reading frame beginning at residue 74 encodes a protein of 219 amino acids with a predicted molecular weight of 24,340 kd.
  • the predicted polypeptide is unusual in that it bears four major hydrophobic segments (doubly underlined), three of which fall in close proximity near the amino terminus of the molecule.
  • the first hydrophobic segment is atypically long for either a signal sequence or a simple transmembrane alpha helix, and contains three cysteine residues and a glycine located in the middle. Both cysteine and glycine have been found to immediately precede the signal cleavage site (von Heijne, Nucleic Acids Res. 14:4683 (1986)), suggesting that the amino terminus of the mature protein begins in the middle of the first hydrophobic domain.
  • the carboxyl terminus located between the third and fourth hydrophobic segments, the carboxyl terminus must lie inside the cell, as well as the short hydrophilic portion between the second and third hydrophobic segments. If the amino terminus is not processed, it must likewise remain intracellular.
  • CD53 is a Type III integral membrane protein related to three other membrane proteins: ME491 antigen, a melanoma protein whose increased expression correlates well with tumor progression (Hotta et al., Cancer Res. 48:2955 (1988)); CD37, an extensively glycosylated antigen predominantly expressed on B cells, but not B cell lineage specific (Schwartz et al., J. Immun. 140:905 (1988); and S5.7, an unglycosylated antigen broadly expressed on cells of hematopoietic lineage (Pressano et al., Cancer Res. 43:4812 (1983)).
  • ME491 antigen a melanoma protein whose increased expression correlates well with tumor progression
  • CD37 an extensively glycosylated antigen predominantly expressed on B cells, but not B cell lineage specific (Schwartz et al., J. Immun. 140:905 (1988); and S5.7, an unglycosylated antigen broadly expressed on cells of hematopoietic
  • coli lac Y permease a type III integral membrane protein which ferries lactose into the bacterial cell.
  • CD53 transcripts in peripheral blood lymphocytes increase in prevalence following mitogenic stimulation by PHA, suggesting that the protein may be involved in the transport of factors essential for cell proliferation.
  • CD53 Among the molecules with broad reactivity in the hemopoietic system, CD53 presently holds the widest reactivity as well as the strictest restriction to hematopoietic cells. Anti-CD53 antibodies are a useful tool for the identification of hematopoietic neoplasms, and may prove helpful for identifying morphologically poorly
  • defined cells for example in spleen or bone marrow primary cultures.

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  • Engineering & Computer Science (AREA)
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Abstract

On a découvert un procédé simple et hautement efficace de clonage d'ADNs complémentaires à partir de banques d'expression mammifère basées sur l'expression transitoire dans des cellules hôtes mammifères. L'invention concerne également de nouveaux vecteurs d'expression permettant l'élaboration hautement efficace de banques d'ADNs complémentaires mammifères. Le procédé de clonage de l'invention ayant été utilisé pour cloner des gènes destinés à des antigènes de surface de cellules de lymphocytes humains, présente une application générale dans le clonage de gènes. On a purifié des antigènes de surface de cellules clonées selon l'invention, et on a déterminé les séquences de nucléotides et d'acides aminés. Ces antigènes présentent une utilité diagnostique et thérapeutique dans des infections d'origine immunitaire chez des mammifères, y compris chez l'homme.
EP91916292A 1990-07-13 1991-07-15 Antigene de surface de cellule cd53 et son utilisation Ceased EP0551301A1 (fr)

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US55375990A 1990-07-13 1990-07-13
US553759 1990-07-13

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JP (3) JPH06504186A (fr)
KR (1) KR100206524B1 (fr)
AU (1) AU658370B2 (fr)
CA (1) CA2087272C (fr)
IE (1) IE912466A1 (fr)
PT (1) PT98326B (fr)
WO (1) WO1992001049A2 (fr)

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EP1006183A1 (fr) * 1998-12-03 2000-06-07 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Récepteurs Fc recombinantes et solubles
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AU8528691A (en) 1992-02-04
IE912466A1 (en) 1992-01-15
CA2087272A1 (fr) 1992-01-14
PT98326B (pt) 1999-01-29
AU658370B2 (en) 1995-04-13
JP2003038179A (ja) 2003-02-12
WO1992001049A2 (fr) 1992-01-23
WO1992001049A3 (fr) 1993-09-30
CA2087272C (fr) 2005-10-11
JPH06504186A (ja) 1994-05-19
KR100206524B1 (ko) 1999-07-01
JP2001157592A (ja) 2001-06-12
PT98326A (pt) 1992-06-30

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