CA2257861A1 - Hexameric fusion proteins and uses therefor - Google Patents

Abstract

A hexameric fusion protein containing a dimeric binding protein provided with a tailpiece from an IgA antibody is described. This fusion protein is useful in therapeutics and vaccines, but is particularly well suited for applications for which the binding protein from which it is derived is unsatisfactory because of low binding affinity or for applications where multivalency is desired. These applications include diagnostics, binding assays and screening assays.

Description

CA 022~7861 1998-12-14 W 097/47732 PCT~US97/12599 HEXAMERIC FUSION PROTEINS AND USES THEREFOR

Related Application This application claims benefit of U.S. Provisional patent applications numbered60/019,934 filed lune 14, 1996, 60/043,948 filed February 19, 1997 and 60/038,915 filed February 21, 1997.

Background of the Invention IgM and IgA are the two classes of human antibodies that form homo-oligomeric structures. By far the most extensively studied of these is IgM.
The classical view of IgM structure is as a pentamer in combination with a single copy of a second proteh~, the J-chain that becomes associated with IgM during its assembly and export. This J-chain can covalently associate with IgM through the formation of a disulfide bond between a cysteine residue in the J chain and a cysteine residue in a short 18 amino acid extension. designated ~tp, from the canonical C-terminal constant region of the heavy chain. This cysteine residue appears to be required for the formation of the IgM
pentamer in association with the J-chain LA C. Davis et al., EMBO J.. _(9):2519-2526 (1989)]. However, a hexameric form of IgM, devoid of the J-chain, was described and characterized over two decades ago and has recently been characterized in more detail in terms of its biochemical and potential biological activities [reviewed in Brewer et al., Immunolo~y Today, 15: 165- 168 ( I 994)] . The production of oligomeric IgG proteins has been achieved by addition of the 18 amino acid IgM tailpiece segment (utp) to the atp corresponding C-termini end of the C~3 region of the IgGl-4 proteins by DNA recombinant technology [R. I. F. Smith and S. L. Morrison, Biotechnolo~y, 12:683-688 (1994); R. I. F.
Smith et al., J. Immunol., 154:2226-2236 (1995)].
Human IgA also has an 18 amino acid tailpiece segment (atp) which bears some sequence homology to utp. In man, there are two a constant region loci which encode distinct sequences, but the tailpiece regions for the al and a2 regions are quite similar, or in some cases reported to be identical l Sequences of Proteins of Immunological Interest, fifth edition, EA Kabat et al., Vol. 1, U.S. Department of Health and Human Services, NIH publication no. 91-3242, (1991)]. However, unlike IgM, IgA occurs most frequently as a monomer antibody, similar to the IgG subclasses, or as a dimer antibody plus one molecule of J-chain [Mestecky and Kilian, Methods in Enzymolo~v, 116:37 75 (1985); T. B. Tomasi, Immun.

CA 022~7861 1998-12-14 W O 97/47732 PCTrUS97/12599 Today, 13 :416-418 (1992)] . Higher oligomers/aggregates of IgA are reported [Mestecky and Kilian. cited above], but these are poorly characterized components in complex mixtures containing other proteins interactive with IgA. Recombinant IgA has been expressed in the presence and absence of theJ chain (Bru~elllanll et al., J. Exp. Med., 166:1351-1361 (1987);
Morton et al., J. Immunol.. 151 :4743-4752 (1993); Carayannopoulos et al., Proc. Natl Acad Sci. USA, 91:8348-8352 (1994); Terskikh etal., Mol. Immunol., 31:1313-1319 (1994)].
The IgA proteins produced in the absence of the J chain were monomeric or dimeric forms by nonreducing SDS/PAGE and appeared as dimers in solution. In one study (Carayannopoulos et al., above), the co-expression of the J-chain led to formation of disulfide linked IgA dimers 10 together with J chain.
The CD28 receptor, a member of the immunoglobulin superfamily of molecules (IgSF) [A.F. Williams and A.N. Barclay, Annu. Rev. Immunol., 6:381-405 (1988)], is a 44 kDa homodimer glycoprotein expressed on the surface of T-lineage cells includingthymocytes and peripheral T cells in the spleen. Iymph node and peripheral blood. CD28 interacts with two different counter-receptors CD80 (also known as B7 and B7.1) ~P. S.
Linsley et al., Proc. Natl. Acad. Sci. USA, 87(13):5031-5035 (1990); G. J. Freeman et al., J.
Exp. Med., 174(3):625-631 (1991)] and CD86 (also called B7.2 and B70) [M. Azuma et al., Nature, 366(6450):76-79 (1993); G. J. Freeman et al., J. Exp. Med., 178(6):2185 2192 (1993); G.J. Freeman et al., Science, 262(5135):909-911 (1993)1, expressed on antigen 20 presenting cells (APCs), to deliver crucial co-stimulatory signals for sll~t~ined activation of T
cells, through its association via the cytoplasmic domain with PI3-kinase [F. Pages et al., Nature, 369(6478):327-329 (1994); P. H. Stein et al., Molecul. & Cell. Biol., 14(5):3392 3402 (1994)] and other signalling pathways [K.E. Truitt et al., J. Immunol., 155:4702-4710 (1995);J.A.Nunesetal.,J.Biol.Chem..271(3):1591-1598(1996);H.Schweideretal., Eur. J. Immunol., 25: 1044- 1050 (1995)1. Both CD80 [P. S. Linsley et al., J. Exp. Med., 174(3):561 -569 (1991)] and CD86 lAzuma et al., cited above; Freeman et al., 1993, cited above; Freeman et al., 1993, cited above] also recognize CTLA-4 [J.F. Brunet et al., Nature, 328(6127):267-270 (1987)], a homolog of CD28, expressed transiently and at low receptor density on activated CD8+ and CD4+ T cells.
Antagonism of CD28 interactions with the CD80 or CD86 counter-receptors using CTLA4-Ig fusion proteins or antibodies directed against CD80 and CD86 inhibits T cell activation in vitro, suppresses humoral and cellular immune responses in vivo, inhibits graft rejection and the progression of autoimmune diseases in vivo [reviewed in J. A. Bluestone, CA 022~7861 1998-12-14 llullily~ 2:555-559 (1995); Harlan etal., Clin. Immunol. and Immunopath.. 75(2):99-111 (1995)]. Thus, CD28 is a target for development of immunosuppressive agents. To identify small molecule antagonists, a rapid and reproducible assay is desirable for the screening of synthetic compounds, natural products, and peptides. Particularly desirable is a protein based assay which would isolate the receptor and its counter-receptor from interference by other components of cell-based assays, and which is additionally adaptable to automation.
The affinity of the interaction of CD28 with both counter receptors is quite low [P. S. Linsley etal., Immunity, 1:793-801 (1994)], with an approximate Kd of 200 nM for the binding of a soluble CD80-Ig fusion protein to an immobilized CD28-Ig fusion protein [P. S. Linsley et al., J. Exp. Med., 173(3):721-730 (1991)]. This low affinity hampers development of a sensitive protein binding assays amenable to screening many compounds.
What is needed is a method for increasing the avidity of binding proteins, particularly those with low affinity, for use in screening and diagnostic assays, therapeutics, and vaccines.

Summary of the Invention In one aspect, the present invention provides a hexameric fusion protein which provides increased binding activity as compared to the protein from which it is derived and methods of making same. This fusion protein is particularly useful in binding assays and may be readily purified.
The h~x~rnPric fusion protein of the invention contains a dimeric binding protein and a tailpiece (atp) characterized by the activity of the tailpiece from the C-terminus of the heavy chain of an IgA antibody. In one embodiment, the binding protein is a natively dimeric binding protein or a functional fragment thereof. In another embodiment, the binding protein is recombinantly engineered to have a dimeric form. This is preferably achieved by fusion of a protein fragment which contains the extracellular domain of a selected binding protein to an Fc fragment. These binding proteins, when provided with the atp, assemble into homo- or hetero-hexamers .
In yet another aspect, the present invention provides a polynucleotide sequence encoding a stable hexameric fusion protein of the invention.
In a further aspect, the present invention provides a vector comprising the above-described polynucleotide sequence and a sequence controlling expression of the fusion protein in a selected host cell.

CA 022~7861 1998-12-14 W O 97/47732 PCT~US97/12599 In still another aspect, the present invention provides a reco--,bh~dlll host cell containing the above-described vector.
In a further aspect, the present invention provides methods of producing and purifying a stable hexameric fusion protein by providing a host cell containing the stable 5 hexameric fusion protein of the invention, recovering the stable hexameric fusion protein, and purifying the recovered protein. The strands of the fusion protein are preferably co-produced and assembled in the host cell.
In still a further aspect, the present invention provides a pharm~euti~l composition containing a stable hexameric fusion protein or a DNA sequence encoding the stable 10 hexameric fusion protein of the invention and a pharmaceutically acceptable carrier.
In yet another aspect, the present invention provides for screening for ligands to a hexameric fusion protein of the invention. Also provided are assays for inhibitors of hexameric binding protein/ligand interaction.
Other aspects and advantages of the present invention are described further in the 1~ following detailed description of the preferred embodiments thereof.

Brief Description of the Drawings Fig. 1 is a schematic representation of the hexameric CD80-Igatp protein of the invention. The regions of the molecule corresponding to the CD80 extracellular domain, the 20 IgGI hinge, CH2, and CH3 domains, and the atp segment are indi~ tt~ The letter "S" in the diagram indicates the positions of predicted disulfide bonds between cysteine residues.
Fig. 2 is a plasmid map illustrating the expression construct for the CD80-Igatpprotein of the invention. The plasmid is 7,167 base pairs in size. Beginning at residue 1 in a clockwise manner: "cmv pro" is the major late CMV promoter for transcription of the 25 downstream CD80-Igatp coding sequence; "CD80" encodes the signal peptide and extracellular domain of human CD80; "Fc" encodes the hinge, CH2, and CH3 regions of human IgGI; "atp" encodes the human atp segment; "BGH" is the polyadenylation signal region from the bovine growth hormone gene; "betaglobin" is the mouse major b-globin promoter; "dhfr" encodes the mouse dhfr(dihydrofolate reductase) protein; "SV40" is the 30 SV40 early polyadenylation region; and "ori" and "amp" are the bacterial origin of replication and beta l~rt:~m~ce gene, respectively, from the common cloning plasmid pBR322. The corresponding plasmids CD86Fcatplink and CTLA4Fcatplink were constructed for theexpression of the CD86-Igatp and CTLA4-Igatp proteins (see Figs. 5 and 6).

CA 022~7861 1998-12-14 Fig. 3A-3H is the complete DNA sequence of the CD80Fcatplink plasmid [SEQ ID
NO: I] shown in Fig. 2.
Fig. 4A-4D is the DNA and encoded protein [SEQ ID NOS: 2 and 3] sequences for the CD80-Igatp region in the vector CD80-Fcatplink. Bolded regions show restriction sites 5 for reference to Fig. 2 and the initiation codon, mature processing site, hinge region, and C-- terminal atp segment.
Fig. SA-5B is the DNA and encoded protein sequences [SEQ ID NOS: 4 and 5] for the extracellular domain of CD86 in the vector CD86Fcatplink. The sequence outside of the Kpn I and Eag I sites is the same as for CD80Fcatplink (see Figs. 3A-3H and 4A-4D).
Fig. 6A-6C is the DNA and encoded protein sequences [SEQ ID NOS: 6 and 7] for the CMV promoter and the extracellular domain of CTLA-4 in the vector CTLA4-Fcatplink.
The sequence 5' to base 514 and 3' of the Eag I site is the same as for CD80Fcatplink.
Fig. 7 is a profile for chromatography of CD80-Igatp on a Superdex 200 column.
The first peak eluting at about 45 min is the hexameric protein complex while the second peak migrates at the position observed for monomeric CD80-Ig. The inset shows a coomassie stained pattern for the purified CD80-Igatp protein on SDS/PAGE under reducing (R) and nonreducing (NR) conditions.
Fig. 8 is a chart showing equilibrium sedimentation ~main panel) and sedimentation velocity (inset) analytical centrifugation of the CD80-Igatp protein with a modeled fit to a hexamer/(hexamer)2 equilibrium. The upper graph shows the residuals for the equilibrium sedimentation centrifugation.
Fig. 9 is a line graph illustrating the binding of biotinylated CD80-Igatp (labeled B7-FcA) to CD28-Ig immobilized at three different concentrations in an ELISA format.
Binding was inhibited by the mAb CD28.1 or by CTLA4-Ig.

2~ Fig. 10 is a line graph illustrating the binding of biotinylated CD80-Igatp, CD86-Igatp, and CD80-Ig compared to immobilized CD28-Ig in an ELISA format.
Fig. 11 is a line graph illustrating the binding of biotinylated CD80-Igatp, CD86-Igatp, and CD80-Ig compared to immobilized CTLA4-Ig in an ELISA format.
Fig.12 is a line graph illustrating the competition of biotinylated CD80-Igatp binding to immobilized CD28-Ig (coated at 200 mg/ml) by CD80-Igatp itself, CD80-Ig, CTLA4-Ig, and CD28.2 MAb.

.. .. ~

CA 022~7861 1998-12-14 Fig. 13A is a line graph illustrating the binding of CD80-Igatp to wild-type andmutant immobilized CD28-muIg2a proteins.
Fig. 13B is a line graph illustrating the binding of CD86-Igatp to wild-type andmutant immobilized CD28-mulg2a proteins.
Fig. 13C is a line graph illustrating the binding of rabbit polyclonal antisera to wild-type and mutant immobilized CD28-muIg2a proteins.
Fig. 14 is a chart illustrating sequentially the binding of CD80-Ig and CD80-Igatp to CD28-Ig immobilized on a biosensor chip as measured by surface plasmon resonance.
Fig. 15 is a chart illustrating the binding of CD80-Igatp and CD86-Igatp to CD28-Ig immobilized on a biosensor chip as measured by surface plasmon resonance.
Figs. 16A and 16B are line graphs illustrating the binding of CD80-Igatp and CD86-Igatp, respectively, to cells expressing human CD28 on their surface in the presence or absence of a CD28 monoclonal antibody that inhibits this interaction.
Fig. 17 is a bar chart illustrating the level of IL-2 production by PCD28.1 cells treated with monomeric and hexameric CD80 (labeled B7.1 -Ig and B7.1-IgA, respectively) and CD86 (labeled B7.2-Ig and B7.2-IgA, respectively) Ig fusion proteins. The proteins were used (I) alone in solution, (2) alone immobilized through goat anti-human antibody (GAH), or (3) immobilized in combination with immobilized CD3 mAb. Controls were GAH alone, or with CD3 mAb, and the CD28 IgM rnAb 248.23.2. IL-2 levels were determined by CTLL-2 bioassay using known amounts of IL-2 as a standard (inset).
Fig. 18 is a bar chart illustrating the level of IL-2 production by DC27.CD28wt cells treated as described in Fig. 17.
Fig. 19 is a bar chart illustrating IL-2 promoter activity in PCD28.1 cells stimulated as described in Fig.17. IL-2 promoter activity was measured by induction of 13-galactosidase activity which serves as a reporter gene under the control of an IL-2 promoter.
Figs. 20A and 20B are bar graphs respectively showing the induction of the IL-2 promoter, and IL-2 production by CD28 expressing cells incubated with CD80-Igatp, CD86-Igc~tp, or CD80-Ig.
Fig. 20C is a bar graph showing the levels of IL-2 production induced with soluble CD80-Igatp and CD86-Igatp hl comparison to that induced by immobilized antibody to CD3.

____ _.. _ . .. .. T

CA 022~7861 1998-12-14 WO g7/47732 PCT/US97/12599 Fig. 21 is a bar chart illustrating inhibition of biotinylated CD80-Igatp binding to immobilized CD28-Ig by individual compounds in the BM-34 test set. The percent inhibition range is plotted against the number of compounds showing that range of inhibition.
Fig. 22 is a profile for Superose 6 chromatography of the chimeric derivative of the 5 Epo receptor antibody I C8 (here labeled ''anti-EPOr-IgGI ") and the atp construct of the same antibody (labeled ''anti-EPOr-IgGIatp'') with binding activity to an immobilized EPOr-Ig protein shown in the inset.

Detailed Description of the Invention The invention provides an hexameric fusion protein useful in therapeutic and immunogenic compositions. The hexameric fusion protein of the invention is particularly well suited for applications for which the binding protein from which it is derived is unsatisfactory because of low binding affinitylavidity and for other applications where multivalency is desired. These applications include diagnostics, binding assays, screening 15 assays and cellular responses based on receptor cross-linking. Also provided are compositions and methods for production and purification of these fusion proteins.
The invention further provides methods of producing stable hexameric fusion proteins, by providing a selected binding protein with an IgA tailpiece (atp) or a functional equivalent thereof. The inventors have found that addition of the atp from the natively 20 monomeric or dimeric IgA, surprisingly, provides the resulting fusion protein with the ability to form stable hexamers.

I. Fusion Proteins As used herein, a hexameric fusion protein of the invention contains a dimeric 25 binding protein which has been provided at its carboxy terminus with a tailpiece (atp) characterized by having the activity of the tailpiece from the C-terminus of the heavy chain of an IgA antibody. This tailpiece, when attached to each monomer of the dimeric binding protein, provides the resulting fusion protein with the ability to form stable hexamers, i.e., the hexameric fusion proteins of the invention do not undergo any appreciable dissociation in 30 solution (e.g., phosphate buffered saline) at room temperature.
In a particularly preferred embodiment, the fusion proteins of the invention are homo-hexamers. However, where desired, hetero-hexamers comprising two different fusion proteins may be constructed.

CA 022~7861 1998-12-14 The binding proteins useful in the invention include full-length proteins and fragments thereof which are characterized by the binding ability of the full-length protein, i.e., the fragment which has the ability to bind to the counter-receptors or other ligands of the selected binding protein. Such binding proteins may be derived from a protein or protein complex which natively dimerizes for biological activity, or may be genetically engineered as described herein. Examples of suitable natively dimeric binding proteins are those with carboxyl termini situated such that addition of the atp to the carboxyl terminus of each polypeptide chain, with or without a linker, allows juxtaposition of the atp chains. One of skill may readily select such native dimeric proteins or dimeric protein complexes, which include, for example, IgG, IgD, or IgE antibodies, Fab fr~gmentc, Fab2 fragments, Ig-Fc fragments, Ig fusion proteins, and the extracellular domains of cell surface proteins such as the at~ chain of a T cell receptor, CD28 and CTLA4, CD8 oc/~ hetorodimers and ol/a homodimers, and the a/,~ chain of integrin proteins and various cytokine receptors (e.g., IL3, IL5, etc.). These binding proteins are available from a variety of commercial and academic sources. Alternatively, these sequences may be chemically syn~hPsi7Pd As discussed above, a selected binding protein may be engineered to be dimeric. For example, a protein fragment comprising a binding domain of a selected monomeric binding protein may be attached to an Ig-Fc fragment which forms dimers. Desirably, the binding protein is selected from surface glycoproteins from the immunoglobulin supergene family and their ligands. For example, in a currently preferred embodiment, the binding protein is selected from CTLA-4 (whose extracellular domain can be expressed as a monomer or dimer) and its counter-receptors CD80 and CD86. However, other proteins, including other binding proteins, are known to those of skill in the art and may be used in the construction of a hexameric fusion protein of the invention. Although a currently preferred embodiment of this invention provides hexameric immunoglobulin fusion proteins, which are exemplified herein, this invention is not so limited. For example, a binding protein may be genetically modified to alter its activity. For example, engineered, mutant forms of IL4 have been described that retain high affinity for its receptor but lack normal agonist activity and serve as antagonists of IL-4 mer~ ed function lsee, e.g., N. Kruse et al, EMBO J.~ 3237-3244 (1992) and W096/04388 (Feb. 15, 1996)]. Such a mutant would be useful iA a hexameric IL4-Ig fusion protein according to the invention, serving as an antagonist of IL4 function.
The protein fragment used to construct a dimeric binding protein contains at least a fragment of the extracellular domain of the selected binding protein. For functional binding ~ T

CA 022~7861 1998-12-14 activity, this extracellular fragment preferably contains the sequences required for binding, which can be readily determined by one of skill in the art. In a preferred embodiment, which makes use of a eukaryotic production system, the protein fragment also contains an export leader sequence which is native to the binding protein select~l However, other export leader 5 sequences which are capable of exporting the protein may be substituted by one of skill in the art. In one exemplary embodiment, where the target is CD28, the protein fragment is the native leader and extracellular domain from CD80 or CD86. The fragments can be obtained from proteins such as CD80 ~P. S. Linsley et al., J. Exp. Med., 173(3):721 730 (1991);
Truneh et al., Mol. Immunol.. 33(3):321-334 (1996); J. E. Ellis et al., J. Immunol., 56:2700-2709 (1996)], and CD86 [P. S. Linsley et al., Immunity, I :793-801 (1994); J. E. Ellis et al., cited above; P. S. Linsley et al., J. Exp. Med.~ 174:561-569 (1991)]. In another embodiment, where the target is CD80 or CD86, the protein fragment is the native leader and extracellular domain from CTLA-4 or CD28.
The Fc fragment used in the construction of the hexameric fusion protein may be from any antibody subclass, except IgA. Thus~ the Fc fragment may be derived from the IgG, IgD, or IgE subclass. When the Fc fragment is derived from an IgG antibody, any of the human isotypes, i.e., IgG" IgG2, IgG3, and IgG4, may be selected. Further, the parental IgG antibody may be mutated to reduce binding to complement or Ig-Fc receptors [see, e.g., A.R. Duncan et al., Nature, 332:563-564 (1988); A. ~. Duncan and G. Winter, Nature, 332:738 (1988); M.-L. Alegre et al., J. Immunol., 148:3461 3468 (1992); M-H Tao et al., J.
Exp. Med., 178:661 667 (1993); V. Xu et la, J. Biol. Chem., 269:3469-3474 (1994)]. When the Ig-Fc fragment is derived from IgM, it desirably contains the hinge/CH2/CH3/CH4 sequence, but not the naturally occuring 18 amino acid tailpiece (~ltp).
Optionally, the C-terminal end of the IgG, CH3 domain of the Fc fragment may be modified by conventional techniques to contain a restriction enzyme site for convenient cloning of the tailpiece segments (i.e., the peptide of the invention). Such modifications are described in more detail in the examples below, and are well known to those of skill in the art.
The peptide used to construct the fusion protein of the invention is derived from tailpiece located at the C-terminus of the heavy chain of an IgA antibody. In a preferred embodiment, this peptide is 18 residues in length and is the octp segment of the human IgAI
heavy chain or a functional equivalent thereof. One particularly suitable peptide is:
PTHVNVSVVMAEVDGTCY [SEQ ID NO: 3]. If desired, this peptide may be modified to remove the glycosylation site by changing I or 2 amino acids at residues 5-7 (NVS). For ",~ ~

CA 022~7861 1998-12-14 W O 97/47732 PCT~US97/12599 example, the N (asparagine) may be to changed to Q (glutamine) and/or the S (serine) may be changed to A (alanine). Additionally, up to about 4 amino acid residues of the human IgA
CH3 domain may be retained. Alternatively, functional equivalents of the human IgAI atp may be select~l Suitable functional equivalents include, for example, gorilla IgG1, human IgA2, rabbit IgA, and mouse IgA. Such functional equivalents may also be modified by removal of glycosylation sites. As described herein~ this peptide is linked, directly or indirectly, to the binding protein (e.g., the Ig-Fc fragment) and provides the fusion protein of the invention with the ability to assemble into a stable hexamer.
The fusion protein may contain a linker sequence. Optionally, such a linker may be located between the binding protein (e.g., the Ig-Fc fragment) and the atp peptide. This linker is preferably an amino acid sequence between about I and 20 amino acid residues, and more preferably between about I and 12 amino acid residues, in length. Other appropriate or desired linkers may be readily selected by one of skill in the art. Although currently less desired, one of skill in the art may substitute other linkers for the preferred amino acid sequence linkers described above.
Three currently preferred embodiments of the fusion proteins of the invention are described herein, CD80-Igatp, CD86-Igatp and CTLA4-Igatp. These proteins are composed of the native leader and extracellular domains of the CD80 (B7. 1), the CD86 (B7.2, B70), and the CTLA-4 surface glycoproteins, respectively, linked to the hinge/CH2/CH3 region of the heavy chain of human IgG, (Fc fragment) and terminating in a short tail piece segment from human IgA I (atp). Another example of a hexameric protein of the invention is an IgG
antibody, where the atp is joined directly to the carboxy terminus of the heavy chain and a light chain is paired with this heavy chain. The atp hexameric antibody and Ig fusion proteins of the invention are advantageous over IgM antibodies and IgM fusion proteins in that the hexamers of the invention are readily purified on commercially available chromatography supports and are more efficiently expressed.
These constructs may be made using known techniques. A detailed description of the construction of these exemplary fusion proteins of the invention is provided in the examples below.
Briefly, each chain of a dimeric binding protein is selected or constructed. Forexample, one preferred binding protein is a recombinant immunoglobulin containing the native leader and extracellular domain fused to an Ig-Fc fragment from the selected human IgG antibody. The atp is added, optionally by introducing a convenient restriction CA 022~7861 1998-12-14 endonuclease site near the C-terminus of the binding protein (e.g., an Fc region) using silent mutations of the coding sequence and then cloning a synthetic oligonucleotide into this site that encodes the tailpiece segment. The tailpiece segment is matched to that of the human a- 1 chain. The tailpiece provides the fusion protein with the ability to form hexamers and the resulting construct is the hexameric fusion protein of the invention. A schematic l~p~csel~ ion of the predicted hexamer for an exemplary fusion construct of the invention, CD80-Igatp, is shown in Fig. 1.
Preferably, the fusion proteins of the invention are produced using recombinant techniques. Desirably, the nucleic acid sequences may be fused and the fusion protein expressed in vitro in a suitable host cell. Alternatively, the fusion proteins of the invention are produced by separately expressing, or co-expressing the nucleic acid sequences encoding the protein fragments and atp fragment of the invention and fusing the expressed products.
Suitably, the resulting fusion protein forms hexamers. These production techniques are discussed in more detail below.
II. Polynucleotide Sequences~ Expression and Purification The present invention further encompasses polynucleotide sequences encoding the fusion proteins of the invention. In addition to the DNA coding strand, the nucleic acid sequences of the invention include the DNA (including complementary DNA) sequence ~ s~ g the non-coding strand and the messenger RNA sequence. Variants of these nucleic acids of the invention include variations due to the degeneracy of the genetic code and are encompassed by this invention. Such variants may be readily identified and/or constructed by one of skill in the art. Further, the polynucleotide sequences may be modified by adding readily assayable tags to facilitate quantitation, where desirable.
To produce recombinant fusion proteins of this invention, the DNA sequences of the invention are inserted into a suitable expression system, preferably a eukaryotic system.
Desirably, a recombinant vector is constructed in which the polynucleotide sequence encoding at least one chain of the fusion protein (i.e., the binding protein/atp) is operably linked to a heterologous expression control sequence pelllliL~ g expression of the fusion protein of the invention. Numerous types of appropriate expression vectors and host cell systems are known in the art for expression, including, e.g., mammalian, yeast, bacterial, fungal, drosophila, and baculovirus expression.

CA 022~7861 1998-12-14 W O 97/47732 PCTrUS97112599 The transformation of one or more of these vectors into appropriate host cells results in expression of the fusion proteins of the invention. Other appropriate expression vectors, of which numerous types are known in the art, can also be used for this purpose.
Such production methods permit assembly of the hexameric fusion protein of the invention by the host cell. Typically, such methods will provide a homo-hPx~m~ric fusion protein. However, in another embodiment, hexameric fusion proteins of mixed specificity may be produced by co-expression of different fusion proteins (i.e., binding protein/atp). For example, two fusion proteins recognizing non-competing sites on the same molecule can be co-expressed resulting in hexamers that can bind to two sites on the same molecule, resulting in higher binding avidity than for each fusion protein alone or as a homogenous hexamer.
Alternatively, the two fusion proteins can bind to two distinct molecules presented on the same, or different surfaces (e.g., expressed on the same or different cells).

Suitable host cells or cell lines for transfection by this method include m~rnm~ n cells, such as Human 293 cells, Chinese hamster ovary cells (CHO), the monkey COS-1 cell line, murine L cells or murine 3T3 cells derived from Swiss, Balb-c or NIH mice. Suitable mammalian host cells and methods for transformation, culture, amplification, screening, and product production and purification are known in the art. [See, e.g., Gething and Sambrook, Nature, 293:620-625 (1981), or alternatively, Kaufman et al., Mol. Cell. Biol., 5(7):1750-1759 (1985) or Howley et al., U. S. Patent 4,419,446]. Another suitable m~mm~ n cell line is the CV- l cell line.
Other host cells include insect cells, such as Spodoptera frugipedera (Sf9) cells.
Methods for the construction and transformation of such host cells are well-known. [See, e.g.
Miller et al., Genetic En~ineerin~, 8:277-298 (Plenum Press 1986) and l~r~.~nces cited therein].
Although less preferred, also useful as host cells for the vectors of the present invention are bacterial cells. For example, the various strains of E. coli (e.g., HB101, MC1061) are well-known as host cells in the field of biotechnology. Various strains of B.
subtilis, Pseudomoltas, other bacilli and the like may also be employed in this method.
Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the proteins of the present invention. Other fungal cells may also be employed as expression systems.

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CA 022~7861 1998-12-14 WO 97/47732 PCT/US97tl2599 Thus, the present invention provides a method for producing a fusion protein of the invention which involves transforming a host cell, preferably a eukaryote, with at least one expression vector containing a recombinant polynucleotide encoding a fusion protein under the control of a transcriptional regulatory sequence. e.g., by conventional means such as 5 transfection or electroporation. The transformed host cell is then cultured under suitable conditions that allow expression of the fusion protein. The expressed and assembled fusion protein is then recovered, isolated, and purified from the culture medium by appropriate means known to one of skill in the art. In a preferred embodiment, the fusion proteins are assembled by the host cell following co-production of one or more of the fusion proteins of 10 the invention. Alternatively, the hexameric fusion protein may be assembled following recovery from the host cell.
Advantageously, the fusion proteins of the invention can be readily purified using conventional techniques. For example. hexameric Ig fusion proteins of the invention may be readily purified on high affinity, high capacity supports based on protein A and protein G.
15 Such resins are commercially available [Pharmacia Inc.; Bioprocessing Ltd.].
Although less preferred, the hexameric fusion protein may be produced in insoluble form. For example, the proteins may be isolated following cell Iysis in soluble form, or extracted in guanidine chloride.

20 III. Pharmaceutical Compositions and Methods of Use Thereof The fusion proteins of this invention or DNA sequences encoding them may be formulated into pharm~eutir~l compositions and administered using a therapeutic or immunogenic regimen compatible with the particular formulation. Pharmaceutical compositions within the scope of the present invention include compositions containing a 25 protein of the invention in an effective amount to have the desired physiological effect.
Suitable formulations for parenteral ~rlmini.~ration include aqueous solutions of the active compounds in water-soluble or water-dispersible form, e.g., saline. Alternatively, suspensions of the active compounds may be administered in suitable conventional lipophilic carriers or in liposomes. In still another alternative, adjuvants may be desired, particularly 30 where the composition is to be used as an immunogen.
The compositions may be supplemented by active pharmaceutical ingredients, wheredesired. Optional antibacterial, antiseptic, and antioxidant agents in the compositions can perform their ordinary functions. The pharmaceutical compositions of the invention may CA 022~7861 1998-12-14 W O 97/47732 PCTrUS97/12599 further contain any of a number of suitable viscosity enhancers, stabilizers, excipients and auxiliaries which facilitate processing of the active compounds into preparations that can be used pharmaceutically. Preferably, these preparations, as well as those pl~pa~lions (liccncced below, are designed for parenteral admini.ctration. However, compositions ~lesif~ned 5 for oral or rectal a~lmini.ctration are also considered to fall within the scope of the present invention.
As used herein, the terms "suitable amount" or "effective amount" means an amount which is effective to treat or prevent the conditions referred to below. A preferred dose of a pharm~relltic~l composition containing a fusion protein of this invention is generally effective 10 above about 0.1 mg fusion protein of the invention per kg of body weight (mg/kg), and preferably from about } mg/kg to about 100 mg/kg. These doses may be administered with a frequency necessary to achieve and m~int~in satisfactory fusion protein levels. Although a preferred range has been described above, determination of the effective amounts for treatment or prophylaxis of a particular condition may be determined by those of skill in the I ~ art.
Particularly, pharmaceutical compositions containing the hexameric antibody/atp fusion proteins of the invention are useful as antagonists for the 7 transmembrane (7 TMR) class of cell surface receptors, since such receptors are often arrayed in many copies on cell surfaces and the aggregation of such receptors does not lead to intracelluar cign:llling 20 (agonism) as can occur for many other types of cell surface receptors. For example, lminictration of a pharmaceutical compositions containing a hexameric antibody/atp fusion protein of the invention blockades chemokine receptors, a subfamily of the 7 TMR, and inhibits chemotaxis and activation of target cells such as eosinophils. A second example is CTLA4-Igatp. CTLA4-Ig is a potent inhibitor of CD80 and CD86 driven stimulation of 25 T-cells through their interaction with CD28. In animal models, CTLA4-Ig has shown benefit in several autoimmune diseases and transplantation.
Since the CD80 and CD86 antigens recognized by CTLA4-Ig are arrayed in many copies on the cell surface, an atp hexameric form of CTLA4-Ig may provide a more potent antagonist than the standard Ig fusion protein. In another embodiment, a pharmaceutical 30 composition of the invention containing Igatp fusion proteins of the invention may be used for removal of complement components or components of the blood coagulation cascade to retard clotting.

CA 022~7861 1998-12-14 In one aspect, the invention provides a method for antagonizing cell surface CD80-and CD86-mediated stimulation of CD28 positive cells by administering to the cells a hexameric fusion protein CTLA4-lgatp. This may be performed in vivo, by ~Aminic~ering a pharmzl~elltic~l composition containing this hexameric fusion protein. In another aspect, the~ invention provides a method for stim~ ting (agonist activity) CD28+ T cells by g the CD80- or CD86-hexameric fusion protein to the cells in culture resulting in stiml-l~tion of IL-2 production from these cells. These proteins may be used alone, or in combination with other stim--l~t-)rs of T-cells (e.g., antibodies directed against the T cell receptor-CD3 complex.) In another embodiment, the compositions of the invention containing Ig-Fc-containing fusion proteins are useful for in vivo clearance of soluble ligands, in view of the fact that hexamerization of the Fc domain enhances interaction with complement components and Fc receptors. Thus, ligands bound to the hexameric fusion protein of the invention are efficiently cleared from circulation.
The hexameric fusion proteins of the invention can also serve as agonists, particularly in situations where aggregation can induce a desired response. For example, aggregation is essential for signal transduction through many cell surface receptors - either as a con.ceqllPnce of multivalent presentation of the receptor ligand (eg., a counter receptor on a the surface of a second cell) or through changes induced upon ligand binding, or both. An 20 example of signalling through a cell surface receptor induced by cross-linking through recognition of its counter-receptor on a second cell is CD28 recognition by CD80 or CD86.
Thus, the invention further provides a method for stimulating CD28 positive cells by ~Arnini.c~ring to CD28 positive cells CD80-Igatp and/or CD86-Igatp. Examples of soluble ligands inducing signal transduction through binding to their receptors are EGF and 25 growth hormone and both result in receptor di...e~i~ation. For these receptors, dimerization induced through antibody binding also can lead to activation [Schreiber etal., Proc. Natl.
Acad. Sci. USA, 78:7535 (1981), Fuh etal., Science, 256: 1677 (1992)]. Hexamericantibodies against such receptors or hexameric ligand-Ig fusion proteins for these receptors are expected to be more efficient stimulators than the standard dimeric antibodies or ligand Ig 30 fusion proteins. For example, the pharmaceutical compositions containing the hexameric antibodies or cytokine-Ig fusion proteins of the invention are useful in inducing signal transduction in receptors for hematopoietic cytokines, such as erythropoietin, thymopoietin and growth stim--l~tory factor.

CA 022~7861 1998-12-14 W O 97/47732 PCTrUS97/12S99 Also provided is a method for sup~ sh~g CTLA-4 positive cells by ~rlmini.ct~ringCD80-Igatp and/or CD86-Igatp to CTLA4 positive cells. This may be performed in vivo, by ~dmini.ctration of a pharmaceutical composition containing the hexameric proteins.
Alternatively, the hexameric proteins are added to CTLA4 positive T-cells in culture resulting S in inhibition of IL-2 production from these cells.
In yet another aspect, hexameric Ig-fusion proteins of the invention can also serve as enh~nred immunogens for the fused protein fragment due to efficient, receptor-m~ ted updake for antigen processing and presentation or efficient interaction with proteins of the complement system. Enhanced immunogenicity is desirable for the efficient generation of 10 polyclonal and monoclonal anlibodies and for therapeutic vaccination. Thus, the invention further provides a method of imml-ni7.ing using the pharm~.elltic~l composition of the invention.

IV. Assavs The hexameric fusion proteins of the invention are useful in in vitro assays formeasuring the binding of the fusion protein to a selected ligand and for identifying the native or synthetic ligand for the binding proteins. Such a ligand includes the native ligand or counter-receptor to the binding protein from which the hexameric fusion protein is derived.
For example, where the fusion protein is derived from CD80 or CD86, the ligand may be CD28 or CTLA-4. Alternatively, the ligand may be a derivative of the native counter-receptor, a peptide, peptide-like compound, or a chemical compound which interacts with the fusion protein.
The hexameric fusion proteins may be used for in vivo assays, including, for example imaging. See, e.g., S. M. Larson et al., Acta Oncolo~ica, 32(7-8):709-715 (1993); R.
DeJageretal.,SeminarsinNuclearMedicine.23(2):165-179(Apr.1993).
Alternatively, a fusion protein of the invention may be used to screen for new ligands.
The use of the fusion proteins of this invention in such an assay is particularly well suited for identifying cell surface or multivalent ligands.
Suitable assay methods may be readily determined by one of skill in the art. Forexample, an ELISA format may be utilized in which the selected ligand is immobilized, directly or indirectly (e.g., via an anti-ligand antibody) to a suitable surface.
Where desired, and depending on the assay selected, the hexameric fusion proteinmay be immobilized on a suitable surface. Such immobilization surfaces are well known.

CA 022~7861 1998-12-14 W O 97147732 PCT~US97/12599 For example, a wettable inert bead may be used in order to facilitate multivalent interaction with the hexameric fusion proteins of the invention.
Further, the methods of the invention are readily adaptable to combinatorial technology, where multiple molecules are contained on an immobilized support system.
S Thus, the fusion proteins of the invention permit screening of chemical compound and peptide based libraries where these agents are presented in a multivalent format compatible with more than one subunit of the hexamer. Monomeric interactions of this type are routinely in the mM
range and thus may not be readily detected with monomeric proteins. Advantageously, the avidity of the hexameric fusion proteins of the invention permit direct binding.Typically, the surface containing the immobilized ligand is permitted to come into contact with a solution containing the fusion protein and binding is measured using an appropriate detection system. Suitable detection systems include the streptavidin horse-radish peroxidase conjugate, direct conjugation by a tag, e.g., fluorescein. Other systems are well known to those of skill in the art. This invention is not limited by the detection system used.
The assay methods described herein are also useful in screening for inhibition of the interaction between a hexameric fusion protein of the invention (and thus, the binding protein from which it is derived) and its ligand(s). For example, one may screen for inhibitors of CD80 and CD86 binding to CD28 and CTLA-4. In a preferred method, a solution containing the suspected inhibitors is contacted with an immobilized recombinant CD28 or CTLA-4 20 protein substantially simultaneously with contacting the immobilized ligand with the solution containing the hexameric CD8()- or CD86-Igoctp protein. The solution containing the inhibitors may be obtained from any appropriate source, including, for example, extracts of supernatants from culture of bioorganisms, extracts from organisms collected from natural sources, chemical compounds, and mixtures thereof. In another variation, the inhibitor 25 solution may be added prior to or after addition of the CD80- or CD86-Ig~ctp proteins to the immobilized CD28 or CTLA-4 protein. Similar methods may be performed using otherhexameric fusion proteins of the invention and their respective ligands.
The large size of the Igatp fusion proteins is also advantageous for biophysical assay methods dependent on diffusion or rotation of the protein target in solution, such as for 30 example, fluorescence polarization, fluorescence correlation spectroscopy and anisotropic analytical methods.

CA 022~7861 1998-12-14 W 097/47732 PCTrUS97/12599 These examples illustrate the preferred methods for preparing and using the fusion proteins of the invention. These examples are illustrative only and do not limit the scope of the invention.

5 Example 1 - Production and characterization of exemplary atp Ig fusion proteins The following describes the production of CD80-Igatp, CD86-Igatp, and CTLA4-Igatp. Further, for comparison, a construct containing the human IgM tailpiece added to the C-terminus of CD80-Ig was also prepared. This construct. designated CD80-Igutp, differs in amino acid sequence from the o~tp derivative as follows:
CH3 Tailpiece SEQ ID NO:
IgG I SLSPGK (none) 9 ,utp SLSTGK PTLYNVSLVMSDTAGTCY 25 and 10 atp SLSAGK PTHVNVSVVMAEVDGTCY 26and 11 A. Construction of Recombinant I~: Bindin~ Protein Fragment/Fc Fusions The pHbactCd28neo vector for expression of CD28 was previously described [D.
Couez et al., Molecul. Immunol., 31 (1):47-57 (1994)]. For expression of CD80, the coding sequence was cloned by PCR and inserted into a derivative [Dr. F. Letourneur, NIH] of pCDLSRa296 [Y. Takebe et al., Molecul. & Cell. Biol., 8(1):466-472 (1988)] as described [C. A. Fargeas et al., J. Exp. Med.~ 182:667-675 (1995)].
The vector COSFcLink [A. Truneh et a~., Mol. Immunol., 33(3):321-334 (1996)]
was constructed for expression of proteins C-terminally fused to a human IgGI Fc region under the transcriptional control of the major late promoter of CMV. The dhfr cassette in this vector permits selection for gene amplification in response to methotrexate. The coding sequences for the native leader and extracellular domain peptide of CD28 and CD80 were grafted onto a human IgG I heavy chain Fc region in the vector COSFcLink, beginning at the start of the hinge region, in a manner similar to that previously described for CD28 and CD80 [P.S. Linsley et al., J. Exp. Med.~ 174(3):561-569 (1991)]. The Fc region in this vector was derived from the human plasma leukemia cell line ARH-77 [ATCC CRL 1621]
and contains a mutation of cysteine to alanine in the upper hinge region (SEQ ID NO: 27 EPKSA, where the mutation is underscored). The CD28 and CD80 sequences were cloned as KpnI - Eag I fragments by PCR from the vectors described above and inserted into the corresponding sites in COSFcLink. The resulting vectors are termed CD28FcLink and CA 022~7861 1998-12-14 CD80FcLink, respectively. For CD28-Ig, the junction of receptor/Fc fragment (immunoglobulin junction) is SEQ ID NO: 12 --GPSKP/EPKSA-- and the mature processed N-terminal sequence is SEQ ID NO: 13 NKIL --. For CD80-Ig, the immunoglobulin junction is SEQ ID NO: 14 -HFPDq/EPKSA-- and the mature processed N-5 terminal sequence is VIHV-- (Fig. 4A-4D). The lower case "q" in CD80 represents the ~ul~slilulion of ~,lul~n~h~e for the native asparagine.
CD86-Ig, the corresponding binding protein/Fc construct for CD86 collL~inillg the native signal peptide of CD86 (B70) [M. Azuma etal., Nature, 366:76-79 (1993)], was constructed using methods essentially identical to those described above. The signal and 10 extracellular sequences were PCR cloned from a plasmid containing the CD86 (B70) coding region that was obtained by reverse transcriptase/PCR cloning from human B-cell RNA
based on the sequence described by M. Azuma etal. (above). Sequence analysis confirmed identity of this cloned CD86 (B70) region with that of Azuma etal. (above). The amino acid sequence at the junction to the Fc region is: SEQ ID NO: 16 --PPPDHepksa-- where 15 capital and lower case letters indicate CD86 and Fc sequences respectively. The mature processed N-terminal sequence is SEQ ID NO: 17 LKIQ -- (Fig.5A-SB).
CTLA4-Ig, the corresponding binding protein/Fc construct for human CTLA4 containing the native signal peptide of CTLA4 [P. Dariavach etal., Eur J Immunol, 18:
1901-1905 (1988); Harper etal., J Immunol, 147: 1037-1044 (1991)] was constructed in a 20 similar manner. HuC4.32, a pCDM8 plasmid containing the cDNA sequence for human CTLA4 (Harper etal., above) was provided by the laboratory of P. Golstein (Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, 13288 Marseille Cedex 9, France).For PCR cloning of the extracellular domain, the 5' primer was positioned in the pCDM8 vector. [Abberent cloning led to deletion of about 140 bp upstream of the EcoRI site relative 25 to CD80Fclink (compare Fig. 6 with Fig. 3, below).] The amino acid sequence at the junction spanning the end of the CTLA4 extracellular domain and the hinge region is: SEQ
ID NO: 18 --EPCPDSDAepksa-- where capital and lower case letters indicate CTLA4 and Fc sequences respectively and the underlined alanine residue indicates its substitution for phenylalanine in the native CTLA4 sequence. The mature processed N-terminal sequence is 30 SEQ ID NO: 19 MHVA-- (Fig. 6A-6C).
Hexameric forms of the CTLA4, CD80 and CD86 recombinant Ig proteins were created by addition of a .se.quenre encoding the 18 amino acid tail piece region of human IgAI

CA 022~7861 1998-12-14 heavy chain to the C-terminus of the CH3 domain in the expression vectors described above.
These methods are described in detail below.
B. Construction of Hexameric Fusion Proteins For convenience, a Hind III site was introduced into the CH3 domain of CD80FcLink [spanning the 3rd base of the codon for Leu44 1 [EU numbering, E.A. Kabat et al., cited above] through the 2nd base of the codon for L443]. The Hind III site was introduced by standard PCR methods (eg., PCR Protocols: A Guide to Methods and Applications, Innis etal., eds, 1990~ using the following oligonucleotides:
5' oligo (positioned in the hinge region of the vector): SEQ ID NO: 20 Eagl cccaaatcggcc~acaaaact 3' oligo (spanning the C-terminus of CH3): SEQ ID NO: 21 XbaI HindIII
tcagcgagc~rt~ra~tcatttaCCcggagac~g~aggctcttctgcgt The PCR fragments were isolated by agarose gel electrophoresis and purified on Spin Bind columns (FMC Corp). The fragment was digested with Eag I and Xba I and cloned into similarly digested CD80FcLink vector and colonies were screened for the newly created Hind III site, yielding the vector CD80FcLink-Hd.
To introduce the atp sequence, a synthetic oligonucleotide linker encoding this sequence was cloned between the newly created Hind III site and the Xba I site in CD80FcLink-Hd. The complementary oligonucleotides for the linker sequence were:
(5') SEQ ID NO: 22 a~cttgtctgcgggt~rcc~r.cc~tgtcaatgtgtctgttgtç~tggc (Hind III adaptor) ggaggtggacggcacctgctactgatagt (5') SEQ ID NO: 23 ct~ct:~trAgtagcaggtgccgtccacctccgcc~tg~r~r~g~r (Xba I adaptor) ~rat~g~ratgggtggrtttacccgcagaca ... ... ,, ,........ --- I ' CA 022~7861 1998-12-14 5 mg of each linker was denatured at 70~C for 10 minutes. The reactions were cooled to room temperature for 20 minutes. The concentration of linker was titrated from 50 to 5 ng using 1000 ng of gel purified CD80FcLink-Hd vector, digested with Hind III/Xba I. Several 5 colonies from each ligation condition were screened for the presence of the atp linker by PCR
and coJlrhll'ed by DNA sequencing.
A schematic representation of the resulting vector, CD80Fcatplink, is shown in Fig. 2 and the complete DNA sequence is given in Fig. 3A-3H. The vector sequence may differ in some sites from the actual plasmid, but would be function. Introduction of the 10 CD80-Igatp coding region into other standard mammalian expression vectors (e.g., pBK-CMV from Stratagene. La Jolla, CA) will give suitable results and can be modified appropriately, e.g., by introduction of a dhfr gene, by one of skill in the art.The vectors for expression of CD86-Igatp as derived from the corresponding Ig expression vector by replacing the Fc coding region with the Fc-atp region from CD80-15 Igatp. The Fc segment of CD86Fclink ~as excised by cleavage with Eag I (in the hingeregion) and Xba I (following the C-terminus of CH3) and replaced with the corresponding fragment of CD80Fcatplink to give the expression vector CD86Fcatplink. The vector for expression of CTLA4-Igatp was derived by replacing a SpeI-EagI fragment in CD80Fcatplink with the corresponding fragment from CTLA4Fclink to give the expression 20 vector CTLA4Fcatplink. The SpeI site is at base 46 in the CMV promoter region. The sequences of the CD86 and CTLA4 constructs in the region differing from CD80Fcatplink are given in Figs. S and 6.
By a similar approach a vector encoding CD28-Igatp could be prepared starting the CD28Fclink vector described in part A above, or a similar construct encoding an altered 25 version of the CD28 extracellular sequence.
C. Production and purification The CD28-Ig, CD80-Ig and CD86-Ig proteins were produced in CHO cells and purified as described in A. Truneh et al., Mol Immunol, 33: 321-334 (1996) and in I.
Kariv et al., l Immunol. 157: 29-38 (1996). The CTLA4-Ig protein was produced and 30 purified in a similar manner, using the vector construct described above in part A of this section. The Igoctp fusion proteins were shown to be produced upon transfection of the Fcatplink vectors into COS-7 cells following standard procedures for transfection of COS
cells (eg., Current Protocols in Molecular Immunology, edited by F.M. Ausubel etal. 1988, , .. . ~, . . , .. . , ~,~

CA 022~7861 1998-12-14 W O 97/47732 PCTrUS97112599 John Wiley & Sons, vol 1, section 9.1) and for immunoblot analysis (eg., JR Jackson et al., J.
Immunologv, 154:3310-3319 (1995)) with rabbit polyclonal anti-sera prepared against various derivatives of the CD80, CD86, and CTLA4 proteins or goat anti-human Fc antibody. The atp and lltp constructs of CD80-lg were compared in terms of theirS efficiency of expression and oligomerization. As determined by SDS/PAGE and immunoblot analysis, the CD80-Ig~tp construct did not express as well as the atp construct of the invention (not shown). The atp and ,utp proteins were purified from the COS cell supernatants by capture on Prosep A (Bioprocessing, Ltd., Consett County Durham, U.K.) and their state of oligomerization ex~mined by analytical size exclusion chromatography on a 3.2 X 30 mm Superose 6 column run on a Smart System HPLC (Pharmacia Biotech, Piscataway NJ). Both proteins showed a similar profile of a dominant large MW species eluting in the molecular weight range of IgM, consistent with formation of a hexameric structure, and a smaller fraction that eluted at the same size as CD80-Ig itself (not shown).
However, the fraction of apparent hexamer in the o~tp construct was higher (about 80%) than for the ,utp construct (about 60%). Both the higher level of expression and the greater efficiency of oligomer formation indicated that the atp construct of the invention was superior to the ~tp derivative. Subsequently, the CD86-Igatp and the CTLA4-Igatpproteins were produced in COS cells at about the same level observed for the C~80-Igatp protein (0. l - 0.2 ug/ml). The CD80- and CD86-Igatp proteins were then produced in a CHO cell system (A. Truneh et al., Mol Immunol. 33: 321-334 (1996)) at levels of 5-10 mg/L. This level of production is comparable to other highly expressed proteins (e.g.
antibodies) produced in the same manner in this system.
These results indicate that development of standard amplified CHO cell lines with high production levels of hexamer (50 mg/L or greater) is feasible. A procedure for transfection and amplification in CHO cells is described in P. Hensley et al., J. Biol. Chem., 269:23949-23958 (1994)). Briefly, a total of 30 ug of linearized plasmid DNA (e.g.
CD80Fcatplink) is electroporated into lxlO' cells. The cells are initially selected in nucleoside-free medium in 96 well plates. After three to four weeks, media from growth positive wells is screened for expression - e.g., in an ELISA format using an antibody directed against the Fc region of human IgG1. The highest expressing colonies are expanded and selected in increasing concentrations of methotrexate for amplification of the transfected vectors. If a commercial vector like pBK-CMV (noted above) is used, a dhfr gene should be CA 022~7861 1998-12-14 W O97/47732 rCT~US97/12599 introduced into this plasmid or provided on a second co-transfecting plasmid to allow selection of amplification in methotrexate.
The proteins produced in CHO cells were purified by protein A affinity and size exclusion chromatography. For the CD80-lg h~ r, thirty liters of conditioned medium 5 containing CD80-Igatp were chromatographed on a Protein A Sepharose Fast Flow column (Pl,~-""acia) at 20 ml/min. The column (5.0 x 11.6 cm; 225 ml) were preequilihrated in 20 mM sodium phosphate, 150 mM NaCl, pH 7.5 (PBS). After loading, the column was washed with 1.8 L of PBS to baseline absorbance. CD80-Igatp was eluted with 0.1 M
sodium citrate, pH 3.0 at 10 ml/min. The elL~ate was neutralized immP~ ly with 1 M Tris-l O HCl, pH 8Ø After filtration with a Sterivex GV filter (Millipore) using a 60 ml syringe, CD80-Igatp was concentrated using an Amicon stirred cell and a YM100 membrane to 1.3 mg/ml. CD80-Igatp was frozen using a dry ice ethanol bath and stored at -70~C.
To separate hexamer from monomer, 10 ml of the concentrated CD80-Igatp was chromatographed on a Superdex 200 column (2.6 x 60 cm; Pharmacia) at 2.5 ml/min. The first peak (eluted at about 45 minutes) containing the majority (about gO%) of the 280 nm absorbing material was pooled (20 ml; 0.6 mg/ml), frozen as before and stored at -70 (Figure 7). This material eluted at approximately the position of thyroglobulin (~700,000 Da.) just behind the void volume. A minor peak at about 57 minutes corresponded to "monomer"
CD80-Ig. The integrity of the CD80-Igatp in the peak fractions is shown by the single 20 band observed in coomassie stained SDS/PAGE gel run under reducing conditions (lane R
in the inset in Figure 7). The diffuse nature of the band is characteristic of highly glycosylated proteins and is thus expected for CD80-Igatp which contains 10 consensus N-linked glycosylation sites per polypeptide chain. Under nonreducing conditions, all of the protein migrates as high molecular weight species (lane NR in Figure 7, insert). This 25 dominant fraction migrated as a symmetrical peak at a MW consistent with a hexamer with a lesser amount of a species that migrated at the size observed for the monomeric CD80-Ig protein (i.e., the Ig homodimer). N-terminal amino acid sequence analysis revealed identity to the previous analysis of CD80-Ig and to that described by others [G. J. Freeman etal., 174(3):625 631 (1991)]. The CD86-Igatp protein was purified in a similar manner. The 30 CTLA4-Igatp protein was expressed in COS cells, but not further characterized.

CA 022~7861 1998-12-14 D. Protein characterization - Molecular Size The size exclusion chromatography noted above during purification was consistent with formation of a homogeneous hexameric species cont~h~ g six CD80-Ig subunits. The size and homogeneity of the CD80-lgatp protein produced in CHO cells was 5 also investigated by analytical ultracentrifugation. Equilibrium sedimPntAtion data for CD80-Igatp in PBS, pH 7.4 is shown in Fig. 8, lower panel. The sample was sedimented at 6000 rpm for 87 hours at 25 ~C in a Beckman XL-A analytical ultracentrifuge. The weight average molecular weight for a fit to all the data was 1,125,000 +/- 5,000 Da. The expected molecular mass of the hexamer of 864,000, assuming 2000 Da. for each N-linked 10 glycosylation site. The data could also be fitted to a hexamer <-> (hexamer)2 model with a Kd of ~ 2 x 10~7 M. The curves in the lower panel are for the fitted distribution of hexamer and (hexamer)2. The sum of these two curves fits the observed data well. Inclusion of terms for a monomer (131 kDa) did not improve the fit. The distribution of residuals (fitted-observed data) for the fit of the monomer dimer model to the data is shown in the 15 upper panel of Fig 8. The residuals are small and random, indicating a good fit. For a description of the analysis see W. Chan et al., Folding and Design~ 1(2): 77-89 (1996). The lower panel inset shows g(s*) analysis of velocity sedimentation data of the protein taken in the absorption mode. Data was collected at 30,000 rpm at 22 ~C. The data could be fitted to two species, one of 19.4 S and one of 26.7 S which could be the hexamer and 20 (hexamer)2 species. For g(s*) data analysis, see W. F. Stafford, Current Opinion in Biotechnolo~y ,8(1): 14-24 (1997) .
The size and extent of covalent association of the CD80- and CD86-Igatp proteinswere examined by SDS/PAGE. Under reducing conditions all of the protein migrated in a diffuse band at about the same size as the corresponding standard Ig constructs, as shown 25 for the CD80-Igatp protein in the inset in Figure 7. Under nonreducing conditions in a 4%
gel, the Igatp constructs migrated as very diffuse bands in the size range of IgM with little material co-migrating with the corresponding Ig constructs at about 150,000 Da (not shown). These results indicate that most of the individual polypeptide chains in the Igatp proteins are covalently joined through cystine bonds, consistent with the described disulfide 30 bond formation among the cysteine residues in the ~ tailpiece segment of IgM [A. C. Davis et al., EMBO J.~ 8(9): 2519-2526 (1989)]. The diffuse nature of the high molecular weight ___ _._ T

CA 022~7861 1998-12-14 bands may reflect incomplete disulfide bond formation but also is expected since a hexamer form of CD80- or CD86-Igatp would contain 120 potential N-linked glycosylation sites.
E. Protein Characterization - Bindin~ properties In several assay formats the hexameric CD80- and CD86-Igatp proteins were distinguished from the corresponding standard Ig fusion proteins by their markedly higher binding avidity to CD28 when it was presented in a multivalent array.
1) Binding to immobilized CD28-Ig in an ELISA format For this assay format, the CD80-Igatp protein was biotinylated for simplicity of assay and for ease of detection since the CD28 protein absorbed to the plate wells was also a human Ig fusion construct. Biotinylation was carried out essentially as described in Avidin-Biotin Chemistry: A handbook, M. D. Savage etal., Pierce Chemical Company (1992). In several preparations of the protein, the molar ratio of biotin/CD80-Ig monomer was about 10: 1. All steps of the assay after coating were carried out at room temperature.
The wells of 96 well microtiter plates (Immunlon 4, Dynatech Laboratories) were coated with CD28-lg (1, 2, or 4 ,ug/ml) in 100 ~I/well of 0.1 M sodium bicarbonate, pH 9.4 and inrllh~t~d overnite @ 4~C. The wells were washed with PBS (phosphate buffered saline) and blocked with 0.5% gelatin in PBS for 1 hour. Following an additional PBS wash, biotinylated CD80-Igatp was serially diluted in PBS containing 1 mg/ml BSA, 0.05% Tween directly in the wells in a final volume of 0. I ml and incubated for 1 hour. The wells were washed with PBS and bound CD80-Igatp protein was measured by the addition of 0.1 ml of strepavidin-HRP (streptavidin conjugated with horseradish peroxidase (Southern Biotech)) at a I :2000 dilution for I hour,followed by washing and color development with 100 ~11 ABTS
substrate (Kierkegaard and Perry Laboratories Inc., Maryland) and measurement ofabsorbance at 405 nm. In some cases the color reactions were arrested by addition of 100 ~1 of I % SDS prior to measurement of absorbance. A plot of CD80-Igatp binding versus concentration of added protein is shown in Fig. 9. In this figure, "CD28-Fc", "CTLA4-Fc", and "B7-FcA" denote CD28-Ig, CTLA4-Ig, and CD80-Igatp, respectively. These curves ~Fig. 9) indicate that concentration dependent binding of biotinylated CD80-Igatp was inhibited by simultaneous addition of the CD28.1 MAb (a murine MAb to human CD28 that inhibits binding of CD80 to CD28; Nunes etal., Int. Immunol., 5:311-315 (1993)) or CTLA4-Ig protein (here labeled as CTLA4-Fc). Under the same conditions, biotinylated CD80-Ig itself showed little binding and only at much higher concentrations (Figure 10).
In the same format biotinylated CD86-Igatp also showed good binding to CD28-Ig (Fig CA 022~7861 1998-12-14 W O 97/47732 PCTrUS97/12599 10). All three biotinylated proteins showed good binding to immobilized CTLA4-Ig (Fig.
11), as expected because of the higher affinity of this interaction [P. S. Linsley et. al., Immunity 1: 793-801 (1994), and see part 4 of this example below], and the rank order of binding was the same as observed with immobilized CD28-Ig.
The specificity of the binding reaction was demonstrated by the expected hierarchical competition of binding with (I) CTLA4-Ig, (2) CD28.2 [Nunes et a/., 1993, cited above], a murine MAb to human CD28 that inhibits binding of CD80 to CD28, (3) unlabeled CD80-and CD86-Igatp proteins, (4) and the expected much weaker inhibition by the monomeric CD80-Ig fusion protein. One example is shown in Fig. 12. Briefly, microtiter wells were coated with 2 ~g/ml CD28-Ig and biotinylated CD80-Igatp was added at a concentration of 50 ~lg/ml, followed immediately by the indicated amounts of llnl~heled CD80-Igatp (B7FcA), CD80-Ig (B7Ig), CTLA4-Ig, or the MAb CD28.2. At 50 llg/ml, the biotinylated CD80-Igatp gives about 50% saturation of OD4~5 (see Fig. 9). CD80-Ig was much less efficient than CD80-Igatp in blocking binding, consistent with the expected lower affinity/avidity of the CD80-Ig protein for the immobilized CD28-Ig protein. The controls gave the expected results - the CD28.2 MAb blocked the binding site on CD28 and similarly, CTLA4-Ig blocked the binding sites on CD80-Igoctp.
Other assay formats are possible. A second example utilizes a CD28-muIg fusion protein constructed in a manner analogous to CD28-Ig except that the Ig region was derived from mouse Ig2a instead of human IgG I . More particularly, the protein was expressed using the vector CosCD28mFc2aLink, which is comparable to the CosCD28FcLink vector (described above), except that the human IgGl-Fc region was replaced with that of mouse IgG2a beginning at the Eag I site in the hinge sequence [described in I. Kariv et al., J.
Immunol.. 157:29-38 (1996)]. The amino acid sequence in the resulting hybrid hinge region 25 is as follows: SEQ ID NO: 24 --GPSKPepl~s~lKP--, where capital letters correspond to the end of CD28 sequence, lower case letters are residues from the human IgG, hinge region, underlined lower case letters are a 2 residue substitution introduced to create an Eag I site, and bold capital letters indicate the beginning of murine IgG2a hinge region.
The CD28-muIg protein was indirectly immobilized in wells using goat anti-mouse 30 Fc antibody and then CD80-Igatp binding was carried out similarly to that described above.
More specifically, CD28-muIg proteins containing wild-type or mutant CD28 sequences and, at equal concentrations, were captured on goat anti-mouse IgG antibody coated 96 well plates. The plates were washed with lx PBS, blocked with 0.5% gelatin-PBS for I hour, and CA 022~7861 1998-12-14 then inr.ub~d with either biotinylated CD80- or CD86-Igatp for 45 min. The plates were washed and Igatp fusion protein was qll~nti~tP.d~c described above. . This assay was used to examine the effects of mutations in CD28 on binding to CD80 and CD86, as illustrated in Figs. 13A and 13B (I. Kariv etal., J. Immunol., 157:29-38 (1996)). Each of the mutant S CD28-muIg2a proteins was captured on the goat anti-mouse IgG coated wells and the binding of biotinylated CD80 - Igatp (Fig. 13A) or CD86 - Igatp (Fig. 13B) was measured.Equivalent capture of each of the CD28-muIg2a proteins was verified by the c~ bl~
binding of polyclonal rabbit CD28 antisera to each of the proteins (Fig. 13C).

2) Binding to immobilized CD28-Ig in a biosensor assay format The binding of CD80-Igatp or CD80-Ig to immobilized CD28 were compared by surface plasmon resonance analysis using a BIAcore instrument, following procedures similar to that described for other proteins [K. Johanson et. al., J. Biol. Chem, 270: 9459-9471 (1995), and references therein].
For comparison of CD80-lg and CD80-Igatp, approximately 4000 RU of CD28-Ig were immobilized onto a BIAcore CMS sensor surface (BIAcore, Piscataway, NJ) by covalent attachment to the surface through its amines. Covalent attachment was achieved by firstly activating the surface with a l: 1 mixture of 0. I M solution of N-hydroxysuccinamide and 0.1 M l-ethyl-3-(3-dimethylaminopropyl)carbodiimide. A
solution of CD28-Ig 50 ug/ml in 0.01 M sodium acetate pH 4.7 was then passed over the surface. Unreacted N-hydroxysuccinamide esters were then deactivated with I M
ethanolamine pH 8.5. The surface was equilibrated with running buffer composed of 20 mM HEPES 150 mM NaCI, pH 7.2, 3 mM EDTA and 0.005% Tween 20. The CD80-Ig and CD80-Igatp (20 ug/ml) diluted in running buffer were injected over the surface (60 ul) with a flowrate of 10 ul/min. The results show that CD80-Ig dissociates very rapidly from the CD28-Ig coated surface, whereas the rate of dissociation for CD80-Igatp is about three orders of m~eni~lcle slower (Figure 14).
For comparison of CD80-Igatp and CD86-Igatp binding with immobilized CD28-Ig, solutions of CD80-Igatp and CD86-Igatp (S ug/ml) were prepared in running buffer described above. Sample solutions were injected (60 ul) at 10 ul/min. Between samples, the surface was regenerated with a 30 ul injection of Gentle elution buffer (Pierce Chemicals, Rockville, ILL). The results show that like CD80-Igatp above, CD86-Igatp dissociates slowly from the CD28-Ig surface (Figure 15). The off-rate for CD86-Igatp CA 022~7861 1998-12-14 W 097/47732 PCT~US97tl2599 appears higher than that for the CD80 construct, consistent with the weaker binding of this protein in the ELISA (part 1, above) and cell binding (part 3, below) formats and the lower intrinsic affinity measured by calorimetry (part 4, below).
3) Binding to cells expressing cell-surface CD28 By flow cytometry, both CD80- and CD86-Igatp show specific binding to CD28 positive cells (Figs. 16A and 16B), whereas no binding is observed with CD80- or CD86-Ig themselves (not shown). Non-adherent CD28 expressing cells (PCD28.1.s2.1) were used for this assay format. PCD28.1.S2.1 cells were created by transfection of PE30.2 cells [D.
Emilie et al., Eur. J.. Immunol., L:1619-1624 (1989)] with a vector for expression of human CD28 [D. Couez et al., Molecular Immunolo~y, 31 :47-57 (1994)]. All incubations were carried out on ice. Unlabelled CD80- and CD86-Igatp or CD80- and CD86-Ig were incubated with the cells in binding buffer consisting of PBS w/o Ca+2/Mg+2, 0.2% bovine serum albumin and 0.1 % sodium azide. After washing twice in binding buffer, bound fusion proteins were detected with a I :2000 dilution of a goat anti-human polyclonal antibody labeled with FITC (Flourescein isothiocyanate, Southern Biotech). Following two additional washes in binding buffer, the cells were resuspended in binding buffer and analyzed on a FACSort analyzer (Becton-Dickinson) using a 488nm laser. Low non-specific binding was shown by incubating the cells with a 200 fold excess of a CD28 monoclonal antibody 28.1 prior to ("block" curves, Figure C), or at the same time as ("competition" curves, Figs. 16A and 16B), or by addition of CTLA4-Ig with the CD80 and CD86 fusion proteins (not shown).

4) Binding to CD28-Ig and CTLA4-Ig in solution The solution binding properties of the hexameric and standard Ig fusion proteins of CD80 and CD86 were compared using isothermal titration calorimetry, essentially as described previously for other proteins [K. Johanson et. al., J. Biol. Chem. 270: 9459-9471 (1995), and references therein]. The binding to CTLA4-Ig is summarized in the following table:

CA 022~7861 1998-12-14 Table I
Direct comparison of CTLA4-Ig binding properties of CD80 and CD86 Ig fusion proteins as measured by titration calorimetry at 2 temperatures s Experimental Kd, nM atMolarRatio ~H, Kd ConstructTel-lp~ldtlJt~ T~ ldtul~; (CTLA4-Ig kcal/mol nM at per construct) 37~C
CD80-Ig 37~C 5.4 0.72 -32+ 2 5 CD80- 37~C 5.9 4.02 -36 + 2 6 Igatp CD86-lg 37~C 38 0.80 -33 + 3 40 CD86- 37~C 20 2.72 -37 + 4 20 Igatp CD80-lg 44~C 4.9 0.60 -36 + 4 2 CD80- 44~C 8.1 3.79 -38 + 3 2 Igatp CD86-lg 44~C 71 0.79 -34 + 4 22 CD86- 44~C 38 2.83 -41 + 4 9 Igatp The error in Kd's is about a factor of 2 and the error in molar binding ratio's is 10-20%. Kd values at 37~C were either measured directly at 37~C or were corrected for temperature differences using the van't Hoff equation, as described in M. L. Doyle et. al., J. Mol.
10 Reco~nition, 2: 65-74 (1996). Concentrations were defined by absorbance at 280 nm using the following: A) molecular masses of 90,059 (CTLA4-Ig), 127,000 (CD80-Ig), 810,000 - (CD80-Igatp), 140,000 (CD86-Ig), and 890,000 (CD86-Igatp) and B) calculated extinction coefficients of 1.22 (CTLA4-lg), 1.10 (CD80-Ig and CD80-Igatp), and I .03 (CD86-Ig and CD86-Igatp). The molecular weights for CTLA4-Ig, CD80-Ig, and CD86-Ig were 15 determined by mass spectral analysis. The molecular masses of CD80-Igatp and CD86-Igatp were estimated as 6x the mass of the respective Ig proteins plus 40,000 Da.
contributed by the twelve tailpiece segments.
Direct comparison of the CTLA4-Ig binding to CD80-Ig, CD80-Igatp, CD86-Ig, and CD86-Igatp constructs in solution phase by isothermal titration calorimetry CA 022~7861 1998-12-14 demonstrates several features. First, the affinities of the Ig versus Ig-atp constructs are equivalent in solution. This suggests that, as expected, solution binding affinities of the atp constructs do not benefit from avidity effects like they do in ELISA and cell binding assays.
Second, the enthalpy changes which accompany the molecular interactions of the Ig and Igatp constructs are also the same and support the view that the molecular details of the interactions are the same. Third, the titration equivalence points for CTLA4-Ig binding to the CD80 and CD86 Ig versus Igatp constructs indicate that all these reagents were 250%
active during the calorimetry assay. With regard to this latter point, comparison of the Igatp and Ig constructs shows a ratio of about 6 for CD80, indicating about equivalent binding activity for the CD80 domains in both constructs. The lower ratio for the corresponding CD80-Igatp protein indicates some loss of activity in this preparation.
Interactions of CD28-Ig with either CD80- or CD86-Ig were not detected in solution by calorimetry, suggesting an affinity of interaction weaker than I uM. This lower affinity for CD28 than for CTLA4 is in agreement with other reports [P. S. Linsley et. al., Immunity 1: 793-801 (1994)]. CD28-Ig also did not show detectable binding to CD80- or CD86-Igatp, which is consistent with the solution affinities of the atp constructs not benefiting from avidity effects.

Example 2 - Demonstration of agonist activity for the CD80- and CD86 I~atp protein A. CTLL-2 bioassay for detection of IL-2 levels The CD80- and CD86-Igatp proteins were compared to the coll~s~.ol.ding CD80 and CD86-Ig proteins to determine their ability to stimulate cells expressing human CD28 using two murine T-cell hybridoma cell lines expressing human CD28, PCD28.1.s2.1 and DCL27CD28wt.s2. The PCD28.1.s2.1 cell line was described in Example I, part 3.
The DCL27CD28wt.s2 cell line was created by transfection of the DC27 cell line [F. Pages etal., Nature, 369:327-329 (1994); F. Pages etal., J. Biol. Chem., 271(16):9403-9409 (1996)] with the same CD28 expression vector used for the PCD28.1.s2.1 cells [D. Couez et al., Molecular Immunolo~y, 31 :47-57 (1994)1. These cell lines were examined for their ability to produce IL-2 in response to activation with CD80- and CD86-Ig in comparison with the corresponding Igatp fusion proteins. 96-well plates were coated with or without a CD3 antibody together with the CD80 and CD86 fusion proteins. This was accomplished by first incubating the plates with a previously determined suboptimal concentration of hamster anti-human CD3 antibody (MAb 145-2CI I, Boerhinger-Mannheim Biochemicals) for two _ .

CA 022C,7861 1998-12-14 hours at room temperature (RT) or with just buffer alone, washing the plates with PBS, adding goat anti-human Ig heavy chain (GAH-lgHc, Sigma Chemical Co.) for an additional two hours at RT, washing again and coating with dirl~lclll concentrations of the CD80 or CD86 fusion protein for 16-18 hours at 4~C, washing again, and finally blocking for 30 min.
with 0.2% BSA-PBS. T cells (lxlO5/well) were added in 150,ul medium into duplicate wells. For comparison, the soluble fusion proteins and the 248.23.2 CD28 MAb (IgM) [A.
Morretta, University of Genova, Italy] were added to non-coated wells. T cells were in~.uh"~.d in the wells for 24 hours at 37~C, and supernatants were collected and evaluated for IL-2 levels in a standard CTLL-2 bioassay [S.M. Gillis etal., J. Immunol., 120:2027 (1978)]. Briefly, lx104 IL-2 dependent CTLL-2 cells (ATCC)/well in 75 ~11 medium were added to an equal volume of test supernatant and incubated for 24 hours at 37~C. The cells were pulsed with 10,ul of 5 mg/ml MTT (Sigma Chemical Co.) for 4 hours, and Iysed with 100 m] 10% SDS/O.OlN HCI solution for 14-16 hours. OD570 readings were converted into ng/ml of IL-2 based on a standard curve generated by treating cells with known concentrations of IL-2.
In all assays, the CD80- and CD86-Igoctp proteins were more efficient stim~ ors of the CD28 T-cells than the corresponding monomeric Ig constructs (Figs. 17 and 18). The soluble hexameric proteins induced IL-2 production in the absence of CD3 cro.~.clinking (GAH), whereas under the same conditions, no activity was observed with CD80- or CD86-Ig themselves. A similar level of IL-2 induction was observed with the oligomeric CD28 IgM
antibody 248.23.2. Cross-linking of the hexameric CD80 and CD86 proteins with GAH
antibody increased the IL-2 response relative to the absence of cross-linker, but still did not give a response for the monomeric Ig constructs. In the presence of CD3 antibody, the differences between the hexameric and monomeric Ig fusion proteins were minimz~l, being about 2-fold or less.
B. Fluorescein di-b-D-galactosidase (FDG) assay for detection of IL-2 promoter activity A second assay for agonist activity measured induction of IL-2 promoter activity, rather than production of IL-2 protein. The PCD28.1.S2.1 cells described above also contain lacZ fused to the IL-2 promoter. Thus, the PCD28.1.S2.1 cell line provides a convenient system for measuring IL-2 promoter activity upon CD28-m~ t~.d T cell simulation. T cells were activated as described above for the CTLL-2 assay, spun down, resuspended in 50 Ill of media + 50,LLI of PBS, Iysed with 10 ~11 of 20% Triton X-100, and .

CA 022~7861 1998-12-14 WO 97/47732 PCTrUS97tl2599 supplemented with 25 ~11 of 10 mM FDG (Molecular Probes), a fluorogenic substrate for b-galactosidase. Hydrolysis of FDG first yields fluorescein monogalactoside (F~IG) and then the highly fJuorescent product fluorescein. Cell lysates were incubated with FDG for 60 min., and the levels of fluorescence were measured by Fluoroscan (MTX Lab Systems, Inc).
The results of these assays (Fig. 19) were similar to those described above for IL-2 production. The primary difference was that low levels of IL-2 promoter activity were observed for the monomeric Ig proteins.
C. Stimulation of CD28 cells by CD80- and CD86-Igatp proteins in solution In the above examples (parts A and B), the Ig and Igatp proteins showed the 10 greatest activity when captured on the surface of the microtiter wellHowever, the CD80-and CD86-Igatp proteins were also able to stimulate CD28 cells when added directly to the cells in solution, whereas no response was observed with the corresponding standard Ig fusion proteins. CD80-and CD86-Igatp showed a dose dependent stimulation of IL-2promoter activity (Fig 20A) and IL-2 production (Fig 20B) when added to PC28.1.s2.1 cells. In contrast. no stimulation was observed with CD80-Ig (Figs. 20A and 20B) or CD86-Ig (not shown). IL-2 promoter activity and IL-2 levels were measured similarly to that described in parts A and B above, except that proliferation of the reader CTLL-2 cells was measured by 3H-thymidine incorporation. The level of response at near saturation levels of CD80- and CD86-Igatp proteins (] ug/ml) was comparable to that observed for stimulation through cross-linking of CD3 with immobilized CD3 antibody (Fig 20C). The specificity of the response to CD80- and CD86-Igatp was confirmed by complete blockade with the addition of CTLA4-Ig (not shown).
In summary. the results from these assays show that the CD80- and CD86- Igatp proteins have agonist activity under conditions where little or no activity was observed for the corresponding monomeric Ig proteins.

Example 3 - Compound screen assay for identifying small molecule antagonists of the interaction between CD28 and CD80 An ELISA format was used to identify small molecule antagonists of CD80 and CD86 binding to CD28 by screening a large bank of chemical compounds and naturalproducts. The assay was carried out as in the format described in Example I, part E. l, except that immediately following addition of the biotinylated CD80-Igatp (222 ng/ml in a volume of 90 ~1), dilutions of test compound were added (10,~LI). The compounds were ., . .,,. . ~ ~

CA 022~7861 1998-12-14 dissolved at 100x assay concentration in dimethyl sulfoxide (DMSO) and subsequently diluted in 50%DMSO/50% H2O to a lOX working stock. The assay was not sensitive (<10%
alteration of signal) to DMSO at concentrations of 5% or less.
Results from one test assay are summarized in Fig. 21 and Table II. The BM-34 5 test set consists of 968 compounds in two formats - as individual compounds and as 88 multimixes with 11 individual compounds in each multimix sample. Both BM-34 fonnats were assayed (at a concentration of 200 ~g/ml for each multimix sample and 20 ,ug/ml for individual compounds) for inhibition of biotinylated CD80-Igatp binding to immobilized CD28-lg in 96 well plates. Results for setting a 70% or 85% cutoff for inhibition are shown 10 in Table II. In Fig. 21, the percent inhibition range is plotted against the number of compounds showing the indicated range of inhibition. The low percentage of compounds showing activity in the 80-90 % range of inhibition makes this a suitable threshold for rapid screening.
Table II
CD80-Igatp Screen Assay Results BM-34 Test Compound Set Result 70 % Cutoff 85 % Cutoff Hits on Multimix plates 7 + Multimix Samples with + Compound 5/7 1/1 - Multimix Samples with + Compound 8 0 As illustrated in this table, eight of the mixes gave 70% or greater inhibition.Deconvolution by assay of the individual compounds from these 8 mixes at 20 ~,lg/ml confirmed that there was a compound with comparable activity. The two other mixes that failed to confirm had one or more compounds with activity very close to the 70% inhibition CA 022~7861 1998-12-14 W O 97/47732 PCTrUS97tl2599 observed in the original multimix assay. Selecting a higher cutoff of 85% gave only one multimix hit and that was confirmed in the assay of individual compounds. Further evidence of reproducibility and selectivity was that only eight compounds from mixes below the 70%
cutoff showed > 70% inhibition when assayed individually. Selectivity was further increased 5 by reducing the concentration of the multimix samples to 100 ~lg/ml and the individual co~ uu,.ds to 10 ,ug/ml. This corresponds to about a 30 ~M concentration for thecompounds since their average MW is 300-400 daltons. At 100 ,ug/ml, mllltimix samples showed a desired shift to lower average inhibition (90% of the mixtures gave 60% or less inhibition) while retaining an acceptable hit rate at a high level of inhibition (4% of the 10 mixtures giving 70% or greater inhibition). Through the use of this assay, small molecule inhibitors of the interaction of CD80 with CD28 can be identified.

Example 4 - atp-mediated oligomerization of a mouse/human I~Gl chimeric antibody.
To examine the generalization of atp-mediated hexamer formation of the Fc region15 of human IgG, the atp segment was introduced into a chimeric antibody containing heavy and light chain variable regions from the mouse monoclonal antibody IC8 and the human kappa and IgG] constant regions. IC8 is directed against the human EPO (erythropoeitin) receptor. The atp sequence was introduced onto the heavy chain of the antibody by replacing the Eco RVSac II fragment of CD80Fcatplink with the Eco RI/Sac II fragment of 20 EpoR(CH)IgG1-PCN, a vectorcontaining the heavy chain of the chimeric lC8 antibody, to give the vector EpoR(CH)Fcatplink. In both vectors, Eco RI cleaves between the CMV
promoter and the start of the N-terminal signal sequences and Sac II cleaves at a conserved site in constant region 2 of the human heavy chain.
Test samples of the hexameric mAb were produced in COS-7 cells upon co-25 transfection of EpoR(CH)Fcatplink and a vector for expression of the light chimeric lightchain, following procedures described above in Example 1, part C. Initially, 5 T150 flasks were co-transfected with the two vectors and 300 ml of conditioned media were collected.
The hexameric antibody was purified by affinity chromatography on Protein A. Purity was about 90% as determined by coomassie staining of the sample as analyzed by reducing 30 SDS/PAGE. Under nonreducing conditions on SDS/PAGE, the antibody migrated in the size range of IgM (not shown).
The sample was further characterized by analytical size exclusion chromatographyon a 3.2 X 30 mm Superose 6 column run on a Smart System HPLC (Pharmacia Biotech, ~ T

CA 022~7861 1998-12-14 W O 97/47732 PCTrUS97/12599 Piscataway NJ). The major peak (Fig. 22) corresponds to binding activity, as monitored in an ELISA using a recombinant human EPO receptor Ig fusion protein (EPOr-Ig), and eluted at a size consistent with hexamer formation (anti-EPOr-IgGlatp). The parental chimeric antibody (anti-EPOr-IgGl) elutes substantially later from the column and is represented in 5 the figure by the dashed lines.
These results indicate that addition of the atp segment to the human IgG1 constant region leads to formation of hexameric antibody.
Numerous modifications and variations of the present invention are inchlrl~d in the above-identified specification and are expected to be obvious to one of skill in the art. Such 10 modifications and alterations to the compositions and processes of the present invention are believed to be encompassed in the scope of the claims appended hereto.

~ . . .

CA 022~786l l998-l2-l4 SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Sweet, Raymond W.
Truneh, Alemseged Chaikin, Margery A.
Lyn, Sally D.P.

(ii) TITLE OF INVENTION: Hexameric Fusion Proteins and Uses Therefor (iii) NUMBER OF SEQUENCES: 27 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: SmithKline Beecham Corporation -Corporate Patents (B) STREET: 709 Swedeland Road (C) CITY: King of Prussia (D) STATE: Pennsylvania (E) COUNTRY: USA
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(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
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(B) FILING DATE: 13-June-1997 (C) CLASSIFICATION:

PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE: 14-June-1996 (C) CLASSIFICATION: 60/019,934 PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US
(B) FILING DATE: 19-FEB-1997 (C) CLASSIFICATION: 60/043,948 CA 022~786l l998-l2-l4 PRIOR APPLICATION DATA:
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(B) FILING DATE: 21-FEB-1997 (C) CLASSIFICATION: 60/038,915 (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Gimmi, Edward R.
(B) REGISTRATION NUMBER: 38,891 (C) REFERENCE/DOCKET NUMBER: P50496 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 610-270-5015 (B) TELEFAX: 610-270-5090 (2) INFORMATION FOR SEQ ID NO:1:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7167 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

CA 022~786l l998-l2-l4 W O 97/47732 PCTrUS97/12599 AAAGAAGTGG CAACGCTGTC CTGTGGTCAC AA~ lll~lG TTGAAGAGCT GGCACAAACT 900 T

CA 022~786l l998-l2-l4 W O 97147732 PCTrUS97/12599 ~"lG~l"l~ CC ATTCCTGAGA AGAATCGACC TTTAAAGGAC AGAATTAATA TAGTTCTCAG 3180 CA 022~7861 1998-12-14 AAGGATCATG CAGGAATTTG AAAGTGACAC ~lllllCCCA GAAATTGATT TGGGGAAATA 3420 CTTATAATGG TTACAAATAA AGCAATAGCA TCACAAATTT CACAAATAAA GCAlllllll 3660 __.

CA 022~7861 1998-12-14 W O 97/47732 PCT~US97112599 4]

. ~ . . ..

CA 022~786l l998-l2-l4 ACCGCTGGTA GCGGTGGTTT llll~lll'GC AAGCAGCAGA TTACGCGCAG AAAAAAAGGA 6000 TCTCAAGAAG ATCCTTTGAT ~''l''l''l''l'~''l'ACG GGGTCTGACG CTCAGTGGAA CGAAAACTCA 6060 TCCGGTTCCC AACGATCAAG GCGAGTTACA TGATCCCCCA l~ll'~'lGCAA AAAAGCGGTT 6600 ATTGGAAAAC ~ll~ll'CGGG GCGAAAACTC TCAAGGATCT TACCGCTGTT GAGATCCAGT 6900 CA 022~786l l998-l2-l4 (2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1560 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:
~A) NAME/KEY: CDS
(B) LOCATION: 63..1538 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly CA 022~786l l998-l2-l4 W 097/47732 PCT~US97/12599 Asp Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser Lys Leu Asp Phe Asn Met Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Gln Glu Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro CA 022~786l l998-l2-l4 W O 97/47732 PCT~US97/12599 Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe ........ .. ~.. ........ ..... .. .

CA 022~786l l998-l2-l4 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Ala Gly Lys Pro Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp Gly Thr Cys Tyr (2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 492 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUE~CE DESCRIPTION: SEQ ID NO:3:

Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr Leu Asn Phe Phe Gln Leu Leu Val Leu Ala Gly Leu Ser His Phe Cys Ser Gly Val Ile His Val Thr Lys Glu Val Lys Glu Val Ala Thr Leu Ser Cys Gly His Asn Val Ser Val Glu Glu Leu Ala Gln Thr Arg Ile Tyr Trp Gln Lys Glu Lys Lys Met Val Leu Thr Met Met Ser Gly Asp CA 022~7861 1998-12-14 W O 97/47732 PCT~US97/12599 Met Asn Ile Trp Pro Glu Tyr Lys Asn Arg Thr Ile Phe Asp Ile Thr Asn Asn Leu Ser Ile Val Ile Leu Ala Leu Arg Pro Ser Asp Glu Gly Thr Tyr Glu Cys Val Val Leu Lys Tyr Glu Lys Asp Ala Phe Lys Arg Glu His Leu Ala Glu Val Thr Leu Ser Val Lys Ala Asp Phe Pro Thr Pro Ser Ile Ser Asp Phe Glu Ile Pro Thr Ser Asn Ile Arg Arg Ile Ile Cys Ser Thr Ser Gly Gly Phe Pro Glu Pro His Leu Ser Trp Leu Glu Asn Gly Glu Glu Leu Asn Ala Ile Asn Thr Thr Val Ser Gln Asp Pro Glu Thr Glu Leu Tyr Ala Val Ser Ser hys Leu Asp Phe Asn Met Thr Thr Asn His Ser Phe Met Cys Leu Ile Lys Tyr Gly His Leu Arg Val Asn Gln Thr Phe Asn Trp Asn Thr Thr Lys Gln Glu His Phe Pro Asp Gln Glu Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val Hi s Asn Ala Lys Thr Lys Pro Arg Glu .... " , ... ....

CA 022~786l l998-l2-l4 W 097/47732 PCTrUS97/12599 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Ala Gly Lys Pro Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp Gly Thr Cys Tyr (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 831 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA

CA 022~786l l998-l2-l4 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 52..831 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Asp Pro Gln Cys Thr Met Gly Leu Ser Asn Ile Leu Phe Val Met Ala Phe Leu Leu Ser Gly Ala Ala Pro Leu Lys Ile Gln Ala Tyr Phe Asn Glu Thr Ala Asp Leu Pro Cys Gln Phe Ala Asn Ser Gln Asn Gln Ser Leu Ser Glu Leu Val Val Phe Trp Gln Asp Gln Glu Asn Leu Val Leu Asn Glu Val Tyr Leu Gly Lys Glu Lys Phe Asp Ser Val His Ser Lys Tyr Met Gly Arg Thr Ser Phe Asp Ser Asp Ser Trp Thr Leu Arg Leu His Asn Leu Gln Ile Lys Asp Lys Gly Leu Tyr Gln Cys Ile Ile His His Lys Lys Pro Thr Gly Met Ile Arg Ile His Gln Met Asn Ser Glu Leu , . . . . .

CA 022~786l l998-l2-l4 W 097/47732 PCTrUS97/12599 Ser Val Leu Ala Asn Phe Ser Gln Pro Glu Ile Val Pro Ile Ser Asn Ile Thr Glu Asn Val Tyr Ile Asn Leu Thr Cys Ser Ser Ile His Gly Tyr Pro Glu Pro Lys Lys Met Ser Val Leu Leu Arg Thr Lys Asn Ser Thr Ile Glu Tyr Asp Gly Ile Met Gln Lys Ser Gln Asp Asn Val Thr Glu Leu Tyr Asp Val Ser Ile Ser Leu Ser Val Ser Phe Pro Asp Val Thr Ser Asn Met Thr Ile Phe Cys Ile Leu Glu Thr Asp Lys Thr Arg Leu Leu Ser Ser Pro Phe Ser Ile Glu Leu Glu Asp Pro Gln Pro Pro Pro Asp His Glu Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 260 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein CA 022~786l l998-l2-l4 W O 97t47732 PCTtUS97/12599 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Met Asp Pro Gln Cys Thr Met Gly Leu Ser Asn Ile Leu Phe Val Met Ala Phe Leu Leu Ser Gly Ala Ala Pro Leu Lys Ile Gln Ala Tyr Phe Asn Glu Thr Ala Asp Leu Pro Cys Gln Phe Ala Asn Ser Gln Asn Gln Ser Leu Ser Glu Leu Val Val Phe Trp Gln Asp Gln Glu Asn Leu Val Leu Asn Glu Val Tyr Leu Gly Lys Glu Lys Phe Asp Ser Val His Ser Lys Tyr Met Gly Arg Thr Ser Phe Asp Ser Asp Ser Trp Thr Leu Arg Leu His Asn Leu Gln Ile Lys Asp Lys Gly Leu Tyr Gln Cys Ile Ile His His Lys Lys Pro Thr Gly Met Ile Arg Ile His Gln Met Asn Ser Glu Leu Ser Val Leu Ala Asn Phe Ser Gln Pro Glu Ile Val Pro Ile Ser Asn Ile Thr Glu Asn Val Tyr Ile Asn Leu Thr Cys Ser Ser Ile His Gly Tyr Pro Glu Pro Lys Lys Met Ser Val Leu Leu Arg Thr Lys Asn Ser Thr Ile Glu Tyr Asp Gly Ile Met Gln Lys Ser Gln Asp Asn Val Thr Glu Leu Tyr Asp Val Ser Ile Ser Leu Ser Val Ser Phe Pro Asp Val Thr Ser Asn Met Thr Ile Phe Cys Ile Leu Glu Thr Asp Lys CA 022~786l l998-l2-l4 W O 97/47732 PCTrUS97/12599 Thr Arg Leu Leu Ser Ser Pro Phe Ser Ile Glu Leu Glu Asp Pro Gln Pro Pro Pro Asp His Glu Pro Lys Ser Ala Asp Lys Thr His Thr Cys Pro Pro Cys Pro (2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1104 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 601..1104 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

CACCCCATTG ACGTCAATGG GA~ll~lll TGGCGACTCA CTATAGGAGT TCCCAAGCTT 540 _,. .~, , - - 1 CA 022~786l l998-l2-l4 Met Ala Cys Leu Gly Phe Gln Arg His Lys Ala Gln Leu Asn Leu Ala Ala Arg Thr Trp Pro Cys Thr Leu Leu Phe Phe Leu Leu Phe Ile Pro Val Phe Cys Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys Ala Ala Thr Tyr Met Thr Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser ....

CA 022~786l l998-l2-l4 W O 97/47732 PCTrUS97/12599 Asp Ala Glu Pro Lys Ser Ala Asp (2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 168 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Met Ala Cys Leu Gly Phe Gln Arg His Lys Ala Gln Leu Asn Leu Ala Ala Arg Thr Trp Pro Cys Thr Leu Leu Phe Phe Leu Leu Phe Ile Pro Val Phe Cys Lys Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys Ala Ala Thr Tyr Met Thr Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly CA 022~786l l998-l2-l4 W O 97t47732 PCTrUS97/l2599 Asn Gly Thr Gln Ile T~r Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Ala Glu Pro Lys Ser Ala Asp (2) INFORMATION FOR SEQ ID NO:8:

~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Pro Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp Gly Thr Cys Tyr (2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: singie (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEOUENCE DESCRIPTION: SEQ ID NO:9:

Ser Leu Ser Pro Gly Lys (2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single CA 022~7861 1998-12-14 W O 97/47732 PCTrUS97/12599 (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l0:

Pro Thr Leu Tyr Asn Val Ser Leu Val Met Ser Asp Thr Ala Gly Thr l 5 l0 15 Cys Tyr ~2) INFORMATION FOR SEQ ID NO:ll:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:

Pro Thr His Val Asn Val Ser Val Val Met Ala Glu Val Asp Gly Thr l 5 l0 15 Cys Tyr (2) INFORMATION FOR SEQ ID NO:12:

~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: l0 amino acids ~B) TYPE: amino acid ~C) STRANDEDNESS: single ~D) TOPOLOGY: unknown ~ii) MOLECULE TYPE: protein (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:12:

Gly Pro Ser Lys Pro Glu Pro Lys Ser Ala l 5 l0 (2) INFORMATION FOR SEQ ID NO:13:

CA 022~786l l998-l2-l4 W O 97/47732 PCTrUS97/12599 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Asn Lys Ile Leu (2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

His Phe Pro Asp Gln Glu Pro Lys Ser Ala (2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Val Ile His Val .. . . .

CA 022~786l l998-l2-l4 (2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Pro Pro Pro Asp His Glu Pro Lys Ser Ala (2) INFORMATION FOR SEQ ID NO:17:

~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

Leu Lys Ile Gln (2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Glu Pro Cys Pro Asp Ser Asp Ala Glu Pro Lys Ser Ala CA 022~786l l998-l2-l4 ~2) INFORMATION FOR SEQ ID NO:l9:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Met His Val Ala (2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 57 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

CA 022~7861 1998-12-14 W O 97/47732 PCTrUS97/12599 (2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 79 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: protein CA 022~7861 1998-12-14 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

Gly Pro Ser Lys Pro Glu Pro Lys Ser Ala Gly Ile Lys Pro (2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

Ser Leu Ser Thr Gly Lys (2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:

Ser Leu Ser Ala Gly Lys (2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: not relevant (ii) MOLECULE TYPE: protein W O 97/47732 PCTrUS97112599 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

Glu Pro Lys Ser Ala l 5 T

Claims (44)

What is claimed is:

1. A hexameric fusion protein comprising:
(a) a dimeric binding protein and (b) a tailpiece .alpha.tp) characterized by having the activity of the tailpiecefrom the C-terminus of the heavy chain of an IgA antibody.

2. The fusion protein according to claim 1, wherein the dimeric binding protein is selected from the group consisting of:
(a) a protein fragment comprising the extracellular domain of a selected monomeric binding protein or a functional fragment thereof fused to an Ig-Fc fragment selected from the group consisting of an Fc fragment from an IgG antibody, an Fc fragment from an IgD antibody, an Fc fragment from an IgE antibody, and an Fc fragment from an IgM antibody excluding the µtp; and (b) a naturally dimeric binding protein or a fragment thereof having the binding ability of said dimeric protein.

3. The fusion protein according to claim 2 further comprising a leader suitable for expression and processing of the fusion protein.

4. The fusion protein according to claim 2 wherein the protein fragment consists of the native leader and extracellular domains selected from the group consisting of CD80, CTLA-4 and CD86.

5. The fusion protein according to claim 2 wherein the dimeric binding protein is a Ig-Fab fragment and the heavy chain is joined to the Ig-Fc fragment.

6. The fusion protein according to claim 2 wherein the Ig-Fc fragment is from an IgG antibody selected from the group of human isotypes consisting of IgG1, IgG2, IgG3, IgG4, and IgG binding mutants.

7. The fusion protein according to claim 2 wherein the Ig-Fc fragment is from a human IgG1 antibody.

8. The fusion protein according to claim 1 wherein the .alpha.tp is the tailpiece of an antibody selected from the group consisting of human IgA1, human IgA2, rabbit IgA, mouse IgA, and gorilla IgG.

9. The fusion protein according to claim 8 wherein the .alpha.tp has the sequence SEQ ID NO: 10 PTHVNVSVVMAEVDGTCY.

10. The fusion protein according to claim 8 wherein the .alpha.tp has been modified to remove the N-linked glycosylation site.

11. The fusion protein according to claim 1 further comprising a linker of between 1 to about 20 amino acids in length, said linker located between the binding protein and the .alpha.tp.

12. The fusion protein according to claim 1 which is a homo-hexamer.

13. The fusion protein according to claim 1 which is a hetero-hexamer.

14. A polynucleotide sequence encoding a hexameric fusion protein comprising:
(a) a dimeric binding protein and (b) a tailpiece (.alpha.tp) characterized by having the biological activity of the tailpiece from the C-terminus of the heavy chain of an IgA antibody.

15. The polynucleotide sequence according to claim 14, wherein the dimeric binding protein is selected from the group consisting of:
(a) a protein fragment comprising the extracellular domain of a selected monomeric binding protein fused to an Ig-Fc fragment selected from the group consisting of an Fc fragment from an IgG antibody, an Fc fragment from an IgD antibody, an Fc fragment from an IgE antibody, and an Fc fragment from an IgM antibody excluding the µtp; and (b) a naturally dimeric binding protein or a fragment thereof having the binding ability of said protein.

16. The polynucleotide sequence according to claim 15 further comprising a leader suitable for expression and processing of the fusion protein.

17. The polynucleotide sequence according to claim 15 wherein the protein fragment consists of the native leader and extracellular domains selected from the group consisting of CD80, CTLA-4 and CD86.

18. The polynucleotide sequence according to claim 15 wherein the dimeric protein is a Ig-Fab fragment and the heavy chain is joined to the Ig-Fc fragment.

19. The polynucleotide sequence according to claim 15 wherein the Ig-Fc fragment is from an IgG antibody selected from the group of human isotypes consisting of IgG1, IgG2, IgG3, IgG4, and IgG binding mutants.

20. The polynucleotide sequence according to claim 15 wherein the Ig-Fc fragment is from a human IgG1 antibody.

21. The polynucleotide sequence according to claim 14 wherein the .alpha.tp is the tailpiece of an antibody selected from the group consisting of human IgA1, human IgA2, rabbit IgA, mouse IgA, and gorilla IgG.

22. The polynucleotide sequence according to claim 21 wherein the .alpha.tp has the sequence SEQ ID NO: 10 PTHVNVSVVMAEVDGTCY.

23. The polynucleotide sequence according to claim 21 wherein the .alpha.tp has been modified to remove the N-linked glycosylation site.

24. The polynucleotide sequence according to claim 14, wherein the fusion protein further comprises a linker of between 1 to about 20 amino acids in length, said linker located between the binding protein and the .alpha.tp.

25. A vector comprising a polynucleotide sequence encoding:
(a) a polynucleotide sequence according to claim 14; and (b) sequences controlling expression of the fusion protein in a selected host cell.

26. A recombinant host cell comprising the vector of claim 25.

27. A pharmaceutical composition comprising an hexameric fusion protein according to claim 1 in a pharmaceutically acceptable carrier.

28. A pharmaceutical composition comprising a polynucleotide sequence according to claim 14 in a pharmaceutically acceptable carrier.

29. A diagnostic reagent comprising a detectable label and an hexameric fusion protein according to claim 1.

30. A diagnostic reagent comprising a detectable label and a polynucleotide sequence according to claim 14.

31. A method for producing a hexameric fusion protein comprising the steps of:
(a) providing a dimeric binding protein; and (b) attaching to each monomer of said binding protein a tailpiece (.alpha.tp) characterized by having the biological activity of the tailpiece from the C-terminus of the heavy chain of an IgA antibody.

32. A method of purifying a hexameric fusion protein comprising:
(a) providing a selected host cell according to claim 26;
(b) recovering the stable hexameric fusion protein; and (c) purifying the recovered fusion protein.

33. The method according to claim 32, wherein said fusion protein comprises IgG or a fragment thereof, and said purification step comprises the step of applying said fusion protein to a Protein A or Protein G column.

34. A method for screening for a ligand which binds to a hexameric fusion protein according to claim 1, comprising the steps of:
(a) providing the hexameric fusion protein;
(b) permitting a test sample to come into contact with the hexameric fusion protein; and (c) detecting binding between the fusion protein and any ligand in the test sample.

35. The method according to claim 34 wherein the fusion protein is immobilized to a surface.

36. The method according to claim 34 wherein the fusion protein is in solution.

37. The method according to claim 34, wherein the fusion protein is selected from the group consisting of CD80-Ig.alpha.tp and CD86-Ig.alpha.tp.

38. A method for screening for a compound that inhibits the interaction between a selected binding protein and a ligand, said method comprising the step of (a) providing a known ligand for said binding protein;
(b) providing a hexameric fusion protein according to claim 1;
(c) contacting the known ligand with a test solution;
(d) contacting the known ligand with the hexameric fusion protein;
(e) detecting inhibition of binding of the hexameric fusion protein; and (f) optionally isolating the compound which binds to the hexameric protein.

39. The method according to claim 38, wherein the ligand is selected from the group consisting of CD28 and CTLA-4 and the hexameric fusion protein is selected from the group consisting of CD80-Ig.alpha.tp and CD86-Ig.alpha.tp.

40. A method for stimulating CD28 positive cells comprising the step of administering to CD28 positive cells a hexameric fusion protein selected from the group consisting of CD80-Ig.alpha.tp and CD86-Ig.alpha.tp.

41. A method for suppressing CTLA-4 positive cells comprising the step of administering to CTLA-4 positive cells a hexameric fusion protein selected from the group consisting of CD80-Ig.alpha.tp and CD86-Ig.alpha.tp.

42. A method for antagonizing cell surface CD80- and CD86-mediated stimulation of CD28 positive cells by administering to said cells a hexameric fusion protein CTLA4-Ig.alpha.tp.

43. A method for immunizing an animal comprising the method of administering to the animal an effective amount of a pharmaceutical compositions according to claim 27.

44. A method for immunizing an animal comprising the method of administering to the animal an effective amount of a pharmaceutical compositions according to claim 28.