EP1021537A1 - Molecules associees au recepteur des lymphocytes t (tram) et leur mode d'utilisation - Google Patents

Molecules associees au recepteur des lymphocytes t (tram) et leur mode d'utilisation

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Publication number
EP1021537A1
EP1021537A1 EP98951056A EP98951056A EP1021537A1 EP 1021537 A1 EP1021537 A1 EP 1021537A1 EP 98951056 A EP98951056 A EP 98951056A EP 98951056 A EP98951056 A EP 98951056A EP 1021537 A1 EP1021537 A1 EP 1021537A1
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EP
European Patent Office
Prior art keywords
tram
protein
nucleic acid
seq
acid molecule
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP98951056A
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German (de)
English (en)
Inventor
Burkhart Schraven
Eddy Bruyns
Anne Marie-Cardine
Henning Kirchgessner
Andrej Shevchenko
Matthias Mann
Stefan Meuer
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BASF SE
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BASF SE
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Publication of EP1021537A1 publication Critical patent/EP1021537A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

Definitions

  • the primary signal that initiates human T cell activation is delivered through the interaction of the clonotypic T cell receptor (TcR) with its natural ligand, the antigen/MHC complex.
  • TcR clonotypic T cell receptor
  • the TcR is composed of disulfide linked polymorphic heterodimers (a/ ⁇ , ⁇ / ⁇ ) which noncovalently associate with the CD3 molecules (CD3- ⁇ , - ⁇ , - ⁇ ) as well as with ⁇ -chains (Meuer, S.C. et al. (1983) Nature 303:808; Meuer, S.C. et al. (1983) Science 222:1239; Meuer, S.C. et al. (1983) J. Exp. Med.
  • PTKs protein tyrosine kinases
  • the major candidates for these kinases were members of the src family (p56 lck and p59 f y n ) (Strauss, B.D. and Weiss, A. (1992) Cell 70:585; Stein, P.L. et al. (1992) Cell 70:741) as well as the recently cloned syk related tyrosine kinase ZAP70 (Chan, A.C. et al. (1991) Proc. Natl. Acad. Sci. USA 88:9166; Chan, A.C. et al. (1992) Cell 71:649).
  • CD3 molecules noncovalently associate with p59fy n (Samelson, L.E. et al. (1990) Proc. Natl. Acad. Sci. USA 87:4358) while tyrosine phosphorylated ⁇ chains interact with ZAP70 (Chan, A.C. et al. (1991) Proc. Natl. Acad. Sci. USA 88:9166; Chan, A.C. et al. (1992) Cell 21:64; Iwashima, M. et al. (1994) Science 263:1136).
  • the CD3- ⁇ and ⁇ -chains are of particular interest. Both molecules alone are capable of transmitting external signals into the intracellular environment independently of expression of CD3- ⁇ and CD3-6 (Wegener, A. et al. (1992) Cell 68:83). This applies to activation of the tyrosine kinase pathway as well as to further downstream events, e.g. lymphokine secretion.
  • a short peptide motif present in the cytoplasmic domains of both CD3- ⁇ and ⁇ has been defined which seems to be responsible for the triggering capacity of these molecules (Romeo, C. et al. (1992) Cell 68:889).
  • This motif was recently name ITAM (Immunoreceptor Tyrosine Based Activation Motif) and comprises the amino acid sequence Tyr-Xaa-Xaa-Leu-(Xaa) 6 . 8 -Tyr-Xaa-Xaa-Leu (SEQ ID. NO: 7). While each of the ⁇ -chains possesses three ITAM-motifs, the CD3- ⁇ chains as well as the other components of the CD3-complex only have one ITAM-motif in their intracellular domains.
  • ITAM Immunoreceptor Tyrosine Based Activation Motif
  • T cell Receptor Associated Molecules This application provides additional molecules that associate with the TcR/CD3/ ⁇ complex, referred to as T cell Receptor Associated Molecules, or TRAMS.
  • T cell Receptor Associated Molecules or TRAMS.
  • TRAMS T cell Receptor Associated Molecules
  • pp29/30 The preferential binding of pp29/30 to the TcR/CD3- complex was used to isolate the protein from lysates of HPB-ALL cells by immunoprecipitation using CD3- ⁇ mAb.
  • Purified pp29/30 was digested with trypsin and the resulting peptides were subjected to microsequencing employing the technique of nano-electrospray-tandem-mass-spectrometry. Based on the peptide sequences, cDNA clones coding for pp29/30 were isolated and the encoded proteins molecularly characterized.
  • TRAM-1 and TRAM-2 The nucleotide and predicted amino acid sequence of a human TRAM-1 are shown in SEQ ID NOs: 1 and 2, respectively.
  • the nucleotide and predicted amino acid sequence of an alternate form of TRAM- 1 are shown in SEQ ID NO: 5 and 6, respectively.
  • the nucleotide and predicted amino acid sequence of a human TRAM-2 are shown in SEQ ID NOs: 3 and 4, respectively.
  • one aspect of the invention pertains to isolated nucleic acid molecules encoding TRAM-1 or TRAM-2, or fragments thereof.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a TRAM-1 or TRAM-2 protein.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence that is homologous to the amino acid sequence of SEQ ID NO: 2 or 4 and associates with the TcR/CD3/ ⁇ -complex.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence homologous to the nucleotide sequence of SEQ ID NO: 1 or 3 and encoding a protein that associates with the TcR/CD3/ ⁇ -complex.
  • the invention provides an isolated nucleic acid molecule which hybridizes under high stringency conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or 3.
  • the invention provides an isolated nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or 3, or the coding regions thereof (nucleotides 130-690 of SEQ ID NO: 1 or nucleotides 88-678 of SEQ ID NO: 3) or the nucleotide sequence of SEQ ID NO: 5.
  • the invention provides an isolated nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 2, 4 or 6. Isolated nucleic acid molecules encoding TRAM-1 or TRAM-2 fusion proteins and isolated antisense nucleic acid molecules are also encompassed by the invention.
  • vectors such as recombinant expression vectors, containing an nucleic acid molecule of the invention and host cells into which such vectors have been introduced.
  • a host cell is used to produce a TRAM-1 or TRAM-2 protein by culturing the host cell in a suitable medium. If desired, the TRAM-1 or TRAM-2 protein can be then isolated from the host cell or the medium.
  • Still another aspect of the invention pertains to isolated TRAM-1 and TRAM-2 proteins, or portions thereof.
  • the invention provides an isolated TRAM-1 or TRAM-2 protein, or a portion thereof that interacts with the TcR/CD3/ ⁇ - complex.
  • the invention provides an isolated protein which comprises an amino acid sequence homologous to the amino acid sequence of SEQ ID NO: 1
  • the invention provides an isolated protein comprising the amino acid sequence of SEQ ID NO: 2, 4 or 6.
  • TRAM-1 and TRAM-2 fusion proteins are also encompassed by the invention.
  • TRAM-1 and TRAM-2 proteins of the invention can be used to prepare anti-TRAM-1 and anti-TRAM-2 antibodies, respectively.
  • the invention further provides antibodies that specifically binds a TRAM-1 or TRAM-2 protein.
  • the antibodies are monoclonal.
  • the antibodies are polyclonal.
  • the antibodies are labeled with a detectable substance.
  • the TRAM-1 or TRAM-2 -encoding nucleic acid molecules of the invention can be used to prepare nonhuman transgenic animals which contain cells carrying a transgene encoding TRAM-1 or TRAM-2 protein, or a portion thereof. Accordingly, such transgenic animals are also provided by the invention.
  • the invention the
  • TRAM-1 or TRAM-2 transgene carried by the transgenic animal alters an endogenous gene encoding endogenous TRAM-1 or TRAM-2 protein ⁇ e.g., a homologous recombinant animal).
  • Another aspect of the invention pertains to methods for detecting the presence of
  • TRAM activity e.g., TRAM-1 or TRAM-2 protein or mRNA
  • TRAM-1 or TRAM-2 protein or mRNA TRAM activity in a biological sample.
  • the biological sample is contacted with an agent capable of detecting TRAM-1 or TRAM-2 protein (such as a labeled anti-TRAM-1 or TRAM-2 antibody) or TRAM-1 or TRAM-2 mRNA (such as a labeled nucleic acid probe capable of hybridizing to TRAM- 1 or TRAM-2 mRNA) such that the presence of TRAM activity is detected in the biological sample.
  • an agent capable of detecting TRAM-1 or TRAM-2 protein such as a labeled anti-TRAM-1 or TRAM-2 antibody
  • TRAM-1 or TRAM-2 mRNA such as a labeled nucleic acid probe capable of hybridizing to TRAM- 1 or TRAM-2 mRNA
  • Still another aspect of the invention pertains to methods for modulating TRAM activity in a cell.
  • the cell is contacted with an agent that modulates TRAM-1 or TRAM-2 activity such that TRAM activity in the cell is modulated.
  • the agent inhibits TRAM- 1 or TRAM-2 activity.
  • the agent stimulates TRAM- 1 or TRAM-2 activity.
  • the agent modulates the activity of TRAM- 1 or TRAM-2 protein ⁇ e.g., the agent can be an antibody that specifically binds to TRAM-1 or TRAM-2 protein).
  • the agent modulates transcription of a TRAM-1 or TRAM-2 gene or translation of a TRAM-1 or TRAM-2 mRNA ⁇ e.g., the agent can be a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of a TRAM-1 or TRAM-2 mRNA or a TRAM-1 or TRAM-2 gene).
  • Still another aspect of the invention pertains to methods for identifying agents that modulate the association between a TRAM protein and the TcR/CD3/ ⁇ -complex.
  • TRAM-1 or TRAM-2 is contacted with the TcR/CD3/ ⁇ -complex, or a T cell molecule such as CD2, CD3, CD4, CD5, CD8, Lck or Fyn, in the presence and absence of a test compound.
  • the degree of interaction between TRAM- 1 /TRAM -2 and the TcR/CD3/ ⁇ -complex, or the T cell molecule is then determined in the presence and absence of the test compound.
  • a modulatory agent is identified based upon the ability of the test compound to increase or decrease ⁇ e.g., stimulate or inhibit) the degree of interaction between TRAM-l/TRAM-2 and the TcR/CD3/ ⁇ -complex, or the T cell molecule (as compared to the degree of interaction in the absence of the test compound).
  • Figure 7 is a photograph of a two-dimensional gel analysis of in v/tro-labeled phosphoproteins that coprecipitate with the accessory receptor molecule CD2 under mild detergent conditions.
  • the phosphorylated 29-30 kDa protein spot (pp29/30) is indicated by a question mark.
  • the identical pattern of phosphoproteins was detectable in CD3, CD4, CD5 and CD8 immunoprecipitates prepared under the same experimental conditions (see Example 1 ).
  • Figures 2A-2F are photographs of two-dimensional gel analyses of the phosphoproteins identified in Figure 1 reprecipitated with either anti- ⁇ (panel 2A); anti- CD3- ⁇ (panel 2B); anti-LPAP (panel 2C), anti-CD5 (panel 2D), anti-p56 lck (panel 2E); and anti-p59 f y n (panel 2F).
  • Figure 3 is a photograph of a two-dimensional gel analysis of a large scale preparation of in v tro-labeled phosphoproteins coprecipitated with CD3. The position of pp29/30 is indicated. The purified pp29/30 was recovered from the gel and subjected to tryptic digestion followed by nano-electrospray-tandem-mass-spectrometry.
  • FIG. 4 is a schematic diagram of the TRAM-1 protein, comprising an eight amino acid extracellular domain, a 19 amino acid transmembrane domain and a 159 amino acid cytoplasmic domain that contains the repeated tyrosine motif EDTPIYGNL (amino acids 58-66 of SEQ ID NO: 2) and ETQMCYASL (amino acids 105-113 of SEQ ID NO: 2). The two regions encompassing the microspray derived peptide sequences are also indicated.
  • FIG. 5 is a schematic diagram of the TRAM-2 protein, comprising a 22 amino acid leader, an 18 amino acid extracellular domain that includes an N-linked glycosylation, a 20 amino acid transmembrane domain and a 136 amino acid cytoplasmic domain that contains a first repeated tyrosine motif EEVPLYGNL (amino acids 85-93 of SEQ ID NO: 4) and EEVMCYTSL (amino acids 122-130 of SEQ ID NO: 4) and a second repeated tyrosine motif PVKYSEV (amino acids 145-151 of SEQ ID NO: 4) and PELYASV (amino acids 166-172 of SEQ ID NO: 4).
  • the region encompassing the microspray derived peptide sequence is also indicated.
  • FIGS 6A-6B are photographs of Northern blot analyses depicting the expression of TRAM- 1 (Figure 6A) or TRAM-2 ( Figure 6B) mRNA in the following human tissues: spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leucocytes (PBL), heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas.
  • Figure 7 A is a photograph of an immunoprecipitation/immunoblotting experiment in which HPB-ALL cell lysates were immunoprecipitated with mAbs to either phosphotyrosine (lanes 1 and 2), CD2 (lane 3), CD3- ⁇ (lane 4), CD4 (lane 5), CD8 (lane 6), CD45 (lane 7), CD28 (lane 8) or HLA-I (lane 9), the immunoprecipitates were transferred to nitrocellulose membranes followed by immunoblotting with an anti- TRAM-1 antisera.
  • the lysate from lane 2 was treated with pervanadate, whereas the lysates from lanes 1 and 3-9 were not treated with pervanadate.
  • Figure 7B depicts indirect immunofluorescence results for HPB-ALL cells treated with either media (Med.) or the CD3- ⁇ mAb 2Ad2A2 (TcR mod.) followed by mAbs to either CD3, CD8 or HLA-I, demonstrating modulation of CD3, but not CD8 or HLA-I, from the T cell surface following CD3- ⁇ mAb 2Ad2A2 treatment.
  • Figure 7C is a photograph depicting the results of a comodulation experiment in which HPB-ALL cells were either untreated (lanes 1 and 3) or treated with the CD3- ⁇ mAb 2 Ad2A2 to induce TcR modulation (lanes 2 and 4) and expression of either TRAM-1 (lanes 1 and 2) or MAP-kinase (lanes 3 and 4) was detected in post-nuclear lysates.
  • the densitometric analysis of the individual protein bands is shown on the bottom of each lane.
  • Figures 8A-8D are photographs depicting the results of an experiment in which post-nuclear lysates from HPB-ALL cells that were either unstimulated (Fig. 8A and 8C) or stimulated by co-crosslinking of CD3 and CD4 (Fig. 8B and 8D) were immunoprecipitated with anti-TRAM-1 antisera and then immunoblotted with either an anti-PTYR mAb (Fig. 8 A and 8B) or anti-TRAM-1 antisera (Fig. 8C and 8D), showing phosphorylation of TRAM- 1 in response to co-crosslinking of CD3 and CD4.
  • Figure 9 A is a photograph depicting the results of a time-course experiment in which post-nuclear lysates from HPB-ALL cells that were stimulated by co-crosslinking of CD3 and CD4 were immunoprecipitated with anti-TRAM-1 antisera and immunoblotted with anti-PTYR mAb, showing rapid phosphorylation of TRAM- 1 on tyrosine upon CD3/C4 co-crosslinking.
  • Figure 9B is another photograph depicting the results of the time-course experiment of Figure 9 A in which post-nuclear lysates from HPB-ALL cells that were stimulated by co-crosslinking of CD3 and CD4 were immunoblotted with anti-TRAM-1 antisera, demonstrating that identical amounts of TRAM- 1 were examined at each time point.
  • T cell Receptor Associated Molecules proteins that associate with the T cell receptor complex, termed T cell Receptor Associated Molecules, or TRAMS.
  • Immunprecipitations using antibodies to various T cell surface antigens identified a 29-30 kDa complex, termed pp29/30 (see Example 1). Large scale purification of this complex, followed by tryptic digestion and microsequencing led to the identification of several peptide sequences present in the complex (see Example 2).
  • TRAM-1 and TRAM-2 are disulfide linked transmembrane dimers that contain repeated tyrosine motifs in their cytoplasmic domains and that show lymphoid restricted expression of their mRNAs (see Examples 3 and 4).
  • the TRAM-1 protein has been shown to preferentially associate with and comodulate with the TcR/CD3/ ⁇ complex (see Example 5).
  • the TRAM-1 protein undergoes rapid phosphorylation upon T cell stimulation (see Example 6). So that the invention may be more readily understood, certain terms are first defined.
  • TRAM is intended to encompass both TRAM-1 and TRAM-2.
  • the term “TRAM-1 protein” is intended to encompass proteins that share the distinguishing structural and functional features (described further herein) of the TRAM- 1 protein of SEQ ID NO: 2.
  • the term “TRAM-2 protein” is intended to encompass proteins that share the distinguishing structural and functional features (described further herein) of the TRAM-2 protein of SEQ ID NO: 4.
  • SH2 domain refers to a protein domain, typically of about 100 amino acids in length and conserved among a variety of cytoplasmic signaling proteins that binds phosphotyrosine containing peptides.
  • SH2 domains See Koch, CA. et al. (1991) Science 252:668-674 (which also discloses and compares the amino acid sequences of many different SH2 domains).
  • nucleic acid molecule is intended to include DNA molecules ⁇ e.g. , cDNA or genomic DNA) and RNA molecules ⁇ e.g. , mRNA).
  • the nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • an "isolated nucleic acid molecule” refers to a nucleic acid molecule that is free of gene sequences which naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived ⁇ i.e., genetic sequences that are located adjacent to the gene for the isolated nucleic molecule in the genomic DNA of the organism from which the nucleic acid is derived).
  • an isolated TRAM nucleic acid molecule typically contains less than about 10 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived, and more preferably contains less than about 5, kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of naturally flanking nucleotide sequences.
  • An "isolated" TRAM nucleic acid molecule may, however, be linked to other nucleotide sequences that do not normally flank the TRAM sequences in genomic DNA (e.g., the TRAM nucleotide sequences may be linked to vector sequences).
  • an "isolated" nucleic acid molecule such as a cDNA molecule, also may be free of other cellular material. However, it is not necessary for the TRAM nucleic acid molecule to be free of other cellular material to be considered “isolated” (e.g., a TRAM DNA molecule separated from other mammalian DNA and inserted into a bacterial cell would still be considered to be “isolated”).
  • hybridizes under high stringency conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences having substantial homology (e.g., typically greater than 70% homology) to each other remain stably hybridized to each other.
  • a preferred, non-limiting example of high stringency conditions are hybridization in a hybridization buffer that contains 6X sodium chloride/ sodium citrate (SSC) at a temperature of about 45°C for several hours to overnight, followed by one or more washes in a washing buffer containing 0.2 X SSC, 0.1% SDS at a temperature of about 50-65°C.
  • SSC sodium chloride/ sodium citrate
  • homologous as used in the context of amino acid sequences (e.g., when one amino acid sequence is said to be X% homologous to another amino acid sequence) is intended to encompass both amino acid identity and similarity between the two sequences.
  • sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of one protein for optimal alignment with the other protein).
  • amino acid residues at corresponding amino acid positions are then compared and when a position in one sequence is occupied by the same or a similar amino acid residue as the corresponding position in the other sequence, then the molecules are homologous at that position.
  • Computer algorithms known in the art can be used to optimally align the two amino acid sequences to be compared and to define similar amino acid residues.
  • BLAST Basic Local Alignment Search Tool
  • the Basic Local Alignment Search Tool (BLAST) algorithm (described in Altschul, S.F. et al. (1990) J. Mol. Biol. 215:403- 410) is used to compare the two amino acid sequences to thereby determine the percent homology between the two sequences.
  • nucleotide sequences e.g., when one nucleotide sequence is said to be X% homologous to another nucleotide sequence
  • sequences are aligned for optimal comparison purposes ⁇ e.g., gaps may be introduced in the sequence of one nucleic acid molecule for optimal alignment with the other nucleic acid molecule).
  • nucleic acid bases at corresponding nucleotide positions are then compared and when a position in one sequence is occupied by the same nucleic acid base as the corresponding position in the other sequence, then the molecules are homologous at that position.
  • the percent homology between two sequences therefore, is a function of the number of identical positions shared by two sequences ⁇ i.e.,
  • % homology # of identical positions/total # of positions x 100).
  • Computer algorithms known in the art can be used to optimally align the two nucleotide sequences to be compared.
  • BLAST Basic Local Alignment Search Tool
  • BLAST Basic Local Alignment Search Tool
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature ⁇ e.g., encodes a natural protein).
  • an "antisense” nucleic acid comprises a nucleotide sequence which is complementary to a “sense” nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule, complementary to an mRNA sequence or complementary to the coding strand of a gene. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • coding region refers to regions of a nucleotide sequence comprising codons which are translated into amino acid residues
  • noncoding region refers to regions of a nucleotide sequence that are not translated into amino acids ⁇ e.g., 5' and 3' untranslated regions
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors ⁇ e.g., non-episomal mammalian vectors
  • vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors".
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector may be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors ⁇ e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • the term "host cell” is intended to refer to a cell into which a nucleic acid of the invention, such as a recombinant expression vector of the invention, has been introduced.
  • the terms "host cell” and “recombinant host cell” are used interchangeably herein. It should be understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • transgenic animal refers to a non-human animal, preferably a mammal, more preferably a mouse, in which one or more of the cells of the animal includes a "transgene".
  • transgene refers to exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome of the mature animal, for example directing the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal.
  • a "homologous recombinant animal” refers to a type of transgenic non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell of the animal, e.g., an embryonic cell of the animal, prior to development of the animal.
  • an “isolated protein” refers to a protein that is substantially free of other proteins, cellular material and culture medium when isolated from cells or produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • an “isolated” TRAM-1 protein of the invention is substantially free of TRAM-2 protein.
  • an “isolated” TRAM-2 protein of the invention is substantially free of TRAM- 1 protein.
  • antibody is intended to include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as Fab and F(ab') 2 fragments.
  • Arginine AGA, ACG, CGA, CGC, CGG, CGT
  • Glycine Gly, G
  • GGC GGG, GGT
  • Isoleucine (lie, I) ATA, ATC, ATT
  • Phenylalanine (Phe, F; TTC, TTT
  • Serine (Ser, S) AGC, AGT, TCA, TCC, TCG, TCT
  • nucleotide triplet An important and well known feature of the genetic code is its redundancy, whereby, for most of the amino acids used to make proteins, more than one coding nucleotide triplet may be employed (illustrated above). Therefore, a number of different nucleotide sequences may code for a given amino acid sequence. Such nucleotide sequences are considered functionally equivalent since they result in the production of the same amino acid sequence in all organisms (although certain organisms may translate some sequences more efficiently than they do others). Moreover, occasionally, a methylated variant of a purine or pyrimidine may be found in a given nucleotide sequence. Such methylations do not affect the coding relationship between the trinucleotide codon and the corresponding amino acid.
  • the nucleotide sequence of a DNA or RNA molecule coding for a TRAM protein of the invention can be use to derive the TRAM amino acid sequence, using the genetic code to translate the DNA or RNA molecule into an amino acid sequence.
  • corresponding nucleotide sequences that can encode the TRAM protein can be deduced from the genetic code (which, because of its redundancy, will produce multiple nucleic acid sequences for any given amino acid sequence).
  • description and/or disclosure herein of a TRAM nucleotide sequence should be considered to also include description and/or disclosure of the amino acid sequence encoded by the nucleotide sequence.
  • description and/or disclosure of a TRAM amino acid sequence herein should be considered to also include description and/or disclosure of all possible nucleotide sequences that can encode the amino acid sequence.
  • an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in SEQ ID NO: 1, 3 or 5.
  • the sequences of SEQ ID NO: 1 and 5 correspond to human TRAM-1 cDNAs, whereas the sequence of SEQ ID NO: 3 corresponds to a human TRAM-2 cDNA.
  • the protein encoded by SEQ ID NO: 5 is a differentially spliced form of the protein encoded by SEQ ID NO: 1 in which amino acids 3-39 (encompassing a transmembrane domain) have been deleted.
  • a TRAM-1 cDNA comprises sequences encoding the TRAM-1 protein ⁇ i.e., "the coding region", for example from nucleotides 130-690 of SEQ ID NO: 1), as well as 5' untranslated sequences ⁇ e.g., nucleotides 1 to 129 of SEQ ID NO: 1) and 3' untranslated sequences (e.g., nucleotides 691 to 1680 of SEQ ID NO: 1). In certain embodiments, however, the TRAM-1 nucleic acid molecule may comprise only the coding region of the cDNA (e.g., nucleotides 130-690 of SEQ ID NO: 1).
  • a TRAM-2 cDNA comprises sequences encoding the TRAM-2 protein (/ e .
  • the coding region for example from nucleotides 88-678 of SEQ ID NO: 3). as well as 5' untranslated sequences (e.g., nucleotides 1 to 87 of SEQ ID NO: 3 ) and 3' untranslated sequences (e.g., nucleotides 679-1235 of SEQ ID NO: 3).
  • the TRAM-2 nucleic acid molecule may comprise only the coding region of SEQ ID NO: 3 ⁇ e.g., nucleotides 88-678 of SEQ ID NO: 3).
  • the nucleic acid molecule of the invention can comprise only a portion of the coding region of SEQ ID NO: 1 or 3, for example a fragment encoding a biologically active portion of a TRAM protein.
  • biologically active portion of TRAM is intended to include portions of a TRAM protein that retain the ability to associate with the TcR/CD3/ ⁇ complex.
  • the ability of portions of a TRAM protein to associate with the TcR/CD3/ ⁇ complex can be determined in standard interaction assays, such as immunoprecipitation and immunoblotting assays such as those described further in the Examples.
  • Nucleic acid fragments encoding biologically active portions of a TRAM protein can be prepared by isolating a portion of SEQ ID NO: 1 or 3, expressing the encoded portion of the TRAM protein or peptide ⁇ e.g., by recombinant expression in a host cell) and assessing the ability of the portion to associate with the TcR/CD3/ ⁇ complex.
  • An example of a fragment of the TRAM-1 sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 5, which encodes a differentially spliced form of TRAM- 1 in which the nucleotide sequence encoding amino acids 3-39 of the TRAM-1 protein of SEQ ID NO: 2 have been deleted.
  • the amino acid sequence for this truncated form of TRAM-1 is shown in SEQ ID NO: 6.
  • an isolated nucleic acid fragment of the invention is at least 30 nucleotides in length. More preferably the fragment is at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides in length. In preferred embodiments, an isolated nucleic acid fragment of the invention comprises at least 30 contiguous nucleotides of SEQ ID NO: 1, more preferably at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 contiguous nucleotides of SEQ ID NO: 1.
  • an isolated nucleic acid fragment of the invention comprises at least 30 contiguous nucleotides of SEQ ID NO: 3, more preferably at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 contiguous nucleotides of SEQ ID NO: 3.
  • the invention further encompasses nucleic acid molecules that differ from SEQ ID NO:l or 3 (and fragments thereof) due to degeneracy of the genetic code and thus encode the same TRAM protein as that encoded by SEQ ID NO: 1 or 3. Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO: 2 or 4. Moreover, the invention encompasses nucleic acid molecules that encode portions of SEQ ID NO: 2 or 4, such as biologically active portions thereof.
  • a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l or 3, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • a human TRAM-1 cDNA can be isolated from a cDNA library ⁇ e.g., prepared from human blood cells (commercially available from Stratagene) or from human T lymphocytes or the human T cell line Jurkat) using all or portion of SEQ ID NO: 1 as a hybridization probe and standard hybridization techniques ⁇ e.g., as described in Sambrook, J., et al. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989).
  • a human TRAM-2 cDNA can be isolated using all or a portion of SEQ ID NO: 3 as a hybridization probe.
  • a nucleic acid molecule encompassing all or a portion of SEQ ID NO: 1 or 3 can be isolated by the polymerase chain reaction using oligonucleotide primers designed based upon the sequence of SEQ ID NO: 1 or 3, respectively.
  • mRNA can be isolated from human cells ⁇ e.g., by the guanidinium-thiocyanate extraction procedure of Chirgwin et al.
  • cDNA can be prepared using reverse transcriptase (e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Russia, FL).
  • reverse transcriptase e.g., Moloney MLV reverse transcriptase, available from Gibco/BRL, Bethesda, MD; or AMV reverse transcriptase, available from Seikagaku America, Inc., St. Russia, FL.
  • Synthetic oligonucleotide primers for PCR amplification can be designed based upon the nucleotide sequence shown in SEQ ID NO: 1 or 3.
  • a nucleic acid of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • oligonucleotides corresponding to a TRAM nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • TRAM-1 and TRAM-2 nucleotide sequences shown in SEQ ID NOs: 1 and 3, respectively.
  • DNA sequence polymorphisms that lead to changes in the amino acid sequences of TRAM-1 and/or TRAM-2 may exist within a population ⁇ e.g., the human population).
  • Such genetic polymorphism in TRAM genes may exist among individuals within a population due to natural allelic variation.
  • Such natural allelic variations can typically result in 1-5 % variance in the nucleotide sequence of the a gene.
  • nucleic acid molecules encoding TRAM proteins from other species and thus which have a nucleotide sequence that differs from the human sequences of SEQ ID NO: 1 and 3 but that are related to the human sequence, are intended to be within the scope of the invention.
  • Nucleic acid molecules corresponding to natural allelic variants and nonhuman homologues of the human TRAM cDNAs of the invention can be isolated based on their homology to the human TRAM nucleic acid molecules disclosed herein using the human cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under high stringency hybridization conditions. Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention hybridizes under high stringency conditions to a second nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or 3.
  • the isolated nucleic acid molecule comprises at least 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 contiguous nucleotides of SEQ ID NO: 1 or 3.
  • an isolated nucleic acid molecule of the invention that hybridizes under high stringency conditions to the sequence of SEQ ID NO: 1 or 3 corresponds to a naturally-occurring nucleic acid molecule.
  • the nucleic acid encodes a natural human TRAM protein.
  • the nucleic acid molecule encodes a natural murine homologue of human TRAM protein, such as mouse TRAM protein.
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequence of the TRAM protein (e.g., the sequence of SEQ ID NO: 2 or 4) without altering the functional activity of the TRAM protein, such as its ability to associate with the TcR/CD3/ ⁇ complex or its ability to participate in signal transduction, whereas an "essential" amino acid residue is required for functional activity.
  • Amino acid residues of TRAM proteins that are conserved among signal transduction molecules are predicted to be essential in TRAM proteins and thus are not likely to be amenable to alteration.
  • TRAM-1 and TRAM-2 each contain repeated tyrosine motifs in their cytoplasmic domains that are predicted to be sites of phosphorylation and SH2- binding regions.
  • such repeated tyrosine motifs are found at positions 58-66 and 105-113 of SEQ ID NO: 2, with Tyr 63 and Ty ] 0 being predicted sites of phosphorylation.
  • repeated tyrosine motifs are found at positions 85- 93, 122-130, 145-151 and 166-172 of SEQ ID NO: 4, with Tyr 90 , Tyr 127 , Tyr ]48 and Tyr 1 6 9 being predicted phosphorylation sites.
  • TRAM- 1 and TRAM-2 are not likely to be amenable to mutation.
  • Other amino acid residues may not be essential for TRAM activity and thus are likely to be amenable to alteration.
  • another aspect of the invention pertains to nucleic acid molecules encoding TRAM proteins that contain changes in amino acid residues that are not essential for TRAM activity, e.g., residues outside of the repeated tyrosine motifs (e.g., potential SH2 domain binding sites) and potential phosphorylation sites.
  • Such TRAM proteins differ in amino acid sequence from SEQ ID NO: 2 and 4 yet retain TRAM activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least 60 % homologous to the amino acid sequence of SEQ ID NO: 2 or 4 and associates with a TcR/CD3/ ⁇ complex in T cells.
  • the protein encoded by the nucleic acid molecule is at least 70 % homologous to SEQ ID NO: 2 or 4, more preferably at least 80 % homologous to SEQ ID NO: 2 or 4, even more preferably at least 90 % homologous to SEQ ID NO: 2 or 4, and most preferably at least 95 % homologous to SEQ ID NO: 2 or 4.
  • the isolated nucleic acid molecule comprises a nucleotide sequence at least 60 % homologous to the nucleotide sequence of SEQ ID NO: 1 or 3 and encodes a protein that associates with a TcR/CD3/ ⁇ complex in T cells.
  • the nucleotide sequence is at least 70 % homologous to SEQ ID NO: 1 or 3, more preferably at least 80 % homologous to SEQ ID NO: 1 or 3, even more preferably at least 90 % homologous to SEQ ID NO: 1 or 3, and most preferably at least 95 % homologous to SEQ ID NO: 1 or 3
  • the percent homology between two amino acid sequences e.g., SEQ ID NO: 2 or 4 and a variant form thereof
  • two nucleotide sequences e.g., SEQ ID NO: 1 or 3 and a variant form thereof
  • An isolated nucleic acid molecule encoding a TRAM protein homologous to the protein of SEQ ID NO: 2 or 4 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NO: 1 or 3 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NO: 1 or 3 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Preferably, conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains ⁇ e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine.
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains ⁇ e.g., glycine, asparagine, glutamine, serine,
  • a predicted nonessential amino acid residue in a TRAM protein is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a TRAM coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for their ability to associate with the TcR/CD3/ ⁇ complex to identify mutants that retain the ability to interact with the complex.
  • the encoded mutant protein can be expressed recombinantly in a host cell and the ability of the mutant protein to associate with the TcR/CD3/ ⁇ complex can be determined using interaction assay such as those described in the Examples.
  • Another aspect of the invention pertains to isolated nucleic acid molecules that are related to the TRAM-1 and TRAM-2 nucleic acid molecules disclosed herein and that are obtainable using processes that utilize the nucleic acid molecules, or portions thereof, disclosed herein.
  • the invention provides an isolated nucleic acid molecule obtainable by a process comprising:
  • nucleic acid molecule from the selected population that is at least 1000 nucleotides in length and whose nucleotide sequence is at least 60% homologous to the nucleotide sequence of SEQ ID NO: 1 or 3.
  • step (d) comprises selecting a nucleic acid molecule within the selected population that is at least 1000 nucleotides in length and whose nucleotide sequence is at least 70%, 80%, 90% or 95% homologous to the nucleotide sequence of SEQ ID NO: 1 or 3.
  • the probe/primer can be at least 20, 25, 30, 35, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides in length.
  • the invention also provides an isolated nucleic acid molecule obtainable by a process comprising:
  • nucleic acid molecule from the selected population that is at least 1000 nucleotides in length and whose nucleotide sequence is at least 60% homologous to the nucleotide sequence of SEQ ID NO: 1 or 3.
  • step (d) comprises selecting a nucleic acid molecule within the selected population that is at least 1000 nucleotides in length and whose nucleotide sequence is at least 70%, 80%, 90% or 95% homologous to the nucleotide sequence of SEQ ID NO: 1 or 3.
  • the probe used in step (a) comprises at least 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 contiguous nucleotides of SEQ ID NO: 1 or 3.
  • the invention still further provides an isolated nucleic acid molecule obtainable by a process comprising:
  • first and second primers are a degenerate oligonucleotide primer comprising a nucleotide sequence encoding an amino acid sequence shown in SEQ ID NO: 2 or 4, said first and second primers being at least 15 nucleotides in length;
  • nucleic acid molecule within the selected population that is at least 1000 nucleotides in length and whose nucleotide sequence is at least 60% homologous to the entire nucleotide sequence of SEQ ID NO: 1 or 3.
  • step (d) comprises selecting a nucleic acid molecule within the selected population that is at least 1000 nucleotides in length and whose nucleotide sequence is at least 70%, 80%, 90% or 95% homologous to the nucleotide sequence of SEQ ID NO: 1 or 3.
  • first and/or second primers used in step (a) are at least 20, 25, 30, 35, 40 or 50 nucleotides in length.
  • the degenerate oligonucleotide primer encodes an amino sequence shown within about amino acid positions 1-20 of SEQ ID NO: 2, 4 or 6
  • the degenerate oligonucleotide primer encodes an amino sequence shown within about amino acid positions 166-186 of SEQ ID NO: 2, 176-196 of SEQ ID NO: 4 or 129-149 of SEQ ID NO: 6 (i.e., the carboxy terminal end of SEQ ID NO: 2, 4, or 6).
  • both the first and second primers used in the process are degenerate oligonucleotide primers encoding sequences shown in SEQ ID NO: 2, 4 or 6, preferably wherein the first primer encodes a sequence at the amino terminal end of SEQ ID NO: 2, 4 or 6 and the second primer encodes a sequence at the carboxy terminal end of SEQ ID NO: 2, 4 or 6.
  • the first and/or second primers have a nucleotide sequence found at the 5' end of SEQ ID NO: 1, 3, or 5 (e.g., within the first 60 nucleotides of SEQ ID NO: 1, 3 or 5) or a nucleotide sequence found at the 3' end of SEQ ID NO: 1, 3, or 5 (e.g., within the last 60 nucleotides of SEQ ID NO: 1, 3 or 5).
  • the first primer has a nucleotide sequence from the 5' end of SEQ ID NO: 1, 3 or 5 (e.g., within the first 60 nucleotides) and the second primer has a nucleotide sequence from the 3' end of SEQ ID NO: 1, 3 or 5 (e.g., within the last 60 nucleotides).
  • Probes/primers to be used in the above-described processes can be prepared based on the nucleotide sequences provided herein using standard molecular biology techniques.
  • the sample population of nucleic acid molecules used in step (b) of the processes can be, for example, a pool of mRNAs, a cDNA library or a genomic DNA library, which can be prepared according to standard molecular biology techniques.
  • Hybridization of a probe to the sample population and isolation of molecules that hybridize under high stringency conditions can be performed as described hereinbefore and using hybridization methods well known in the art.
  • amplification of sequences within the sample population using first and second primers can be performed as described hereinbefore and using PCR methods well known in the art.
  • the other primer is a "docking" primer that is complimentary to a fixed sequence within the sample population, such as an oligo dT primer or a primer that hybridizes to fixed vector sequences within the sample population.
  • Preferred PCR methods for use in the processes include 5'- and 3'-RACE.
  • nucleotide sequences of the nucleic acid molecules within the selected population can be determined by, for example, dideoxynucleotide sequencing (manual or automated) or other well known techniques for DNA sequencing. Finally, the degree of homology between nucleotide sequences within the selected population and either SEQ ID NO: 1 or 3 can be determined as described hereinbefore to thus allow for isolation of nucleic acid molecules related to SEQ ID NO: 1 or 3.
  • Another aspect of the invention pertains to isolated nucleic acid molecules that are antisense to the coding strand of a TRAM mRNA or gene.
  • An antisense nucleic acid of the invention can be complementary to an entire TRAM coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a coding region of the coding strand of a nucleotide sequence encoding TRAM-1 (e.g., the entire coding region of SEQ ID NO: 1) or TRAM-2 (e.g., the entire coding region of SEQ ID NO: 3).
  • the antisense nucleic acid molecule is antisense to a noncoding region of the coding strand of a nucleotide sequence encoding TRAM-1 or TRAM-2.
  • an antisense nucleic acid of the invention is at least 300, nucleotides in length. More preferably, the antisense nucleic acid is at least 400, 500, 600, 700, 800, 900 or 1000 nucleotides in length. In preferred embodiments, an antisense of the invention comprises at least 300 contiguous nucleotides of the noncoding strand of SEQ ID NO: 1, more preferably at least 400, 500, 600, 700, 800, 900 or 1000 contiguous nucleotides of the noncoding strand of SEQ ID NO: 1.
  • an isolated nucleic acid fragment of the invention comprises at least 300 contiguous nucleotides of the noncoding strand of SEQ ID NO: 3, more preferably at least 400, 500, 600, 700, 800, 900 or 1000 contiguous nucleotides of the noncoding strand SEQ ID NO: 3.
  • antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing.
  • the antisense nucleic acid molecule may be complementary to the entire coding region of a TRAM mRNA, or alternatively can be an oligonucleotide which is antisense to only a portion of the coding or noncoding region of TRAM mRNA.
  • the antisense oligonucleotide may be complementary to the region surrounding the translation start site of TRAM mRNA.
  • An antisense oligonucleotide can be, for example, about 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid ⁇ e.g., an antisense oligonucleotide can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation ⁇ i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • an antisense nucleic acid of the invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • a ribozyme having specificity for a TRAM-encoding nucleic acid can be designed based upon the nucleotide sequence of a TRAM cDNA disclosed herein ⁇ i.e., SEQ ID NO: 1 or 3).
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the base sequence of the active site is complementary to the base sequence to be cleaved in a TRAM-encoding mRNA.
  • TRAM mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See for example Bartel, D. and Szostak, J.W. (1993) Science 261 : 1411-1418.
  • nucleic acid molecules encoding TRAM fusion proteins.
  • Such nucleic acid molecules comprising at least a first nucleotide sequence encoding a TRAM protein, polypeptide or peptide operatively linked to a second nucleotide sequence encoding a non-TRAM protein, polypeptide or peptide, can be prepared by standard recombinant DNA techniques. TRAM fusion proteins are described in further detail below in subsection IV.
  • the expression vectors of the invention comprise a nucleic acid of the invention in a form that allows for expression of the nucleic acid in a host cell under appropriate conditions, which means that the recombinant expression vectors include one or more regulatory sequences, typically selected on the basis of the host cells to be used for expression, which are operatively linked to the nucleic acid sequence to be expressed.
  • the regulatory sequences of the expression vector direct the transcription of the TRAM-encoding nucleic acid molecule in a host cell carrying the expression vector.
  • the term "operably linked” is intended to mean that the nucleotide sequence of interest (i.e., TRAM-encoding sequence) is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • the term "regulatory sequence” is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell (i.e., the nucleotide sequence is expressed in the host cell upon introduction of the expression vector into the host cell) and those which direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences) or under certain conditions (e.g., inducible regulatory sequences). It will be appreciated by those skilled in the art that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., TRAM proteins, mutant forms of TRAM proteins, TRAM fusion proteins and the like).
  • the recombinant expression vectors of the invention can be designed for expression of TRAM proteins in prokaryotic or eukaryotic cells.
  • TRAM proteins can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector may be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein.
  • Such fusion vectors typically serve three purposes: 1) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S.
  • GST glutathione S-transferase
  • Suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al, (1988) Gene 69:301-315) and pET 1 Id (Studier et al. Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid trp-lac fusion promoter.
  • Target gene expression from the pET 1 Id vector relies on transcription from a T7 gnlO-lac fusion promoter mediated by a coexpressed viral RNA polymerase (T7 gnl). This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident ⁇ prophage harboring a T7 gnl gene under the transcriptional control of the lacUV 5 promoter.
  • nucleic acid sequence of the nucleic acid is altered so that the individual codons for each amino acid are those preferentially utilized in E. coli (Wada et al., (1992) Nuc. Acids Res. 20:21 11-2118).
  • Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.
  • the TRAM expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerivisae include pYepSecl (Baldari. et al, (1987) EMBOJ. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al, (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA).
  • TRAM proteins can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells ⁇ e.g., Sf 9 cells include the pAc series (Smith et al, (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow, V.A., and Summers, M.D., (1989) Virology 170:31-39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBO J. 6:187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type ⁇ e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43 . :235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J.
  • Patent No. 4,873,316 and European Application Publication No. 264,166 Developmentally-regulated promoters are also encompassed, for example the murine hox promoters (Kessel and Gruss (1990) Science 249:374-379) and the ⁇ -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
  • the expression vector is capable of directing expression of the nucleic acid in cells only under certain conditions (e.g., inducible regulatory elements are used to express the nucleic acid).
  • inducible regulatory systems for use in mammalian cells are known in the art, for example systems in which gene expression is regulated by heavy metal ions (see e.g.. Mayo el al (1982) Cell 29:99-108; Brinster et al. (1982) Nature 296:39-42; Searle et al. ( 1985) Mol Cell Biol 5:1480- 1489), heat shock (see e.g., Nouer et al. (1991) in Heat Shock Response, e.d. Nouer, L.
  • the invention further provides a recombinant expression vector comprising a DNA molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription of the DNA molecule) of an RNA molecule which is antisense to a TRAM-1 or TRAM-2 mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • a host cell may be any prokaryotic or eukaryotic cell.
  • TRAM-1 or TRAM-2 protein may be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • CHO Chinese hamster ovary cells
  • COS cells Chinese hamster ovary cells
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and transfection are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. ⁇ Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drugs, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker may be introduced into a host cell on the same vector as that encoding TRAM-1 or TRAM-2 or may be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive, while the other cells die).
  • a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce ⁇ i.e., express) TRAM proteins.
  • the invention further provides methods for producing a TRAM protein using the host cells of the invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding a TRAM protein has been introduced) in a suitable medium until a TRAM protein is produced.
  • the method further comprises isolating the TRAM protein from the medium or the host cell.
  • Recombinant TRAM-1 or TRAM-2 proteins that include the transmembrane domain can be expressed as integral membrane proteins in a recombinant host cell.
  • recombinant forms of TRAM proteins that lack the transmembrane domain e.g., the protein of SEQ ID NO: 6
  • a recombinant TRAM protein can be isolated from the host cell, e.g., by lysing the host cell and recovering the recombinant TRAM protein from the lysate.
  • a host cell of the invention can also be used to produce nonhuman transgenic animals.
  • a host cell of the invention is a fertilized oocyte or an embryonic stem cell into which TRAM-coding sequences have been introduced.
  • Such host cells can then be used to create non-human transgenic animals in which exogenous TRAM sequences have been introduced into their genome or homologous recombinant animals in which endogenous TRAM sequences have been altered.
  • Such animals are useful for studying the function and/or activity of TRAM-1 or TRAM-2 and for identifying and/or evaluating modulators of TRAM- 1 or TRAM-2 activity.
  • another aspect of the invention pertains to nonhuman transgenic animals which contain cells carrying a transgene encoding a TRAM protein or a portion of a TRAM protein.
  • the transgene alters an endogenous gene encoding an endogenous TRAM protein (e.g. , homologous recombinant animals in which an endogenous TRAM gene has been functionally disrupted or "knocked out", or the nucleotide sequence of the endogenous TRAM gene has been mutated or the transcriptional regulatory region of the endogenous TRAM gene has been altered).
  • a transgenic animal of the invention can be created by introducing TRAM-1- or
  • TRAM-2-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the human TRAM-1 cDNA sequence of SEQ ID NO: 1 or the human TRAM-2 cDNA of SEQ ID NO: 3 can be introduced as a transgene into the genome of a nonhuman animal.
  • a nonhuman homologue of the human TRAM-1 or TRAM-2 gene such as a mouse TRAM-1 or TRAM-2 gene, can be isolated based on hybridization to the human TRAM-1 or TRAM-2 cDNA and used as a transgene.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression of the transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to the TRAM transgene to direct expression of TRAM protein to particular cells.
  • transgenic animals via embryo manipulation and microinjection, particularly animals such as mice
  • methods for generating transgenic animals via embryo manipulation and microinjection have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, both by Leder et al, U.S. Patent No. 4,873,191 by Wagner et al. and in Hogan, B., Manipulating tbe Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar methods are used for production of other transgenic animals.
  • a transgenic founder animal can be identified based upon the presence of the TRAM transgene in its genome and/or expression of TRAM mRNA in tissues or cells of the animals.
  • transgenic founder animal can then be used to breed additional animals carrying the transgene.
  • transgenic animals carrying a transgene encoding a TRAM protein can further be bred to other transgenic animals carrying other transgenes.
  • a vector is prepared which contains at least a portion of a TRAM- 1 or TRAM-2 gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, an endogenous TRAM gene.
  • the TRAM gene may be a human gene (e.g.
  • a mouse TRAM-1 or TRAM-2 gene can be isolated from a mouse genomic DNA library using the human TRAM-1 cDNA of SEQ ID NO: 1 or the human TRAM-2 cDNA of SEQ ID NO: 3 as a probe.
  • the mouse TRAM gene then can be used to construct a homologous recombination vector suitable for altering an endogenous TRAM gene in the mouse genome.
  • the vector is designed such that, upon homologous recombination, the endogenous TRAM gene is functionally disrupted ⁇ i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous TRAM gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of the endogenous TRAM protein).
  • the altered portion of the TRAM gene is flanked at its 5' and 3' ends by additional nucleic acid of the TRAM gene to allow for homologous recombination to occur between the exogenous TRAM gene carried by the vector and an endogenous TRAM gene in an embryonic stem cell.
  • the additional flanking TRAM nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5' and 3' ends
  • are included in the vector see e.g., Thomas, K.R. and Capecchi, M. R. (1987) Cell 5 _:503 for a description of homologous recombination vectors).
  • the vector is introduced into an embryonic stem cell line ⁇ e.g., by electroporation) and cells in which the introduced TRAM gene has homologously recombined with the endogenous TRAM gene are selected (see e.g., Li, E. et al. (1992) Cell 69:915).
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A. in
  • the invention provides an isolated preparation of a TRAM protein.
  • the TRAM protein has an amino acid sequence shown in SEQ ID NO: 2, 4 or 6.
  • the TRAM protein is substantially homologous to SEQ ID NO: 2 or 4 and retains the functional activity of the protein of SEQ ID NO: 2 or 4 yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above.
  • the TRAM protein is a protein which comprises an amino acid sequence at least 60 % homologous to the amino acid sequence of SEQ ID NO: 2 or 4 and associates with the TcR/CD3/ ⁇ complex.
  • the protein is at least 70 % homologous to SEQ ID NO: 2 or 4, more preferably at least 80 % homologous to SEQ ID NO: 2 or 4, even more preferably at least 90 % homologous to SEQ ID NO: 2 or 4, and most preferably at least 95 % homologous to SEQ ID NO: 2 or 4.
  • TRAM proteins are preferably produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding the protein is cloned into an expression vector (as described above), the expression vector is introduced into a host cell (as described above) and the TRAM protein is expressed in the host cell. The TRAM protein can then be isolated from the cells by an appropriate purification scheme using standard protein purification techniques. Alternative to recombinant expression, a TRAM polypeptide can be synthesized chemically using standard peptide synthesis techniques. Moreover, native TRAM protein can be isolated from cells (e.g., human T cells or HPB-ALL cells) (e.g., as described in Example 2) or by immunoprecipitation using an anti-TRAM antibody (discussed further below).
  • cells e.g., human T cells or HPB-ALL cells
  • an anti-TRAM antibody discussed further below.
  • a TRAM "fusion protein” comprises a TRAM polypeptide operatively linked to a non-TRAM polypeptide.
  • a "TRAM polypeptide” refers to a polypeptide having an amino acid sequence from a TRAM protein
  • a non-TRAM polypeptide refers to a polypeptide having an amino acid sequence from another protein.
  • the term "operatively linked” is intended to indicate that the TRAM polypeptide and the non-TRAM polypeptide are fused in-frame to each other.
  • the non-TRAM polypeptide may be fused to the N-terminus or C-terminus of the TRAM polypeptide.
  • the fusion protein is a glutathione-S-transferase
  • TRAM-TRAM fusion protein in which the TRAM sequences are fused to the C-terminus of the GST sequences.
  • fusion proteins can facilitate the purification of recombinant TRAM proteins.
  • the fusion protein can comprise only a portion of the TRAM protein, such as an SH2 binding domain.
  • a TRAM fusion protein of the invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a TRAM-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the TRAM polypeptide.
  • An isolated TRAM protein, or fragment thereof, can be used as an immunogen to generate antibodies that bind the TRAM protein using standard techniques for polyclonal and monoclonal antibody preparation.
  • a TRAM-1 or TRAM-2 protein can be used to generate antibodies or, alternatively, an antigenic peptide fragment of TRAM - 1 or TRAM-2 can be used as the immunogen.
  • An antigenic peptide fragment of a TRAM protein typically comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 or 4 and encompasses an epitope of TRAM- 1 or TRAM-2 such that an antibody raised against the peptide forms a specific immune complex with TRAM-1 or TRAM-2.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of the TRAM protein that are located on the surface of the protein, e.g., hydrophilic regions. A standard hydrophobicity analysis of the TRAM-1 protein sequence shown in SEQ ID NO: 2 or the TRAM-2 protein sequence shown in SEQ ID NO: 4 can be performed to identify such hydrophilic regions.
  • TRAM- 1 peptides that can be used as immuogens to elicit anti-TRAM- 1 antibodies are peptides encompassing amino acid positions 174-179 or 152-173 of SEQ ID NO: 2.
  • An example of TRAM-2 peptide that can be used as an immunogen to elicit anti-TRAM-2 antibodies is a peptide encompassing amino acid positions 148-158 of SEQ ID NO: 4.
  • a TRAM immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for examples, recombinantly expressed TRAM protein or a chemically synthesized TRAM peptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.
  • an adjuvant such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent.
  • Immunization of a suitable subject with an immunogenic TRAM preparation induces a polyclonal anti-TRAM antibody response.
  • another aspect of the invention pertains to anti-TRAM- 1 or anti- TRAM-2 antibodies.
  • Polyclonal anti-TRAM antibodies can be prepared as described above by immunizing a suitable subject with a TRAM immunogen.
  • the anti-TRAM antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized TRAM protein.
  • ELISA enzyme linked immunosorbent assay
  • the antibody molecules directed against TRAM can be isolated from the mammal (e.g.
  • antibody -producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol 127:539-46; Brown et al. (1980) J Biol Chem 255:4980-83; Yeh et al. (1976) PNAS 76:2927-31; and Yeh et al.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds a TRAM protein.
  • the immortal cell line ⁇ e.g., a myeloma cell line
  • the immortal cell line is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation of the present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • HAT medium culture medium containing hypoxanthine, aminopterin and thymidine
  • Any of a number of myeloma cell lines may be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma lines are available from the American Type Culture Collection (ATCC), Rockville, Md.
  • ATCC American Type Culture Collection
  • HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG").
  • PEG polyethylene glycol
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody of the invention are detected by screening the hybridoma culture supernatants for antibodies that bind a TRAM protein, e.g., using a standard ELISA assay.
  • a monoclonal anti-TRAM antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with a TRAM protein to thereby isolate immunoglobulin library members that bind the TRAM protein.
  • Kits for generating and screening phage display libraries are commercially available ⁇ e.g., the Pharmacia Recombinant Phage Antibody System. Catalog No. 27-9400-01; and the Stratagene Sur/ZAPTM Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. International Publication No. WO 92/18619; Dower et al. International Publication No. WO 91/17271 ; Winter et al. International Publication WO 92/20791 ; Markland et al. International Publication No. WO 92/15679; Breitling et al. International Publication WO 93/01288; McCafferty et al International Publication No. WO 92/01047; Garrard et al. International Publication No.
  • recombinant anti-TRAM antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non-human portions, which can be made using standard recombinant DNA techniques, are within the scope of the invention.
  • chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al.
  • an anti-TRAM antibodies can be used to isolate TRAM proteins by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-TRAM antibodies can facilitate the purification of natural TRAM proteins from cells and of recombinantly produced TRAM proteins expressed in host cells.
  • an anti-TRAM antibody can be used to detect TRAM protein ⁇ e.g., in a cellular lysate or cell supernatant). Detection may be facilitated by coupling ⁇ i.e., physically linking) the antibody to a detectable substance.
  • anti-TRAM antibodies of the invention are labeled with a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ - galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • suitable radioactive material include 125 !, 131 I, 35 S or 3 H.
  • anti-TRAM antibodies i.e., antibodies that specifically bind a TRAM-1 or TRAM-2 protein
  • compositions suitable for administration can be incorporated into pharmaceutical compositions suitable for administration.
  • Such compositions typically comprise the protein or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents
  • antibacterial agents such as benzyl alcohol or methyl parabens
  • antioxidants such as ascorbic acid or sodium bisulfite
  • chelating agents such as ethylene
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor EL ⁇ M (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a TRAM protein or anti-TRAM antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., a TRAM protein or anti-TRAM antibody
  • dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
  • the tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth or gelatin
  • an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch
  • a lubricant such as magnesium stearate or Sterotes
  • a glidant such as colloidal silicon dioxide
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4.522,81 1.
  • the invention provides a method for detecting the presence of TRAM activity in a biological sample.
  • the method involves contacting the biological sample with an agent capable of specifically detecting TRAM activity (e.g., an agent that specifically detects TRAM-1 or TRAM-2 protein or an agent that specifically detects TRAM-1 or TRAM-2 mRNA) such that the presence of TRAM activity is detected in the biological sample.
  • an agent capable of specifically detecting TRAM activity e.g., an agent that specifically detects TRAM-1 or TRAM-2 protein or an agent that specifically detects TRAM-1 or TRAM-2 mRNA
  • a preferred agent for detecting TRAM-1 mRNA is a labeled nucleic acid probe capable of hybridizing to TRAM-1 mRNA.
  • the nucleic acid probe can be, for example, the TRAM-1 cDNA of SEQ ID NO: 1, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides in length and sufficient to specifically hybridize under high stringency conditions to TRAM-1 mRNA.
  • a preferred agent for detecting TRAM-2 mRNA is a labeled nucleic acid probe capable of hybridizing to TRAM-2 mRNA.
  • the nucleic acid probe can be, for example, the TRAM-2 cDNA of SEQ ID NO: 3, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides in length and sufficient to specifically hybridize under high stringency conditions to TRAM-2 mRNA.
  • a preferred agent for detecting TRAM-1 protein is a labeled antibody capable of specifically binding to TRAM-1 protein.
  • a preferred agent for detecting TRAM-2 protein is a labeled antibody capable of specifically binding to TRAM-2 protein.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab') 2 ) can be used.
  • the term "labeled", with regard to the probe or antibody is intended to encompass direct labeling of the probe or antibody by coupling ⁇ i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids.
  • Methodologies known in the art that can be used for detection of TRAM activity by detecting TRAM nucleic acid include Northern hybridizations and in situ hybridizations.
  • Methodologies known in the art that can be used for detection of TRAM activity by detecting TRAM protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • the invention further provides methods for identifying agents that modulate an interaction between a TRAM protein and a T cell molecule (i.e., screening assays for TRAM modulatory agents).
  • the method comprises:
  • the TRAM protein is a TRAM-1 protein.
  • the TRAM protein is a TRAM-2 protein.
  • T cell molecules that can be used in the assay include CD2, CD3, CD4, CD5, CD8, p56 lck and p59 f y n . Isolated TRAM proteins and T cell molecules may be used in the method, or, alternatively, only portions of the TRAM protein and or T cell molecules may be used.
  • an isolated Lck or Fyn SH2 domain (or a larger subregion of Lck or Fyn that includes the SH2 domain) can be used as the T cell molecule that interacts with the TRAM protein.
  • an isolated SH2 binding domain of TRAM- 1 or TRAM-2 can be used as the TRAM protein that interacts with the T cell molecule.
  • one or both of (i) and (ii) are fusion proteins, such as GST fusion proteins
  • the degree of interaction between (i) and (ii) can be determined, for example, by labeling one of the proteins with a detectable substance ⁇ e.g., a radiolabel), isolating the non-labeled protein and quantitating the amount of detectable substance that has become associated with the non-labeled protein.
  • the assay can be used to identify agents that either stimulate or inhibit the interaction between the TRAM protein and the T cell molecule.
  • An agent that stimulates the interaction between a TRAM protein and a T cell molecule is identified based upon its ability to increase the degree of interaction between (i) and (ii) as compared to the degree of interaction in the absence of the agent, whereas an agent that inhibits the interaction between a TRAM protein and a T cell molecule is identified based upon its ability to decrease the degree of interaction between (i) and (ii) as compared to the degree of interaction in the absence of the agent.
  • Assays systems for identifying agents that modulate SH2 domain-ligand interactions that can be adapted to the TRAM proteins of the invention, and the SH2 domains of Lck and Fyn, in accordance with the present invention are described further in U.S. Patent No. 5,352,660 by Pawson.
  • Yet another aspect of the invention pertains to methods of modulating TRAM activity in a cell.
  • the modulatory methods of the invention involve contacting the cell with an agent that specifically modulates TRAM activity such that TRAM activity in the cell is modulated.
  • the agent may act by modulating the activity of TRAM protein in the cell or by modulating transcription of the TRAM gene or translation of the TRAM mRNA.
  • modulating is intended to include inhibiting or decreasing TRAM activity and stimulating or increasing TRAM activity.
  • a modulatory agent of the invention inhibits TRAM activity.
  • An. inhibitory agent may function, for example, by directly inhibiting TRAM activity, by inhibiting an interaction between TRAM and a T cell molecule, by inhibiting TRAM-mediated signaling, and/or by inhibiting TcR/CD3/ ⁇ /TRAM-mediated signaling.
  • the agent stimulates TRAM activity.
  • a stimulatory agent may function, for example, by directly stimulating TRAM activity, by promoting an interaction between TRAM and a T cell molecule, by promoting TRAM-mediated signaling, and/or by promoting TcR/CD3/ ⁇ /TRAM-mediated signaling.
  • TRAM activity is inhibited in a cell by contacting the cell with an inhibitory agent.
  • Inhibitory agents of the invention can be, for example, intracellular binding molecules that act to inhibit the expression or activity of a TRAM protein.
  • intracellular binding molecule is intended to include molecules that act intracellularly to inhibit the expression or activity of a protein by binding to the protein itself, to a nucleic acid (e.g., an mRNA molecule) that encodes the protein or to a second protein with which the first protein normally interacts.
  • intracellular binding molecules examples include antisense TRAM nucleic acid molecules (e.g., to inhibit translation of TRAM- 1 or TRAM-2 mRNA), intracellular anti-TRAM- 1 or TRAM-2 antibodies ⁇ e.g., to inhibit the activity of TRAM- 1 or TRAM-2 protein), molecules that mimic an SH2 binding site of TRAM- 1 or TRAM-2 (e.g., to inhibit the interaction of TRAM-1 or TRAM-2 with an SH2 domain, such as the Fyn or Lck SH2 domain) and dominant negative mutants of a TRAM- 1 or TRAM-2 protein.
  • antisense TRAM nucleic acid molecules e.g., to inhibit translation of TRAM- 1 or TRAM-2 mRNA
  • intracellular anti-TRAM- 1 or TRAM-2 antibodies ⁇ e.g., to inhibit the activity of TRAM- 1 or TRAM-2 protein
  • molecules that mimic an SH2 binding site of TRAM- 1 or TRAM-2 e.g., to inhibit the interaction of TRAM-1 or TRAM-2 with an SH
  • an inhibitory agent of the invention is an antisense nucleic acid molecule that is complementary to a gene encoding a TRAM protein, or to a portion of said gene, or a recombinant expression vector encoding said antisense nucleic acid molecule.
  • antisense nucleic acids to downregulate the expression of a particular protein in a cell is well known in the art (see e.g., Weintraub, H. et al, Antisense RNA as a molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1) 1986; Askari, F.K. and McDonnell, W.M. (1996) N. Eng. J. Med.
  • An antisense nucleic acid molecule comprises a nucleotide sequence that is complementary to the coding strand of another nucleic acid molecule (e.g., an mRNA sequence) and accordingly is capable of hydrogen bonding to the coding strand of the other nucleic acid molecule.
  • Antisense sequences complementary to a sequence of an mRNA can be complementary to a sequence found in the coding region of the mRNA, the 5' or 3' untranslated region of the mRNA or a region bridging the coding region and an untranslated region ⁇ e.g., at the junction of the 5' untranslated region and the coding region).
  • an antisense nucleic acid can be complementary in sequence to a regulatory region of the gene encoding the mRNA, for instance a transcription initiation sequence or regulatory element.
  • an antisense nucleic acid is designed so as to be complementary to a region preceding or spanning the initiation codon on the coding strand or in the 3' untranslated region of an mRNA.
  • An antisense nucleic acid for inhibiting the expression of TRAM- 1 protein in a cell can be designed based upon the nucleotide sequence encoding the TRAM-1 protein (e.g., SEQ ID NO: 1, or a portion thereof), constructed according to the rules of Watson and Crick base pairing.
  • An antisense nucleic acid for inhibiting the expression of TRAM-2 protein in a cell can be designed based upon the nucleotide sequence encoding the TRAM-2 protein (e.g., SEQ ID NO: 3, or a portion thereof), constructed according to the rules of Watson and Crick base pairing.
  • an antisense nucleic acid can exist in a variety of different forms.
  • the antisense nucleic acid can be an oligonucleotide that is complementary to only a portion of a TRAM gene.
  • An antisense oligonucleotides can be constructed using chemical synthesis procedures known in the art.
  • An antisense oligonucleotide can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g. phosphorothioate derivatives and acridine substituted nucleotides can be used.
  • one or more antisense oligonucleotides can be added to cells in culture media, typically at 200 ⁇ g oligonucleotide/ml.
  • an antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation ⁇ i.e., nucleic acid transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).
  • Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the expression of the antisense RNA molecule in a cell of interest, for instance promoters and/or enhancers or other regulatory sequences can be chosen which direct constitutive, tissue specific or inducible expression of antisense RNA.
  • the antisense expression vector is prepared as described above for recombinant expression vectors, except that the cDNA (or portion thereof) is cloned into the vector in the antisense orientation.
  • the antisense expression vector can be in the form of, for example, a recombinant plasmid, phagemid or attenuated virus.
  • the antisense expression vector is introduced into cells using a standard transfection technique, as described above for recombinant expression vectors.
  • an antisense nucleic acid for use as an inhibitory agent is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region (for reviews on ribozymes see e.g., Ohkawa, J. et al. (1995) J. Biochem. 118:251-258; NASAdsson, S.T. and Eckstein, F. (1995) Trends Biotechnol 13:286-289; Rossi, J.J. (1995) Trends Biotechnol. 13:301-306; Kiehntopf, M.
  • a ribozyme having specificity for a TRAM mRNA can be designed based upon the nucleotide sequence of a TRAM- 1 or TRAM-2 cDNA.
  • a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the base sequence of the active site is complementary to the base sequence to be cleaved in a TRAM-1 or TRAM-2 mRNA. See for example U.S. Patent Nos. 4,987,071 and 5,1 16,742, both by Cech et al.
  • TRAM-1 or TRAM-2 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See for example Bartel, D. and Szostak, J.W. (1993) Science 261 : 1411-1418.
  • inhibitory agent that can be used to inhibit the expression and/or activity of a TRAM protein in a cell is an intracellular antibody specific for the TRAM protein.
  • intracellular antibodies to inhibit protein function in a cell is known in the art (see e.g., Carlson, J. R. (1988) Mol. Cell. Biol. 8:2638-2646; Biocca, S. et al. (1990) EMBOJ. 9:101-108; Werge, T.M. et al. (1990) FEBS Letters 274:193-198; Carlson, J.R. (1993) Proc. Natl. Acad. Sci. USA 90:7427-7428; Marasco, W.A. et al.
  • a recombinant expression vector which encodes the antibody chains in a form such that, upon introduction of the vector into a cell, the antibody chains are expressed as a functional antibody in an intracellular compartment of the cell.
  • an intracellular antibody that specifically binds the TRAM protein is expressed in the cytoplasm of the cell.
  • antibody light and heavy chain cDNAs encoding antibody chains specific for the target protein of interest e.g.. TRAM- 1 or TRAM-2, are isolated, typically from a hybridoma that secretes a monoclonal antibody specific for the TRAM protein.
  • Hybridomas secreting anti-TRAM monoclonal antibodies, or recombinant anti-TRAM monoclonal antibodies can be prepared as described above.
  • a monoclonal antibody specific for a TRAM protein ⁇ e.g., either a hybridoma-derived monoclonal antibody or a recombinant antibody from a combinatorial library
  • DNAs encoding the light and heavy chains of the monoclonal antibody are isolated by standard molecular biology techniques.
  • light and heavy chain cDNAs can be obtained, for example, by PCR amplification or cDNA library screening.
  • cDNA encoding the light and heavy chains can be recovered from the display package ⁇ e.g., phage) isolated during the library screening process.
  • Nucleotide sequences of antibody light and heavy chain genes from which PCR primers or cDNA library probes can be prepared are known in the art. For example, many such sequences are disclosed in Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition. U.S. Department of Health and Human Services, NIH Publication No. 91-3242 and in the "Vbase" human germline sequence database.
  • an intracellular antibody expression vector can encode an intracellular antibody in one of several different forms. For example, in one embodiment, the vector encodes full-length antibody light and heavy chains such that a full-length antibody is expressed intracellularly. In another embodiment, the vector encodes a full-length light chain but only the VH/CH1 region of the heavy chain such that a Fab fragment is expressed intracellularly.
  • the vector encodes a single chain antibody (scFv) wherein the variable regions of the light and heavy chains are linked by a flexible peptide linker ⁇ e.g., (Gly 4 Ser)3) and expressed as a single chain molecule.
  • scFv single chain antibody
  • the expression vector encoding the anti-TRAM intracellular antibody is introduced into the cell by standard transfection methods, as discussed hereinbefore.
  • Other inhibitory agents that can be used to inhibit the activity of a TRAM protein are chemical compounds that inhibit the interaction between TRAM and a T cell molecule (e.g., CD2, CD3, CD4, CD5, CD8, Fyn or Lck). Such compounds can be identified using screening assays that select for such compounds, as described in detail above.
  • SH2 domains are known to interact with phosphotyrosine-containing peptides, with the specificity of a particular SH2 domain for a target binding site being influenced by the amino acid residues surrounding the phosphotyrosine residue (see e.g., Songyang, Z. et al. (1993) Cell 72:767-778).
  • potential SH2 binding sites within TRAM-1 are predicted to comprise amino acid positions 63-66 and 1 10-113 of SEQ ID NO: 2.
  • potential SH2 binding sites within TRAM-2 are predicted to comprise amino acid positions 85-93, 122-130, 145-151 and 166-172 of SEQ ID NO: 4.
  • a competitive inhibitor of TRAM interactions with SH2 domains can be designed based on the amino acid sequence(s) of an SH2 binding site(s) of TRAM- 1 or TRAM-2, such as those described above.
  • such an inhibitory molecule comprises a nonhydrolyzable phosphonopeptide having an appropriate amino acid sequence for recognition by an SH2 domain.
  • the tyrosine residue within the SH2 binding site is replaced with phosphonomethyl-phenylalanine (Pmp), a nonnatural analogue of phosphotyrosine that is resistant to hydrolysis by phosphatases.
  • Pmp phosphonomethyl-phenylalanine
  • Nonhydrolyzable phosphonopeptide inhibitors of SH2 domain interactions can be prepared as described in Domchek, S.M. et al.
  • an inhibitory molecule can comprise a peptidomimetic of the SH2 binding site, such as a benzodiazepine mimetic of a dipeptidyl amide backbone or a boronotyrosine-containing analogue of the phosphotyrosine-containing SH2 binding site (e.g.
  • an inhibitory agent of the invention is an inhibitory form of a TRAM protein, also referred to herein as a dominant negative inhibitor.
  • a dominant negative inhibitor can be a form of a TRAM protein that retains the ability to interact with an SH2 domain of a target protein (e.g., Fyn or Lck) but that lacks one or more other functional activities such that the dominant negative form of the TRAM protein cannot participate in normal signal transduction.
  • This dominant negative form of a TRAM protein may be, for example, a mutated form of a TRAM protein in which the SH2 binding site that interacts with a target SH2 domain is conserved but in which one or more amino acid residues elsewhere within the TRAM protein are mutated, or in which one or more potential phosphorylation sites are mutated.
  • Such dominant negative TRAM proteins can be expressed in cells using a recombinant expression vector encoding the mutant TRAM protein, which is introduced into the cell by standard transfection methods.
  • nucleotide sequences encoding amino acid residues 63-66 and 110-1 13 of SEQ ID NO: 2 are conserved, whereas at least one amino acid residue within the TRAM-1 protein is mutated or deleted. Additionally or alternatively, one or more of the potential phosphorylation sites (e.g., Tyr 63 and/or Tyr j 10 ) can be mutated or deleted.
  • nucleotide sequences encoding amino acid residues 85-93, 122-130, 145- 151 and 166-172 of SEQ ID NO: 4 are conserved, whereas at least one amino acid residue within the TRAM-2 protein is mutated or deleted.
  • one or more of the potential phosphorylation sites e.g., Tyr 90 , Tyr j2 7, Tyr 1 8 and/or Tyrj 69
  • Mutation or deletion of specific codons within the cDNA can be performed using standard mutagenesis methods.
  • the mutated cDNA is inserted into a recombinant expression vector, which is then introduced into a cell to allow for expression of the mutated TRAM protein.
  • the ability of the mutant TRAM protein to associate with the TcR/CD3/ ⁇ complex can be assessed using assays such as those described in the Examples (see e.g., Examples 1 and 2).
  • the effect of the mutant TRAM protein on T cell signal transduction can be assessed, for example, by expressing the mutant TRAM protein in T cells in culture ⁇ e.g., peripheral blood T cells or Jurkat cells), stimulating the T cells ⁇ e.g., using anti-CD3 antibodies) and measuring at least one indicator of T cell activation (e.g., calcium flux, tyrosine phosphorylation, IL-2 production).
  • a mutant form of TRAM that retains the ability to associate with the TcR/CD3/ ⁇ complex but that interferes with normal T cell signal transduction when expressed in the T cell can be selected as a dominant negative inhibitor of TRAM activity.
  • inhibitory agents that can be used to inhibit the activity of a TRAM protein are chemical compounds that inhibit the interaction between TRAM and a T cell molecule. Such compounds can be identified using screening assays that select for such compounds, as described in detail above.
  • TRAM activity is stimulated in a cell by contacting the cell with a stimulatory agent.
  • stimulatory agents include active TRAM proteins, and nucleic acid molecules encoding TRAM proteins, that are introduced into the cell to increase TRAM activity in the cell.
  • a preferred stimulatory agent is a nucleic acid molecule encoding a TRAM protein, wherein the nucleic acid molecule is introduced into the cell in a form suitable for expression of the active TRAM protein in the cell.
  • a TRAM cDNA is first introduced into a recombinant expression vector using standard molecular biology techniques, as described herein.
  • a TRAM cDNA can be obtained, for example, by amplification using the polymerase chain reaction (PCR) or by screening an appropriate cDNA library as described herein using the nucleotide sequences provided in SEQ ID NO: 1 (for TRAM-1) and SEQ ID NO: 3 (for TRAM-2). Following isolation or amplification of a TRAM-encoding cDNA, the DNA molecule is introduced into an expression vector and transfected into target cells by standard methods, as described herein.
  • PCR polymerase chain reaction
  • TRAM protein Other stimulatory agents that can be used to stimulate the activity of a TRAM protein are chemical compounds that promote the interaction between TRAM and a T cell molecule. Such compounds can be identified using screening assays that select for such compounds, as described in detail above.
  • the modulatory methods of the invention can involve the use of one or more additional agents that modulate T cell activation.
  • the modulatory methods of the invention can involve the use of an agent that modulates TRAM activity in combination with an agent that modulates tyrosine phosphorylation in T cells (e.g., an agent that inhibits protein tyrosine kinase activity, such as herbimycin A, or a derivative or analogue thereof), an agent that modulates intracellular calcium levels in T cells (e.g.
  • a calcium ionophore a calcium ionophore
  • a phorbol ester e.g., PMA
  • a cytokine that modulates T cell activation e.g., IL-2 and/or IL-4
  • Various agents that modulate T cell activation are known in the art.
  • the modulatory methods of the invention can be performed in vitro ⁇ e.g., by culturing the cell with the agent or by introducing the agent into cells in culture) or, alternatively, in vivo ⁇ e.g., by administering the agent to a subject or by introducing the agent into cells of a subject, such as by gene therapy).
  • cells can be obtained from a subject by standard methods and incubated ⁇ i.e., cultured) in vitro with a modulatory agent of the invention to modulate TRAM activity in the cells.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • Specific cell populations can be depleted or enriched using standard methods. For example, monocytes/macrophages can be isolated by adherence on plastic. T cells can be enriched for example, by positive selection using antibodies to T cell surface markers, for example by incubating cells with a specific primary monoclonal antibody (mAb), followed by isolation of cells that bind the mAb using magnetic beads coated with a secondary antibody that binds the primary mAb. Specific cell populations (e.g., T cells) can also be isolated by fluorescence activated cell sorting according to standard methods. Monoclonal antibodies to T cell-specific surface markers known in the art and many are commercially available. If desired, cells treated in vitro with a modulatory agent of the invention can be readministered to the subject.
  • mAb primary monoclonal antibody
  • the modulatory agent can be administered to the subject such that TRAM activity in cells of the subject is modulated.
  • subject is intended to include living organisms in which an immune response can be elicited.
  • Preferred subjects are mammals. Examples of subjects include humans, monkeys, dogs, cats, mice, rats, cows, horses, goats and sheep.
  • the agents can be introduced into cells of the subject using methods known in the art for introducing nucleic acid ⁇ e.g., DNA) into cells in vivo. Examples of such methods include:
  • Naked DNA can be introduced into cells in vivo by directly injecting the DNA into the cells (see e.g., Acsadi et al (1991) Nature 332:815-818; Wolff et al. (1990) Science 247:1465-1468).
  • a delivery apparatus e.g., a "gene gun” for injecting DNA into cells in vivo can be used.
  • Such an apparatus is commercially available (e.g., from BioRad).
  • Receptor-Mediated DNA Uptake Naked DNA can also be introduced into cells in vivo by complexing the DNA to a cation, such as polylysine, which is coupled to a ligand for a cell-surface receptor (see for example Wu, G.
  • Binding of the DNA-ligand complex to the receptor facilitates uptake of the DNA by receptor-mediated endocytosis.
  • a DNA-ligand complex linked to adenovirus capsids which naturally disrupt endosomes, thereby releasing material into the cytoplasm can be used to avoid degradation of the complex by intracellular lysosomes (see for example Curiel et al. (1991) Proc. Natl. Acad. Sci. USA 88:8850; Cristiano et al. (1993) Proc. Natl. Acad. Sci. USA 90:2122-2126).
  • Retroviruses Defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D. (1990) Blood 76:271).
  • a recombinant retrovirus can be constructed having a nucleotide sequences of interest incorporated into the retroviral genome. Additionally, portions of the retroviral genome can be removed to render the retrovirus replication defective. The replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals.
  • retroviruses examples include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art.
  • suitable packaging virus lines include ⁇ Crip, ⁇ Cre, ⁇ 2 and ⁇ Am.
  • Retroviruses have been used to introduce a variety of genes into many different cell types, including epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and Mulligan (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464; Wilson et al.
  • Retroviral vectors require target cell division in order for the retroviral genome (and foreign nucleic acid inserted into it) to be integrated into the host genome to stably introduce nucleic acid into the cell. Thus, it may be necessary to stimulate replication of the target cell.
  • Adenoviruses The genome of an adenovirus can be manipulated such that it encodes and expresses a gene product of interest but is inactivated in terms of its ability to replicate in a normal lytic viral life cycle. See for example Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al (1992) Cell 68:143-155.
  • Suitable adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus ⁇ e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art.
  • Recombinant adenoviruses are advantageous in that they do not require dividing cells to be effective gene delivery vehicles and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89:6482- 6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA 90:2812-2816) and muscle cells (Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-2584).
  • introduced adenoviral DNA (and foreign DNA contained therein) is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA).
  • the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al. cited supra; Haj- Ahmand and Graham (1986) J. Virol. 57:267).
  • Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle. (For a review see Muzyczka et al. Curr. Topics in Micro, and Immunol. (1992) 158:97-129).
  • a variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470; Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988) Mol. Endocrinol 2:32-39; Tratschin et al. (1984) J. Virol. 51:61 1-619; and Flotte et al (1993) J. Biol. Chem. 268:3781-3790).
  • DNA introduced into a cell can be detected by a filter hybridization technique (e.g. , Southern blotting) and RNA produced by transcription of introduced DNA can be detected, for example, by Northern blotting, RNase protection or reverse transcriptase-polymerase chain reaction (RT-PCR).
  • RNA produced by transcription of introduced DNA can be detected, for example, by Northern blotting, RNase protection or reverse transcriptase-polymerase chain reaction (RT-PCR).
  • RT-PCR reverse transcriptase-polymerase chain reaction
  • the gene product can be detected by an appropriate assay, for example by immunological detection of a produced protein, such as with a specific antibody, or by a functional assay to detect a functional activity of the gene product.
  • a modulatory agent such as a chemical compound that modulates the association of a TRAM protein with the TcR/CD3/ ⁇ complex
  • Such compositions typically comprise the modulatory agent and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • Pharmaceutical compositions can be prepared as described above in subsection IV.
  • TRAM proteins associate with the TcR/CD3/ ⁇ complex and that TRAM-1 is comodulated with CD3 upon T cell activation (see Example 6)
  • modulation of TRAM activity in T cells may be beneficial in a variety of clinical situations in which is desirable to modulate T cell immune responses, including immunodeficiencies, infectious diseases (e.g., viral infections), cancer, autoimmune diseases, transplantations ⁇ e.g., graft rejection or graft-versus-host disease) and allergies, as discussed further below.
  • Immunodeficiencies Stimulation of T cell activation through the use of a modulatory agent that modulates TRAM activity may be beneficial in a variety of clinical disorders characterized by general or specific immunodeficiency, including human immunodeficiency virus infection and congenital immunodeficiency diseases.
  • Infectious Diseases Stimulation of T cell activation through the use of a modulatory agent that modulates TRAM activity may be beneficial in a variety of infectious disease, as a means to promote a T cell response against the infectious agent.
  • infectious diseases include bacterial, viral, fungal and parasitic infections.
  • Stimulation of T cell activation through the use of a modulatory agent that modulates TRAM activity may be beneficial in a variety of malignancies, as a means to promote a T cell response against malignant cells.
  • inhibition of T cell activation through use of a modulatory agent that modulates TRAM activity may be beneficial, as a means to inhibit growth or progression of these malignancies.
  • autoimmune disorders Inhibition of T cell activation through the use of a modulatory agent that modulates TRAM activity may be beneficial in a variety of autoimmune disorders, as a means to downregulate T cell response against autoantigens. It is well known in the art that many autoimmune disorders are the result of inappropriate activation of T cells that are reactive against self tissue and that promote the production of cytokines and autoantibodies involved in the pathology of the diseases.
  • Non-limiting examples of autoimmune diseases and disorders having an autoimmune component that may be treated according to the modulatory methods of the invention include diabetes mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis), multiple sclerosis, myasthenia gravis, systemic lupus erythematosis, autoimmune thyroiditis.
  • dermatitis including atopic dermatitis and eczematous dermatitis
  • psoriasis Sjogren's Syndrome, including keratoconjunctivitis sicca secondary to Sjogren's Syndrome
  • alopecia areata, allergic responses due to arthropod bite reactions, Crohn's disease, aphthous ulcer, ulceris, conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, poly
  • the efficacy of a modulatory agent in ameliorating autoimmune diseases can be tested in an animal models of human diseases.
  • animal models include experimental allergic encephalomyelitis as a model of multiple sclerosis, the NOD mice as a model for diabetes, the mrl/lpr/lpr mouse as a model for lupus erythematosus, murine collagen-induced arthritis as a model for rheumatoid arthritis, and murine experimental myasthenia gravis (see Paul ed., Fundamental Immunology, Raven Press, New York, 1989, pp. 840-856).
  • a modulatory ⁇ i.e., stimulatory or inhibitory) agent of the invention is administered to test animals and the course of the disease in the test animals is then monitored by the standard methods for the particular model being used. Effectiveness of the modulatory agent is evidenced by amelioration of the disease condition in animals treated with the agent as compared to untreated animals (or animals treated with a control agent).
  • Transplantation Inhibition of T cell activation through the use of a modulatory agent that modulates TRAM activity may be beneficial in transplantation, as a means to downregulate T cell responses against an allograft or to inhibit graft-versus-host disease. Accordingly, the modulatory methods of the invention can be used both in solid organ transplantation and in bone marrow transplantation.
  • Allergies are mediated through IgE antibodies whose production is regulated by the activity of T cells and the cytokines produced thereby. Accordingly, the modulatory methods of the invention can be used to inhibit T cell activation as a means to downregulate allergic responses.
  • a modulatory agent may be directly administered to the subject or T cells may be obtained from the subject, contacted with an modulatory agent ex vivo, and readministered to the subject. Moreover, in certain situations it may be beneficial to coadminister to the subject the allergen together with the modulatory agent or cells treated with the modulatory agent to desensitize the allergen-specific response.
  • the modulatory methods of the invention may be used for other purposes.
  • the modulatory methods that result in increased T cell activation can be used in the production of T cell cytokines in vitro.
  • the modulatory methods of the invention may be applied to vaccinations to promote T cell responses to an antigen of interest in a subject. That is, a modulatory agent of the invention may be used in combination with a vaccine to promote T cell responses against the vaccinating antigen.
  • proteins that coprecipitate with the accessory T cell molecules CD2, CD3, CD4, CD5 and CD8 were analyzed.
  • a 29-30 kD protein (referred to as pp29/30) was identified.
  • Proteins were immunoprecipitated from postnuclear lysates employing 25 ⁇ l of packed CNBr-Sepharose beads (Pharmacia, Uppsala, Sweden) coupled with protein-A purified anti-CD2 mAb AIDC2.1.1A (IgG2a. 6 mg of purified antibody were coupled to 1 ml of packed beads). The immunoprecipitates were subsequently washed three times with washing buffer I (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM NaF, 1 mM PMSF, 0.1% Brij58).
  • an in vitro kinase reaction was carried out by resuspending the lysates in 40 ⁇ l kinase buffer (20 mM Tris-HCl pH 7.5, 10 mM MnCl 2 , 0.1% Brij58) supplemented with 10 ⁇ Ci of p2p]- ⁇ -ATP (Amersham, Braunschweig, FRG), allowing the labelling reactions to proceed for 20 minutes at room temperature and then stopping the reaction by addition of 1 ml washing buffer II (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM EDTA, 0.1% Brij58). The precipitates were washed six times in washing buffer II.
  • the beads were resuspended in 80 ⁇ l of lysis buffer supplemented with 8 M urea and 1% Triton X- 100 and incubated for 10 minutes at 37° C to allow release of the immunoprecipitated proteins. Beads were spun down by centrifugation, the supernatant was then collected and supplemented with 10 ⁇ l of 9x O'Farrell buffer (18% NP40 (Sigma), 3.6% ampholines pH 6-10 (Pharmacia), 0.27 M DTT) and subjected to two-dimensional gel electrophoresis, with isoelectric focusing (IEF) in the first dimension followed by reducing SDS-PAGE (10%) in the second dimension, as described further below.
  • 9x O'Farrell buffer 18% NP40 (Sigma), 3.6% ampholines pH 6-10 (Pharmacia), 0.27 M DTT
  • Two dimensional gel analysis was carried out according to O'Farrell with minor modifications. Briefly, 1 mm tube gels (pH gradient from 3.5 to 7.5) were subjected to a pre-run step (15 minutes at 200 V, 20 minutes at 300 V and 30 minutes at 400 V). The sample was then loaded on top of the gel and overlayed with the upper tank buffer (100 mM NaOH). The buffer for the lower tank was 0.085% H 3 PO 4 . Isoelectric focusing was performed for 1 hour at 200 V, 1 hour at 300 V, 15.25 hours at 400 V and 1.5 hours at 800V.
  • the gel tube was then equilibrated for 20 minutes at room temperature in 4 ml of Ivan Lefkowitz buffer (120 mM Tris-HCl pH 6.8, 2% SDS, 50 mM DTT, 10% glycerol and 0.02% bromophenol blue) and thereafter loaded on a SDS-PAGE (10%) gel in order to separate the proteins in the second dimension according to their molecular weight. In vitro labeled proteins were then detected by autoradiography.
  • Ivan Lefkowitz buffer 120 mM Tris-HCl pH 6.8, 2% SDS, 50 mM DTT, 10% glycerol and 0.02% bromophenol blue
  • FIG. 1 The results for CD2 coprecipitates are shown in Figure 1 , in which the phosphorylated 29-30 kD phosphoprotein (pp29/30) is indicated by a questionmark.
  • the pp29/30 polypeptide represents the reduced component of a 58-60 kDa disulfide linked dimer that runs off diagonal on a two-dimensional non-reducing/reducing SDS- PAGE.
  • an identical pattern of phosphoproteins to that seen in Figure 1 was detectable in CD3, CD4, CD5 and CD8 immunoprecipitates prepared under the same experimental conditions.
  • HPB-ALL cells 5 x 10 9 HPB-ALL cells were washed once in TBS and lysed in 70 ml of 1% Brij58 lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% Brij58, 1 mM
  • Immunocomplexes were washed four times in washing buffer I (described in Example 1). Approximately 5-10% of the sample was subjected to an in vitro kinase assay to allow for identification of the proteins of interest by autoradiography. Thus, 50 ⁇ l of washed beads were separated and resuspended in 80 ⁇ l of kinase buffer (20 mM Tris-HCl pH 7.5, 10 mM MnCl 2 , 0.1% Brij58) supplemented with 20 ⁇ Ci of [ 32 P]- ⁇ - ATP and the in vitro labeling carried out for 20 minutes at room temperature.
  • kinase buffer (20 mM Tris-HCl pH 7.5, 10 mM MnCl 2 , 0.1% Brij58
  • the in vitro kinase reaction was stopped by addition of 1 ml washing buffer II (20 mM Tris- HCl pH 7.5, 150 mM NaCl, 20 mM EDTA, 0.1% Brij58), the radiolabeled beads were washed six times and were pooled with unlabeled precipitates. The pooled beads were then resuspended for acidic elution in one volume (ca. 5 ml) glycine buffer (100 mM glycine-HCl, pH 2.5) and incubated for 5 minutes at room temperature with agitation.
  • the solution was neutralized by addition of 500 ⁇ l 1.5 M Tris-HCl, pH 8.8, the beads were spun down, the supernatant removed and the beads washed once with 4 ml washing buffer.
  • the bead pellet was then resuspended for basic eluation in one volume (ca. 5 ml) of 100 mM triethylamine, pH 1 1.5 and incubated for 5 minutes at room temperature with agitation.
  • the solution was neutralized by the addition of 500 ⁇ l 1 M Tris-HCl pH 7.5, the beads spun down, the supernatant removed and the beads washed once with ml washing buffer.
  • Both eluates and both washing supernatants were pooled (ca. 17 ml) and the isolated proteins precipitated overnight at -20° C after addition of 2.5 volumes of ice cold acetone.
  • the acetone precipitated proteins were pelleted for 10 minutes at 10,000 g at 4° C, the pellet washed 3 times with ice cold methanol and dissolved- aided by sonication - in 100 ⁇ l lysis buffer supplemented with 1 % Triton X-100.
  • 20 ⁇ l of 5x nonreducing sample buffer was added, incubated for 10 minutes at 37° C and centrifuged for 5 minutes at 10,000 rpm in an Eppendorf microfuge.
  • cDNAs encoding TRAM-1 and TRAM-2 which include peptide sequences corresponding to tryptic fragments of pp29/30 obtained as described in Example 2, were isolated and characterized.
  • Peptides of purified pp29/30 were generated by digestion with trypsin and sequenced by nano-electrospray-tandem-mass-spectrometry.
  • Amino acid sequence information was obtained for three peptides: YSEVV(L/I)DSEPK (SEQ ID NO: 8), (L/I)FG(L/I)(L/I)R (SEQ ID NO: 9) and AM(L/I)VDSFSPEASGAVEEN(L/I) HDDTHK (SEQ ID NO: 10).
  • the mas spectrometry method cannot discriminate between the amino acids leucine and isoleucine since they have identical mass).
  • TRAM-2 A cDNA encoding a protein that includes the peptide sequence YSEVV(L/I)DSEPK (SEQ ID NO: 8) was isolated and sequenced in its entirety.
  • a schematic diagram of the TRAM-2 protein is shown in Figure 5.
  • the open reading frame of this cloned cDNA contained the coding sequence for the tryptic peptide of SEQ ID NO: 8, at amino acid positions 148-158 of the TRAM-2 protein of SEQ ID NO: 4.
  • the TRAM-2 open reading frame did not contain the peptide sequences of the tryptic fragments shown in SEQ ID NO: 9 or 10, thus indicating that the pp29/30 protein spot was actually composed of two individual dimeric proteins of very similar molecular weight.
  • TRAM-1 a second gene (coding for a protein referred to as TRAM-1) was isolated and sequenced in its entirety.
  • the nucleotide and predicted amino acid sequence of this clone are shown in SEQ ID NOs: 1 and 2, respectively.
  • a schematic diagram of the TRAM-1 protein is shown in Figure 4.
  • the open reading frame of this cloned cDNA contained the coding sequence for the tryptic peptide of SEQ ID NO: 9, at amino acid positions 174-179 of the TRAM-1 protein of SEQ ID NO: 2.
  • the open reading frame of this cloned cDNA contained a coding sequence essentially corresponding to the tryptic peptide of SEQ ID NO: 10 (at positions 152-173 of the TRAM-1 protein of SEQ ID NO: 2), except that an alanine codon was missing, a glutamine codon was present instead of glycine, and PIR codons were present instead of THK.
  • the errors in the amino acid sequences of the microsequenced pp29/30 peptide is due to the fact that the amino acids alanine and glycine possess the same molecular mass as glutamine and that the two peptides THK and PIR have the same molecular mass.
  • TRAM-1 protein is expressed as a disulfide-linked dimeric transmembrane molecule. It has an apparent molecular weight (according to SDS-PAGE) of 29-30 kD and a calculated MW of 21 kD. It has a calculated isoelectric point of 5.1.
  • the expression pattern of TRAM- 1 mRNA is restricted to lymphoid tissues (T cells and NK cells; see Example 4). TRAM-1 associates with the TcR/CD3/ ⁇ complex (see Example 5) and shows rapid induction of tyrosine phosphorylation on multiple tyrosine residues upon T cell receptor mediated T cell activation (see Example 6).
  • TRAM- 1 contains a repeated tyrosine motif, EDTPIYGNL at amino acid positions 58-66 of SEQ ID NO: 2 and ETQMCYASL at amino acid positions 105-113 of SEQ ID NO: 2, in which the tyrosines represent potential phosphorylation sites for src-family protein tyrosine kinases.
  • TRAM-1 mRNA At least two forms of the TRAM-1 mRNA exist (thought to result from differential splicing), which potentially lead to the expression of two isoforms of the protein: a transmembrane form and a soluble form.
  • the transmembrane form corresponds the protein of SEQ ID NO: 2.
  • the nucleotide and predicted amino acid sequence for the soluble form are shown in SEQ ID NO: 5 and 6, respectively.
  • the overall structure of the TRAM-1 protein of SEQ ID NO: 2 comprises an extracellular domain of about amino acids 1-8 of SEQ ID NO: 2, a transmembrane domain of about amino acids 9-27 of SEQ ID NO: 2 and a cytoplasmic domain of about amino acids 28- 186 of SEQ ID NO: 2.
  • the TRAM-1 protein of SEQ ID NO: 6 differs from the TRAM- 1 protein of SEQ ID NO: 2 in that amino acid residues 3-39 of SEQ ID NO: 2 are deleted.
  • the TRAM-2 protein of SEQ ID NO: 6 lacks a transmembrane domain.
  • Further characterization of the TRAM-2 protein revealed the following distinguishing structural and functional characteristics: The protein is expressed as a disulfide-linked dimeric transmembrane molecule. It has an apparent molecular weight (according to SDS-PAGE) of 29-30 kD and a calculated MW of 18 kD. It has a calculated isoelectric point of 5.7.
  • TRAM-2 mRNA The expression pattern of TRAM-2 mRNA is restricted to cells of the lymphoid compartment, but is expressed in both B lymphocytes and T lymphocytes (see Example 4).
  • the overall structure of TRAM-2 comprises a signal peptide of about amino acids 1-22 of SEQ ID NO: 4, an extracellular domain of about amino acids 23-40 of SEQ ID NO: 4, a transmembrane domain of about amino acids 41-61 of SEQ ID NO: 4 and a cytoplasmic domain of about amino acids 62-196 of SEQ ID NO: 4.
  • the cytoplasmic domain of TRAM-2 contains two repeated tyrosine motifs, in which the tyrosines represent potential phosphorylation sites for src-family protein tyrosine kinases.
  • the first repeated tyrosine motif corresponds to EEVPLYGNL at amino acid positions 85-93 of SEQ ID NO: 4 and EEVMCYTSL at amino acid positions 122-130 of SEQ ID NO: 4.
  • the second repeated tyrosine motif corresponds to PVKYSEV at amino acid positions 145-151 of SEQ ID NO: 4 and PELYASV at amino acid positions 166-172 of SEQ ID NO: 4.
  • TRAM-2 has been found to be a glycoprotein with N-linked sugars.
  • TRAM- 1 and TRAM-2 mRNA in human tissues were examined by standard Northern blot analyses.
  • Pre-prepared Northern blot membranes containing 2 ⁇ g poly(A) + -RNA of the following tissues: spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leucocytes (PBL), heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas, were purchased from Clontech.
  • the membranes were prehybridized and hybridized at 65° C in 50 mM Tris-HCl (pH 7.5), 500 mM NaCl, 1 mM EDTA, 0.1% Na 4 P 2 O 7 , 10% dextransulfate, 1% casein, 1% SDS and 250 ⁇ g/ml salmon sperm DNA, overnight with the following probes: for TRAM-1, a probe encompassing nucleotides 436-619 of the TRAM-1 cDNA and for TRAM-2, a probe encompassing nucleotides 1-788 of the TRAM-2 cDNA.
  • T cell lysates were immunoprecipitated with mAbs to a variety of T cell antigens (e.g., CD2, CD3- ⁇ , CD4, CD8, CD45, CD28 and HLA-1), followed by immunoblotting of the immunoprecipitates with an anti-TRAM- 1 antisera to determine whether TRAM-1 complexes with any of these T cell antigens. Association of TRAM- 1 with CD3- ⁇ of the T cell receptor complex was demonstrated. Additionally, comodulation of TRAM- 1 and CD3- ⁇ following treatment with an anti-CD3 mAb was demonstrated.
  • T cell antigens e.g., CD2, CD3- ⁇ , CD4, CD8, CD45, CD28 and HLA-1
  • nuclei were removed by centrifugation for 15 minutes at 4° C and immunoprecipitations were performed on postnuclear lysates using 75 ⁇ l of packed CNBr-Sepharose beads covalently coupled with Protein-A purified mAbs (6 mg of purified antibody/ml of packed beads).
  • anti-phosphotyrosine PY72, IgGl; anti-CD2: AICD2.1.1A, IgG2a; anti-CD3: OKT-3, IgG2a; anti-CD4: AICD4.1, IgGl; anti-CD8: AICD8.1, IgGl; anti-CD45: AICD45.2, IgGl ; anti-CD28: 15B9E9, IgGl ; and anti-HLA-class I: W6.32, IgG2a.
  • the immunoprecipitates were washed four times (three times 5 ml, once with 1 ml) in washing buffer I (the composition of which is described in Example 1).
  • the immunoprecipitated proteins were released with 40 ⁇ l of washing buffer I supplemented with 8 ⁇ l of 5x SDS-PAGE sample buffer.
  • the samples were run on a SDS-PAGE (14%) and subsequently blotted onto a nitrocellulose membrane (Hybond C, Amersham).
  • the membrane was blocked with 5% non-fat dried milk for 1 hour at room temperature and then incubated for an additional hour with a 1 :500 (v/v) dilution of crude TRAM-1 antiserum that was generated in rabbits immunized with a KHL-coupled synthetic peptide corresponding to the amino acid sequence of a mass-spectrometry derived TRAM-1 peptide sequence.
  • TBS-T TBS/0.2% TWEEN20
  • POD peroxidase
  • goat-anti-rabbit antibody Jackson Immunol.
  • FIG. 7A The results of this coprecipitation/immunoblotting experiment are shown in Figure 7A.
  • the blot demonstrates that TRAM-1 selectively coprecipitates with CD3- ⁇ mAb and, following pervanadate treatment of the cells (lane 2), also with an anti-PTYR mAb.
  • the immunoprecipitates shown in lanes 1 and 3-9 of Figure 7A were performed from non-treated (i.e., no pervanadate) cells.
  • the remaining untreated or TcR/CD3 -modulated cells were lysed at a density of 0.5 x 10 6 cells/30 ⁇ l of NP40 lysis buffer. Following removal of nuclei by centrifugation, an appropriate volume of 5x reducing sample buffer was added and the solution was incubated for 5 minutes at 95° C Lysates corresponding to 1 x 10 6 cells were then separated on reducing SDS-PAGE (14%) and blotted onto nitrocellulose.
  • TRAM-1 antiserum 10 ⁇ g/ml
  • UBI polyclonal rabbit anti-MAP-kinase serum
  • FIG. 7C Lanes 1 and 2 show TRAM-1 expression (lane 1 is without TcR modulation; lane 2 is with TcR modulation). Lanes 3 and 4 show MAP-kinase expression (lane 3 is without TcR modulation; lane 4 is with TcR modulation).
  • HPB-ALL cells were stimulated in vitro employing a mixture of biotinylated CD3 and CD4 mAbs that were crosslinked with avidin (Sigma).
  • Protein-A purified monoclonal CD3- ⁇ mAb (OKT-3) and CD4 mAb (AICD4.1 ) were biotinylated using a standard protocol (see e.g., Pierce catalogue).
  • Postnuclear lysates were subjected to immunoprecipitation employing polyclonal anti-TRAM-1 serum at a 1 :500 v/v final dilution for 30 minutes on ice. Immunoprecipitates were collected using protein-A sepharose beads and washed four times in washing buffer I.
  • precipitated proteins were released in Triton X- 100 lysis buffer supplemented with 8 M urea as described in Example 1. Following two-dimensional gel electrophoresis, proteins were blotted onto nitrocellulose and the blots were first probed with anti-PTYR mAb (4G10, UBI, Lake Placid, NY, 1 ⁇ g/ml). Subsequently, blots were stripped and reprobed with anti-TRAM-1 antiserum (1 :500 v/v dilution).
  • HPB-ALL cells were stimulated with anti-CD3 and anti-CD4 as described above for either 30 seconds or 1, 2, 5, 10, 20 or 40 minutes. The cells were then lysed in 1 ml of NP40-containing lysis buffer. Postnuclear lysates corresponding to 1 x 10 6 cell equivalents were supplemented with 5x reducing sample buffer and separated on SDS-PAGE (14%), followed by anti-TRAM- 1 immunoblotting to demonstrate that identical amounts of TRAM- 1 were analyzed for each time point of the experiment, the results of which are shown in Figure 9B.
  • TRAM-1 was detected using affinity -purified anti-TRAM- 1 antiserum at a concentration of 10 ⁇ g/ml. The remaining cell lysates were subjected to anti-TRAM-1 immunoprecipitation followed by anti- PTYR immunoblotting as described above, the results of which are shown in Figure 9A. The results demonstrate that TRAM-1 becomes tyrosine phosphorylated as rapidly as 30 seconds following engagement of the CD3- ⁇ and CD4 molecules and that the level of phosphorylation slowly declines beyond 2 minutes of activation.

Abstract

L'invention concerne des molécules d'acide nucléique codant pour des protéines qui s'associent au complexe du récepteur des lymphocytes T et qui ont été dénommées TRAM-1 et TRAM-2. Outre ces molécules, l'invention concerne également des molécules d'acide nucléique antisens, des vecteurs d'expression recombinants contenant une molécule d'acide nucléique de l'invention, des cellules hôtes dans lesquelles ont été introduits des vecteurs d'expression et des animaux transgéniques non humains porteurs d'un transgène TRAM. L'invention concerne aussi des protéines et des peptides TRAM-1 et TRAM-2, des protéines de fusion TRAM-1 et TRAM-2 et des anticorps anti-TRAM-1 et anti-TRAM-2. L'invention concerne enfin des techniques qui permettent d'utiliser les compositions de TRAM de l'invention, notamment des techniques qui permettent de détecter une activité TRAM dans un échantillon biologique, des techniques qui permettent de moduler l'activité TRAM dans une cellule et des techniques qui permettent d'identifier des agents pouvant moduler une interaction entre une protéine TRAM et les molécules des lymphocytes T.
EP98951056A 1997-10-10 1998-10-13 Molecules associees au recepteur des lymphocytes t (tram) et leur mode d'utilisation Withdrawn EP1021537A1 (fr)

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US10055540B2 (en) 2015-12-16 2018-08-21 Gritstone Oncology, Inc. Neoantigen identification, manufacture, and use
US11264117B2 (en) 2017-10-10 2022-03-01 Gritstone Bio, Inc. Neoantigen identification using hotspots
US11885815B2 (en) 2017-11-22 2024-01-30 Gritstone Bio, Inc. Reducing junction epitope presentation for neoantigens

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AU2451199A (en) * 1998-01-07 1999-07-26 Human Genome Sciences, Inc. 36 human secreted proteins
GB0604170D0 (en) * 2006-03-02 2006-04-12 Antitope Ltd T cell assays
PT1989544E (pt) * 2006-03-02 2011-09-26 Antitope Ltd Ensaios com células t

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Publication number Priority date Publication date Assignee Title
US10055540B2 (en) 2015-12-16 2018-08-21 Gritstone Oncology, Inc. Neoantigen identification, manufacture, and use
US10847252B2 (en) 2015-12-16 2020-11-24 Gritstone Oncology, Inc. Neoantigen identification, manufacture, and use
US10847253B2 (en) 2015-12-16 2020-11-24 Gritstone Oncology, Inc. Neoantigen identification, manufacture, and use
US11183286B2 (en) 2015-12-16 2021-11-23 Gritstone Bio, Inc. Neoantigen identification, manufacture, and use
US11264117B2 (en) 2017-10-10 2022-03-01 Gritstone Bio, Inc. Neoantigen identification using hotspots
US11885815B2 (en) 2017-11-22 2024-01-30 Gritstone Bio, Inc. Reducing junction epitope presentation for neoantigens

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