WO1991017170A1 - Multimeric gelsolin fusion constructs - Google Patents

Abstract

This invention relates to multimeric and hetero-multimeric gelsolin fusion constructs, compositions containing them and methods using them. More particularly, this invention relates to multimeric gelsolin fusion constructs in which at least two gelsolin fusion polypeptides are bound to vesicles containing polyphosphoinositides. This invention also relates to gelsolin fusion polypeptides which comprise gelsolin moieties linked to functional moieties and in particular, to CD4-gelsolin fusion polypeptides comprising an amino acid sequence for a human CD4 protein linked to a gelsolin moiety.

Description

MULTIMERIC GELSOLIN FUSION CONSTRUCTS

TECHNICAL FIELD OF INVENTION

This invention relates to multimeric and hetero-multi eric gelsolin fusion constructs, compositions containing them and methods using them. More particularly, this invention relates to multimeric gelsolin fusion constructs in which at least two gelsolin fusion polypeptides are bound to vesicles containing polyphosphoinositides. This invention also relates to gelsolin fusion polypeptides wliich comprise gelsolin moieties linked to functional moieties and, in particular, to CD4-gelsolin fusion polypeptides comprising an amino acid seguence for a human CD4 protein linked to a gelsolin moiety.

BACKGROUND ART

The rapid development of biotechnologies has led to novel delivery and carrier systems for pharmaceuticals, vaccines, diagnostics ani 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 serum 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. Scientists are seeking to identify new molecules, including proteins, that they can advantageously develop into these systems.

Gelsolin is a protein found in mammals and other vertebrates [H.L. Yin and T.P. Stossel, "Control of Cytoplasmic Actin Gel-sol Transformation by Gelsolin, a Calcium-dependent Regulatory Protein", Nature. 281. pp. 583-86 (1979); F.S. Southwick and M.J. DiNubile, "Rabbit Alveolar Macrophages Contain a Ca²⁺- sensitive, 41,000-dalton Protein Which Reversibly Blocks the 'Barbed' Ends of Actin Filaments but Does not Sever Them", J. Biol. Chem.. 261. pp. 14191-95 (1986) ; T. Ankenbauer et al. , "Proteins Regulating

Actin Assembly in Oogenesis and Early Embryogenesis of Xenopus laevis; Gelsolin Is the Major Cytoplasmic Actin-binding Protein", J. Cell Biol.. 107. pp. 1489-98 (1988); H.L. Yin et al. , "Identification of Gelsolin, a Ca²⁺-dependent Regulatory Protein of Actin Gel-sol

Transformation and Its Intracellular Distribution in a Variety of Cells and Tissues", J. Cell. Biol.. 91. pp. 901-06 (1980); C.W. Dieffenbach et al., "Cloning of Murine Gelsolin and Its Regulation During Differentiation", J. Biol. Chem.. 264. pp. 13281-88

(1989)]. In mammals, gelsolin occurs in two forms — a cytoplasmic form and a serum form. Gelsolin regulates the activity of actin, a major protein involved in cell structure and movement. Actin is a globular protein with a slightly elongated shape that can polymerize into filaments. Polymerization occurs when the "barbed" end of one actin monomer binds non-covalently and reversibly to the "pointed" end of another. Inside most cells, monomers and short filaments exist in a fluid-like "sol" state until the monomers are activated to polymerize into filaments and the filaments, in turn, are activated to crosslink, producing a firmer "gel" phase that forms part of the cellular cytoskeleton. Investigators have observed that in the presence of calcium ion, gelsolin prevents the transition of monomers and filaments from gel phase to sol phase.

Gelsolin acts on actin in three ways. First, it severs the noncovalent bonds between the actin monomers that compose actin filaments ("severing") . Second, it binds to the barbed end of actin filaments and prevents elongation of the filament from that end ("capping") . Third, it binds to actin monomers and promotes the formation of actin filaments by providing a nucleus for polymerization ("nucleation") . The result is a steady state which favors short actin filaments unable to support the gel phase [P.A. Janmey et al., "Interactions of Gelsolin and Gelsolin-actin Complexes with Actin. Effects of Calcium on Actin Nucleation, Filament Severing, and End Blocking", Biochemistry. 24. pp. 3714-23 (1985)].

Gelsolin's actin-severing function is stoichiometric: one gelsolin molecule binds to two monomers on the actin filament, breaks the filament, and remains bound to both monomers. The binding of gelsolin to one of the monomers is Ca⁺⁺ dependent, and chelating agents such as EGTA cause dissociation of gelsolin from only one monomer.

Scientists have identified two phosphatidyl inositol phosphate phospholipids that bind to and regulate the function of gelsolin. They are phosphatidylinositol 4-monophosphate (PIP) and phosphatidylinositol 4,5-biphosphate (PIP₂) [P.A. Janmey et al., "Polyphosphoinositide Micelles and Polyphosphoinositide-containing Vesicles Dissociate Endogenous Gelsolin-actin Complexes and Promote Actin Assembly from the Fast-growing End of Actin Filaments Blocked by Gelsolin", J. Biol. Chem.. 262, pp. 12228-36 (1987) , P.A. Janmey and T.P. Stossel, "Modulation of Gelsolin Function by Phosphatidylinositol 4,5- biphosphate", Nature. 325. pp. 362-64 (1987) and P.A. Janmey and T.P. Stossel, "Gelsolin-phosphoinositide

Interaction", J. Biol. Chem.. 264. pp. 4825-31 (1989)]. These polyphosphoinositides are minor membrane phospholipids that play a role in signal transduction in cells [B. Alberts et al. , Molecular Biology of the Cell. Second Edition, Garland Publishing, Inc. , New York, New York, pp. 702-703 (1989)]. Together they comprise less than 10% of the total phospholipids of cell membranes, and PIP₂ comprises less than 1%. These two molecules inhibit gelsolin activity by binding to gelsolin and displacing the actin monomers that are bound to it in a non-Ca⁺⁺ dependent manner.

In extensively sonicated aqueous suspensions, both PIP and PIP- form vesicles. PIP, forms small vesicles, also called micelles, of about 80 ran in diameter, that contain about one-hundred PIP, molecules. Each PIP, micelle binds about eight gelsolin molecules. PIP forms larger unilamellar (one- layered) vesicles. Aggregation of PIP into large unilamellar or multimellar vesicles in the presence of millimolar concentrations of Mg⁺⁺ or nonionic detergents decreases the ability of PIP, to inhibit the actin filament-severing function of gelsolin. Incorporation of PIP into mixed vesicles composed of phosphatidyl choline (PC) also decreases this ability, although about a third of maximal activity persists, even in vesicles containing a very high ratio of PC to PIP,• Mixed lipid vesicles whose composition approximates that of the cell membrane (less than 3% PIP,) also inhibit gelsolin activity. Several other polyphosphoinostides which may be constructed, or have already been identified in nature, would also be expected to bind gelsolin.

The cDNA for human plasma gelsolin encodes a protein of 755 amino acids plus a 27 amino acid signal sequence [Kwiatkowski et al., "Plasma and Cytoplasmic Gelsolins Are Encoded by a Single Gene and Contain a Duplicated Actin-binding Domain", Nature. 323, pp. 455- 58 (1986)]. This cDNA sequence accounts for both the plasma and serum forms of gelsolin, which are the result of alternative transcriptional initiation sites and message processing from a single gene, 70 kb long [D. Kwiatkowski et al., "Genomic Organization and Biosynthesis of Secreted and Cytoplasmic Forms of Gelsolin", J. Cell Biol.. 106. pp. 375-84 (1988)]. The difference between the plasma and cytoplasmic forms is a 25 amino-acid residue extension on plasma gelsolin. This appears to account for the difference in relative molecular weight between the proteins as assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) , 93 kD and 90 kD, respectively.

Investigators have identified several functional domains of gelsolin [H.L. Yin et al. , "Identification of a Polyphosphoinositide-modulated Domain in Gelsolin Which Binds to the Sides of Actin Filaments", J. Cell Biol. _f 106, pp. 805-12 (1988) and D. Kwiatkowski et al., "Identification of Critical Functional and Regulatory Domains in Gelsolin", J. Cell Biol.. 108. pp. 1717-26 (1989)]. The gelsolin cDNA contains a strong tandem repeat that divides the molecule into two roughly equal halves. These structural halves correspond to two functional halves: The amino-terminal half of the protein contains a Ca⁺⁺- insensitive actin-severing function and the carboxy- terminal half has a Ca⁺⁺-sensitive actin binding domain. Within these two tandem repeats are six domains of weaker homology. The polypeptide has three actin binding sites. Two monomer binding sites are located between residues 26-139 and 407-756 (probably 661-738) and an actin filament binding site is located between residues 151-406. Amino acid residues 732-738 are potentially important for Ca⁺⁺ regulation. Residues 660-738 are important for nucleation. This function probably requires actin binding sites on both halves of the molecule. The severing function resides in residues 1-160, possibly between residues 139-160, with critical dependence on the sequence 150-160 (the first eleven residues of domain two) . The PIP -regulation of gelsolin's severing activity apparently resides within the first 160 residues. Sequences in domains 2 and 3 appear to hide a cryptic Ca⁺⁺-sensitive domain because when they are removed, the severing function of gelsolin becomes Ca⁺⁺ dependent.

Significantly, the amino acid sequence of gelsolin exhibits homology with several other actin binding proteins. It is forty-five percent homologous with villin, found in vertebrate brush border microvilli, which also has a Ca⁺⁺-dependent actin severing function. It is thirty-three percent homologous with severin and fragmin [P. Matsudaira and P. Janmey, "Pieces in the Actin-severing Protein Puzzle", Cell. 54. pp. 139-40 (1988)]. These polypeptides also bind PIP and PIP,«

Despite advances in biotechnology, the need still exists for methods and products which optimize the characteristics and delivery of pharmaceuticals, vaccines, diagnostics and bioactive molecules — including polyvalency, affinity for a single target particle, serum half-life, bioactivity and, in some cases, immunogenicity. Furthermore, systems in which the component parts may be easily varied would be especially useful because they would allow one to test for species with optimal characteristics.

SUMMARY OF THE INVENTION

This invention solves these problems by providing multimeric and hetero-multimeric gelsolin fusion constructs. A multimeric gelsolin fusion construct is a vesicle comprising at least one polyphosphoinosi ide, such as PIP or PIP, to which gelsolin fusion polypeptides are bound. Gelsolin fusion polypeptides comprise gelsolin moieties linked to functional moieties which may be pharmaceutical agents, vaccine agents, diagnostic agents or other bioactive molecules. Hetero-multimeric gelsolin fusion constructs comprise at least two different functional moieties or gelsolin moieties.

Gelsolin is a particularly attractive candidate for attachment to lipid vesicles because it binds specifically and with great affinity to polyphosphoinositides. Other proteins, related to gelsolin, which also specifically bind polyphosphoinositides may also be employed. Some examples are villin, frag in, severin, profilin, cofilin, Cap42(a) , gCap39, CapZ and destrin. Lipocortin (annexin) and DNasel are other molecules that bind polyphosphoinositides. Proteins that specifically bind other lipids may also be used, as well as proteins that bind lipids non-specifically.

The fusion constructs of this invention advantageously utilize the ability of polyphosphoinositide vesicles to bind multiple copies of gelsolin fusion polypeptides. Consequently, in contrast to monomeric molecules, the bioactive molecules linked to them as functional moieties are characterized by one or more of the following: polyvalency, increased serum half-life, affinity for target particles or cells, greater bioactivity or immunogenicity, and targetability.

The present invention also provides gelsolin fusion polypeptides. Gelsolin fusion polypeptides comprise gelsolin moieties fused or chemically coupled to a functional moiety. In particular, this invention provides CD4-gelsolin fusion polypeptides.

The lipid composition of a vesicle may also be varied to permit the production of vesicles varying in fluidity, size, the number of gelsolin molecules that will bind to it and the rate of degradation in the blood stream.

Depending upon the choice of functional moiety, multimeric and hetero-multimeric gelsolin fusion constructs are characterized by 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 fusion proteins to particular antigens. When targeted in this manner, multimeric gelsolin fusion constructs are useful to block the binding of viruses to cells that results in infection, or the binding of cells to other cells that, for example, characterizes pathologic inflammation. Due to the multivalency of the fusion constructs of this invention, we believe that they possess greater affinity for the target than monovalent molecules. In one embodiment of this invention, 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.

When hetero-multimeric fusion constructs comprise gelsolin fusion polypeptides having combinations of recognition molecules and toxins, anti-retroviral agents or radionuclides, they are useful as therapeutic agents which search out and destroy their target. Multimeric gelsolin fusion constructs with recognition molecules are also useful for signal enhancement in diagnostic assays. As large, multimeric molecules, they present many binding sites for reporter molecules, such as horseradish peroxidase-conjugated antibodies. Alternatively, they may take the form of hetero-multimeric constructs, possessing both recognition molecules and multiple reporter groups. When the functional moiety is one or more immunogen from one or more infectious agent, the fusion proteins of this invention are useful in vaccines.

Also, multimeric gelsolin fusion constructs may be employed as agents with increased bioactivity when the functional group is an enzyme, substrate, or inhibitor.

This invention also provides multimeric gelsolin fusion constructs that are liposomes whose constituents include polyphosphoinositides and that contain bioactive agents in their interiors. This invention further provides DNA sequences that encode gelsolin fusion polypeptides, recombinant DNA molecules comprising them and unicellular host cells transformed with them. And this invention provides methods for producing these fusion polypeptides by culturing such hosts.

This invention also provides compositions comprising any of the above-identified fusion polypeptides or proteins that are useful as therapeutic, prophylactic or diagnostic agents. Multimeric CD4-gelsolin fusion constructs may be used in diagnosing, preventing and treating AIDS, ARC or HIV infection. BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1F ("Figure 1") (SEQ ID NO:l) depict the DNA sequence and deduced amino acid sequence of human gelsolin as set forth in D.J. Kwiatkowski et al.. Nature, 323, pp. 455-58 (1986). The negatively numbered amino acids correspond to the signal sequence, which is absent from the mature polypeptide. Throughout this specification, references to human gelsolin by amino acid sequence or DNA sequence correspond to the coordinate system set forth in this figure.

Figure 2 depicts the functional regions of human gelsolin amino acid sequence.

Figures 3A-3D ("Figure 3") (SEQ ID NO:2) depict the DNA sequence and deduced amino acid sequence of human CD4 DNA. Nucleotides 1-75 are derived from plasmid pl70.2. Nucleotides 76-741 are derived from plasmid pCD4-gelsolin. Nucleotides 742 to 1377 are derived from pl70.2. Throughout this specification, references to CD4 by amino acid or DNA sequence 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 Figure 3.

Figure 5 depicts the DNA sequences of the oligomers used in the processes set forth in the examples of this application. The gelsolin sequences in this figure are derived from SEQ ID N0:1. ACE 144 is SEQ ID NO:3. ACE 145 is SEQ ID NO:4. T4 AID-133 is SEQ ID NO:5. T4AID-134 is SEQ ID NO:6. T4AID-137 is SEQ ID NO:7. T4AID-176 is SEQ ID NO:8. T4AID-176 is SEQ ID NO:9. Figure 6 depicts the construction of plasmid pCD4-gelsolin. Figures 7A-7B ("Figure 7") (SEQ ID NO:10) depicts the DNA sequence and deduced amino acid sequence of pCD4-gelsolin.

Figure 8 is a restriction map of pCD4-gelsolin.

Figure 9 depicts the construction of plasmid pDC219.

Figures 10A-10F ("Figure 10") (SEQ ID NO:11) depict the DNA sequence of p218-8. Figure 11 depicts the construction of plasmid pλP I80cys.

Figures 12A-12I ("Figure 12") (SEQ ID NO:12) depict the DNA sequence of pBG39l.

Figures 13A-13H ("Figure 13") (SEQ ID NO:13) depict the DNA sequence of pEX46.

DETAILED DESCRIPTION OF THE INVENTION

"Human plasma gelsolin" refers to a polypeptide having the amino acid sequence depicted in Figure 1 (SEQ ID NO:l) from amino acids -27 to +755. It should be understood that polypeptide expression often involves post-translational modifications such as cleavage of the signal sequence, intramolecular disulfide bonding, glycosylation and the like. The use of the term, human plasma gelsolin, contemplates such modifications to the amino acid sequence of Figure 1 (SEQ ID N0:1). The term also includes gelsolin obtained from natural, recombinant or synthetic sources.

"Multimeric gelsolin fusion constructs" and "hetero-multimeric gelsolin fusion constructs" each comprise gelsolin fusion polypeptides bound to a vesicle of aggregated phospholipids. A "gelsolin fusion polypeptide" comprises a gelsolin moiety bound to a functional moiety. "Functional moieties" may be polypeptides ("polypeptide moieties") or chemical compounds ("chemical moieties") . Throughout this application, specific gelsolin fusion polypeptides are referred to by the name of the functional moiety. For example, we call a gelsolin fusion polypeptide having CD4 as the functional moiety, CD4-gelsolin fusion polypeptide. Hetero-multimeric gelsolin fusion constructs comprise at least two different functional moieties or gelsolin moieties.

When the functional moiety is a polypeptide, gelsolin fusion polypeptides may be produced by chemical crosslinking or genetic fusion. Genetic fusion involves creating a hybrid DNA sequence in which the DNA sequence encoding the polypeptide is fused to the 5' end or 3' end of a DNA sequence encoding the gelsolin moiety. Upon expression in an appropriate host, this hybrid DNA sequence produces a gelsolin fusion polypeptide in which the polypeptide moiety is fused to the N-terminus or C-terminus of the gelsolin moiety. A "gelsolin moiety" as used herein is gelsolin or a fragment thereof that specifically binds to a polyphosphoinositide. Preferably, the gelsolin moiety will be derived from human plasma gelsolin. A gelsolin moiety preferably includes amino acids +150 to +160 of Figure 1 (SEQ ID NO:l). As demonstrated herein, the polypeptide containing amino acids +150 to +169 of Figure 1 (SEQ ID N0:1) has the ability to bind PIP . We believe that gelsolin derived from non-human vertebrates may also be useful according to this invention. The structure of gelsolin is highly conserved in evolution and gelsolin from non-human mammals may not be immunogenic in humans.

Lipid binding proteins ("LBPs") other than gelsolin are also known to exist. These proteins, or fragments of them that bind to particular lipids, are useful as LBP moieties (similarly to gelsolin moieties) to produce LBP fusion polypeptides that bind to vesicles containing the particular lipid. This creates multimeric or hetero-multimeric LBP fusion constructs. Gelsolin-related proteins that specifically bind polyphosphoinositides include villin, severin, fragmin, profilin, cofilin, Cap42(a) , gCap39, CapZ and destrin [E. Andre et al., "Severin, Gelsolin, and Villin Share a Homologous Sequence in Regions Presumed to Contain F-actin Severing Domains", J. Biol. Chem.. 263. pp. 722-27 (1988); W.L. Bazari et al., "Villin Sequence and Peptide Map Identify Six Homologous Domains", Proc. Natl. Acad. Sci.. USA. 85. pp. 4986-90 (1988) ; C. Ampe et al. , "The Primary Structure of Human Platelet Profilin: Reinvestigation of the Calf Spleen Sequence", FEBS Letters. 228. pp. 17-21 (1988) ; D.J. Kwiatkowski and G.A.P. Bruns, "Human Profilin", J. Biol. Chem.. 263, pp. 5910-15 (1988); I. Lassing and U. Lindberg, "Specificity of the Interaction Between Phosphatidylinositol 4,5-biphosphate and the Profilin: Actin Complex", J. Cell. Biochem.. 37. pp. 255-67 (1988) ; C. Ampe and J. Vandekerckhove, "The F-actin Capping Proteins of Physarum polvcephalum". EMBO. J. , .6, pp. 4149-57 (1987); I. Lassing and U. Lindberg, "Specific Interaction between Phosphatidylinositol 4,5- biphosphate and Profilactin", Nature. 314. pp. 472-74 (1985), F.-X. Yu et al., "gCap39, a Calcium Ion- and Polyphosphoinositide-regulated Actin Capping Protein", Science, 250, pp. 1413-15 (1990) ; and N. Yonezawa et al., "Inhibition of the Interactions of Cofilin, Destrin and Deoxyribonuclease I with Actin by

Phosphoinositides", J. Biol. Chem.. 265, pp. 8382-86 (1990)]. Other LBPs that specifically bind polyphosphoinositides are lipocortin [K. Machoczek et al. , "Lipocortin I and Lipocortin II Inhibit Phosphoinositide and Polyphosphoinositide-specific

Phospholipase C" FEBS Letters. 251. pp. 207-12 (1989)] and DNase I [J.A. Cooper et al., "The Role of Actin Polymerization in Cell Motility", Ann. Rev. Phys.. 53. pp. 585-605 (1991)]. Protein kinase C is also an LBP which binds to some phospholipids. DNA sequences encoding gelsolin moieties are derived from DNA sequences encoding gelsolin. Several methods are available to obtain these DNA sequences. First, one can chemically synthesize the gelsolin gene or a degenerate version of it using a commercially available chemical synthesizer. Figure l (SEQ ID NO:l) sets forth a DNA sequence for gelsolin. The coding region encompasses nucleotides +1 to +2360.

Second, one can isolate a cDNA sequence encoding gelsolin by screening a cDNA library. Many screening methods are known to the art. For example, colonies may be screened by nucleic acid hybridization with oligonucleotide probes. Probes may be prepared by chemically synthesizing an oligonucleotide having part of the known DNA sequence of gelsolin. Alternatively, cDNA libraries may be constructed in expression vectors, such as λgtll, and the colonies screened with anti-gelsolin antibodies.

Third, one can isolate a cDNA encoding gelsolin or a gelsolin moiety by amplifying DNA with polymerase chain reaction (PCR) . We describe this process in Example I.

The DNA sequence encoding the gelsolin moiety may then be fused to a DNA sequence encoding the polypeptide moiety. DNA sequences for the polypeptide moieties useful in this invention are available from many sources. These include DNA sequences described in the literature and DNA sequences for particular polypeptides obtained by any of the conventional molecular cloning techniques. A wide array of polypeptides are useful to produce the gelsolin fusion polypeptides of this invention. Those most useful include polypeptides that are advantageously administered in multimeric form. For example, viral receptors, cell receptors or cell ligands are useful because they typically bind to particles or cells exhibiting many copies of the receptor. Fusion constructs containing these fusion polypeptides are useful in therapies that involve the inhibition of viral-cell or cell-cell binding. Useful viral-cell receptors include ICAMl, a rhinovirus receptor; the polio virus receptor [J. M. White and D.R. 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 ICAMl, ELAMl, VCAMl and VCAMlb and their lymphocyte counterparts (ligands) LFA1, CDX 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. Osborn et al., "Direct Expression Cloning of Vascular Cell Adhesion Molecule 1: A Cytokine-induced Endothelial Protein that Binds to Lymphocytes", Cell. 59. pp. 1203-11 (1989); CA. Hession et al., "Endothelial Cell- leukocyte Adhesion Molecules (ELAMs) and Molecules Involved in Leukocyte Adhesion (MILAs)", WO 90/13300]. Other lymphocyte associated antigens, such as LFA2 (CD2) and LFA3 (both members of the CD11/CD18 family) and PAGEM are also useful.

Bacterial immunogens, parasitic immunogens and viral immunogens may be used as polypeptide moieties to produce multimeric or hetero-multimeric gelsolin fusion constructs useful as vaccines. Bacterial sources of these immunogens include those responsible for bacterial pneumonia and r^eumocystis 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, cytomegalovirus, 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.g., rubella; flaviviruses, e.g., yellow fever virus, Dengue fever viruses, Japanese encephalitis virus, Tick-borne encephalitis virus and hepatitis C virus; coronaviruses, e.g., human coronaviruses; paramyxoviruses, e.g., parainfluenza 1, 2, 3 and 4 and mumps; morbilliviruses, e.g., measles virus; pneumovirus, e.g., respiratory syncytial virus; vesiculoviruses, e.g., vesicular stomatitis virus; lyssaviruses, e.g., rabies virus; orthomyxoviruses, e.g., influenza A and B; bunyaviruses e.g., LaCrosse virus; phleborviruses, e.g., Rift Valley fever virus; nairoviruses, e.g., Congo hemorrhagic fever virus; hepadnaviridae, e.g., hepatitis B; arenaviruses, e.g., lcm virus, Lassa virus and Junin virus; retroviruses, e.g., HTLV I, HTLV II, HIV I and HIV II; enteroviruses, e.g., polio virus 1, 2 and 3, coxsackie viruses, echoviruses, human enteroviruses, hepatitis A virus, hepatitis E virus, and Norwalk virus; rhinoviruses e.g., human rhinovirus; and filoviridae, e.g., Marburg (disease) virus and Ebola virus.

Immunoglobulins or fragment thereof that bind to a target molecule may also be employed as functional moieties. Immunoglobulin molecules are bivalent, but multimeric immunoglobulin-gelsolin fusion constructs, which are multivalent, may demonstrate increased affinity or avidity for the target. Investigators have also made use of single domain antibodies (dAbs) [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 Huse et al. and use individual domains as functional moieties in multimeric or hetero-multimeric gelsolin fusion constructs according to this invention [W.D. Huse et al. , "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda", Science. 246. pp. 1275-81 (1989)]. See also A. Skerra and A. Pluckthun "Assembly of a Functional Immunoglobulin Fv Fragment in Escherichia coli". Science. 240. pp. 1038-43 (1988)].

According to this invention, multimeric gelsolin fusion constructs may be produced in which the functional moiety is an enzyme, enzyme substrate or enzyme inhibitor. We believe that such agents will exhibit greater bioactivity than monomeric molecules because multimers have a higher density of the moiety and will exhibit increased catalytic rate. For example, we believe that a multimeric gelsolin fusion construct with tissue plasminogen activator would have greater clot-dissolving catalytic activity than its onovalent counterpart. Similarly, we believe that a multimeric gelsolin fusion construct with hirudin would demonstrate greater anti-coagulant activity than hirudin alone.

Other useful functional moieties include, but are not limited to, polypeptides such as cytokines, including the various IFN-α's, particularly α2, α5, cκ8,

IFN-β and IFN-₇, the various interleukins, including IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7 and IL-8 and the tumor necrosis factors, TNF-α, and β. In addition, functional moieties include monocyte colony stimulating factor (M-CSF) , granulocyte colony stimulating factor (G-CSF) , granulocyte macrophage colony stimulating factor (GM-CSF) , erythropoietin, platelet-derived growth factor (PDGF) , and human and animal hormones, including growth hormones and insulin.

According to one embodiment of this invention, multimeric gelsolin fusion constructs comprise CD4-gelsolin fusion polypeptides. CD4 is the receptor on those white blood cells, T-lymphocytes, which recognizes HIV, the causative agent of AIDS and ARC [P.J. Maddon et al., "The T4 Gene Encodes the AIDS Virus Receptor and Is Expressed in the Immune System and the Brain", Cell. 47. pp. 333-48 (1986)]. Specifically, CD4 recognizes the HIV viral surface protein, gpl20 and gpl60. In CD4-gelsolin fusion polypeptides the functional moiety is a polypeptide moiety comprising full length CD4 or a fragment thereof, preferably soluble CD4. Use of the term, CD4, in this specification may refer to full length CD4 or fragments of CD4, unless specified.

A DNA sequence encoding full length human CD4 polypeptide and its deduced amino acid sequence is set forth in Figure 3 (SEQ ID NO:2). (See also P.J. Maddon et al., "The Isolation and Nucleotide Sequence of a cDNA Encoding the T Cell Surface Protein T4: A New Member of the Immunoglobulin Gene Family", Cell. 42. pp. 93-104 (1985).) Based upon its deduced primary structure, the CD4 polypeptide is divided into functional domains as follows: Amino Acid Coordinates

Structure/Proposed Location In Figure 3

Hydrophobic/Secretory Signal -25 to -1

First Immunoglobulin-related +1 to +107 domain/Extracellular

Second Immunoglobulin-related +108 to +177 domain/Extracellular

Third Immunoglobulin-related +178 to +293 domain/Extracellular

Fourth Immunoglobulin-related domain/Extracellular +294 to +370

Hydrophobic/Transmembrane +371 to +391 Sequence

Very Hydrophilic/ +392 to +431 Intracytoplasmic

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 [S.J. Clark et al., "Peptide and Nucleotide Sequences of Rat CD4 (W3/25) Antigen: Evidence for Derivation from a Structure with Four Immunoglobulin-related Domains", Proc. Natl. Acad. Sci._f USA. 84. pp. 1649-53 (1987) ] .

These domains also correspond roughly to structural domains of the CD4 protein due to intra- domain disulfide bonding. Thus, disulfide bonds join amino acids at positions +16 and +84 in the first immunoglobulin-related domain, amino acids +130 and +159 of the second immunoglobulin-related domain, and amino acids +303 and +345 of the fourth immunoglobulin- related domain. Figure 4 depicts the domain structure of the full length human CD4 protein.

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)]. These 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 and gpl60. By acting as soluble virus receptors, 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 and gpl60. These include fragments of CD4 lacking the transmembrane domain, amino acids +371 to +391 of Figure 3 (SEQ ID NO:2). Such fragments may be truncated forms of CD4 or be fusion proteins in which the fourth immunoglobulin-related domain is joined directly to the hydrophilic cytoplasmic domain. We shall refer herein to a CD4 polypeptide which includes amino acids +1 to +X of Figure 3 (SEQ ID NO:2), and optionally including an N-terminal methionine or f-methionine, as "CD4(X)^M. When a CD4 polypeptide is engineered to include a carboxy-terminal cysteine, we shall refer to the polypeptide as "CD4(XCys)".

For example, referring now to Figure 3 (SEQ ID NO:2) , 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. Most preferably, 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 im unoglobulin-like domains preferably will include at least amino acids +1 to +345 [CD4(345)] and at most amino acids +1 to +375 [CD4(375)]. Any of these molecules may optionally include the CD4 signal sequence, amino acids -23 to -1 of Figure 3 (SEQ ID NO:2) . Also, these molecules may have a modified methionine residue preceding amino acid, +1.

Soluble CD4 proteins useful in the fusion polypeptides and methods of this invention may be produced in a variety of ways. According to the coordinate system in Figure 3 (SEQ ID NO:2), the amino terminal amino acid of mature CD4 protein isolated from T cells is lysine, encoded at nucleotides 136 to 139 of Figure 3 (SEQ ID NO:2) . [D.R. Littman et al., "Corrected CD4 Sequence", Cell. 55. p. 541 (1988).]

Soluble CD4 proteins also include those in which amino acid +1 is asparagine, +62 is arginine and +229 is phenylalanine. Therefore, when we refer to CD4, we intend to include amino acid sequences including any or all of these substitutions. Soluble CD4 polypeptides may be produced by conventional recombinant techniques involving 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 and Serial No. 141,649, filed January 7, 1988, and PCT patent application Serial No. PCT/US88/02940, filed September 1, 1988, and published as PCT patent application WO 89/01940, the disclosures of which are hereby incorporated by reference.

Microorganisms and recombinant DNA molecules characterized by DNA sequences coding for soluble CD4 proteins are exemplified by cultures described in PCT patent application WO 89/01940. They were deposited in the In Vitro International, Inc. culture collection, in Linthicum, Maryland, USA on September 2, 1987 and identified as:

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. Additionally, such microorganisms and recombinant DNA molecules are exemplified by cultures deposited in the In Vitro International, Inc. culture collection on August 24, 1988 and identified as: 211-11: E.coli A89/pBG211-ll - IVI 10183 214-10: E.coli A89/pBG214-10 - IVI 10184 215-7 : E.coli A89/pBG215-7 - IVI 10185.

Multimeric CD4-gelsolin fusion constructs comprising CD4-gelsolin fusion polypeptides may be used in pharmaceutical compositions and methods to treat humans having AIDS, ARC, HIV infection, or antibodies to HIV. Accordingly, they may be used to lessen the immuno-compromising effects of HIV infection or to prevent the incidence and spread of HIV infection. In addition, these 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 gelsolin fusion polypeptides are useful for producing multimeric gelsolin fusion constructs. The preferred process for using these DNA sequences involves expressing the gelsolin fusion polypeptide in an appropriate host, isolating the polypeptide, and binding it to a vesicle comprising a polyphosphoinositide.

As is well known in the art, for expression of the DNA sequences of this invention, 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, of course, includes the provision of a translational start signal in the correct reading frame upstream of the DNA sequence. If a particular DNA sequence being expressed does not begin with an ATG, the start signal will result in an additional amino acid — methionine (or f-methionine in bacteria) — being located at the N-terminus of the product. While such methionyl-containing product may be employed directly in the compositions and methods of this invention, it is usually more desirable to remove the methionine before use. Methods are known to those of skill in the art to remove such N-terminal methionines from polypeptides expressed with them. For example, certain hosts and fermentation conditions permit removal of substantially all of the N-terminal methionine in vivo. Expression in other hosts requires in vitro removal of the N-terminal methionine.

However, such in vivo and in vitro methods are well known in the art.

A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, 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 A, e.g., NM989, and other DNA phages, e.g., M13 and filamentous single stranded DNA phages, yeast plasmids, such as the 2μ plasmid or derivatives thereof, and vectors derived from combinations of plasmids and phage DNAs, such as plasmids which have been modified to employ phage DNA or other expression control sequences. In addition, 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. Such 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 λ, 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 α-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. For 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. Neither will all hosts function equally well with the same expression system. However, one of skill in the art may make a selection among these vectors, expression control sequences, and hosts without undue experimentation and without departing from the scope of this invention. For example, in selecting a vector, the host must be considered because the vector must replicate in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered. In selecting 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 regards 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.

Within these parameters, one of skill in the art may select various 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.

According to one embodiment of this invention, a plasmid comprising a DNA sequence encoding a CD4-gelsolin fusion polypeptide operatively linked to a AP promoter expression control sequence is expressed in E.coli to produce a CD4-gelsolin fusion polypeptide.

The 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. One can also produce gelsolin 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)]. Another method useful for producing gelsolin fusion polypeptides, in addition to genetic fusion and chemical synthesis, is by chemically coupling the functional moiety to the gelsolin moiety. This method is useful for both chemical moieties and polypeptide moieties.

Several approaches are available for chemically coupling the gelsolin moiety to a polypeptide moiety. The preferable strategy is to identify or create sites on the polypeptide moiety through which it may be selectively linked to the gelsolin moiety without destroying the activity of the polypeptide moiety. Glycoproteins, such as CD4, have limited numbers of sugars that are useful as cross- linking sites. The sugars may be oxidized to aldehydes and an aldehyde then reacted with an amine group on the gelsolin moiety to create an aldehyde-amine linkage. [P.K. Nakane and A. Kawaoi, "Peroxidase Labelled Antibody: A New Method of Conjugation", J. Histochem. Cvtochem.. 22, p. 1084 (1984) and T.-H. Liao et al.,

"Modification of Sialyl Residues of Sialolycoprotein(s) of the Human Erythrocyte Surface", J. Biol. Chem.. 248. pp. 8247-53 (1973)]. CD4 has two functional glycosylation sites at amino acids +269 to +271 and +298 to +300 (see SEQ ID NO:3). These are outside the gpl20 binding region, which is within the first 113 amino acids of the protein [B.H. Chao et al., "A 113- amino Acid Fragment of CD4 Produced in Escherichia coli Blocks Human Immunodeficiency Virus-induced Cell Fusion", J. Biol. Chem.. 264. pp. 5812-17 (1989)].

Therefore, coupling CD4 through the carbohydrate should not interfere with function. Alternatively, CD4 may be genetically engineered to eliminate one of the glycosylation sites. This would increase selectivity during linkage. We describe aldehyde-amine linkages in Example II using CD4. Protein chemists have also developed specific chemistries for covalently coupling polypeptides through thiol groups. A polypeptide moiety having a free thiol may be linked to a gelsolin moiety containing a cysteine either by direct formation of a disulfide bond or indirectly through a ho o- bifunctional crosslinker. One example of a homo- bifunctional crosslinker is bismaleimidohexane (BMH) which has thiol-reactive maleimide groups and forms covalent bonds with free thiols. These methods require the construction of a gelsolin moiety with a cysteine at the amino- or carboxy-terminus. Peptide synthesizers (Example II, Section 2) are useful for in these constructions. If the polypeptide moiety does not have a free thiol group, such a group may be introduced. For example, the polypeptide may be bound to a thiol- containing amine. More particularly, an oxidized sugar on the polypeptide moiety may be reacted with the amine as described above.

Also, a cysteine may be introduced into the amino acid sequence of the polypeptide moiety by site- directed mutagenesis.

Alternatively, the polypeptide moiety and the gelsolin moiety may be crosslinked through hetero- bifunctional crosslinking agents. These are chosen so that one of the functional groups binds to a group on the polypeptide moiety and the other binds to the thiol on the gelsolin moiety. For example, a succinimide group could bind to an amine group on the polypeptide moiety and a thiol-reactive group, such as a maleimide or an activated thiol could bind to a cysteine on the gelsolin moiety.

We describe methods involving thiol linkage in Example III using CD4. The Pierce Co.

Immunotechnology Catalogue and Handbook Volume 1 §§ E4-E12, E41-E48 and E31-E40 describes many useful homo- and hetero-bifunctional crosslinkers, thiol- containing amines and molecules with reactive groups. Other methods useful for coupling both polypeptide and chemical moieties include, for example, those employing glutaraldehyde [M. Reichlin, "Use of Glutaraldehyde as a Coupling Agent for Proteins and Peptides", Methods In Enzymology. 70, pp. 159-65 (1980) ] , N-ethyl-N'-(3-dimethylaminopropyl)- carbodiimide [T.L. Goodfriend et al., "Antibodies to Bradykinin and Angiotensin: A Use of Carbodiimides in Immunology", Science. 144. pp. 1344-46 (1964)] or a mixture of N-ethy1-N'-(3-dimethylaminopropy1)- carbodiimide and a succinylated carrier [M.H. Klapper and I.M. Klotz, "Acylation with Dicarboxylic Acid Anhydrides", Methods In Enzymology, 25. pp. 531-36 (1972)]. Since chemical coupling is not limited to one site on the gelsolin moiety, it is possible to couple more than one functional moiety to each gelsolin moiety.

Multimeric and hetero-multimeric gelsolin fusion constructs according to this invention may be produced by binding gelsolin fusion polypeptides to phospholipids aggregated into a vesicle. The vesicle must comprise at least one phospholipid that binds to gelsolin, but may contain others as well. The phosphatidylinositols, PIP and PIP₂, are preferable components of the vesicle because they bind to gelsolin. To be effective the vesicles preferably contain at least 3% of PIP or I _2' Other lipids that may comprise the vesicle include, but are not limited to, phosphatidylcholme (PC) , phosphatidyl ethanolamine (PE) , phosphatidylserine (PS) . One may also create vesicles containing detergents such as Triton. The production of phospholipid vesicles is well known to the art [D.M. Haverstick and M. Glaser, "Visualization of Ca²⁺-induced Phospholipid Domains", Proc. Natl. Acad. Sci.. USA. 64. pp. 4475-79 (1987)]. For example, dried lipids are mixed with water and the mixture is sonicated, producing vesicles. PIP should be sonicated more thoroughly than PIP₂ in order to obtain vesicles of similar size and binding. The gelsolin fusion polypeptide is then added and allowed to bind to the vesicles. The resulting product is a multimeric gelsolin fusion construct. The fact that a vesicle may comprise many different lipids and detergents allows great flexibility in engineering a fusion construct with desired characteristics. For example, one may produce vesicles that bind different numbers of gelsolin fusion polypeptides by varying the lipid composition of the starting materials to create larger vesicles, or by increasing the percentage of PIP or PIP₂ in the vesicle. Also, one may alter the half-life of the functional moiety. We expect that these vesicles will be subject to eventual degradation by lipases. By altering the lipid composition of the vesicle, one could vary the degradation rate of the vesicle.

When phospholipid vesicles containing cavities are prepared in the presence of a bioactive molecule, such as those illustrated herein, that molecule will come to be enclosed within the vesicles. Accordingly, it is possible to produce a multimeric gelsolin fusion construct that encloses within it a bioactive agent. These liposomes may fuse with cell membranes, delivering their contents to cells and adding the gelsolin fusion polypeptide to the cell membrane.

Hetero-multimeric gelsolin fusion constructs comprise at least two different functional moieties or two different gelsolin moieties. For example, hetero- multimeric gelsolin fusion constructs may comprise two different polypeptide moieties, two different chemical moieties or both a polypeptide moiety and a chemical moiety.

Hetero-multimeric gelsolin fusion constructs are especially useful when the properties of the different moieties complement one another. For example, it is possible to combine receptors that bind to a particular target particle or cell with toxins or anti-retroviral agents in fusion proteins according to this invention to produce targeted toxic or anti- retroviral agents. Polypeptides useful as toxins include, but are not limited to, ricin, abrin, angiogenin, Pseudomonas Exotoxin A, pokeweed antiviral protein, saponin, gelonin and diphtheria 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 gelsolin fusion constructs 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 functional moiety which is a recognition molecule, such as an immunoglobulin or a fragment thereof (Fab, dAb) that binds to the target molecule [See Ward et al., supra] and a second functional moiety, which is a reporter group, such as a radionuclide, an enzyme (such as horseradish peroxidase) or a fluorescent or chemiluminescent marker. Typically, the reporter group will be bound directly to the reporter group; for example, HRP is bound directly to the immunoglobulin. Many reporter groups may be coupled to a multimeric gelsolin fusion constructs thereby enhancing the signal. These constructs may be used, for example, to replace antibodies as the recognition molecules that contact the sample in ELISA-type assays, or as in vivo imaging agents.

Hetero-multimeric gelsolin fusion constructs according to this invention may also be used as multi- vaccines. For example, one may produce such constructs using several different antigenic determinants from the same infective agent. Also, one can produce constructs comprising antigenic determinants from several infective agents, such as polio, measles, mumps and others used for childhood vaccination.

The pharmaceutical compositions of this invention typically comprise a pharmaceutically effective amount of a multimeric gelsolin fusion construct 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.

The pharmaceutical 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 infusible 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. Generally, 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 constructs of this invention may be administered by conventional routes of administration, such as parenteral, subcutaneous, intravenous, intramuscular or intralesional routes. It will be understood that conventional doses will vary depending upon the particular molecular moiety involved. In order that this invention may be better understood, the following examples are set forth. These examples are for the purposes of illustration only, and are not to be construed as limiting the scope of the invention in any manner. In the examples that follow, the molecular biology techniques employed, such as cloning, cutting with restriction enzymes, isolating DNA fragments, filling out with Klenow enzyme and deoxyribonucleotides triphosphate (dXTP) , ligating, transforming E.coli and the like are conventional protocols exemplified and further described in J. Sambrook et al., Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) .

EXAMPLE I - PRODUCTION OF A CD4-GELSOLIN FUSION POLYPEPTIDE BY GENETIC FUSION

1. Cloning of pCD4-Gelsolin

We constructed a plasmid expression vector containing a DNA sequence encoding a CD4-gelsolin fusion polypeptide and used it to transform E.coli. The coding region contains a DNA sequence for CD4(181) fused to the 5' end of 140 bp fragment encoding a 12 amino-acid spacer and amino acids 150-173 of gelsolin. This includes the PIP, binding domain. We constructed the plasmid as follows. (See Figure 6.) First, we produced a DNA sequence containing the human gelsolin PIP, binding domain. The PIP, binding domain is encompassed within amino acids +150 to +169 (nucleotides 541-600) of Figure 1 (SEQ ID N0:1). We created this DNA sequence from the plasmid pMID which contains the cDNA human gelsolin-encoding sequence of Figure 1 (SEQ ID NO:l). (Plasmid pMID was the gift of David Kwiatkowski, Harvard Medical School, Boston, Massachusetts.) We amplified a cDNA sequence for the PIP, binding domain using polymerase chain reaction (PCR) (Sambrook et al. , Chapter 14) . We carried out all amplifications using Tag DNA polymerase and primers prephosphorylated with T4 polynucleotide kinase and ATP. We used the oligonucleotide ACE 144 (SEQ ID NO:3) as the sense primer (which hybridizes to the anti-sense strand) and ACE 145 (SEQ ID NO:4) as the anti-sense primer. (See Figure 5.) We filled out the amplified fragments with Klenow enzyme and dXTP. This produced blunt-ended 140 bp DNA fragments having a BgJ.II site near the 5' end and an EcoRI site near the 3' end. The fragments encoded gelsolin amino acids +143 through +173 (see SEQ ID NO:l).

Then we digested an intermediate plasmid, pNN03, with EcoRV and dephosphorylated the ends to prevent recircularization. Plasmid pNN03 is derived from pUC13 by the incorporation of a polylinker.

(Pharmacia PL Biochemicals) . We subcloned the 140 bp fragments into this plasmid. We called the resulting plasmid pGell.

We then inserted the BallI/EcoRI DNA fragment encoding the gelsolin PIP, binding domain from pGell into a prokaryotic expression vector containing a DNA sequence encoding CD4(181) and derived from pEX56. Plasmid pEX56 encodes CD4(181) fused in- frame to the 5' end of a DNA insert encoding Pseudomonas endotoxin. The insert is bordered by EcoRI sites at the 5' and 3' ends and contains a BgJ.II site at the junction of the CD4-endotoxin sequence. The Pseudomonas endotoxin gene has been altered to remove the ribosome binding region. Plasmid pEX56 is created by site-directed mutagenesis of pEX46 (Example III, section 2 and Figure 13 (SEQ ID NO:13) with oligonucleotide T4-AID 176 (Figure 5, SEQ ID NO:9). [The plasmid is described in co-pending PCT application PCT/US89/04584, incorporated herein by reference.] We digested a first sample of pEX56 with EcoRI and Bglll and isolated the 613 bp fragment that encodes CD4(181). Then we digested a second sample of pEX56 with EcoRI. dephosphorylated the fragments, and isolated the 3922 bp fragment representing the pEX56 vector portion. We ligated together the 3922 bp EcoRI fragment, the 613 bp EcoRI/Bglll fragment and the 140 bp Bglll/EcoRI fragment. We used this ligation mixture to transform E.coli JA221 [ATCC 33875] by standard CaCl procedures. (See Sambrook. Chapter 1.82.) We identified the plasmids pCD4-gelsolin and pαCD4- gelsolin (opposite orientation and therefore non- expressing) by restriction digests of mini-plasmid DNA preparations. The plasmid map of pCD4-gelsolin is shown in Figure 8. The DNA sequence and predicted amino acid sequence of the CD4-gelsolin fusion polypeptide obtained is shown in Figure 7 (SEQ ID

NO:10). We have deposited an isolate of pCD4-gelsolin with In Vitro International, IVI-10253.

2. Expression of CD4-Gelsolin

We transformed E.coli JA221 and E.coli A89 (an htpR^" protease deficient mutant) with pCD4-gelsolin and pαCD4-gelsolin. E.coli A89 is a tetracycline- sensitive mutant of E.coli SG936 [ATCC 39624]. We then tested the cultures for the production of CD4-gelsolin. Our results showed that pCD4-gelsolin, but not pαCD4- gelsolin, produced a polypeptide of the molecular weight predicted for CD4-gelsolin.

We grew 5 ml overnight cultures in LB + 12.5 μg/ml tetracycline at 30°C. We diluted the overnight cultures 1:10 into LB + 12.5 μg/ml tetracycline and grew the cultures until the optical density at 550 nm was between 1 and 1.5. We then added the culture to an equal volume of LB + 12.5 μg/ml tetracycline at 42°C. After two hours we harvested the cells, lysed them, and analyzed the contents for a protein band corresponding to the size expected for a CD4-gelsolin fusion molecule by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) . We thus identified a protein having molecular weight of about 28 kD.

We have isolated this protein using the protocol of Example III, section 2b.

EXAMPLE II CHEMICAL CROSS-LINKING OF A GELSOLIN

MOIETY TO CD4 VIA ALDEHYDE-AMIDE LINKAGE

We cross-linked CD4(375) (a gift of Biogen, Inc. , Cambridge, Massachusetts) to a gelsolin moiety by oxidizing sugars on the CD4 glycoprotein to aldehydes and then reacting an aldehyde with an amine on the gelsolin moiety to create an aldehyde-amine linkage.

1. Oxidation of CD4(375)

We dialyzed 100 μM CD4(375) protein against 0.1 M sodium acetate pH 5.0 at 4°C. We incubated the preparation at 23°C for 1 hour with 1 mM aqueous sodium periodate and immediately desalted on a P6DG column (BioRad, Richmond, California) that was equilibrated in 10 mM sodium acetate pH 5.0, 100 mM NaCl. We stored the oxidized CD4(375) at 4°C for subsequent use or at -70°C for long term storage. We monitored the extent of oxidation by measuring incorporation of tritiated sodium borohydride. Typically 8-10 aldehydes per CD4(375) were generated. To confirm that oxidation did not interfere with the CD4(375) function, we assessed the ability of the modified protein to bind gpl20 in an ELISA format. We coated IMMULON II® plates (Dynatech Laboratories, Chantilly, Virginia) , with gpl20 (a gift of Biogen, Inc., and commercially available from American Bio-Technologies, Inc., Cambridge, Massachusetts), added CD4(375) or oxidized CD4(375), and then determined the binding with a reporter system using OKT4 antibody (available from Ortho Diagnostics

Systems, Raritan, New Jersey) that was conjugated with horseradish peroxidase. There was no difference in binding of soluble CD4 protein or oxidized CD4 to gpl20. Upon amino acid analysis, both samples were also found to be similar with no apparent effect of oxidation on individual amino acids.

2. Reaction of Oxidized CD4(375) with the Gelsolin Moiety. GEL1

We synthesized a gelsolin moiety, GEL1, using an Applied Biosystems 430A peptide synthesizer. GEL1 has the amino acid sequence Gly-Tyr-Gly-Lys-His-Val- Val-Pro-Asn-Glu-Val-Val-Val-Gln-Arg-Leu-Phe-Gln-Val- Lys-Gly-Arg-Arg (SEQ ID NO:14). The final twenty amino acids constitute the PIP -binding sequence of gelsolin, amino acids +150 to +169 (see SEQ ID NO:l). To crosslink GEL1 with CD4(375), we incubated varying concentrations of GEL1 overnight at 23°C with 10 μM oxidized CD4(375) in the presence of 50 mM MES, pH 6.5, and 5 mM sodium cyanoborohydride. We tested the sample for crosslinking by SDS-

PAGE. Samples were either analyzed directly by staining with Coomassie brilliant blue or by Western blotting using an antiserum raised in rabbits against GEL1. The immunogen consisted of GEL1 crosslinked to Keyhole limpet hemocyanin with glutaraldehyde.

We found a dose dependent increase in the molecular weight of CD4 treated with GEL1, indicating that the protein had become modified. At low peptide concentrations, there was little effect on the mobility of CD4(375) but when incubated with 1 mM GEL1, approximately 50% of the CD4(375) migrated with an increased apparent molecular weight that is consistent with it containing one GEL1 peptide per CD4(375). When CD4(375) was incubated with 10 mM GEL1, all of the protein shifted to a high-molecular weight form. We observed a series of bands that likely correspond to moieties with one, two, and three gelsolin moieties per CD4(375). The need for a large molar excess of GEL1 over CD4(375) to drive the crosslinking reaction is consistent with the results obtained for modifying periodate oxidized CD4 with other amino-containing reagents as well. (See Example III.)

To verify that GEL1 had been crosslinked to CD4(375), we analyzed selected fractions by Western blotting using antibodies against GEL1. A prominent immunoreactive band was observed in the sample after crosslinking. This band is absent from the Western blot of an untreated CD4 sample.

3. Analysis of the CD4-Gelsolin Fusion Polypeptide

We demonstrated above that the crosslinking chemistry did not affect the ability of CD4(375) to bind gpl20. We have further established that CD4(375)- gelsolin fusion polypeptides bind to PIP, vesicles. We assayed the ability of CD4(375)-gelsolin to associate with PIP or PIP, vesicles using an aggregation assay similar to that described by Janmey et al., "Phosphoinositide Micelles and Polyphosphoinositide-containing Vesicles Dissociate Endogenous Gelsolin-actin Complexes and Promote Actin Assembly From the Fast-growing End of Actin Filaments Blocked by Geloslin", J. Biol. Chem.. 262. pp. 12228- 36 (1987) . In the assay, the amount of protein used is appropriately adjusted to take into account the molecular weight of the CD4-gelsolin fusion polypeptide. Mg⁺⁺ causes micelles of pure polyphosphoinositides to aggregate into larger vesicles, increasing the turbidity of the solution. However, gelsolin inhibits this aggregation. We found that CD4(375)-gelsolin behaved like the GELl peptide in this assay. Recombinant sCD4, alone, had no activity in this assay.

Because the junction between the gelsolin peptide fragment and the spacer is unnatural, it may be necessary to change the composition or length of the spacer region in order to optimize function. This involves resynthesizing the gelsolin peptide fragment with other sequences added at either the amino or carboxy terminus of the polypeptide. The coupling chemistry would not be affected. Alternatively, it may be advantageous to change selected amino acids from the binding sequence in order to change the affinity of the fusion polypeptide for PIP,_'

EXAMPLE III STRATEGIES FOR CROSSLINKING CD4 THROUGH THIOL GROUPS

We describe here three strategies for crosslinking the CD4 polypeptide moiety with a gelsolin moiety through thiol groups. They involve the modification of the CD4 protein to contain a cysteine, a free thiol or a thiol-reactive group.

!• Introducing a Free Thiol into CD4

First, a thiol group may be introduced into CD4 using thiol-containing amines, such as cysteine, cystamine or glutathione. An aldehyde is introduced into CD4 and then one creates an aldehyde-amine linkage (see Example II) . Once the thiol-containing CD4 is generated, it can be selectively crosslinked to the gelsolin moiety. We incubated periodate oxidized CD4(375) (0.5 mg/ml) overnight at 23°C in 50 mM MES, pH 6.5, 5 mM sodium cyanoborohydride with 20 mM of either cysteine, oxidized cystamine or oxidized glutathione to create CD4(cysteine) , CD4(cystamine) , and

CD4(glutathione) . We treated the samples with 40 mM DTT for 40 minutes at 23°C. We then dialyzed them against storage buffer (10 mM sodium acetate, pH 5.0, 100 mM NaCl) . We monitored the extent of modification with Ellman's reagent. Briefly, we diluted the samples into 100 μl of 100 mM sodium phosphate pH 8.0, 0.5 mM DTNB and measured the absorbance after 5 minutes at 410 nm. We calibrated the samples against a standard curve that was developed with reduced glutathione. Both cystamine and glutathione treatments resulted in three to five groups per CD4. For subsequent studies, the preparations were concentrated to 5 mg/ml using a CENTRICON-10® filtration unit (Amicon, Danvers, Massachusetts) . These molecules may be bound to gelsolin moieties through the thiol groups using ho o- bifunctional crosslinking agents with two thiol- reactive groups, such as BMH or o- or p-phenylene dimaleimide. We believe that this method will result in crosslinking because treatment of CD4(cystamine) with these agents induced the formation of CD4 dimers and higher molecular weight complexes. With sub- stoichiometric amounts of crosslinker we were able to drive crosslinking of CD4 to greater than 50%. A similar strategy will be used with the cysteine- containing gelsolin moiety where a dimaleimide agent will be used to generate crosslinking complexes.

Alternatively, the moieties may be crosslinked through disulfide bonds using conventional techniques. 2. Introducing a Free Cysteine into CD4 by Site-Specific Mutagenesis

Second, a free cysteine may be introduced in the primary sequence of CD4 through genetic engineering. Crosslinking to the gelsolin moiety is then directed using the methods of section 1 of this example. We describe herein the construction and isolation of two truncated forms of CD4 engineered to contain cysteine residues at their C-termini.

a. Construction of pDC219 and Expression of CD4(lllCys)

To produce CD4(lllCys) we constructed the expression plasmid pDC219. (See Figure 9.) We began with p218-8, a plasmid in which the AP_T promoter controls the expression of CD4(111). This plasmid is described in PCT patent application WO 89/0194, p. 77/93, Figure 28. The DNA seguence for p218-8 is depicted in Figure 10 (SEQ ID NO:11). We digested a first sample of p218-8 with PstI and Bglll and isolated the 3645 bp fragment. We then digested a second sample of p218-8 with PstI and EcoRI and isolated the 269 bp fragment. We digested a third sample of p218-8 with EcoRI and BSPMI and isolated the 395 bp fragment. We isolated these fragments by electrophoresing the digests on agarose gels, cutting out the relevant bands and electroeluting the DNA fragments. We precipitated the electroeluted DNA fragments with ethanol, centrifuged the mixture to pellet the DNA fragments and resuspended the fragments in 10 mM Tris-HCl, pH 8.0, 1 mM Na₂EDTA.

We phosphorylated oligonucleotides T4AID-133 (SEQ ID NO:5) and T4AID-134 (SEQ ID NO:6) (Figure 5) using bacteriophage T4 polynucleotide kinase. These oligonucleotides contain a Bglll recognition sequence. Then we ligated the purified DNA fragments and the oligonucleotides.

We used the reaction mixture to transform E.coli DHL We selected colonies that grew at 30°C, 12.5 μg/ml tetracycline and analyzed them for the correct sequences by digestion with Bglll. We subjected those plasmid DNAs which had the additional Bglll site to DNA sequence analysis. Thus we obtained pDC219. To produce CD4(lllCys), we transformed A89 cells with pDC219 and fermented the cells at a 10 liter scale. (We achieved an expression level of 13%.) We stored cells as frozen cell pellets.

To isolate CD4(lllCys) we thawed 50 g frozen whole cells, suspended them in 20 mM Tris pH 7.5, 1 mM EDTA, 0.4 mg/ml lysozyme, and mixed with a Polytron (Brinkman Instruments, Westbury, N.Y.). We stirred the cell slurry at room temperature for one hour, then passed it three times through a prechilled Manton Gaulin French press (550 setting) . We chilled the lysate on ice between each passage. We pelleted particulates in a SA600 rotor for 15 minutes at 10,000 rpm. We washed the resulting pellet twice with a 1:4 dilution in 20 mM Tris pH 9.0 and pelleted it as before. (All ratios given are whole cell weight to buffer volume.) We then washed the pellet with a 1:4 dilution in 20 mM Tris pH 9.0 containing 0.5 M NaCl and spun down the pellet using previous conditions by resuspending with a Polytron. We extracted the final pellet in a 1:4 dilution of extraction buffer (7 M urea, 20 mM Tris 9.0, 10 mM /3-mercaptoethanol) and stirring at room temperature for 15 minutes. We removed debris by centrifugation in a SA600 rotor at 15,000 G for 30 minutes. We diluted the clarified supernatant 1:4 with fresh extraction buffer and passed it over a Fast S cation exchange column (Pharmacia) pre-equilibrated with extraction buffer at a column ratio of 1 gm whole cells to 4 ml resin. We washed the column extensively with extraction buffer. We then eluted the protein with salt steps of half column volume of extraction buffer containing 0.05 M, 0.075 M, 0.1 M, 0.15 M and 0.2 M NaCl, respectively. CD4(lllCys) routinely eluted in the 0.15 M NaCl step.

We pooled the CD4(lllCys) peak and diluted it to an absorbance of under O.D. 0.5 at 280 nm. Then we dialyzed the sample overnight, 1:100 V:V, with one change, against 3 M urea, 20 mM Tris pH 7.5. We diluted the dialysate to 1 M urea with the 20 mM Tris pH 7.5, and filtered it through 0.45 μ sterile filter unit. We bound CD4 from the filtrate to 6C6-Sepharose for one hour at 4°C with rocking. 6C6 is a monoclonal antibody developed at Biogen that recognizes CD4 and blocks CD4 binding to gpl20. Alternatively, one may use anti-Leu-3a, a monoclonal available from Becton- Dickinson, Mountain View, California. Then we poured the slurry into a column and washed with 2 x 0.5 column volumes 50 mM Tris pH 7.5, 0.5 M NaCl (wash 2), and 2 x 0.5 column volumes of wash 1 buffer (wash 3) . CD4(lllCys) was eluted from the resin with a series of 0.1 column volume additions of 50 mM glycine, pH 3.0, 250 mM NaCl. We neutralized the eluate by the addition of 2 M Tris pH 9.0 to 50 mM.

The resulting affinity purified protein was 90% CD4(lllCys) monomer with contaminating multimeric bands. When run under reducing conditions these additional bands collapsed into the monomer, indicating they were disulfide forms of the protein. From 1 gm wet weight of cells we recovered between 0.5 to 0.75 mg of CD4(lllCys). We assayed the gpl20 binding activity and found it to be about half the specific activity that is observed for full length CD4. We carried out biotinylation studies using maleimidobutyryl biocytin (MBB) to test the susceptibility of the engineered cysteine to modification with the maleimide. We monitored biotin labeling on Western blots using avidin-conjugated HRP to track the biotin. Specific biotin labeling of CD4(lllCys) was observed when fresh samples were analyzed; however, the efficiency of labeling decreased with time as the samples aged.

b. Construction of λP 180cys and

Expression of CD4(l80Cys)

To produce CD4(180Cys), we constructed the expression plasmid AP_i180Cys, in which a AP_Li promoter controls the expression of a DNA sequence encoding CD4(180Cys). (See Figure 11.)

We began with plasmid pBG391, an animal cell expression vector that expresses CD4(375). (The DNA sequence of this plasmid is set forth in Figure 12 (SEQ ID NO:12)). We cleaved pBG391 with StuI. StuI cuts the CD4 gene at the codon for amino acid 182. We phosphorylated oligonucleotides T4AID-137 (SEQ ID NO:7) and T4AID-138 (SEQ ID NO:8) (Figure 5) and ligated into the Stul-cleaved pBG391. This generated pBG398C2. We identified pBG398C2 by the presence of a BamHI site, generated at the junction of the StuI site and T4AID- 137.

Then we cleaved pBG398C2 with SacI and Bglll and isolated the 490 bp fragment. We cleaved pEX46 with SacI and BamHI and isolated the large fragment. (The DNA sequence of pEX46 is set forth in Figure 13 (SEQ ID N0:13)). Then we ligated the two fragments together. This generated plasmid λP_ Li180cys.

In 10 liter fermentations, CD4(180Cys) was expressed at about 5% of the total cell protein. We suspended fermentation cells at 8 ml/gm cell wet weight in 20 mM Tris-HCl, 1 mM Na₂EDTA, pH 7.7, broke them in two passes through a French press and washed them twice with 20 ml/gm cell wet weight of •5 l M guanidine-HCl, 1 M urea, 15 mM sodium acetate, pH 5 followed by two washes in 20 mM Tris-HCl, 1 mM Na EDTA, pH 7.7. We extracted the washed pellet with 25 ml/gm cell wet weight of 6 M guanidine-HCl, 20 mM Tris-HCl, 10 mM DTT, pH 7.7 overnight at room 10 temperature. We spun the suspension for 45 minutes in a SS-34 rotor at 20,000 rpm. We diluted the supernatant 1:60 into cold 20 mM Tris-HCl, pH 7.7 and added BSA to a final concentration of 0.5 mg/ml.

To generate microgram amounts of the protein, 15 we concentrated the diluted extract by ultrafiltration using a PM10® membrane (Amicon) followed by affinity purification on 6C6-Sepharose 4B. Alternatively, CD4(180Cys) may be prepared as follows: The pH of the diluted extract obtained as described above is lowered 20 to 7.0 with HCl and loaded at 1% vol/vol onto a Fast S column equilibrated in 20 mM Tris-HCl, pH 7.0. Bound protein is washed with 5 column volumes of equilibration buffer and eluted with 0.2 M NaCl in the same buffer. The elution pool is diluted with one 25 volume of 20 mM Tris-HCl, pH 7.7 and loaded on a

6C6-Sepharose 4B column. The bound protein is washed and eluted from the affinity column in 50 mM glycine, 250 mM NaCl, pH 3.0. The elution fractions are neutralized with 1/15 volume of 0.5 M HEPES pH 7.5, 30 pooled according to the A„_or, profile and stored at 4°C. One may bind CD4(lllcys) or CD4(180cys) to a v thiol-containing gelsolin moiety using the chemistries described in section 1 of this example. 3. Hetero-bifunctional Crosslinking Agents

According to a third method, CD4 may also be crosslinked with a cysteine-containing gelsolin moiety using a hetero-bifunctional crosslinking agent. Such crosslinkers include succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) , m-maleimidobenzoyl- N-hydroxysuccinimide ester (MBS) , or N-succinimidyl 3- (2-pyridyldithiol) proprionate (SPDP) . The succinimidyl arms of these crosslinkers bind to primary amines in CD4. The reactive thiol (maleimide) of SMCC and MBS and the activated thiol of SPDP react with the thiol from the cysteine in the gelsolin moiety to form the covalent linkage.

To carry out the reaction with SMCC and MBS, the crosslinker is incubated with CD4 for 0.5 hours at pH 6.0 at 23°C. Unreacted crosslinker is then removed on a desalting column. SPDP is used as described in the Pharmacia Co. Users Manual. A gelsolin moiety having a free terminal cysteine is then added. The mixture is incubated for 3 hours at 23°C, creating the covalent linkage. Unreacted gelsolin moiety is removed on a desalting column.

The extent and specificity of the modification can be analyzed as described in Example II. The lysine content of CD4 is high; therefore reactions with lysine would not provide much specificity. However, by limiting the amount of crosslinker added, it may be possible to direct crosslinking to one or a small number of lysines that are particularly reactive.

Alternatively, one may bind the reactive thiol group of the hetero-bifunctional crosslinker to a thiol group introduced into CD4 and then bind the succinimidyl arm to an amine in the gelsolin moiety. EXAMPLE IV - MULTIMERIC GELSOLIN FUSION CONSTRUCT

We have shown that CD4-gelsolin fusion polypeptides retain affinity for gpl20 and that they bind PIP₂ vesicles through the gelsolin moiety. This demonstrates that the chemistry we have developed to produce multimeric gelsolin fusion constructs is sound. As a next step, we produced and tested a multimeric CD4(375)-gelsolin fusion construct.

Multimeric gelsolin fusion constructs comprising CD4-gelsolin fusion polypeptides were produced using methods that involve binding the fusion polypeptides to PIP₂ vesicles.

PIP vesicles were produced in the following manner. ^pIP₂ may be obtained as a lyophilized solid (Sigma Chemical Co., St. Louis, Missouri). Water was added to the dried sample to a concentration of 1 to 3 mg/ml and the mixture was sonicated for between 30 seconds to 2 minutes at maximum intensity in a Heat Systems - Ultrasonics, Inc. (Farmingdale, New York) W185® apparatus or its equivalent until an optically clear solution formed. These samples were kept at 4°C and used within a week or they were stored frozen for future use. For storage, the samples were divided into aliquots, frozen in liquid nitrogen and stored at -70° until use. Prior to use, the samples were thawed quickly under a stream of warm water and sonicated for 30 minutes at room temperature in a water bath sonicator.

CD4-gelsolin fusion polypeptides were then added to lipid at a 5 to 10 molar excess of lipid over protein and the mixture was incubated at room temperature for about five minutes.

We tested the ability of the multimeric CD4(375)-gelsolin fusion construct to bind gpl20 in an ELISA-type assay. Briefly, we coated plates with gpl20, added the fusion construct and assayed for binding using anti-CD4 as the reporter antibody. We did not detect binding of the multimeric CD4(375)- gelsolin fusion construct to gpl20. We also tested the biological activity of the fusion construct in a viral replication assay similar to the one described in co-pending United States application 07/583,022 (incorporated herein by reference) . Briefly, we incubated the fusion construct with HIV, added cells from a T-cell line, and measured the incidence of infection. Multimeric CD4(375)- gelsolin fusion construct did not block infection in this assay.

As a result, we found that rsCD4, itself, binds to PIP₂ vesicles and that in doing so, its ability to bind gpl20 is inactivated. Recombinant sCD4 has pockets of positive charge that cause it to bind to cation exchange matrices with high avidity at neutral pH. Since PIP vesicles, like cation exchange matrices, possess high negative charge, we believe that the binding of rsCD4 to PIP₂ vesicles is due to its ionic character.

Therefore, one may produce multimeric CD4- gelsolin fusion constructs that bind gpl20 by altering the charge of the CD4 moiety so that it no longer binds PIP, vesicles. The first one-hundred-thirteen amino acids of rsCD4, which contain the gpl20 binding domain, contain sixteen basic amino acid residues: thirteen lysine residues and three arginine residues. Using site specific mutagenesis, one may alter one or more of these into histidine, a basic, but less polar amino acid, or into neutral amino acids. Among these alternate versions of CD4, one may select molecules that bind gpl20 but do not bind PIP₂ vesicles. We believe that these alternate versions of CD4 would be useful to produce multimeric CD4-gelsolin fusion constructs that possess gpl20 binding ability.

Although they do not bind gpl20, multimeric CD4(181)-gelsolin fusion constructs have other uses. For example, they are useful as immunogens to elicit α- CD4 antibodies. In diagnostic assays, they are useful to detect the presence of α-CD4 in a sample. A percentage of patients infected with HIV exhibit α-CD4 antibodies. Positive charge at neutral pH and high salt concentration is uncommon among proteins. Accordingly, we do not believe that many proteins other than CD4 would exhibit deactivation when employed to produce multimeric-gelsolin fusion constructs according to this invention. Nevertheless, the ionic character and lipid-binding properties of potential functional moieties are factors to be considered in predicting the ultimate biological activity and characteristics of multimeric gelsolin fusion constructs produced using them.

Microorganisms and recombinant DNA molecules according to this invention are exemplified by cultures deposited in the In Vitro International, Inc. culture collection, in Linthicum, Maryland, USA on May 4, 1990, and identified as: pCD4-gelsolin IVI-10253 pl70.2 IVI-10252.

While we have hereinbefore described a number of embodiments of this invention, it is apparent that our basic embodiments can be altered to provide other embodiments which utilize the processes and compositions of this invention. Therefore, it will be appreciated that the scope of this invention includes all alternative embodiments and variations which are defined in the foregoing specification and by the claims appended hereto; and the invention is not to be limited by the specific embodiments which have been presented herein by way of example.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: PEPINSKY, R. BLAKE ROSA, MARGARET D. STOSSEL, THOMAS P.

(ii) TITLE OF INVENTION: MULTIMERIC GELSOLIN FUSION CONSTRUCTS

(iii) NUMBER OF SEQUENCES: 14

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: FISH & NEAVE

(B) STREET: 875 Third Avenue

(C) CITY: New York

(D) STATE: New York

(E) COUNTRY: United States of America

(F) ZIP: 10022

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 07/520,368

(B) FILING DATE: 04-MAY-1990

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Haley Jr., James F.

(B) REGISTRATION NUMBER: 27,794

(C) REFERENCE/DOCKET NUMBER: B144CIP

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (212) 715-0600

(B) TELEFAX: (212) 715-0634

(C) TELEX: 14-8367 (2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2588 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNΞSS: double

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATGGCTCCGC ACCGCCCCGC GCCCGCGCTG CTTTGCGCGC TGTCCCTGGC GCTGTGCGCG 60

CTGTCGCTGC CCGTCCGCGC GGCCACTGCG TCGCGGGGGG CGTCCCAGGC GGGGGCGCCC 120

CAGGGGCGGG TGCCCGAGGC GCGGCCCAAC AGCATGGTGG TGGAACACCC CGAGTTCCTC 180

AAGGCAGGGA AGGAGCCTGG CCTGCAGATC TGGCGTGTGG AGAAGTTCGA TCTGGTGCCC 240

GTGCCCACCA ACCTTTATGG AGACTTCTTC ACGGGCGACG CCTACGTCAT CCTGAAGACA 300

GTGCAGCTGA GGAACGGAAA TCTGCAGTAT GACCTCCACT ACTGGCTGGG CAATGAGTGC 360

AGCCAGGATG AGAGCGGGGC GGCCGCCATC TTTACCGTGC AGCTGGATGA CTACCTGAAC 420

GGCCGGGCCG TGCAGCACCG TGAGGTCCAG GGCTTCGAGT CGGCCACCTT CCTAGGCTAC 480

TTCAAGTCTG GCCTGAAGTA CAAGAAAGGA GGTGTGGCAT CAGGATTCAA GCACGTGGTA 540

CCCAACGAGG TGGTGGTGCA GAGACTCTTC CAGGTCAAAG GGCGGCGTGT GGTCCGTGCC 600

ACCGAGGTAC CTGTGTCCTG GGAGAGCTTC AACAATGGCG ACTGCTTCAT CCTGGACCTG 660

GGCAACAACA TCCACCAGTG GTGTGGTTCC AACAGCAATC GGTATGAAAG ACTGAAGGCC 720

ACACAGGTGT CCAAGGGCAT CCGGGACAAC GAGCGGAGTG GCCGGGCCCG AGTGCACGTG 780

TCTGAGGAGG GCACTGAGCC CGAGGCGATG CTCCAGGTGC TGGGCCCCAA GCCGGCTCTG 840

CCTGCAGGTA CCGAGGACAC CGCCAAGGAG GATGCGGCCA ACCGCAAGCT GGCCAAGCTC 900

TACAAGGTCT CCAATGGTGC AGGGACCATG TCCGTCTCCC TCGTGGCTGA TGAGAACCCC 960

TTCGCCCAGG GGGCCCTGAA GTCAGAGGAC TGCTTCATCC TGGACCACGG CAAAGATGGG 1020

AAAATCTTTG TCTGGAAAGG CAAGCAGGCA AACACGGAGG AGAGGAAGGC TGCCCTCAAA 1080

ACAGCCTCTG ACTTCATCAC CAAGATGGAC TACCCCAAGC AGACTCAGGT CTCGGTCCTT 1140

CCTGAGGGCG GTGAGACCCC ACTGTTCAAG CAGTTCTTCA AGAACTGGCG GGACCCAGAC 1200

CAGACAGATG GCCTGGGCTT GTCCTACCTT TCCAGCCATA TCGCCAACGT GGAGCGGGTG 1260 CCCTTCGACG CCGCCACCCT GCACACCTCC ACTGCCATGG CCGCCCAGCA CGGCATGGAT 1320

GACGATGGCA CAGGCCAGAA ACAGATCTGG AGAATCGAAG GTTCCAACAA GGTGCCCGTG 1380

GACCCTGCCA CATATGGACA GTTCTATGGA GGCGACAGCT ACATCATTCT GTACAACTAC 1440

CGCCATGGTG GCCGCCAGGG GCAGATAATC TATAACTGGC AGGGTGCCCA GTCTACCCAG 1500

GATGAGGTCG CTGCATCTGC CATCCTGACT GCTCAGCTGG ATGAGGAGCT GGGAGGTACC 1560

CCTGTCCAGA GCCGTGTGGT CCAAGGCAAG GAGCCCGCCC ACCTCATGAG CCTGTTTGGT 1620

GGGAAGCCCA TGATCATCTA CAAGGGCGGC ACCTCCCGCG AGGGCGGGCA GACAGCCCCT 1680

GCCAGCACCC GCCTCTTCCA GGTCCGCGCC AACAGCGCTG GAGCCACCCG GGCTGTTGAG 1740

GTATTGCCTA AGGCTGGTGC ACTGAACTCC AACGATGCCT TTGTTCTGAA AACCCCCTCA 1800

GCCGCCTACC TGTGGGTGGG TACAGGAGCC AGCGAGGCAG AGAAGACGGG GGCCCAGGAG 1860

CTGCTCAGGG TGCTGCGGGC CCAACCTGTG CAGGTGGCAG AAGGCAGCGA GCCAGATGGC 1920

TTCTGGGAGG CCCTGGGCGG GAAGGCTGCC TACCGCACAT CCCCACGGCT GAAGGACAAG 1980

AAGATGGATG CCCATCCTCC TCGCCTCTTT GCCTGCTCCA ACAAGATTGG ACGTTTTGTG 2040

ATCGAAGAGG TTCCTGGTGA GCTCATGCAG GAAGACCTGG CAACGGATGA CGTCATGCTT 2100

CTGGACACCT GGGACCAGGT CTTTGTCTGG GTTGGAAAGG ATTCTCAAGA AGAAGAAAAG 2160

ACAGAAGCCT TGACTTCTGC TAAGCGGTAC ATCGAGACGG ACCCAGCCAA TCGGGATCGG 2220

CGGACGCCCA TCACCGTGGT GAAGCAAGGC TTTGAGCCTC CCTCCTTTGT GGGCTGGTTC 2280

CTTGGCTGGG ATGATGATTA CTGGTCTGTG GACCCCTTGG ACAGGGCCAT GGCTGAGCTG 2340

GCTGCCTGAG GAGGGGCAGG GCCCACCCAT GTCACCGGTC AGTGCCTTTT GGAACTGTCC 2400

TTCCCTCAAA GAGGCCTTAG AGCGAGCAGA GCAGCTCTGC TATGAGTGTG TGTGTGTGTG 2460

TGTGTTGTTT CTTTTTTTTT TTTTTACAGT ATCCAAAAAT AGCCCTGCAA AAATTCAGAG 2520

TCCTTGCAAA ATTGTCTAAA ATGTCAGTGT TTGGGAAATT AAATCCAATA AAAACATTTT 2580

GAAGTGTG 2588 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1377 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

ATGAACCGGG GAGTCCCTTT TAGGCACTTG CTTCTGGTGC TGCAACTGGC GCTCCTCCCA 60

GCAGCCACTC AGGGAAAGAA AGTGGTGCTG GGCAAAAAAG GGGATACAGT GGAACTGACC 120

TGTACAGCTT CCCAGAAGAA GAGCATACAA TTCCACTGGA AAAACTCCAA CCAGATAAAG 180

ATTCTGGGAA ATCAGGGCTC CTTCTTAACT AAAGGTCCAT CCAAGCTGAA TGATCGCGCT 240

GACTCAAGAA GAAGCTTGTG GGACCAAGGA AACTTTCCCC TGATCATCAA GAATCTTAAG 300

ATAGAAGACT CAGATACTTA CATCTGTGAA GTGGAGGACC AGAAGGAGGA GGTGCAATTG 360

CTAGTGTTCG GATTGACTGC CAACTCTGAC ACCCACCTGC TTCAGGGGCA GAGCCTGACC 420

CTGACCTTGG AGAGCCCCCC TGGTAGTAGC CCCTCAGTGC AATGTAGGAG TCCAAGGGGT 480

AAAAACATAC AGGGGGGGAA GACCCTCTCC GTGTCTCAGC TGGAGCTCCA GGATAGTGGC 540

ACCTGGACAT GCACTGTCTT GCAGAACCAG AAGAAGGTGG AGTTCAAAAT AGACATCGTG 600

GTGCTAGCTT TCCAGAAGGC CTCCAGCATA GTCTACAAGA AAGAGGGGGA ACAGGTGGAG 660

TTCTCCTTCC CACTCGCCTT TACAGTTGAA AAGCTGACGG GCAGTGGCGA GCTGTGGTGG 720

CAGGCGGAGA GGGCTTCCTC CTCCAAGTCT TGGATCACCT CTGACCTGAA GAACAAGGAA 780

GTGTCTGTAA AACGGGTTAC CCAGGACCCT AAGCTCCAGA TGGGCAAGAA GCTCCCGCTC 840

CACCTCACCC TGCCCCAGGC CTTGCCTCAG TATGCTGGCT CTGGAAACCT CACCCTGGCC 900

CTTGAAGCGA AAACAGGAAA GTTGCATCAG GAAGTGAACC TGGTGGTGAT GAGAGCCACT 960

CAGCTCCAGA AAAATTTGAC CTGTGAGGTG TGGGGACCCA CCTCCCCTAA GCTGATGCTG 1020

AGCTTGAAAC TGGAGAACAA GGAGGCAAAG GTCTCGAAGC GGGAGAAGGC GGTGTGGGTG 1080

CTGAACCCTG AGGCGGGGAT GTGGCAGTGT CTGCTGAGTG ACTCGGGACA GGTCCTGCTG 1140

GAATCCAACA TCAAGGTTCT GCCCACATGG TCGACCCCGG TGCAGCCAAT GGCCCTGATT 1200

GTGCTGGGGG GCGTCGCCGG CCTCCTGCTT TTCATTGGGC TAGGCATCTT CTTCTGTGTC 1260 AGGTGCCGGC ACCGAAGGCG CCAAGCAGAG CGGATGTCTC AGATCAAGAG ACTCCGCAGT 1320 GAGAAGAAGA CCTGCCAGTG CCCTCACCGG TTTCAGAAGA CATGTAGCCC CATTTGA 1377

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 37 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: AGATCTACGG GGGCGTGGCA TCAGGATTCA AGCACGT 37

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: GAATTCTTAG GCACGGACCA CACGCCG 27

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GGGGTGTTGA TAGTAAGATC TTGCA 25

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: AGATCTTACT ATCAAGA 17

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ATCCCTGTCC GTAGAAGCTT ATCGAT 26

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: ATCGATAAGC TTCTACGGAC AGGGAT 26

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 46 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GGAGGACCAG AAAGAAGAAG TTCAGCTGCT GGTTTTCGGA TTGACT 46

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 654 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

ATGAAAAAAG TAGTACTGGG CAAAAAAGGG GATACAGTGG AACTGACCTG TACAGCTTCC 60

CAGAAGAAGA GCATACAATT CCACTGGAAA AACTCCAACC AGATAAAGAT TCTGGGAAAT 120

CAGGGCTCCT TCTTAACTAA AGGTCCATCC AAGCTGAATG ATCGCGCTGA CTCAAGAAGA 180

AGCTTGTGGG ACCAAGGAAA CTTTCCCCTG ATCATCAAGA ATCTTAAGAT AGAAGACTCA 240

GATACTTACA TCTGTGAAGT GGAGGACCAG AAAGAAGAAG TTCAGCTGCT GGTTTTCGGA 300

TTGACTGCCA ACTCTGACAC CCACCTGCTT CAGGGGCAGA GCCTGACCCT GACCTTGGAG 360

AGCCCCCCTG GTAGTAGCCC CTCAGTGCAA TGTAGGAGTC CAAGGGGTAA AAACATACAG 420

GGGGGGAAGA CCCTCTCCGT GTCTCAGCTG GAGCTCCAGG ATAGTGGCAC CTGGACATGC 480

ACTGTCTTGC AGAACCAGAA GAAGGTGGAG TTCAAAATAG ACATCGTGGT GCTAGCTTTC 540

CAGAAGGGGA AGATCTACGG GGGCGTGGCA TCAGGATTCA AGCACGTGGT ACCCAACGAG 600

GTGGTGGTGC AGAGACTCTT CCAGGTCAAA GGGCGGCGTG TGGTCCGTGC CTAA 654

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4309 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

GAATTCTTAG ACTTAGTTAA ATTGCTAACT TTATAGATTA CAAAACTTAG GAAATCGATT 60

TGGATGAAAA AAGTAGTACT GGGCAAAAAA GGGGATACAG TGGAACTGAC CTGTACAGCT 120

TCCCAGAAGA AGAGCATACA ATTCCACTGG AAAAACTCCA ACCAGATAAA GATTCTGGGA 180

AATCAGGGCT CCTTCTTAAC TAAAGGTCCA TCCAAGCTGA ATGATCGCGC TGACTCAAGA 240

AGAAGCTTGT GGGACCAAGG AAACTTTCCC CTGATCATCA AGAATCTTAA GATAGAAGAC 300

TCAGATACTT ACATCTGTGA AGTGGAGGAC CAGAAGGAGG AGGTGCAATT GCTAGTGTTC 360

GGATTGACTG CCAACTCTGA CACCCACCTG CTTCAGGGGT GATAGTAAGA TCCTGCAGCC 420

CAGCTTGGGG ACCCTAGAGG TCCCCTTTTT TATTTTGAAT TGGGAGATCC CAATTCTCAT 480

GTTTGACAGC TTATCATCGA TAAGCTAGCT TTAATGCGGT AGTTTATCAC AGTTAAATTG 540

CTAACGCAGT CAGGCACCGT GTATGAAATC TAACAATGCG CTCATCGTCA TCCTCGGCAC 600

CGTCACCCTG GATGCTGTAG GCATAGGCTT GGTTATGCCG GTACTGCCGG GCCTCTTGCG 660

GGATATCGTC CATTCCGACA GCATCGCCAG TCACTATGGC GTGCTGCTAG CGCTATATGC 720

GTTGATGCAA TTTCTATGCG CACCCGTTCT CGGAGCACTG TCCGACCGCT TTGGCCGCCG 780

CCCAGTCCTG CTCGCTTCGC TACTTGGAGC CACTATCGAC TACGCGATCA TGGCGACCAC 840

ACCCGTCCTG TGGATTCTCT ACGCCGGACG CATCGTGGCC GGCATCACCG GCGCCACAGG 900

TGCGGTTGCT GGCGCCTATA TCGCCGACAT CACCGATGGG GAAGATCGGG CTCGCCACTT 960

CGGGCTCATG AGCGCTTGTT TCGGCGTGGG TATGGTGGCA GGCCCCGTGG CCGGGGGACT 1020

GTTGGGCGCC ATCTCCTTGC ACGCACCATT CCTTGCGGCG GCGGTGCTCA ACGGCCTCAA 1080

CCTACTACTG GGCTGCTTCC TAATGCAGGA GTCGCATAAG GGAGAGCGTC GTCCGATGCC 1140

CTTGAGAGCC TTCAACCCAG TCAGCTCCTT CCGGTGGGCG CGGGGCATGA CTATCGTCGC 1200

CGCACTTATG ACTGTCTTCT TTATCATGCA ACTCGTAGGA CAGGTGCCGG CAGCGCTCTG 1260 GGTCATTTTC GGCGAGGACC GCTTTCGCTG GAGCGCGACG ATGATCGGCC TGTCGCTTGC 1320

GGTATTCGGA ATCTTGCACG CCCTCGCTCA AGCCTTCGTC ACTGGTCCCG CCACCAAACG 1380

TTTCGGCGAG AAGCAGGCCA TTATCGCCGG CATGGCGGCC GACGCGCTGG GCTACGTCTT 1440

GCTGGCGTTC GCGACGCGAG GCTGGATGGC CTTCCCCATT ATGATTCTTC TCGCTTCCGG 1500

CGGCATCGGG ATGCCCGCGT TGCAGGCCAT GCTGTCCAGG CAGGTAGATG ACGACCATCA 1560

GGGACAGCTT CAAGGATCGC TCGCGGCTCT TACCAGCCTA ACTTCGATCA CTGGACCGCT 1620

GATCGTCACG GCGATTTATG CCGCCTCGGC GAGCACATGG AACGGGTTGG CATGGATTGT 1680

AGGCGCCGCC CTATACCTTG TCTGCCTCCC CGCGTTGCGT CGCGGTGCAT GGAGCCGGGC 1740

CACCTCGACC TGAATGGAAG CCGGCGGCAC CTCGCTAACG GATTCACCAC TCCAAGAATT 1800

GGAGCCAATC AATTCTTGCG GAGAACTGTG AATGCGCAAA CCAACCCTTG GCAGAACATA 1860

TCCATCGCGT CCGCCATCTC CAGCAGCCGC ACGCGGCGCA TCTCGGGGGA TGATCAGCTG 1920

CCTCGCGCGT TTCGGTGATG ACGGTGAAAA CCTCTGACAC ATGCAGCTCC CGGAGACGGT 1980

CACAGCTTGT CTGTAAGCGG ATGCCGGGAG CAGACAAGCC CGTCAGGGCG CGTCAGCGGG 2040

TGTTGGCGGG TGTCGGGGCG CAGCCATGAC CCAGTCACGT AGCGATAGCG GAGTGTATAC 2100

TGGCTTAACT ATGCGGCATC AGAGCAGATT GTACTGAGAG TGCACCATAT GCGGTGTGAA 2160

ATACCGCACA GATGCGTAAG GAGAAAATAC CGCATCAGGC GCTCTTCCGC TTCCTCGCTC 2220

ACTGACTCGC TGCGCTCGGT CGTTCGGCTG CGGCGAGCGG TATCAGCTCA CTCAAAGGCG 2280

GTAATACGGT TATCCACAGA ATCAGGGGAT AACGCAGGAA AGAACATGTG AGCAAAAGGC 2340

CAGCAAAAGG CCAGGAACCG TAAAAAGGCC GCGTTGCTGG CGTTTTTCCA TAGGCTCCGC 2400

CCCCCTGACG AGCATCACAA AAATCGACGC TCAAGTCAGA GGTGGCGAAA CCCGACAGGA 2460

CTATAAAGAT ACCAGGCGTT TCCCCCTGGA AGCTCCCTCG TGCGCTCTCC TGTTCCGACC 2520

CTGCCGCTTA CCGGATACCT GTCCGCCTTT CTCCCTTCGG GAAGCGTGGC GCTTTCTCAA 2580

TGCTCACGCT GTAGGTATCT CAGTTCGGTG TAGGTCGTTC GCTCCAAGCT GGGCTGTGTG 2640

CACGAACCCC CCGTTCAGCC CGACCGCTGC GCCTTATCCG GTAACTATCG TCTTGAGTCC 2700

AACCCGGTAA GACACGACTT ATCGCCACTG GCAGCAGCCA CTGGTAACAG GATTAGCAGA 2760

GCGAGGTATG TAGGCGGTGC TACAGAGTTC TTGAAGTGGT GGCCTAACTA CGGCTACACT 2820

AGAAGGACAG TATTTGGTAT CTGCGCTCTG CTGAAGCCAG TTACCTTCGG AAAAAGAGTT 2880 GGTAGCTCTT GATCCGGCAA ACAAACCACC GCTGGTAGCG GTGGTTTTTT TGTTTGCAAG 2940

CAGCAGATTA CGCGCAGAAA AAAAGGATCT CAAGAAGATC CTTTGATCTT TTCTACGGGG 3000

TCTGACGCTC AGTGGAACGA AAACTCACGT TAAGGGATTT TGGTCATGAG ATTATCAAAA 3060

AGGATCTTCA CCTAGATCCT TTTCAGATCT CCCGATCTTT AGCTGTCTTG GTTTGCCCAA 3120

AGCGCATTGC ATAATCTTTC AGGGTTATGC GTTGTTCCAT ACAACCTCCT TAGTACATGC 3180

AACCATTATC ACCGCCAGAG GTAAAATAGT CAACACGCAC GGTGTTAGAT ATTTATCCCT 3240

TGCGGTGATA GATTTAACGT ATGAGCACAA AAAAGAAACC ATTAACACAA GAGCAGCTTG 3300

AGGACGCACG TCGCCTTAAA GCAATTTATG AAAAAAAGAA AAATGAACTT GGCTTATCCC 3360

AGGAATCTGT CGCAGACAAG ATGGGGATGG GGCAGTCAGG CGTTGGTGCT TTATTTAATG 3420

GCATCAATGC ATTAAATGCT TATAACGCCG CATTGCTTAC AAAAATTCTC AAAGTTAGCG 3480

TTGAAGAATT TAGCCCTTCA ATCGCCAGAG AAATCTACGA GATGTATGAA GCGGTTAGTA 3540

TGCAGCCGTC ACTTAGAAGT GAGTATGAGT ACCCTGTTTT TTCTCATGTT CAGGCAGGGA 3600

TGTTCTCACC TAAGCTTAGA ACCTTTACCA AAGGTGATGC GGAGAGATGG GTAAGCACAA 3660

CCAAAAAAGC CAGTGATTCT GCATTCTGGC TTGAGGTTGA AGGTAATTCC ATGACCGCAC 3720

CAACAGGCTC CAAGCCAAGC TTTCCTGACG GAATGTTAAT TCTCGTTGAC CCTGAGCAGG 3780

CTGTTGAGCC AGGTGATTTC TGCATAGCCA GACTTGGGGG TGATGAGTTT ACCTTCAAGA 3840

AACTAATTAG GGATAGCGGT CAGGTGTTTT TACAACCACT AAACCCACAG TACCCAATGA 3900

TCCCATGCAA TGAGAGTTGT TCCGTTGTGG GGAAAGTTAT CGCTAGTCAG TGGCCTGAAG 3960

AGACGTTTGG CTGATCGGCA AGGTGTTCTG GTCGGCGCAT AGCTGATAAC AATTGAGCAA 4020

GAATCTTCAT CGGGGCTGCA GCCCACGATG CGTCCGGCGT AGAGGATCTC TCACCTACCA 4080

AACAATGCCC CCCTGCAAAA AATAAATTCA TATAAAAAAC ATACAGATAA CCATCTGCGG 4140

TGATAAATTA TCTCTGGCGG TGTTGACATA AATACCACTG GCGGTGATAC TGAGCACATC 4200

AGCAGGACGC ACTGACCACC ATGAAGGTGA CGCTCTTAAA ATTAAGCCCT GAAGAAGGGC 4260

AGCATTCAAA GCAGAAGGCT TTGGGGTGTG TGATACGAAA CGAAGCATT 4309 (2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6151 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

GAATTAATTC CAGCTTGCTG TGGAATGTGT GTCAGTTAGG GTGTGGAAAG TCCCCAGGCT 60

CCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA GTCAGCAACC AGGTGTGGAA 120

AGTCCCCAGG CTCCCCAGCA GGCAGAAGTA TGCAAAGCAT GCATCTCAAT TAGTCAGCAA 180

CCATAGTCCC GCCCCTAACT CCGCCCATCC CGCCCCTAAC TCCGCCCAGT TCCGCCCATT 240

CTCCGCCCCA TGGCTGACTA ATTTTTTTTA TTTATGCAGA GGCCGAGGCC GCCTCGGCCT 300

CTGAGCTATT CCAGAAGTAG TGAGGAGGCT TTTTTGGAGG GGTCCTCCTC GTATAGAAAC 360

TCGGACCACT CTGAGACGAA GGCTCGCGTC CAGGCCAGCA CGAAGGAGGC TAAGTGGGAG 420

GGGTAGCGGT CGTTGTCCAC TAGGGGGTCC ACTCGCTCCA GGGTGTGAAG ACACATGTCG 480

CCCTCTTCGG CATCAAGGAA GGTGATTGGT TTATAGGTGT AGGCCACGTG ACCGGGTGTT 540

CCTGAAGGGG GGCTATAAAA GGGGGTGGGG GCGCGTTCGT CCTCACTCTC TTCCGCATCG 600

CTGTCTGCGA GGGCCAGCTG TTGGGCTCGC GGTTGAGGAC AAACTCTTCG CGGTCTTTCC 660

AGTACTCTTG GATCGGAAAC CCGTCGGCCT CCGAACGGTA CTCCGCCACC GAGGGACCTG 720

AGCGAGTCCG CATCGACCGG ATCGGAAAAC CTCTCGAGAA AGGCGTCTAA CCAGTCACAG 780

TCGCAAGGTA GGCTGAGCAC CGTGGCGGGC GGCAGCGGGT GGCGGTCGGG GTTGTTTCTG 840

GCGGAGGTGC TGCTGATGAT GTAATTAAAG TAGGCGGTCT TGAGACGGCG GATGGTCGAG 900

GTGAGGTGTG GCAGGCTTGA GATCGATCTG GCCATACACT TGAGTGACAA TGACATCCAC 960

TTTGCCTTTC TCTCCACAGG TGTCCACTCC CAGGTCCAAC TGGATCCAAG CTTCGACTCG 1020

AGGAATTCCC CGAAGGAACA AAGCACCCTC CCCACTGGGC TCCTGGTTGC AGAGCTCCAA 1080

GTCCTCACAC AGATACGCCT GTTTGAGAAG CAGCGGGCAA GAAAGACGCA AGCCCAGAGG 1140

CCCTGCCATT TCTGTGGGCT CAGGTCCCTA CTGGCTCAGG CCCCTGCCTC CCTCGGCAAG 1200

GCCACAATGA ACCGGGGAGT CCCTTTTAGG CACTTGCTTC TGGTGCTGCA ACTGGCGCTC 1260 CTCCCAGCAG CCACTCAGGG AAAGAAAGTG GTGCTGGGCA AAAAAGGGGA TACAGTGGAA 1320

CTGACCTGTA CAGCTTCCCA GAAGAAGAGC ATACAATTCC ACTGGAAAAA CTCCAACCAG 1380

ATAAAGATTC TGGGAAATCA GGGCTCCTTC TTAACTAAAG GTCCATCCAA GCTGAATGAT 1440

CGCGCTGACT CAAGAAGAAG CTTGTGGGAC CAAGGAAACT TTCCCCTGAT CATCAAGAAT 1500

CTTAAGATAG AAGACTCAGA TACTTACATC TGTGAAGTGG AGGACCAGAA GGAGGAGGTG 1560

CAATTGCTAG TGTTCGGATT GACTGCCAAC TCTGACACCC ACCTGCTTCA GGGGCAGAGC 1620

CTGACCCTGA CCTTGGAGAG CCCCCCTGGT AGTAGCCCCT CAGTGCAATG TAGGAGTCCA 1680

AGGGGTAAAA ACATACAGGG GGGGAAGACC CTCTCCGTGT CTCAGCTGGA GCTCCAGGAT 1740

AGTGGCACCT GGACATGCAC TGTCTTGCAG AACCAGAAGA AGGTGGAGTT CAAAATAGAC 1800

ATCGTGGTGC TAGCTTTCCA GAAGGCCTCC AGCATAGTCT ATAAGAAAGA GGGGGAACAG 1860

GTGGAGTTCT CCTTCCCACT CGCCTTTACA GTTGAAAAGC TGACGGGCAG TGGCGAGCTG 1920

TGGTGGCAGG CGGAGAGGGC TTCCTCCTCC AAGTCTTGGA TCACCTTTGA CCTGAAGAAC 1980

AAGGAAGTGT CTGTAAAACG GGTTACCCAG GACCCTAAGC TCCAGATGGG CAAGAAGCTC 2040

CCGCTCCACC TCACCCTGCC CCAGGCCTTG CCTCAGTATG CTGGCTCTGG AAACCTCACC 2100

CTGGCCCTTG AAGCGAAAAC AGGAAAGTTG CATCAGGAAG TGAACCTGGT GGTGATGAGA 2160

GCCACTCAGC TCCAGAAAAA TTTGACCTGT GAGGTGTGGG GACCCACCTC CCCTAAGCTG 2220

ATGCTGAGTT TGAAACTGGA GAACAAGGAG GCAAAGGTCT CGAAGCGGGA GAAGGCGGTG 2280

TGGGTGCTGA ACCCTGAGGC GGGGATGTGG CAGTGTCTGC TGAGTGACTC GGGACAGGTC 2340

CTGCTGGAAT CCAACATCAA GGTTCTGCCC ACATGGTCGA CCCCGGTGCA GCCAATGGCC 2400

CTGATTTGAG ATCTTTGTGA AGGAACCTTA CTTCTGTGGT GTGACATAAT TGGACAAACT 2460

ACCTACAGAG ATTTAAAGCT CTAAGGTAAA TATAAAATTT TTAAGTGTAT AATGTGTTAA 2520

ACTACTGATT CTAATTGTTT GTGTATTTTA GATTCCAACC TATGGAACTG ATGAATGGGA 2580

GCAGTGGTGG AATGCCTTTA ATGAGGAAAA CCTGTTTTGC TCAGAAGAAA TGCCATCTAG 2640

TGATGATGAG GCTACTGCTG ACTCTCAACA TTCTACTCCT CCAAAAAAGA AGAGAAAGGT 2700

AGAAGACCCC AAGGACTTTC CTTCAGAATT GCTAAGTTTT TTGAGTCATG CTGTGTTTAG 2760

TAATAGAACT CTTGCTTGCT TTGCTATTTA CACCACAAAG GAAAAAGCTG CACTGCTATA 2820

CAAGAAAATT ATGGAAAAAT ATTCTGTAAC CTTTATAAGT AGGCATAACA GTTATAATCA 2880 TAACATACTG TTTTTTCTTA CTCCACACAG GCATAGAGTG TCTGCTATTA ATAACTATGC 2940

TCAAAAATTG TGTACCTTTA GCTTTTTAAT TTGTAAAGGG GTTAATAAGG AATATTTGAT 3000

GTATAGTGCC TTGACTAGAG ATCATAATCA GCCATACCAC ATTTGTAGAG GTTTTACTTG 3060

CTTTAAAAAA CCTCCCACAC CTCCCCCTGA ACCTGAAACA TAAAATGAAT GCAATTGTTG 3120

TTGTTAACTT GTTTATTGCA GCTTATAATG GTTACAAATA AAGCAATAGC ATCACAAATT 3180

TCACAAATAA AGCATTTTTT TCACTGCATT CTAGTTGTGG TTTGTCCAAA CTCATCAATG 3240

TATCTTATCA TGTCTGGATC CTCTACGCCG GACGCATCGT GGCCGGCATC ACCGGCGCCA 3300

CAGGTGCGGT TGCTGGCGCC TATATCGCCG ACATCACCGA TGGGGAAGAT CGGGCTCGCC 3360

ACTTCGGGCT CATGAGCGCT TGTTTCGGCG TGGGTATGGT GGCAGGCCCG TGGCCGGGGG 3420

ACTGTTGGGC GCCATCTCCT TGCATGCACC ATTCCTTGCG GCGGCGGTGC TCAACGGCCT 3480

CAACCTACTA CTGGGCTGCT TCCTAATGCA GGAGTCGCAT AAGGGAGAGC GTCGACCGAT 3540

GCCCTTGAGA GCCTTCAACC CAGTCAGCTC CTTCCGGTGG GCGCGGGGCA TGACTATCGT 3600

CGCCGCACTT ATGACTGTCT TCTTTATCAT GCAACTCGTA GGACAGGTGC CGGCAGCGCT 3660

CTGGGTCATT TTCGGCGAGG ACCGCTTTCG CTGGAGCGCG ACGATGATCG GCCTGTCGCT 3720

TGCGGTATTC GGAATCTTGC ACGCCCTCGC TCAAGCCTTC GTCACTGGTC CCGCCACCAA 3780

ACGTTTCGGC GAGAAGCAGG CCATTATCGC CGGCATGGCG GCCGACGCGC TGGGCTACGT 3840

CTTGCTGGCG TTCGCGACGC GAGGCTGGAT GGCCTTCCCC ATTATGATTC TTCTCGCTTC 3900

CGGCGGCATC GGGATGCCCG CGTTGCAGGC CATGCTGTCC AGGCAGGTAG ATGACGACCA 3960

TCAGGGACAG CTTCAAGGAT CGCTCGCGGC TCTTACCAGC CTAACTTCGA TCACTGGACC 4020

GCTGATCGTC ACGGCGATTT ATGCCGCCTC GGCGAGCACA TGGAACGGGT TGGCATGGAT 4080

TGTAGGCGCC GCCCTATACC TTGTCTGCCT CCCCGCGTTG CGTCGCGGTG CATGGAGCCG 4140

GGCCACCTCG ACCTGAATGG AAGCCGGCGG CACCTCGCTA ACGGATTCAC CACTCCAAGA 4200

ATTGGAGCCA ATCAATTCTT GCGGAGAACT GTGAATGCGC AAACCAACCC TTGGCAGAAC 4260

ATATCCATCG CGTCCGCCAT CTCCAGCAGC CGCACGCGGC GCATCTCGGG CCGCGTTGCT 4320

GGCGTTTTTC CATAGGCTCC GCCCCCCTGA CGAGCATCAC AAAAATCGAC GCTCAAGTCA 4380

GAGGTGGCGA AACCCGACAG GACTATAAAG ATACCAGGCG TTTCCCCCTG GAAGCTCCCT 4440

CGTGCGCTCT CCTGTTCCGA CCCTGCCGCT TACCGGATAC CTGTCCGCCT TTCTCCCTTC 4500 GGGAAGCGTG GCGCTTTCTC AATGCTCACG CTGTAGGTAT CTCAGTTCGG TGTAGGTCGT 4560

TCGCTCCAAG CTGGGCTGTG TGCACGAACC CCCCGTTCAG CCCGACCGCT GCGCCTTATC 4620

CGGTAACTAT CGTCTTGAGT CCAACCCGGT AAGACACGAC TTATCGCCAC TGGCAGCAGC 4680

CACTGGTAAC AGGATTAGCA GAGCGAGGTA TGTAGGCGGT GCTACAGAGT TCTTGAAGTG 4740

GTGGCCTAAC TACGGCTACA CTAGAAGGAC AGTATTTGGT ATCTGCGCTC TGCTGAAGCC 4800

AGTTACCTTC GGAAAAAGAG TTGGTAGCTC TTGATCCGGC AAACAAACCA CCGCTGGTAG 4860

CGGTGGTTTT TTTGTTTGCA AGCAGCAGAT TACGCGCAGA AAAAAAGGAT CTCAAGAAGA 4920

TCCTTTGATC TTTTCTACGG GGTCTGACGC TCAGTGGAAC GAAAACTCAC GTTAAGGGAT 4980

TTTGGTCATG AGATTATCAA AAAGGATCTT CACCTAGATC CTTTTAAATT AAAAATGAAG 5040

TTTTAAATCA ATCTAAAGTA TATATGAGTA AACTTGGTCT GACAGTTACC AATGCTTAAT 5100

CAGTGAGGCA CCTATCTCAG CGATCTGTCT ATTTCGTTCA TCCATAGTTG CCTGACTCCC 5160

CGTCGTGTAG ATAACTACGA TACGGGAGGG CTTACCATCT GGCCCCAGTG CTGCAATGAT 5220

ACCGCGAGAC CCACGCTCAC CGGCTCCAGA TTTATCAGCA ATAAACCAGC CAGCCGGAAG 5280

GGCCGAGCGC AGAAGTGGTC CTGCAACTTT ATCCGCCTCC ATCCAGTCTA TTAATTGTTG 5340

CCGGGAAGCT AGAGTAAGTA GTTCGCCAGT TAATAGTTTG CGCAACGTTG TTGCCATTGC 5400

TGCAGGCATC GTGGTGTCAC GCTCGTCGTT TGGTATGGCT TCATTCAGCT CCGGTTCCCA 5460

ACGATCAAGG CGAGTTACAT GATCCCCCAT GTTGTGCAAA AAAGCGGTTA GCTCCTTCGG 5520

TCCTCCGATC GTTGTCAGAA GTAAGTTGGC CGCAGTGTTA TCACTCATGG TTATGGCAGC 5580

ACTGCATAAT TCTCTTACTG TCATGCCATC CGTAAGATGC TTTTCTGTGA CTGGTGAGTA 5640

CTCAACCAAG TCATTCTGAG AATAGTGTAT GCGGCGACCG AGTTGCTCTT GCCCGGCGTC 5700

AACACGGGAT AATACCGCGC CACATAGCAG AACTTTAAAA GTGCTCATCA TTGGAAAACG 5760

TTCTTCGGGG CGAAAACTCT CAAGGATCTT ACCGCTGTTG AGATCCAGTT CGATGTAACC 5820

CACTCGTGCA CCCAACTGAT CTTCAGCATC TTTTACTTTC ACCAGCGTTT CTGGGTGAGC 5880

AAAAACAGGA AGGCAAAATG CCGCAAAAAA GGGAATAAGG GCGACACGGA AATGTTGAAT 5940

ACTCATACTC TTCCTTTTTC AATATTATTG AAGCATTTAT CAGGGTTATT GTCTCATGAG 6000

CGGATACATA TTTGAATGTA TTTAGAAAAA TAAACAAATA GGGGTTCCGC GCACATTTCC 6060

CCGAAAAGTG CCACCTGACG TCTAAGAAAC CATTATTATC ATGACATTAA CCTATAAAAA 6120 TAGGCGTATC ACGAGGCCCT TTCGTCTTCA A 6151

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5727 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: circular

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

GAATTCTTAC ACTTAGTTAA ATTGCTAACT TTATAGATTA CAAAACTTAG GAAATCGATT 60

TGGATGAAAA AAGTAGTACT GGGCAAAAAA GGGGATACAG TGGAACTGAC CTGTACAGCT 120

TCCCAGAAGA AGAGCATACA ATTCCACTGG AAAAACTCCA ACCAGATAAA GATTCTGGGA 180

AATCAGGGCT CCTTCTTAAC TAAAGGTCCA TCCAAGCTGA ATGATCGCGC TGACTCAAGA 240

AGAAGCTTGT GGGACCAAGG AAACTTTCCC CTGATCATCA AGAATCTTAA GATAGAAGAC 300

TCAGATACTT ACATCTGTGA AGTGGAGGAC CAGAAGGAGG AGGTGCAATT GCTAGTGTTC 360

GGATTGACTG CCAACTCTGA CACCCACCTG CTTCAGGGGC AGAGCCTGAC CCTGACCTTG 420

GAGAGCCCCC CTGGTAGTAG CCCCTCAGTG CAATGTAGGA GTCCAAGGGG TAAAAACATA 480

CAGGGGGGGA AGACCCTCTC CGTGTCTCAG CTGGAGCTCC AGGATAGTGG CACCTGGACA 540

TGCACTGTCT TGCAGAACCA GAAGAAGGTG GAGTTCAAAA TAGACATCGT GGTGCTAGCT 600

TTCCAGAAGG GGAAGATCTT TCCCGAGGGC GGCAGCCTGG CCGCGCTGAC CGCGCACCAG 660

GCTTGCCACC TGCCGCTGGA GACTTTCACC CGTCATCGCC AGCCGCGCGG CTGGGAACAA 720

CTGGAGCAGT GCGGCTATCC GGTGCAGCGG CTGGTCGCCC TCTACCTGGC GGCGCGGCTG 780

TCGTGGAACC AGGTCGACCA GGTGATCCGC AACGCCCTGG CCAGCCCCGG CAGCGGCGGC 840

GACCTGGGCG AAGCGATCCG CGAGCAGCCG GAGCAGGCCC GTCTGGCCCT GACCCTGGCC 900

GCCGCCGAGA GCGAGCGCTT CGTCCGGCAG GGCACCGGCA ACGACGAGGC CGGCGCGGCC 960

AACGCCGACG TGGTGAGCCT GACCTGCCCG GTCGCCGCCG GTGAATGCGC GGGCCCGGCG 1020

GACAGCGGCG ACGCCCTGCT GGAGCGCAAC TATCCCACTG GCGCGGAGTT CCTCGGCGAC 1080

GGCGGCGACG TCAGCTTCAG CACCCGCGGC ACGCAGAACT GGACGGTGGA GCGGCTGCTC 1140

CAGGCGCACC GCCAACTGGA GGAGCGCGGC TATGTGTTCG TCGGCTACCA CGGCACCTTC 1200

CTCGAAGCGG CGCAAAGCAT CGTCTTCGGC GGGGTGCGCG CGCGCAGCCA GGACCTCGAC 1260 GCGATCTGGC GCGGTTTCTA TATCGCCGGC GATCCGGCGC TGGCCTACGG CTACGCCCAG 1320

GACCAGGAAC CCGACGCACG CGGCCGGATC CGCAACGGTG CCCTGCTGCG GGTCTATGTG 1380

CCGCGCTCGA GCCTGCCGGG CTTCTACCGC ACCAGCCTGA CCCTGGCCGC GCCGGAGGCG 1440

GCGGGCGAGG TCGAACGGCT GATCGGCCAT CCGCTGCCGC TGCGCCTGGA CGCCATCACC 1500

GGCCCCGAGG AGGAAGGCGG GCGCCTGGAG ACCATTCTCG GCTGGCCGCT GGCCGAGCGC 1560

ACCGTGGTGA TTCCCTCGGC GATCCCCACC GACCCGCGCA ACGTCGGCGG CGACCTCGAC 1620

CCGTCCAGCA TCCCCGACAA GGAACAGGCG ATCAGCGCCC TGCCGGACTA CGCCAGCCAG 1680

CCCGGCAAAC CGCCGCGCGA GGACCTGAAG TAACTGCCGC GACCGGCCGG CTCCCTTCGC 1740

AGGAGCCGGC CTTCTCGGGG CCTGGCCATA CATCAGGTTT TCCTGATGCC AGCCCAATCG 1800

AATATGAATT CTCATCGATT TCCATGGGAT CCTGCAGCCC AGCTTGGGGA CCCTAGAGGT 1860

CCCCTTTTTT ATTTTTTGAA TTGGGAGATC CAATTCTCAT GTTTGACAGC TTATCATCGA 1920

AGCTAGCTTT AATGCGGTAG TTTATCACAG TTAAATTGCT AACGCAGTCA GGCACCGTGT 1980

ATGAAATCTA ACAATGCGCT CATCGTCATC CTCGGCACCG TCACCCTGGA TGCTGTAGGC 2040

ATAGGCTTGG TTATGCCGGT ACTGCCGGGC CTCTTGCGGG ATATCGTCCA TTCCGACAGC 2100

ATCGCCAGTC ACTATGGCGT GCTGCTAGCG CTATATGCGT TGATGCAATT TCTATGCGCA 2160

CCCGTTCTCG GAGCACTGTC CGACCGCTTT GGCCGCCGCC CAGTCCTGCT CGCTTCGCTA 2220

CTTGGAGCCA CTATCGACTA CGCGATCATG GCGACCACAC CCGTCCTGTG GATTCTCTAC 2280

GCCGGACGCA TCGTGGCCGG CATCACCGGC GCCACAGGTG CGGTTGCTGG CGCCTATATC 2340

GCCGACATCA CCGATGGGGA AGATCGGGCT CGCCACTTCG GGCTCATGAG CGCTTGTTTC 2400 GGCGTGGGTA TGGTGGCAGG CCCCGTGGCC GGGGGACTGT TGGGCGCCAT CTCCTTGCAC 2460 GCACCATTCC TTGCGGCGGC GGTGCTCAAC GGCCTCAACC TACTACTGGG CTGCTTCCTA 2520 ATGCAGGAGT CGCATAAGGG AGAGCGTCGT CCGATGCCCT TGAGAGCCTT CAACCCAGTC 2580 AGCTCCTTCC GGTGGGCGCG GGGCATGACT ATCGTCGCCG CACTTATGAC TGTCTTCTTT 2640 ATCATGCAAC TCGTAGGACA GGTGCCGGCA GCGCTCTGGG TCATTTTCGG CGAGGACCGC 2700 TTTCGCTGGA GCGCGACGAT GATCGGCCTG TCGCTTGCGG TATTCGGAAT CTTGCACGCC 2760 CTCGCTCAAG CCTTCGTCAC TGGTCCCGCC ACCAAACGTT TCGGCGAGAA GCAGGCCATT 2820 ATCGCCGGCA TGGCGGCCGA CGCGCTGGGC TACGTCTTGC TGGCGTTCGC GACGCGAGGC 2880 TGGATGGCCT TCCCCATTAT GATTCTTCTC GCTTCCGGCG GCATCGGGAT GCCCGCGTTG 2940

CAGGCCATGC TGTCCAGGCA GGTAGATGAC GACCATCAGG GACAGCTTCA AGGATCGCTC 3000

GCGGCTCTTA CCAGCCTAAC TTCGATCACT GGACCGCTGA TCGTCACGGC GATTTATGCC 3060

GCCTCGGCGA GCACATGGAA CGGGTTGGCA TGGATTGTAG GCGCCGCCCT ATACCTTGTC 3120

TGCCTCCCCG CGTTGCGTCG CGGTGCATGG AGCCGGGCCA CCTCGACCTG AATGGAAGCC 3180

GGCGGCACCT CGCTAACGGA TTCACCACTC CAAGAATTGG AGCCAATCAA TTCTTGCGGA 3240

GAACTGTGAA TGCGCAAACC AACCCTTGGC AGAACATATC CATCGCGTCC GCCATCTCCA 3300

GCAGCCGCAC GCGGCGCATC TCGGGGGATG ATCAGCTGCC TCGCGCGTTT CGGTGATGAC 3360

GGTGAAAACC TCTGACACAT GCAGCTCCCG GAGACGGTCA CAGCTTGTCT GTAAGCGGAT 3420

GCCGGGAGCA GACAAGCCCG TCAGGGCGCG TCAGCGGGTG TTGGCGGGTG TCGGGGCGCA 3480

GCCATGACCC AGTCACGTAG CGATAGCGGA GTGTATACTG GCTTAACTAT GCGGCATCAG 3540

AGCAGATTGT ACTGAGAGTG CACCATATGC GGTGTGAAAT ACCGCACAGA TGCGTAAGGA 3600

GAAAATACCG CATCAGGCGC TCTTCCGCTT CCTCGCTCAC TGACTCGCTG CGCTCGGTCG 3660

TTCGGCTGCG GCGAGCGGTA TCAGCTCACT CAAAGGCGGT AATACGGTTA TCCACAGAAT 3720

CAGGGGATAA CGCAGGAAAG AACATGTGAG CAAAAGGCCA GCAAAAGGCC AGGAACCGTA 3780

AAAAGGCCGC GTTGCTGGCG TTTTTCCATA GGCTCCGCCC CCCTGACGAG CATCACAAAA 3840

ATCGACGCTC AAGTCAGAGG TGGCGAAACC CGACAGGACT ATAAAGATAC CAGGCGTTTC 3900

CCCCTGGAAG CTCCCTCGTG CGCTCTCCTG TTCCGACCCT GCCGCTTACC GGATACCTGT 3960

CCGCCTTTCT CCCTTCGGGA AGCGTGGCGC TTTCTCAATG CTCACGCTGT AGGTATCTCA 4020

GTTCGGTGTA GGTCGTTCGC TCCAAGCTGG GCTGTGTGCA CGAACCCCCC GTTCAGCCCG 4080

ACCGCTGCGC CTTATCCGGT AACTATCGTC TTGAGTCCAA CCCGGTAAGA CACGACTTAT 4140

CGCCACTGGC AGCAGCCACT GGTAACAGGA TTAGCAGAGC GAGGTATGTA GGCGGTGCTA 4200

CAGAGTTCTT GAAGTGGTGG CCTAACTACG GCTACACTAG AAGGACAGTA TTTGGTATCT 4260

GCGCTCTGCT GAAGCCAGTT ACCTTCGGAA AAAGAGTTGG TAGCTCTTGA TCCGGCAAAC 4320

AAACCACCGC TGGTAGCGGT GGTTTTTTTG TTTGCAAGCA GCAGATTACG CGCAGAAAAA 4380

AAGGATCTCA AGAAGATCCT TTGATCTTTT CTACGGGGTC TGACGCTCAG TGGAACGAAA 4440

ACTCACGTTA AGGGATTTTG GTCATGAGAT TATCAAAAAG GATCTTCACC TAGATCCTTT 4500 TCAGATCTCC CGATCTTTAG CTGTCTTGGT TTGCCCAAAG CGCATTGCAT AATCTTTCAG 4560

GGTTATGCGT TGTTCCATAC AACCTCCTTA GTACATGCAA CCATTATCAC CGCCAGAGGT 4620

AAAATAGTCA ACACGCACGG TGTTAGATAT TTATCCCTTG CGGTGATAGA TTTAACGTAT 4680

GAGCACAAAA AAGAAACCAT TAACACAAGA GCAGCTTGAG GACGCACGTC GCCTTAAAGC 4740

AATTTATGAA AAAAAGAAAA ATGAACTTGG CTTATCCCAG GAATCTGTCG CAGACAAGAT 4800

GGGGATGGGG CAGTCAGGCG TTGGTGCTTT ATTTAATGGC ATCAATGCAT TAAATGCTTA 4860

TAACGCCGCA TTGCTTACAA AAATTCTCAA AGTTAGCGTT GAAGAATTTA GCCCTTCAAT 4920

CGCCAGAGAA ATCTACGAGA TGTATGAAGC GGTTAGTATG CAGCCGTCAC TTAGAAGTGA 4980

GTATGAGTAC CCTGTTTTTT CTCATGTTCA GGCAGGGATG TTCTCACCTA AGCTTAGAAC 5040

CTTTACCAAA GGTGATGCGG AGAGATGGGT AAGCACAACC AAAAAAGCCA GTGATTCTGC 5100

ATTCTGGCTT GAGGTTGAAG GTAATTCCAT GACCGCACCA ACAGGCTCCA AGCCAAGCTT 5160

TCCTGACGGA ATGTTAATTC TCGTTGACCC TGAGCAGGCT GTTGAGCCAG GTGATTTCTG 5220

CATAGCCAGA CTTGGGGGTG ATGAGTTTAC CTTCAAGAAA CTAATTAGGG ATAGCGGTCA 5280

GGTGTTTTTA CAACCACTAA ACCCACAGTA CCCAATGATC CCATGCAATG AGAGTTGTTC 5340

CGTTGTGGGG AAAGTTATCG CTAGTCAGTG GCCTGAAGAG ACGTTTGGCT GATCGGCAAG 5400

GTGTTCTGGT CGGCGCATAG CTGATAACAA TTGAGCAAGA ATCTTCATCG GGGCTGCAGC 5460

CCACGATGCG TCCGGCGTAG AGGATCTCTC ACCTACCAAA CAATGCCCCC CTGCAAAAAA 5520

TAAATTCATA TAAAAAACAT ACAGATAACC ATCTGCGGTG ATAAATTATC TCTGGCGGTG 5580

TTGACATAAA TACCACTGGC GGTGATACTG AGCACATCAG CAGGACGCAC TGACCACCAT 5640

GAAGGTGACG CTCTTAAAAT TAAGCCCTGA AGAAGGGCAG CATTCAAAGC AGAAGGCTTT 5700

GGGGTGTGTG ATACGAAACG AAGCATT 5727 (2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Gly Tyr Gly Lys His Val Val Pro Asn Glu Val Val Val Gin Arg Leu 1 5 10 15

Phe Gin Val Lys Gly Arg Arg 20

Claims

CLAIMS We claim:

1. A recombinant DNA molecule comprising a DNA sequence encoding a gelsolin fusion polypeptide comprising a first DNA sequence encoding a polypeptide moiety and a second DNA sequence comprising a gelsolin moiety.

2. The recombinant DNA molecule according to claim 1, wherein the gelsolin moiety is derived from human plasma gelsolin.

3. The recombinant DNA molecule according to claim 2, wherein the gelsolin moiety comprises amino acids +1 to +169 of Figure 1 (SEQ ID NO:l).

4. The recombinant DNA molecule according to claim 3, wherein the gelsolin moiety comprises amino acids +150 to +169 of Figure 1 (SEQ ID NO:l).

5. The recombinant DNA molecule according to claim 1, wherein the polypeptide moiety is selected from the group consisting of viral receptors, cell receptors, cell ligands, bacterial immunogens, parasitic immunogens, viral immunogens, immunoglobulins or fragments thereof that bind to target molecules, enzymes, enzyme inhibitors, enzyme substrates, cytokines, growth factors, colony stimulating factors, hormones and toxins.

6. The recombinant DNA molecule according to claim 5, wherein the polypeptide moiety is a soluble CD4 protein.

7. The recombinant DNA molecule according to claim 6, wherein the soluble CD4 protein is selected from the group consisting of CD4(111), CD4(lllCys), CD4(180cys), CD4(181), CD4(183), CD4(187), CD4(345) and CD4(375) .

8. The recombinant DNA molecule according to claim 7 which is pCD4-gelsolin.

9. The recombinant DNA molecule according to claim 5, wherein the polypeptide moiety is a cell receptor or a cell ligand selected from the group consisting of ICAMl, ELAMl, VCAMl, VCAMlb, LFA3, CDX and VLA4.

10. The recombinant DNA molecule according to claim 1, wherein the DNA sequence encoding a gelsolin fusion polypeptide is operatively linked to an expression control sequence.

11. The recombinant DNA molecule according to claim 10, wherein the expression control sequence is selected from the group consisting of 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 λ, the control regions of fd coat protein, the promoter for 3- phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, the promoters of the yeast α-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.

12. A recombinant DNA molecule comprising a DNA sequence encoding a lipid binding protein fusion polypeptide comprising a first DNA sequence encoding a polypeptide moiety and a second DNA sequence encoding a lipid binding protein moiety.

13. The recombinant DNA molecule according to claim 12, wherein the lipid binding protein moiety is selected from the group consisting of protein kinase C, lipocortin, severin, villin, fragmin, profilin, cofilin, Cap42(a), gCap39, Cap2, destrin and DNase I.

14. A unicellular host transformed with a recombinant DNA molecule according to claim 1 or 12.

15. The unicellular host according to claim 14, selected from the group consisting of E.coli. Pseudomonas. Bacillus. Streptomvces. 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.

16. The unicellular host according to claim 15, said host being a COS-7 cell or a CHO cell.

17. A lipid binding protein fusion polypeptide comprising a functional moiety and a lipid binding protein moiety.

18. The lipid binding protein fusion protein according to claim 17, wherein the lipid binding protein is selected from the group consisting of villin, severin, fragmin, profilin, cofin, Cap42(a), gCap39, Cap2 and destrin.

19. The lipid binding protein fusion polypeptide according to claim 17, wherein the lipid binding protein is selected from the group consisting of protein kinase C, lipocortin and DNase I.

20. A gelsolin fusion polypeptide comprising a functional moiety and a gelsolin moiety.

21. The gelsolin fusion polypeptide according to claim 20, wherein the functional moiety is a polypeptide moiety.

22. The gelsolin fusion polypeptide according to claim 20, wherein said functional moiety is selected from the group consisting of viral receptors, cell receptors, cell ligands, bacterial immunogens, parasitic immunogens, viral immunogens, immunoglobulins or fragments of them that bind to target molecules, enzymes, enzyme inhibitors, enzyme substrates, cytokines, growth factors, colony stimulating factors, hormones and toxins.

23. The gelsolin fusion polypeptide according to claim 22, wherein said functional moiety is a soluble CD4 protein.

24. The gelsolin fusion polypeptide according to claim 23, wherein the soluble CD4 protein is selected from the group consisting of CD4(111), CD4(lllcys) CD4(180cys) CD4(181), CD4(183), CD4(187), CD4(345), CD4(375), CD4(Cystamine) , CD (Cysteine) and CD4(Glutathione) .

25. The gelsolin fusion polypeptide according to claim 22, wherein said functional moiety is a cell receptor or a cell ligand selected from the group consisting of ICAMl, ELAMl, VCAMl, VCAMlb, LFA3, CDX and VLA4.

26. The gelsolin fusion polypeptide according to claim 21, wherein the C-terminus of the polypeptide moiety is fused to the N-terminus of the gelsolin moiety.

27. The gelsolin fusion polypeptide according to claim 21, wherein the polypeptide moiety is chemically coupled to the gelsolin moiety.

28. The gelsolin fusion polypeptide according to claim 27, wherein the polypeptide moiety is chemically coupled to the gelsolin moiety through an aldehyde-amine linkage.

29. The gelsolin fusion polypeptide according to claim 27, wherein the polypeptide moiety is chemically coupled to the gelsolin moiety through a thiol group.

30. The gelsolin fusion polypeptide according to claim 27, wherein the polypeptide moiety comprises an amino-terminal or carboxy-terminal cysteine.

31. The gelsolin fusion polypeptide according to claim 20, wherein said functional moiety is selected from the group consisting of toxins, anti- retroviral agents, enzyme substrates and enzyme inhibitors.

32. The gelsolin fusion polypeptide according to claim 31, wherein the functional moiety is AZT.

33. The gelsolin fusion polypeptide according to claim 20, comprising a reporter group selected from the group consisting of enzymes, radionuclides, fluorescent markers and chemiluminescent markers.

34. A gelsolin fusion construct comprising a gelsolin fusion polypeptide and a vesicle comprising a polyphosphoinositide, said construct being multimeric or hetero-multimeric.

35. The gelsolin fusion construct according to claim 34, wherein the polyphosphoinositide is PIP or

PIP₂.

36. The gelsolin fusion construct according to claim 35, said construct comprising a CD4-gelsolin fusion polypeptide.

37. The gelsolin fusion construct according to claim 36, wherein said CD4-gelsolin fusion polypeptide is CD4(181)-gelsolin fusion polypeptide.

38. The gelsolin fusion construct according to claim 34, selected from the group consisting of ELAMl-gelsolin fusion polypeptides, VCAMl-gelsolin fusion polypeptides, VCAMlb-gelsolin fusion polypeptides, ICAMl-gelsolin fusion polypeptides, CDX- gelsolin fusion polypeptides, VLA4-gelsolin fusion polypeptides and LFA3-gelsolin fusion polypeptides.

39. The hetero-multimeric gelsolin fusion construct according to claim 34, said construct comprising a first functional moiety selected from the group consisting of viral receptors, cell receptors and cell ligands, and a second functional moiety selected from the group consisting of toxins and anti- retroviral agents.

40. The hetero-multimeric gelsolin fusion construct according to claim 34, said construct comprising a recognition molecule and a reporter group.

41. The hetero-multimeric gelsolin fusion construct according to claim 34, said construct comprising at least two immunogens.

42. The gelsolin fusion construct according to claim 34, said construct comprising a vesicle that consists essentially of PIP or PIP-,-

43. The gelsolin fusion construct according to claim 34, wherein the vesicle comprises lipids selected from the group consisting of PC, PE and PS.

44. The gelsolin fusion construct according to claim 34, said construct comprising a mixed lipid vesicle.

45. The gelsolin fusion construct according to claim 34, wherein the vesicle comprises a detergent.

46. The gelsolin fusion construct according to claim 34, wherein said vesicle contains a bioactive agent.

47. A lipid binding protein fusion construct comprising a lipid binding protein fusion polypeptide and a vesicle comprising a lipid capable of binding to said lipid binding protein fusion polypeptide, said construct being multimeric or hetero-multimeric.

48. The lipid binding protein fusion construct according to claim 47, wherein the lipid binding protein is selected from the group consisting of villin, severin, fragmin, profilin, cofilin, Cap42(a), gCap39, Cap2 and destrin.

49. The lipid binding protein fusion construct according to claim 47, wherein the lipid binding protein is protein kinase C, lipocortin or DNase I.

50. A method for producing a multimeric or hetero-multimeric gelsolin fusion polypeptide comprising the step of transforming a unicellular host with a recombinant DNA molecule comprising a DNA sequence encoding a gelsolin fusion polypeptide operatively linked to an expression control sequence.

51. A method for treating a patient having AIDS, ARC, HIV infection or antibodies to HIV comprising the step of administering to the patient a therapeutically effective amount of a multimeric or hetero-multimeric CD4-gelsolin fusion construct.

52. The method according to claim 51 wherein the fusion construct comprises a toxin or an anti- retroviral agent.

53. A method for identifying the presence of a target molecule in a sample comprising the step of contacting the sample with a hetero-multimeric gelsolin fusion construct according to claim 40.

54. A method for identifying the presence of a target molecule in vivo comprising the step of administering to a patient an effective amount of a hetero-multermic gelsolin fusion construct according to claim 40.