CA1340869C - T cell activation markers - Google Patents
T cell activation markersInfo
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- CA1340869C CA1340869C CA000591285A CA591285A CA1340869C CA 1340869 C CA1340869 C CA 1340869C CA 000591285 A CA000591285 A CA 000591285A CA 591285 A CA591285 A CA 591285A CA 1340869 C CA1340869 C CA 1340869C
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- Prior art keywords
- ser
- val
- ala
- leu
- pro
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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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
- C07K14/52—Cytokines; Lymphokines; Interferons
Abstract
A polypeptide designated H400 and its encoding nucleic acid are provided as markers specific for activated human T cells. Activated T cells are detected immunochemically by monoclonal antibodies specific for H400 or for its immunogenic peptide fragments. Activated T cells are also detected by nucleic acid probes directed to messenger RNA encoding the H400 protein.
Description
T C'.ELL ACTIVATION MARKERS
Field of the Invention The invention relates generally to methods and compositions for monitoring the status of the immune system; more particularly, the invention provides compositions for detecting activated T-cells by nucleic acid hybridization probes and/or immunochemical assays.
BACKGROUND
Regulation of normal immunologic reactivity involves the '~~alance between positive and negative influences exerted by various subsets of T cells.
Abnormally hyperactive and hypoactive T cells are believed to be associated with several immune disorders, including AIDS (hypoactivity and destruction of the helper T cell subset), and a variety of autoimmune disorders, such as MHC-associated autoimmune disease (e. g. lupus erythem.atosus) which is believed to be caused by excessive production of gamma interferon by T cells.
T cells have been identified by certain phenotypic celLl surface markers. For example, the surface molecule T8, which is present on human T cells of the cytotoxic subset, can be detected by immunofluoresc;ent staining using murine anti-human monoclonal ant:ibodic~s, such as OKT8 (Ortho Diagnostics, Raritan, NJ) or Leu -2 (Becton-Dickinson, Mountain View, CA). However, such markers are, at best, only useful in measuring the size of subpopulations; they do not necessarily imply that the cells are secreting products associated with an activated state.
Many diagnostic assays have been developed which are based either on immunochemical detection of proteins: e.g., U.S. patents 4,562,003, 4,474,892, and 4,427,782; or on th.e detection of nucleic acids, either RNA or DNA, present in or produced by target cells:
e.g., Pettersson et al., Immunology Today, Vol. 6, pgs.
268-272.(1985), Falkow et al., U.S. Patent 4,358,535, and Gillespi.e e~t al., U.S. Patent 4,483.,92. Thewlatter assays use nucleic acid probes, usually fluorescently labeled or radioactively labeled DNAs or RNAs, which can be preferenti~~lly hybridized to complementary target nucleic acids in appropriately prepared samples or tissues. The assays can take a variety of forms: e.g.
RNA blotting: Thomas, Proc. Natl. Acad. Sci., Vol. 77, pgs. 5201-5205 (1980); dot hybridization: White et al., J. Biol. Chem _, Vol. 257, pgs. 8569-8572 (1982); Southern Blotting: Southern, J. Mol. Biol., Vol. 98, pgs. 503-517; and in situ hybridization: Pinkel et al., Proc.
Natl. Acad. S<:i., Vol. 83, pgs. 2934-2938 (1986); and Angerer et al.,, Gen~stic Engineering, Vol. 7 pgs. 43-65 (1985).
In view o:E the present lack of convenient direct methodic for measuring abnormal T cell activation, the availability of sensitive immunochemical assays or nucleic acid ~>robes for detecting activated T cells would provide useful. diagnostic tools.
SUt~It4ARY OF THE INVENTION
The invent: ion includes a mature human protein produced by activated T cells, designated herein as H400, ~.3~0869 and immuno~~enic :peptide fragments thereof, all of which are useful for generating antibodies employed in immunochem:ical assays for activated T cells. The , amino acid sequence corresponding to the open reading frame encoding H~400 is given in Formula I:
Lys - Leu Cys - Val Thr - - Leu - Ser - - Val -Leu - Leu Met - Leu Val - - Ala - Phe - - Ala -Cys - Ser Pro - Ala Leu - - Ala - Pro - - Ser -Met - Gly Ser - Asp Pro - - Thr - Ala - - Pro -Cys - Cys Phe - Ser Tyr Thr - Arg Glu - - - - -,. - , Ala - Ser Ser - Asn ~ Phe - - Val Asp - , , -Val Tyr - ~ Tyr Glu ~Thr Ser Ser - Leu Cys.
~- - '- - - -Ser - Gln Pro - Ala Val - - Phe - Gln - - Val -Thr - Lys Arg Ser Lys Gln Val Cys ~- - - - - - -Ala - Asp Pro Ser Glu Ser - Trp Val - - - - - -Gln - Glu Tyr Val Tyr Asp Leu Glu ~- - - - - - -Leu - Asn .
Formula I
The present invention relates to a process for producing the po7_ypeptide of Formula I which comprises at least ome of t:ransfecting and/transforming a cell or bacterium with ~an expression vector containing a DNA sequence which encodes said polypeptide;
Culturing said cell or bacterium under conditions wherein the polypeptide is expressed by said cell or bacterium; and iso:Lating said polypeptide.
The invention further includes monoclonal antibodies specific for the mature H400 protein and its immunogenic ~>eptide fragments, and nucleic acids encoding H400 and peptide fragments thereof useful in' the construction of nucleic acid probes for H400 messenger RNA (mF;NA). The preferred nucleotide ~~~o~s~
- 3a -sequence oi: the open reading frame encoding Fi400 is given in Formula II:
ATG - AAG CTC TGC GTG ACT GTC CTG -- - - - - -TCT - CTC - CTC - ATG - CTA - GTA - GCT - GCC -TTC - TGC TCT CCA GCG CTC TCA GCA -- - - - - -CCA - ATG - GGC - TCA - GAC - CCT - CCC - ACC -GCC - TGC TGC TTT TCT TAC ACC CGA -- - - - - -GAA - GCT - TCC - TCG - AAC - TTT - GTG - GTA -GAT - TAC TAT GAG ACC AGC AGC CTC -- - - - - -".~..
;t. .a ;--.,:~
.~3~~~~g TGC - TCt~- CAG - CCA GCT GTG - GTA - TTC
- - -CAA ACC - AA,A- AGA AGC AAG - CAA GTC
- - - - -TGT GC'.~ - GA'T- CCC AGT GAA - TCC - TGG
- - - -GTC CA(s - GAG TAC GTG TAT GAC CTG
- - - - - - -GAA CT(i - AAC - ( - TGA
) .
Formula II
As used herein, the term "mature" in reference to protein H9E00 means the secreted form of H400, that is, the form of Ft400 that results from proteolytic cleavage of the signal peptide.
As,used herein, "immunogenic peptide" in.
reference to H4Q0' means a peptide ~ ( T) which cons~ists~ bf from 6 to 40 amino acids having a sequence identical to an equally long portion of mature H400 and (2) which in conjugation with a carrier protein is capable of eliciting antibodies which cross-react with mature H400.
As used herein, "nucleic acid fragment" means a nucleic acid which consists of from about 18 to 264 nucleotides having a sequence complementary to an equal length portion of the nucleic acid defined by Formula II.
A plasmid, designated herein as pcD(SRa)-H400, containing a ~~DNA insert capable of encoding H400 has been deposited (in its E. coli JM101 host) with the American Type Culture Collection (Rockville, MD) under accession number 67614.
Brief Description of the Drawings Figure 1 diagrammatically illustrates the plasmid pcD(SR a)-H4~00 and selected restriction sites.
Figure 2 Lists the nucleotide sequence of the cDNA insert of pcD(SRa)-H400 and indicates the amino acid sequence corre~spond:ing to the largest open reading frame.
~3~0~6~
_5_ DE'.~AILED DESCRIPTION OF THE INVENTION
ThE~ invention is based on the discovery of a novel protein, H400, which is produced specifically by activated T c:ells. The protein H400 is encoded by the largest open reading frame of the cDNA insert of pcD(SRa)-H400. ThE~ invention is directed to compounds useful in det.ectinc~ cells which either produce the H400 protein or produce mRNA transcripts capable of encoding the H400 protein. These compounds include the mature protein H400, immunogenic peptide fragments thereof, monoclonal antibodies specific for mature H400 or its immunogenic,peptide~ fragments, nucleic.acids capable of encoding H400, and,nucle.ic acid fragments fo,r constructing nucleic acid probes specific for mRNA that encodes H400.
I. Production of Mature H400 H400 can be produced by expressing a nucleotide sequence encoding it in a suitable expression system.
Possible types of host cells include, but are not limited to, bacterial, yeast, insect, mammalian, and the like.
Selecting an expression system, and optimizing protein production thereby, involves the consideration and balancing of many factors, including (1) the nature of the protein to be expressed, e.g. the protein may be poisonous to :;ome host organisms, it may be susceptible to degradation by host proteases, or it may be expressed in inactive conformations or in insoluble form in some hosts, ( 2) the nature of the messenger RNA (mRNA) corresponding to this protein of interest, e.g. the mRNA
may have sequences particularly susceptible to host endonucleases,. which drastically reduce the functional lifetime of the mRNA, or the mRNA may form secondary structures that mask the start codon or ribosome binding site, thereby inhibiting initiation of translation in some hosts, (3) the selection, availability, and arrangement of hosl:-compatible expression control sequences in the 3"- and 5'-regions flanking the coding region -- these inc:lude promoters, 5'- and 3'- protector sequences, ribosome binding sites, transcription terminators, enhanc;ers, polyadenylate addition sites, cap sites, intron-splice sites, and the like, (4) whether the protein has a ~~ecretion signal sequence which can be processed by the host, or whether an expression control sequence encoding a. signal sequence endogenous to the host must be spliced onto the region encoding the mature . protein, (5) the a.vailable~modes and efficiencies of transfection or transformation.of the host, and whether transient or stable expression is desired, (6) the scale and cost of the host culture system desired for expressing th~~ protein, (7) whether, and what type of, posttranslati~~nal modifications are desired, e.g. the extent and ki~~nd of glycosylation desired may affect the choice of host, ( 8 ) the ease with which the expressed protein can be separated from proteins and other materials of the hose cells and/or culture medium, e.g.
in some cases it ma:y be desirable to express a fusion protein with ~~ specialized signal sequence to aid in later purification steps (e. g. Sassenfeld et al., Biotechnology,, January 1984), (9) the stability and copy number of a p<~rticular vector in a selected host, e.g.
Hofschneider Eat al., eds. Gene Cloning in Organisms Other than E. Coli I;Springer Verlag, Berlin, 1982), and (10) similar factors known to those skilled in the art.
Mane revi<~ws are available which provide guidance for making choices and/or modifications of specific expression systems in light of the recited factors: e.g. de Boer and Shepard, "Strategies for Optimizing Foreign Gene Expression in Escherichia coli", pgs. 205-247, in Kroon, ed., Genes: Structure and ~.~44~~~
_, _ Expression (~iohn W:iley & Sons, New York, 1983), review several E. coli expression systems; Kucherlapati et al., Critical Reviews in Biochemistry, Vol. 16, Issue 4, pgs.
349-379 (198~E), and Banerji et al., Genetic Engineering, Vol. 5, pgs. 19-31 (1983) review methods for transfecting and transforming mammalian cells; Reznikoff and Gold, eds., Maximizing Gene Expression (Butterworths, Boston, 1986) review selected topics in gene expression in _E.
coli, yeast, and mammalian cells; and Thilly, Mammalian Cell Technolo~r (Butterworths, Boston, 1986) reviews mammalian expression systems.
Likewise, many reviews are available which .describe techniques and conditions .for ~linki.ng and/or manipulating specific cDNAs and expression control ,sequences to create and/or modify expression vectors suitable for use with the present invention: e.g.
Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, N.Y., 1982); Glover, DNA
Cloning: A Practical Approach, Vol. I and II (IRL Press, Oxford, 1985); and Perbal, A Practical Guide to Molecular Cloning (John Wiley & Sons, N.Y., 1984). Generally, within an expression vector various sites may be selected for insertion of the cDNA of the invention. These sites are usually designated by the restriction endonuclease which cuts them and are well recognized by those of skill in the art. Various methods for inserting DNA sequences into these sii:es to form recombinant DNA molecules are also well known. These include, for example, dG-dC or dA-dT tailing" direct ligation, synthetic linkers, exonuclease and pol:ymerase-linked repair reactions followed by 1~_gation, or extension of the DNA strand with DNA polymerase and an appropriate single-stranded template followed by ligation.
Often a vE~ctor containing the cDNA of the invention must: be obtained in large quantities before 13~~~6~
_8_ transfection and/o:r transformation of cells in a host culture can hake place. For this purpose the vector is often replicated without significant expression in an organism (the cloning host) other than the one ffinally used for expression. In such cases, after propagation, the vectors acre separated from the cloning host using standard techniques, e.g. as disclosed by Maniatis et al.
(cited above).
Preferably H400 is produced by expressing the H400 cDNA carried by pcD(SRa)-H400 'in a host cell suitable for pcD vectors, e.g. COS monkey cells .. (available from the ATCC under accession numbers CRL 1650 ., or CRL~16fi1,),-C127.mouse mammary tumor cell (available from ATCC under accession number CRL 1616), or mouse L
cells. Eukaryotic hosts are capable of cleaving the signal peptide from the freshly translated H400 polypeptide to form the mature H400 polypeptide.
Eukaryotic hosts are also capable of producing other posttranslati~an modifications, such as glycosylation, which may be ,~ntigenically important.
Production of H400 in a bacterial expression system requires that the cleavage site of the signal peptide be dei_ermined, either for direct expression of a nucleic acid encoding the mature H400 polypeptide or for linking such <~ nucleic acid to a bacterial sequence encoding an endogenous signal peptide. Empirical rules have been developed for making such predications with a high degree of: accuracy: e.g. von Heijne, Eur. J.
Biochem., Vol" 133, pgs. 17-21 (1983); J. Mol. Biol., Vol. 184, pgs. 99-105 (1985); Nucleic Acids Research, Vol. 14, pgs. 4683-4690 (1986); and Perlman et al., _J.
Mol. Biol., Vol. 16'1, pgs. 391-409 (1983). von Heijne's rules indicate that mature H400 begins with the alanine of position 23 with respect to the td-terminal amino acid of Formula I; the predicted mature sequence is illustrated below in Formula III.
~~~~86~
_g_ Mature H400 is produced as follows by COS7 cells transiently itransfected with pcD(SRa)-H400. One day prior to transiEection, approximately 106 COS 7 monkey cells are seeded onto individual 100 mm plates in Dulbecco's me>dif ie<9 Eagle's medium (DME ) containing 10$
fetal calf serum and 2 mM glutamine. To perform the transfection, the rnedium is aspirated from each plate and replaced with 4 ml of DME containing 50 mM Tris.HCl pH
7.4, 400 ug/ml DEAF;-Dextran and 50 yg of the plasmid DNAs to be tested. The plates are incubated for four hours at 37°C, then the DNA--containing medium is removed, and the plates are washed twice with 5 ml of serum-free DM F;. DME
is added balk to the plates which ~ are then incubated .for an additional 3 hrs at 37°C. The plates are washed once with DME, and then DME containing 4~ fetal calf serum, 2 mM glutamine, penicillin and streptomycin is added. The cells are then incubated for 72 hrs at 37°C. The growth medium is collected and H400 purified therefrom using standard methods.
II. Immunogenic Peptides of H400 The present invention includes peptides derived from mature H400 which form immunogens when conjugated with a carrier. The term immunogen as used herein refers to a substance which is capable of causing an immune response. Th~~ term carrier as used herein refers to any substance whi~~h when chemically conjugated to a peptide of the invention permits a host organism immunized with the resulting conjugate to generate antibodies specific for the conjugated peptide. Carriers include red blood cells, bacter:iophages, proteins, and synthetic particles such as agarose beads. Preferably carriers are proteins, such as serum albumin, gamma-globulin, keyhole limpet hemocyanin, thyroglobulin, ovalbumin, fibrinogen, or the like.
~3~0869 Peptides of the invention are defined in terms of their positions within the following amino acid sequence of mature H400:
( Ala - Pro - r4et- Gly Ser - Asp - Pro - Pro ) (9) Thr - Ala - Cys Cys Phe - Ser - Tyr Thr - - - -( Arch - Glu - Ala - Ser er - Asn - Phe - Val ) (25) Val. - Asp - Tyr Tyr Glu - Thr Ser Ser - - - - -(33) Leu - Cys - Ser - Gln Pro - Ala - Val - Val - -( Phe~ - Gln Thr Lys Arg Ser Lys Gln ) (49) Val. - Cy:a- Ala Asp Pro - Ser - Glu Ser - - - -( Tr~~ Val. Gln Glu Tyr Val, Tyr Asp 57.) - - - - - - - -(65) Leu. - Glu ~ Leu Asn. .
.-Formula III
Amino acids of Formula III are numbered with respect to the N-terminus of the mature H400 polypeptide. Thus, SerS is serine at position 5.
Peptides are def ine~d as fragments of the mature H400 polypeptide. Thus, the peptide designated herein by SerS - Cysll is the peptide Ser-Asp-Pro-Pro-Thr-Ala-Cys.
Groups of peptides of the invention are also deffined in terms of fragments of the mature H400 polypeptide. Thus, the group designated herein as [SerS - Cys11~5-6 consists of all 5- and 6- amino acid peptides (i.e. all 5-mers and 6-mers) whose sequences are identical to all or part of the peptide SerS - Cysll. That is, the group consists of the following five peptides: Ser-Asp-Pro-Pro-Thr-Ala, Asp-Pro-Pro-T:hr-Ala-Cys, Ser-Asp-Pro-Pro-Thr, Asp-Pro-Pro-Thr-Ala, ~~nd Pro-Pro-Thr-Ala-Cys.
Generally, the invention includes all 6-mer to 24-mer peptidE~ fragments of mature H400, i.e.
[Alai - Asn6816-24' Preferably, the invention includes 6-mer to 24-mE:r peptide fragments which include the N-terminal or C-terminal sequences of the mature H400 l3~os~~
polypeptide, or which correspond to sequences of the mature H400 poly,pepticle that have higher-than-average hydrophilicity, as determined by Hopp-Woods analysis or related analysis technique, e.g. Hopp and Woods, Proc.
Natl. Acad. Sci., Vol. 78, pgs. 3824-3828 (1981); or Kyte and Doolittle, J. Mol. Biol., Vol. 157, pgs. 105-132 ( 1982) . The latter set of preferred peptides of the invention (those having higher-than-average hydrophilicity) includes the following groups:
[Alai - Ser20J6-20 and [Thr43 - Asn6816-24' Standard symbols are used throughout for amino acids: e.g. Cohen, "Nomenclature and Symbolism of.alpha-Amino Acids':, Mgt:liods in Enzymology, ~Vol. 106, pgs. 3-17 (Academic Press, N.Y., 1984).
Peptides of the invention can be synthesized by standard techniques, E~.g. Stewart and Young, Solid Phase Pep tide Synthesis, 2nc! Ed. (Pierce Chemical Company, Rockford, IL, 1984). Preferably a commercial automated synthesizer is used, e.g. Vega Biochemicals (Tucson, AZ) model 296A or B, or Applied Biosystems, Inc. (Foster Ci-ty, CA) model 430A.
Peptides of the invention can be assembled by solid-phase synthesis on a cross-linked polystyrene support starting from the carboxyl terminal residue and adding amino acids in a stepwise fashion until the entire chain has been formed. We performed the synthesis on a fully automated peptide synthesizer (Applied Biosystems, Inc. model 430A) . Thc: following references are guides to the chemistry employed during synthesis: Plerrifield, J.
Amer. Chem. Soc., Vol. 85, pg. 2149 (1963); Kent et al., pg. 185, in Peptides 1984, Ragnarsson, Ed. (Almquist and Weksell, Stockholm, 1'984); Kent et al., pg. 217 in Peptide Chemistry 84, Izumiya, Ed. (Protein Research Foundation, B.H. Osaka, 1985); Merrifield, Science, Vol.
232, pgs. 341-347 (1986).
In solid-state synthesis it is most important to eliminate synthetic by-products, which are primarily termination, deletion, or modification peptides. Most side reactions can be eliminated or minimized by use of clean, well characterized resins, clean amino acid derivatives, clean solvents, and the selection of proper coupling and cleavage methods and reaction conditions:
e.g. Barany and ~9errifield, The Peptides, Cross and Meienhoter, Eds.,.~Vol. 2, pgs.~ 1-284 (Academic Press, New Xork, 1979). It is important to monitor coupling reactions ~ to .detE~rmine that they proceed to 'completion so that deletion peptides lacking one or more residues will be avoided. The quantitative ninhydrin reaction is useful for that purpose, Sarin et al. Anal. Biochem., Vol. 117, pg. 14'1 (1981). Na-t-butyloxycarbonyl-amino acids were used with appropriate side chain protecting groups stable to the conditions of chain assembly but labile to strong acids. After assembly of the protected peptide chain, the protecting groups were removed arid the peptide anchorinca-bond was cleaved by the use of low and then high concenl_ratio~ns of anhydrous hydrogen fluoride in the presence of a t,hioester scavenger: Tam et al., J.
Amer. Chem. Soc., Vol. 105, pg. 6442 (1983).
Side chain protecting groups used were Asp(OBzl), Glu(OBzl), Ser(Bzl), Thr(Bzl), Lys(C1-Z), Tyr ( Br-Z ) , Arg ( N~'Tos ) , Cys ( 4-hleBz 1 ) , and H is ( ImUNP ) .
(Bzl - benzyl: T~~s - t;oluenesulfonyl; DNP -dinitrophenyl; Im = imidazole; Z - benzyloxycarbonyl.) The remaining amino acids had no protecting groups on their side chains. Al.l the amino acids were obtained from Peninsula Laboratories, except the tBoc-His(ImDNP), which was from Chemical Dynamics and was recrystallized from ethanol before use. [tBoc - N a-t-butyloxy-~3~0~~6~
carbonyl.] E'or each cycle the tBoc-Na-protected peptide-resin was exposed i~o 65 percent trifluoroacetic acid ( from Eastman Kodak ) (distilled before use ) in dichloromethame (D(:M) (Mallenckrodt): first for 1 minute and then for 13 minutes to remove the Na-protecting group. The p~eptide~-resin was washed with DCM and then neutralized twice with 10 percent diisopropylethylamine (DIEA) (Aldrich) in dimethylformamide (DMF) (Applied Biosystems), for 1 minute each. Neutralization was followed by washings with DMF. Coupling was performed with the preformed symmetric anhydride of the amino acid ._ in DMF for 16 minutes. T.he performed symmetric anhydride was preparad on the~_. synthesizer by dissvlving~ .2 mmol of amino acid in 6 ml of DCM and adding 1 mmol of ,dicyclohexycarbodiimide (Aldrich) in 2 ml of DCM. After minutes, the activated amino acid was transferred to a separate vessel and the DCM was evaporated off by purging with a continuous stream of nitrogen gas. The DCM was replaced by DI~IF (6 ml total) at various stages during the purging. After the first coupling, the peptide-resin was washed with D~~M, 10 percent DIEA in DCM, and then with DCM. For rec~~upling, the same amino acid and the activating agent, dicyclohexylcarbodiimide, were transferred sequentially to the reaction vessel. After activation in situ and coupling for 10 minutes, sufficient DMF was .added to make a 50 percent De~4F-DCM
mixture, and i:.he coupling was continued for 15 minutes.
Arginine was coupled as a preformed ester with hydroxybenzotriazol~s (Aldrich) in DMF for 60 minutes and then recouple<i in tile same manner as the other amino acids. Asparagine .and glutamine were coupled twice as preformed hydroxybenzotriazole esters in DMF, 40 minutes for each coupling. For all residues, the resin was washed after t:he second coupling and a sample was automatically taken for monitoring residual uncoupled a-13~08~9 amine by quantitativEa ninhydrin reactin: Sarin et al.
(cited above).
The general. technique of linking synthetic peptides to a carrier is described in several references: e.g. Wa7.ter and Doolittle, "Antibodies Against Synthetic Peptides," in Genetic Engineering, (Setlow et al., eds.), Vol. 5, pgs. 61-91 (Plenum Press, N.Y., 1983); Green et. al., Cell, Vol. 28, pgs. 477-487 (1982); Lerner et al.., Proc. Natl. Acad. Sci.~, Vol. 78, pgs. 3403-3407 (1981); Shimizu et al., U.S. Patent 4,474,754; and Ganfield et al., U.S. Patent 4,311,639.
Al~o; teghniquers employed to link haptens to carriers are essentially the same as the above-referenced techniques, e.g. chapter 20 in Tijsseu, Practice and Theory of Enzyme Immunoassays (Elsevier, New York, 1985).
The four me>st commonly used methods for attaching a peptide t:o a carrier use (1) glutaraldehyde for amino coupling, e~.g. as disclosed by Kagan and Glick, in Jaffe and Beh rman, eds. Methods of Hormone Radioimmunoassa ~, pgs~. 328-329 (Academic Press, N.Y., 1979), and Walt~ar et al., Proc. Natl. Acad. Sci., Vol.
77, pgs. 5197-5200 (1.980); (2) water-soluble carbodiimides f~~r carboxyl-to-amino coupling, e.g. as disclosed by Ho;are et. al., J. Biol. Chem., Vol. 242, pgs.
2447-2453 (1967); (?'~) bis-diazobenzidine (BDB) for tyrosine-to-tyrosine sidechain coupling, e.g. as disclosed by Ba;~siri et al., pgs. 46-47, in Methods of Hormone Radioimmunoass~, Jaffe and Behrman, eds. (cited above), and by l~lalter et al. (cited above); and (4) maleimidobenzoy:L-N-hydroxysuccinimide ester (MBS) for coupling cysteine (or other sulfhydryls) to amino groups, e.g. as disclosE~d by Kitagawa et al., J. Biochem.
(Tokyo), Vol. 7!7, pgs. 233-239 (1976), and by Lerner et al. (cited abovE:).
y ~~~~Da6~
A genera:L rule for selecting an appropriate method for coupling a given peptide to a carrier can be stated as follows: the amino acid chosen for coupling should occur only once in the sequence, preferably at the appropriate end of the segment. For example, BDB should not be used if a tyrosine residue occurs in the main part of a sequence chosE:n for its potentially antigenic character. S~imilanly, centrally located lysines rule out the glutaraldehyde method, and the occurrence of aspartic or glutamic acid will frequently exclude the carbodiimide method. On the other hand, suitable amino acid residues can.be positioned apt either end of a chosen. sequence segment as attachment ~ sites, whether . or. not .they v naturally occur there in the chosen protein sequence.
A segment. including the actual amino or carboxy terminus of mature H400 will preferably be attached to the carrier at the other end of the segment, leaving the terminus free. A segment lacking both actual termini may cause a problem in that its free end is not a natural terminus of mature H400. The problem can be remedied, to some extent, by acetylating the a-amino group and then attaching the peptide by way of its carboxy terminus.
The coupling efficiency of a given peptide to the carrier protein is conveniently measured by using a radioactively labeled peptide, prepared either by using a radioactive amino acid for one step of the synthesis or by labeling the completed peptide by the iodination of a tyrosine resi~~ue. The presence of tyrosine in the peptide also ,allows one to set up a sensitive radioimmune assay, if desirable. Therefore, it may be desirable to introduce tyrosine as a terminal residue if it is not part of the pE~ptide sequence def fined by the nat five H400 polypeptide.
Prei_erred carriers are proteins, and preferred protein carriE~rs include bovine serum albumin, .~3~~~~g myoglobulin, ovalbumin (OVA), keyhole limpet hemocyanin (KLH), or the like.
Peptides can be linked to KLH through cysteines by MBS as disclosed', by Liu et al., Biochemistry, Vol. 18, pgs. 690-697 (1979). The peptides are dissolved in phosphate-buffered saline (pH 7.5), 0.1 M sodium borate buffer (pH 9.0) or 1.0 M sodium acetate buffer (pH
4.0). The pH for the dissolution of the peptide is chosen to optimize peptide solubility. The content of free cysteine for soluble peptides is determined by the method of Ellman, Pvrch. Biochem. Biophys., Vol. 82, pg.
707.7 ( 1959 ) .
Fpr each peptide, 4 mg KLH ~ in' 0.25 ml of 10. mM
sodium phosphate buffer (pH 7.2) is reacted with 0.7 mg ,MBS (dissolved in dimethyl formamide) and stirred for 30 min at room temperature. The MBS is added dropwise to ensure that the local concentration of formamide is not too high, since KLH is insoluble in >30~ formamide. The reaction product, B:LH-P9B, is then passed through Sephadex G-25 equilibrated with 50 mfZ sodium phosphate buffer (pH
6.0) to remove free P1BS; KLH recovery from peak fractions of the column eluat.e' (monitored by OD280) is estimated to be approximately 8C1~.
KLH-P9B is. then reacted with 5 mg peptide dissolved in 1 ml of the chosen buffer. The pH is adjusted to 7-7.5 and the reaction is stirred for 3 hr at room temperature. Coupling efficiency is monitored with radioactive peptides by dialysis of a sample of the conjugate against phosphate-buffered saline, and ranges f rom 8$ to 60 ~ .
III. Monoclenal Antibodies and Immunochemical Assays Monoclonal antibodies are produced against mature' H400 c~r pept:ide-carrier conjugates by standard techniques: e.g. as disclosed by Campbell in Monoclonal B
_A_ntibody Technology (Elsevier, New York, 1984); Hurrell, ed. Monoclonal 'Hybrid,oma Antibodies: Techniques and Applications (C:RC Press, Boca Raton, FL, 1982); Schreier et al. Hybridom,a Techniques (Cold Spring Harbor Laboratory, New York, 1980); U.S. Patent 4,562,003; or the like. For monoclonal antibody production, the first. step is to immunize a host animal to obtain a source of B lymphocytes. The B lymphocytes are fused with an appropriate immortalizing cell line to form hybridomas that secrete monoclonal antibodies.
Immortalizing cell lines are usually tumor cell,lines, such as myeloma,s. Preferably, the host animals are rodents, and the immortalizing cell line also is preferably derived from rodent cells, especially from the same rodent spe~~ies. After formation, hybridomas are screened for these producing antibodies against the peptide of the invention. Immunization, harvesting of lymphocytes, and cell fusion are all techniques well known in the art. Immunization is carried out by a regimen of repe;~ted injections into the host animal of the purified pe~~tide-carrier conjugate, usually mixed with a suitable adjuvant. Immunization can be optimized by varying several factors, including the amount of antigen used fo,r the primary injection and subsequent boosts, the route of injection, the time schedule for injecting and b:Leeding, and the use of adjuvant, e.g.
Freund's comple~~e or incomplete adjuvant. Techniques for fusion are also well known in the art, and in general involve mixing the cells with a fusing agent such as, most commonly, ~~olyethylene glycol.
Succe:;sful hybridoma formation is assessed and selected by standard procedures such as, for example, HAT
selection. From among successful hybridomas, those successfully secreting the desired antibody are selected ..
by assaying the culture medium for their presence.
Ordinarily this is done using immunoreaction-based assays, for example, Western blot, ELISA, or RIA
assays. The antibodies can be recovered from the medium using standard protein purification techniques.
"Twa site" or "sandwich" immunoassays are the preferred immunoassays of the invention, e.g. as disclosed in U.;S. Pat.ent 4,486,530. Such assays entail the use of two ~~ifferent sets of anti-H400 antibodies, at least one of which consists of a monoclonal antibody of the invention. Antibodies from one of the two sets are attached xo the surface of a solid-phase support. The attached antibodies are then exposed to a sample suspected of containing H400. The H400 molecules bind to the attached antibodies. Next, the second set of antibodies is applied to the bound H400, and binds to one or more antigenic determinants distinct from that (or those ) to which the first set of antibodies is (or are ) bound. The H40~~ is then detected by an indirect or direct signal-g~:nerating means associated with the second set of antibodies. for example, the antibodies can be directly conjug;~ted t.o a signal-generating moiety, such as an enzyme, r,~re earth chelator, or an organic dye.
Or, they can be indirectly linked to one or more signal-generating moieties through additional antibodies, or high affinity c~~mplex.es, such as the avidin-biotin complexes. Qua:ntitat.ive measures of H400 concentration are made by com;paring~ the signal generated by the sample to signals generated by H400 standards containing known concentrations of H400.
The invention includes kits of reagents for use in immunoassays, particularly sandwich immunoassays.
Such kits include (l.) a solid-phase support, (2) a first antibody which is monoclonal and which is capable ~f, ~3~pg~~
of binding to a first antigenic determinant of H400, (3) a second antibody selected from a monoclonal antibody capable of binding to a second antigenic determinant of H400 and a polyclonal antibody specific for H400 (referred to herein as a "polyclonal antibody composition"), and (4) a signal-generation means associated with one of the three antibodies, preferably with the second antibody. Depending on the particular embodiment, kits may include a selection of two of the three anti-H400 antibody types, either a monoclonal antibody specific f:or a first antigenic determinant and a monoclonal antibody specific for a second antigenic determinants or,a monoclonal antibody specifis for a first or second antigenic determinant and a polyclonal antibody composition. The antibodies may be in solution or in lyophilized form. One of the sets of antibodies may come pre-attached to the solid support, or may be applied to the surface of the solid support only when the kit is to be used. The signal-generating means may come preassociated with one of the two antibody types, or may require combination with one or more components, e.g.
buffers, antibody-enzyme conjugates, enzyme substrates, or the like, prior to use. Many types of signal-generating means are available and could make up one or more components of a kit. Various signal generating means are dis~~losed by Tijssen, Practice and Theory of Enzyme Immunoassays.(Elsevier, Amsterdam, 1985). Kits of the invention may also include additional reagents, e.g.
blocking reagents for reducing nonspecific binding to the solid-phase surface, washing reagents, enzyme substrates, and the like. The solid-phase surface may be in the form of microtiter plates, microspheres, or the like, composed of polyvinyl ~~hloride, polystyrene, or similar materials suitable for immobilizing proteins. Preferably, an enzyme which catalyzes the formation of a fluorescent or colored product is a component of the signal-generating means.. More preferably, the enzyme is selected from peroxidase, alkaline phosphatase, and beta-galact:osidase. Substrates and reaction conditions for these E~nzyme:~ are well known in the art, e.g.
Tijssen (cited above). , IV. Nucleic Acid Probes Nucleic acid probes of the invention are constructed from nucleotide sequences determined by the nucleic: acid.of Formula II. The nature of the probe and _Lts associated nucleic acid can vary depending on the particular hybridization assay employed. One method of measuring H400 mRNA is by cytoplasmic dot hybridization as described by White et al., J. Biol. Chem., Vol. 257, pgs. 8569-8572 (1982), and by Gil:lespie et al., U.S. patent 4,483,920. Other approaches include in situ hybridization, e.g. Angerer et al., Genetic :Enaineerina, Vol. 7, pgs. 43-65 (1985), an~3 dot :blotting using purified RNA, e.g.
chapter 6 :in Ham~es et al., eds., Nucleic Acid Hvbridizat:ion: A Practical Approach (IRL Press, Washington, D.C., 1985). Generally, cytoplasmic dot hybridization involves anchoring mRNA from a cell or tissue sample onto a solid-phase support, e.g.
nitrocellulose, hybridizing a DNA probe to the anchored mRNA, and removing probe sequences nonspecifically bound to the solid-phase support or forming mismatched hybrids with the mRNA so that only probe sequences forming substantially perfect hybrids with target mRNA.s remain. The amount of DNA probe remaining is a measure of the amount of target mRNA
anchored to the solid-phase support. The amount of DNA probe remaining is determined by the signal generated. by its. label.
I3~~8~~
Several standard techniques are available for labeling single anc! double stranded nucleic acid fragments. 'They include incorporation of radioactive labels, e.g. Harper et al., Chromosoma, Vol. 83, pgs.
431-439 (1989); direct attachment of fluorescent labels, e.g. Smith et al., Nucleic Acids Research, Vol. 13, pgs.
2399-2412 (1985), and Connolly et al., Nucleic Acids Research, Vol. 13, pgs. 4485-4502 (1985); and various chemical modifications of the nucleic acid fragments that render them detectable immunochemically or by other affinity reactions: e.g. Tchen et al., Proc. Natl. Acad.
Sci., Vol. 81, pgs. 3466-3470 (1984); Richardson et al., Nucleic Acids Resea.rch~, Vo.l . 11, ~pgs. 6167-6184. ( 1983 ) ;
Langer et al., Proc. Natl. Acad. Sci., Vol. 78, pgs.
6633-6637 (1981); E~rigati et al., Virology, Vol. 126, pgs. 32-50 (1983); Broker et al., Nucleic Acids Research, Vol. 5, pgs. 363-384 (1978); and Bayer et al., Methods of Biochemical Analysis, Vol. 26, pgs. 1-45 (1980).
Preferably probes are prepared by nick translation of a fragment of pcD(SRa)-H400 containing all or the major portion of the H400 coding region. For example, pcD(SRa)-H400 is amplified in JM101, mixed with ethidium bromide, isolated by equilibrium density gradient centrifugation in cesium chloride, and digested with BamHI; and the restriction fragment carrying the entire coding region for H400 is then separated by gel electrophoresis. The fragment containing the coding region (which is readily identified by a size marker) is extracted from the gel and subjected to nick-translation in the presence of one or more kinds of labeled nucleotides: ~~.g. Maniatis et al., Molecular Cloning: A
Laboratory Pianual (Cold Spring Harbor Laboratory, New York, 1982); or Brigati et al. (cited above). After removal of un:incorporated nucleotides, the probe is applied to thE: anchored mRNA at a concentration in the range of between about 1 and 50 ng/ml (with a specif is activity in the range of about 1-2 x 108 cpm/ ~ probe).
Peripheral blood lymphocytes (PBLs) (which contain about 80~ T cells) are used as a source of T
cells for the assay. PBLs are obtained by standard techniques, e.g. Boyum, Scand. J. Clin. Lab. Invest , Vol. 21 (S~appl. 97), pg. 77 (1968). If desired, a fraction o:E cells enriched in T cells (to about 95$) can be obtained using standard techniques, e.g. eliminating B
cells by resetting: Gmelig-Meyling et al., Vox San ., Vol. 33, pct. 5 (1977). Generally, PBLs are obtained from fresh blood. by density gradient centrifugation in Ficoll-Fiypaque. F?re~erably mRNA from PBLs is anchored for hybridization to the probe by the following protocol.
Isolated PE~Ls are lysed by suspending in a lysis buffer (0.14 M NaC'.1, 1.5 mM MgCl2, 10 mM Tris-HC1 pH 8.6, 0.5~
Nonidet P-9:(~'~(a nonionic detergent, e.g. from Sigma)) at 4°C at a final concentration of 1x108 cells/ml. The suspension is vortexed for 10 sec and the nuclei are pelleted (13,000 g, 2.5 min). The resulting cytoplasmic lysates are then transferred to a sterile 1.5 ml tube containing 0.3 volumes of 20x SSC (lx SSC=0.15 ti NaCl, 0.015 M trisodium. citrate (standard saline citrate)) and 0.2 volumes of 37~ (w/w) formaldehyde. The mixture is then incubated at 60°C for 15 min and stored in aliquots at -70°C. For analysis, 15 ul of each sample is titered by serial three-fold dilutions in 15x SSC into a 96-well flat-bottomed microtiter plate (Falcon, Becton Dickinson, Oxnard, CA) in a 0.1 ml. Each dilution is applied with suction to ~~ sheet of Nytran* (a modified nylon support available from Sclzleicher and Schuell, Keene, NH; 0.45 pore size ) :~uppor~ted on a f i lter paper ( Whatman* 3mmChr, P~hatman Inc", Cli:Eton, NJ) utilizing a 96 hold Minifold apparatus (:~chleicher and Schuell). The Nytran paper is then baked 1;80°C, 2 hours) and treated with a * Trade mark ~~~O~s~9 prehybridization solution consisting of 50$ formamide (BRL, Gaithersburg, ML)) 6x SSC, 50 ~/ml E. cola tRNA
(Sigma), U.2$ (w,/v) a<<ch of ficoll (Mw=400,000), polyvinylpyrolli~done, and bovine serum albumin (BSA).
The probe is applied t:o the Nytran support at a concentration of about. 50 ng probe/ml of prehybridization solution. Following hybridization, the support is washed twice for 15 min each at room temperature in 2x SSC, then twice for 30 min each at 60°C in 2x SSC/0.5$ SDS. The support is then ~axpose~d to film using an intensifying screen and quant:itated, by scanning with a laser densitometer (e.c~. Ultroscan*XL, LKB Instruments Inc., Gaittiersburg, MD ) .
If cytoplasmic.dot hybridization lacks sufficient sensii_ivity, preferably the RNA is first extracted from the PBLs prior to blotting. for example, RNA may be extra c;ted by the guanidinium thiocyanate method disclosed by Chirgwin et: al, in Biochemistry, Vol.
18, pgs. 5294-5259 (1979).
Applicants have deposited E. coli JM101 carrying pcD(SRa)-H400 with the American Type Culture Collection, Rockville, ~iD, USA (ATCC), under accession number 67614. This deposit was made under the Budapest Treaty relating t:o the Deposit of f4icroorganisms.
* Trade mark w xG.~.
Field of the Invention The invention relates generally to methods and compositions for monitoring the status of the immune system; more particularly, the invention provides compositions for detecting activated T-cells by nucleic acid hybridization probes and/or immunochemical assays.
BACKGROUND
Regulation of normal immunologic reactivity involves the '~~alance between positive and negative influences exerted by various subsets of T cells.
Abnormally hyperactive and hypoactive T cells are believed to be associated with several immune disorders, including AIDS (hypoactivity and destruction of the helper T cell subset), and a variety of autoimmune disorders, such as MHC-associated autoimmune disease (e. g. lupus erythem.atosus) which is believed to be caused by excessive production of gamma interferon by T cells.
T cells have been identified by certain phenotypic celLl surface markers. For example, the surface molecule T8, which is present on human T cells of the cytotoxic subset, can be detected by immunofluoresc;ent staining using murine anti-human monoclonal ant:ibodic~s, such as OKT8 (Ortho Diagnostics, Raritan, NJ) or Leu -2 (Becton-Dickinson, Mountain View, CA). However, such markers are, at best, only useful in measuring the size of subpopulations; they do not necessarily imply that the cells are secreting products associated with an activated state.
Many diagnostic assays have been developed which are based either on immunochemical detection of proteins: e.g., U.S. patents 4,562,003, 4,474,892, and 4,427,782; or on th.e detection of nucleic acids, either RNA or DNA, present in or produced by target cells:
e.g., Pettersson et al., Immunology Today, Vol. 6, pgs.
268-272.(1985), Falkow et al., U.S. Patent 4,358,535, and Gillespi.e e~t al., U.S. Patent 4,483.,92. Thewlatter assays use nucleic acid probes, usually fluorescently labeled or radioactively labeled DNAs or RNAs, which can be preferenti~~lly hybridized to complementary target nucleic acids in appropriately prepared samples or tissues. The assays can take a variety of forms: e.g.
RNA blotting: Thomas, Proc. Natl. Acad. Sci., Vol. 77, pgs. 5201-5205 (1980); dot hybridization: White et al., J. Biol. Chem _, Vol. 257, pgs. 8569-8572 (1982); Southern Blotting: Southern, J. Mol. Biol., Vol. 98, pgs. 503-517; and in situ hybridization: Pinkel et al., Proc.
Natl. Acad. S<:i., Vol. 83, pgs. 2934-2938 (1986); and Angerer et al.,, Gen~stic Engineering, Vol. 7 pgs. 43-65 (1985).
In view o:E the present lack of convenient direct methodic for measuring abnormal T cell activation, the availability of sensitive immunochemical assays or nucleic acid ~>robes for detecting activated T cells would provide useful. diagnostic tools.
SUt~It4ARY OF THE INVENTION
The invent: ion includes a mature human protein produced by activated T cells, designated herein as H400, ~.3~0869 and immuno~~enic :peptide fragments thereof, all of which are useful for generating antibodies employed in immunochem:ical assays for activated T cells. The , amino acid sequence corresponding to the open reading frame encoding H~400 is given in Formula I:
Lys - Leu Cys - Val Thr - - Leu - Ser - - Val -Leu - Leu Met - Leu Val - - Ala - Phe - - Ala -Cys - Ser Pro - Ala Leu - - Ala - Pro - - Ser -Met - Gly Ser - Asp Pro - - Thr - Ala - - Pro -Cys - Cys Phe - Ser Tyr Thr - Arg Glu - - - - -,. - , Ala - Ser Ser - Asn ~ Phe - - Val Asp - , , -Val Tyr - ~ Tyr Glu ~Thr Ser Ser - Leu Cys.
~- - '- - - -Ser - Gln Pro - Ala Val - - Phe - Gln - - Val -Thr - Lys Arg Ser Lys Gln Val Cys ~- - - - - - -Ala - Asp Pro Ser Glu Ser - Trp Val - - - - - -Gln - Glu Tyr Val Tyr Asp Leu Glu ~- - - - - - -Leu - Asn .
Formula I
The present invention relates to a process for producing the po7_ypeptide of Formula I which comprises at least ome of t:ransfecting and/transforming a cell or bacterium with ~an expression vector containing a DNA sequence which encodes said polypeptide;
Culturing said cell or bacterium under conditions wherein the polypeptide is expressed by said cell or bacterium; and iso:Lating said polypeptide.
The invention further includes monoclonal antibodies specific for the mature H400 protein and its immunogenic ~>eptide fragments, and nucleic acids encoding H400 and peptide fragments thereof useful in' the construction of nucleic acid probes for H400 messenger RNA (mF;NA). The preferred nucleotide ~~~o~s~
- 3a -sequence oi: the open reading frame encoding Fi400 is given in Formula II:
ATG - AAG CTC TGC GTG ACT GTC CTG -- - - - - -TCT - CTC - CTC - ATG - CTA - GTA - GCT - GCC -TTC - TGC TCT CCA GCG CTC TCA GCA -- - - - - -CCA - ATG - GGC - TCA - GAC - CCT - CCC - ACC -GCC - TGC TGC TTT TCT TAC ACC CGA -- - - - - -GAA - GCT - TCC - TCG - AAC - TTT - GTG - GTA -GAT - TAC TAT GAG ACC AGC AGC CTC -- - - - - -".~..
;t. .a ;--.,:~
.~3~~~~g TGC - TCt~- CAG - CCA GCT GTG - GTA - TTC
- - -CAA ACC - AA,A- AGA AGC AAG - CAA GTC
- - - - -TGT GC'.~ - GA'T- CCC AGT GAA - TCC - TGG
- - - -GTC CA(s - GAG TAC GTG TAT GAC CTG
- - - - - - -GAA CT(i - AAC - ( - TGA
) .
Formula II
As used herein, the term "mature" in reference to protein H9E00 means the secreted form of H400, that is, the form of Ft400 that results from proteolytic cleavage of the signal peptide.
As,used herein, "immunogenic peptide" in.
reference to H4Q0' means a peptide ~ ( T) which cons~ists~ bf from 6 to 40 amino acids having a sequence identical to an equally long portion of mature H400 and (2) which in conjugation with a carrier protein is capable of eliciting antibodies which cross-react with mature H400.
As used herein, "nucleic acid fragment" means a nucleic acid which consists of from about 18 to 264 nucleotides having a sequence complementary to an equal length portion of the nucleic acid defined by Formula II.
A plasmid, designated herein as pcD(SRa)-H400, containing a ~~DNA insert capable of encoding H400 has been deposited (in its E. coli JM101 host) with the American Type Culture Collection (Rockville, MD) under accession number 67614.
Brief Description of the Drawings Figure 1 diagrammatically illustrates the plasmid pcD(SR a)-H4~00 and selected restriction sites.
Figure 2 Lists the nucleotide sequence of the cDNA insert of pcD(SRa)-H400 and indicates the amino acid sequence corre~spond:ing to the largest open reading frame.
~3~0~6~
_5_ DE'.~AILED DESCRIPTION OF THE INVENTION
ThE~ invention is based on the discovery of a novel protein, H400, which is produced specifically by activated T c:ells. The protein H400 is encoded by the largest open reading frame of the cDNA insert of pcD(SRa)-H400. ThE~ invention is directed to compounds useful in det.ectinc~ cells which either produce the H400 protein or produce mRNA transcripts capable of encoding the H400 protein. These compounds include the mature protein H400, immunogenic peptide fragments thereof, monoclonal antibodies specific for mature H400 or its immunogenic,peptide~ fragments, nucleic.acids capable of encoding H400, and,nucle.ic acid fragments fo,r constructing nucleic acid probes specific for mRNA that encodes H400.
I. Production of Mature H400 H400 can be produced by expressing a nucleotide sequence encoding it in a suitable expression system.
Possible types of host cells include, but are not limited to, bacterial, yeast, insect, mammalian, and the like.
Selecting an expression system, and optimizing protein production thereby, involves the consideration and balancing of many factors, including (1) the nature of the protein to be expressed, e.g. the protein may be poisonous to :;ome host organisms, it may be susceptible to degradation by host proteases, or it may be expressed in inactive conformations or in insoluble form in some hosts, ( 2) the nature of the messenger RNA (mRNA) corresponding to this protein of interest, e.g. the mRNA
may have sequences particularly susceptible to host endonucleases,. which drastically reduce the functional lifetime of the mRNA, or the mRNA may form secondary structures that mask the start codon or ribosome binding site, thereby inhibiting initiation of translation in some hosts, (3) the selection, availability, and arrangement of hosl:-compatible expression control sequences in the 3"- and 5'-regions flanking the coding region -- these inc:lude promoters, 5'- and 3'- protector sequences, ribosome binding sites, transcription terminators, enhanc;ers, polyadenylate addition sites, cap sites, intron-splice sites, and the like, (4) whether the protein has a ~~ecretion signal sequence which can be processed by the host, or whether an expression control sequence encoding a. signal sequence endogenous to the host must be spliced onto the region encoding the mature . protein, (5) the a.vailable~modes and efficiencies of transfection or transformation.of the host, and whether transient or stable expression is desired, (6) the scale and cost of the host culture system desired for expressing th~~ protein, (7) whether, and what type of, posttranslati~~nal modifications are desired, e.g. the extent and ki~~nd of glycosylation desired may affect the choice of host, ( 8 ) the ease with which the expressed protein can be separated from proteins and other materials of the hose cells and/or culture medium, e.g.
in some cases it ma:y be desirable to express a fusion protein with ~~ specialized signal sequence to aid in later purification steps (e. g. Sassenfeld et al., Biotechnology,, January 1984), (9) the stability and copy number of a p<~rticular vector in a selected host, e.g.
Hofschneider Eat al., eds. Gene Cloning in Organisms Other than E. Coli I;Springer Verlag, Berlin, 1982), and (10) similar factors known to those skilled in the art.
Mane revi<~ws are available which provide guidance for making choices and/or modifications of specific expression systems in light of the recited factors: e.g. de Boer and Shepard, "Strategies for Optimizing Foreign Gene Expression in Escherichia coli", pgs. 205-247, in Kroon, ed., Genes: Structure and ~.~44~~~
_, _ Expression (~iohn W:iley & Sons, New York, 1983), review several E. coli expression systems; Kucherlapati et al., Critical Reviews in Biochemistry, Vol. 16, Issue 4, pgs.
349-379 (198~E), and Banerji et al., Genetic Engineering, Vol. 5, pgs. 19-31 (1983) review methods for transfecting and transforming mammalian cells; Reznikoff and Gold, eds., Maximizing Gene Expression (Butterworths, Boston, 1986) review selected topics in gene expression in _E.
coli, yeast, and mammalian cells; and Thilly, Mammalian Cell Technolo~r (Butterworths, Boston, 1986) reviews mammalian expression systems.
Likewise, many reviews are available which .describe techniques and conditions .for ~linki.ng and/or manipulating specific cDNAs and expression control ,sequences to create and/or modify expression vectors suitable for use with the present invention: e.g.
Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, N.Y., 1982); Glover, DNA
Cloning: A Practical Approach, Vol. I and II (IRL Press, Oxford, 1985); and Perbal, A Practical Guide to Molecular Cloning (John Wiley & Sons, N.Y., 1984). Generally, within an expression vector various sites may be selected for insertion of the cDNA of the invention. These sites are usually designated by the restriction endonuclease which cuts them and are well recognized by those of skill in the art. Various methods for inserting DNA sequences into these sii:es to form recombinant DNA molecules are also well known. These include, for example, dG-dC or dA-dT tailing" direct ligation, synthetic linkers, exonuclease and pol:ymerase-linked repair reactions followed by 1~_gation, or extension of the DNA strand with DNA polymerase and an appropriate single-stranded template followed by ligation.
Often a vE~ctor containing the cDNA of the invention must: be obtained in large quantities before 13~~~6~
_8_ transfection and/o:r transformation of cells in a host culture can hake place. For this purpose the vector is often replicated without significant expression in an organism (the cloning host) other than the one ffinally used for expression. In such cases, after propagation, the vectors acre separated from the cloning host using standard techniques, e.g. as disclosed by Maniatis et al.
(cited above).
Preferably H400 is produced by expressing the H400 cDNA carried by pcD(SRa)-H400 'in a host cell suitable for pcD vectors, e.g. COS monkey cells .. (available from the ATCC under accession numbers CRL 1650 ., or CRL~16fi1,),-C127.mouse mammary tumor cell (available from ATCC under accession number CRL 1616), or mouse L
cells. Eukaryotic hosts are capable of cleaving the signal peptide from the freshly translated H400 polypeptide to form the mature H400 polypeptide.
Eukaryotic hosts are also capable of producing other posttranslati~an modifications, such as glycosylation, which may be ,~ntigenically important.
Production of H400 in a bacterial expression system requires that the cleavage site of the signal peptide be dei_ermined, either for direct expression of a nucleic acid encoding the mature H400 polypeptide or for linking such <~ nucleic acid to a bacterial sequence encoding an endogenous signal peptide. Empirical rules have been developed for making such predications with a high degree of: accuracy: e.g. von Heijne, Eur. J.
Biochem., Vol" 133, pgs. 17-21 (1983); J. Mol. Biol., Vol. 184, pgs. 99-105 (1985); Nucleic Acids Research, Vol. 14, pgs. 4683-4690 (1986); and Perlman et al., _J.
Mol. Biol., Vol. 16'1, pgs. 391-409 (1983). von Heijne's rules indicate that mature H400 begins with the alanine of position 23 with respect to the td-terminal amino acid of Formula I; the predicted mature sequence is illustrated below in Formula III.
~~~~86~
_g_ Mature H400 is produced as follows by COS7 cells transiently itransfected with pcD(SRa)-H400. One day prior to transiEection, approximately 106 COS 7 monkey cells are seeded onto individual 100 mm plates in Dulbecco's me>dif ie<9 Eagle's medium (DME ) containing 10$
fetal calf serum and 2 mM glutamine. To perform the transfection, the rnedium is aspirated from each plate and replaced with 4 ml of DME containing 50 mM Tris.HCl pH
7.4, 400 ug/ml DEAF;-Dextran and 50 yg of the plasmid DNAs to be tested. The plates are incubated for four hours at 37°C, then the DNA--containing medium is removed, and the plates are washed twice with 5 ml of serum-free DM F;. DME
is added balk to the plates which ~ are then incubated .for an additional 3 hrs at 37°C. The plates are washed once with DME, and then DME containing 4~ fetal calf serum, 2 mM glutamine, penicillin and streptomycin is added. The cells are then incubated for 72 hrs at 37°C. The growth medium is collected and H400 purified therefrom using standard methods.
II. Immunogenic Peptides of H400 The present invention includes peptides derived from mature H400 which form immunogens when conjugated with a carrier. The term immunogen as used herein refers to a substance which is capable of causing an immune response. Th~~ term carrier as used herein refers to any substance whi~~h when chemically conjugated to a peptide of the invention permits a host organism immunized with the resulting conjugate to generate antibodies specific for the conjugated peptide. Carriers include red blood cells, bacter:iophages, proteins, and synthetic particles such as agarose beads. Preferably carriers are proteins, such as serum albumin, gamma-globulin, keyhole limpet hemocyanin, thyroglobulin, ovalbumin, fibrinogen, or the like.
~3~0869 Peptides of the invention are defined in terms of their positions within the following amino acid sequence of mature H400:
( Ala - Pro - r4et- Gly Ser - Asp - Pro - Pro ) (9) Thr - Ala - Cys Cys Phe - Ser - Tyr Thr - - - -( Arch - Glu - Ala - Ser er - Asn - Phe - Val ) (25) Val. - Asp - Tyr Tyr Glu - Thr Ser Ser - - - - -(33) Leu - Cys - Ser - Gln Pro - Ala - Val - Val - -( Phe~ - Gln Thr Lys Arg Ser Lys Gln ) (49) Val. - Cy:a- Ala Asp Pro - Ser - Glu Ser - - - -( Tr~~ Val. Gln Glu Tyr Val, Tyr Asp 57.) - - - - - - - -(65) Leu. - Glu ~ Leu Asn. .
.-Formula III
Amino acids of Formula III are numbered with respect to the N-terminus of the mature H400 polypeptide. Thus, SerS is serine at position 5.
Peptides are def ine~d as fragments of the mature H400 polypeptide. Thus, the peptide designated herein by SerS - Cysll is the peptide Ser-Asp-Pro-Pro-Thr-Ala-Cys.
Groups of peptides of the invention are also deffined in terms of fragments of the mature H400 polypeptide. Thus, the group designated herein as [SerS - Cys11~5-6 consists of all 5- and 6- amino acid peptides (i.e. all 5-mers and 6-mers) whose sequences are identical to all or part of the peptide SerS - Cysll. That is, the group consists of the following five peptides: Ser-Asp-Pro-Pro-Thr-Ala, Asp-Pro-Pro-T:hr-Ala-Cys, Ser-Asp-Pro-Pro-Thr, Asp-Pro-Pro-Thr-Ala, ~~nd Pro-Pro-Thr-Ala-Cys.
Generally, the invention includes all 6-mer to 24-mer peptidE~ fragments of mature H400, i.e.
[Alai - Asn6816-24' Preferably, the invention includes 6-mer to 24-mE:r peptide fragments which include the N-terminal or C-terminal sequences of the mature H400 l3~os~~
polypeptide, or which correspond to sequences of the mature H400 poly,pepticle that have higher-than-average hydrophilicity, as determined by Hopp-Woods analysis or related analysis technique, e.g. Hopp and Woods, Proc.
Natl. Acad. Sci., Vol. 78, pgs. 3824-3828 (1981); or Kyte and Doolittle, J. Mol. Biol., Vol. 157, pgs. 105-132 ( 1982) . The latter set of preferred peptides of the invention (those having higher-than-average hydrophilicity) includes the following groups:
[Alai - Ser20J6-20 and [Thr43 - Asn6816-24' Standard symbols are used throughout for amino acids: e.g. Cohen, "Nomenclature and Symbolism of.alpha-Amino Acids':, Mgt:liods in Enzymology, ~Vol. 106, pgs. 3-17 (Academic Press, N.Y., 1984).
Peptides of the invention can be synthesized by standard techniques, E~.g. Stewart and Young, Solid Phase Pep tide Synthesis, 2nc! Ed. (Pierce Chemical Company, Rockford, IL, 1984). Preferably a commercial automated synthesizer is used, e.g. Vega Biochemicals (Tucson, AZ) model 296A or B, or Applied Biosystems, Inc. (Foster Ci-ty, CA) model 430A.
Peptides of the invention can be assembled by solid-phase synthesis on a cross-linked polystyrene support starting from the carboxyl terminal residue and adding amino acids in a stepwise fashion until the entire chain has been formed. We performed the synthesis on a fully automated peptide synthesizer (Applied Biosystems, Inc. model 430A) . Thc: following references are guides to the chemistry employed during synthesis: Plerrifield, J.
Amer. Chem. Soc., Vol. 85, pg. 2149 (1963); Kent et al., pg. 185, in Peptides 1984, Ragnarsson, Ed. (Almquist and Weksell, Stockholm, 1'984); Kent et al., pg. 217 in Peptide Chemistry 84, Izumiya, Ed. (Protein Research Foundation, B.H. Osaka, 1985); Merrifield, Science, Vol.
232, pgs. 341-347 (1986).
In solid-state synthesis it is most important to eliminate synthetic by-products, which are primarily termination, deletion, or modification peptides. Most side reactions can be eliminated or minimized by use of clean, well characterized resins, clean amino acid derivatives, clean solvents, and the selection of proper coupling and cleavage methods and reaction conditions:
e.g. Barany and ~9errifield, The Peptides, Cross and Meienhoter, Eds.,.~Vol. 2, pgs.~ 1-284 (Academic Press, New Xork, 1979). It is important to monitor coupling reactions ~ to .detE~rmine that they proceed to 'completion so that deletion peptides lacking one or more residues will be avoided. The quantitative ninhydrin reaction is useful for that purpose, Sarin et al. Anal. Biochem., Vol. 117, pg. 14'1 (1981). Na-t-butyloxycarbonyl-amino acids were used with appropriate side chain protecting groups stable to the conditions of chain assembly but labile to strong acids. After assembly of the protected peptide chain, the protecting groups were removed arid the peptide anchorinca-bond was cleaved by the use of low and then high concenl_ratio~ns of anhydrous hydrogen fluoride in the presence of a t,hioester scavenger: Tam et al., J.
Amer. Chem. Soc., Vol. 105, pg. 6442 (1983).
Side chain protecting groups used were Asp(OBzl), Glu(OBzl), Ser(Bzl), Thr(Bzl), Lys(C1-Z), Tyr ( Br-Z ) , Arg ( N~'Tos ) , Cys ( 4-hleBz 1 ) , and H is ( ImUNP ) .
(Bzl - benzyl: T~~s - t;oluenesulfonyl; DNP -dinitrophenyl; Im = imidazole; Z - benzyloxycarbonyl.) The remaining amino acids had no protecting groups on their side chains. Al.l the amino acids were obtained from Peninsula Laboratories, except the tBoc-His(ImDNP), which was from Chemical Dynamics and was recrystallized from ethanol before use. [tBoc - N a-t-butyloxy-~3~0~~6~
carbonyl.] E'or each cycle the tBoc-Na-protected peptide-resin was exposed i~o 65 percent trifluoroacetic acid ( from Eastman Kodak ) (distilled before use ) in dichloromethame (D(:M) (Mallenckrodt): first for 1 minute and then for 13 minutes to remove the Na-protecting group. The p~eptide~-resin was washed with DCM and then neutralized twice with 10 percent diisopropylethylamine (DIEA) (Aldrich) in dimethylformamide (DMF) (Applied Biosystems), for 1 minute each. Neutralization was followed by washings with DMF. Coupling was performed with the preformed symmetric anhydride of the amino acid ._ in DMF for 16 minutes. T.he performed symmetric anhydride was preparad on the~_. synthesizer by dissvlving~ .2 mmol of amino acid in 6 ml of DCM and adding 1 mmol of ,dicyclohexycarbodiimide (Aldrich) in 2 ml of DCM. After minutes, the activated amino acid was transferred to a separate vessel and the DCM was evaporated off by purging with a continuous stream of nitrogen gas. The DCM was replaced by DI~IF (6 ml total) at various stages during the purging. After the first coupling, the peptide-resin was washed with D~~M, 10 percent DIEA in DCM, and then with DCM. For rec~~upling, the same amino acid and the activating agent, dicyclohexylcarbodiimide, were transferred sequentially to the reaction vessel. After activation in situ and coupling for 10 minutes, sufficient DMF was .added to make a 50 percent De~4F-DCM
mixture, and i:.he coupling was continued for 15 minutes.
Arginine was coupled as a preformed ester with hydroxybenzotriazol~s (Aldrich) in DMF for 60 minutes and then recouple<i in tile same manner as the other amino acids. Asparagine .and glutamine were coupled twice as preformed hydroxybenzotriazole esters in DMF, 40 minutes for each coupling. For all residues, the resin was washed after t:he second coupling and a sample was automatically taken for monitoring residual uncoupled a-13~08~9 amine by quantitativEa ninhydrin reactin: Sarin et al.
(cited above).
The general. technique of linking synthetic peptides to a carrier is described in several references: e.g. Wa7.ter and Doolittle, "Antibodies Against Synthetic Peptides," in Genetic Engineering, (Setlow et al., eds.), Vol. 5, pgs. 61-91 (Plenum Press, N.Y., 1983); Green et. al., Cell, Vol. 28, pgs. 477-487 (1982); Lerner et al.., Proc. Natl. Acad. Sci.~, Vol. 78, pgs. 3403-3407 (1981); Shimizu et al., U.S. Patent 4,474,754; and Ganfield et al., U.S. Patent 4,311,639.
Al~o; teghniquers employed to link haptens to carriers are essentially the same as the above-referenced techniques, e.g. chapter 20 in Tijsseu, Practice and Theory of Enzyme Immunoassays (Elsevier, New York, 1985).
The four me>st commonly used methods for attaching a peptide t:o a carrier use (1) glutaraldehyde for amino coupling, e~.g. as disclosed by Kagan and Glick, in Jaffe and Beh rman, eds. Methods of Hormone Radioimmunoassa ~, pgs~. 328-329 (Academic Press, N.Y., 1979), and Walt~ar et al., Proc. Natl. Acad. Sci., Vol.
77, pgs. 5197-5200 (1.980); (2) water-soluble carbodiimides f~~r carboxyl-to-amino coupling, e.g. as disclosed by Ho;are et. al., J. Biol. Chem., Vol. 242, pgs.
2447-2453 (1967); (?'~) bis-diazobenzidine (BDB) for tyrosine-to-tyrosine sidechain coupling, e.g. as disclosed by Ba;~siri et al., pgs. 46-47, in Methods of Hormone Radioimmunoass~, Jaffe and Behrman, eds. (cited above), and by l~lalter et al. (cited above); and (4) maleimidobenzoy:L-N-hydroxysuccinimide ester (MBS) for coupling cysteine (or other sulfhydryls) to amino groups, e.g. as disclosE~d by Kitagawa et al., J. Biochem.
(Tokyo), Vol. 7!7, pgs. 233-239 (1976), and by Lerner et al. (cited abovE:).
y ~~~~Da6~
A genera:L rule for selecting an appropriate method for coupling a given peptide to a carrier can be stated as follows: the amino acid chosen for coupling should occur only once in the sequence, preferably at the appropriate end of the segment. For example, BDB should not be used if a tyrosine residue occurs in the main part of a sequence chosE:n for its potentially antigenic character. S~imilanly, centrally located lysines rule out the glutaraldehyde method, and the occurrence of aspartic or glutamic acid will frequently exclude the carbodiimide method. On the other hand, suitable amino acid residues can.be positioned apt either end of a chosen. sequence segment as attachment ~ sites, whether . or. not .they v naturally occur there in the chosen protein sequence.
A segment. including the actual amino or carboxy terminus of mature H400 will preferably be attached to the carrier at the other end of the segment, leaving the terminus free. A segment lacking both actual termini may cause a problem in that its free end is not a natural terminus of mature H400. The problem can be remedied, to some extent, by acetylating the a-amino group and then attaching the peptide by way of its carboxy terminus.
The coupling efficiency of a given peptide to the carrier protein is conveniently measured by using a radioactively labeled peptide, prepared either by using a radioactive amino acid for one step of the synthesis or by labeling the completed peptide by the iodination of a tyrosine resi~~ue. The presence of tyrosine in the peptide also ,allows one to set up a sensitive radioimmune assay, if desirable. Therefore, it may be desirable to introduce tyrosine as a terminal residue if it is not part of the pE~ptide sequence def fined by the nat five H400 polypeptide.
Prei_erred carriers are proteins, and preferred protein carriE~rs include bovine serum albumin, .~3~~~~g myoglobulin, ovalbumin (OVA), keyhole limpet hemocyanin (KLH), or the like.
Peptides can be linked to KLH through cysteines by MBS as disclosed', by Liu et al., Biochemistry, Vol. 18, pgs. 690-697 (1979). The peptides are dissolved in phosphate-buffered saline (pH 7.5), 0.1 M sodium borate buffer (pH 9.0) or 1.0 M sodium acetate buffer (pH
4.0). The pH for the dissolution of the peptide is chosen to optimize peptide solubility. The content of free cysteine for soluble peptides is determined by the method of Ellman, Pvrch. Biochem. Biophys., Vol. 82, pg.
707.7 ( 1959 ) .
Fpr each peptide, 4 mg KLH ~ in' 0.25 ml of 10. mM
sodium phosphate buffer (pH 7.2) is reacted with 0.7 mg ,MBS (dissolved in dimethyl formamide) and stirred for 30 min at room temperature. The MBS is added dropwise to ensure that the local concentration of formamide is not too high, since KLH is insoluble in >30~ formamide. The reaction product, B:LH-P9B, is then passed through Sephadex G-25 equilibrated with 50 mfZ sodium phosphate buffer (pH
6.0) to remove free P1BS; KLH recovery from peak fractions of the column eluat.e' (monitored by OD280) is estimated to be approximately 8C1~.
KLH-P9B is. then reacted with 5 mg peptide dissolved in 1 ml of the chosen buffer. The pH is adjusted to 7-7.5 and the reaction is stirred for 3 hr at room temperature. Coupling efficiency is monitored with radioactive peptides by dialysis of a sample of the conjugate against phosphate-buffered saline, and ranges f rom 8$ to 60 ~ .
III. Monoclenal Antibodies and Immunochemical Assays Monoclonal antibodies are produced against mature' H400 c~r pept:ide-carrier conjugates by standard techniques: e.g. as disclosed by Campbell in Monoclonal B
_A_ntibody Technology (Elsevier, New York, 1984); Hurrell, ed. Monoclonal 'Hybrid,oma Antibodies: Techniques and Applications (C:RC Press, Boca Raton, FL, 1982); Schreier et al. Hybridom,a Techniques (Cold Spring Harbor Laboratory, New York, 1980); U.S. Patent 4,562,003; or the like. For monoclonal antibody production, the first. step is to immunize a host animal to obtain a source of B lymphocytes. The B lymphocytes are fused with an appropriate immortalizing cell line to form hybridomas that secrete monoclonal antibodies.
Immortalizing cell lines are usually tumor cell,lines, such as myeloma,s. Preferably, the host animals are rodents, and the immortalizing cell line also is preferably derived from rodent cells, especially from the same rodent spe~~ies. After formation, hybridomas are screened for these producing antibodies against the peptide of the invention. Immunization, harvesting of lymphocytes, and cell fusion are all techniques well known in the art. Immunization is carried out by a regimen of repe;~ted injections into the host animal of the purified pe~~tide-carrier conjugate, usually mixed with a suitable adjuvant. Immunization can be optimized by varying several factors, including the amount of antigen used fo,r the primary injection and subsequent boosts, the route of injection, the time schedule for injecting and b:Leeding, and the use of adjuvant, e.g.
Freund's comple~~e or incomplete adjuvant. Techniques for fusion are also well known in the art, and in general involve mixing the cells with a fusing agent such as, most commonly, ~~olyethylene glycol.
Succe:;sful hybridoma formation is assessed and selected by standard procedures such as, for example, HAT
selection. From among successful hybridomas, those successfully secreting the desired antibody are selected ..
by assaying the culture medium for their presence.
Ordinarily this is done using immunoreaction-based assays, for example, Western blot, ELISA, or RIA
assays. The antibodies can be recovered from the medium using standard protein purification techniques.
"Twa site" or "sandwich" immunoassays are the preferred immunoassays of the invention, e.g. as disclosed in U.;S. Pat.ent 4,486,530. Such assays entail the use of two ~~ifferent sets of anti-H400 antibodies, at least one of which consists of a monoclonal antibody of the invention. Antibodies from one of the two sets are attached xo the surface of a solid-phase support. The attached antibodies are then exposed to a sample suspected of containing H400. The H400 molecules bind to the attached antibodies. Next, the second set of antibodies is applied to the bound H400, and binds to one or more antigenic determinants distinct from that (or those ) to which the first set of antibodies is (or are ) bound. The H40~~ is then detected by an indirect or direct signal-g~:nerating means associated with the second set of antibodies. for example, the antibodies can be directly conjug;~ted t.o a signal-generating moiety, such as an enzyme, r,~re earth chelator, or an organic dye.
Or, they can be indirectly linked to one or more signal-generating moieties through additional antibodies, or high affinity c~~mplex.es, such as the avidin-biotin complexes. Qua:ntitat.ive measures of H400 concentration are made by com;paring~ the signal generated by the sample to signals generated by H400 standards containing known concentrations of H400.
The invention includes kits of reagents for use in immunoassays, particularly sandwich immunoassays.
Such kits include (l.) a solid-phase support, (2) a first antibody which is monoclonal and which is capable ~f, ~3~pg~~
of binding to a first antigenic determinant of H400, (3) a second antibody selected from a monoclonal antibody capable of binding to a second antigenic determinant of H400 and a polyclonal antibody specific for H400 (referred to herein as a "polyclonal antibody composition"), and (4) a signal-generation means associated with one of the three antibodies, preferably with the second antibody. Depending on the particular embodiment, kits may include a selection of two of the three anti-H400 antibody types, either a monoclonal antibody specific f:or a first antigenic determinant and a monoclonal antibody specific for a second antigenic determinants or,a monoclonal antibody specifis for a first or second antigenic determinant and a polyclonal antibody composition. The antibodies may be in solution or in lyophilized form. One of the sets of antibodies may come pre-attached to the solid support, or may be applied to the surface of the solid support only when the kit is to be used. The signal-generating means may come preassociated with one of the two antibody types, or may require combination with one or more components, e.g.
buffers, antibody-enzyme conjugates, enzyme substrates, or the like, prior to use. Many types of signal-generating means are available and could make up one or more components of a kit. Various signal generating means are dis~~losed by Tijssen, Practice and Theory of Enzyme Immunoassays.(Elsevier, Amsterdam, 1985). Kits of the invention may also include additional reagents, e.g.
blocking reagents for reducing nonspecific binding to the solid-phase surface, washing reagents, enzyme substrates, and the like. The solid-phase surface may be in the form of microtiter plates, microspheres, or the like, composed of polyvinyl ~~hloride, polystyrene, or similar materials suitable for immobilizing proteins. Preferably, an enzyme which catalyzes the formation of a fluorescent or colored product is a component of the signal-generating means.. More preferably, the enzyme is selected from peroxidase, alkaline phosphatase, and beta-galact:osidase. Substrates and reaction conditions for these E~nzyme:~ are well known in the art, e.g.
Tijssen (cited above). , IV. Nucleic Acid Probes Nucleic acid probes of the invention are constructed from nucleotide sequences determined by the nucleic: acid.of Formula II. The nature of the probe and _Lts associated nucleic acid can vary depending on the particular hybridization assay employed. One method of measuring H400 mRNA is by cytoplasmic dot hybridization as described by White et al., J. Biol. Chem., Vol. 257, pgs. 8569-8572 (1982), and by Gil:lespie et al., U.S. patent 4,483,920. Other approaches include in situ hybridization, e.g. Angerer et al., Genetic :Enaineerina, Vol. 7, pgs. 43-65 (1985), an~3 dot :blotting using purified RNA, e.g.
chapter 6 :in Ham~es et al., eds., Nucleic Acid Hvbridizat:ion: A Practical Approach (IRL Press, Washington, D.C., 1985). Generally, cytoplasmic dot hybridization involves anchoring mRNA from a cell or tissue sample onto a solid-phase support, e.g.
nitrocellulose, hybridizing a DNA probe to the anchored mRNA, and removing probe sequences nonspecifically bound to the solid-phase support or forming mismatched hybrids with the mRNA so that only probe sequences forming substantially perfect hybrids with target mRNA.s remain. The amount of DNA probe remaining is a measure of the amount of target mRNA
anchored to the solid-phase support. The amount of DNA probe remaining is determined by the signal generated. by its. label.
I3~~8~~
Several standard techniques are available for labeling single anc! double stranded nucleic acid fragments. 'They include incorporation of radioactive labels, e.g. Harper et al., Chromosoma, Vol. 83, pgs.
431-439 (1989); direct attachment of fluorescent labels, e.g. Smith et al., Nucleic Acids Research, Vol. 13, pgs.
2399-2412 (1985), and Connolly et al., Nucleic Acids Research, Vol. 13, pgs. 4485-4502 (1985); and various chemical modifications of the nucleic acid fragments that render them detectable immunochemically or by other affinity reactions: e.g. Tchen et al., Proc. Natl. Acad.
Sci., Vol. 81, pgs. 3466-3470 (1984); Richardson et al., Nucleic Acids Resea.rch~, Vo.l . 11, ~pgs. 6167-6184. ( 1983 ) ;
Langer et al., Proc. Natl. Acad. Sci., Vol. 78, pgs.
6633-6637 (1981); E~rigati et al., Virology, Vol. 126, pgs. 32-50 (1983); Broker et al., Nucleic Acids Research, Vol. 5, pgs. 363-384 (1978); and Bayer et al., Methods of Biochemical Analysis, Vol. 26, pgs. 1-45 (1980).
Preferably probes are prepared by nick translation of a fragment of pcD(SRa)-H400 containing all or the major portion of the H400 coding region. For example, pcD(SRa)-H400 is amplified in JM101, mixed with ethidium bromide, isolated by equilibrium density gradient centrifugation in cesium chloride, and digested with BamHI; and the restriction fragment carrying the entire coding region for H400 is then separated by gel electrophoresis. The fragment containing the coding region (which is readily identified by a size marker) is extracted from the gel and subjected to nick-translation in the presence of one or more kinds of labeled nucleotides: ~~.g. Maniatis et al., Molecular Cloning: A
Laboratory Pianual (Cold Spring Harbor Laboratory, New York, 1982); or Brigati et al. (cited above). After removal of un:incorporated nucleotides, the probe is applied to thE: anchored mRNA at a concentration in the range of between about 1 and 50 ng/ml (with a specif is activity in the range of about 1-2 x 108 cpm/ ~ probe).
Peripheral blood lymphocytes (PBLs) (which contain about 80~ T cells) are used as a source of T
cells for the assay. PBLs are obtained by standard techniques, e.g. Boyum, Scand. J. Clin. Lab. Invest , Vol. 21 (S~appl. 97), pg. 77 (1968). If desired, a fraction o:E cells enriched in T cells (to about 95$) can be obtained using standard techniques, e.g. eliminating B
cells by resetting: Gmelig-Meyling et al., Vox San ., Vol. 33, pct. 5 (1977). Generally, PBLs are obtained from fresh blood. by density gradient centrifugation in Ficoll-Fiypaque. F?re~erably mRNA from PBLs is anchored for hybridization to the probe by the following protocol.
Isolated PE~Ls are lysed by suspending in a lysis buffer (0.14 M NaC'.1, 1.5 mM MgCl2, 10 mM Tris-HC1 pH 8.6, 0.5~
Nonidet P-9:(~'~(a nonionic detergent, e.g. from Sigma)) at 4°C at a final concentration of 1x108 cells/ml. The suspension is vortexed for 10 sec and the nuclei are pelleted (13,000 g, 2.5 min). The resulting cytoplasmic lysates are then transferred to a sterile 1.5 ml tube containing 0.3 volumes of 20x SSC (lx SSC=0.15 ti NaCl, 0.015 M trisodium. citrate (standard saline citrate)) and 0.2 volumes of 37~ (w/w) formaldehyde. The mixture is then incubated at 60°C for 15 min and stored in aliquots at -70°C. For analysis, 15 ul of each sample is titered by serial three-fold dilutions in 15x SSC into a 96-well flat-bottomed microtiter plate (Falcon, Becton Dickinson, Oxnard, CA) in a 0.1 ml. Each dilution is applied with suction to ~~ sheet of Nytran* (a modified nylon support available from Sclzleicher and Schuell, Keene, NH; 0.45 pore size ) :~uppor~ted on a f i lter paper ( Whatman* 3mmChr, P~hatman Inc", Cli:Eton, NJ) utilizing a 96 hold Minifold apparatus (:~chleicher and Schuell). The Nytran paper is then baked 1;80°C, 2 hours) and treated with a * Trade mark ~~~O~s~9 prehybridization solution consisting of 50$ formamide (BRL, Gaithersburg, ML)) 6x SSC, 50 ~/ml E. cola tRNA
(Sigma), U.2$ (w,/v) a<<ch of ficoll (Mw=400,000), polyvinylpyrolli~done, and bovine serum albumin (BSA).
The probe is applied t:o the Nytran support at a concentration of about. 50 ng probe/ml of prehybridization solution. Following hybridization, the support is washed twice for 15 min each at room temperature in 2x SSC, then twice for 30 min each at 60°C in 2x SSC/0.5$ SDS. The support is then ~axpose~d to film using an intensifying screen and quant:itated, by scanning with a laser densitometer (e.c~. Ultroscan*XL, LKB Instruments Inc., Gaittiersburg, MD ) .
If cytoplasmic.dot hybridization lacks sufficient sensii_ivity, preferably the RNA is first extracted from the PBLs prior to blotting. for example, RNA may be extra c;ted by the guanidinium thiocyanate method disclosed by Chirgwin et: al, in Biochemistry, Vol.
18, pgs. 5294-5259 (1979).
Applicants have deposited E. coli JM101 carrying pcD(SRa)-H400 with the American Type Culture Collection, Rockville, ~iD, USA (ATCC), under accession number 67614. This deposit was made under the Budapest Treaty relating t:o the Deposit of f4icroorganisms.
* Trade mark w xG.~.
Claims (7)
1. A process for producing a polypeptide having the following amino acid sequence:
Lys - Leu - Cys - Val - Thr - Val - Leu - Ser -Leu - Leu - Met - Leu - Val - Ala - Ala - Phe -Cys - Ser - Pro - Ala - Leu - Ser - Ala - Pro -Met - Gly - Ser - Asp - Pro - Pro - Thr - Ala -Cys - Cys - Phe - Ser - Tyr - Thr - Arg - Glu -Tyr - Tyr - Glu - Thr - Ser - Ser - Leu - Cys -Ser - Gln - Pro - Ala - Val - Val - Phe - Gln -Thr - Lys - Arg - Ser - Lys - Gln - Val - Cys -Ala - Asp - Pro - Ser - Glu - Ser - Trp - Val -Gln - Glu - Tyr - Val - Tyr - Asp - Leu - Glu -Leu - Asn comprising:
at least one of transfecting and transforming a prokaryotic or eukaryotic cell with an expression vector containing a DNA sequence which encodes said polypeptide;
culturing said prokaryotic or eukaryotic cell under conditions wherein the polypeptide is expressed by said prokaryotic or eukaryotic cell; and isolating said polypeptide.
Lys - Leu - Cys - Val - Thr - Val - Leu - Ser -Leu - Leu - Met - Leu - Val - Ala - Ala - Phe -Cys - Ser - Pro - Ala - Leu - Ser - Ala - Pro -Met - Gly - Ser - Asp - Pro - Pro - Thr - Ala -Cys - Cys - Phe - Ser - Tyr - Thr - Arg - Glu -Tyr - Tyr - Glu - Thr - Ser - Ser - Leu - Cys -Ser - Gln - Pro - Ala - Val - Val - Phe - Gln -Thr - Lys - Arg - Ser - Lys - Gln - Val - Cys -Ala - Asp - Pro - Ser - Glu - Ser - Trp - Val -Gln - Glu - Tyr - Val - Tyr - Asp - Leu - Glu -Leu - Asn comprising:
at least one of transfecting and transforming a prokaryotic or eukaryotic cell with an expression vector containing a DNA sequence which encodes said polypeptide;
culturing said prokaryotic or eukaryotic cell under conditions wherein the polypeptide is expressed by said prokaryotic or eukaryotic cell; and isolating said polypeptide.
2. A process for producing a polypeptide having the following amino acid sequence:
Ala - Pro - Met - Gly - Ser - Asp - Pro - Pro -Thr - Ala - Cys - Cys - Phe - Ser - Tyr - Thr -Arg - Glu - Ala - Ser - Ser - Asn - Phe - Val -Val - Asp - Tyr - Tyr - Glu - Thr - Ser - Ser -Leu - Cys - Ser - Gln - Pro - Ala - Val - Val -Phe - Gln - Thr - Lys - Arg - Ser - Lys - Gln -Val - Cys - Ala - Asp - Pro - Ser - Glu - Ser -Trp - Val - Gln - Glu - Tyr - Val - Tyr - Asp -Leu - Glu - Leu - Asn comprising:
at least one of transfecting and transforming a prokaryotic or eukaryotic cell with an expression vector containing a DNA sequence which encodes said polypeptide;
culturing said prokaryotic or eukaryotic cell under conditions wherein the polypeptide is expressed by said prokaryotic or eukaryotic cell; and isolating said polypeptide.
Ala - Pro - Met - Gly - Ser - Asp - Pro - Pro -Thr - Ala - Cys - Cys - Phe - Ser - Tyr - Thr -Arg - Glu - Ala - Ser - Ser - Asn - Phe - Val -Val - Asp - Tyr - Tyr - Glu - Thr - Ser - Ser -Leu - Cys - Ser - Gln - Pro - Ala - Val - Val -Phe - Gln - Thr - Lys - Arg - Ser - Lys - Gln -Val - Cys - Ala - Asp - Pro - Ser - Glu - Ser -Trp - Val - Gln - Glu - Tyr - Val - Tyr - Asp -Leu - Glu - Leu - Asn comprising:
at least one of transfecting and transforming a prokaryotic or eukaryotic cell with an expression vector containing a DNA sequence which encodes said polypeptide;
culturing said prokaryotic or eukaryotic cell under conditions wherein the polypeptide is expressed by said prokaryotic or eukaryotic cell; and isolating said polypeptide.
3. A process for producing a polypeptide selected from polypeptides Ala1 - Ser20 and Thr43 - Asn68 as defined by Formula III:
(1) Ala - Pro - Met - Gly - Ser - Asp - Pro - Pro -(9) Thr - Ala - Cys - Cys - Phe - Ser - Tyr - Thr -(17) Arg - Glu - Ala - Ser - Ser - Asn - Phe - Val -(25) Val - Asp - Tyr - Tyr - Glu - Thr - Ser - Ser -(33) Leu - Cys - Ser - Gln - Pro - Ala - Val - Val -(41) Phe - Gln - Thr - Lys - Arg - Ser - Lys - Gln -(49) Val - Cys - Ala - Asp - Pro - Ser - Glu - Ser -(57) Trp - Val - Gln - Glu - Tyr - Val - Tyr - Asp -(65) Leu - Glu - Leu - Asn which comprises the steps of:
at least one of transfecting and transforming a prokaryotic or eukaryotic cell with an expression vector containing a DNA sequence which encodes said polypeptide;
culturing said prokaryotic or eukaryotic cell under conditions wherein the polypeptide is expressed by said prokaryotic or eukaryotic cell; and isolating said polypeptide.
(1) Ala - Pro - Met - Gly - Ser - Asp - Pro - Pro -(9) Thr - Ala - Cys - Cys - Phe - Ser - Tyr - Thr -(17) Arg - Glu - Ala - Ser - Ser - Asn - Phe - Val -(25) Val - Asp - Tyr - Tyr - Glu - Thr - Ser - Ser -(33) Leu - Cys - Ser - Gln - Pro - Ala - Val - Val -(41) Phe - Gln - Thr - Lys - Arg - Ser - Lys - Gln -(49) Val - Cys - Ala - Asp - Pro - Ser - Glu - Ser -(57) Trp - Val - Gln - Glu - Tyr - Val - Tyr - Asp -(65) Leu - Glu - Leu - Asn which comprises the steps of:
at least one of transfecting and transforming a prokaryotic or eukaryotic cell with an expression vector containing a DNA sequence which encodes said polypeptide;
culturing said prokaryotic or eukaryotic cell under conditions wherein the polypeptide is expressed by said prokaryotic or eukaryotic cell; and isolating said polypeptide.
4. The polypeptide produced by the process according to claim 1, 2 or 3.
5. A nucleic acid selected from the group consisting of nucleic acids encoding polypeptides produced according to any one of claims 1, 2 and 3.
6. The nucleic acid of claim 5 which is capable of encoding the polypeptide of the open reading frame defined by the following amino acid sequence:
Lys - Leu - Cys - Val - Thr - Val - Leu - Ser -Leu - Leu - Met - Leu - Val - Ala - Ala - Phe -Cys - Ser - Pro - Ala - Leu - Ser - Ala - Pro -Met - Gly - Ser - Asp - Pro - Pro - Thr - Ala -Cys - Cys - Phe - Ser - Tyr - Thr - Arg - Glu -Ala - Ser - Ser - Asn - Phe - Val - Val - Asp -Tyr - Tyr - Glu - Thr - Ser - Ser - Leu - Cys -Ser - Gln - Pro - Ala - Val - Val - Phe - Gln -Thr - Lys - Arg - Ser - Lys - Gln - Val - Cys -Ala - Asp - Pro - Ser - Glu - Ser - Trp - Val -Gln - Glu - Tyr - Val - Tyr - Asp - Leu - Glu -Leu - Asn.
Lys - Leu - Cys - Val - Thr - Val - Leu - Ser -Leu - Leu - Met - Leu - Val - Ala - Ala - Phe -Cys - Ser - Pro - Ala - Leu - Ser - Ala - Pro -Met - Gly - Ser - Asp - Pro - Pro - Thr - Ala -Cys - Cys - Phe - Ser - Tyr - Thr - Arg - Glu -Ala - Ser - Ser - Asn - Phe - Val - Val - Asp -Tyr - Tyr - Glu - Thr - Ser - Ser - Leu - Cys -Ser - Gln - Pro - Ala - Val - Val - Phe - Gln -Thr - Lys - Arg - Ser - Lys - Gln - Val - Cys -Ala - Asp - Pro - Ser - Glu - Ser - Trp - Val -Gln - Glu - Tyr - Val - Tyr - Asp - Leu - Glu -Leu - Asn.
7. The nucleic acid of claim 6 defined by the following nucleotide sequence:
ATG - AAG - CTC - TGC - GTG - ACT - GTC - CTG
TCT - CTC - CTC - ATG - CTA - GTA - GCT - GCC
TTC - TGC - TCT - CCA - GCG - CTC - TCA - GCA
CCA - ATG - GGC - TCA - GAC - CCT - CCC - ACC
GCC - TGC - TGC - TTT - TCT - TAC - ACC - CGA
GAA - GCT - TCC - TCG - AAC - TTT - GTG - GTA
GAT - TAC - TAT - GAG - ACC - AGC - AGC - CTC
TGC - TCC - CAG - CCA - GCT - GTG - GTA - TTC
CAA - ACC - AAA - AGA - AGC - AAG - CAA - GTC
TGT - GCT - GAT - CCC - AGT - GAA - TCC - TGG
GTC - CAG - GAG - TAC - GTG - TAT - GAC - CTG
GAA - CTG - ACC - (TGA).
ATG - AAG - CTC - TGC - GTG - ACT - GTC - CTG
TCT - CTC - CTC - ATG - CTA - GTA - GCT - GCC
TTC - TGC - TCT - CCA - GCG - CTC - TCA - GCA
CCA - ATG - GGC - TCA - GAC - CCT - CCC - ACC
GCC - TGC - TGC - TTT - TCT - TAC - ACC - CGA
GAA - GCT - TCC - TCG - AAC - TTT - GTG - GTA
GAT - TAC - TAT - GAG - ACC - AGC - AGC - CTC
TGC - TCC - CAG - CCA - GCT - GTG - GTA - TTC
CAA - ACC - AAA - AGA - AGC - AAG - CAA - GTC
TGT - GCT - GAT - CCC - AGT - GAA - TCC - TGG
GTC - CAG - GAG - TAC - GTG - TAT - GAC - CTG
GAA - CTG - ACC - (TGA).
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/157,743 US5001230A (en) | 1988-02-18 | 1988-02-18 | T cell activation markers |
| US157,743 | 1988-02-18 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1340869C true CA1340869C (en) | 2000-01-04 |
Family
ID=22565071
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000591285A Expired - Fee Related CA1340869C (en) | 1988-02-18 | 1989-02-16 | T cell activation markers |
Country Status (9)
| Country | Link |
|---|---|
| US (1) | US5001230A (en) |
| EP (2) | EP0329363B1 (en) |
| JP (1) | JP2910861B2 (en) |
| AT (1) | ATE87655T1 (en) |
| AU (1) | AU3192889A (en) |
| CA (1) | CA1340869C (en) |
| DE (1) | DE68905633T2 (en) |
| ES (1) | ES2046462T3 (en) |
| WO (1) | WO1989007646A1 (en) |
Families Citing this family (34)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5705612A (en) * | 1984-10-18 | 1998-01-06 | Institut Pasteur And Centre National De La Recherche Scientifique | NEF peptide encoded by human immunodefiency virus type 1 (HIV-1) |
| AU582288B2 (en) * | 1986-03-07 | 1989-03-16 | Damon Biotech Inc. | Vector and method for achieving high level expression in eukaryotic cells |
| US5637488A (en) * | 1987-06-01 | 1997-06-10 | The United States Of America As Represented By The Department Of Health And Human Services | HIV protease gene and method for its expression |
| DE3878468T2 (en) * | 1987-12-23 | 1993-06-09 | Upjohn Co | CHIMARENE GLYCOPROTEINS, CONTAINING IMMUNOGENIC SEGMENTS OF THE HUMAN RESPIRATORY SYNCYTIAL VIRUS. |
| JPH04503300A (en) * | 1988-10-06 | 1992-06-18 | デイナ・フアーバー・キヤンサー・インステイテユート | H. SAIMIRI-HTLV-X region vector |
| IL92685A0 (en) * | 1988-12-16 | 1990-09-17 | Us Commerce | Dna encoding lymphokine/cytokine like proteins,cells transformed thereby and production of said proteins and antibodies against them |
| JP2903414B2 (en) * | 1989-05-31 | 1999-06-07 | 味の素株式会社 | Acid-fast bacterium secretion expression vector and acid-fast bacterium |
| ES2150188T3 (en) * | 1989-07-07 | 2000-11-16 | Unilever Nv | PROCEDURE FOR THE PREPARATION OF A PROTEIN THROUGH A FUNGUS TRANSFORMED BY MULTICOPY INTEGRATION OF A VECTOR OF EXPRESSION. |
| US6004776A (en) * | 1989-07-07 | 1999-12-21 | Unilever Patent Holdings, B.V. | Process for preparing a protein by a fungus transformed by multicopy integration of an expression vector |
| AU649228B2 (en) * | 1989-07-28 | 1994-05-19 | United States of America, as represented by the Secretary, U.S. Department of Commerce, The | Efficient directional genetic cloning system |
| US5541083A (en) * | 1989-10-24 | 1996-07-30 | The Regents Of The University Of California | Method for producing secretable glycosyltransferases and other golgi processing enzymes |
| FR2656800B1 (en) * | 1990-01-08 | 1992-05-15 | Roussy Inst Gustave | NEW PROTEINS PRODUCED BY HUMAN LYMPHOCYTES, DNA SEQUENCE ENCODING THESE PROTEINS AND PHARMACEUTICAL AND BIOLOGICAL APPLICATIONS. |
| US5976877A (en) * | 1990-01-08 | 1999-11-02 | Institut National De La Sante Et De La Recherche Medicale (Inserm) | Proteins produced by human lymphocytes DNA sequence encoding these proteins and their pharmaceutical and biological uses |
| FR2656866B1 (en) * | 1990-01-10 | 1992-05-15 | Cis Bio Int | PORPHYRIN DERIVATIVES AND METALLOPORPHYRINS POTENTIALLY COUPLED TO A BIOLOGICALLY ACTIVE MOLECULE, AND PHARMACEUTICAL COMPOSITION CONTAINING THEM. |
| US5652118A (en) * | 1991-03-11 | 1997-07-29 | Creative Biomolecules, Inc. | Nucleic acid encoding a novel morphogenic protein, OP-3 |
| US5403581A (en) * | 1991-07-12 | 1995-04-04 | Hoffmann-La Roche Inc. | Coccidiosis vaccines |
| US6008342A (en) * | 1991-07-12 | 1999-12-28 | Roche Vitamins Inc. | DNA encoding eimeria antigen |
| US5413927A (en) * | 1991-09-03 | 1995-05-09 | North Carolina State University | Feline immunodeficiency virus isolate NCSU1Lb |
| WO1993011244A1 (en) * | 1991-11-29 | 1993-06-10 | Weyerhaeuser Company | Cyclic di-guanylate metabolic enzymes |
| TW318189B (en) * | 1991-12-06 | 1997-10-21 | American Cyanamid Co | |
| US5721349A (en) * | 1992-02-26 | 1998-02-24 | Vanderbilt University | Vacuolating toxin-deficient H. pylori |
| US5792751A (en) * | 1992-04-13 | 1998-08-11 | Baylor College Of Medicine | Tranformation of cells associated with fluid spaces |
| IL108707A (en) * | 1993-02-19 | 1999-06-20 | Weiner David B | Pharmaceutical compositions and diagnostic kits comprising hiv protein vpr or proteins that bind to vpr |
| US5597707A (en) * | 1993-04-15 | 1997-01-28 | Bristol-Myers Squibb Company | Tumor associated antigen recognized by the murine monoclonal antibody L6, its oligonucleotide sequence and methods for their use |
| AU1373395A (en) * | 1993-12-15 | 1995-07-03 | Trustees Of The University Of Pennsylvania, The | Vpr receptor protein |
| US5653686A (en) * | 1995-01-13 | 1997-08-05 | Coulter Corporation | Closed vial transfer method and system |
| US6087486A (en) * | 1996-01-29 | 2000-07-11 | The Trustees Of The University Of Pennsylvania | Nucleotide sequences encoding vpr receptor protein |
| US6060587A (en) | 1996-01-29 | 2000-05-09 | The Trustees Of The University Of Pennsylvania | Cellular receptor for HIV-1 VPR essential for G2/M phase transition of the cell cycle |
| WO1998054226A1 (en) * | 1997-05-29 | 1998-12-03 | New Zealand Pastoral Agriculture Research Institute Limited | Processes for production of immunoglobulin a in milk |
| US6121021A (en) * | 1997-12-16 | 2000-09-19 | Connaught Laboratories Limited | Constitutive expression of non-infectious HIV-like particles |
| US6087128A (en) * | 1998-02-12 | 2000-07-11 | Ndsu Research Foundation | DNA encoding an avian E. coli iss |
| US6448078B1 (en) | 1998-10-09 | 2002-09-10 | The Trustees Of The University Of Pennsylvania | Cellular receptor for HIV-1 Vpr essential for G2/M phase transition of the cell cycle |
| CA2376245A1 (en) * | 1999-07-29 | 2001-02-08 | Dyax Corp. | Binding moieties for fibrin |
| TWI240632B (en) | 2001-07-30 | 2005-10-01 | Epix Medical Inc | Purified peptides for peptide-based multimeric targeted contrast agents |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4358535A (en) * | 1980-12-08 | 1982-11-09 | Board Of Regents Of The University Of Washington | Specific DNA probes in diagnostic microbiology |
| US4483920A (en) * | 1982-05-17 | 1984-11-20 | Hahnemann University | Immobilization of message RNA directly from cells onto filter material |
| EP0194626A3 (en) * | 1985-03-15 | 1988-05-18 | Sloan-Kettering Institute For Cancer Research | Method for transformation and direct selection of transformants in eucaryotic cells |
| US4788137A (en) * | 1985-10-29 | 1988-11-29 | Dana-Farber Cancer Institute, Inc. | Detection of activated T-cells |
| DE3650184T2 (en) * | 1985-12-03 | 1995-05-11 | T Cell Sciences Inc | CELL-FREE T-CELL ANTIGUE RECEPTOR AND CLINICAL USE. |
| DE260880T1 (en) * | 1986-09-11 | 1988-11-03 | Dana-Farber Cancer Institute, Inc., Boston, Mass., Us | METHOD FOR TRIGGERING THE CYTOTOXICITY. |
-
1988
- 1988-02-18 US US07/157,743 patent/US5001230A/en not_active Expired - Lifetime
-
1989
- 1989-02-13 AT AT89301341T patent/ATE87655T1/en not_active IP Right Cessation
- 1989-02-13 AU AU31928/89A patent/AU3192889A/en not_active Abandoned
- 1989-02-13 EP EP89301341A patent/EP0329363B1/en not_active Expired - Lifetime
- 1989-02-13 DE DE8989301341T patent/DE68905633T2/en not_active Expired - Fee Related
- 1989-02-13 JP JP1502671A patent/JP2910861B2/en not_active Expired - Fee Related
- 1989-02-13 WO PCT/US1989/000488 patent/WO1989007646A1/en not_active Application Discontinuation
- 1989-02-13 EP EP89902878A patent/EP0415930A1/en active Pending
- 1989-02-13 ES ES198989301341T patent/ES2046462T3/en not_active Expired - Lifetime
- 1989-02-16 CA CA000591285A patent/CA1340869C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| US5001230A (en) | 1991-03-19 |
| JP2910861B2 (en) | 1999-06-23 |
| EP0415930A1 (en) | 1991-03-13 |
| ES2046462T3 (en) | 1994-02-01 |
| EP0329363A1 (en) | 1989-08-23 |
| JPH03504316A (en) | 1991-09-26 |
| ATE87655T1 (en) | 1993-04-15 |
| EP0329363B1 (en) | 1993-03-31 |
| DE68905633T2 (en) | 1993-07-08 |
| WO1989007646A1 (en) | 1989-08-24 |
| DE68905633D1 (en) | 1993-05-06 |
| AU3192889A (en) | 1989-09-06 |
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