WO1998024895A1 - Recepteur appartenant a la famille de recepteurs tnf/ngf - Google Patents

Recepteur appartenant a la famille de recepteurs tnf/ngf Download PDF

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WO1998024895A1
WO1998024895A1 PCT/EP1997/006252 EP9706252W WO9824895A1 WO 1998024895 A1 WO1998024895 A1 WO 1998024895A1 EP 9706252 W EP9706252 W EP 9706252W WO 9824895 A1 WO9824895 A1 WO 9824895A1
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gitr
gly
cys
pro
cell
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PCT/EP1997/006252
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English (en)
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Carlo Riccardi
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Pharmacia & Upjohn S.P.A.
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Priority to AU53207/98A priority Critical patent/AU5320798A/en
Publication of WO1998024895A1 publication Critical patent/WO1998024895A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95

Definitions

  • the present invention relates to a previously unknown receptor protein, named GITR (Glucocorticoid Induced TNFR- family Related protein) and previously coded D4 , which shares structural and biological characteristics with other members of the tumor necrosis factor/nerve growth factor receptor (TNFR/NGFR) family. Also disclosed are splicing variants of GITR. BACKGROUND OF THE INVENTION The proteins belonging to the tumor necrosis factor/nerve growth factor receptor (TNFR/NGFR) family play a crucial role in cell activation, differentiation and death. The signals initiated with the triggering of these receptors by a corresponding family of structurally related ligand ⁇ , are required for the normal development and function of the immune system. Excessive signaling through some of the receptors can cause severe inflammatory reaction, tissue injury and shock. Mutation of genes corresponding to the ligands or to the receptors can cause characteristic disturbances of lymphocytes, derangement of the immune response or autoimmune disease.
  • GITR Glucocorticoid Induce
  • members of the TNFR/NGFR family are classified as type I transmembrane proteins characterized in the extracellular portion by the presence of 3-5 similar motifs (the cystein pseudorepeats C-x(4,6) - [FYH] -x(5,10) -C-x(0,2) -C-x(2,3) -C- x(7,ll) -C-x(4,6) - [DNEQSKP] -x(2) -C) , which have recently been recognized as true domains (Banner D. ., D'Arcy A., Janes W., Gentz R., Schoenfeld H-J., Broger C, Loetscher H. & Lesslauer .
  • Both ligand and receptor are transmembrane proteins (with the only exception of TNF and lymphotoxin- ⁇ ) and the receptor/ligand interaction takes place following cell- to-cell contact.
  • Each member of the family binds to one specific receptor (with the only exception of TNF and lymphotoxin- ⁇ and TRAIL) .
  • TNFR/NGFR family the receptors mainly inducing apoptosis (TNFR-p55, Fas, CAR1, DR3 and the TRAIL receptors family) and the receptors mainly stimulating cell proliferation, differentiation and activation (TNFR- p75, CD40, CD30, CD27, 4-IBB and 0X40) .
  • some receptors e.g. CD40
  • CD40 inhibit cell death.
  • the apoptosis inducing receptors contain a 60- residue cytoplasm sequence known as the "death domain" , required for the transduction of an apoptotic signal .
  • Humans with mutations of the Fas gene have lymphadenopathy, splenomegaly and signs of autoimmunity at an early age.
  • Two poxvirus gene products (T2 and A53R) have been shown to encode soluble, secreted forms of TNFR. These TNFR-like proteins form a complex with (and thereby inactive) host-produced TNF.
  • TRAIL-R3 lacks the splicing domain and it has been recently suggested that TRAIL-R3b expression protects normal cells from TRAIL induced apoptosis (Trisha Gura (1997) Science 277, 768) .
  • the syndrome of X-linked immunodeficiency (high levels of IgM and low or absent levels of other immunoglobulins) is caused by a mutation in the CD40L.
  • CD30 was originally described as a marker in the Hodgkin's lymphoma, because overexpressed in these cancerous cells (Schwab U., Stein H, Gerdes J, Lemke H, Kirchner H, Schaadt M & Diehl V (1982) Nature 299: 65- 68) .
  • CD30 triggering is involved in promoting HIV replication (Del Prete G., Maggi E, Pizzolo G, Romagnani S (1995) Immunol . today 16(2): 76-80).
  • CD40/CD4OL, CD27/CD70 and 4-1BB/4-1BBL interactions costimulate T-cells activated through the T cell receptor (TCR) /CD3 complex in a way similar to CD28/CD80 interaction.
  • TCR T cell receptor
  • the receptors are induced following antigenic stimulation and play a role during T-cell activation. It appears therefore evident that these receptor proteins are involved in many diseases. Different strategies can be used in different diseases. Stimulation of these receptors can be useful when lymphocyte activation is needed (e.g., in oncologic patients). Inhibition of these receptors can be helpful when a decrease of lymphocyte reactivity is needed (e.g. autoimmune diseases). Finally, when tumor cells overexpress one of these receptors, this can be used as a tool to target these cells ( e.g. by immunotoxins) , to inhibit tumor cell proliferation and/or to monitor the response to chemotherapy (absence of minimal residual disease and early diagnosis of relapse) . There is therefore the need to gain a deeper insight into the biological mechanisms regulated by or involving the TNFR/NGFR family.
  • GITR a new member of the TNFR/NGFR family
  • splicing variant it is meant each of the different forms of the GITR receptor deriving from the alternative splicing of the primary transcript.
  • genomic DNA of eukaryotes is organized in regions called exons and regions called introns .
  • the genomic DNA is transcribed into a primary transcript (nuclear mRNA) containing exons and introns .
  • Introns are subsequently excised and the coding sequences are simultaneously linked by a splicing complex to form the mature mRNA (cytoplasmic mRNA) .
  • the organization of the genomic DNA into exons and introns offers the potentiality for generating a series of related proteins by splicing a nascent RNA transcript in different ways. This process is known as alternative splicing and it is a means of forming a set of proteins that are variants of a basic motif .
  • the protein of the invention find a useful application in several diagnostic as well as therapeutic fields.
  • the protein of the invention can be used as a probe to isolate ligands to GITR.
  • GITR can be used for lymphocyte activity stimulation and cell death rescue. These goals are accomplished by a variety of means: a fusion protein comprising the extracellular portion of GITR could be used to trigger the corresponding ligand; alternatively GITR can be transfected through viral vectors or encapsulated plasmids in unresponsive or low-level responsive T- lymphocytes (such as tumor infiltrating T-lymphocytes) ; lymphocytes can be treated with agonist antibodies or with a peptide mimicking the intracytoplasmic domain of GITR thus activating the intracellular pathways physiologically initiated by triggering the receptor.
  • GITR soluble fusion protein of GITR acting as a decoy target
  • a mutated GITR construct can be transfected through viral vectors or encapsulated plasmids to act as cell linked decoy target
  • lymphocytes can be treated with a peptide binding the intracytoplasmic domain of GITR (and possibly 4-IBB and CD27) thus inhibiting the intracellular pathways physiologically initiated by triggering these receptors .
  • GITR can be used to suppress the growth of tumor cells overexpressing GITR: in this case, approaches similar to those described above to suppress the lymphocyte activity can be followed; moreover, GITR antibodies conjugated with toxins (such as ricin, saporin, momordin) can be used as immunotoxins which target specifically GITR overexpressing tumor cells.
  • toxins such as ricin, saporin, momordin
  • a similar approach has been used successfully to cure SCID mice with human xenografted CD30+ anaplastic large-cell lymphoma (Pasqualucci L., Wasik M., Teicher B.A., Flenghi L., Bolognesi A., Stirpe F., Polito L., Falini B., Kadin M.E.
  • an increase of the host defense against tumor can be accomplished by transfecting GITR or its ligand into the patient cell in vitro and subsequently reinfusing the transfected cells into the patient.
  • a similar approach has been used with another system which is crucial in co ⁇ timulation of T-cells (CD28/B7) .
  • Many tumors lack expression of B7-1 and this has been suggested to contribute to the failure of immune recognition of these diseases.
  • transfection of CD28 ligands (B7-1 or B7-2) in the tumor cells causes the rejection of the tumor and the mice develop protective immunity against subsequent challenge with B7-K-) (or B7-2(-)) untransfected tumors: (e.g. Matulonis U.A. et al . Blood (1995) 85(9); 2507-2515).
  • B7 transfection does not elicit rejection.
  • transfection of other coaccessory molecules may be useful.
  • the present invention discloses a novel member of the TNFR/NGFR family, designated GITR, and its ⁇ plicing variants GITR-B and GITR-C.
  • the present invention provides an isolated single or double stranded polynucleotide, typically DNA, having a nucleotide sequence which comprises: (a) a nucleotide sequence selected from the group consisting of (i) the sequence from nucleotide position 46 to nucleotide position 729 of SEQ ID NO. 1;
  • a preferred embodiment is a DNA molecule.
  • the polynucleotide is an RNA molecule.
  • a DNA molecule of the present invention is contained in an expression vector.
  • the expression vector preferably further comprises an enhancer-promoter operatively linked to the polynucleotide.
  • the DNA molecule in the vector is one of the sequences of SEQ ID NO. 1, SEQ ID NO. 4 and SEQ ID NO . 6.
  • the present invention still further provides for a host cell transformed with an expression vector of this invention.
  • the host may be a prokaryotic or a eukaryotic cell.
  • Example of a preferred prokaryotic host cell is E. coli
  • preferred hosts are yeast or insect cells.
  • the invention provides an isolated and purified polypeptide which is coded for by a nucleotide sequence selected from the group consisting of: (a) the sequence from nucleotide position 46 to nucleotide position 729 of SEQ ID NO. 1; the sequence from nucleotide position 46 to nucleotide position 930 of SEQ ID NO. 4; the sequence from nucleotide position 46 to nucleotide position 714 of SEQ ID NO.
  • the invention provides a recombinant process for the expression of a polypeptide according to the invention, which process comprises inserting a said polynucleotide of the invention into an appropriate expression vector, transfecting the expression vector into an appropriate host, growing the transfected host in a suitable culture medium and purifying the said polypeptide from the culture medium.
  • the present invention provides isolated and purified polynucleotides that encode the GITR receptor and its splicing variants, vectors containing these polynucleotides, host cells transformed with these vectors, a process of making the GITR receptor or its splicing variants using the above polynucleotides and vectors, and isolated and purified recombinant GITR receptor as well as its splicing variants.
  • amino acid sequences are presented in the amino to carboxy direction, from left to right.
  • the amino and carboxy groups are not presented in the sequence .
  • the nucleotide sequences are presented by single strand only, in the 5' to 3 ' direction, from left to right. Nucleotides and amino acids are represented in the manner recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by three letters code.
  • the present invention provides isolated and purified polynucleotides that encode the GITR receptor from mouse and its splicing variants.
  • a polynucleotide of the present invention is an isolated single or double stranded polynucleotide having a nucleotide sequence which comprises : (a) a nucleotide sequence selected from the group consisting of (i) the sequence from nucleotide position 46 to nucleotide position 729 of SEQ ID NO. 1; (ii) the sequence from nucleotide position 46 to nucleotide position 930 of SEQ ID NO. 4; and (iii) the sequence from nucleotide position 46 to nucleotide position 714 of SEQ ID NO. 6;
  • a polynucleotide of the invention may thus consi ⁇ t essentially of sequence (a) , (b) , (c) or (d) .
  • a preferred polynucleotide is a DNA molecule.
  • the polynucleotide is an RNA molecule.
  • the nucleotide sequences and deduced amino acid sequence of the mouse GITR gene and of its splicing variants herein disclosed are set forth in SEQ ID NOs . 1, 4 and 6.
  • the nucleotide sequences of SEQ ID NOs. 1, 4 and 6 represent full length DNA clones of the sense strands of the mouse GITR gene and of its splicing variants GITR-B and GITR-C. All the isolated clones share a common extracytoplasmic sequence. In particular they have three cysteine pseudorepeats with the following structures:
  • the first pseudorepeat is similar to the first pseudorepeat of TNFRII (p75) which is considered to be the reference of the whole TNFR/NGFR family;
  • the second pseudorepeat is similar to the third pseudorepeat of TNFRII (p75) ;
  • the third pseudorepeat is similar to the fourth pseudorepeat of TNFRII (p75) .
  • TNFRII cysteine pseudorepeat
  • GITR lacks that pseudorepeat.
  • This second pseudorepeat of TNFRII is defined by the following motif: x-C-x(0, 1) - [DEP] -x (2 , 3) -
  • RNA derives from the splicing of 5 exons.
  • the boundary exon- intron and intron-exon are in agreement with the splicing rule.
  • the start codon and the stop codon for GITR protein synthesis are located in the first exon and in the fifth exon, respectively.
  • the fourth exon contains the sequence coding for the transmembrane domain, whereas the sequence coding for the cytoplasmic domain is contained in the fourth and the fifth exon. From the analysis of numerous clones isolated with the library screening (performed to isolate full-length GITR) it was found that some of them resulted to be different from GITR. In fact, between exon 4 and exon 5, 11 bases more were present (belonging to the 3 ' end of intron 4)
  • exon 5 is 11 bp longer (at the 5' end) than the exon 5 found in GITR.
  • the protein putatively coded by this clone called GITR-B, is different from GITR in the cytoplasmic domain (compare SEQ ID NOs. 2 and 5), due to the reading frame shift with respect to GITR. This quite long cytoplasmic domain does not have significant homology with other known proteins .
  • GITR-C SEQ ID NO. 6
  • the intron between exon 4 and exon 5 is not spliced out.
  • GITR-C is another GITR splicing.
  • the protein putatively coded by GITR-C is different from GITR and GITR-B in the cytoplasmic domain, due to the addition of 67 bp of intron 4 and a reading frame shift with respect to GITR and GITR-B (compare SEQ ID NOs. 2, 5 and 7) .
  • This cytoplasmic domain does not have significant homology with other known proteins .
  • the present invention also contemplates analogous DNA sequences which hybridize under stringent conditions to the DNA sequences set forth above.
  • An analogous polypeptide may thus incorporate from 1 to 20, for example from 1 to 15 or from 1 to 10, such conservative substitutions. There may be 1, 2, 3, 4 or 5 conservative substitutions.
  • Table 1 set ⁇ out conservative sub ⁇ titutions which may be made. Amino acids in the same line may be sub ⁇ tituted for each other:
  • the present invention also contemplates naturally occurring allelic variations and mutations of the DNA sequences set forth above so long as those variation ⁇ and mutation ⁇ code, on expression, for a GITR receptor.
  • the present invention includes further splicing variants a ⁇ defined above which can be identified with the aid of the information provided herein.
  • DNA sequences coding for a GITR receptor of any species of origin are also part of the present invention.
  • the DNA sequences code for proteins of mammalian origin; more preferably the DNA sequences code for the mouse GITR protein.
  • the present invention contemplates those other DNA molecules which, on expression, encode the polypeptides of SEQ ID NOs. 2, 5 or 7.
  • Tyrosine Tyr Y UAC UAU A ⁇ is well known in the art, codons constitute triplet ⁇ equences of nucleotides in mRNA molecules and, as such, are characterized by the base uracil (U) in place of base thymidine (T) (which is present in DNA molecules) .
  • the mouse GITR receptor of the present invention includes proteins homologous to, and having es ⁇ entially the ⁇ ame biological propertie ⁇ a ⁇ , the protein coded for by the nucleotide ⁇ equence herein di ⁇ clo ⁇ ed.
  • Thi ⁇ definition is intended to encompass natural allelic variants of GITR sequence, in particular those deriving from the various splicing variants of the GITR receptor.
  • DNA which encodes the GITR receptor may be obtained by ⁇ creening of cDNA or genomic DNA libraries with oligonucleotide probes generated from the GITR receptor gene sequence information provided herein. Probes may be labeled with a detectable group such as a fluorescent group, a radioactive atom or a chemiluminescent group in accordance with known procedure ⁇ and used in conventional hybridization assay ⁇ , a ⁇ de ⁇ cribed by, for example, Maniati ⁇ et al . Molecular cloning: a laboratory manual, Second Edition, Cold Spring Harbor Pre ⁇ , Cold Spring Harbor, NY (1989) .
  • the GITR gene ⁇ equence may alternatively be recovered by use of the polymerase chain reaction (PCR) procedure, with the PCR oligonucleotide primers produced from the GITR receptor sequence ⁇ provided herein. See U. S. Pat. Nos. 4,683,195 to Mullis et al . and U.S. Pat. No. 4,683,202 to Mulli ⁇ .
  • the PCR reaction provide ⁇ a method for ⁇ electively increasing the concentration of a particular nucleic acid sequence even when that ⁇ equence has not been previously purified and i ⁇ pre ⁇ ent only in a ⁇ ingle copy in a particular ⁇ ample.
  • the method can be u ⁇ ed to amplify either ⁇ ingle- or double- ⁇ tranded DNA.
  • the essence of the method involves the use of two oligonucleotides probe ⁇ to serve a ⁇ primer ⁇ for the template-dependent, polymerase mediated replication of a desired nucleic acid molecule.
  • the recombinant DNA molecules of the present invention can be produced through any of a variety of means well known to the experts in the art and disclo ⁇ ed by, for example, Maniati ⁇ et al . Molecular cloning: a laboratory manual, Second Edition, Cold Spring Harbor Pre ⁇ , Cold Spring Harbor, NY (1989) .
  • Vector ⁇ are used herein either to amplify DNA encoding the GITR receptor and/or to expre ⁇ DNA which encode ⁇ the GITR receptor.
  • An expression vector is a replicable DNA construct in which a DNA sequence encoding GITR receptor is operably linked to suitable control sequences capable of effecting the expression of the GITR receptor enzyme in a suitable host. DNA region ⁇ are operably linked when they are functionally related to each other.
  • a promoter i ⁇ operably linked to a coding sequence if it controls the transcription of the ⁇ equence.
  • Amplification vectors do not require expres ⁇ ion control domain . All that i ⁇ needed i ⁇ the ability to replicate in a host, usually conferred by an origin of replication, and a ⁇ election gene to facilitate recognition of transformants .
  • DNA sequences encoding GITR receptor or its splicing variants may be recombined with vector DNA in accordance with conventional techniques, including blunt-ended or staggered-ended termini for ligation, restriction enzyme digestion to provide appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphata ⁇ e treatment to avoid unde ⁇ iderable joining, and ligation with appropriate liga ⁇ e ⁇ . Techniques for such manipulation are di ⁇ clo ⁇ ed by Maniati ⁇ et al . Molecular cloning: a laboratory manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) and are well known in the art .
  • Expre ⁇ ion of the cloned ⁇ equence occurs when the expression vector is introduced into an appropriate host cell. If a prokaryotic expres ⁇ ion vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expre ⁇ ing the cloned ⁇ equence ⁇ , for example E. coli . Similarly, if an eukaryotic expre ⁇ ion vector is employed, then the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequence.
  • a yeast ho ⁇ t may be employed, for example S. cerevisiae .
  • in ⁇ ect cells may be used, in which case a baculovirus vector sy ⁇ tem may be appropriate.
  • Another alternative ho ⁇ t i ⁇ a mammalian cell line for example COS-1 cells.
  • control sequences include a transcriptional promoter, an optional operator sequence to control transcription, a sequence encoding suitable mRNA ribosomal binding, for example a Shine-Dalgarno sequence, and sequences which control the termination of transcription and translation.
  • Vector ⁇ u ⁇ eful for practi ⁇ ing the pre ⁇ ent invention include pla ⁇ mids, viruses (including phages) , retroviru ⁇ e ⁇ , and integrable DNA fragment ⁇ (i. e. fragments integrable into the host genome by homologous recombination) .
  • the vectors replicate and function independently of the ho ⁇ t genome, or may, in ⁇ ome instances, integrate into the genome itself .
  • Expres ⁇ ion vector ⁇ should contain a promoter which is recognized by the host organism.
  • the promoter sequences of the present invention may be either prokaryotic, eukaryotic or viral.
  • suitable prokaryotic sequences include the P R and P L promoters of bacteriophage lambda (The bacteriophage Lambda, Hershey, A. D., Ed., Cold Spring Harbor Pres ⁇ , Cold Spring Harbor, NY (1973) ; Lambda II, Hendrix, R. W., Ed., Cold Spring Harbor Pre ⁇ s, Cold Spring Harbor, NY (1980) ) ; the trp, recA, heat ⁇ hock, and lacZ promoter ⁇ of E. Coli and the SV40 early promoter (Benoi ⁇ t, C.
  • Shine-Dalgarno sequence i ⁇ concerned preferred example ⁇ of suitable regulatory sequence ⁇ are repre ⁇ ented by the Shine-Dalgarno of the replica ⁇ e gene of the phage MS-2 and of the gene ell of bacteriophage lambda.
  • the Shine-Dalgarno sequence may be directly followed by the DNA encoding GITR receptor and result in the expre ⁇ ion of the mature GITR protein.
  • the DNA encoding GITR may be preceded by a DNA ⁇ equence encoding a carrier peptide sequence.
  • a fusion protein is produced in which the N- terminu ⁇ of GITR i ⁇ fu ⁇ ed to a carrier peptide, which may help to increa ⁇ e the protein expre ⁇ ion levels and intracellular ⁇ tability, and provide ⁇ imple mean ⁇ of purification.
  • a preferred carrier peptide includes one or more of the IgG binding domains of protein A which are ea ⁇ ily purified to homogeneity by affinity chromatography e. g. on IgG-coupled Sepharose.
  • a DNA sequence encoding a recognition site for a proteolytic enzyme ⁇ uch a ⁇ enterokina ⁇ e, factor X or procollagenase may immediately precede the sequence for GITR to permit cleavage of the fusion protein to obtain the mature GITR protein.
  • a suitable expres ⁇ ion vector include ⁇ an appropriate marker which allow ⁇ the screening of the transformed host cells. The transformation of the selected host is carried out using any one of the various technique ⁇ well known to the expert in the art and described in Maniatis et al . Molecular cloning: a laboratory manual, Second Edition, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989) .
  • One further embodiment of the invention i ⁇ a prokaryotic ho ⁇ t cell tran ⁇ formed with the ⁇ aid expre ⁇ ion vector and able to produce, under appropriate culture conditions, the GITR receptor of the invention.
  • Cultures of cells derived from multicellular organisms are a desirable host for recombinant GITR synthe ⁇ i ⁇ .
  • any eukaryotic cell culture is workable, whether from vertebrate or invertebrate cell culture, including insect cells. Propagation of such cells in cell culture has become a routine procedure. See Tis ⁇ ue Culture, Academic Press, Kruse and Patterson, eds . (1973) .
  • Example ⁇ of useful host cell lines are HeLa cell ⁇ , CHO and COS cell line ⁇ .
  • the tran ⁇ criptional and translational control sequence ⁇ in expre ⁇ ion vectors to be used in transforming vertebrate and invertebrate cells are often provided by viral sources, for example, commonly used promoter ⁇ are derived from Adenoviru ⁇ 2, polyoma and SV40. See, e. g. U. S. Pat. No. 4,599, 308.
  • An origin of replication may be provided either by construction of the vector to include an exogenous origin or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient.
  • a selectable marker is dihydrofolate reductase (DHFR) or thymidine kinase. See U. S. Pat. No. 4,399,216.
  • DHFR dihydrofolate reductase
  • Cloned genes and vectors of the present invention are useful to transform cell ⁇ which do not ordinarly expres ⁇ GITR to thereafter expres ⁇ thi ⁇ receptor. Such cell ⁇ are u ⁇ eful a ⁇ intermediates for making recombinant GITR preparations useful for drug screening.
  • ⁇ tructural data deriving from the analy ⁇ is of the deduced amino acid sequences of the DNAs of the pre ⁇ ent invention are u ⁇ eful to de ⁇ ign new drugs, more specific and therefore with a higher pharmacological potency.
  • Variants of the GITR receptor protein of the present invention (obtained as described above) could be present in different tis ⁇ ues and/or organs, and might represent potential new pharmacological targets to develop more ⁇ pecific drug ⁇ .
  • Cloned genes of the present invention are u ⁇ eful for ⁇ creening for re ⁇ triction fragment length polymorphi ⁇ m (RFLP) a ⁇ ociated with certain di ⁇ order ⁇ .
  • RFLP fragment length polymorphi ⁇ m
  • Oligonucleotide ⁇ derived from the GITR DNA ⁇ equence or from the DNA sequences of its splicing variants disclo ⁇ ed in the pre ⁇ ent invention are u ⁇ eful a ⁇ diagnostic tools for probing GITR gene expression in various tissues.
  • ti ⁇ ue can be probed in si tu with oligonucleotide probe ⁇ carrying detectable group ⁇ by conventional autoradiography technique ⁇ to investigate native expression of this receptor or pathological condition ⁇ relating thereto.
  • RNA-free RNA were retrotranscribed (M-MLV reverse transcripta ⁇ e from GIBCO-BRL) by using an anchored primer T in _AC. 40 Cycles of PCR were performed using T 1X AC and the OPA 5 ' CGCGGAGGTG3 ' , SEQ ID NO: 3. Three independent ⁇ amples of untreated 3D0 cells were compared with 3 ⁇ ample ⁇ of 3h and 24h DEX-treated 3D0 cell ⁇ , by running a polyacrylamide gel.
  • the radioactive band ⁇ present in each of the short- or long-term treated samples and absent in each of the untreated cells were cloned by using TA- cloning kit (Invitrogen) and con ⁇ idered for further re ⁇ earch.
  • the cloned DNA corresponding to GITR cDNA was about 400 bp long.
  • a library screening was performed in order to obtain the full length cDNA.
  • a primary and secondary screening of a mouse T-cell (M30, CD4+) cDNA library (Stratagene) cloned unidirectionally in the Uni-ZAP XR vector was performed following the standard procedures (Sambrook K., Fritsch, E.F., & Maniati ⁇ , T. (1989) Molecular Cloning ed ⁇ C. Nolan (Cold Spring Harbor Laboratory Pre ⁇ , New York)) .
  • the 18 po ⁇ itive phages were in vivo excised through the ExA ⁇ i ⁇ t/SORL ⁇ y ⁇ tem, following the manufacturer' ⁇ in ⁇ truction .
  • Po ⁇ itive bacterial clones were PCR screened and, from among the longest in ⁇ ert ⁇ , three were cho ⁇ en for ⁇ equencing. The three clone ⁇ had identical sequences .
  • GITR cDNA has a 684 bp open reading frame (ORF) , beginning at nucleotide position 46 and extending to a TGA termination codon at po ⁇ ition 730. Three in-frame ATG ⁇ are found between po ⁇ i ion 46 and po ⁇ ition 79. The fir ⁇ t, at po ⁇ ition 46, i ⁇ ⁇ urrounded by a ⁇ equence (AGCACTATGG) in good agreement with the consensus sequence for initiation of translation in eukaryotes (Kozak) . The termination codon is followed by a 3' untranslated region of 276 bp . A canonical polyadenylation signal is present 18 bp before the poly-A tail.
  • ORF open reading frame
  • the protein putatively coded by GITR mRNA is a cysteine- rich protein 228 amino acid long. Two hydrophobic regions were found, probably representing the ⁇ ignal peptide and a tran ⁇ membrane domain.
  • the site of cleavage of the signal peptide might be between Gly and Gin (amino acid ⁇ 19 and 20 re ⁇ pectively in SEQ ID NOS: 1 and 2) de ⁇ pite the unu ⁇ ual pre ⁇ ence of A ⁇ p at amino acid position 17 (SEQ ID NOS: 1 and 2).
  • the transmembrane domain might ⁇ pan between po ⁇ ition 154 and po ⁇ ition 176.
  • GITR can be cla ⁇ ified a ⁇ a type I transmembrane protein with 153 amino acids forming the extracellular domain and 52 amino acids forming the intracellular domain.
  • the molecular weight of the native protein calculated on the ba ⁇ i ⁇ of the cDNA sequence is 25334 Da and thi ⁇ weight i ⁇ consistent to that obtained after in vitro translation of the cloned GITR cDNA ( ⁇ ee Example 3) .
  • the predicted molecular weight of the putative mature protein before further po ⁇ t-translational modifications is equal to 23321 Da and its isoelectric point is equal to 6.46.
  • the GITR amino acid sequence displays significant homologies with the 4-IBB receptor which belongs to the TNF/NGFR family.
  • the extracellular domain of molecules belonging to the TNF/NGFR family is characteri ⁇ ed by cy ⁇ teine p ⁇ eudorepeats whose functional properties have been defined.
  • the canonical cysteine pseudo-repeat is formed by cysteine 1 (Cl) that forms a disulfide bridge with cysteine 2 (C2), cysteine 3 (C3) that forms a di ⁇ ulfide bridge with cysteine 5 (C5) and cysteine 4 (C4) that forms a disulfide bridge with cy ⁇ teine 6 (C6) .
  • the first p ⁇ eudorepeat from Cy ⁇ at po ⁇ ition 29 to Cy ⁇ at po ⁇ ition 60, although atypical, has some features similar to that of the first pseudorepeat of several proteins belonging to the TNF/NGFR family (CD30, CD27, TNFR p-55 and p-75, LT ⁇ R, Fas, NGFR, CD40, OX40). It is formed by Cl, C2 , C3 and C5. C6 i ⁇ al ⁇ o present but, ⁇ ince C4 i ⁇ not pre ⁇ ent, C6 ⁇ hould not form a disulfide bridge in this pseudorepeat .
  • TNFR/NGFR family (TNFR p75, CD40, LT ⁇ R, CD30 and 4-1BB) .
  • the third cysteine pseudorepeat from Cys at position 103 to Cys at po ⁇ ition 141 lack ⁇ C3 and C5 and ⁇ how ⁇ exten ⁇ ive homologie ⁇ with the p ⁇ eudorepeat number 4 of ⁇ everal members of the TNFR/NGFR family (OX40, 4-IBB, CD40 and TNFR p75) .
  • the cytoplasm domain of GITR has a high similarity with the intracellular domain of murine and human CD27 and 4- 1BB (see Table 3), ⁇ o that it could be hypothe ⁇ ized that 4-1BB, CD27 and GITR define a cytopla ⁇ mic domain ("life domain") of the TNFR/NGFR family which i ⁇ different from the TNFR-Fa ⁇ "death domain". Thu ⁇ , GITR should activate intracellular pathways similar to those activated by CD27 and 4 -IBB. We are the first to de ⁇ cribe this domain since GITR i ⁇ similar to both CD27 and 4-1BB, while CD27 and 4- 1BB have a lower degree of similarity.
  • the "life domain” should have a functional meaning since the similarity among the extracellular domain of GITR, CD27 and 4 -IBB are much lower (at the same level of the other member of the family) and thus the common derivation from an ancestral gene can be excluded.
  • Example 3 In vitro translation of GITR cDNA.
  • 949 bp DNA coding for GITR were cloned into pCR3 (Invitrogen) from which the portion coding for resi ⁇ tance to Geneticin had been removed.
  • 1 ⁇ g of the re ⁇ ulting plasmid was added together with the translation system and 40 ⁇ Ci [ 35 S]methionine (Amersham Life Science International) and translation wa ⁇ allowed to proceed for 90 minute ⁇ at 30 °C according to manufacturer' ⁇ instruction ⁇ .
  • the product was analyzed by electrophoresi ⁇ in 15% SDS- PAGE gel ⁇ , followed by tran ⁇ fer to nitrocellulo ⁇ e (Bioblot NK, Costar) for 5 hours at 250 mA at 4°C in 25 mM Tris/glycine, pH 8.3, and 20% v/v methanol . After transfer the radioactive protein was revealed by autoradiography for 1 day. The molecular weight of the expressed product was consistent with the predicted molecular weight of 25334 Da.
  • Lymphocytes from thymus, spleen and lymph nodes expres ⁇ ed low or undetectable mRNA level ⁇ of GITR as demon ⁇ trated by PCR or Northern blotting re ⁇ pectively .
  • Treatment of lymphocytes from lymph nodes for 1-5 days with anti-CD3 antibodies, with ConA or TPA plus Ca-ionophore caused an up-modulation of GITR mRNA. Similar re ⁇ ult ⁇ were obtained with thymocytes and splenocytes .
  • TNF/NGFR family are involved in lymphocyte activation and are able to induce or inhibit cell death by apopto ⁇ i ⁇ .
  • GITR expres ⁇ ion we transfected cells of the hybridoma T cell line 3DO with an expres ⁇ ion vector in which the GITR cDNA is expres ⁇ ed under the control of the CMV promoter.
  • a ⁇ controls we also transfected cells with the empty vector (clones pCR3/l-6) . or the same vector expressing the same GITR sequence but in the anti-sen ⁇ e direction (RTIG, clone ⁇ RTIG/l-6) .
  • RT-PCR reverse-tran ⁇ criptase PCR
  • the parental plasmid u ⁇ ed in the tran ⁇ fection experiments was pCR3 (Invitrogen) .
  • 979 bp DNA coding for GITR in the sense or antisense (RTIG) orientation wa ⁇ al ⁇ o cloned into a pCR3 plasmid from which the portion coding for resistance to Geneticin had been removed (pCR3/G-) .
  • 3DO cells were cotransfected with 5 ⁇ g of pCR3 and 15 ⁇ g of pCR3/G- to increa ⁇ e the amount of GITR or RTIG in the Geneticin re ⁇ istant cells.
  • Pla ⁇ mid preps were made with Qiagen's Maxiprep plasmid DNA kit. 3DO cells were electroporated at 300 mA, 960 ⁇ F in the presence of plasmid and cultured for 48h in the standard medium. Then Geneticin (0.5 mg/ml) was added to the cell culture and 200 ⁇ l of the cell ⁇ uspension were plated in 96-well ⁇ plate ⁇ (3 for each tran ⁇ fection) . Following 10-15 day ⁇ , no more than 15% of the well ⁇ presented alive growing cells. These cells were con ⁇ idered clone ⁇ and PCR screened for the expres ⁇ ion of exogenou ⁇ GITR or RTIG. The six best clones were considered for functional studie ⁇ . Logarithmically growing cell ⁇ were cultured in 96-well plate ⁇ (5xl0 5 cells/ml) coated overnight with anti-mouse
  • CD3e mAb ⁇ (Pharmingen, San Diego, CA) (10 ⁇ g/ml) . 24 h later apopto ⁇ i ⁇ wa ⁇ measured by flow cytometry a ⁇ de ⁇ cribed (Migliorati G., Nicoletti I., Pagliacci M.C., D'Adamio L. & Riccardi C. (1993) Blood 81, 1352-1358) and a ⁇ below ⁇ pecified. Briefly, after culturing, cell ⁇ were centrifuged and the pellet ⁇ were gently re ⁇ u ⁇ pended in
  • hypotonic PI ⁇ olution 50 ⁇ g/ml in 0.1% sodium citrate plus 0.1% Triton X-100, Sigma, St. Louis, MO,
  • the nuclei traversed the light beam of a 488 nm Argon laser.
  • a 560 nm dichroid mirror (DM 570) and a 600 nm band pass filter (band width 35 nm) were used to collect the red fluorescence due to PI DNA staining, and the data were recorded in logarithmic scale in a Hewlett Packard (HP 9000, model 310) computer.
  • the percentage of apoptotic cell nuclei (sub-diploid DNA peak in the DNA fluore ⁇ cence hi ⁇ togram) wa ⁇ calculated with ⁇ pecific FACSCAN re ⁇ earch ⁇ oftware (Lysis II, Becton Dickinson) .
  • clone ⁇ expressing antisense RTIG RNA were more sensitive to anti-CD3- induced apoptosis (apoptosis between 80 and 93% as compared to 50-60% of pCR3 control clones; P ⁇ 0.01) ⁇ ugge ⁇ ting that anti ⁇ ense expression may have inhibited the low levels of endogenous RTIG expres ⁇ ion.
  • GITR can modulate T cell apopto ⁇ i ⁇ triggered by T-cell receptor (TCR) /CD3 complex.
  • nuc normal untransfected clone ⁇ (fir ⁇ t control)
  • pCR3 empty vector tran ⁇ fected clone ⁇ (second control]
  • GITR sense GITR transfected clones
  • RTIG antisen ⁇ e GITR tran ⁇ fected clone ⁇
  • TRIzol LS reagent Gibco- BRL, Life TECH ⁇ , Pai ⁇ ley, Scotland
  • DNAse RNAse-free Promega
  • GITR-C was obtained with several primers located on exon 4 (forward) and on exon 5 (reverse) . However, the product obtained could derive by a contaminating DNA (de ⁇ pite DNAse treatment) , since the sequence obtained was identical to the genomic sequence of GITR.
  • an RT-PCR was performed by using a forward primer located on exon 2
  • NAME PHARMACIA & UPJOHN S.p.A.
  • CTGCTCCCCT CAACAGTGGC GGAAGTGGGT GTATGAGAGC GGTGAGTTAC GATTGGGCCC 964

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Abstract

La présente invention concerne une protéine de récepteur inconnue dans l'art antérieur, dénommée GITR (protéine liée à la famille TNFR induite par glucocorticoïde) qui partage des caractéristiques biologiques et structurelles avec d'autres membres de la famille (TNFR/NGFR) des récepteurs du facteur de nécrose tumorale/ de croissance du nerf. L'invention traite aussi de variantes d'épissage du GITR. L'invention a aussi pour objet des séquences de polynucléotides codant le récepteur du GITR et ses variantes d'épissage, des vecteurs comprenant ces séquences de polynucléotides, des cellules hôtes transformées par ce vecteur et un processus de recombinaison permettant de produire les protéines selon l'invention.
PCT/EP1997/006252 1996-12-02 1997-11-08 Recepteur appartenant a la famille de recepteurs tnf/ngf WO1998024895A1 (fr)

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999040196A1 (fr) * 1998-02-09 1999-08-12 Genentech, Inc. Nouveaux homologues recepteurs du facteur necrosant des tumeurs et acides nucleiques codant ceux-ci
US6503184B1 (en) 1997-10-21 2003-01-07 Human Genome Sciences, Inc. Human tumor necrosis factor receptor-like proteins TR11, TR11SV1 and TR11SV2
WO2003006058A1 (fr) * 2001-07-12 2003-01-23 Wyeth Marqueurs differentiels cd25+ et leurs utilisations
US6689607B2 (en) 1997-10-21 2004-02-10 Human Genome Sciences, Inc. Human tumor, necrosis factor receptor-like proteins TR11, TR11SV1 and TR11SV2
US7618632B2 (en) 2003-05-23 2009-11-17 Wyeth Method of treating or ameliorating an immune cell associated pathology using GITR ligand antibodies
US7812135B2 (en) 2005-03-25 2010-10-12 Tolerrx, Inc. GITR-binding antibodies
EP1947183B1 (fr) * 1996-08-16 2013-07-17 Merck Sharp & Dohme Corp. Antigène de surface de cellule de mammifère; agents chimiques relatifs
US8591886B2 (en) 2007-07-12 2013-11-26 Gitr, Inc. Combination therapies employing GITR binding molecules
US9228016B2 (en) 2014-06-06 2016-01-05 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (GITR) and uses thereof
US9464139B2 (en) 2013-08-30 2016-10-11 Amgen Inc. GITR antigen binding proteins and methods of use thereof
US11213586B2 (en) 2015-11-19 2022-01-04 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (GITR)
US11408889B2 (en) 2015-06-03 2022-08-09 Bristol-Myers Squibb Company Anti-GITR antibodies for cancer diagnostics
US11685787B2 (en) 2017-05-16 2023-06-27 Bristol-Myers Squibb Company Treatment of cancer with anti-GITR agonist antibodies

Citations (1)

* Cited by examiner, † Cited by third party
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WO1998006842A1 (fr) * 1996-08-16 1998-02-19 Schering Corporation Antigenes de surface de cellules mammaliennes et reactifs qui y sont lies

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WO1998006842A1 (fr) * 1996-08-16 1998-02-19 Schering Corporation Antigenes de surface de cellules mammaliennes et reactifs qui y sont lies

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FIORUCCI S ET AL: "Isolated guinea pig gastric chief cells express tumour necrosis factor receptors coupled with the sphingomyelin pathway.", GUT, (1996 FEB) 38 (2) 182-9. JOURNAL CODE: FVT. ISSN: 0017-5749., XP002061243 *
NOCENTINI G ET AL: "A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, (1997 JUN 10) 94 (12) 6216-21. JOURNAL CODE: PV3. ISSN: 0027-8424., XP002061244 *

Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1947183B1 (fr) * 1996-08-16 2013-07-17 Merck Sharp & Dohme Corp. Antigène de surface de cellule de mammifère; agents chimiques relatifs
US6503184B1 (en) 1997-10-21 2003-01-07 Human Genome Sciences, Inc. Human tumor necrosis factor receptor-like proteins TR11, TR11SV1 and TR11SV2
US6689607B2 (en) 1997-10-21 2004-02-10 Human Genome Sciences, Inc. Human tumor, necrosis factor receptor-like proteins TR11, TR11SV1 and TR11SV2
JP2002502607A (ja) * 1998-02-09 2002-01-29 ジェネンテク・インコーポレイテッド 新規な腫瘍壊死因子レセプター相同体及びそれをコードする核酸
WO1999040196A1 (fr) * 1998-02-09 1999-08-12 Genentech, Inc. Nouveaux homologues recepteurs du facteur necrosant des tumeurs et acides nucleiques codant ceux-ci
WO2003006058A1 (fr) * 2001-07-12 2003-01-23 Wyeth Marqueurs differentiels cd25+ et leurs utilisations
US7618632B2 (en) 2003-05-23 2009-11-17 Wyeth Method of treating or ameliorating an immune cell associated pathology using GITR ligand antibodies
US7812135B2 (en) 2005-03-25 2010-10-12 Tolerrx, Inc. GITR-binding antibodies
US8388967B2 (en) 2005-03-25 2013-03-05 Gitr, Inc. Methods for inducing or enhancing an immune response by administering agonistic GITR-binding antibodies
US10570209B2 (en) 2005-03-25 2020-02-25 Gitr, Inc. Methods for inducing or enhancing an immune response by administering agonistic glucocorticoid-induced TNFR-family-related receptor (GITR) antibodies
US9028823B2 (en) 2005-03-25 2015-05-12 Gitr, Inc. Methods of inducing or enhancing an immune response in a subject by administering agonistic GITR binding antibodies
US10030074B2 (en) 2005-03-25 2018-07-24 Gitr, Inc. Methods of inducing or enhancing an immune response in a subject having cancer by administering GITR antibodies
US9493572B2 (en) 2005-03-25 2016-11-15 Gitr, Inc. GITR antibodies and methods of inducing or enhancing an immune response
US9241992B2 (en) 2007-07-12 2016-01-26 Gitr, Inc. Combination therapies employing GITR binding molecules
US8591886B2 (en) 2007-07-12 2013-11-26 Gitr, Inc. Combination therapies employing GITR binding molecules
US9464139B2 (en) 2013-08-30 2016-10-11 Amgen Inc. GITR antigen binding proteins and methods of use thereof
US9745379B2 (en) 2014-06-06 2017-08-29 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (GITR) and uses thereof
US9228016B2 (en) 2014-06-06 2016-01-05 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (GITR) and uses thereof
US10465010B2 (en) 2014-06-06 2019-11-05 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (GITR) and uses thereof
US10501550B2 (en) 2014-06-06 2019-12-10 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (GITR) and uses thereof
US11084881B2 (en) 2014-06-06 2021-08-10 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (GITR) and uses thereof
US11802162B2 (en) 2014-06-06 2023-10-31 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (GITR) and uses thereof
US11408889B2 (en) 2015-06-03 2022-08-09 Bristol-Myers Squibb Company Anti-GITR antibodies for cancer diagnostics
US11213586B2 (en) 2015-11-19 2022-01-04 Bristol-Myers Squibb Company Antibodies against glucocorticoid-induced tumor necrosis factor receptor (GITR)
US11685787B2 (en) 2017-05-16 2023-06-27 Bristol-Myers Squibb Company Treatment of cancer with anti-GITR agonist antibodies

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