WO1991011461A1 - Proteines de fusion de proteines liantes c4 - Google Patents

Proteines de fusion de proteines liantes c4 Download PDF

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Publication number
WO1991011461A1
WO1991011461A1 PCT/US1991/000567 US9100567W WO9111461A1 WO 1991011461 A1 WO1991011461 A1 WO 1991011461A1 US 9100567 W US9100567 W US 9100567W WO 9111461 A1 WO9111461 A1 WO 9111461A1
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c4bp
protein
polypeptide
multimeric
human
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PCT/US1991/000567
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Mark P. Pasek
Gunther Winkler
Theresa R. Liu
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Biogen, Inc.
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Priority to CA002049964A priority Critical patent/CA2049964A1/fr
Publication of WO1991011461A1 publication Critical patent/WO1991011461A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • C07K14/70514—CD4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

  • This invention relates to multimeric and hetero-multimeric C4 binding protein (C4bp) fusion proteins and compositions and methods using them. More particularly, this invention relates to multimeric C4bp fusion proteins which are aggregates or assemblies of C4bp monomers linked to functional moieties. It also relates to C4bp fusion polypeptides and in particular CD4-C4bp fusion polypeptides comprising an amino acid sequence for a soluble human CD4 protein fused to a C4bp monomer having, preferably, four short consensus repeat regions.
  • C4bp C4 binding protein
  • bioactive molecules In light of rapidly developing biotechnologies, researchers are producing novel delivery and carrier systems for pharmaceuticals, vaccines, diagnostics and other bioactive molecules. Optimally, these systems enhance the properties of the molecules they carry, complement those molecules with characteristics they lack and combine useful characteristics of different molecules. Of particular interest to researchers are the isefum half-life of bioactive molecules, their affinity for target particles and cells, targetability of bioactive molecules, bioactivity, immunogenicity and the ability to administer or deliver several molecules simultaneously.
  • Human C4 binding protein is a molecule possessing many attractive characteristics as a delivery vehicle for bioactive molecules. Human C4bp is involved in the human complement system — a group of immune system proteins whose functions include lysing invading cells, activating phagocytic cells and facilitating the clearance of foreign substances from the system. It regulates the action of proteins in this system, particularly C4 protein. Structurally, hC4bp is a flexible, disulfide-bonded molecule expected to have long serum half-life and the ability to target bioactive molecules to the lymph nodes. The serum form of hC4bp has a molecular weight of about 590 kD.
  • hC4bp On reducing SDS gels, hC4bp produces a strong band at about 70 kD, indicating a disulfide-bonded multimeric protein.
  • human C4bp appears as a structure with seven monomeric tentacles [B. Dahlback et al., "Visualization of Human C4b- Binding Protein and Its Complexes with Vitamin K- Dependent Protein S and Complement Protein C4b", Proc. Natl. Acad. Sci.. USA. 80. pp. 3461-65 (1983)].
  • a cDNA encoding the C4bp monomer has been cloned and characterized [L.P. Chung et al., "Molecular Cloning and Characterization of the cDNA Coding for C4b-Binding Protein of the Classical Pathway of the Human Complement System", Biochem. J.. 230. pp. 133-41 (1985) ] .
  • Chun ⁇ et al. refers to hC4bp as a polypeptide of 549 amino acids.
  • the polypeptide predicted from the DNA sequence has a molecular weight of about 61.5 kD, rather than 70 kD as actually measured on reducing SDS gels.
  • the difference in molecular weight apparently is due to glycosylation of the serum form of the polypeptide.
  • the first 491 amino acids from the N-terminus of the Chung et al. sequence are divisible into eight domains called short consensus repeat regions (SCRs) of about sixty amino acids each. These regions are designated, from N-terminus to C-terminus, SCR8 to SCRl.
  • SCR domains are defined by the amino acids of Figure 1 of this application as follows: SCR8 - +1 to +61; SCR7 - +62 to +123; SCR6 - +124 to +187; SCR5 - +188 to +247; SCR4 - +248 to +313; SCR3 - +314 to +374; SCR2 - +375 to +432; SCRl - +433 to +491.
  • These domains which share significant sequence homology, each contain four similarly situated cysteine residues. These cysteine residues form intra-domain disulfide bonds in a regular pattern [J. Janatova et al., "Disulfide Bonds Are Localized Within the Short Consensus Repeat Units of Complement Regulatory
  • hC4bp In addition to the eight SCR domains, hC4bp also has a 58 amino acid sequence at the C-terminus, the C4bp core, which bears no homology to the other regions of the protein. This region is responsible for assembly of the molecule into a multimer. According to one model, the cysteine at position +498 of one C4bp monomer forms a disulfide bond with the cysteine at position +510 of another monomer.
  • human C4bp contains another subunit, a 45 kD polypeptide which is linked by disulfide bonds to the C4bp heptamer core [A. Hillarp and B. Dahlback, "Novel Subunit in C4b-Binding Protein Required for Protein S Binding", J. Biol. Chem.. 263. pp. 12759-64 (1988)].
  • This subunit binds protein S, a protein involved in the regulation of blood clotting. When bound to protein S, protease C catalyses the transformation of clotting factors VIII and V from the active to inactive forms.
  • C4bp also exists in mammals other than humans. It has been isolated from both mouse and guinea pig [S.J. Lintin et al., "Derivation of the Sequence of the Signal Peptide in Human C4b-protein and Interspecies Cross-hybridization of the C4bp cDNA Sequence", FEBS Letters. 232. pp. 328-332 (1988)]. Analysis of mouse C4bp indicates that it contains contiguous SCRs, as does human C4bp. Mouse C4bp, however, has only six SCRs within each C4bp monomer and the multimer is held together by non-covalent bonds.
  • Multimeric C4bp fusion proteins are aggregates or assemblies of C4bp monomers linked to functional moieties which may be pharmaceutical agents, vaccine agents, diagnostic agents or other bioactive molecules.
  • Hetero-multimeric C4bp fusion proteins contain combinations of different C4bp monomers, different functional moieties, or combinations of both.
  • This invention also provides multimeric and hetero- multimeric non-human C4bp fusion proteins.
  • C4bp fusion polypeptides comprise C4bp monomers fused or chemically coupled to a functional moiety.
  • this invention provides the fusion polypeptide CD (187)-C4bp(SCR4) .
  • This invention also relates to multimeric C4bp fusion proteins comprising monomeric C4bp fusion polypeptides.
  • this invention further provides DNA sequences encoding C4bp fusion polypeptides, recombinant DNA molecules comprising those sequences and unicellular host cells transformed with those molecules. This invention also provides methods for producing these fusion polypeptides by cultur ⁇ ng such hosts.
  • compositions comprising C4bp fusion polypeptides or proteins that are useful as therapeutic, prophylactic or diagnostic agents, particularly in diagnosing, preventing and treating AIDS, ARC and HIV infection.
  • the fusion proteins of this invention advantageously utilize various features of hC4bp, including its multimeric nature, its large size, the flexibility of its tentacles and its ability to migrate through the lymph nodes. Consequently, the bioactive molecules linked to C4bp monomers as functional moieties in such fusion proteins are characterized by one or more of the following: polyvalency, increased serum half-life, increased affinity for target particles or cells, greater bioactivity or immunogenicity and targetability.
  • multimeric and hetero-multimeric C4bp fusion proteins have many uses.
  • Recognition molecules such as those containing the antigen binding site of antibodies, viral receptors or cell receptors, are useful as functional moieties to target C4bp fusion proteins to particular antigens.
  • multimeric C4bp fusion proteins are useful to block the binding of viruses to cells, thereby preventing viral infection.
  • C4bp fusion proteins may also be used to inhibit cell to cell binding such as that which characterizes pathologic inflammation. Due to the multivalency and conformational flexibility of the fusion proteins of this invention, we believe that they possess greater affinity for the target than monovalent or rigid multivalent molecules.
  • the functional moiety is the receptor on human lymphocytes, CD4, which is the target of the HIV virus — the causative agent of AIDS and ARC.
  • recognition molecules are used in conjunction with toxins, anti-retroviral agents or radionuclides in hetero-multimeric C4bp fusion proteins according to this invention, those proteins become - 7 -
  • C4bp fusion proteins having recognition molecules are also useful for signal enhancement in diagnostic assays.
  • reporter molecules such as horseradish peroxidase-c ⁇ njugated antibodies.
  • they m y take the form of hetero- 13,rs, possessing both recognition molecules for the target and mul ⁇ tip ⁇ e reporter groups.
  • C4bp fusion protein is on ⁇ or more immunogen from infectious agents, the proteins of this invention are useful in vaccines. And when the functional group is an enzyme, substrate, or inhibitor, the multimeric C4bp fusion proteins may function as agents with increased bioactivity.
  • the present invention also provides recombinant human C4bp and processes for production of that protein.
  • Figures 1A-1C depict the DNA sequence and deduced amino ac ⁇ d. sequence of- human C4bp polypeptide derived from pJOD.C4bp.3. The negatively numbered amino acids correspond " to the signal sequence, which is absent from the mature polypeptide. Throughout this application, references to C4bp by amino acid formula correspond to the coordinate system set forth in this figure.
  • Figure 2 depicts the structure of an SCR domain. It portrays an amino acid sequence of a short consensus repeat (SCR) region connected to adjacent SCRs. Each amino aci-ct" is represented by a circle. As described, infra, each SCR is held together by two disulfide bonds between cysteines 1 and 3 and between cysteines 2 and 4, as depicted in this figure. The loop is depicted as the amino acid sequence between cysteines 1 and 4, inclusive. The joints are depicted as the amino acid sequences between two connected loops.
  • SCR short consensus repeat
  • Figures 3A-3B depict the nucleotide sequence and deduced amino acid sequence of human CD4 protein.
  • Nucleotides 1 to 636 are derived from pJOD.sCD4.Y187.SnaBl.
  • Nucleotides 637 to 1377 are derived from pl70.2.
  • the amino acids are numbered from -25 to 375.
  • references to CD4 by amino acid formula correspond to the coordinate system of this figure, unless otherwise specified.
  • Figure 4 depicts the domain structure of human CD4 protein. The numbered amino acids are cysteine residues involved in disulfide bonding according to Figures 3A-3B.
  • Figures 5A-5B depict the DNA sequences of oligomers C4bp.l to C4bp.20, SCR.l, SCR.4, SCR.8,
  • left to right designates 5 1 to 3'.
  • Figures 6A-6H depict the construction of plasmids pJOD.C4bp and pJOD.sCD4.Y187.SnaBl.
  • Figure 7 depicts the construction of a plasmid containing a sequence encoding a CD4-C4bp fusion polypeptide according to this invention.
  • a "CD4-C4bp fusion polypeptide" comprises amino acid sequences of human CD4 protein and C4bp.
  • the top strand depicts pJOD.sCD4 including the adenovirus major late promoter (Ad MLP) ; the 5' untranslated sequence (5 1 UTS); the ATG initiation codon and signal sequence encoding region; the region encoding human CD4 protein through the codon for tyrosine (TAC(187)); the SnaBl site (TACGTA) ; the B ⁇ lll site (AGATCT) ; and the SV40 polyadenylation control sequence.
  • the bottom strand depicts pJ0D.C4bp, including the region encoding SCR8- SCR1, the core and'termination codon and che MIS gene polyadenylation control sequence.
  • Figure 8 depicts the results of purification of recombinant human C4bp (rhC4bp) and the serum form of human C4bp (serum) by HPLC.
  • Figure 9 depicts illustrative embodiments of C4bp fusion polypeptides and proteins according to this invention.
  • Figure 10 is.a table summarizing the antibodies used in ⁇ LISA assays 1-9, described herein.
  • C4 binding protein (“C4bp”) refers to a polypeptide having the amino acid sequence depicted in
  • Figure 1 from amino acids -32 to +549. It should be understood that expression of polypeptides often involves post-translational modifications, such as cleavage of the signal" sequence, intramolecular disulfide bonding, glycosylation and the like. Accordingly, the term,*C4 binding protein, also contemplates such modifications "to the amino acid sequence of C4bp. It also encompasses naturally occurring genetic polymorphisms. The term also includes C4 binding proteins from natural, recombinant or synthetic sources.
  • Multimeric C4bp fusion proteins and “hetero-multimeric C4bp fusion proteins” each comprise aggregates or assemblies of C4bp fusion polypeptides.
  • C4bp fusion polypeptides comprise a C4bp monomer bound to a functional moiety.
  • “Functional moieties” may be polypeptides ("polypeptide moieties”) or chemical compounds (“chemical moieties”) .
  • One may produce multimeric C4bp fusion proteins by genetic fusion, chemical synthesis, or chemical coupling techniques.
  • the functional moiety is a polypeptide
  • genetic fusion involves, for example, creating a hybrid DNA sequence encoding the C4bp fusion polypeptide in which the 3' end of a DNA sequence encoding the polypeptide is ligated to the 5 1 end of a DNA sequence encoding a. C4bp monomer. Upon expression in an appropriate host, this hybrid DNA sequence will produce a C4bp fusion polypeptide that will assemble into a multimer.
  • a “C4bp monomer” as used herein is a polypeptide comprising a C4bp core or, more preferably, a sequence of at least one SCR fused to the N-terminus of a C4bp core.
  • a "C4bp core” encompasses, at a minimum, amino acids +498 to +549 of Figure 1 and, preferably, amino acids +492 to +549.
  • an "SCR” is a polypeptide fragment of C4bp.
  • An SCR comprises, at a minimum, a loop and, at a maximum, a loop and two joints.
  • a “loop” comprises the amino acid sequence encompassed by the first and fourth cysteines of the eight SCR domains as defined above.
  • the loops encompass amino acids +2 to +60 of SCR8, +65 to +122 of SCR7, +127 to +186 Of SCR6, +191 to +246 of SCR5, +251 to +312 of SCR4, +316 to +375 of SCR3, +378 to +432 of SCR2 and +446 to +490 of SCRl.
  • a "joint" comprises the amino acid sequences between and (in the cases of SCR8 and SCRl) outside the loops.
  • each loop is bonded to another loop via a joint.
  • SCRs having joints are preferable to those that do not have joints because it is unlikely that two loops bonded without a joint will be as flexible as those bonded through a joint.
  • an SCR comprises the amino acid sequence of the SCR domains defined above. It should be understood that one could make minor alterations in the ammo acid sequence of an SCR, for example by adding a few amino acids to the short loops of SCRl and SCR8.
  • the C4bp monomers of this invention include any sequence of SCRs, including SCRs strung together at random. However, it is an object of this invention to produce proteins least likely to evoke an immune response against the C4bp monomer. Therefore, more preferably, the amino acid sequence of the C4bp monomer corresponds to at least a fragment of the amino acid sequence of mature_ * C4bp, which is not normally immunogenic.
  • the C4bp monomer, C4bp(SCR8) corresponds to the mature C4bp polypeptide.
  • C4bp(SCR4) corresponds to amino acids +248 to +549 of Figure 1.
  • C4bp(SCRl) corresponds to amino acids +433 to +549 of Figure 1.
  • the C4bp monomer, C4bp(SCR4) is most preferable.
  • C4bp monomers include variable numbers of SCRs. At a minimum, there may be no SCRs. At a maximum, C4bp monomers' may contain about 32 SCRs, about as many as the longest known repeating unit molecule, CR1, which has 30 domains [L.B. Vogelstein, "Isolation of NH2-terminal CR1 (CD35) Clones and Expression of Recombinant Human GR1", FASEB J.. 2 , p. A1832 #8921 (1988) ].
  • a C4bp monomer containing more than eight SCRs corresponds more preferably to the amino acid sequence of mature C4bp fused to at least a fragment of the same.
  • a sixteen SCR monomer may comprise SCR8-SCR1 fused to SCR8-SCR1.
  • DNA sequences encoding C4bp monomers are derived from DNA sequences encoding C4bp.
  • Several methods are available to obtain these DNA sequences.
  • Chung et al. identifies those codons as GGT, TGC and GAA.
  • a cDNA sequence encoding the C4bp polypeptide by screening a cDNA library.
  • Many screening methods are known to those of skill in the art. For example, one can screen colonies by nucleic acid hybridization with oligonucleotide probes. Probes can be prepared by chemically synthesizing an oligonucleotide having part of the known DNA sequence of C4bp.
  • probes can be prepared by chemically synthesizing an oligonucleotide having part of the known DNA sequence of C4bp.
  • cDNA libraries in expression vectors, such as ⁇ gtll and screen the colonies with anti-hC4bp antibodies.
  • DNA sequence encoding the C4bp monomer may then be fused to a DNA sequence encoding a functional moiety, such as a polypeptide moiety.
  • DNA sequences for polypeptides useful in this invention are available from many sources. These include DNA sequences described in the literature and DNA sequences encoding particular polypeptides obtained by any of conventional molecular cloning techniques.
  • This invention also contemplates non-human C4bp fusion proteins comprising non-human C4bp fusion polypeptides.
  • the C4bp monomers comprise C4bp cores and SCRs derived from the amino acid sequence of a non-human C4bp. Any non-human C4bp having monomeric units that assemble into a multimer are useful for this purpose.
  • Such C4bp multimers exist in the guinea pig and mouse [Lintin et al. , supra] .
  • Mouse C4bp is preferable, because its amino acid sequence is known to contain contiguous SCRs.
  • polypeptides are useful to produce the C4bp fusion proteins or fusion polypeptides of this invention. Those most useful include polypeptides that are advantageously administered in multimeric form.
  • viral receptors or cell receptors or ligands are useful, because they typically bind to particles or cells exhibiting many copies of the receptor. Fusion proteins containing these polypeptides are useful in therapies that involve inhibiting viral-cell or cell-cell binding.
  • Useful viral-cell receptors include ICAM1, a rhinovirus receptor; the polio virus receptor [J. White and D. Littman, "Viral Receptors of the Immunoglobulin Superfamily", Cell. 56, pp. 725-28 (1989)] and, most preferably, CD4, the HIV receptor.
  • Cell-cell receptors or ligands include members of the vascular cell adhesion molecule family, such as ICAM1, ⁇ LAM1, and VCAM1 and VCAMlb and their lymphocyte counterparts (ligands) ; the lymphocyte associated antigens, LFA1, LFA2 (CD2) and LFA3, members of the CD11/CD18 family, and VLA4. These molecules are involved in pathologic inflammation [M.P. Bevilacqua et al., "Identification of an Inducible Endothelial-leukocyte Adhesion Molecule", Proc. Natl. Acad. Sci. , USA. 84. pp. 9238- 42 (1987); L.
  • Bacterial immunogens, parasitic " immunogens and viral immunogens are useful as polypeptide moieties to create multimeric or hetero-multimeric C4bp fusion proteins useful as vaccines.
  • Bacterial sources of these immunogens include those responsible for bacterial pneumonia and pneu ocystis pneumonia.
  • Parasitic sources include malarial parasites, such as Plasmodium.
  • Viral sources include poxviruses, e.g., cowpox virus and orf virus; herpes viruses, e.g., herpes simplex virus type 1 and 2, B-virus, varicella- zoster virus, cytomeg lovirus, and Epstein-Barr virus; adenoviruses, e.g., mastadenovirus; papovaviruses, e.g., papillomaviruses, and polyomaviruses such as BK and JC virus; parvoviruses, e.g., adeno-associated virus; reoviruses, e.g., reoviruses 1, 2 and 3; orbiviruses, e.g., Colorado tick fever; rotaviruses, e.g., human rotaviruses; alphaviruses, e.g., Eastern encephalitis virus and Venezuelan encephalitis virus; rubiviruses, e.
  • this invention provides C4bp fusion polypeptides comprising a polypeptide moiety comprising viral polypeptides having hepatitis B virus e antigenicity.
  • a DNA sequence encoding hepatitis B virus e antigens (“HBeAg") is described in L. Mimms et al., "Production, Purification, and Immunological Characterization of a Recombinant DNA- derived Hepatitis B e Antigen" , Viral Hepatitis and Liver Disease, pp. 248-251 Alan R. Liss, Inc. (1988) .
  • the amino acids encoded by this sequence correspond to the amino-terminal 144 amino acids of Hepatitis B Virus core antigen ("HBcAg") , (subtype adw.) .
  • a DNA sequence encoding HBeAg includes the sequence corresponding to amino' a ⁇ ids 1 to 144 of HBcAg, as set forth in M. Pasek et al., "Hepatitis B Virus Genes and Their Expression in E_ coli", Nature, 282. pp. 575-579 (1979) .
  • a DNA sequence which encodes HBeAg may also be obtained according to the processes set forth in Murray et al., U.S. patent 4/758,507. We shall refer herein to a DNA sequence encoding, or a polypeptide having, HBeAg amino acids numbers 2 (Asp) to X as "HBeAg(2- X)".
  • Immunoglobulin molecules are bivalent, but immunoglobulin-C4bp fusion proteins will be multivalent and may demonstrate increased affinity or avidity for the target. It has been demonstrated that single domain antibodies (dAbs) are useful [E.S. Ward et al., "Binding Activities of a Repertoire of Single Immunoglobulin Variable Domains Secreted from Escherichia coli.” Nature, 341. pp. 544-46 (1989)]. One can generate monoclonal Fab fragments recognizing specific antigens using the technique of W.D. Huse et al.
  • Multimeric C4bp fusion proteins with hirudin, C-terminal hirudin peptides (described in PCT patent application WO 90/03391, incorporated herein by reference) and molecules based on hirudin structure (i.e., hirulogs, described in U.S. patent application 549,388, filed July 6, 1990, incorporated herein by reference) may display greater anti-coagulant activity than monomers of these polypeptides.
  • polypeptides such as cytokines, including the various IFN- ⁇ 's, particularly ⁇ 2, ⁇ 5, 08, IFN- ⁇ and IFN- , the various interleukins, including IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7 and Hi-8 and the tumor " necrosis factors, TNF- ⁇ , and ⁇ .
  • functional moieties include, for example-,' monocyte colony stimulating factor (M-CSF) , granulocyte colony stimulating factor (G-CSF * ', g anulocyte macrophage colony stimulating factor (GM-CSF) , erythropoietin, platelet-derived growth factor (PDGF) and human and animal hormones, including growth hormones and insulin.
  • M-CSF monocyte colony stimulating factor
  • G-CSF * ' granulocyte colony stimulating factor
  • GM-CSF granulocyte colony stimulating factor
  • GM-CSF granulocyte colony stimulating factor
  • PDGF platelet-derived growth factor
  • multimeric C4bp fusion proteins comprise CD4-C4bp fusion polypeptides.
  • CD4 is the receptor on those white blood cells, T-lymphocytes, which recognize HIV, the causative agent of AIDS and ARC [P.J.
  • CD4 recognizes the HIV vi aL' surface protein, gpl20/160.
  • the functional moiety is a polypeptide moiety comprising CD4 or a fragment thereof, preferably soluble CD4.
  • T Cell Surface Protein T4 A New Member of the Immunoglobulin Gene Family", Cell,, 42, pp. 93- 104 (1985) ; D.Rl Littman et all, "Corrected CD4 Sequence", Cell, 55. p. 541 (1988)]. Base upon its deduced primary structure, the CD4 protein is divided into functional domains as follows:
  • the first immunoglobulin-related domain can be further resolved into a variable-related (V) region and joint- related (J) region, beginning at about amino acid +95.
  • V variable-related
  • J joint- related
  • FIG. 4 depicts the domain structure of the human CD4 protein of Figures 3A-3B. - 19 -
  • Soluble CD4 proteins have been constructed by truncating the full length CD4 protein at amino acid +375, to eliminate the transmembrane and cytoplasmic domains. Such proteins have been produced by recombinant DNA techniques and are referred to as recombinant soluble CD4 (rsCD4) [R.A. Fisher et al., "HIV Infection Is Blocked In Vitro by Recombinant Soluble CD4", Nature..331. pp. 76-78 (1988); Fisher et al., PCT patent application WO 89/01940 (incorporated herein by reference)].
  • soluble CD4 proteins advantageously interfere with the CD4 + lymphocyte/HIV interaction by blocking or competitive binding mechanisms which inhibit HIV infection of cells expressing the CD4 protein.
  • the first immunoglobulin- related domain is sufficient -to bind gpl20/160.
  • soluble CD4 proteins are useful as antiviral therapeutics to inhibit HIV binding to CD4 + lymphocytes and virally induced syncytia formation.
  • the CD4 polypeptides useful in this invention include all CD4 polypeptides which bind to or otherwise inhibit gpl20/160 ⁇ These include fragments of CD4 lacking the transmembrane domain, amino acids +371 to +391 of Figures 3A-3B.
  • Such fragments may be truncated forms of CD4 or may be fusion proteins in which the fourth immunoglobulin-related domain is joined directly to the hydrophilic cytoplasmic domain.
  • soluble CD4 proteins preferably will contain enough of a domain to allow an intra-domain disulfide bond but not enough to include the first cysteine of the next iiif_munoglobulin domain. Within this range, certain amino acid sequences bind gpl60/120 with greater affinity than others.
  • CD4(X) a CD4 polypeptide Which includes amino acids +1 to +X of Figures 3A-3B, and optionally including an N-terminal methionine
  • a soluble CD4 protein containing the first immunoglobulin-like domain preferably will contain at least amino acids +1 to +84 and at most amino acids +1 to +129. Most preferably, a soluble CD4 protein comprises amino acids +1 to +111 [CD4(111)].
  • a soluble CD4 protein containing the first two immunoglobulin- like domains preferably will include at least amino acids +1 to +159 and at most amino acids +1 to +302. More preferably, a soluble CD4 protein will include at least amino acids +1 to +175 and at most amino acids +1 to +190.
  • a soluble CD4 protein will include amino acids +1 to +181 [CD4(181)], amino acids +1 to +183 [CD4(183)], or amino acids +1 to +187 [CD4(187)].
  • a soluble CD4 protein which includes the first four immunoglobulin-like domains preferably will include at least amino acids +1 to +345 and at most amino acids +1 to +375 [CD4(375)]. Any of these molecules may optionally include the CD4 signal sequence, amino acids -25 to -1 of Figures 3A-3B. Also, these molecules may have a methionine residue optionally preceding amino acid +1 of Figures 3A-3B.
  • Soluble CD4 proteins useful in the fusion polypeptides and methods of this invention may be produced in a variety of ways.
  • the amino terminal amino acid of mature CD4 protein isolated from T cells is lysine, located at nucleotide 136 of Figure 3 [D.R. Littman et al., supra1.
  • Soluble CD4 proteins also include those in which amino acid +62 is arginine, encoded by CGG, and those in which amino acid +229 is phenylalanine, encoded by TTT. Therefore, when we refer to CD4, we intend to include amino acid sequences including one or both of these substitutions.
  • Soluble CD4 polypeptides may be produced by conventional techniques of oligonucleotide directed mutagenesis and restriction digestion, followed by insertion of linkers, or by digesting full-length CD4 protein with enzymes.
  • Soluble CD4 proteins include those produced by recombinant techniques according to the processes set forth in copending, * commonly assigned United States patent applications Serial No. 094,322, filed September 4, 1987, Serial No. 141,649, filed January 7, 1988 and Serial No.
  • EC100 E.COli JM83/pEC100 - IVI 10146 BG377: E.coli MC1061/pBG377 - IVI 10147 BG380: E.coli MC1061/pBG380 - IVI 10148 BG381: E.coli MC1061/pBG381 - IVI 10149.
  • Such microorganisms and recombinant DNA molecules are also exemplified by cultures deposited in the In Vitro International, Inc. culture collection on January 6, 1988 and identified as: BG-391: E.COli MC1061/pBG391 - IVI 10151 BG-392: E.COli MC1061/pBG392 - IVI 10152 BG-393: E.coli MC1061/pBG393 - IVI 10153 BG-394: E.coli MC1061/pBG394 - IVI 10154 BG-396: E.COli MC1061/pBG396 - IVI 10155 203-5 : E.COli SG936/p203-5 - IVI 10156.
  • Multimeric C4bp fusion proteins comprising CD4-C4bp fusion polypeptides are useful in a variety of pharmaceutical compositions and methods.
  • CD4-C4bp fusion proteins advantageously inhibit HIV binding to T4 + lymphocytes by virtue of their competitive binding characteristics.
  • the CD4-C4bp fusion proteins may be used in pharmaceutical compositions and methods to treat humans having AIDS, ARC, HIV infection, or antibodies to HIV. They are also useful to lessen the immuno-compromising effects of HIV infection or to prevent incidence and spread of HIV infection.
  • these CD4-C4bp fusion proteins and methods may be used for treating AIDS-like diseases caused by retroviruses, such as simian immunodeficiency viruses, in mammals, including humans.
  • DNA sequences encoding C4bp fusion polypeptides are useful for producing multimeric C4bp fusion proteins. The preferred process involves expressing such DNA sequences in a host that will properly assemble the expressed polypeptides into a multimer. - 23 -
  • the DNA sequence should be operatively linked to an expression control sequence in an appropriate expression vector and employed in that expression vector to transform an appropriate unicellular host.
  • Such operative linking of a DNA sequence of this invention to an expression control sequence includes the provision of a translation start signal in the correct reading frame upstream of the DNA sequence. If-a particular DNA sequence being expressed does not begin with a methionine, the start signal will result in an additional amino acid — methionine — being located at the N-terminus of the.product. While such a methionyl- containing product may be employed directly in the compositions and method ' s of this invention, it is usually more desirable ⁇ to remove the methionine before use.
  • useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known derivatives of SV40 and known bacterial plasmids, e.g., plasmids from E.coli including colEl, pCRl, pBR322, pMB9 and their derivatives, wider host range plasmids, e.g., RP4; phage DNAs, e.g., the numerous derivatives of phage ⁇ , e.g., NM989, and other DNA phages, e.g., M13 and filamentous single stranded DNA phages; yeast plasmids, such as the 2 ⁇ plasmid or derivatives thereof; and vectors derived from combinations of plasmids and phage DNAs, such as plasmids which have been modified to employ
  • any of a wide variety of expression control sequences sequences that control the expression of a DNA sequence when operatively linked to it — may be used in these vectors to express the DNA sequences of this invention.
  • useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3- phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast ⁇ -mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • a wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention.
  • These hosts include well known eukaryotic and prokaryotic hosts, such as strains of E.coli. Pseudomonas. Bacillus, Streptomyces. fungi, such as yeasts, and animal cells, such as CHO and mouse cells, African green monkey cells, such as COS-1, COS-7, BSC 1, BSC 40, and BMT 10, insect cells, and human cells and plant cells in tissue culture.
  • animal cell expression we prefer CHO cells and COS-7 cells. It should of course be understood that not all vectors and expression control sequences will function equally well to express the DNA sequences of this invention.
  • an expression control sequence a variety of factors should also be considered. These include, for example, the relative strength of the system, its controllability and its compatibility with the particular DNA sequence of this invention, particularly as regard ⁇ potential secondary structures. Unicellular hosts should!be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for on expression by the DNA sequences of this-.invention to them, their secretion characteristics, their ability to fold proteins correctly, their fermentation requirements and the ease of purification of the products coded on expression by the DNA -sequences of this invention.
  • vector/expression control system/host combinations that will express the DNA sequences of this invention on fermentation or in large scale animal culture, e.g., CHO cells or COS-7 cells.
  • a DNA sequence encoding a CD4-C4bp fusion polypeptide inserted into plasmid pJOD-S (described herein) and expressed in COS-7 or CHO cells produces fusion polypeptides which naturally assemble into heptameric CD4-C4bp fusion proteins.
  • polypeptides and proteins produced on expression of the DNA sequences of this invention may be isolated from fermentation or animal cell cultures and purified using any of a variety of conventional methods. One of skill in the art may select the most appropriate isolation and purification techniques without departing from the scope of this invention.
  • C4bp fusion polypeptides by chemical synthesis using conventional peptide synthesis techniques, such as solid phase synthesis [R.B. Merrifield, "Solid Phase Peptide Synthesis. I. The Synthesis Of A Tetrapeptide", J. Am. Chem. Soc.. 83. pp. 2149-54 (1963)].
  • Multimeric C4bp fusion proteins may then be produced in vitro by forming intra- and inter-C4bp fusion polypeptide disulfide bonds.
  • Another method useful for producing multimeric C4bp fusion proteins, in addition to genetic fusion and chemical synthesis is by chemically coupling a functional moiety to the C4bp monomer. This method is useful for both chemical moieties or polypeptide moieties.
  • One may couple the functional moiety to individual C4bp monomers or to C4bp monomers already assembled into a multimer, for example, hC4bp itself or multimeric recombinant hC4bp.
  • Hetero-multimeric C4bp fusion proteins comprise combinations of different C4bp monomers, different functional moieties, or combinations of both.
  • hetero-multimeric C4bp fusion proteins may comprise combinations of more than one C4bp monomer (i.e., C4bp.SCR4 and C4bp.SCR8) with one type of functional moiety, one type of C4bp monomer with combinations of more than one type of functional moiety (i.e., a recognition molecule and a reporter group), combinations of more than one type of C4bp monomer with combinations of more than one type of functional moiety and combinations in which not all of the C4bp monomers are fused or chemically coupled to functional moieties.
  • a hetero-multimeric C4bp fusion protein comprising two different" polypeptide moieties may advantageously be produced by expressing DNA sequences encoding the two different polypeptides in a single host. Upon expression in an appropriate system, the polypeptides will assemble into multimeric fusion proteins containing more than one type of functional moiety.
  • hetero-multimers characterized by polypeptide and chemical moieties, or two different chemical moieties may also be produced.
  • a moiety may be chemically coupled to polypeptides before or after assembly.
  • Hetero-multimeric C4bp fusion proteins are especially useful when the properties of the different moieties complement one another.
  • Polypeptides useful as toxins include, but are not limited to, ricin, abrin, angiogenin, Pseudomonas Exo- toxin A, pokeweed antiviral protein, saporin, gelonin and diptheria toxin, or toxic portions thereof.
  • Useful anti-retroviral agents include suramin, azidothymidine (AZT) , dideoxycytidine and glucosidase inhibitors such as castanospermine, deoxynojirimycin and derivatives thereof.
  • Hetero-multimeric C4bp fusion proteins according to this invention are also useful as diagnostic agents to identify the presence of a target molecule in a sample or in vivo.
  • Such proteins comprise one f nctional moiety which is a recognition molecule, such as an immunoglobulin or a fragment thereof (Fab, dAb) that binds to the target molecule
  • reporter group such as a radionuclide, an enzyme (such as horseradish peroxidase) or a fluorescent or chemiluminescent marker.
  • a reporter group such as a radionuclide, an enzyme (such as horseradish peroxidase) or a fluorescent or chemiluminescent marker.
  • Hetero-multimeric C4bp fusion proteins according to this invention may also be used as multi- vaccines.
  • such fusion proteins may be constructed using several different antigenic determinants from the same infective agent.
  • Multimeric C4bp fusion proteins according to this invention also include the normally associated protein S-binding subunit of human C4bp. Such proteins are produced upon transformation of a host with a first DNA sequence encoding a C4bp fusion polypeptide and a second DNA sequence encoding the protein S-binding subunit. Upon expression of these DNA sequences, the C4bp polypeptides will assemble into a multimer associated with the protein S-binding subunit.
  • compositions of this invention typically comprise a pharmaceutically effective amount of a C4bp fusion protein of this invention and a pharmaceutically acceptable carrier.
  • Therapeutic methods of this invention comprise the step of treating patients in a pharmaceutically acceptable manner with those compositions. These compositions may be used to treat any mammal, including humans.
  • compositions of this invention may be in a variety of forms. These include, for example, solid, semi-solid and liquid dosage forms, such as tablets, pills, powders, liquid solutions or suspensions, liposomes, suppositories, injectable and infusable solutions and sustained release forms. The preferred form depends on the intended mode of administration and therapeutic application.
  • the compositions also preferably include conventional pharmaceutically acceptable carriers and adjuvants which are known to those of skill in the art.
  • the pharmaceutical compositions of the present invention may be formulated and administered using methods and compositions similar to those used for pharmaceutically important polypeptides such as, for example, alpha interferon.
  • the fusion proteins of this invention may be stored in lyophilized form, reconstituted with sterile water just prior to administration, and administered by conventional routes of administration such as parenteral, subcutaneous, intravenous, intramuscular or intralesional routes.
  • An effective dosage may be in the range of about 10-100 ⁇ g/kg body weight/day, it being recognized that lower and higher doses may also be useful. It will be understood that conventional doses will vary depending upon the particular molecular moiety involved.
  • DNA sequences encoding C4bp fusion polypeptides in somatic gene therapy. This involves, for example, inserting DNA sequences into retroviral-based vectors suitable for - 31 -
  • patients with AIDS or ARC could be treated as follows. First, one would prepare a retrov ⁇ rus characterized by a DNA sequence encoding a CD4-C4bp fusion polypeptide. Then, one would isolate T cells from the patient and infect them, in vitro. with the retrovirus. One wduld then reintroduce these cells into the patient, where the vector will express and the cell will secrete CD4-C4bp polypeptide.
  • pJOD-10 As described in European patent application 343,783, pJOD-10 may be prepared as follows. Plasmid pSV2-DHFR, (ATCC 37146, from the American Type Culture Collection) [S. Subra ani et al., "Expression of the Mouse Dihydrofolate Reductase Complementary Deoxyribonucleic Acid in Simian Virus 40 Vectors", Molec. Cell. Biol. f 1, pp. 854-64 (1981)] was digested with Apal and EcoRI and the 4420 bp fragment was isolated. Then, a synthetic double stranded DNA sequence having an Apal overhang, a DNA sequence encoding nucleotides +190 to +233 of the human gastrin gene [K.
  • plasmids containing the insert in the proper orientation by hybridizing with 32 P-labelled synthetic oligonucleotide splint probes C4bp.9 and C4bp.l0.
  • Splint probes are 30 base long synthetic oligonucleotides that hybridize across the point of fusion between an insert and a vector.
  • pJOD.C4bp We have deposited one isolate of this plasmid, pJ0D.C4bp.3.
  • Recombinant human C4bp differs from the serum form in that it lacks a protein S-binding subunit.
  • 530 kD representing a C4bp heptamer bound to the 45 kD protein S-binding subunit.
  • the rhC4bp produced a band of about 490 kD, the predicted molecular weight of a heptameric C4bp protein not including the protein S-binding subunit.
  • HRP horseradish peroxidase
  • COS-7 cells was present as a heptamer exposing correctly folded immune epitopes.
  • the elution profile of hC4bp peaked soifiewhat higher than 700 kD, in contrast to the expected 530 k . This may have resulted from non-cova ⁇ ent binding of the molecule to its natural ligands, C4 and protein S.
  • Double stranded Xhol linkers having the sequence 5' CCTCGAGG were ligated to the ends with T4 DNA ligase.
  • the mixture was digested with Xhol and the 1407 bp fragment encoding rsCD4 was isolated.
  • pJOD-Sjj 'described in Example I was digested with Sail and phosphorylated with alkaline phosphatase.
  • the resulting 1407 bp fragment was ligated into the Sail-digested pJOD-S with T4 DNA ligase. This produced pJ0DsCD4.
  • P-labelled oligonucleotide splint probe 312.36 We named such plasmids pJOD.sCD4.Y187.SCR5.
  • pJOD.sCD4.Y187.SCR5. We called the fusion polypeptide expressed by such plasmids, CD4(187)-C4bp(SCR5) .
  • CD4(187)-C4bp(SCR5) We obtained two isolates of those plasmids, pJOD.sGD4.Y187.SCR5.1 and pJOD.SCD4.Y187.SCR5.2.
  • CD4(187)-C4bp(SCRl) We called the fusion polypeptide expressed by such plasmids, CD4(187)-C4bp(SCRl) .
  • CD4(187)-C4bp(SCRl) We obtained three isolates of those plasmids, pJ0D.sCD4.Y187.SCRl.1, pJOD.sCD4.Y187.SCRl.2 and pJ0D.sCD4.Y187.SCR1.3.
  • C4bp(SCR4) by both immunodetection on Western blots and by ELISA assay.
  • Immunodetection of the conditioned-media on Western blot confirmed that the transformed cells had produced CD4(187)-C4bp(SCR4) and further indicated that the polypeptides had assembled into heptamers.
  • 6C6 antibody 6C6 antibody.
  • anti-hC4bp,* also detected a high molecular weight form of protein.
  • the anti-h"CD4 monoclonal failed to detect any protein in the control samples.
  • CD4(187)-C4bp(SCR4) >actually had been expressed as a fusion polypeptide and'assembled to a heptameric form.
  • CD4(187)-C4bp(SCR4) which had been expressed in COS-7 cells in the presence of 35 S-labelled cysteine (New England Nuclear) .
  • CD4 (187)-C4bp(SCR4) using conventional column ctiromatography techniques.
  • the CD4-C4bp fusion protein may be partially purified on FAST S® alone.
  • FAST S® chromatography we adjusted the conditioned medium to pH 8.0 with sodium hydroxide and filtered it through a 5 ⁇ m prefilter and a 0.45 ⁇ m filter.
  • Zn chelate columns (40 ml, ID 25 mm) sequentially. We carried out all procedures on these columns at 4°C and at a flow rate of 0.4 ml/cm 2 hr. Each wash, as we describe, was carried out with 80 ml of buffer.
  • We prepared the Cu chelate column by loading chelating SEPHAROSE® with Cu ions using an aqueous 50 mM CuCl solution. We washed the column twice, first with 500 mM Tris, 500 mM NaCl, pH 8.0, followed by with 10 mM Tris, 500 mM NaCl, pH 8.0. Then we loaded the eluate from the FAST Q® column on the Cu chelate column.
  • CD4(187)- C4bp(SCR4) we then examined the purified CD4(187)- C4bp(SCR4) by electron microscopy. To do this, we mixed the CD4-C4bp fusion protein with glycerol, sprayed it on a carbon-coated grid and rotary shadowed it with platinum using conventional techniques. At high magnification, the molecule appeared to have a "spider-like" shape, with multiple rod-like arms extending from its center.
  • CD4(187)-C4bp(SCR4) As an alternative to the column chromatography purification described above, we also purified CD4(187)-C4bp(SCR4) as follows.
  • 1D7 is a monoclonal antibody that binds to the second immunoglobulin-related domain of CD4 (1D7 was a gift from Patricia Chisholm of Biogen, Inc.).
  • C4bp(SCR4) to bind to gpl20 of the HIV virus by means of two types of assays: ELISA assays and a syncytia blocking assay.
  • ELISA assay We performed an ELISA assay (ELISA 3) that demonstrated the ability of CD4(187)-C4bp(SCR4) to bind gpl20.
  • ELISA 3 ELISA assay
  • ELISA 3 ELISA assay
  • gpl20 was coated Immulon II plates with gpl20 by adding 50 ⁇ l/well of a 5 ⁇ g/ml solution of gpl20 (commercially available from American Bio- Technologies, Inc., Cambridge, Massachusetts) in PBS and incubating the plates overnight at 4°C. After removing the coating solution, we blocked non-specific binding by adding 200 ⁇ l/well of 2% non-fat dry milk in PBS and incubated the plates for at least 30 minutes at room temperature.
  • HIV-infected cells which express gpl20 on their surface, fuse with CD4-expressing cells to form multinucleate cells (syncytia) .
  • Molecules that bind to gpl20 tend to block ⁇ the formation of syncytia.
  • CD4(187)-C4bp(SCR4) was tested the ability of CD4(187)-C4bp(SCR4) to block HIV-1 replication in vitro in a microreplication assay, essentially as described in M. Robert-Guroff et al.. Nature, 316. pp. 72-7.4 (1985), however we performed the incubation at 37°C rather than 4°C as described therein.
  • CD4(187)-C4bp(SCR4) two different preparations of CD4(187)-C4bp(SCR4) .
  • One preparation was conditioned cell culture fluid from a stable CHO cell line (Example V, section B, infra) which synthesizes and secretes CD4(187)-C4bp(SCR4) .
  • the other preparation was CD4(187)-C4bp(SCR4) , partially purified from conditioned culture fluid from a transformed CHO cell line on a FAST S® column. After infection at 37°C for 30 minutes, we added 15 ⁇ l aliquots in triplicate to 200 ⁇ l of RPMI-20% FCS in microtiter plates. We incubated the plates at 37°C in 5% CO and examined them 4 to 8 days later for syncytia formation, a signal for active infection. 55 -
  • multimeric CD4(187)-C4bp(SCR4) blocked HIV-1 infection at a concentration 100 times less than the concentration of recombinant soluble CD4 necessary to block HIV-1 infection.
  • 300 pM CD4(187)- C4bp(SCR4) both partially purified and from cell culture medium
  • about 30 nM recombinant soluble CD4 was necessary to obtain the same result.
  • There was a falloff in protection against HIV-1 infection of C8166 cells at about 100 pM for CD4(187)-C4bp(SCR4) and about 10 nM for recombinant soluble CD4.
  • Recombinant HIV-1 was obtained by transfecting colon carcinoma cell line SW480 with CaP0 4 -precipitated pNL4-3. (Both the cell line and the plasmid are available from the AIDS Research and Reference Reagent Program, NIH, Bethesda, Maryland.)
  • CD4(187)-C4bp(SCR5) , CD4(187)-C4bp(SCR3) AND CD4(187)-C4bpfSCRl) We performed several ELISA assays to test for the production of CD4(187)-C4bp(SCR8) , CD4(187)- C4bp(SCR5), CD4(187)-C4bp(SCR3) and CD4(187)- C4bp(SCRl).
  • ELISA 5 we carried out the fifth assay (ELISA 5) in the same way as ELISA 4, except that we used the antibody 5A8 as the detection antibody. Antibody 5A8 does not block CD4 binding to gpl20 and it recognizes domain 2 of CD4 (see Figure 4) . Another monoclonal antibody having such characteristics might also be useful in, this assay.
  • ELISA 6 we performed the sixth assay (ELISA 6) as in ELISA 3, except that we used the anti-C4bp onoclonals 051-198 or 051-28 (Quidel, San Diego, California). In ELISA 5 (plate coated with gpl20, 5A8 used as detection antibody) all three isolates gave positive results. This indicates that the cells produced a protein comprising CD4( 87).
  • CD4(187)-C4bp(SCRl) can assemble into a multimer.
  • ELISA 7 plates coated with 1D7, 5A8 used as detection antibody
  • conditioned medium from COS-7 cells transformed with pJOD.sCD4.Y187.SCR5.1, pJOD.SCD4.Y187.SCR5.2, pJOD.CD4.Y187.SCR3.2 and pJOD.sCD4.Y187.SCR3.3. All assays gave a strong positive result.
  • HBeAg-C4bp hepatitis B virus e antigen-C4bp
  • pJOD.C4bp.2 an isolate of pJ0D.C4bp (Example I) between the DNA sequences encoding the C4bp signal sequence and the amino terminus of SCR8 (amino acid +1 of Figure 1) . This created a site into which we could insert DNA sequences encoding HBeAg epitopes.
  • PCR fragments encoding various HBeAg sequences were inserted into the Xbal site to create polypeptides in which HBeAg sequences were sandwiched between the DNA - 59 -
  • a unique Xbal" site into pJOD.C4bp.2 via gapped mutagenesis as follows.
  • the oligomer had the sequence:
  • Plasmid 8.1.5 is characterized by a DNA sequence encoding HBeAg. It is also referred to as pHBV139A fPasek et al.. supra] . (Plasmid 8.1.5 was a gift of Professor Kenneth Murray, University of Edinburgh, Scotland) .
  • Plasmid pAMG ATCC 45020, as a PCR template. Plasmid pAMG is characterized by a DNA sequence derived from HBV subculture ADW and encodes HBeAg.
  • plasmids contained DNA sequences encoding the following fusion polypeptides: HBeAg(2-148)- C4bp(SCR8); HBeAg(2-138)-C4bp(SCR8) ; HBeAg(2-100)- C4bp(SCR8) and HBeAg(2-89)-C4bp(SCR8) , respectively.
  • constructs may be altered by replacing the C4bp signal sequence with the hepatitis B virus precore signal sequence to insure proper processing of the primary translation product.
  • Microorganism ⁇ and recombinant DNA molecules according to this ihvenH_ion are exemplified by cultures deposited in the In Vitro international, Inc. culture collection, in Linthicum, Maryland, USA on January 24, 1990, and identified as:

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Abstract

Protéines de fusion (C4bp) de protéines liantes C4 multimériques et hétéro-multimériques et compositions et procédés utilisant lesdites protéines. La présente invention concerne plus particulièrement des protéines de fusion C4bp multimériques qui sont des agrégats ou des assemblages de monomères C4bp liées à des fractions fonctionnelles. Elle concerne également des polypeptides de fusion C4bp et en particulier des polypeptides de fusion CD4-C4bp comprenant une séquence d'acides aminés d'une protéine CD4 humaine soluble enchaînée à un monomère C4bp ayant, de préférence, quatre régions de répétition de consensus court.
PCT/US1991/000567 1990-01-26 1991-01-28 Proteines de fusion de proteines liantes c4 WO1991011461A1 (fr)

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Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994022477A1 (fr) * 1993-03-26 1994-10-13 Progenics Pharmaceuticals, Inc. Vaccins derives de vih-1, compositions d'anticorps correspondantes et leur utilisation therapeutique et prophylactique
EP0723555A1 (fr) * 1993-09-24 1996-07-31 Cytomed, Inc. Proteines chimeriques avec activation de complement en bloc
US5547853A (en) * 1991-03-12 1996-08-20 Biogen, Inc. CD2-binding domain of lymphocyte function associated antigen 3
FR2736916A1 (fr) * 1995-07-21 1997-01-24 Univ Paris Curie Proteines hetero-multimeriques recombinantes du type alpha-beta c4bp
US5928643A (en) * 1991-03-12 1999-07-27 Biogen, Inc. Method of using CD2-binding domain of lymphocyte function associated antigen 3 to initiate T cell activation
WO1999054954A2 (fr) * 1998-04-15 1999-10-28 Wolfgang Bergter Substances pharmaceutiques radio-immunologiques a base de cd4 destinees au traitement de l'infection par le vih
WO2000069907A1 (fr) * 1999-05-14 2000-11-23 Medical Research Council Echafaudage proteinique interne et utilisation de ce dernier pour multimeriser des polypeptides monomeres
US6551595B1 (en) 1998-03-03 2003-04-22 The Trustees Of The University Of Pennsylvania Smallpox inhibitor of complement enzymes (SPICE) protein and methods of inhibiting complement activation
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EP1795540A1 (fr) 2005-11-30 2007-06-13 Imaxio Complexes multiples d'antigènes et d'un adjuvant
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US10815290B2 (en) 2015-11-10 2020-10-27 Fred Hutchinson Cancer Research Center NKG2D decoys
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US5547853A (en) * 1991-03-12 1996-08-20 Biogen, Inc. CD2-binding domain of lymphocyte function associated antigen 3
US5728677A (en) * 1991-03-12 1998-03-17 Biogen, Inc. Methods of inhibiting T-cell dependent proliferation of peripheral blood lymphocytes using the CD2-binding domain of lymphocyte function associated antigen 3
US5914111A (en) * 1991-03-12 1999-06-22 Biogen Inc. CD2-binding domain of lymphocyte function associated antigen-3
US7323171B2 (en) 1991-10-07 2008-01-29 Astellas Us Llc Methods of treating skin conditions using inhibitors of the CD2/LFA-3 interaction
WO1994022477A1 (fr) * 1993-03-26 1994-10-13 Progenics Pharmaceuticals, Inc. Vaccins derives de vih-1, compositions d'anticorps correspondantes et leur utilisation therapeutique et prophylactique
US5886163A (en) * 1993-03-26 1999-03-23 Progenics Pharmaceuticals, Inc. HIV-1 antigens, antibody compositions related thereto, and therapeutic and prophylactic uses thereof
EP0723555A1 (fr) * 1993-09-24 1996-07-31 Cytomed, Inc. Proteines chimeriques avec activation de complement en bloc
US5679546A (en) * 1993-09-24 1997-10-21 Cytomed, Inc. Chimeric proteins which block complement activation
US5851528A (en) * 1993-09-24 1998-12-22 Cytomed, Inc. Methods of inhibiting complement activation
EP0723555A4 (fr) * 1993-09-24 1999-04-28 Cytomed Inc Proteines chimeriques avec activation de complement en bloc
WO1997004109A1 (fr) * 1995-07-21 1997-02-06 Universite Pierre Et Marie Curie (Paris Vi) PROTEINES HETERO-MULTIMERIQUES RECOMBINANTES DU TYPE α - β C4BP
FR2736916A1 (fr) * 1995-07-21 1997-01-24 Univ Paris Curie Proteines hetero-multimeriques recombinantes du type alpha-beta c4bp
US7504483B2 (en) 1995-07-21 2009-03-17 Universite Pierre Et Marie Curie (Paris Vi) α-β C4BP-type recombinant heteromultimeric proteins
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WO1999054954A3 (fr) * 1998-04-15 2000-01-20 Wolfgang Bergter Substances pharmaceutiques radio-immunologiques a base de cd4 destinees au traitement de l'infection par le vih
WO2000069907A1 (fr) * 1999-05-14 2000-11-23 Medical Research Council Echafaudage proteinique interne et utilisation de ce dernier pour multimeriser des polypeptides monomeres
US7034132B2 (en) 2001-06-04 2006-04-25 Anderson David W Therapeutic polypeptides, nucleic acids encoding same, and methods of use
US7858095B2 (en) 2001-07-24 2010-12-28 Astellas Us Llc Method for treating or preventing sclerotic disorders using CD-2 binding agents
EP1483397A2 (fr) * 2002-02-11 2004-12-08 The Trustees of The University of Pennsylvania Molecules oligomeriques et leurs applications
EP1483397A4 (fr) * 2002-02-11 2005-06-01 Univ Pennsylvania Molecules oligomeriques et leurs applications
WO2004020639A3 (fr) * 2002-08-14 2004-04-22 Avidis Sa Production de proteines de fusion multimeres utilisant un echafaudage c4bp
WO2004016283A3 (fr) * 2002-08-14 2004-06-03 Avidis Sa Complexes multimeres d'antigenes et d'adjuvants
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WO2005014654A2 (fr) * 2003-08-12 2005-02-17 Avidis Sa Complexes multimeres d'antigenes et d'un adjuvant
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WO2005051414A1 (fr) * 2003-11-26 2005-06-09 Avidis Sa Utilisation de la zone nucleique c4bp comme agoniste de cd40
WO2005077976A3 (fr) * 2004-02-13 2006-03-09 Avidis Sa Domaines bispirales
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US7662921B2 (en) 2004-05-07 2010-02-16 Astellas Us Llc Methods of treating viral disorders
EP1790358A1 (fr) * 2005-11-23 2007-05-30 Université de Reims Champagne-Ardennes Constructions protéiques concues pour cibler et lyser des cellules
US9644033B2 (en) 2005-11-23 2017-05-09 Universite De Reims Champagne Ardenne Protein constructs designed for targeting and lysis of cells
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US9181311B2 (en) 2009-10-16 2015-11-10 Isis Innovation Limited Mycobacterial vaccines
WO2013010998A3 (fr) * 2011-07-15 2013-04-04 Fundació Institut D'investigació Biomèdica De Bellvitge (Idibell) Compositions et procédés d'immunomodulation
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US10342863B2 (en) 2015-03-24 2019-07-09 Fred Hutchinson Cancer Research Center Engineered and multimerized human immunodeficiency virus envelope glycoproteins and uses thereof
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US10815290B2 (en) 2015-11-10 2020-10-27 Fred Hutchinson Cancer Research Center NKG2D decoys
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