WO2002046231A2 - Caml-binding peptides - Google Patents
Caml-binding peptides Download PDFInfo
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- WO2002046231A2 WO2002046231A2 PCT/CA2001/001769 CA0101769W WO0246231A2 WO 2002046231 A2 WO2002046231 A2 WO 2002046231A2 CA 0101769 W CA0101769 W CA 0101769W WO 0246231 A2 WO0246231 A2 WO 0246231A2
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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
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
- C12N2710/00011—Details
- C12N2710/10011—Adenoviridae
- C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
- C12N2710/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- This application relates to modulation of apoptosis and immune response.
- Viral infection is a cellular injury, and can result in the induction of programmed cell death (apoptosis) of the host cell.
- Many viruses particularly persistent DNA viruses modify the apoptotic response of a cell to allow continued virus replication.
- Apoptosis can be induced by the members of the TNF receptor super-family such as Fas (APO-1 or CD95) and p55 Tumor Necrosis Factor Receptor (p55 TNFR) as well as the death domain-containing receptors 3, 4 and 5 (DR3, DR4 and DR5, respectively).
- the intracellular factors responsible for death of the cell are highly conserved across species and are the target of viral inhibitors of apoptosis.
- adenovirus E1B 55K SV40 Large T antigen and human papiUoma virus E6 inhibit p53-mediated lysis.
- the cellular survival factor Bcl-2 is mimicked by adenovirus E1B 19K, Epstein-Barr virus BHRF1 and African swine fever virus LMW5-HL.
- ICE Interleukin lb Converting Enzymes
- caspases are blocked by baculovirus p35 and crmA, the cowpox serpin protein.
- the adenovirus E3/10.4K and E3/14.5K proteins downregulate surface Fas, while the Inhibitors of Apoptosis (IAP) family of baculovirus and mammalian homologues interact with the TNF- ⁇ receptor associated factors (TRAFs) therefore blocking the signalling cascade that leads to the recruitment of caspases.
- IAP Inhibitors of Apoptosis
- TNFs TNF- ⁇ receptor associated factors
- FADD-like interleukin- lbeta- converting enzyme FLICE
- caspase-8 FlEC-like interleukin- lbeta- converting enzyme
- vFLIPs viral- FLICE-inhibitory proteins
- Adenovirus (Ad) is a very common human pathogen that results in persistent infections of the respiratory or gastrointestinal tract. Persistent infections stem from an elaborate evasion of the host defense mechanisms. The adenovirus genes responsible for immune evasion map to the Early 3 (E3) region of the Ad genome. The persistence, ease of infection and weak pathogenesis have made adenovirus suitable as vectors for gene therapy.
- Ad gene transfer vectors are the most efficient technique available for in vivo gene transduction.
- the genetic makeup of the original vectors was designed to accommodate large fragments of DNA for the transduced gene, to the expense of areas of the adenoviral genome that were considered dispensable.
- the E3 region was one of the first areas to be replaced.
- the 6.7K protein encoded by the E3 region does not have any significant homology to any other known proteins. It is well conserved between group C Ad2 and Ad5 adenovirus and between group B Ad3, Ad7 and Ad35 adenovirus.
- the Ad2 E3/6.7K protein (Wils ⁇ n-Rawls et al., (1990) Virology 178:204-212) has been shown to be an integral membrane protein localized to the endoplasmic reticulum (ER) (Wilson- Rawls and Wold, (1993) Virology 195:6-15).
- the protein is present in two forms, one unglycosylated with an apparent molecular weight of 8kDa and one glycosylated with an apparent weight of 14kDa.
- the protein though targeted to the ER, does not have a cleavable signal sequence, but it has a hydrophobic central region that could act as a signal anchor (Wilson-Rawls et ⁇ /.,(1994) Virology 201 :66-76).
- Ad vectors The major impediment for the success of Ad vectors as well as all the other gene transfer technologies is the unexpectedly strong immune response to cells infected by a modified Adenovirus.
- the strong immune response to modified Ad vectors appears to be mediated by the circulating cytokine Tumor Necrosis Factor (TNF) ⁇ and by the innate immune response.
- TNF Tumor Necrosis Factor
- the negative effects of an immune response might be alleviated by implementing immunomodulatory proteins that allow the vector and the transduced cells to survive the immune response.
- the evasion of immune response is also a central impediment to the establishment of successful transplant technology as well as the treatment of autoimmune and neurodegenerative diseases.
- Apoptosis of the affected organ is often the result of neurodegenerative inflammatory disease. Factors that prevent apoptosis could lead to better therapies for these conditions.
- Cell culture reactor expression systems are typically limited only by the ability of cells to grow and produce proteins of interest. As cells grow, they reach densities where protein production stops and producer cells undergo apoptosis in response to factors that are currently poorly characterized. There is potential for improving protein yield by avoiding the apoptotic response of cells grown in culture by including an antiapoptotic protein in the makeup of the cell.
- CAML also known as "calcium-signal modulating cyclophilin ligand"; United States Patent No's. 5,523,227 and 5,969,102
- CAML affects calcineurin in response to an extrinsic signal and has been shown to be important in lymphocyte activation.
- CAML is involved in activation of transcription factors in lymphocytes, including T-cell transcription factor (NF-AT) and is important in modulation of immune response or modulation of apoptosis.
- NF-AT T-cell transcription factor
- TACI transmembrane activator and CAML interactor
- TACI is reported to have a N-terminal extracellular domain, a centrally located transmembrane domain, and a C-terminal intracellular domain. Investigation of sequence homology with respect to TACI only revealed homology between portions of the N-terminal domain of TACI (representing a TNFR_NGFR repeat motif in the regions of amino acids 33-104 of human TACI),and a number of growth factor receptors (United States Patent No. 5,969,102). In the 293 amino acid human sequence of TACI, the transmembrane domain is thought to reside at about amino acids 167-186.
- adenovirus E3/6.7L prevents inflammation, apoptosis and the cellular damage response following viral infection.
- the presence of the E3/6.7K protein correlates with reduced inflammatory response in the lungs of virally infected mice.
- Transfected cells that express the E3/6.7K protein are now shown to be protected against apoptosis induced by TNF- ⁇ .
- TNF- ⁇ induced release of arachidonic acid is significantly reduced in cells expressing transfected E3/6.7K.
- Efflux of calcium ions from the ER is also reduced in the presence of E3/6.7K. Therefore, the mechanism of action of E3/6.7K does involve maintenance of calcium ion homeostasis.
- the release of calcium ions from the ER is known to be important for the generation of mediators of inflammation, apoptosis and of the cellular damage response.
- E3/6.7K has no sequence homology to any of the previously described inhibitors of apoptosis.
- E3/6.7K therefore represents a member of a new class of modulators of apoptosis (particularly in lymphocytes) and the immune response, and which are useful as modulators of inflammation.
- This new class of modulators act through alteration of Ca + influx and thus may be inhibiting or promoting in their effects.
- E3/6.7K binds to CAML and inhibits Ca 2+ influx, thus resulting in inhibition of apoptosis and inflammation. Binding by E3/6.7K fragments to full length CAML has been determined by immunoprecipitation and by yeast two-hybrid assays.
- E3/6.7K capable of interacting with CAML
- Such E3/6.7K domains are collectively termed herein "6.7-effector domain" (SED).
- SED 6.7-effector domain
- part of this domain shares homology with the putative transmembrane domain of TACI, contrary to the previous published reports with respect to TACI binding to CAML.
- Examples of the CAML-binding domains in TACI which correspond to SED include domains of about 28 amino acids in length found from about amino acids 163-189 of human TACI and from about amino acids 126-152 or 153 of mouse TACI.
- CAML-binding motif With knowledge of the CAML-binding domain of TACI and E3/6.7K, a CAML- binding motif has been determined and has been employed to identify known proteins which share this motif, thereby making such proteins available for use in CAML-binding.
- One aspect of the invention involves the provision of and use of CAML-binding motifs that may be used to confer CAML-binding function on a ligand or other moiety intended to bind to CAML.
- Such motifs may in themselves modulate CAML function or may be used to facilitate CAML modulation by permitting binding of a CAML modulating ligand or moiety to CAML.
- the motifs may also be used to permit binding of moieties which are not intended to modulate CAML function (such as a CAML specific labeling moieties).
- CAML-binding motifs may comprise sequences of 26, 27 or 28 amino acids, such as follows:
- LALLCLRVAACCTHVCTYCQLFKRWG (from Ad2 E3/6.7K) SEQ ID NO:l; LTLLCLRLAACCVHICIYCQLFKRWG (from Ad5 E3/6.7K) SEQ ID NO:2;
- VLILCYLYTPCCAYLVTLCCWFI ⁇ KWG (from Ad3 E3/1.6K) SEQ ID NO:3;
- VYSTLGLCLCAVLCCFLVAVACFLKKR (from human TACI) SEQ ID NO:4;
- LYCTLGVCLCAIFCCFLVALASFLRRRG (from murine TACI) SEQ ID NO:5.
- the CAML-binding motif of this invention may comprise a sequence having any one of the following consensus sequences, wherein: a "x" represents any amino acid with the number of amino acids (or range of possible numbers of amino acids) being indicated by a bracketed number or numbers following "x"; and, single letter amino acid abbreviations within square brackets represent alternative amino acids at a single position: (a) C-C-x(2)-[FILV]-[ACN]-x(2)-[CS]-x(3)-[KR]-[KR]; (b) C-C-x(2)-[ILV]-[ACV]-x(2)-[CS]-x(3)-[KR]-[KR];
- CAML-binding motifs of the invention may be derived from or used as part of known proteins or fragments of known proteins, including those set out in the following list in which the accession number of the database reference is followed by the name and source of the protein, together with identification of the amino acid sequence and its position within the protein corresponding to consensus sequence (b) above.
- SWISS-PROT E316_ADE03 P11320 Early E3 16 KDA glycoprotein Human adenovirus type 3 129-142 CCAYLVILCCWFKK; SEQ ID NO:6 [2] SWISS-PROT: SY61_DISOM P24505
- Synaptotagmin A (synaptic vesicle protein O-P65-A) Discopyge ommata (Electric ray) 79-92 CCFCICKKCLLKKK; SEQ ID NO:7 [3] SWISS-PROT: SY62_DISOM P24506 Synaptotagmin B (synaptic vesicle protein O-P65-B)
- SWISS-PROT SYT1_CHICK P47191 Synaptotagmin I (P65) Gallus gallus (chicken) 77-90 CCFCLCKKCLFKKK; SEQ ID NO: 10 [6] SWISS-PROT: SYT1JHUMAN P21579
- Rattus norvegicus (rat)
- Rattus norvegicus (rat) 82-95 CCFCICKKCCCKKK; SEQ ID NO: 15
- TrEMBL Q64830 Q64830
- TrEMBL Q9Q8F7 Q9Q8F7 M147R Myxoma virus
- Peptides of this invention may comprise an amino acid sequence corresponding to the above-described CAML-binding motifs combined with additional amino acids selected in order for the peptide to affect CAML function.
- Peptides of this invention will have a minimum of about 14 amino acids. While no specific maximum length of peptides (including proteins comprising peptides) of this invention is contemplated and may comprise (for example) up to about 300 amino acids, peptides of this invention selected or employed for purposes of CAML-binding will typically have a maximum length of about 100, preferably less than 100 amino acids.
- peptides of this invention will have a number of amino acids selected from a number from 14-60, more preferably from 18-60, more preferably from 20-60, more preferably from 25-60, more preferably from 25-50.
- peptides of this invention may consist of from about 20 to about 75 amino acids; or, from about 25 to about 60 amino acids; or, from about 26 to about 40 amino acids; or, from about 26 to about 35 amino acids; or, from about 26 to about 30 amino acids. Any combination of these examples of minimum and maximum lengths may be employed.
- This invention does not include the use of previously identified, native (e.g. full length) human and murine TACI proteins or known fragments of such proteins for
- CAML-binding CAML modulation, or modulation of apoptosis or immune response.
- this invention does include peptides and the use of peptides derived from TACI as described herein.
- TACI derived proteins and fragments may comprise or consist of the 27 and 28 amino acid CAML-binding peptides from human and murine TACI described above and the CAML-binding motifs comprising amino acids 176-189 of human TACI or amino acids 139-152 of murine TACI as described above.
- compositions of matter consisting only or essentially of: native E3/6.7K protein, native human and murine TACI, known proteins identified at items [2]-[10], [12] and [13] above or known fragments of these proteins.
- this invention includes compositions of matter consisting of the CAML-binding peptides, sequences and motifs of this invention, as defined herein.
- This invention includes an isolated CAML-binding peptide comprising a sequence of amino acids defined by the motif: C-C-x(2)-[FILV]-[ACV]-x(2)-[CS]-x(3)-[KR]-[KR], wherein: x represents any amino acid; a bracketed numeral represents the number of or a range of numbers of any amino acid represented by x; a single letter represents a specific amino acid identified by standard single letter amino acid code; and, a bracketed set of two or more single letters represents alternate amino acids at a single position; providing that the ligand does not comprise more than 100 contiguous amino acids of native TACI.
- This invention provides for methods for targeted binding of a moiety or ligand to CAML, including for modulation of immune response and for modulation of apoptosis, which methods make use of the CAML-binding peptides of this invention.
- This invention provides a method for binding a ligand to CAML, comprising combining CAML with a ligand, the ligand comprising a peptide defined by the amino acid motif:
- x represents any amino acid
- a bracketed numeral represents the number of or a range of numbers of any amino acid represented by x
- a single letter other than x represents a specific amino acid identified by standard single letter amino acid code
- a bracketed set of two or more single letters represents alternate specific amino acids at a single position; providing that the ligand does not comprise more than 100 contiguous amino acids of native TACI.
- This invention also provides nucleic acids and nucleic acid vectors encoding the
- CAML-binding peptides of this invention for use in treatment (such as gene therapy or to minimize transplant rejection), and for use in recombinant expression of the peptides of this invention or proteins comprising the peptides of this invention. Also included are cells comprising nucleic acids and vectors according to this invention.
- This invention provides methods for effecting targeted binding of a ligand or other moiety to CAML, including for inhibiting or inducing apoptosis or treatment of inflammation comprising treating the cell, a mammal comprising the cell, or a tissue comprising the cell, with a medicament comprising a CAML-binding peptide of this invention.
- the treating may comprise administering a nucleic acid encoding a peptide of this invention whereby the peptide is expressed in the cell.
- the administering may be by a viral vector comprising the nucleic acid, with the proviso that if the vector is adenovirus or myxoma virus, the nucleic acid is other than a naturally occurring nucleic acid from E3 of adenovirus, or the nucleic acid is under the transcription control of a promotor not found in the selected virus.
- the methods of this invention may be employed for treatment of a mammalian patient suffering from a degenerative (e.g. neurodegenerative) disease, an immunodeficiency, or an inflammatory disease by modulation of immune response or apoptosis.
- a degenerative disease e.g. neurodegenerative
- an immunodeficiency e.g. an immunodeficiency
- an inflammatory disease e.g. an inflammatory fibrosis
- This invention also provides for methods of modulating immune response or programmed cell death in a tissue or cell population in a patient comprising: (a) withdrawing tissue or a cell from the patient, (b) treating the tissue or cells with an apoptosis modulating moiety comprising a CAML-binding peptide of this invention; and
- This invention also provides medicaments, pharmaceutical compositions and medical devices comprising a CAML-binding peptide of this invention, a carrier suitable for facilitating delivery of the peptide to a cell and optionally other active components such as a moiety intended to affect CAML activity. Also provided is a nucleic acid comprising a non-naturally occurring adenovirus E3 nucleic acid capable of encoding a
- CAML-binding peptide of this invention CAML-binding peptide of this invention.
- This invention also provides recombinant virus comprising a nucleic acid encoding a CAML-binding peptide of this invention with the proviso that if the virus is adenovirus or myxoma virus, the nucleic acid is other than a naturally occurring adenovirus E3 nucleic acid or myxoma virus nucleic acid; or, the nucleic acid is under the transcriptional control of a promoter not from the selected virus.
- This invention also provides: the use of a CAML-binding peptide of this invention, a nucleic acid encoding said peptide or a vector comprising said nucleic acid, for treatment, including for inducing or inhibiting apoptosis, for modulating an immune or inflammatory response; and, the use of a CAML-binding peptide of this invention, a nucleic acid encoding said peptide or a vector comprising said nucleic acid for the preparation of a medicament, pharmaceutical composition or implantable medical device or material, for such uses.
- This invention also provides an assay for selecting an agent capable of modulating activity of CAML which comprises: combining a CAML-binding peptide with a sample suspected of comprising such an agent or with a putative agent; and, determining whether such CAML activity is modulated. Determining whether CAML activity is modulated may be done by known means for assessing CAML activity. Combining of CAML and a CAML-binding peptide may occur in vitro (e.g. in a cell extract) or may be done in vivo, as in a cell that is a cell that is rescued from apoptosis or an immune response. The combining in an assay of this invention may include a coupling of a peptide of this invention to an agent or putative CAML modulating agent.
- Figure 1 A chart showing alignment of a nucleic acid sequence which is capable of encoding a E3/6.7K protein corresponding to that of Adenovirus serotype Ad2 wild-type(Wt) (SEQ ID NO:20); and the polymerase chain reaction (PCR) nucleic acid product expected (SEQ ID NO: 21) when a forward primer (FP - SEQ ID NO: 22) and a reverse primer (RP - SEQ ID NO: 23) are used to amplify the wild-type sequence(Wt). Start codons are underlined.
- the nucleic acids shown in bold in the forward primer (FP) represent a modification to provide a Kozak consensus sequence.
- FIG. 1 A chart showing alignment of E3/6.7K amino acid sequences from the Ad2 (SEQ ID NO: 24) and Ad5 (SEQ ID NO: 25) Adenovirus serotypes.
- the Ad2 E3/6.7K amino acid sequence is 61 amino acids in length and the Ad5 E3/6.7K amino acid sequence is 63 amino acids in length.
- E3/6.7K protein, peptide or polypeptide includes a protein or fragment thereof encoded by a nucleic acid as depicted in Figure lor an Ad2 or Ad5 adenovirus serotype protein or fragment thereof as depicted in Figure 2.
- the Ad5 protein shown in Figure 2 is about 7. IK.
- Modulation of apoptosis including inhibition of apoptosis or rescue of a cell from apoptosis may be determined by various methods known in the art, including assays which directly measure apoptosis or which measure the activity of TNF- ⁇ , such as those described herein.
- the isolated nucleic acid molecule depicted in Figure 1 a nucleic acid molecule encoding a CAML-binding peptide as defined herein or a nucleic acid molecule complementary to those described above, may be incorporated into a vector suitable for expression in a host cell or to act as a transformation vector, such as to introduce the nucleic acid into cells of a mammal to be treated.
- Suitable vectors for such purposes include retroviruses and adenoviruses.
- transfection vector comprising a CAML- binding peptide encoding nucleic acid molecule
- Techniques for the formation of the transfection vector comprising a CAML- binding peptide encoding nucleic acid molecule are well-known in the art, and are generally described in "Working Toward Human Gene Therapy," Chapter 28 in Recombinant DNA, 2nd Ed , Watson, J.D. ci al., eds., New York: Scientific American Books, pp. 567-581 (1992), and in the references cited therein.
- Various promoters may be used to enhance expression in host cells or gene expression in specific tissues. For example, in neuronal tissue the neuron-specific enolase promoter (Ad-NSE) and in Lymphocytes the lck promoter could be used for gene therapy methods.
- Ad-NSE neuron-specific enolase promoter
- Lymphocytes the lck promoter could be used for gene therapy methods.
- CAML-binding peptides have potential uses in tissue and organ transplantation, for example, to render them less susceptible to apoptosis.
- Peptides of this invention may also be used in the transplantation of genetically modified cells, or tissue or organs comprising such cells, capable of expressing the inhibiting protein; it most particularly is directed to methods of transplanting modified xenogeneic or allogeneic cells, tissue or organs; recombinant genes, proteins and vectors for accomplishing same; and the cells, tissue or organs, as well as non-human transgenic or somatic recombinant animals, so modified.
- Appropriate methods of inserting foreign cells or nucleic acids into animal tissue include microinjection, embryonic stem (ES) cell manipulation, electroporation, cell gun, transfection-k, transduction, retroviral infection, etc.
- Nucleic acids can be inserted into germ cells (e.g. fertilized ova) to produce transgenic non-human animals bearing the gene, which is then passed on to offspring.
- Nucleic acids can also be inserted into somatic/body cells to provide somatic recombinants, from whom the gene is not passed on to offspring. Transcription of DNA may be made subject to an inducible promoter, so that expression of a recombinant protein can be delayed for a suitable period of time prior to grafting.
- DNA may be inserted into a particular locus, e.g. the thrombomodulin or P-selectin locus and the construct introduced into embryonic stem (ES) cells, with the resulting progeny expressing the construct in vascular endothelium.
- Retroviral vectors and in particular replication-defective retroviral vectors lacking one or more of the gag, pol, and env sequences required for retroviral replication, are well-known to the art and may be used to transform endothelial cells.
- the ability of adenoviruses to attach to cells at low ambient temperatures is an advantage in the transplant setting which, can facilitate gene transfer during cold preservation.
- p65RHD vectors can be inserted by direct infection of cells, tissues or organs in situ.
- the vessels of an organ such as a kidney can be temporarily clamped off from the blood circulation, and the blood vessels perfused with a solution comprising a transmissible vector construct containing a CAML-binding peptide for a time sufficient for the gene to be inserted into cells of the organ; and on removal of the clamps, blood flow can then be restored to the organ and its normal functioning resumed.
- Cell modification can be carried out ex vivo.
- Cell populations can be removed from the donor or patient, genetically modified by insertion of a vector, and then implanted into the patient or a syngeneic or allogeneic recipient.
- an organ can be removed from a donor, subjected ex vivo to the perfusion step described above, and the organ can be re-grafted into the donor or implanted into a different recipient of the same or different species.
- DNA encoding a peptide of this invention will be placed under the control of a constitutive or inducible promoter.
- An advantage of employing an inducible promoter for transplantation purposes is that the desired high level transcription/expression of the active gene/protein can be delayed for a suitable period of time before grafting.
- transcription can be obtained on demand in response to a predetermined stimulus, such as, e.g. the presence of tetracycline in the cellular environment.
- a tetracycline-inducible promoter which is suitable for use in the invention is disclosed by Furte et al, PEAS US 91 (1994) 9302-9306.
- a promoter system where transcription is initiated by the withdrawal of tetracycline is described by Gossen and Bujard, PEAS URSA 90 (1992) 5547-51.
- a peptide according to the invention or a derivative thereof such as a chimeric peptide (including proteins) comprising a CAML-binding peptide may be administered as a pharmaceutical composition which may be formulated according to various methods.
- a pharmaceutical composition which may be formulated according to various methods.
- such a formulation may be a solution or suspension.
- peptides can also be formulated for therapeutic administration as tablets, pills, capsules, sustained release formulations or powders.
- the preparation of therapeutic compositions which comprise polypeptides as active ingredients is well understood in the art.
- such compositions are prepared in injectable form, e.g. as liquid solutions or suspensions.
- Peptides to be used according to this invention may be synthesized using standard techniques such as those described in Bodansky, M. Principles of Peptide Synthesis (1993) Springer Verlag, Berlin. Automated peptide synthesizers are commercially available (e.g. Advanced ChemTech Model 396; Milligen/Biosearch 9600). Peptides may be purified by high pressure liquid chromatography and analyzed by mass spectrometry. One or more modifying groups may be attached to such a peptide by standard methods, for example by modification of amino, carboxyl, hydroxyl or other suitable reactive groups on an amino acid side chain or at either terminus of a peptide (e.g. Greene, T.W. and Wuts, P.G.M.
- Peptides may also be prepared according to standard recombinant techniques using a nucleic acid molecule encoding the peptide.
- a nucleotide sequence encoding a desired peptide may be determined pursuant to the genetic code and an oligonucleotide having this sequence may be synthesized by standard DNA synthesis methods (e.g. using automated DNA synthesizer) or by deriving such DNA from a natural gene or cDNA using standard molecular biology techniques such as site-directed mutagenesis, polymerase chain reaction, and/or restriction enzyme digestion.
- standard DNA synthesis methods e.g. using automated DNA synthesizer
- deriving such DNA from a natural gene or cDNA using standard molecular biology techniques such as site-directed mutagenesis, polymerase chain reaction, and/or restriction enzyme digestion.
- production of recombinant adenovirus and TACI proteins is known in the art, including from literature described herein.
- nucleic acids according to this invention may be incorporated into a recombinant vector. Accordingly, this invention also provides such vectors comprising the nucleic acid molecules of this invention.
- vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors may include circular double stranded plasmids and viral vectors. Certain vectors are capable of autonomous replication in a host cell such as vectors of bacterial origin and episomal mammalian vectors.
- vectors such as non-episomal mammalian vectors may be integrated into the genome of a host cell upon introduction into the host cell and thereby may be replicated along with the host cell genome.
- Certain vectors may be capable of directing the expression of genes to which they have been operatively linked and are referred to as expression vectors.
- a nucleotide sequence encoding a peptide of or to be used in this invention may be operatively linked to one or more regulatory sequences selected on the basis of the host cells to be used for expression. This means that the sequences encoding the peptide are linked to a regulatory sequence in a manner that allows for expression of the peptide.
- regulatory sequences may include promoters, enhancers, polyadenylation signals and other expression control elements such as are described in Goddel; Gene Expression Technology: Methods in Enzymology 185 (1990) Academic Press, San Diego, California. Regulatory sequences may direct constituative expression in many types of host cells or may direct expression only in certain tissues or cells.
- Regulatory elements may direct expression in a regulatable manner such as only in the presence of an inducing agent.
- Suitable expression vectors for adenovirus proteins and for TACI are known in the art, including references referred to herein.
- Peptides, polypeptides and proteins to be used according to this invention may comprise sequences of amino acids not derived from the natural source for a dedicated CAML-binding peptide employed (e.g. fusion or chimeric protein).
- such proteins may comprise a peptide of this invention fused to a peptide that facilitates transfer across a cell membrane or fused to a peptide that affects or facilitates modulation of CAML activity.
- derivatives of peptides of this invention including derivatives intended to enhance the immunogenicity, biological activity, or pharmacokinetic properties of the peptide or protein comprising the peptide.
- peptides of this invention may be modified by labeling or by coupling to another agent intended to facilitate detection or recovery of the peptide or its binding partners, including CAML.
- labeling include coupling to an enzyme or a detectable label such as a metallic, radioactive, or fluorescent element.
- modification to affect pharmacokinetic properties include modification of N or C termini (e.g.
- amide group or a D-amino acid to reduce the ability of a peptide to act as a substrate for a carboxypeptidase or a aminopeptidase, or myristoylation to improve accessibility to a cell interior.
- parenteral administration examples include intravenous, subcutaneous and intramuscular routes.
- Intravenous administration can be used to obtain acute regulation of peak plasma concentrations of the drug as might be needed for example to treat acute episodes of airway hyper-responsiveness.
- Improved half-life and targeting of the drug to the airway epithelia may be aided by entrapment of the drug in liposomes. It may be possible to improve the selectivity of liposomal targeting to the airways by incorporation of ligands into the outside of the liposomes that bind to airway-specific macromolecules.
- intramuscular or subcutaneous depot injection with or without encapsulation of the drug into degradable microspheres e.g.
- poly (DL-lactide-co-glycolide) may be used to obtain prolonged sustained drug release as may be necessary to suppress the development of airway hyper-responsiveness.
- an i.p. implanted reservoir and septum such as the Percuseal system available from Pharmacia.
- Improved convenience and patient compliance may also be achieved by the use of either injector pens (e.g. the Novo Pin or Q-pen) or needle-free jet injectors (e.g. from Bioject, Mediject or Becton Dickinson). Prolonged zero-order or other precisely controlled release such as pulsatile release can also be achieved as needed using implantable pumps.
- Examples include the subcutaneously implanted osmotic pumps available from ALZA, such as the ALZET osmotic pump.
- Nasal delivery may be achieved by incorporation of the protein drug into bioadhesive particulate carriers ( ⁇ 200 ⁇ m) such as those comprising cellulose, polyacrylate or polycarbophil, in conjunction with suitable absorption enhancers such as phospholipids or acylcarnitines.
- bioadhesive particulate carriers ⁇ 200 ⁇ m
- suitable absorption enhancers such as phospholipids or acylcarnitines.
- Available systems include those developed by DanBiosys and Scios Nova.
- Oral delivery may be achieved by incorporation of a drug into enteric coated capsules designed to release the drug into the colon where digestive protease activity is low. Examples include the OROS-CTVOsmetTM. and PULSINCAP.TM.
- Micron- sized particles may be delivered to the distal alveolar surface using dry powder inhalers similar in principle to those designed by Inhale, Dura, Fisons (Spinhaler), Glaxo (Rotahaler) or Astra (Turbohaler) propellant-based metered dose inhalers.
- Solution formulations with or without liposomes may be delivered using ultrasonic nebulizers.
- inflammation caused by implants or surgical procedures examples include: restenosis, senile calcific aortic stenosis, balloon angioplasty induced inflammation, intimal hyperplasia (a cause of vascular restenosis), atherosclerosis, benign prostate hyperplasia, hysteroscopically delivered fallopian tube fertilization and sterilization aided therapies, inflammation caused by catheters, inflammation caused by the use of mesh or other implants for hernia repair, inflammation caused by the use of mesh in the surgical repair of rectocoele and rectal prolapses, urological stress, incontinence, inflammation caused by surgical uterine suspensions, inflammation caused by tapes, staples and sutures, and inflammation resulting from vascular grafts (including peripheral, coronary artery and bypass grafts).
- treatment may include provision of peptides or nucleic acids of this invention on an implantable medical device including beads, tape, mesh, gauze, membranes, appliances such as stents, and the like.
- implantable medical device including beads, tape, mesh, gauze, membranes, appliances such as stents, and the like.
- Methods for associating (e.g. coating) of peptides or nucleic acids with such devices or materials are known, including those described in: WO 98/16268, WO 00/23123, US 6,140,128, US 4,610,692, US 6,117,456, US 4,946,686, Tarr, E.R. (1997) Biomed Sci Instrum, 33:143-8, and Abrams, L. (1994) Biomed Sci Instrum, 30:169-74.
- Percutaneous transluminal coronary angioplasty is widely used as the primary treatment modality in many patients with coronary artery disease, to reduce obstruction and improving coronary flow.
- PTCA Percutaneous transluminal coronary angioplasty
- the present invention includes therapeutic methods comprising the administration of a therapeutic agent comprising or in addition to a CAML-binding protein of this invention to a procedurally traumatized mammalian vessel (e.g., by an angioplasty procedure).
- the therapeutic agent is an E3/6.7K or TACI derived peptide or polynucleotide of this invention.
- Preferred therapeutics in the practice of the present invention include, for example The SED domain of E3/6.7K or analogs or derivatives thereof.
- the administration of a therapeutic agent of the invention is effective to biologically stent the vessel, inhibit or reduce vascular remodeling of the vessel, inhibit or reduce vascular smooth muscle cell proliferation, or any combination thereof.
- the administration of the therapeutic agent preferably is carried out during the procedure, which traumatizes the vessel, e.g., during the angioplasty or other vascular surgical procedure.
- the invention also provides therapeutic compositions and dosage forms adapted for use in the present method, as well as kits containing them.
- this invention includes methods for biologically stenting a traumatized mammalian blood vessel.
- the method comprises administering to the blood vessel an effective amount of a therapeutic agent to biologically stent the vessel.
- biological stenting means the fixation of the vascular lumen in a dilated state near its maximal systolic diameter, e.g., the diameter achieved following balloon dilation and maintained by systolic pressure.
- the method may comprise the administration of an effective amount of an E3/6.7K peptide or polynucleotide into the blood vessel.
- the peptide or polynucleotide is dispersed in a pharmaceutically acceptable liquid carrier, viral vector or liposome mediated gene delivery preferably administered locally via a catheter.
- Th peptide or polynucleotide may be dispersed in a pharmaceutically acceptable liquid carrier at about 0.001 to about 25 mu.g per ml of aqueous vehicle.
- a portion of the amount administered penetrates to at least about 6 to 9 cell layers of the inner tunica media of the vessel and so is effective to biologically stent the vessel.
- the invention also includes therapeutic methods comprising inhibiting diminution of vessel lumen diameter by administering to a traumatized vessel of a mammal an effective amount of a CAML-binding peptide.
- an E3/6.7K or TACI derived peptide or polynucleotide is administered via an implantable device, wherein the implantable device is not a catheter, which has a first and a second expansile member, i.e., balloons, which are disposed on opposite sides of the vessel area to be treated in order to isolate the portion of the vessel to be treated prior to peptide or polynucleotide administration.
- the isolated portion of the vessel is not washed to remove blood prior to peptide or polynucleotide administration ("bloodless angioplasty").
- Blood angioplasty bloodless angioplasty
- isolated does not mean occlusive contact of the actual treatment area by the catheter balloon, which is preferred in the practice of the present invention.
- the invention further includes methods for inhibiting or reducing diminution in vessel lumen volume in a traumatized mammalian blood vessel.
- the method may comprise administering to the blood vessel of a mammal an effective amount of peptide or polynucleotide of this invention, wherein the peptide or polynucleotide is in sustained release dosage form.
- the peptide or polynucleotide is administered in situ, by means of an implantable device, wherein the peptide or polynucleotide is releasably embedded in, coated on, or embedded in and coated on, the implantable device.
- a crystalline peptide may be releasably embedded in, or dispersed in, a adventitial wrap, e.g., a silicone membrane.
- the invention further includes therapeutic methods comprising administering to a traumatized mammalian blood vessel a sustained release dosage form comprising microparticles or nanoparticles comprising a peptide or polynucleotide of this invention or analogs thereof.
- a sustained release dosage form comprising a SED peptide is preferably administered via an implantable device, which is not a catheter used to perform bloodless angioplasty.
- the amount administered will be effective to inhibit or reduce diminution in vessel lumen area of the mammalian blood vessel.
- the sustained release dosage form preferably comprises microparticles of 4 to about 50 microns in diameter.
- the sustained release dosage form can also preferably comprise about 2 to about 50, and more preferably greater than 3 and less than 10, microns in diameter.
- preferred sizes include about 10 to about 5000, more preferably about 20 to about 500, and more preferably about 50 to about 200, nanometers.
- methods comprising administering to a mammalian blood vessel a dosage form of peptide of this invention or an analog thereof in a non-liquid vehicle or matrix effective inhibit or reduce diminution in vessel lumen area of the mammalian blood vessel.
- the dosage form is a substantially solid dosage form.
- solid form does not include microparticles, nanoparticles, and the like.
- the non- liquid vehicle or matrix preferably includes, but is not limited to, a gel, paste, gauze or a membrane, which comprises the peptide.
- kits comprising, preferably separately packaged, at least one implantable device adapted for the in situ delivery of at least one peptide or polynucleotide of this invention to a site in the lumen of a traumatized mammalian vessel and at least one unit dosage form of a therapeutic agent comprising the peptide or polynucleotide in a liquid vehicle adapted for delivery by said device.
- the administration of at least a portion of the unit dosage form to the traumatized vessel is intended to be effective to biologically stent the vessel, inhibit or reduce the vascular remodeling of the vessel, inhibit or reduce vascular smooth muscle cell proliferation, or any combination thereof.
- kits comprising, preferably separately packaged, an implantable device adapted for the delivery of at least one therapeutic agent to a site in the lumen of a traumatized mammalian vessel and a unit dosage form comprising at least one peptide or polynucleotide of this invention, wherein the administration of at least a portion of the unit dosage form is effective to cause CAML-binding and actions of the therapeutic agent to inhibit or reduce diminution in vessel lumen diameter of the vessel.
- the device could be a catheter, having a first and a second expansile member, which are disposed on opposite sides of the region to be treated so as to isolate a portion of the vessel to be treated. Alternatively, the isolated portion of the vessel is not washed to remove blood prior to administration.
- the invention also includes pharmaceutical compositions suitable for administration by means of an implantable device.
- the composition may comprise an amount of a SED peptide or analog thereof effective to inhibit or reduce stenosis or restenosis of a mammalian vessel traumatized by a surgical procedure and a pharmaceutically acceptable non-liquid release matrix for said SED peptide.
- the release matrix comprises a gel, paste, gauze or membrane.
- the invention also includes therapeutic devices.
- a therapeutic shunt comprising an amount of a peptide or polynucleotide of this invention effective to facilitate inhibition of stenosis or reduce restenosis following placement of the therapeutic shunt as a result of CAML interaction.
- Another embodiment of the invention comprises therapeutic artificial graft comprising an amount of a SED peptide or analog thereof to inhibit stenosis or reduce restenosis following placement of the graft.
- Yet another embodiment of the invention comprises a therapeutic adventitial wrap comprising an amount of a SED peptide or polynucleotide effective to inhibit stenosis or reduce restenosis following placement of the wrap.
- the amount of a pharmaceutical composition according to this invention to be employed will depend on the recipient and the condition being treated. The requisite amount may be determined without undue experimentation by protocols known to those skilled in the art. Alternatively, the requisite amount may be calculated, based on a determination of the amount of tryptase which, must be inhibited in order to treat the condition.
- Wild Type Ad5 (Ad5wt) was obtained from the American Type Culture Collection (Rockville, Maryland, USA) and dl739, E3/6.7K-deleted viral mutant (dl739) was according to Brady et al. (1992), J. Nirol. 66:5914-5923. These two adenovirus group C viruses share a great degree of similarity, but differ in the expression of E3/6.7K protein, which is deleted in dl739. Both viral serotypes were propagated in monolayer culture of A549 cells grown in Minimal Essential Media (Gibco BRL Life Technologies Inc., Gaithersburg, Maryland, USA) supplemented with 10% Fetal Calf Serum (FCS).
- FCS Fetal Calf Serum
- mice Inoculation of airway ducts and viral plaque assays.
- Two groups of 24 mice were anaesthetized with Halothane.
- One group of mice were infected intranasally with 10 7 pfu of Ad5wt in 60 ⁇ l of culture media while the other group of mice was infected intranasally with 10 7 pfu of dl739 in 60 ⁇ l of culture media.
- Six animals were infected with sterile- culture media alone.
- Six animals from each of the two groups were sacrificed with an overdose of Halothane 2 hours, 1, 3 and 7 days post infection (p.i.). Two sham infected ammals were sacrificed on days 1, 3 and 7 days p.i..
- the left lung was removed and frozen in liquid nitrogen for use in viral plaque assays.
- the right Jung was inflated with 4% paraformaldehyde in PBS pH7.4 (0.149 M NaCl, 0.012 M Na2HPO4, 0.004M KH2PO4) and embedded in paraffin.
- Viral titer Viral plaque assays were used to quantitate the amount of replicating virus in mouse lungs. Approximately 200mg of lung was homogenized on ice in 1ml sterile MEM with a polytron. The homogenate was spun for 2 min at 10,000g while the supernatant was removed and stored at -70°C. The lung homogenate supernatant titer was determined by plaque assay on A549 cell monolayer cultures grown in MEM/10%FBS on six well plates using decimal dilutions from 10-1 to 10-6 in MEM from the supernatant of animal lung homogenate from all time groups.
- the histopathologic grades were 0 - no inflammation, 1 - mild inflammation. 2 - moderate inflammation, 3 - severe inflammation for each feature.
- the scores for each feature were summed to give a total inflammatory score with maximum being 9 for each animal.
- a mean inflammatory score was calculated for each animal by dividing the total score by 3. The mean and standard deviation was calculated for each experimental group.
- Plasmid constructs cDNA for E3/6.7K was obtained by amplifying by PCR the region coding for the E3/6.7K ORF from a vector carrying the Ad2 E3 region using SEQ ID NO:22 and 23 as primers ( Figure 1). The PCR product was cloned in the Xhol site of the BPV based cDNA expression vector pBCMGSneo (Karasuyama and Melchers, (1988), Eur. J. Immunol. 18:97-104) and sequenced to ensure accuracy.
- the reaction cocktail contained template DNA, 0.5 ⁇ M of each primer forward and reverse, 250 ⁇ M of each nucleotide, 5U of Pfu polymerase (Canadian Life Technologies, 2270 Industrial St., Burlington , Ontario) in IX Pfu Buffer.
- the reaction conditions are: melting of double stranded DNA at 95°C for 30sec, followed by annealing at 57°C for 30 sec, followed by a 30 sec. ramp to 72 and continued elongation for an additional 30sec. 30 cycles of the above PCR reaction produced sufficient DNA in most cases.
- the newly generated cDNA for E3/6.7K contained modifications (highlighted in bold in Figure 1) which are not found in naturally occurring E3 nucleic acid. Both modifications enhance translation initiation at the start site of E3/6.7K and provide for increased production of the protein in a transformed cell.
- the forward primer provides the start site of E3/6.7K with an optimal upstream Kozak consensus sequence.
- the reverse primer was modified to replace the naturally occurring TGA-Stop codon with an Ochre-Stop codon (TAA). The latter modification eliminates the start site of E3/19K, which overlaps with the sequence of E3/6.7K in the naturally occurring E3 nucleic acid and decreases translation efficiency of E3/6.7K from the natural sequence.
- pBD-6.7 a bait vector containing E3/6.7
- pGBKT7 CLONTECH
- pAD-CAML was constructed by subcloning full length CAML in frame into the C terminus of the GAL4 activation domain of the prey vector pGADT7 (CLONTECH).
- pAD-CAML(201) was constructed by subcloning amino acids 1 to 201 of CAML in frame into the C terminus of the GAL4 activation domain of the prey vector pGADT7. The fidelity of the constructs was confirmed by sequencing.
- pBD-6.7 and pAD-CAML or pAD- CAML(201) were transformed together into Saccharomyces cerevisiae AH109 using the Lithium Acetate Yeast Transformation procedure as described by Gietz, D., et al. (1992) Nucleic Acids Res., 20:1425.
- Control cotransformation of pBD-53 / pAD-T and pBD- Lam / pAD-T were also done.
- pBD-53 / pAD-T encode fusions between the GAL4 DNA-BD and AD and murine p53 and SV40 large T-antigen, respectively.
- p53 and large T-antigen interact in a yeast two-hybrid assay.
- pBD-Lam encodes a fusion of the DNA- BD with human lamin C and provides a negative control for an interaction with pAD-T.
- pBD containing the TRP1 gene was selected on synthetic drop out media lacking tryptophan (SD-T).
- pAD contains the LEU2 gene and was selected for on SD-L (leucine).
- SD-LTHX leucine/- tryptophan -histidine deficient/X- ⁇ -Gal supplemented media
- SD-AHLTX adenine/- histidine/-leucine/-tryptophan deficient /X- ⁇ -Gal supplemented media
- the transformed yeast can only grow on SD-LTHX or SD-AHLTX if a protein interaction occurs.
- Yeast transformed with pBD-6.7 and pAD-CAML or pBD-6.7 and pAD- CAML(201) were able to grow on both SD-LTHX or SD-AHLTX indicating that the reporter enzymes necessary to grow on deficient media are synthesized which indicates that the proteins E3/6.7K and CAML interact.
- U937 human histiocytic lymphoma cells obtained from ATCC (CRL 1593) were maintained in RPMI 1640, 10% FCS, 2mM L-glutamine, lOmM HEPES, lOOU/ml penicillin and lOO ⁇ g/ml streptomycin in an atmosphere of 5% CO2 and 100% humidity.
- Cells were transfected with the appropriate construct by using the DMRIE-C cationic lipid reagent available from Life Technologies using the manufacturer's protocol. Transfected cells were maintained in medium containing geneticin G-418 sulphate at a final concentration of 800 ⁇ g/ml. Media and supplements were purchased from Life Technologies.
- a total of 2xl0 7 cells were labelled for one hour in prewarmed methionine/cysteine-free media containing 0.5mCi/ml [35S]- Cysteine and 0.2 mCi/ml (Amersham) [35S]-Methionine (Amersham) at a concentration of 5x10 6 cells /ml.
- Cells were washed and then lysed on ice in freshly made lysis buffer containing 1% TritonX-100, 1% BSA (bovine serum albumin), lmM iodoacetamide, ImM PMSF, 2.5TIU/ml aprotinin, 0.01M Tris pH8.0, 0.14M NaCl.
- cell lysate equivalent to 105 cells was denatured in SDS-PAGE loading buffer and loaded on 10% glycine SDS-PAGE gel system, separated and blotted onto a Immobilon-P PVDF membrane (Millipore) and probed with cPLA2 rabbit polyclonal antiserum (Cayman Chemical). The signal was detected via horse radish peroxidase-conjugated, goat antirabbit antiserum and by chemiluminescence using the ECL kit (Biorad).
- Arachidonic acid release assays Cells were grown at low density in 10% Hyclone FCS, RPMI 1640, 2mM L-glutamine, lOmM HEPES for several days then harvested and washed twice in PBS, 1% BSA. Approximately 5xl0 6 cells (5xl0 5 cells/ml) were labelled for 20hrs in same media as above supplemented with 0.4 ⁇ Ci/ml [3H] arachidonic acid [5,6,8,9,11,12,14,15-3H(N)] (O.lmCi/ml stock; New England Nuclear).
- the assay was set up in triplicate and the cells were stimulated either with media alone or with 20ng/ml human rTNF- ⁇ (2000U/ml) (Boehringer, Mannheim), or with lO ⁇ g/ml cycloheximide or with a combination of 20ng/ml TNF- ⁇ and lO ⁇ g/ml cycloheximide. After 20 hours of treatment the cells were centrifuged and lOO ⁇ l of supernatant out of 500 ⁇ l total was mixed with 3ml scintillation fluid and counted. For each cell line three samples were lysed in lysis buffer and the lysate was used to determine the total counts of incorporated [3H] Arachidonic Acid. The counts per minute of released [3H] arachidonic acid were expressed as a percentage of the average of total incorporated [3H] arachidonic acid.
- Annexin V-FACS apoptosis assay.
- Annexin N-FITC (PharMingen) was used to determine the binding of Annexin N to externalized phosphatidyl serine. The protocol followed was based on the manufacturers Annexin N-FITC staining protocol. Cells were grown at low density in 10% Hyclone FCS, RPMI 1640, 2mM L-glutamine, lOmM HEPES for several days then 5xl0 6 cells were harvested and washed twice in PBS.
- Cells resuspended in above media were treated for 7 hours with media alone or with lOOng/ml (10,000 U/ml) human rT ⁇ F- ⁇ or with 200 ⁇ g/ml cycloheximide or with a combination of 100 ng/ml T ⁇ F- ⁇ and 200 ⁇ g/ml cycloheximide.
- the cells were resuspended at lxlO 6 cells/ml in lxBinding Buffer (lOmMHepes/ ⁇ aOH, pH7.4, 140mM ⁇ aCl, 2.5mM CaC12).
- lxl 0 5 cells were combined with 5 ⁇ l of Annexin N-FITC.
- SV5 backbone previously described (Chen (1997) PNAS) has been successfully used to transduce in vivo the dystrophin gene.
- the backbone lacks the El and E2 region. Without these two regions the SV5 Ad vector is replication defective and therefore safer to use as well as it elicits a reduced inflammatory response.
- the cDNA encoding E3/6.7K under the control of the actin promoter and the CMV enhancer was added to SV5 and used to rescue a new vector called SV5-6.7, which will incorporate E3/6.7K as an immunomodulatory protein.
- Production of Producer Cells resistant to apoptosis Creation of hybridoma, Chinese hamster ovary (CHO) or insect cells that are resistant to apoptosis follow the same procedure as the transfection of U937 cells outlined above except at the end of the selection in G-418, the cells are sorted or clonally expanded in order to screen for the expression of the protein of interest.
- CHO Chinese hamster ovary
- insect cells that are resistant to apoptosis follow the same procedure as the transfection of U937 cells outlined above except at the end of the selection in G-418, the cells are sorted or clonally expanded in order to screen for the expression of the protein of interest.
- E3/6.7K Results in more Persistent Viral Titers and a reduction of the inflammatory response.
- the presence of E3/6.7K results in more persistent viral titers during the course of infection by comparing mice infected with the E3/6.7K deletion virus (dl739) to mice infected with the wild type virus (Ad5wt).
- the titers of dl739 were significantly higher than Ad5 wild type one day after inoculation (pOIOOl). Over time, the titers of dl739 decreased as the virus was cleared (pO.OOl). In contrast Ad5wt titers did not change significantly over the 7 day experimental period.
- E3/6.7K Mediated Arachidonic Acid Release Is Reduced in the Presence of E3/6.7K.
- E3/6.7K can affect the cellular response to inflammatory cytokines.
- a U937 cell line was transfected with the cDNA for E3/6.7K and expression of E3/6.7K was confirmed using immunoprecipitation with a polyclonal rabbit antiserum raised against an E3/6.7K C-terminal derived peptide and SDS-PAGE electrophoresis.
- the U937 cells transfected with E3/6.7K cDNA decreased [3H] arachidonic acid release by 50% when compared with U937 cells transfected with vector alone (U937neor) when stimulated with TNF- ⁇ .
- TNF- ⁇ induced apoptosis was assayed by measuring by measuring the externalization of phosphatidyl serine using FITC labelled Annexin V according to Martin et al., (1995), J. Exp. Med. 182:1545-1556.
- Cells expressing E3/6.7K show a 55% reduction in percentage of apoptotic cells compared with U937neor following stimulation with TNF- ⁇ .
- the U937-E3/6.7K cells show a 65% reduction in apoptosis compared to U937neor following an augmented stimulation with a combination of TNF- ⁇ and CHX.
- the presence of E3/6.7K decreased the apoptotic response in U937 cells upon stimulation with TNF- ⁇ or a combination of TNF- ⁇ and CHX.
- cPLA2 Is Cleaved to a 78kDa Form Following TNF- ⁇ Induction.
- the expression of cPLA2 in U937 cells following induction with TNF- ⁇ was assayed.
- the cPLA2 antiserum recognized two forms of the enzyme: one larger form of approximately HOkDa; and a second form of 78kda.
- U937neor cells and U937-E3/6.7K with regards to the ratio of the 1 lOkDa versus the 78kDa forms of cPLA2.
- the yeast two hybrid system takes advantage of the GAL4 transcriptional activator which consists of two separable and functionally essential domains: a DNA binding domain and a domain that activates transcription.
- a bait gene fragment of the E3/6.7K gene
- BD GAL4 DNA-binding domain
- a prey gene A prey gene
- AD GAL4 activation domain
- E3/6.7 K interacts with full length human and mouse CAML and more particularly, with the same domain of CAML that TACI has been previously shown to interact (the 1-201 residue domain of CAML).
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US10/433,919 US20040219514A1 (en) | 2000-12-07 | 2001-12-07 | Caml-binding peptides |
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CA 2325610 CA2325610A1 (en) | 2000-12-07 | 2000-12-07 | Caml modulation proteins |
CA002335411A CA2335411A1 (en) | 2001-03-02 | 2001-03-02 | Medical devices for modulation of apoptosis and immune response |
CA2,335,411 | 2001-03-02 | ||
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2001
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