EP0975355A2 - Proteines de fusion hexameres et utilisations associees - Google Patents

Proteines de fusion hexameres et utilisations associees

Info

Publication number
EP0975355A2
EP0975355A2 EP97935005A EP97935005A EP0975355A2 EP 0975355 A2 EP0975355 A2 EP 0975355A2 EP 97935005 A EP97935005 A EP 97935005A EP 97935005 A EP97935005 A EP 97935005A EP 0975355 A2 EP0975355 A2 EP 0975355A2
Authority
EP
European Patent Office
Prior art keywords
fusion protein
protein
fragment
binding
igαtp
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97935005A
Other languages
German (de)
English (en)
Inventor
Margery Ann Chaikin
Sally Doreen Patricia Lyn
Raymond Whitney Sweet
Alemseged Truneh
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
SmithKline Beecham Corp
Original Assignee
SmithKline Beecham Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by SmithKline Beecham Corp filed Critical SmithKline Beecham Corp
Publication of EP0975355A2 publication Critical patent/EP0975355A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70521—CD28, CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

  • IgM and IgA are the two classes of human antibodies that form homo-ohgome ⁇ c structures By far the most extensively studied of these is IgM
  • IgM structure is as a pentamer in combination with a single copy of a second protein, the J-chain that becomes associated with IgM du ⁇ ng its assembly and export This J-chain can covalently associate with IgM through the formation of a disulfide bond between a cysteine residue in the J chain and a cysteine residue in a short 18 amino acid extension, designated ⁇ tp, from the canonical C-terminal constant region of the heavy chain
  • ⁇ tp short 18 amino acid extension
  • Human IgA also has an 18 amino acid tailpiece segment ( ⁇ tp) which bears some sequence homology to utp
  • ⁇ tp 18 amino acid tailpiece segment
  • the tailpiece regions for the ⁇ l and ⁇ 2 regions are quite similar, or in some cases reported to be identical
  • IgM IgA occurs most frequently as a monomer antibody, similar to the IgG subclasses, or as a dimer antibody plus one molecule of J-chain [Mestecky and Kilian, Methods in Enzymology, 1 16 37-75 (1985), T B Tomasi, Immun Today.
  • the IgA proteins produced in the absence of the J chain were monomeric or dimeric forms by nonreducing SDS/PAGE and appeared as dimers in solution.
  • the co-expression of the J-chain led to formation of disulfide linked IgA dimers together with J chain.
  • the CD28 receptor a member of the immunoglobulin superfamily of molecules (IgSF) (A.F. Williams and A.N. Barclay, Annu. Rev. Immunol.. 6:381-405 ( 1988)], is a 44 kDa homodimer glycoprotein expressed on the surface of T-lineage cells including thymocytes and peripheral T cells in the spleen, lymph node and peripheral blood.
  • CD28 interacts with two different counter-receptors CD80 (also known as B7 and B7.1 ) [P. S.
  • CD80 P. S. Linslev et ai. J. Exp. Med.. 174(3):561-569 (1991)
  • CD86 Azuma et ai, cited above; Freeman et ai, 1993, cited above ; Freeman et ai, 1993, cited above] also recognize CTLA-4 [J.F. Brunei et ai, Nature.
  • CTLA4-Ig fusion proteins or antibodies directed against CD80 and CD86 inhibits T cell activation in vitro, suppresses humoral and cellular immune responses in vivo, inhibits graft rejection and the progression of autoimmune diseases in vivo [reviewed in J. A. Bluestone, Immunity. 2:555-559 (1995); Harlan et ai, Clin. Immunol, and Immunopafh.. 75(2):99-l 1 1 (1995)].
  • CD28 is a target for development of immunosuppressive agents.
  • a rapid and reproducible assay is desirable for the screening of synthetic compounds, natural products, and peptides.
  • What is needed is a method for increasing the avidity of binding proteins, particularly those with low affinity, for use in screening and diagnostic assays, therapeutics, and vaccines.
  • the present invention provides a hexameric fusion protein which provides increased binding activity as compared to the protein from which it is derived and methods of making same.
  • This fusion protein is particularly useful in binding assays and may be readily purified.
  • the hexameric fusion protein of the invention contains a dimeric binding protein and a tailpiece ( ⁇ tp) characterized by the activity of the tailpiece from the C-terminus of the heavy chain of an IgA antibody.
  • the binding protein is a natively dimeric binding protein or a functional fragment thereof.
  • the binding protein is recombinantly engineered to have a dimeric form. This is preferably achieved by fusion of a protein fragment which contains the extracellular domain of a selected binding protein to an Fc fragment.
  • the present invention provides a polynucleotide sequence encoding a stable hexameric fusion protein of the invention.
  • the present invention provides a vector comprising the above- described polynucleotide sequence and a sequence controlling expression of the fusion protein in a selected host cell.
  • the present invention provides a recombinant host cell containing the above-described vector
  • the present invention provides methods of producing and purifying a stable hexameric fusion protein by providing a host cell containing the stable hexameric fusion protein of the invention, recovering the stable hexameric fusion protein, and purifying the recovered protein
  • the strands of the fusion protein are preferably co-produced and assembled in the host cell
  • the present invention provides a pharmaceutical composition containing a stable hexameric fusion protein or a DNA sequence encoding the stable hexameric fusion protein of the invention and a pharmaceutically acceptable earner
  • the present invention provides for screening for ligands to a hexameric fusion protein of the invention Also provided are assays for inhibitors of hexameric binding protem/ligand interaction
  • Fig 1 is a schematic representation of the hexameric CD80-Ig ⁇ tp protein of the invention
  • the regions of the molecule corresponding to the CD80 extracellular domain, the IgGl hinge, CH2, and CH3 domains, and the ⁇ tp segment are indicated
  • the letter "S" in the diagram indicates the positions of predicted disulfide bonds between cysteine residues
  • Fig 2 is a plasmid map illustrating the expression construct for the CD80-Ig ⁇ tp protein of the invention
  • the plasmid is 7,167 base pairs in size Beginning at residue 1 in a clockwise manner
  • “cmv pro” is the major late CMV promoter for transcription of the downstream CD80-Ig ⁇ tp coding sequence
  • “CD80” encodes the signal peptide and extracellular domain of human CD80
  • “Fc” encodes the hinge
  • ⁇ tp encodes the human ⁇ tp segment
  • BGH is the polyadenylation signal region from the bovine growth hormone gene
  • betaglobin is the mouse major b-globm promoter
  • “dhfr” encodes the mouse dhfr(d ⁇ hydrofolate reductase) protein
  • "SV40” is the SV40 early polyadenylation region
  • "on” and “amp” are the bacterial origin of replication and beta lactamase
  • Fig. 4A-4D is the DNA and encoded protein [SEQ ID NOS: 2 and 3] sequences for the CD80-Ig ⁇ tp region in the vector CD80-Fc ⁇ tplink.
  • Bolded regions show restriction sites for reference to Fig. 2 and the initiation codon, mature processing site, hinge region, and C- terminal ⁇ tp segment.
  • Fig. 5A-5B is the DNA and encoded protein sequences [SEQ ID NOS: 4 and 5] for the extracellular domain of CD86 in the vector CD86Fc ⁇ tplink.
  • the sequence outside of the Kpn I and Eag I sites is the same as for CD80Fc ⁇ tplink (see Figs. 3A-3H and 4A-4D).
  • Fig. 6A-6C is the DNA and encoded protein sequences [SEQ ID NOS: 6 and 7] for the CMV promoter and the extracellular domain of CTLA-4 in the vector CTLA4-Fc ⁇ tplink.
  • the sequence 5' to base 514 and 3' of the Eag I site is the same as for CD80Fc ⁇ tplink.
  • Fig. 7 is a profile for chromatography of CD80-Ig ⁇ tp on a Superdex 200 column.
  • the first peak eluting at about 45 min is the hexameric protein complex while the second peak migrates at the position observed for monomeric CD80-Ig.
  • the inset shows a coomassie stained pattern for the purified CD80-Ig ⁇ tp protein on SDS/PAGE under reducing (R) and nonreducing (NR) conditions.
  • Fig. 8 is a chart showing equilibrium sedimentation (main panel) and sedimentation velocity (inset) analytical centrifugation of the CD80-Igatp protein with a modeled fit to a hexamer/(hexamer)2 equilibrium.
  • the upper graph shows the residuals for the equilibrium sedimentation centrifugation.
  • Fig. 9 is a line graph illustrating the binding of biotinylated CD80-Ig ⁇ tp (labeled B7-FcA) to CD28-Ig immobilized at three different concentrations in an ELISA format. Binding was inhibited by the mAb CD28.1 or by CTLA4-lg.
  • Fig. 10 is a line graph illustrating the binding of biotinylated CD80-Ig ⁇ tp, CD86-
  • Fig. 11 is a line graph illustrating the binding of biotinylated CD80-Ig ⁇ tp, CD86- Ig ⁇ tp, and CD80-Ig compared to immobilized CTLA4-Ig in an ELISA format.
  • Fig. 12 is a line graph illustrating the competition of biotinylated CD80-lg ⁇ tp binding to immobilized CD28-Ig (coated at 200 mg/ml) by CD80-Ig ⁇ tp itself, CD80-Ig, CTLA4-Ig, and CD28.2 MAb.
  • Fig. 13A is a line graph illustrating the binding of CD80-Ig ⁇ tp to wild-type and mutant immobilized CD28-muIg2a proteins.
  • Fig. 13B is a line graph illustrating the binding of CD86-Ig ⁇ tp to wild-type and mutant immobilized CD28-muIg2a proteins.
  • Fig. 13C is a line graph illustrating the binding of rabbit polyclonal antisera to wild- type and mutant immobilized CD28-muIg2a proteins.
  • Fig. 14 is a chart illustrating sequentially the binding of CD80-Ig and CD80-Ig ⁇ tp to CD28-Ig immobilized on a biosensor chip as measured by surface plasmon resonance.
  • Fig. 15 is a chart illustrating the binding of CD80-Ig ⁇ tp and CD86-Ig ⁇ tp to CD28- lg immobilized on a biosensor chip as measured by surface plasmon resonance.
  • Figs. 16A and 16B are line graphs illustrating the binding of CD80-Ig ⁇ tp and CD86-Ig ⁇ tp, respectively, to cells expressing human CD28 on their surface in the presence or absence of a CD28 monoclonal antibody that inhibits this interaction.
  • Fig. 17 is a bar chart illustrating the level of IL-2 production by PCD28.1 cells treated with monomeric and hexameric CD80 (labeled B7.1-Ig and B7.1-IgA, respectively) and CD86 (labeled B7.2-Ig and B7.2-lgA, respectively) lg fusion proteins.
  • the proteins were used ( 1) alone in solution, (2) alone immobilized through goat anti-human antibody (GAH), or (3) immobilized in combination with immobilized CD3 mAb. Controls were GAH alone, or with CD3 mAb, and the CD28 IgM mAb 248.23.2.
  • IL-2 levels were determined by CTLL-2 bioassay using known amounts of IL-2 as a standard (inset).
  • Fig. 18 is a bar chart illustrating the level of IL-2 production by DC27.CD28wt cells treated as described in Fig. 17.
  • Fig. 19 is a bar chart illustrating IL-2 promoter activity in PCD28.1 cells stimulated as described in Fig.17. IL-2 promoter activity was measured by induction of ⁇ -galactosidase activity which serves as a reporter gene under the control of an IL-2 promoter.
  • Figs. 20A and 20B are bar graphs respectively showing the induction of the IL-2 promoter, and IL-2 production by CD28 expressing cells incubated with CD80-Ig ⁇ tp, CD86-Ig ⁇ tp, or CD80-Ig.
  • Fig. 20C is a bar graph showing the levels of IL-2 production induced with soluble CD80-Ig ⁇ tp and CD86-Ig ⁇ tp in comparison to that induced by immobilized antibody to CD3.
  • Fig. 21 is a bar chart illustrating inhibition of biotinylated CD80-Ig ⁇ tp binding to immobilized CD28-Ig by individual compounds in the BM-34 test set. The percent inhibition range is plotted against the number of compounds showing that range of inhibition.
  • Fig. 22 is a profile for Superose 6 chromatography of the chimeric derivative of the Epo receptor antibody 1 C8 (here labeled "anti-EPOr-IgG j ”) and the ⁇ tp construct of the same antibody (labeled "anti-EPOr-IgG ] ⁇ tp”) with binding activity to an immobilized EPOr-Ig protein shown in the inset.
  • the invention provides an hexameric fusion protein useful in therapeutic and immunogenic compositions.
  • the hexameric fusion protein of the invention is particularly well suited for applications for which the binding protein from which it is derived is unsatisfactory because of low binding affinity/avidity and for other applications where multivalency is desired. These applications include diagnostics, binding assays, screening assays and cellular responses based on receptor cross-linking. Also provided are compositions and methods for production and purification of these fusion proteins.
  • the invention further provides methods of producing stable hexameric fusion proteins, by providing a selected binding protein with an IgA tailpiece ( ⁇ tp) or a functional equivalent thereof.
  • ⁇ tp IgA tailpiece
  • the inventors have found that addition of the ⁇ tp from the natively monomeric or dimeric IgA, su ⁇ risingly, provides the resulting fusion protein with the ability to form stable hexamers.
  • a hexameric fusion protein of the invention contains a dimeric binding protein which has been provided at its carboxy terminus with a tailpiece ( ⁇ tp) characterized by having the activity of the tailpiece from the C-terminus of the heavy chain of an IgA antibody.
  • This tailpiece when attached to each monomer of the dimeric binding protein, provides the resulting fusion protein with the ability to form stable hexamers, i.e., the hexameric fusion proteins of the invention do not undergo any appreciable dissociation in solution (e.g., phosphate buffered saline) at room temperature.
  • the fusion proteins of the invention are homo- hexamers.
  • hetero-hexamers comprising two different fusion proteins may be constructed.
  • the binding proteins useful in the invention include full-length proteins and fragments thereof which are characterized by the binding ability of the full-length protein, 1 e , the fragment which has the ability to bind to the counter-receptors or other ligands of the selected binding protein
  • binding proteins may be derived from a protein or protein complex which natively dimenzes for biological activity, or may be genetically engineered as described herein.
  • suitable natively dimeric binding proteins are those with carboxyl termini situated such that addition of the ⁇ tp to the carboxyl terminus of each polypeptide chain, with or without a linker, allows juxtaposition of the ⁇ tp chains
  • native dimenc proteins or dimeric protein complexes include, for example, IgG, IgD, or IgE antibodies, Fab fragments, Fab 2 fragments, Ig-Fc fragments, lg fusion proteins, and the extracellular domains of cell surface proteins such as the ⁇ / ⁇ chain of a T cell receptor, CD28 and CTLA4, CDS ⁇ / ⁇ hetorodimers and ⁇ / ⁇ homodimers, and the ⁇ / ⁇ chain of intcg ⁇ n proteins and various cytokine receptors (e.g , IL3, IL5, etc ) These binding proteins are available from a variety of commercial and academic sources Alternatively, these sequences may be chemically synthesized
  • a selected binding protein may be engineered to be dimeric
  • a protein fragment comprising a binding domain of a selected monomeric binding protein may be attached to an Ig-Fc fragment which forms dimers
  • the binding protein is selected from surface glycoproteins from the immunoglobulin supergene family and their ligands
  • the binding protein is selected from CTLA-4 (whose extracellular domain can be expressed as a monomer or dimer) and its counter-receptors CD80 and CD86
  • CTLA-4 whose extracellular domain can be expressed as a monomer or dimer
  • other proteins, including other binding proteins are known to those of skill in the art and may be used in the construction of a hexame ⁇ c fusion protein of the invention
  • a currently preferred embodiment of this invention provides hexameric immunoglobulin fusion proteins, which are exemplified herein, this invention is not so limited.
  • a binding protein may be genetically modified to alter its activity
  • engineered, mutant forms of IL4 have been described that retain high affinity for its receptor but lack normal agonist activity and serve as antagonists of IL-4 mediated function [see, e g , N Kruse et al, EMBO J . J_l 3237-3244 (1992) and WO96/04388 (Feb 15, 1996)]
  • Such a mutant would be useful in a hexameric IL4-Ig fusion protein according to the invention, serving as an antagonist of IL4 function
  • the protein fragment used to construct a dimeric binding protein contains at least a fragment of the extracellular domain of the selected binding protein
  • this extracellular fragment preferably contains the sequences required for binding, which can be readily determined by one of skill in the art.
  • the protein fragment also contains an export leader sequence which is native to the binding protein selected.
  • other export leader sequences which are capable of exporting the protein may be substituted by one of skill in the art.
  • the protein fragment is the native leader and extracellular domain from CD80 or CD86.
  • the fragments can be obtained from proteins such as CD80 [P. S. Linsley et ai, J. Exp. Med..
  • the protein fragment is the native leader and extracellular domain from CTLA-4 or CD28.
  • the Fc fragment used in the construction of the hexameric fusion protein may be from any antibody subclass, except IgA.
  • the Fc fragment may be derived from the IgG, IgD, or IgE subclass.
  • any of the human isotypes i.e., IgGi, IgG 2 , IgG3. and IgG 4 , may be selected.
  • the parental IgG antibody may be mutated to reduce binding to complement or Ig-Fc receptors fsee, e.g., A.R. Duncan et ai, Nature. 332:563-564 (1988); A. R. Duncan and G. Winter, Nature.
  • Ig-Fc fragment is derived from IgM, it desirably contains the hinge/CH2/CH3/CH4 sequence, but not the naturally occuring 18 amino acid tailpiece ( ⁇ tp).
  • the C-terminal end of the IgG] CH3 domain of the Fc fragment may be modified by conventional techniques to contain a restriction enzyme site for convenient cloning of the tailpiece segments (i.e., the peptide of the invention). Such modifications are described in more detail in the examples below, and are well known to those of skill in the art.
  • the peptide used to construct the fusion protein of the invention is derived from tailpiece located at the C-terminus of the heavy chain of an IgA antibody.
  • this peptide is 18 residues in length and is the ⁇ tp segment of the human IgAl heavy chain or a functional equivalent thereof.
  • One particularly suitable peptide is: PTHVNVSVVMAEVDGTCY [SEQ ID NO: 3].
  • this peptide may be modified to remove the glycosylation site by changing 1 or 2 amino acids at residues 5-7 (NVS). For example, the N (asparagme) may be to changed to Q (glutamine) and/or the S (senne) may be changed to A (alanine).
  • Suitable functional equivalents include, for example, gorilla IgGl, human IgA2, rabbit IgA, and mouse IgA Such functional equivalents may also be modified by removal of glycosylation sites As described herein, this peptide is linked, directly or indirectly, to the binding protein (e.g , the Ig-Fc fragment) and provides the fusion protein of the invention with the ability to assemble into a stable hexamer
  • the fusion protein may contain a linker sequence
  • a linker may be located between the binding protein (e g , the Ig-Fc fragment) and the ⁇ tp peptide
  • This linker is preferably an ammo acid sequence between about 1 and 20 amino acid residues, and more preferably between about 1 and 12 amino acid residues, in length
  • Other appropriate or desired linkers may be readily selected by one of skill in the art Although currently less desired, one of skill in the art may substitute other linkers for the preferred amino acid sequence linkers described above
  • CD80-Ig ⁇ tp Three currently preferred embodiments of the fusion proteins of the invention are described herein, CD80-Ig ⁇ tp, CD86-Ig ⁇ tp and CTLA4-Ig ⁇ tp
  • CD80-Ig ⁇ tp CD86-Ig ⁇ tp
  • CTLA4-Ig ⁇ tp These proteins are composed of the native leader and extracellular domains of the CD80 (B7 1 ), the CD86 (B7 2, B70), and the CTLA-4 surface glycoproteins, respectively, linked to the h ⁇ nge/CH2/CH3 region of the heavy chain of human IgGi (Fc fragment) and terminating in a short tail piece segment from human IgAl ( ⁇ tp)
  • Another example of a hexameric protein of the invention is an IgG antibody, where the ⁇ tp is joined directly to the carboxy terminus of the heavy chain and a light chain is paired with this heavy chain
  • the ⁇ tp hexameric antibody and lg fusion proteins of the invention
  • each chain of a dimeric binding protein is selected or constructed
  • one preferred binding protein is a recombinant immunoglobulin containing the native leader and extracellular domain fused to an Ig-Fc fragment from the selected human IgG antibody
  • the ⁇ tp is added, optionally by introducing a convenient restriction endonuclease site near the C-terminus of the binding protein (e.g., an Fc region) using silent mutations of the coding sequence and then cloning a synthetic oligonucleotide into this site that encodes the tailpiece segment.
  • the tailpiece segment is matched to that of the human ⁇ - 1 chain.
  • the tailpiece provides the fusion protein with the ability to form hexamers and the resulting construct is the hexameric fusion protein of the invention.
  • a schematic representation of the predicted hexamer for an exemplary fusion construct of the invention, CD80-Ig ⁇ tp, is shown in Fig. 1.
  • the fusion proteins of the invention are produced using recombinant techniques.
  • the nucleic acid sequences may be fused and the fusion protein expressed in vitro in a suitable host cell.
  • the fusion proteins of the invention are produced by separately expressing, or co-expressing the nucleic acid sequences encoding the protein fragments and ⁇ tp fragment of the invention and fusing the expressed products.
  • the resulting fusion protein forms hexamers.
  • the present invention further encompasses polynucleotide sequences encoding the fusion proteins of the invention.
  • the nucleic acid sequences of the invention include the DNA (including complementary DNA) sequence representing the non-coding strand and the messenger RNA sequence.
  • Variants of these nucleic acids of the invention include variations due to the degeneracy of the genetic code and are encompassed by this invention. Such variants may be readily identified and/or constructed by one of skill in the art.
  • the polynucleotide sequences may be modified by adding readily assayable tags to facilitate quantitation, where desirable.
  • the DNA sequences of the invention are inserted into a suitable expression system, preferably a eukaryotic system.
  • a recombinant vector is constructed in which the polynucleotide sequence encoding at least one chain of the fusion protein (i.e., the binding protein/ ⁇ tp) is operably linked to a heterologous expression control sequence permitting expression of the fusion protein of the invention.
  • appropriate expression vectors and host cell systems are known in the art for expression, including, e.g., mammalian, yeast, bacterial, fungal, drosophila, and baculovirus expression. The transformation of one or more of these vectors into appropriate host cells results in expression of the fusion proteins of the invention
  • Other appropriate expression vectors of which numerous types are known in the art, can also be used for this pu ⁇ ose
  • hexamenc fusion proteins of mixed specificity may be produced by co-expression of different fusion proteins (1 e , binding protein/ ⁇ tp)
  • fusion proteins (1 e , binding protein/ ⁇ tp)
  • two fusion proteins recognizing non-competing sites on the same molecule can be co-expressed resulting in hexamers that can bind to two sites on the same molecule, resulting in higher binding avidity than for each fusion protein alone or as a homogenous hexamer
  • the two fusion proteins can bind to two distinct molecules presented on the same, or different surfaces (e g , expressed on the same or different cells)
  • Suitable host cells or cell lines for transfection by this method include mammalian cells, such as Human 293 cells, Chinese hamster ovary cells (CHO), the monkey COS-1 cell line, mu ⁇ ne L cells or mu ⁇ ne 3T3 cells derived from Swiss, Balb-c or NIH mice Suitable mammalian host cells and methods for transformation, culture, amplification, screening, and product production and purification are known in the art [See, e g , Gething and Sambrook, Nature. 293 620-625 (1981), or alternatively, Kaufman et al , Mol Cell Biol.. 5(7).1750- 1759 (1985) or Howley et ai , U S. Patent 4,419,446] Another suitable mammalian cell line ⁇ s the CV-1 cell line
  • host cells include insect cells, such as Spodoptera frugipedera (Sf9) cells
  • Spodoptera frugipedera Sf9 cells
  • Methods for the construction and transformation of such host cells are well-known [See, e g Miller et al , Genetic Engineering. 8 277-298 (Plenum Press 1986) and references cited therein].
  • the present invention provides a method for producing a fusion protein of the invention which involves transforming a host cell, preferably a eukaryote, with at least one expression vector containing a recombinant polynucleotide encoding a fusion protein under the control of a transcriptional regulatory sequence, e.g., by conventional means such as transfection or electroporation.
  • the transformed host cell is then cultured under suitable conditions that allow expression of the fusion protein.
  • the expressed and assembled fusion protein is then recovered, isolated, and purified from the culture medium by appropriate means known to one of skill in the art.
  • the fusion proteins are assembled by the host cell following co-production of one or more of the fusion proteins of the invention.
  • the hexameric fusion protein may be assembled following recovery from the host cell.
  • the fusion proteins of the invention can be readily purified using conventional techniques.
  • hexameric lg fusion proteins of the invention may be readily purified on high affinity, high capacity supports based on protein A and protein G.
  • Such resins are commercially available [Pharmacia Inc.; Bioprocessing Ltd.].
  • the hexameric fusion protein may be produced in insoluble form.
  • the proteins may be isolated following cell lysis in soluble form, or extracted in guanidine chloride.
  • fusion proteins of this invention or DNA sequences encoding them may be formulated into pharmaceutical compositions and administered using a therapeutic or immunogenic regimen compatible with the particular formulation.
  • Pharmaceutical compositions within the scope of the present invention include compositions containing a protein of the invention in an effective amount to have the desired physiological effect.
  • Suitable formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble or water-dispersible form, e.g., saline.
  • suspensions of the active compounds may be administered in suitable conventional lipophilic carriers or in liposomes.
  • adjuvants may be desired, particularly where the composition is to be used as an immunogen.
  • compositions may be supplemented by active pharmaceutical ingredients, where desired.
  • Optional antibacterial, antiseptic, and antioxidant agents in the compositions can perform their ordinary functions.
  • the pharmaceutical compositions of the invention may further contain any of a number of suitable viscosity enhancers, stabilizers, excipients and auxiliaries which facilitate processing of the active compounds into preparations that can be used pharmaceutically.
  • these preparations, as well as those preparations discussed below, are designed for parenteral administration.
  • compositions designed for oral or rectal administration are also considered to fall within the scope of the present invention.
  • suitable amount or “effective amount” means an amount which is effective to treat or prevent the conditions referred to below.
  • a preferred dose of a pharmaceutical composition containing a fusion protein of this invention is generally effective above about 0.1 mg fusion protein of the invention per kg of body weight (mg/kg), and preferably from about 1 mg/kg to about 100 mg/kg. These doses may be administered with a frequency necessary to achieve and maintain satisfactory fusion protein levels. Although a preferred range has been described above, determination of the effective amounts for treatment or prophylaxis of a particular condition may be determined by those of skill in the art.
  • compositions containing the hexameric antibody/ ⁇ tp fusion proteins of the invention are useful as antagonists for the 7 transmembrane (7 TMR) class of cell surface receptors, since such receptors are often arrayed in many copies on cell surfaces and the aggregation of such receptors does not lead to intracelluar signalling (agonism) as can occur for many other types of cell surface receptors.
  • administration of a pharmaceutical compositions containing a hexameric antibody/ ⁇ tp fusion protein of the invention blockades chemokine receptors, a subfamily of the 7 TMR, and inhibits chemotaxis and activation of target cells such as eosinophils.
  • CTLA4-Ig ⁇ tp CTLA4-Ig is a potent inhibitor of CD80 and CD86 driven stimulation of T-cells through their interaction with CD28. In animal models, CTLA4-Ig has shown benefit in several autoimmune diseases and transplantation.
  • an ⁇ tp hexameric form of CTLA4-Ig may provide a more potent antagonist than the standard lg fusion protein.
  • a pharmaceutical composition of the invention containing lg ⁇ tp fusion proteins of the invention may be used for removal of complement components or components of the blood coagulation cascade to retard clotting.
  • the invention provides a method for antagonizing cell surface CD80- and CD86-mediated stimulation of CD28 positive cells by administering to the cells a hexameric fusion protein CTLA4-Ig ⁇ tp.
  • the invention provides a method for stimulating (agonist activity) CD28+ T cells by administering the CD80- or CD86-hexameric fusion protein to the cells in culture resulting in stimulation of IL-2 production from these cells.
  • These proteins may be used alone, or in combination with other stimulators of T-cells (e.g., antibodies directed against the T cell receptor-CD3 complex.)
  • the compositions of the invention containing Ig-Fc-containing fusion proteins are useful for in vivo clearance of soluble ligands, in view of the fact that hexamerization of the Fc domain enhances interaction with complement components and Fc receptors.
  • ligands bound to the hexameric fusion protein of the invention are efficiently cleared from circulation.
  • the hexameric fusion proteins of the invention can also serve as agonists, particularly in situations where aggregation can induce a desired response.
  • aggregation is essential for signal transduction through many cell surface receptors - either as a consequence of multi valent presentation of the receptor ligand (eg., a counter receptor on a the surface of a second cell) or through changes induced upon ligand binding, or both.
  • An example of signalling through a cell surface receptor induced by cross-linking through recognition of its counter-receptor on a second cell is CD28 recognition by CD80 or CD86.
  • the invention further provides a method for stimulating CD28 positive cells by administering to CD28 positive cells CD80-Ig ⁇ tp and/or CD86-Ig ⁇ tp.
  • soluble ligands inducing signal transduction through binding to their receptors are EGF and growth hormone and both result in receptor dimerization.
  • dimerization induced through antibody binding also can lead to activation [Schreiber et ai, Proc. Natl. Acad. Sci. USA. 78:7535 (1981), Fuh et ai. Science. 256: 1677 (1992)].
  • Hexameric antibodies against such receptors or hexameric ligand-Ig fusion proteins for these receptors are expected to be more efficient stimulators than the standard dimeric antibodies or ligand lg fusion proteins.
  • the pharmaceutical compositions containing the hexameric antibodies or cytokine-Ig fusion proteins of the invention are useful in inducing signal transduction in receptors for hematopoietic cytokines, such as erythropoietin, thymopoietin and growth stimulatory factor.
  • a method for suppressing CTLA-4 positive cells by administering CD80-Ig ⁇ tp and/or CD86-Ig ⁇ tp to CTLA4 positive cells. This may be performed in vivo, by administration of a pharmaceutical composition containing the hexameric proteins. Alternatively, the hexameric proteins are added to CTLA4 positive T-cells in culture resulting in inhibition of IL-2 production from these cells.
  • hexameric Ig-fusion proteins of the invention can also serve as enhanced immunogens for the fused protein fragment due to efficient, receptor-mediated updake for antigen processing and presentation or efficient interaction with proteins of the complement system.
  • Enhanced immunogenicity is desirable for the efficient generation of polycional and monoclonal antibodies and for therapeutic vaccination.
  • the invention further provides a method of immunizing using the pharmaceutical composition of the invention.
  • the hexameric fusion proteins of the invention are useful in in vitro assays for measuring the binding of the fusion protein to a selected ligand and for identifying the native or synthetic ligand for the binding proteins.
  • a ligand includes the native ligand or counter-receptor to the binding protein from which the hexameric fusion protein is derived.
  • the ligand may be CD28 or CTLA-4.
  • the ligand may be a derivative of the native counter- receptor, a peptide, peptide-like compound, or a chemical compound which interacts with the fusion protein.
  • the hexameric fusion proteins may be used for in vivo assays, including, for example imaging. See, e.g., S. M. Larson et ai, Acta Oncologica. 32(7-8):709-715 ( 1993); R. DeJager et ai. Seminars in Nuclear Medicine. 23(2): 165- 179 (Apr. 1993).
  • a fusion protein of the invention may be used to screen for new ligands.
  • the use of the fusion proteins of this invention in such an assay is particularly well suited for identifying cell surface or multivalent ligands.
  • Suitable assay methods may be readily determined by one of skill in the art.
  • an ELISA format may be utilized in which the selected ligand is immobilized, directly or indirectly (e.g., via an anti-ligand antibody) to a suitable surface.
  • the hexameric fusion protein may be immobilized on a suitable surface.
  • immobilization surfaces are well known.
  • a wettable inert bead may be used in order to facilitate multivalent interaction with the hexameric fusion proteins of the invention.
  • the methods of the invention are readily adaptable to combinatorial technology, where multiple molecules are contained on an immobilized support system.
  • the fusion proteins of the invention permit screening of chemical compound and peptide based libraries where these agents are presented in a multivalent format compatible with more than one subunit of the hexamer. Monomeric interactions of this type are routinely in the mM range and thus may not be readily detected with monomeric proteins.
  • the avidity of the hexameric fusion proteins of the invention permit direct binding.
  • the surface containing the immobilized ligand is permitted to come into contact with a solution containing the fusion protein and binding is measured using an appropriate detection system.
  • Suitable detection systems include the streptavidin horse-radish peroxidase conjugate, direct conjugation by a tag, e.g., fluorescein. Other systems are well known to those of skill in the art. This invention is not limited by the detection system used.
  • the assay methods described herein are also useful in screening for inhibition of the interaction between a hexameric fusion protein of the invention (and thus, the binding protein from which it is derived) and its ligand(s). For example, one may screen for inhibitors of CD80 and CD86 binding to CD28 and CTLA-4.
  • a solution containing the suspected inhibitors is contacted with an immobilized recombinant CD28 or CTLA-4 protein substantially simultaneously with contacting the immobilized ligand with the solution containing the hexameric CD80- or CD86-Ig ⁇ tp protein.
  • the solution containing the inhibitors may be obtained from any appropriate source, including, for example, extracts of supernatants from culture of bioorganisms, extracts from organisms collected from natural sources, chemical compounds, and mixtures thereof.
  • the inhibitor solution may be added prior to or after addition of the CD80- or CD86-Ig ⁇ tp proteins to the immobilized CD28 or CTLA-4 protein. Similar methods may be performed using other hexameric fusion proteins of the invention and their respective ligands.
  • the large size of the Ig ⁇ tp fusion proteins is also advantageous for biophysical assay methods dependent on diffusion or rotation of the protein target in solution, such as for example, fluorescence polarization, fluorescence correlation spectroscopy and anisotropic analytical methods.
  • fluorescence polarization fluorescence polarization
  • fluorescence correlation spectroscopy fluorescence correlation spectroscopy
  • anisotropic analytical methods such as for example, fluorescence polarization, fluorescence correlation spectroscopy and anisotropic analytical methods.
  • CD80-Ig ⁇ tp The following describes the production of CD80-Ig ⁇ tp, CD86-Ig ⁇ tp, and CTLA4- Ig ⁇ tp. Further, for comparison, a construct containing the human IgM tailpiece added to the C-terminus of CD80-Ig was also prepared. This construct, designated CD80-Igutp, differs in amino acid sequence from the ⁇ tp derivative as follows: CH3 Tailpiece SEQ ID NO:
  • IgGl SLSPGK (none) 9 ⁇ tp SLS1GK PTLYNVSLVMSDTAGTCY 25 and 10 ⁇ tp SLSAGK PTHVNVSVVMAEVDGTCY 26 and 1 1
  • the pHbactCd28neo vector for expression of CD28 was previously described [D. Couez et ai, Molecul. Immunol., 3_L(l ):47-57 (1994)].
  • CD80 the coding sequence was cloned by PCR and inserted into a derivative [Dr. F. Letourneur, NIH] of pCDLSR ⁇ 296 [Y. Takebe et ai, Molecul. & Cell. Biol.. 8(l):466-472 (1988)] as described [C. A. Fargeas et ai, J. Exp. Med., 182:667-675 (1995)].
  • the vector COSFcLink [A. Truneh et ai, Mol. Immunol.. 33(3):321 -334 ( 1996)] was constructed for expression of proteins C-terminally fused to a human IgGl Fc region under the transcriptional control of the major late promoter of CMV.
  • the dhfr cassette in this vector permits selection for gene amplification in response to methotrexate.
  • the coding sequences for the native leader and extracellular domain peptide of CD28 and CD80 were grafted onto a human IgGl heavy chain Fc region in the vector COSFcLink, beginning at the start of the hinge region, in a manner similar to that previously described for CD28 and CD80 [P.S. Linsley et ai, J. Exp.
  • the Fc region in this vector was derived from the human plasma leukemia cell line ARH-77 [ATCC CRL 1621] and contains a mutation of cysteine to alanine in the upper hinge region (SEQ ID NO: 27
  • CD28 and CD80 sequences were cloned as Kpnl - Eag I fragments by PCR from the vectors described above and inserted into the corresponding sites in COSFcLink. The resulting vectors are termed CD28FcLink and CD80FcLink, respectively.
  • CD28-Ig the junction of receptor/Fc fragment (immunoglobulin junction) is SEQ ID NO: 12 --GPSKP/EPKSA- and the mature processed N-terminal sequence is SEQ ID NO: 13 NKIL -.
  • the immunoglobulin junction is SEQ ID NO: 14 -HFPDq/EPKS A- and the mature processed N- terminal sequence is VIHV-- (Fig. 4A-4D).
  • the lower case "q" in CD80 represents the substitution of glutamine for the native asparagine.
  • CD86-Ig the corresponding binding protein/Fc construct for CD86 containing the native signal peptide of CD86 (B70) [M. Azuma et ai, Nature. 366:76-79 (1993)], was constructed using methods essentially identical to those described above.
  • the signal and extracellular sequences were PCR cloned from a plasmid containing the CD86 (B70) coding region that was obtained by reverse transcriptase/PCR cloning from human B-cell RNA based on the sequence described by M. Azuma et al. (above). Sequence analysis confirmed identity of this cloned CD86 (B70) region with that of Azuma et al. (above).
  • the amino acid sequence at the junction to the Fc region is: SEQ ID NO: 16 -PPPDHepksa- where capital and lower case letters indicate CD86 and Fc sequences respectively.
  • the mature processed N-terminal sequence is SEQ ID NO: 17 LKIQ - (Fig. 5A-5B).
  • CTLA4-Ig the corresponding binding protein/Fc construct for human CTLA4 containing the native signal peptide of CTLA4 [P. Dariavach et ai, Eur 1 Immunol. 18: 1901-1905 (1988); Harper et ai. J Immunol. 147: 1037-1044 (1991)] was constructed in a similar manner.
  • HuC4.32 a pCDM8 plasmid containing the cDNA sequence for human CTLA4 (Harper et ai, above) was provided by the laboratory of P. Golstein (Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, 13288 Marseille Cedex 9, France).
  • the 5' primer was positioned in the pCDM8 vector.
  • the amino acid sequence at the junction spanning the end of the CTLA4 extracellular domain and the hinge region is: SEQ ID NO: 18 -EPCPDSDAepksa- where capital and lower case letters indicate CTLA4 and Fc sequences respectively and the underlined alanine residue indicates its substitution for phenylalanine in the native CTLA4 sequence.
  • the mature processed N-terminal sequence is SEQ ID NO: 19 MHVA-- (Fig. 6A-6C).
  • Hexameric forms of the CTLA4, CD80 and CD86 recombinant lg proteins were created by addition of a sequence encoding the 18 amino acid tail piece region of human IgAl heavy chain to the C-terminus of the CH3 domain in the expression vectors described above. These methods are described in detail below.
  • Hind III site was introduced into the CH3 domain of CD80FcLink [spanning the 3rd base of the codon for Leu441 [EU numbering, E.A. Kabat et ai, cited above] through the 2nd base of the codon for L443].
  • the Hind III site was introduced by standard PCR methods (eg., PCR Protocols: A Guide to Methods and Applications, Innis et ai, eds, 1990) using the following oligonucleotides: 5' oligo (positioned in the hinge region of the vector): SEQ ID NO: 20
  • the PCR fragments were isolated by agarose gel electrophoresis and purified on Spin Bind columns (FMC Co ⁇ ). The fragment was digested with Eag I and Xba I and cloned into similarly digested CD ⁇ OFcLink vector and colonies were screened for the newly created Hind III site, yielding the vector CD80FcLink-Hd.
  • a synthetic oligonucleotide linker encoding this sequence was cloned between the newly created Hind III site and the Xba I site in CD80FcLink-Hd.
  • the complementary oligonucleotides for the linker sequence were: (5') SEQ ID NO: 22 agcttgtctgcgggtaaacccacccatgtcaatgtgtctgttgtcatggc (Hind III adaptor)
  • acattgacatgggtgggtttacccgcagaca 5 mg of each linker was denatured at 70°C for 10 minutes. The reactions were cooled to room temperature for 20 minutes. The concentration of linker was titrated from 50 to 5 ng using 1000 ng of gel purified CD80FcLink-Hd vector, digested with Hind III/Xba I. Several colonies from each ligation condition were screened for the presence of the ⁇ tp linker by PCR and confirmed by DNA sequencing.
  • FIG. 2 A schematic representation of the resulting vector, CD80Fc ⁇ tplink, is shown in Fig. 2 and the complete DNA sequence is given in Fig. 3A-3H.
  • the vector sequence may differ in some sites from the actual plasmid, but would be function.
  • Introduction of the CD80-Ig ⁇ tp coding region into other standard mammalian expression vectors e.g., pBK- CMV from Stratagene. La Jolla, CA
  • pBK- CMV from Stratagene. La Jolla, CA
  • the vectors for expression of CD86-Ig ⁇ tp as derived from the corresponding lg expression vector by replacing the Fc coding region with the Fc-atp region from CD80- Ig ⁇ tp.
  • the Fc segment of CD86Fclink was excised by cleavage with Eag I (in the hinge region) and Xba I (following the C-terminus of CH3) and replaced with the corresponding fragment of CD80Fcatplink to give the expression vector CD86Fcatplink.
  • the vector for expression of CTLA4-Ig ⁇ tp was derived by replacing a Spel-EagI fragment in CD80Fcatplink with the corresponding fragment from CTLA4Fclink to give the expression vector CTLA4Fcatplink.
  • the Spel site is at base 46 in the CMV promoter region.
  • the sequences of the CD86 and CTLA4 constructs in the region differing from CD80Fcatplink are given in Figs. 5 and 6.
  • CD28-Ig ⁇ tp a vector encoding CD28-Ig ⁇ tp could be prepared starting the CD28Fclink vector described in part A above, or a similar construct encoding an altered version of the CD28 extracellular sequence.
  • the CD28-Ig, CD80-Ig and CD86-lg proteins were produced in CHO cells and purified as described in A. Truneh et al., Mol Immunol. 33: 321-334 (1996) and in I. Kariv et al., J Immunol. 157: 29-38 (1996).
  • the CTLA4-Ig protein was produced and purified in a similar manner, using the vector construct described above in part A of this section.
  • the Ig ⁇ tp fusion proteins were shown to be produced upon transfection of the Fcatplink vectors into COS-7 cells following standard procedures for transfection of COS cells (eg., Current Protocols in Molecular Immunology, edited by F.M. Ausubel et al.
  • CD80-Ig were compared in terms of their efficiency of expression and oligomerization. As determined by SDS/PAGE and immunoblot analysis, the CD80-Ig ⁇ tp construct did not express as well as the ⁇ tp construct of the invention (not shown).
  • the ⁇ tp and ⁇ tp proteins were purified from the COS cell supernatants by capture on Prosep A (Bioprocessing, Ltd., Consett County Durham, U.K.) and their state of oligomerization examined by analytical size exclusion chromatography on a 3.2 X 30 mm Superose 6 column run on a Smart System HPLC (Pharmacia Biotech, Piscataway NJ). Both proteins showed a similar profile of a dominant large MW species eluting in the molecular weight range of IgM, consistent with formation of a hexameric structure, and a smaller fraction that eluted at the same size as CD80-Ig itself (not shown).
  • the fraction of apparent hexamer in the ⁇ tp construct was higher (about 80%) than for the ⁇ tp construct (about 60%). Both the higher level of expression and the greater efficiency of oligomer formation indicated that the ⁇ tp construct of the invention was superior to the ⁇ tp derivative.
  • the CD86-Ig ⁇ tp and the CTLA4-Ig ⁇ tp proteins were produced in COS cells at about the same level observed for the CD80-Ig ⁇ tp protein (0.1 - 0.2 ug/ml).
  • the CD80- and CD86-Ig ⁇ tp proteins were then produced in a CHO cell system (A. Truneh et al.. Mol Immunol. 13: 321-334 (1996)) at levels of 5-10 mg/L. This level of production is comparable to other highly expressed proteins (e.g. antibodies) produced in the same manner in this system.
  • media from growth positive wells is screened for expression - e.g., in an ELISA format using an antibody directed against the Fc region of human IgGl .
  • the highest expressing colonies are expanded and selected in increasing concentrations of methotrexate for amplification of the transfected vectors. If a commercial vector like pBK-CMV (noted above) is used, a dhfr gene should be introduced into this plasmid or provided on a second co-transfecting plasmid to allow selection of amplification in methotrexate.
  • the proteins produced in CHO cells were purified by protein A affinity and size exclusion chromatography.
  • For the CD80-Ig hexamer thirty liters of conditioned medium containing CD80-Ig ⁇ tp were chromatographed on a Protein A Sepharose Fast Flow column (Pharmacia) at 20 ml/min. The column (5.0 x 1 1.6 cm; 225 ml) were preequilibrated in 20 mM sodium phosphate, 150 mM NaCl, pH 7.5 (PBS). After loading, the column was washed with 1.8 L of PBS to baseline absorbance. CD80-Ig ⁇ tp was eluted with 0.1 M sodium citrate, pH 3.0 at 10 ml/min.
  • the eluate was neutralized immediately with 1 M Tris- HC1, pH 8.0.
  • CD80-Ig ⁇ tp was concentrated using an Amicon stirred cell and a YM100 membrane to 1.3 mg/ml.
  • CD80-Ig ⁇ tp was frozen using a dry ice ethanol bath and stored at -70°C.
  • CD80-Ig ⁇ tp The integrity of the CD80-Ig ⁇ tp in the peak fractions is shown by the single band observed in coomassie stained SDS/PAGE gel run under reducing conditions (lane R in the inset in Figure 7).
  • the diffuse nature of the band is characteristic of highly gly cosy lated proteins and is thus expected for CD80-Ig ⁇ tp which contains 10 consensus N- linked glycosylation sites per polypeptide chain. Under nonreducing conditions, all of the protein migrates as high molecular weight species (lane NR in Figure 7, insert).
  • the size exclusion chromatography noted above during purification was consistent with formation of a homogeneous hexameric species containing six CD80-Ig subunits
  • the size and homogeneity of the CD80-Ig ⁇ tp protein produced in CHO cells was also investigated by analytical ultracent ⁇ fugation Equilibrium sedimentation data for
  • CD80-Ig ⁇ tp in PBS, pH 7.4 is shown in Fig. 8, lower panel The sample was sedimented at
  • the lower panel inset shows g(s*) analysis of velocity sedimentation data of the protein taken in the absorption mode Data was collected at 30,000 rpm at 22 °C The data could be fitted to two species, one of 19 4 S and one of 26 7 S which could be the hexamer and (hexamer)2 species
  • g(s*) data analysis see W F Stafford, Current Opinion in B ⁇ otechno.ogy.8m 14-24 (1997)
  • the CD80-Ig ⁇ tp protein was biotinylated for simplicity of assay and for ease of detection since the CD28 protein absorbed to the plate wells was also a human lg fusion construct. Biotinylation was carried out essentially as described in Avidin-Biotin
  • the wells of 96 well microtiter plates (Immunlon 4. Dynatech Laboratories) were coated with CD28-Ig (1 , 2, or 4 ⁇ g/ml) in 100 ⁇ l/well of 0.1 M sodium bicarbonate, pH 9.4 and incubated overnite @ 4°C.
  • the wells were washed with PBS (phosphate buffered saline) and blocked with 0.5% gelatin in PBS for 1 hour.
  • biotinylated CD80-Ig ⁇ tp was serially diluted in PBS containing 1 mg/ml BSA, 0.05% Tween directly in the wells in a final volume of 0.1 ml and incubated for 1 hour.
  • CD80-Ig ⁇ tp protein was measured by the addition of 0.1 ml of strepavidin-HRP (streptavidin conjugated with horseradish peroxidase (Southern Biotech)) at a 1 :2000 dilution for 1 hour,followed by washing and color development with 100 ⁇ l ABTS substrate (Kierkegaard and Perry Laboratories Inc., Maryland) and measurement of absorbance at 405 nm. In some cases the color reactions were arrested by addition of 100 ⁇ l of 1 % SDS prior to measurement of absorbance. A plot of CD80-Ig ⁇ tp binding versus concentration of added protein is shown in Fig. 9.
  • CD28-Fc denote CD28-Ig, CTLA4-Ig, and CD80-Ig ⁇ tp, respectively.
  • CD28.1 MAb a murine MAb to human CD28 that inhibits binding of CD80 to CD28; Nunes et ai. Int. Immunol.. 5:31 1-315 (1993)) or
  • CTLA4-Ig protein (here labeled as CTLA4-Fc).
  • biotinylated CD80-Ig itself showed little binding and only at much higher concentrations ( Figure 10).
  • biotinylated CD86-Ig ⁇ tp also showed good binding to CD28-Ig (Fig 10).
  • All three biotinylated proteins showed good binding to immobilized CTLA4-Ig (Fig. 1 1), as expected because of the higher affinity of this interaction [P. S. Linsley et. al., Immunity 1 : 793-801 ( 1994), and see part 4 of this example below], and the rank order of binding was the same as observed with immobilized CD28-Ig.
  • microtiter wells were coated with 2 ⁇ g/ml CD28-Ig and biotinylated CD80-Ig ⁇ tp was added at a concentration of 50 ⁇ g/ml, followed immediately by the indicated amounts of unlabeled CD80-Ig ⁇ tp (B7FcA), CD80-Ig (B7Ig), CTLA4-Ig, or the MAb CD28.2.
  • B7FcA unlabeled CD80-Ig ⁇ tp
  • B7Ig CD80-Ig
  • CTLA4-Ig or the MAb CD28.2.
  • the biotinylated CD80- Ig ⁇ tp gives about 50% saturation of OD40 5 (see Fig. 9).
  • CD80-Ig was much less efficient than CD80-lg ⁇ tp in blocking binding, consistent with the expected lower affinity/avidity of the CD80-Ig protein for the immobilized CD28-Ig protein.
  • the controls gave the expected results - the CD28.2 MAb blocked the binding site on CD28 and similarly, CTLA4-Ig blocked the binding sites on CD80-Ig ⁇ tp.
  • a second example utilizes a CD28-muIg fusion protein constructed in a manner analogous to CD28-Ig except that the lg region was derived from mouse Ig2a instead of human IgG 1. More particularly, the protein was expressed using the vector CosCD28mFc2aLink, which is comparable to the CosCD28FcLink vector (described above), except that the human IgGl-Fc region was replaced with that of mouse IgG2a beginning at the Eag I site in the hinge sequence [described in I. Kariv et ai, L Immunol. 157:29-38 (1996)].
  • the amino acid sequence in the resulting hybrid hinge region is as follows: SEQ ID NO: 24 -GPSKPepksaglKP-, where capital letters correspond to the end of CD28 sequence, lower case letters are residues from the human IgGi hinge region, underlined lower case letters are a 2 residue substitution introduced to create an Eag I site, and bold capital letters indicate the beginning of murine IgG2a hinge region.
  • CD28-m «Ig protein was indirectly immobilized in wells using goat anti-mouse Fc antibody and then CD80-Ig ⁇ tp binding was carried out similarly to that described above. More specifically, CD28-m «Ig proteins containing wild-type or mutant CD28 sequences and, at equal concentrations, were captured on goat anti-mouse IgG antibody coated 96 well plates. The plates were washed with lx PBS, blocked with 0.5% geiatin-PBS for 1 hour, and then incubated with either biotinylated CD80- or CD86-Ig ⁇ tp for 45 min. The plates were washed and lg ⁇ tp fusion protein was quantitatedas described above. .
  • Figs. 13A and 13B This assay was used to examine the effects of mutations in CD28 on binding to CD80 and CD86, as illustrated in Figs. 13A and 13B (I. Kariv et ai. J. Immunol.. 157:29-38 (1996)).
  • Each of the mutant CD28-muIg2a proteins was captured on the goat anti-mouse IgG coated wells and the binding of biotinylated CD80 - Ig ⁇ tp (Fig. 13A) or CD86 - Ig ⁇ tp (Fig. 13B) was measured. Equivalent capture of each of the CD28-muIg2a proteins was verified by the comparable binding of polycional rabbit CD28 antisera to each of the proteins (Fig. 13C).
  • CD80-Ig ⁇ tp or CD80-Ig were compared by surface plasmon resonance analysis using a BlAcorc instrument, following procedures similar to that described for other proteins [K. Johanson et. al., J. Biol. Chem. 270: 9459-9471 ( 1995), and references therein] .
  • BIAcore CM5 sensor surface BIAcore, Piscataway, NJ
  • Covalent attachment was achieved by firstly activating the surface with a 1 : 1 mixture of 0.1 M solution of N- hydroxysuccinamide and 0.1 M l-ethyl-3-(3-dimethylaminopropyl)carbodiimide. A solution of CD28-Ig 50 ug/ml in 0.01 M sodium acetate pH 4.7 was then passed over the surface. Unreacted N-hydroxysuccinamide esters were then deactivated with 1 M ethanolamine pH 8.5. The surface was equilibrated with running buffer composed of 20 mM HEPES 150 mM NaCl, pH 7.2, 3 mM EDTA and 0.005% Tween 20.
  • CD80-Ig and CD80-Igatp (20 ug/ml) diluted in running buffer were injected over the surface (60 ul) with a flowrate of 10 ul/min.
  • the results show that CD80-Ig dissociates very rapidly from the CD28-Ig coated surface, whereas the rate of dissociation for CD80-Ig ⁇ tp is about three orders of magnitude slower (Figure 14).
  • CD80-Ig ⁇ tp and CD86-Ig ⁇ tp binding were prepared in running buffer described above. Sample solutions were injected (60 ul) at 10 ul/min. Between samples, the surface was regenerated with a 30 ul injection of Gentle elution buffer (Pierce Chemicals, Rockville, ILL). The results show that like CD80-Ig ⁇ tp above, CD86-Ig ⁇ tp dissociates slowly from the CD28-Ig surface ( Figure 15).
  • Kd's The error in Kd's is about a factor of 2 and the error in molar binding ratio's is 10-20% Kd values at 37°C were either measured directly at 37°C or were corrected for temperature differences using the van't Hoff equation, as described in M L Doyle et al , J Mol Recognition .
  • the titration equivalence points for CTLA4-Ig binding to the CD80 and CD86 lg versus Ig ⁇ tp constructs indicate that all these reagents were >50% active during the calorimetry assay.
  • comparison of the Ig ⁇ tp and lg constructs shows a ratio of about 6 for CD80, indicating about equivalent binding activity for the CD80 domains in both constructs.
  • the lower ratio for the corresponding CD80-Ig ⁇ tp protein indicates some loss of activity in this preparation.
  • CD28-Ig Interactions of CD28-Ig with either CD80- or CD86-Ig were not detected in solution by calorimetry, suggesting an affinity of interaction weaker than 1 uM. This lower affinity for CD28 than for CTLA4 is in agreement with other reports [P. S. Linsley et. al., Immunity 1: 793-801 (1994)]. CD28-Ig also did not show detectable binding to CD80- or CD86-Ig ⁇ tp, which is consistent with the solution affinities of the ⁇ tp constructs not benefiting from avidity effects.
  • Example 2 Demonstration of agonist activity for the CD80- and CD86 Ig ⁇ tp protein A. CTLL-2 bioassav for detection of IL-2 levels
  • the CD80- and CD86-Ig ⁇ tp proteins were compared to the corresponding CD80 and CD86-Ig proteins to determine their ability to stimulate cells expressing human CD28 using two murine T-cell hybridoma cell lines expressing human CD28, PCD28.1.s2.1 and DCL27CD28wt.s2.
  • the PCD28.1.s2.1 cell line was described in Example 1 , part 3.
  • the DCL27CD28wt.s2 cell line was created by transfection of the DC27 cell line [F. Pages et ai, Nature. 369:327-329 ( 1994); F. Pages et ai, J. Biol. Chem..
  • soluble fusion proteins and the 248.23.2 CD28 MAb (IgM) [A. Morretta, University of Genova, Italy] were added to non-coated wells. T cells were incubated in the wells for 24 hours at 37°C, and supernatants were collected and evaluated for IL-2 levels in a standard CTLL-2 bioassay [S.M. Gillis et ai, J. Immunol.. 120:2027 (1978)]. Briefly, lxlO 4 IL-2 dependent CTLL-2 cells (ATCQ/well in 75 ⁇ l medium were added to an equal volume of test supernatant and incubated for 24 hours at 37°C.
  • the cells were pulsed with 10 ⁇ l of 5 mg/ml MTT (Sigma Chemical Co.) for 4 hours, and lysed with 100 ml 10% SDS/0.01N HC1 solution for 14-16 hours.
  • OD 570 readings were converted into ng/ml of IL-2 based on a standard curve generated by treating cells with known concentrations of IL-2.
  • the CD80- and CD86-Ig ⁇ tp proteins were more efficient stimulators of the CD28 T-cells than the corresponding monomeric lg constructs (Figs. 17 and 18).
  • the soluble hexameric proteins induced IL-2 production in the absence of CD3 crosslinking (GAH), whereas under the same conditions, no activity was observed with CD80- or CD86- lg themselves.
  • a similar level of IL-2 induction was observed with the oligomeric CD28 IgM antibody 248.23.2.
  • Cross-linking of the hexameric CD80 and CD86 proteins with GAH antibody increased the IL-2 response relative to the absence of cross-linker, but still did not give a response for the monomeric lg constructs.
  • the differences between the hexameric and monomeric lg fusion proteins were minimal, being about 2-fold or less.
  • FDG Fluorescein di-b-D-galactosidase
  • PCD28.1.S2.1 cells described above also contain lacZ fused to the IL-2 promoter.
  • the PCD28.1.S2.1 cell line provides a convenient system for measuring IL-2 promoter activity upon CD28-mediated T cell simulation.
  • T cells were activated as described above for the CTLL-2 assay, spun down, resuspended in 50 ⁇ l of media + 50 ⁇ l of PBS, lysed with 10 ⁇ l of 20% Triton X-100, and supplemented with 25 ⁇ l of 10 mM FDG (Molecular Probes), a fluorogenic substrate for b- galactosidase.
  • FDG fluorescein monogalactoside
  • FMG fluorescein monogalactoside
  • Fig. 19 The results of these assays (Fig. 19) were similar to those described above for IL-2 production. The primary difference was that low levels of IL-2 promoter activity were observed for the monomeric lg proteins.
  • the lg and Ig ⁇ tp proteins showed the greatest activity when captured on the surface of the microtiter well.
  • the CD80- and CD86-Ig ⁇ tp proteins were also able to stimulate CD28 cells when added directly to the cells in solution, whereas no response was observed with the corresponding standard lg fusion proteins.
  • CD80-and CD86-Ig ⁇ tp showed a dose dependent stimulation of IL-2 promoter activity (Fig 20A) and IL-2 production (Fig 20B) when added to PC28.1.s2.1 cells. In contrast, no stimulation was observed with CD80-Ig (Figs. 20A and 20B) or
  • CD86-Ig (not shown). IL-2 promoter activity and IL-2 levels were measured similarly to that described in parts A and B above, except that proliferation of the reader CTLL-2 cells was measured by • 'H-thymidine incorporation.
  • results from these assays show that the CD80- and CD86- Ig ⁇ tp proteins have agonist activity under conditions where little or no activity was observed for the corresponding monomeric lg proteins.
  • Example 3 Compound screen assay for identifying small molecule antagonists of the interaction between CD28 and CD80
  • An ELISA format was used to identify small molecule antagonists of CD80 and CD86 binding to CD28 by screening a large bank of chemical compounds and natural products.
  • the assay was carried out as in the format described in Example 1 , part E.1 , except that immediately following addition of the biotinylated CD80-Ig ⁇ tp (222 ng/ml in a volume of 90 ⁇ l), dilutions of test compound were added (10 ⁇ l).
  • the compounds were dissolved at lOOx assay concentration in dimethyl sulfoxide (DMSO) and subsequently diluted in 50%DMSO/50% H 2 0 to a 10X working stock.
  • DMSO dimethyl sulfoxide
  • the assay was not sensitive ( ⁇ 10% alteration of signal) to DMSO at concentrations of 5% or less.
  • Results from one test assay are summarized in Fig. 21 and Table II.
  • the BM-34 test set consists of 968 compounds in two formats - as individual compounds and as 88 multimixes with 1 1 individual compounds in each multimix sample. Both BM-34 formats were assayed (at a concentration of 200 ⁇ g/ml for each multimix sample and 20 ⁇ g/ml for individual compounds) for inhibition of biotinylated CD80-Ig ⁇ tp binding to immobilized CD28-Ig in 96 well plates. Results for setting a 70% or 85% cutoff for inhibition are shown in Table II. In Fig. 21, the percent inhibition range is plotted against the number of compounds showing the indicated range of inhibition. The low percentage of compounds showing activity in the 80-90 % range of inhibition makes this a suitable threshold for rapid screening.
  • Example 4 - ⁇ tp-mcdiated oligomerization of a mouse/human IgG1 chimeric antibody.
  • the ⁇ tp segment was introduced into a chimeric antibody containing heavy and light chain variable regions from the mouse monoclonal antibody 1C8 and the human kappa and IgGl constant regions. 1C8 is directed against the human EPO (erythropoeitin) receptor.
  • the ⁇ tp sequence was introduced onto the heavy chain of the antibody by replacing the Eco Rl/Sac II fragment of CD80Fc ⁇ tplink with the Eco Rl/Sac II fragment of EpoR(CH)IgGl-PCN, a vector containing the heavy chain of the chimeric 1C8 antibody, to give the vector EpoR(CH)Fc ⁇ tplink.
  • Eco RI cleaves between the CMV promoter and the start of the N-terminal signal sequences
  • Sac II cleaves at a conserved site in constant region 2 of the human heavy chain.
  • Test samples of the hexameric mAb were produced in COS-7 cells upon co- transfection of EpoR(CH)Fc ⁇ tplink and a vector for expression of the light chimeric light chain, following procedures described above in Example 1, part C. Initially, 5 T150 flasks were co-transfected with the two vectors and 300 ml of conditioned media were collected. The hexameric antibody was purified by affinity chromatography on Protein A. Purity was about 90% as determined by coomassie staining of the sample as analyzed by reducing SDS/PAGE. Under nonreducing conditions on SDS/PAGE, the antibody migrated in the size range of IgM (not shown).
  • the sample was further characterized by analytical size exclusion chromatography on a 3.2 X 30 mm Superose 6 column run on a Smart System HPLC (Pharmacia Biotech, Piscataway NJ).
  • the major peak corresponds to binding activity, as monitored in an ELISA using a recombinant human EPO receptor lg fusion protein (EPOr-Ig), and eluted at a size consistent with hexamer formation (anti-EPOr-IgG j ⁇ tp).
  • the parental chimeric antibody (anti-EPOr-IgG] ) elutes substantially later from the column and is represented in the figure by the dashed lines.
  • MOLECULE TYPE protein
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 5:
  • GTC TTC TGC AAA GCA ATG CAC GTG GCC CAG CCT GCT GTG GTA CTG GCC 744
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE protein
  • xi SEQUENCE DESCRIPTION : SEQ ID NO : 24 :
  • MOLECULE TYPE protein

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Toxicology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention porte sur une protéine de fusion hexamétrique contenant une protéine fixatrice dimère pourvue d'une queue provenant d'un anticorps IgA. Cette protéine de fusion est utile dans des agents thérapeutiques et dans des vaccins, mais convient particulièrement à des applications dans lesquelles la protéine de fixation dont elle est dérivée n'est pas satisfaisante en raison de sa faible affinité de liaison, ou à des applications pour lesquelles une polyvalence est requise. Ces applications comprennent notamment les diagnostics, les analyses de liaison et les analyses pour le criblage.
EP97935005A 1996-06-14 1997-06-13 Proteines de fusion hexameres et utilisations associees Withdrawn EP0975355A2 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US1993496P 1996-06-14 1996-06-14
US19934P 1996-06-14
US4394897P 1997-02-19 1997-02-19
US43948P 1997-02-19
US3891597P 1997-02-21 1997-02-21
US38915P 1997-02-21
PCT/US1997/012599 WO1997047732A2 (fr) 1996-06-14 1997-06-13 Proteines de fusion hexameres et utilisations associees

Publications (1)

Publication Number Publication Date
EP0975355A2 true EP0975355A2 (fr) 2000-02-02

Family

ID=27361321

Family Applications (1)

Application Number Title Priority Date Filing Date
EP97935005A Withdrawn EP0975355A2 (fr) 1996-06-14 1997-06-13 Proteines de fusion hexameres et utilisations associees

Country Status (5)

Country Link
EP (1) EP0975355A2 (fr)
JP (1) JP2000515731A (fr)
AU (1) AU3804397A (fr)
CA (1) CA2257861A1 (fr)
WO (1) WO1997047732A2 (fr)

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2285692C (fr) * 1997-03-27 2013-05-28 The Council Of The Queensland Institute Of Medical Research Amelioration de la reaction immune au moyen de molecules de ciblage
US6475749B1 (en) * 1999-08-11 2002-11-05 The Regents Of The University Of California Rh hybrid antibody
US7094874B2 (en) 2000-05-26 2006-08-22 Bristol-Myers Squibb Co. Soluble CTLA4 mutant molecules
US6875904B2 (en) 2000-09-20 2005-04-05 The Ohio State University Research Foundation Animal model for identifying agents that inhibit or enhance CTLA4 signaling
WO2003040311A2 (fr) 2001-10-25 2003-05-15 The Government Of The United States Of America As Represented By The Secretary Of Health And Human Services Inhibition efficace de l'entree virale du vih-1 au moyen d'une nouvelle proteine de fusion telle que cd4
CA2516834C (fr) 2003-02-27 2013-07-16 Theravision Gmbh Polypeptides et methodes d'obtention
ES2777778T3 (es) * 2012-05-11 2020-08-06 Medimmune Ltd Variantes de CTLA-4
BR112015000167B1 (pt) * 2012-07-06 2021-11-23 Genmab B.V. Proteína dimérica, proteína, composição, kit de partes e seus usos, bem como método para aumentar a oligomerização em solução de uma proteína dimérica compreendendo um primeiro e segundo polipeptídeo, cada um compreendendo pelo menos as regiões ch2 e ch3 de uma cadeia pesada de igg1 humana e proteína dimérica variante
PT2869845T (pt) * 2012-07-06 2019-12-09 Genmab Bv Proteína dimérica com mutações triplas
SG10201913507SA (en) 2014-05-02 2020-02-27 Momenta Pharmaceuticals Inc Compositions and methods related to engineered fc constructs
KR20180002653A (ko) * 2015-04-07 2018-01-08 제넨테크, 인크. 효능작용 활성을 갖는 항원 결합 복합체 및 사용 방법
KR102462084B1 (ko) 2016-05-23 2022-11-02 모멘타 파머슈티컬스 인코포레이티드 유전자 조작 Fc 작제물에 관한 조성물 및 방법
CN110650748B (zh) 2017-01-06 2024-01-02 动量制药公司 与经工程改造的Fc构建体相关的组合物和方法
MX2021004660A (es) * 2018-10-23 2021-05-28 Igm Biosciences Inc Moleculas de union multivalentes a base de igm- e iga-fc.

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9747732A3 *

Also Published As

Publication number Publication date
JP2000515731A (ja) 2000-11-28
AU3804397A (en) 1998-01-07
CA2257861A1 (fr) 1997-12-18
WO1997047732A2 (fr) 1997-12-18
WO1997047732A3 (fr) 1998-01-29

Similar Documents

Publication Publication Date Title
US20020147326A1 (en) Hexameric fusion proteins and uses therefor
AU2019283892B2 (en) Anti-human papillomavirus 16 E7 T cell receptors
KR100249937B1 (ko) 인간 인터루킨-6 수용체에 대한 재구성 인간 항체
AU740738B2 (en) Humanized antibodies and methods for forming humanized antibodies
AU657788B2 (en) Chimaeric interleukin 5-receptor/immunoglobulin polypeptides
KR100897379B1 (ko) 혈관신생-저해 키메릭 단백질 및 그 사용
US6153190A (en) Erythropoietin receptor antibodies
WO1997047732A2 (fr) Proteines de fusion hexameres et utilisations associees
US20040191257A1 (en) Pharmaceutical compositions comprising herpes virus entry receptor protein
CA2239522A1 (fr) Proteine hybride a interferon-.alpha. et fragment fc d'immunoglobuline a liaison assuree par peptide non immunogene
KR20210104060A (ko) Il-15 조성물 및 이의 사용 방법
EP3431507A1 (fr) Protéine de fusion comprenant un facteur de croissance nerveuse et procédé de préparation et utilisation correspondants
CN112979807B (zh) Bcma结合抗体及其用途
CN102875683B (zh) 长效重组人生长激素的Fc融合蛋白
CN109970866B (zh) 一种cd28双向激活共刺激分子受体及其用途
AU2023202642A1 (en) IL-2 Dependent NK-92 cells with stable Fc receptor expression
JP2554418B2 (ja) ヒトインターロイキン−５受容体のα鎖又はその一部、特にｓｈＩＬ５ＲαをコードするＤＮＡ及びそれを含むベクター、このベクターで形質転換された宿主細胞、ならびにその製造及び利用方法
CN110357959A (zh) Gcgr抗体及其与glp-1的融合蛋白质，以及其药物组合物和应用
US20090131308A1 (en) Splice Variants of ErbB Ligands, Compositions and Uses Thereof
RU2787060C1 (ru) НУКЛЕОТИДНАЯ ПОСЛЕДОВАТЕЛЬНОСТЬ, КОДИРУЮЩАЯ СЛИТЫЙ БЕЛОК, СОСТОЯЩИЙ ИЗ РАСТВОРИМОГО ВНЕКЛЕТОЧНОГО ФРАГМЕНТА ЧЕЛОВЕЧЕСКОГО Dll4 И КОНСТАНТНОЙ ЧАСТИ ТЯЖЕЛОЙ ЦЕПИ ЧЕЛОВЕЧЕСКОГО IgG4
CN109957578A (zh) 双基因表达载体及其制备方法和应用
CN110655577A (zh) APJ抗体及其与Elabela的融合蛋白质，以及其药物组合物和应用
CA3237004A1 (fr) Inhibiteurs doubles de facteur d'activation des lymphocytes b (baff)-ligand a induisant la proliferation (april)
KR20230028509A (ko) 안구 병리의 치료를 위한 dna 구조물
JP2020164461A (ja) 新規転写調節融合ポリペプチドを用いた血管新生の誘導方法

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 19990113

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): BE CH DE ES FR GB IT LI NL

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20040103

REG Reference to a national code

Ref country code: HK

Ref legal event code: WD

Ref document number: 1026617

Country of ref document: HK