AU3899401A - Soluble divalent and multivalent heterodimeric analogs of proteins - Google Patents

Description

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P/00/01l1 Regulation 3.2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Invention title: Soluble dlivalent and mnultivalent heterodimneric analogs of proteins The following statement is a full description of this invention, including the best method of performing it known to us: dxbm M01191694 1vlI 141353 SOLUBLE DIVALENT AND MULTIVALENT HETERODIMERIC ANALOGS OF

PROTEINS

Technical field The present invention is directed to compositions comprising soluble divalent and multivalent heterodimeric analogs of proteins that are involved in immune regulation and methods of making and using the same. The high affinity that these complexes have for their cognate ligands enables them to be effective competitors to T cell receptors and MHC molecules normally involved in transplant rejection and autoimmune disease. Molecules such as divalent T cell receptors may also have an impact on the diagnosis and treatment of cancer in that they may be used to augment antitumor responses, or may be conjugated to toxins which may then be used to help eliminate tumors. Use of such compositions will allow one to accomplish selective immune modulation without compromising the general performance of the immune system.

Background of the Invention Except where the context indicates otherwise, we do not admit that any prior art ooooo information included in this specification was known in Australia or formed part of the common general knowledge in Australia at the priority date.

The process of signal transduction often involves proteins that have extracellular domains, •oo.

transmembrane domains, and intracellular domains. During ligand binding there is often oligomerization of receptor molecules in order to transmit effectively the signal to the •intracellular component of the ceil. The immune system is an excellent example of a signal transduction pathway that works by these methods (Rosen et al. J. Med. Chem. 38: 48-55).

The immune system is a defense system found in most advanced forms of higher vertebrates. A properly functioning lymphatic and immune system distinguishes between self and nonself. A healthy body protects against foreign invaders, such as bacteria, viruses, fungi, and parasites. As the body encounters foreign material (nonself), also known as an antigens, the immune system becomes activated. An antigen is recognized by characteristic shapes or epitopes on its surface. This defense mechanism provides a

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2 means of rapid and highly specific responses that are used to protect an organism against invasion by pathogenic microorganisms. It is the myriad of pathogenic microorganisms that have principally caused the evolution of the immune system to its current form. In addition to protection against infectious agents, specific immune responses are thought to be involved in surveillance against alterations in self antigens as seen in tumor development. Immune responses are also involved in the development of autoimmune disease, AIDS, as well as rejection of transplanted tissues.

Lymphocytes Within the immune system, lymphocytes play a central role. Lymphocyte responses to foreign organisms orchestrate the effector limbs of the immune system, and ultimately, determine the fate of an infection. Lymphocytes can be divided into two main categories, B and T cells. These two types of lymphocytes are specialized in that they have different effector functions and play different roles in the development of specific immune responses. Individual lymphocytes are specialized in that they are committed to respond to 15 a limited set of structurally related antigens. Specificity is conferred by an unique set of cell surface receptors expressed on individual lymphocytes. These receptors interact with soluble proteins, in the case of B cells, and with antigenic peptide/major histocompatibility 0 complex (MHC) molecules in the case of T lymphocytes. The nature of the interaction with their ligands also differs between B and T cells. The antigen receptors produced by B cells, immunoglobulins (Igs), interact with their ligands with a high affinity. In contrast, T cell receptors interact with their ligands with low affinity. Thus, the T cell response is driven by the interaction of many T cell receptors (TcR) on the surface of an individual T cell interacting with multiple antigenic peptide/MHC complexes on the surface of the antigen presenting cell. Thus, these two diverse groups of cell-surface glycoproteins, the TcRs and the MHC glycoproteins, form key components of specificity in the T lymphocyte response to antigens.

T cells are a major regulatory cell of the immune system. Their regulatory functions depend not only on expression of a unique T cell receptor, but also on expression of a variety of accessory molecules and effector functions associated with an individual T cell response. Effector functions include responses such as cytotoxic responses or other responses characterized by secretion of effector molecules, lymphokines. It is this

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3 regulatory function that often goes awry in the development of autoimmune diseases. The different effector functions also play a large role in tissue graft rejection, and can be important in tumor rejection.

T cells respond to antigens in the context of either Class I or Class II MHC molecules.

Cytotoxic T cells respond mainly against foreign antigens in the context of Class I glycoproteins, such as viral-infected cells, tumor antigens and transplantation antigens. In contrast, helper T cells respond mainly against foreign antigens in the context of Class II molecules. Both types of MHC molecules are structurally distinct, but fold into very similar shapes. Each MHC molecule has a deep groove into which a short peptide, or protein fragment, can bind. Because this peptide is not part of the MHC molecule itself, it varies from one MHC molecule to the next. It is the presence of foreign peptides displayed in the MHC groove that engages clonotypic T cell receptors on individual T cells, causing them to respond to foreign antigens.

Antigen-specific recognition by T cells is based on the ability of clonotypic T cell receptor 15 to discriminate between various antigenic-peptides resident in MHC molecules. These receptors have a dual specificity for both antigen and MHC (Zinkemagel et al. Nature 248: 701-702 (1974)). Thus, T cells are both antigen-specific and MHC-restricted. A simple molecular interpretation of MHC-restricted recognition by T cells is that TcRs recognize MHC residues as well as peptide residues in the MHC-peptide complex. Independent of S 20 the exact mechanism of recognition, the clonotypic T cell receptor is the molecule that is both necessary and sufficient to discriminate between the multitude of peptides resident in

MHC.

T cells can be divided into two broad subsets; those expressing a/P TcR and a second set that expresses y/8 TcR. Cells expressing a/p TcR have been extensively studied and are known to comprise most of the antigen-specific T cells that can recognize antigenic peptide/MHC complexes encountered in viral infections, autoimmune responses, allograft rejection and tumor-specific immune responses. Cells expressing a/p TcRs can be further divided into cells that express CD8 accessory molecules and cells that express CD4 accessory molecules. While there is no intrinsic difference between the clonotypic a/p T cell receptors expressed either on CD4 and CD8 positive cells, the accessory molecules largely correlate with the ability of T cells to respond to different classes of MHC molecules. Class I MHC molecules are recognized by CD8+, or cytotoxic, T cells and class II MHC molecules by CD4+, or helper, T cells.

y/8 T cells make up another significant population of T cells seen in circulation as well as in specific tissues. These cells are not well understood; their antigen/MHC specificity is poorly defined and in most cases their ligands are completely unknown. These cells are present in high quantities in certain tissues, including skin and gut epithelium, and are thought to play a significant role in immune responses of those organs. They have also been implicated in autoimmune responses and may be involved in the recognition of heat shock proteins. A general approach to the identification of antigenic complexes, as outlined in the present invention, would greatly facilitate understanding of how these cells influence the development of both normal and abnormal immune responses. There is a large degree of homology between both a/ and y/ 5 TcR expressed in rodents and humans.

This extensive homology has, in general, permitted one to develop murine experimental models from which results and implications may be extrapolated to the relevant human counterpart.

MHC Molecules in Health and Disease Major histocompatibility antigens consist of a family of antigens encoded by a complex of genes called the major histocompatibility complex. In mice, MHC antigens are called H-2 antigens (Histocompatibility-2 antigens). In humans MHC antigens are called HLA antigens (Human-Leukocyte-associated Antigens). The loci that code for MHC glycoproteins are polymorphic. This means that each species has several different alleles at each locus. For example, although a large number of different Class I antigens may be seen in a species as a whole, any individual inherits only a single allele from each parent at each locus, and therefore expresses at most two different forms of each Class I antigen.

In the murine system, the class II MHC molecules are encoded by I-A and I-E loci, and in humans, class II molecules are encoded by the DR, DP and DQ loci. Polymorphism of class II alleles is attributed to the alpha and beta chains and specificities are designated using the nomenclature set forth by the World Health Organization (Immunogenetics (1992) 36:135).

The Role of MHC Molecules Transplantation MHC molecules play an essential role in determining the fate of grafts. Various species display major immunological functional properties associated with the MHC including, but not limited to, vigorous rejection of tissue grafts, stimulation of antibody production, stimulation of the mixed lymphocyte reaction (MLR), graft-versus-host reactions (GVH), cell-mediated lympholysis (CML), immune response genes, and restriction of immune responses. Transplant rejection occurs when skin, organs kidney, liver, lung), or other tissues blood, bone marrow) are transplanted across an MHC incompatibility.

A vigorous graft rejection occurs when the immune system is activated by mismatched transplantation antigens that are present in donor tissue but not in recipient. Graft rejection may occur in the graft itself by exposure of circulating immune cells to foreign antigens, or it may occur in draining lymph nodes due to the accumulation of trapped transplantation antigens or graft cells. Because of the extensive diversity of MHC antigens, numerous Ispecificities are possible during physiological and pathophysiologic immune-related activities, transplantation, viral infections, and tumor development). The recognized HLA specificities are depicted, for example, in a review by Bodmer et al. (In: Dupont B.

Immunobiology of HLA (Volume I) New York: Springer-Verlag (1989)).

The Role of MHC Molecules Autoimmune Response Susceptibility to many autoimmune disease shows a significant genetic component and 20 familial linkage. Most genetic linkages of autoimmune diseases are with certain class II MHC alleles (see Table 1 for Overview). The level of association between a particular disease and an allele at one of the MHC loci is defined by a term called "relative risk".

This term reflects the frequency of the disease in individuals who have the antigen compared to the frequency of the disease among individuals who lack the antigens. For example, there is a strong association with DQP genotype in insulin-dependent diabetes mellitus; the normal DQP sequence has an aspartic acid at position 57, whereas in Caucasoid populations, patients with diabetes most often have valine, serine or alanine at that position.

Table 1: r r Associations of HLA genotype with susceptibility to autoimmune disease Disease HLA allele Relative risk Goodpasture's syndrome DR2 15.9 Multiple Sclerosis DR2 4.8 Graves' disease DR3 3.7 Myasthenia gravis DR3 Systemic lupus DR3 5.8 erythematosus Insulin-dependent DR3 and DR4 3.2 diabetes mellisis Rheumatoid arthritis DR4 4.2 Pemphigus vulgaris DR4 14.4 Addison's disease DR3 8.8 Dermatitis herpetiformis DR3 13.5 Celiac disease DR3 73.0 Hashimoto's thyroiditis DR5 3.2 Regulation of Immune Reponses Interest in analyzing both normal and abnormal T cell-mediated immune responses led to the development of a series of novel soluble analogs of T cell receptors and MHC molecules to probe and regulate specific T cell responses. The development of these reagents was complicated by several facts. First, T cell receptors interact with peptide/MHC complexes with relatively low affinities (Matsui et al Science 254:1788-1891 (1991) Sykulev et al Immunity 1:15-22 (1994) Corr et al Science 265:946-949 (1994)). In order to specifically regulate immune responses, soluble molecules with high affinities/avidities for either T cell receptors or peptide/MHC complexes are needed. However, simply making soluble monovalent analogs of either T

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7 cell receptors or peptide/MHC complexes has not proven to be effective at regulating immune responses with the required specificity and avidity.

To regulate immune responses selectively, investigators have made soluble versions of proteins involved in immune responses. Soluble divalent analogs of proteins involved in regulating immune responses with single transmembrane domains have been generated by several laboratories. Initially, CD4/Ig chimeras were generated (Capon et al Nature 337:525-531 (1989); Bryn et al Nature 344:667-670 (1990)), as well as CR2/Ig chimeras (Hebell et al Science 254:102-105 (1991)). Later it was demonstrated that immune responses could be modified using specific CTLA-4/Ig chimeras (Linsley et al Science 257:7920-795 (1992); U.S. Patent No. 5,434,131; Lenschow et al Science 257:789-791 (1992)). In addition, class I MHC/Ig chimeras were used to modify in vitro allogeneic responses (Dal Porto, supra). However, these examples include only soluble divalent analogs of single transmembrane polypeptide molecules and not chimeric molecules of heterodimeric proteins in which the heterodimer consists of a and P polypeptides that are S 15 both transmembrane polypeptides. The present invention reports the generation of soluble divalent and multivalent heterodimeric analogs of integral membrane protein complexes, which consist of alpha and beta polymorphic integral membrane polypeptides that properly fold to form a functional unit that has potential use in immune modulation.

Previously, replacement of two transmembrane domains in the generation of multivalent S 20 analogs has not been achieved. The challenge of generating these molecules lies in achieving the proper folding and expression of two polypeptides, both of which ordinarily -require transmembrane domains (Figure In addition, soluble multivalent analogs of heterodimeric proteins generally have increased affinity and, therefore, are preferred therapeutic agents. These soluble protein complexes, which consist of a and P polymorphic integral membrane polypeptides that properly fold to form a functional unit, have potential use as immune modulating agents.

Summary of the Invention The present invention accordingly provides in one embodiment an expression vector encoding a fusion protein wherein the fusion protein comprises an immunoglobulin heavy chain, wherein the immunoglobulin heavy chain comprises a variable region, and (ii) an extracellular domain of a first transmembrane polypeptide.

The present invention provides in another embodiment an expression vector encoding a fusion protein wherein said fusion protein comprises an immunoglobulin light chain, wherein the immunoglobulin light chain comprises a variable region, and (ii) an extracellular domain of a transmembrane polypeptide.

In yet a further separate embodiment, the present invention provides an expression vector which encodes a first fusion protein and a second fusion protein, wherein the first fusion protein comprises an extracellular domain of a transmembrane polypeptide and an immunoglobulin heavy chain, wherein the immunoglobulin heavy chain comprises a variable region, and wherein the second fusion protein comprises an extracellular domain of a transmembrane polypeptide and an immunoglobulin light chain.

In another separate embodiment, the present invention provides a method of making each of these expression vectors.

The present invention in another embodiment provides a polynucleotide encoding a fusion protein wherein the fusion protein comprises an immunoglobulin heavy chain, wherein 15 the immunoglobulin heavy chain comprises a variable region, and (ii) an extracellular domain of a first transmembrane polypeptide.

The present invention provides in a further embodiment a polynucleotide encoding a fusion protein wherein said fusion protein comprises an immunoglobulin light chain, wherein the immunoglobulin light chain comprises a variable region, and (ii) an extracellular domain of a transmembrane polypeptide.

In yet a further separate embodiment, the present invention provides a polynucleotide which encodes a first fusion protein and a second fusion protein, wherein the first fusion protein comprises an extracellular domain of a transmembrane polypeptide and an immunoglobulin heavy chain, wherein the immunoglobulin heavy chain comprises a variable region, and wherein the second fusion protein comprises an extracellular domain of a transmembrane polypeptide and an immunoglobulin light chain.

In yet a further separate embodiment, the present invention provides a host cell comprising any of these expression vectors or polynucleotides.

In yet a further separate embodiment, the present invention provides a method of making a molecular complex comprising four fusion proteins.

Brief description of the Drawings Figure 1A. A typical configuration of a heterodimeric double transmembrane protein.

Figure lB. Heterodimeric transmembrane protein made divalent and soluble by covalent linkage of outer-membrane region to antibody.

Figure 1C. Outer-membrane region of MHC class II covalently linked to an antibody.

Figure 1D. A schematic of the chimeric protein showing the TcR a polypeptide (shaded) linked to IgGI heavy chain and TcR P polypeptide (shaded) linked to Ig kappa 10 light chain is shown. The linkers between the chimeric chains consist of short glycine/serine spacers. Presumptive binding sites of two monoclonal antibodies (mAb), H57 (TcR specific) and 1B2 (2C TcR specific), on the putative 2C TcR/Ig structure are also noted.

Figure 2. Map of the expression vector, which encodes soluble divalent heterodimeric 15 proteins. Multi-step construction schematic is shown to depict fusion of a and p polypeptide subunit linked to Ig heavy and light chains to form the chimeric .:Immunoglobulin molecules.

Figure 3. Detail of the DNA sequences introduced into the plasmid construct.

Figure 4. Schematic of TcR/MHC interactions.

Figure 5A-5C. Detection of soluble heterodimeric proteins by ELISA assays.

Figure 6. SDS-PAGE analysis of I-Ek/Ig and 2C TcR/Ig chimeric proteins.

Figure 7. Graph showing that the affinity of soluble divalent 2C TcR/Ig for peptide/H-2 L d complexes is higher than that of soluble monovalent 2C TcR is depicted in Figure 11. RMA S-L d cells were loaded with peptides (QL9 p2Ca; or pMCMV) and subsequently incubated with a constant amount of FITC labeled 30.5.7 Fab and varying concentrations of either 2C TcR/Ig (solid lines) or soluble monovalent 2C TcR, sm2C TcR (dashed line) Binding of FITC-30.5.7 Fab was determined by flow cytometry. Plotted as the maximal (no 2C TcR Analog) 30.5.7 binding vs. the concentration of2C TcR analog. Apparent affinities were determined from a replot of maximal 30.5.7 binding) vs [TcR Analog] see text and Table II for further discussion. Data shown are from one representative experiment that has been repeated at least three times. Each data point is the average of duplicates.

Figure 8. Cells were then harvested as described above and processed for flow cytometry analysis. Cells were stained with either purified mAb, 30.5.7 (Panels or 2C TcR/Ig culture supernatants (Panels E-H) diluted to 20-40 ga g/ml final concentration.

In each panel the histogram of treated cells (solid line) is contrasted with that of cells not treated with any peptide and cultured for one hour at 370 C (broken line). Histograms shown are from one representative experiment that has been repeated at least three times.

Figure 9. A comparison of2C TcR/Ig reactivity versus mAb 30.5.7 reactivity in peptide-stabilized H-2Ld molecules. RMA-S Ld cells were incubated under various conditions. Following overnight incubation of RMA-S Ld cells at 270 C, cells were 15 cultured in the presence or absence of various H-2 Ld binding peptides: no peptide cells maintained at 270 C, (Panel A and tum-, (Panel B and p2Ca, (Panel C and and QL9, (Panel D and were added to cultures as described in section.

Figure 10. Graph demonstrating inhibition of in vitro 2C T cell mediated lysis by soluble 2C TcR/Ig superdimers.

Figure 11. Fluorescence data showing that soluble divalent 2C TcR/Ig interacts with SIY/MHC complexes but not with dEV-8/MHC complexes is depicted in Figure 11. T2 cells transfected with either H-2 Kb, H-2 Kbm 3 or H-2 Kbm ll were incubated overnight at 27 0 C and loaded with peptides dEV-8 SIY or pVSV (aaaaa) as described below. Cells were stained with purified 2C TcR/Ig (-50 mg/ml) and GAM-IgG-RPE as described in Methods, and analyzed by FACS. Resultant histograms are shown; Panel A, T2-Kb cells; B, T2-Kbm 3 C, T2-Kbmll. In the histograms presented 2C TcR/Ig reactivity with either dEV-8 or pVSV (aaaaa) was virtually identical leading to difficulty in discriminating between these two histograms.

Figure 12. 2C CTL mediated lysis on various peptide/MHC targets is depicted in this figure. T2 cells transfected with either H-2 Ld (Panel H-2 Kb (Panel or H-2 Kbm 3 (Panel were chromium labeled as described and then loaded with peptides by incubating at 25 {C for 1.5 hrs. in the presence of variable amounts of peptides: p2Ca and pMCMV (Panel and dEV-8 SIY or pVSV (Panels B and Peptide loaded target cells were then incubated at an effector to target ratio of 10:1 and specific lysis calculated as described below. Data shown are representative of three separate experiments.

Figure 13. Fluorescence data showing modulation of endogenous 2C specific peptide/H-2 Ld complexes on the surface of RENCA cells by y-IFN is depicted in Figure 13. RENCA cells were cultured for 48hrs. with 0 (panels A 5 (panels B (panels C or 50 (panels D units/ml -y-IFN. As described in the results, y-IFN is known to have a direct effect on class I expression, making it necessary to establish background binding of 2C TcR/Ig to y-IFN treated cells. This was accomplished by incubating RENCA cells with saturating amounts of the H-2 L d binding peptide, MCMV, which efficiently displaced the endogenous H-2 Ld bound peptides, including any 2C-reactive peptides. Cells were harvested, stained with 2C TcR/Ig (75 mg/ml), panels A-D, or the mAb 30.5.7 (45 mg/ml), panels E-H, as described below. Cells were subsequently stained with GAM-IgG-RPE and analyzed by FACS. Resultant histograms are shown. Solid lines represent histograms of cultures with no added peptide while dotted lines (aaa) represent histograms from cultures incubated with pMCMV. All 20 experiments were done in duplicate and repeated at least three times. Note the differences in the extents of fluorescence (see the scales on the histograms) upon staining with 2C-TcR/Ig vs. staining with 30.5.7.

Detailed description of the Invention Soluble recombinant divalent and multivalent analogs of heterodimeric proteins were generated that specifically bind to target molecules that regulate activities of the immune system. The construction and expression of soluble recombinant divalent and multivalent analogs of heterodimeric proteins involved linking polypeptide sequences from the heterodimeric proteins to immunoglobulin heavy and light chains. Specifically, soluble recombinant divalent and multivalent analogs of heterodimeric proteins link a polypeptide chain of a heterodimeric transmembrane protein to an immunoglobulin heavy chain and a 12 second polypeptide chain of a heterodimeric transmembrane protein to an immunoglobulin light chain. These soluble hybrid constructs contain two or more binding sites for the same ligand.

"Polypeptide" refers to any polypeptide of a heterodimeric protein. "Polypeptide" may refer to a protein in its entirety, or to a portion thereof. Selected polypeptide sequences will minimally contain any binding site involved in a specific immune response for regulation, including regions of the protein required for proper folding and conformation of the binding site or any other region necessary for the function of the molecule. "Binding site" refers to the domain or sequence of amino acids from the protein of interest that mediates interaction or association with the ligand or target cell. The binding site may be formed from a nonconsecutive sequence of amino acids that associate in a tertiary conformation. A binding site may also be found within the extracellular domains of a glycoprotein. A glycoprotein is a protein that contains at least one carbohydrate group.

SPolypeptide sequences contain about 5 amino acid sequences to about 1000 amino acid 15 sequences. Preferably, the polypeptide sequences contain 200 amino acid sequences or less. Mammalian polypeptides are preferred, and more preferably, human polypeptides from transmembrane proteins. DNA, RNA, and amino acid sequences which have slight oo and non-consequential sequence variations from the actual sequences containing more two or more binding sites for the same ligand are within the scope of the present invention.

20 Conventional abbreviations for amino acids, peptides and their derivatives are used as generally accepted in the peptide art and as recommended by the IUPAC-IUB Commission on Biochemical Nomenclature (European J. Biochem (1984) 138:9-37). "Slight and S: non-consequential" sequence variations mean that the homologous sequences will function in substantially the same manner to produce substantially the same proteins and polypeptides of the present invention. Functionally equivalent polypeptides are also encompassed within this invention. Conservative substitutions may be made in such amino acid sequences without losing biological or chemical functionality.

As used herein the term "soluble" means that the composition of interest is sufficiently soluble at 37 0 C or in bodily fluids, plasma, etc. such that it may be used at the specified range of concentrations required to enable the composition to serve its intended function according to the present invention.

"Divalent" means that the naturally occurring or genetically engineered chimeric protein or polypeptide of interest that has two binding sites for the same ligand. This is in contrast to bifunctional in which a chimeric protein has two binding sites for different ligands on the same polypeptide. Thus all immunoglobulins are both bifunctional and also minimally divalent. There are bifunctional in that they all have at least one binding site for antigen and a separate site for Fc-receptor binding. Immunoglobulins are also minimally divalent in that they have at least two identical but separate binding sites for antigen.

"Multivalent" means that the naturally occurring or genetically engineered chimeric proteins or polypeptides of interest have more than two binding sites for the same ligand.

For example, "multivalent" would encompass IgM and IgA chimeric molecules according to the present invention, which are pentavalent and tetravalent, respectively. In addition, "multivalent" might indicate a composition having more than one chimeric antibody molecule. Since each divalent heterodimeric IgG molecule has two binding sites (divalent), a chimeric antibody complex containing four IgG molecules would have eight antigen binding sites (octavalent). Similar multivalent antibody complexes that are Snon-chimeric have been constructed using methods known in the art. For instance, Sano and Cantor disclose a method for making a multivalent antibody U.S. Patent No. 5,328,985 using streptavidin-proteinA, which has four or more IgG binding sites per molecule. The number of antibody molecules per conjugate molecule is controlled by mixing the 20 streptavidin-Protein A and antibody of interest at an appropriate ratio. Other methods of *o*o conjugating antibodies known in the art could also be used to form soluble multivalent chimeric compositions according to the present invention.

"Linker" refers to the linker region inserted between the immunoglobulin molecules and the heterodimeric polypeptides. The length of the linker sequence will vary depending upon the desired flexibility to regulate the degree of antigen binding and cross-linking.

The "linker" should be considered to be an optional feature. Constructs may be designed such that the heterodimeric polypeptides are directly and covalently attached to the immunoglobulin molecules without an additional linker region. If a linker region is included, this region will preferably contain at least 3 and not more than 30 amino acids.

More preferably, the linker is about 5 and not more than 20 amino acids and most preferably, the linker is less than 10 amino acids. Generally, the linker consists of short glycine/serine spacers, but any known amino acid may be used.

"Immunoglobulin(s) or Ig(s) means a group of proteins that are products of antibody secreting cells. Igs are constructed of one, or several, units, each of which consists of two heavy polypeptide chains and two light polypeptide chains. Each unit possesses two combining sites for antigen. The H and L chains are made up of a series of domains.

The L chains, of which there are two major types (K and consists of two domains. The H chains of Ig molecules are of several types, including p, 6, and y (of which there are several subclasses), a and e. There are eight genetically and structurally identified Ig classes and subclasses as defined by heavy chain isotypes: IgM, IgD, IgG3, IgGI, IgG2b, IgG2a, IgE, and IgA. Further, for example, "IgG" means an immunoglobulin of the G class, and that, "IgG refers to an IgG molecules of subclass 1 of the G class. "Fab" and are fragments of Ig molecules that can be produced by proteolytic digestion of an intact Ig molecule. Digestion of an IgG molecule with papain will produce two Fab fragments and an Fc fragment and digestion with pepsin will produce an F(ab') 2 fragment and subfragments of the Fc portion.

o 15 The "transplantation antigens" referred to in the present invention are molecules 0 0* responsible for graft recognition and rejection. Since the immunological status of the recipient is a critical factor affecting graft survival, diverse antigen systems may be involved in the acceptance/rejection process. These not only include the well recognized HLA system, such as class I and class II MHC molecules, but also include other minor 20 histocompatibility antigens, such asthe ABO blood group system, (including carbohydrates, which includes but is not limited to, disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, oligosaccharides, polysaccharides, and more preferably, 0oo0 the carbohydrate P Galactosyl epitope [3 Gal]), autoantigens on T and B cells, and monocyte/endothelial cell antigens. Since the present invention is primarily concerned with divalent and multivalent heterodimeric compounds comprising two subunit molecules, each generally known in the native state to possess a transmembrane domain, transplantation antigens in the context of the present invention include MHC class II antigens. In clinical applications concerning treatment or therapy to inhibit or reduce graft rejection, selective suppressing antigen soecific responses are targeted. A transplantation antigen may be any class I or class II MHC molecule, or more specifically for humans, any MHC molecules including HLA specificities such as A A1-A74), B B1-B77), C C1-C11), D D1-D26), DR DR1-DR8), DQ DQ1-DQ9) and DP (e.g.

DP1-DP6). More preferably, HLA specificities include Al, A2, A3, All, A23, A24, A28, A33, B7, B8, B35, B44, B53, B60, B62, DR1, DR2, DR3, DR4, DR7, DR8, and DR11 (Zachary et al., Transplant. 62: 272-283). In clinical applications concerning the therapy of autoimmune disease, a transplantation antigen is any MHC class II molecule associated or linked with the disease of interest. Such transplantation antigens particularly include any D and DR allele, but DQ and DP alleles that are shown to be associated with autoimmune disease are also encompassed. Therapeutic applications involve the specific suppression of transplantation antigens using soluble proteins (also referred to as "specific antigen suppressors") of the present invention. In particular, one therapeutic application involves specific suppression of preformed anti-carbohydrate antibody responses using specific antigen suppressors.

"Heterodimeric" means that the protein of interest is comprised of two separate polypeptide chains. In this description we will consider only those polypeptide chains that have transmembrane and intracellular domains. Different classes of heterodimeric transmembrane proteins, which contain a and p polymorphic integral membrane polypeptides that bind each other forming a functional unit involved in immune recognition, include, but are not limited to, proteins such as T cell receptors, and class II MHC molecules, integrins including more than 20 cell surface heterodimers), and cytokine receptors IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, erythropoietin (EPO), 20 leukemia inhibitory factor (LIF), G-CSF, Oncostatin M, ciliary neurotrophic factor growth hormone, and prolactin).

It is also possible for the compositions of the present invention to be prepared such that both the heavy and light immunoglobulin chains are fused to the same extracellular domain the extracellular domain from a class I MHC molecule or glycoprotein). Protein expression and folding would then result in a chimeric homotetrameric composition comprising two light chains and two heavy chains, all fused to the same polypeptide.

"Integrin" refers to a class of proteins defined as having adhesive properties and known to be involved in mediating adhesion between both like and different cells. These molecules are also heterodimeric transmembrane proteins consisting of A and E polypeptides.

"Superdimer" refers to dimers of heterodimeric proteins. This term has been coined to describe what may be the conformation of MHC molecules on the surface of antigen 16 presenting cell. In this application this term will be used to describe only soluble "superdimers", such as the soluble divalent or multivalent versions of either class II MHC or TcR molecules.

"Cytokine" refers to proteins that affect the behavior of other cells. Cytokines made by lymphocytes are often called lymphokines or interleukins, but the generic term "cytokine" is used most often in the literature. Cytokines act on specific "cytokine receptors" on the cells they affect. Cytokine receptors also belong to a family of molecules in which at least two component polypeptides are transmembrane spanning proteins. This system is central in the growth and regulation of many cell types including cells of the immune system.

Cytokines/cytokine receptors include the following examples, but are not limited to this listing: I) hematopoietin family erythropoietin(Epo)/EpoR; IL-2(T-cell growth factor)/CD25, CD122; IL-3 (multicolony CSF)/CD123; IL-4 (BCGF-1, BSF-1)/CD124; IL-5 (BCGF-2)/CD125; IL-6 (INF-3 2 BSF-2, BCDF)/ CD126, Cdwl30; IL-7/CDwl27; IL-9/ IL-9R; IL-11/IL-l 1R, Cdwl30; IL-13 (P600)/IL-13R; IL-15 (T-cell growth factor)/IL-15R; GM-CSF (granulocyte macrophage colony stimulating family)/CDwl 16; OSM (OM, oncostatin M)/OMR, CDwl30; LIF (leukemia inhibitory factor)/LIFR, Cdwl30); II) Interferon Family IFN- y/CD1 19; INF-a/CD118; INF- ,/CDl 18); III) Immunoglobulin Superfamily B7.1 (CD80)/CD28;CTLA-4; B7.2/ CD28, CTLA-4); IV) TNF Family TNF-a (cachectin)/p55, p75, CD120a, CD120b; TNF-P 20 (lymphotoxin, LT, LT- a)/p55, p75, CD120a, CD120b), LT-3), CD40 ligand (CD40-L)/CD40; Fas ligand/CD95 (Fas); CD27 ligand/CD27; CD30 4-1BBL/4-1BB; V) Chemokine Family IL-8 (NAP-1)/CDwl28; MP-1 (MCAP); MIP-la; MIP-11; RANTES); and VI) others (TFG-P; IL-la IL-10; IL-10 (cytokine synthesis inhibitor IL-12 (natural killer cell stimulatory factor); and MIF).

DNA constructs encoding the chimeric compounds of the present invention generally comprise sequences coding for the signal sequence and extracellular domain of one polypeptide of the heterodimeric complex TcR a or 3, or MHC class II a or P) fused to the first amino acid of either the heavy or light chain immunoglobulin variable region sequence. Such a DNA construct results in the expression and secretion of a protein comprising the extracellular portion of the polypeptide of interest at the N terminus (transmembrane regions are not included) spliced to the intact variable region of the 17 immunoglobulin molecule (see Figure Variations or truncations of this general structure in which one or more amino acids are inserted or deleted but which retain the ability to bind to the target ligand are encompassed in the present invention.

Standard Cloning Methods:The techniques for cloning and expressing DNA sequences encoding the amino acid sequences corresponding to binding sites of divalent heterodimeric analogs of integral membrane proteins, such as TcR and MHC molecules, soluble fusion proteins and hybrid fusion proteins consisting of an a and 3 polypeptide subunit, synthesis of oligonucleotides, PCR, transforming cells, constructing vectors, expression systems, and the like are well-established in the art, and skilled artisans are familiar with the standard resource materials for specific conditions and procedures.

In general, various expression systems are well known in the art. Prokaryotes are useful for cloning variant DNA sequences. For example, E. coli strain SR101 (Messing et al Nucl ~Acids Res 9(2):309-321 (1981), E. coli K12 strain 294 (ATTC No. 31446), E. coli B, UM101, and E. coli 01776 (ATTC No. 31537) are particularly useful. Constructs are 15 inserted for expression into vectors containing promoters and control sequences, which are derived from species compatible with the intended host cell. The vector ordinarily, but not necessarily, carries a replication site as well as one or more marker sequences, which are capable of providing phenotypic selection in transformed cells. For example, E. coli is typically transformed using a derivative of pBR322, which is a plasmid derived from an E.

coli species (Bolivar et al Gene 2:95 (1977). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. The pBR322 plasmid, or other microbial plasmid must also contain or be modified to contain promoters and other control elements commonly used in recombinant DNA constructions.

Promoters suitable for use with prokaryotic hosts include, but are not limited to, the beta-lactamase and lactose promoter systems (Chang et al Nature 275:615 (1978); Goeddel et al Nature 281:544 (1979), alkaline phosphatase, the tryptophan (trp) promoter system (Goeddel et al Nucl Acid Res 8:4057 (1980), and hybrid promoters, such as the tac promoter (de Boer et al Proc NatlAcadSci USA 80:21-25 (1983). Other functional bacterial promoters are suitable. Nucleotide sequences called linkers or adaptors are generally known which enable the skilled artisan to operably ligate DNA sequences of interest (Siebenlist et al Cell 20:269 (1980)). Promoters for use in a bacterial system will 18 also contain a Shine-Dalgarno sequence.

In addition to prokaryotes, eukaryotic microbes, such as yeast cultures, are useful as cloning or expression hosts. In particular, Saccharomyces cerevisiae, or common baker's yeast, is commonly used (although other strains are commonly available). For expression in Sarraromyces, the plasmid YRp7, for example, is commonly used (Stinchcomb et al Nature 282:39 (1979). This plasmid already contains the trpl gene, which provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan (ATTC No. 44076). The presence of the trpl lesion as a characertistic of the yeast host cell genome then provides an effective means of selection by growth in the absence of tryptophan. Suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, hexokinase, pyruvate kinase, and glucokinase. Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, acid phosphatase, metallothionenin, for 15 example. Suitable vectors and promoters for use in yeast expression are further described S "in R. Hitzeman et al European Patent Publication No. 73,657A.

i Promoters for controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses, such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV), or from heterologous mammalian promoters, e.g. the beta actin promoter. The early and late promoters of the SV40 virus are conveniently obtained as an V40 restriction fragment, which also contains the SV40 viral origin of replication (Fiers et al Nature 273:113 (1978). The immediate early promoter of the human CMV is conveniently obtained as a HindIII E restriction fragment (Greenaway et al Gene 18:355-360 (1982).

DNA transcription in higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually from 10 to 300bp, that act to increase the transcription initiation capability of a promoter. Enhancers are relatively orientation and position independent having been found 5' and 3' to the transcription unit, within an intron as well as within the coding sequence itself. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, and insulin, for example). Typically, however, one will use an enhancer from a eukaryotic cell virus.

Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the CMV early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human or nucleated cells) may also contain sequences necessary for the termination of transcription, which may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding the desired sequence. The clones containing DNA encoding soluble constructs are transfected into suitable host cells for expression. Depending upon the host cell used, transfection is performed using standard techniques (transfection into mammalian cells is accomplished using DEAE-dextran mediated transfection, CaP0 4 co-precipitation, lipofection, electroporation, or protoplast fusion, and still other procedures known in the art including, S:but not limited to, lysozyme fusion or direct uptake, osmotic or sucrose shock, direct 15 microinjection, indirect microinjection, and/or subjecting cells to electric currents.

Peptides, proteins, or molecules of the present invention may be conjugated to a reporter group, including, but not limited to, a radiolabel 32 a fluorescent label, an enzyme, a substrate, a solid matrix, or a carrier biotin or avidin) for use in the detection of specific levels of molecules or the specific binding activity of particular molecules of the 20 present invention. Hybrid constructs of the present invention may be further modified to include toxins.

Fusion proteins produced from the expression vectors and polynucleotides of the present invention may be included as part of divalent and multivalent heterodimeric compounds which in turn may be used as immune modulating agents in methods of regulating the immune system. For example, immunoregulatory actions that may be activated or inhibited include the ability to stimulate, depress or abrogate the following immune responses: erythroid progenitor production, T-cell proliferation, hematopoiesis production, B-cell activation, class switching IgE switch), Eosinophil growth and differentiation, T-and B-cell growth and differentiation, acute phase reaction, growth ofpre-B cells and pre-T cells, mast cell activity, IL-3 and IL-4 involvement in hematopoiesis, cytokine activation or inhibition; differentiation of myeolomonocytic lineage; cancer cell growth and development; macrophage activation, MHC expression, anti-viral activity, T-cell respones, inflammation, anti-inflammation, endothelial activation, B-cell activation, apoptosis, calcium-independent cytotoxicity; chemotactic activity ofneutrophils, T-cells, eosinophils, and macrphages, fever, cell (macrophage, T-cell, B-cell, neutrophils, eosinophils, natural killer cells) functions, antigen processing, cytotoxicity, and receptor crosslinking. In essence, the hybrid constructs of the present invention selectively enhances, decreases, or abrogates cellular activation, proliferation, anergy (tolerance), or deletion of specific T-cell subsets (Hewitt et al. J. Exp. Med. 175:1493 (1992); Choi et al. Proc. Natl. Acad. Sci.

86:8941 (1989); Kappler et al. Science 244:811 (1989); Minasi et al. J. Exp. Med.

177:1451 (1993); Sundstedt et al. Immunology 82:117 (1994); and White et al. Cell 56:27 (1989).

In addition, those compounds may also be used in the treatment of diseases related to immune dysfunction. Conditions which might benefit from the activation or inhibition of immune responses include, but are not limited to, the following disorders and diseases: autoimmune diseases, such as idiopathic thrombocytopenia purpura, systemic lupus erythematosus, myasthenia gravis, arthritis, autoimmune hemolysis, glomerulonephritis, multiple sclerosis, psoriasis, juvenile diabetes, primary idiopathic myxedema, systemic lupus erythematosus, autoimmune asthma, scleroderma, chronic hepatitis, Addison's disease, hypogonadism, pernicious anemia, vitiligo, alopecia areata, Coeliac disease, 20 autoimmune enteropathy syndrome, idiopathic thrombocytic purpura, acquired spenic atrophy, idiopathic diabetes insipidus, infertility due to antispermatazoan antibodies, sudden hearing loss, sensoneural hearing loss, polymyositis, autoimmune demyelinating diseases, traverse myelitis, ataxic sclerosis, progressive systemic sclerosis, dermatomyositis, polyarteritis nodosa, hemolytic anemia, glomerular nephritis, idiopathic facial paralysis, Pemphigus vulgaris, cryoglobulinemia, and AIDS, Epstein Barr virus associated diseases, such as Sjorgren's Syndrome, rheumatoid arthritis, Burkitt's lymphoma, Hodgkin's disease, virus (AIDS or EBV) associated B cell lymphoma, chronic fatigue syndrome, parasitic diseases, such as Lesihmania and immunosuppressed disease states, such as viral infections following allograft transplantation or AIDS, cancers, chronic active hepatitis diabetes, toxic shock syndrome, food poisoning, and transplant rejection.

Since the vector constructs of the present invention incorporate a signal sequence for the secretion of each member of the chimeric heterodimeric molecule, it is possible that the therapeutic methods of the present invention may also be performed with polynucleotides or vectors designed for gene therapy. The polynucleotide may be DNA or RNA. When the polynucleotide is DNA, it can also be a DNA sequence which is itself non-replicating, but is inserted into a replicating plasmid vector. The polynucleotide may be engineered such that it is not integrated into the host cell genome. Alternatively, the polynucleotide may be engineered for integration into the chromosome provided the expression of the polypeptide may be controlled. Such regulatable gene expression systems having in vivo applicability are known in the art, and may be used in the present invention. For example, selective killing oftransfected cells may be mediated by including in the polynucleotide or vector a gene sequence encoding a cytotoxic peptide such as HSV thymidine kinase (Borrelli et al. Proc. Nat. Acad. Sci. USA 85:7572, 1988). The thymidine kinase gene acts as a suicide gene for transfected cells if the patient is exposed to gancyclovir. Thus, if expression of the encoded peptides of the invention is too high, gancyclovir may be administered to reduce the percentage of cells expressing the peptides.

S 15 The said compounds or more specifically different classes of heterodimeric transmembrane proteins or polynucleotides encoding the same, which contain a and P polymorphic integral membrane polypeptides that bind each other forming a functional unit involved in immune recognition, may be made into pharmaceutical compositions with appropriate pharmaceutically acceptable carriers or diluents, such as a macromolecule, 20 which is soluble in the circulatory system and which is physiologically acceptable where physiological acceptance means that those skilled in the art would accept injection of said carrier into a patient as part of a therapeutic regime. The carrier preferably is relatively stable in the circulatory system with an acceptable plasma half-life for clearance. Suitable carriers include, but are not limited to, water, alcoholic/aqueous solutions, emulsions, or suspensions, including saline and buffered media, and proteins such as serum albumin, heparin, immunoglobulin, polymers such as polyethylene glycol or polyoxyethylated polyols or proteins modified to reduce antigenicity by, for example, derivitizing with polyethylene glycol. Suitable carriers are well known in the art and are described, for example, in U.S. Patents 4,745,180, 4,766,106, and 4,847,325, and references cited therein.

If appropriate, pharmaceutical compositions may be formulated into preparations including, but not limited to, solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, and aerosols, in the usual ways for their respective route of administration. Methods known in the art can be utilized to prevent release or absorption of the composition until it reaches the target organ or to ensure time-release of the composition. A pharmaceutically-acceptable form should be employed which does not ineffectuate the compositions of the present invention. In pharmaceutical dosage forms, the compositions may be used alone or in appropriate association, as well as in combination with, other pharmaceutically-active compounds. For example, in applying the method of the present invention for delivery of a soluble constructs of the invention, or more specifically different classes of heterodimeric transmembrane proteins, which contain aand a/3 integral membrane polypeptides that bind each other forming a functional unit involved in immune recognition, such delivery may be employed in conjunction with other means of treatment of infectious diseases, autoimmunity, cancers, for example. The compounds may be administered alone or in combination with other diagnostic, therapeutic or additional agents. Therapeutic agents may include cytokines or lymphokines, such as IL-2, S 15 a-interferon and interferon-y.

S Accordingly, the pharmaceutical compositions of the present invention can be delivered via various routes and to various sites in an animal body to achieve a particular effect.

Local or system delivery can be accomplished by administration comprising application or instillation of the formulation into body cavities, inhalation, or insufflation of an aerosol, or 20 by parenteral introduction, comprising intramuscular, intravenous, peritoneal, subcutaneous intradermal, as well as topical administration.

The composition of the present invention can be provided in unit dosage form, wherein each dosage unit, a teaspoon, tablet, solution, or suppository, contains a predetermined amount of the composition, alone or in appropriate combination with other pharmaceutically-active agents. The term "unit dosage form" refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the composition of the present invention, alone or in combination with other active agents, calculated in an amount sufficient to produce the desired effect, in association with a pharmaceutically-acceptable diluent, carrier liquid carrier such as a saline solution, a buffer solution, or other physiological aqueous solution), or vehicle, where appropriate. The specifications for the novel unit dosage forms of the present invention depend on the particular effect to be achieved and the particular pharmacodynamics associated with the pharmaceutical composition in the particular host.

Additionally, the present invention specifically provides a method of administering soluble constructs of compounds which include the invention to a host, which comprises administering the composition of the present invention using any of the aforementioned routes of administration or alternative routes known to those skilled in the art and appropriate for the particular application. The "effective amount" of the composition is such as to produce the desired effect in a host which can be monitored using several end-points known to those skilled in the art. For example, one desired effect might comprise effective nucleic acid transfer to a host cell. Such transfer could be monitored in terms of a therapeutic effect, alleviation of some symptom associated with the disease being treated, or further evidence of the transferred gene or expression of the gene within the host, e.g, using PCR, Northern or Southern hybridization techniques, or transcription assays to detect the nucleic acid in host cells, or using immunoblot analysis, Santibody-mediated detection, or particularized assays, as described in the examples, to detect protein or polypeptide encoded by the transferred nucleic acid, or impacted level or function due to such transfer. These methods described are by no means all-inclusive, and further methods to suit the specific application will be apparent to the ordinary skilled S. artisan.

The particular dosages of divalent and multivalent heterodimeric compounds employed for 20 a particular method of treatment will vary according to the condition being treated, the binding affinity of the particular reagent for its target, the extent of disease progression, •"etc. However, the dosage will generally fall in the range of 1 pg/kg to 100 mg/kg of body weight per day. Where the active ingredient of the pharmaceutical composition is a nucleic acid, dosage will generally range from 1 nM to 50 M per kg of body weight. The amounts of each active agent included in the compositions employed in the examples described herein provide general guidance of the range of each component to be utilized by the practitioner upon optimizing the method of the present invention for practice either in vitro or in vivo. Moreover, such ranges by no means preclude use of a higher or lower amount of a component, as might be warranted in a particular application. For example, the actual dose and schedule may vary depending on whether the compositions are administered in combination with other pharmaceutical compositions, or depending on individual differences in pharmacokinetics, drug disposition, and metabolism. Similarly, amounts may vary in vitro applications depending on the particular cell line utilized, e.g., the ability of the plasmid employed to replicate in that cell line. Furthermore, the amount of nucleic acid to be added per cell or treatment will likely vary with the length and stability of the nucleic acid, as well as the nature of the sequence, and is particularly a parameter which needs to be determined empirically, and may be altered due to factors not inherent to the method of the present invention, the cost associated with synthesis, for instance. One skilled in the art can easily make any necessary adjustments in accordance with the necessities of the particular situation.

The following examples merely illustrate the best mode now contemplated for practicing the invention and should not be construed to limit the invention.

Examples *b- Cells and Culture Conditions: RMA-S, RMA-S L T2, T2 Kb, T2 Kbm3, T2 Kbmll, and RENCA cells were maintained by 1:10 passage three times weekly in RPMI-1640 supplemented with 2 mM glutamine, nonessential amino acids, 50 5g/ml of gentamicin, 5x10-5M 2-mercaptoethanol, and 10% fetal calf serum.

Expression of soluble 2C TcR analogs: The details of construction, expression, purification and characterization of soluble divalent 2C TcR/Ig were carried out as described elsewhere (O'Herrin et al manuscript in preparation). Briefly, to generate the o soluble divalent 2C TcR, cDNAs encoding the 2C TcR a and-P chains were genetically linked via a six amino acid glycine/serine spacer to cDNAs encoding IgGI heavy chains and K light chains, respectively (see Figure 1 for protein schematic). Soluble monovalent 2C TcR was made and purified as previously described (Corr et al Science 265:946-949 1994).

Peptide Loading of Cells: RMA-S and T2 cell lines are defective in antigen processing and express functionally empty class I MHC on their cell surface (Spies et al Nature 355:644-646 (1992); Townsend et al Nature 340:443-448 (1989). These empty MHC molecules may be loaded with peptides as described (Catipovic et al Journal of Experimental Medicine 176:1611-1618 (1992); Townsend et al (1989) supra. Briefly, cells (RMA-S, RMA-S L, T2, T2 L d T2 Kb, T2 Kbm3 or T2 KbmI were cultured at 27 0 C overnight. Subsequently, cells were incubated in the presence or absence of various antigenic peptides (100 5M final concentration) for an additional 1.5 hours at 27 0 C and then for one hour at 37 0

C.

RENCA cells were loaded with peptides by incubation with peptides (100 5M final concentration) for >2 hrs. at 37 0 C. Cells were then harvested and processed for FACS analysis as described.

Measurement of the affinity of soluble 2C TcR for H-2 Ld molecules: Affinities of soluble 2C TcR analogs for peptide loaded cells were determined in a competition assay with FITC-30.5.7 Fab similar to one previously described (Schlueter et al Journal of Molecular Biology 256:859-869 (1996). 30.5.7 is a monoclonal antibody that recognizes an epitope near the peptide-exposed face of H-2 Ld; thus 30.5.7 and 2C TcR compete for binding to the peptide exposed face of H-2 Ld. Kd of 30.5.7 Fab for peptide-loaded RMA-S Ld cells were determined as follows. Cells (0.3 x 106/ 0 ml) were loaded with peptide as described above. Subsequently, peptide-loaded or control cells were incubated with varying 15 concentrations of FITC-30.5.7 Fab for 1 hr. at 4 0 C, and then diluted 1:6 with FACS wash buffer (PBS, 1% FCS, .02% NaN 3 immediately prior to analysis by flow cytometry. Kd were estimated from a plot of 1/(mean channel fluorescence) vs. 1/[FITC-30.5.7 Fab].

Affinities of 2C TcR analogs were determined by competition with constant concentrations of FITC-30.5.7 Fab. Cells were loaded with peptide, and subsequently incubated with a constant concentration of FITC-30.5.7 Fab and varying concentration of 2C TcR analogs for 1 hour at 4 0 C. Cells were diluted 1:6 with FACS wash buffer immediately prior to analysis by flow cytometry. Maximal inhibition of FITC-30.5.7 Fab binding was determined by incubation in the presence of 30.5.7 mAb (75mg). Kapp was determined from a plot of maximal inhibition) vs. [2C TCR analog]. Kapp was corrected for the affinity of FITC-30.5.7 Fab for peptide loaded cells according to the equation Kd,TcR=Kapp/(l+([FITC 30.5.7 Fab]/Kd,30.5.7)) (Schlueter et al (1996) supra.

Direct Flow Microfluorimetry Approximately 3 xl05 peptide-loaded or control cells were incubated for 60 min. at 4 0 C with either -50 mg/ml mAb 30.5.7 culture supernatants in a 30-50 ml volume, 50 ml of2C TcR/Ig culture supernatants (10 5g/ml final concentration), or 25-50 mg/ml purified 2C TcR/Ig in a 30 ml volume. Cells were washed once in lx PBS, 1% FBS, 0.02% Na-azide (FACS wash Buffer) and then incubated for an additional min. at 4 0 C in 20 51 of a 1/20 dilution goat anti-mouse IgG-RPE (Southern Biotechnology Associates, Inc.). Cells were subsequently washed once with FACS wash buffer, resuspended in 250 ul FACS wash buffer and analyzed on a Becton Dickinson FACScan flow cytometer.

CTL Assays (Generation of CTL) Splenocytes from 2C TCR transgenic mice (Sha et al Nature 336:73-76 (1988 were resuspended at 1.25 X 106 per ml and stimulated with 1.75 X 106 BALB/c splenocytes that had been exposed to 3,000 cGy radiation. On day 7, the 2C T cell-enriched cultures were restimulated at 5 X 10 5 per ml with 2.5 X 10 6 per ml BALB/c splenocytes. Experiments were performed on this and subsequent stimulationUs on day 4. All subsequent stimulation was performed with 3.75 X 10 5 per ml 2C splenocytes and 2.5 X106 per ml BALB/c cells in the presence of IL-2 (5 U/ml).

Assays were performed in triplicate according to established CTL protocols. Briefly, target cells (2-4 X 106) were incubated with 100 5Ci 5 [Cr] at 37 0 C for Ih. After three washes, cells were added to V-bottom 96 well plates (3 X 103/100 51) and incubated S 15 (25 0 C for 1.5 h) with peptides at the indicated concentrations. 2C T cells (3 X 10 4 /10051) were added to targets and plates were incubated at 37 0 C for 4.5 h. Maximum release was achieved by incubating targets with 5% Triton X 100. Percent specific lysis was calculated from raw data using [(experimental release spontaneous release )/(maximum release spontaneous release)] X 100.

General Construction and Biochemical Characterization of Chimeric Molecules Using immunoglobulin as a backbone, a general system has been designed for expression o* of soluble recombinant divalent analogs of heterodimeric transmembrane proteins (Figured 1B-D and Figure As shown in Figure 2, site-directed mutagenesis was used to insert restriction enzyme sites, such as KpnI and Hind III, into the 5' region of the Ig heavy and light chains, respectively. The enzyme sites were introduced immediately after the leader sequence prior to the start of the mature protein encoding the intact variable domains. This strategy leads to a generic system for expression of chimeric polypeptides and serves as a foundation molecule for construction of soluble divalent analogs of two different classes of heterodimeric proteins. The different classes of heterodimeric transmembrane proteins, which contain a and f/ polymorphic integral membrane polypeptides that bind each other forming a functional unit involved in immune recognition, include, but are not limited to, proteins such as T cell receptors, and class II MHC molecules, integrins, and cytokine receptors.

A multi-step construction was used to genetically fuse a and 8 polypeptides to Ig heavy and light chains to form the chimeric IgG molecules. As chimeric fusion partners consisted ofcDNA encoding a murine IgGI arsonate-specific heavy chain, 93G7, and iK light chain, 91A3 (Haseman et al Proc Natl Acad Sci USA 87:3942-3946 (1990). Both of these Ig polypeptides have been expressed and produce intact soluble intact IgG1 molecules in baculovirus infected cells. cDNA encoding the light chain clone 91A3 was modified by introduction of 5' HindIII site and linker immediately prior to position one amino acid residue, Asp, at the start of the mature protein. A KpnI restriction enzyme endonuclease site was introduced after the stop codon in the mature K polypeptide.

cDNA encoding the 93G7 clone was modified but introduction of a KpnI restriction enzyme endonuclease site immediately prior to 5' to amino acid residue position Glu located at the start of the mature protein, and an SpHI restriction enzyme endonuclease site 3' to the stop codon in the mature IgGI protein.

Example 1: Construction and Expression of Soluble Divalent Class II MHC S, Molecules and T Cell Receptors The difficulty experienced with the construction and expression of soluble heterodimeric integral proteins, such as soluble divalent class II MHC molecules and T cell receptors (TcRs), was overcome by linking a and 3 chain polypeptides to immunoglobulin heavy and light chains (Figures 1 and Using the soluble divalent TcRs, data are presented to show that soluble proteins are high affinity ligands for peptide/MHC complexes.

TcR Rationale and Construction: The 2C TcR was selected to generate soluble divalent TcR analogs. 2C is a well characterized alloreactive, peptide-specific cytotoxic T lymphocyte (CTL) clone (Kranz et al Proc Natl Acad Sci USA 81:573-577 (1984). This clone is specific for a naturally processed endogenous peptide derived from alpha-ketoglutarate dehydrogenase bound by the murine class I molecule H-2Ld (Udaka et al Cell 69:989-998 (1992). The original 2C reactive peptide, called p2Ca, identified as an eight amino acids residue. Both higher and lower affinity variants of p2Ca reactive with 2C cells have been defined (Sykulev, ,41 ii 28 Immunity, supra; Sykulev et al Proc Natl Acad Sci USA 91:11487-11491 (1994) (see Table A clonotypic monoclonal antibody, 1B2, specific for the 2C TcR has also been developed (Kranz Proc. Natl. Acad. Sci. USA 81:573-577 (1984). The TcR conferring 2C specificity has been cloned and transgenic mice expressing 2C TcR have also been derived (Sha et al Nature 336:73-76 (1988); Sha et al Nature 335:271-274 (1988). The above mentioned prior art makes 2C TcRs an excellent model to study.

To generate the soluble divalent TcR, cDNA encoding the TcR a and /3 chains of TcR was genetically linked to cDNA encoding IgG1 heavy chains and /c light chains, respectively. Site-directed mutagenesis was used to introduce restriction endonuclease enzyme sites into the 5' region prior to the leader sequence and into the 3' region of the TcR a and 3 genes immediately preceding the regions encoding the transmembrane domains (see Figures 1 B-D for schematic of proteins, Figure 2 for expression vector and Figure 3 for oligonucleotides used to induce mutations). The sites introduced in the 3' region in the TcR a and 3 cDNA were compatible with the sites introduced into the immunoglobulin (Ig) heavy and light chain cDNA, respectively. For expression, the constructs were cloned into a modified version of baculovirus expression vector, pAcUW51, and other baculovirus expression systems (Kozono et al Nature 369:151-154 (1994). This expression vector has two separate viral promoters, polyhedron and allowing expression of two polypeptides in a single virally infected cell.

The 2C TcR a chain was modified by introduction of a linker and a KpnI restriction enzyme endonuclease site immediately 3' to the Gin residue at interface between the extracellular and transmembrane domains of the a polypeptide. The 5' regions of the genes already expressed the appropriate restriction enzyme endonuclease sites and did not require any additional modifications.

The 2C TcR P chain was modified by introduction of a Xhol site 5' to the start of the signal sequence for the 3 chain and a HindIII restriction enzyme endonuclease site immediately 3' to the Ile residue at interface between the extracellular ad transmembrane domains of the 3 polypeptide.

Class II MHC Rational and Construct: To study class II MHC molecules, the well-characterized murine I-Ek molecule was chosen as a model antigen. Other class II molecules that could have been chosen include murine I-A molecules and human HLA-DR, DP, and DQ molecules. Murine I-Ek is a known restriction element for a model class II antigen moth cytochrome C (MCC). Soluble monovalent versions of relevant TcR and class II MHC/peptide complexes have been generated (Wettstein et al JExp Med 174:219-228 (1991); Lin et al Science 249:251 (1990). T cell responses to this complex have been well characterized (Jorgensen et al Nature 355:224 (1992) and the affinity of specific T cell clones to MCC/I-Ek complexes have been measured (Matsui Proc. Natl. Acad. Sci. 91:12862-12866 (1994). A genetically engineered soluble version of murine I-Ek that was covalently linked to MCC has also been shown to stimulate MCC-specific T cells (Kozono, supra). Thus, this well characterized MHC system was used as a model to study the influence of divalent class II MHC on T cell reactivity.

For expression of soluble divalent class II MHC molecules, cDNA encoding the I-EkB chain was genetically linked to a cDNA encoding an IgG heavy chain. A cDNA encoding an I-E chain was linked to the one encoding the kappa light chain. The 5' amino terminus *of the 3 chain was previously genetically linked via a thrombin cleavage site to the I-E"-restricted antigenic-peptide derived from MCC (81-101) (Kozono, supra).

Site-directed mutagenesis was used to introduce a KpnI restriction enzyme endonuclease enzyme site into the 3' region of the I-E" a immediately preceding the regions encoding the transmembrane domains. The cDNA encoding the I-Ea chain was modified by introduction of a HindIII restriction enzyme endonuclease immediately preceeding the transmembrane domains. The 5' I-E, and I-Es regions of the genes did not require any additional modifications.

General Linker Region Rational and Construction: A linker of six amino acid residues was also added at the junctions between the end of the TcR a and and I-E a and 3 polypeptides and the start of the mature IgG polypeptides. For the junction with Ig 7 1 polypeptides the linker consists of Gly-Gly-Gly-Thr-Ser-Gly. For the junction with Ig K, polypeptides the linker consists of Gly-Ser-Leu-Gly-Gly-Ser. Oligonucleotides used to introduce all the above mutations are described in Figure 3.

.4 g The expression vector used to generate a soluble divalent T cell receptor analog was derived from the baculovirus expression vector pAcUW51 (Pharmingen, California). This vector has two separate viral promoters, polyhedron and P10, allowing one to express both chimeric polypeptide chains in the same cell. To facilitate cloning of different genes into the vector, multiple cloning sites were previously introduced after each of the promoters (Kozono, supra).

Example 2: Detection of Soluble Heterodimeric Proteins Cells infected with baculovirus containing transfer vectors encoding the soluble chimeric Ig constructs described above secrete a soluble chimeric Ig-like molecule detected by specific ELISA assays 4-5 days post infection. For 2C TcR/IgG, the assay was based on a primary antibody specific for murine IgG Fc (plated at 10 g g/ml) and a biotinylated secondary antibody, H57 (used at 1:5000 final dilution), specific for a conformational epitope expressed on the chain of many TcR (Figure 5, Panel A) or biotinylated 1B2 or a monoclonal antibody specific for a clontoypic epitope expressed on 2C TcR (Figure Panel For detection of I-E/IgG chimeric molecules, the same primary antibody was •used and the biotinylated secondary antibody was 14.4.4, which is specific for I-E a chain (Figure 5, Panel Supematants from infected cells were incubated for 1 hour at room temperature. Plates were washed extensively with phosphate buffer saline, incubated with .0 the biotinylated secondary antibody for 1 hour at room temperature. The plates were then washed and incubated with HRP-conjugated strepatvidin (100 ga 1 of a 1:10000 dilution) (Sigma, St. Louis, MO) for 1 hour at room temperature, washed and developed with 3, 3', 5'-Tetramethylbenzidine Dihydrochloride (TMB) substrate for 3-5 minutes.

Supernatants from cells infected with baculovirus containing the 2C TcR/Ig and I-E/Ig transfer vectors were compared to control supernatant from cells infected with the wild type baculovirus.

The chimeric proteins are conformationally intact as shown in Figure 5. The soluble divalent 2C TcR/Ig is reactive with H57, a monoclonal antibody specific for a conformational epitope expressed on most TcR /3 chains as well as with 1B2, the anti-clonotypic monoclonal antibody determinant specific for the 2C TcR as shown in Figures 5A and 5B. Soluble divalent class II molecules are reactive with the conformationally dependent monoclonal antibody specific for a native alpha chain 31 determinant only expressed on intact I-E molecules, monoclonal antibody 14.4.4 as shown in Figure 5C. The immunoglobulin portion of the chimeric molecules is also conformationally intact. It is reactive immunoglobulin specific ELISA, as mentioned above, and can be used to purify the chimeric molecules. Protein G or arsenate-sepharose affinity purification column methods can also be used (data not shown).

The purified material has the expected molecular weights when analyzed by SDA-PAGE as depicted in Figure 6. The chimeric TcRAE/Igo has an apparent molecular weight (MW) of 55,000 and the chimeric TcR a /Ig 1 has an apparent MW of approximately 89,000.

The chimeric I-E a /Ig K has an approximate MW of 44,000 and the chimeric I-E 3 /Ig 7- 1 has an apparent MW of approximately 76,000 (Figure 6).

Example 3: Affinity measurements of soluble divalent TCR interaction with peptide/MHC complexes.

A competitive inhibition assay was developed to measure the affinity of soluble 2C TCR/Ig for peptide/MHC complexes. This assay, similar to one previously used to S 15 determine the affinity of soluble monovalent 2C TCR for peptide/MHC complexes (Schlueter et al Journal of Molecular Biology 256:859-869 (1996), is based on mAb 30.5.7 binding to a region of the a2 helix of H-2 Ld that overlaps with TCR receptor binding (Solheim et al Journal of Immunology 154:1188-1197 (1995); Solheim et al Journal of Immunology 150:800-811 (1993). Briefly, affinities of 30.5.7 Fab fragments for RMA-S Ld cells were determined by direct saturation analysis of 30.5.7 Fab binding to cells analyzed by flow cytometry. Cells were incubated with increasing amounts of FITC labeled 30.5.7 Fab, and Kd's were estimated from a plot of 1/MCF vs. 1/[30.5.7 Fab].

Affinities of 2C TCR analogs were determined by competition of the 2C TCR analog with a constant amount of FITC labeled 30.5.7 Fab fragments for RMA-S Ld cells as described in Methods. Kapp was calculated from a plot of(% maximal 30.5.7 Fab binding)-' vs. [2C TCR analog]. The Kapp was corrected for the affinity of 30.5.7 Fab for RMA-S L d cells according to the equation Kd.TCR=Kapp/(1+{30.5.7 Fab]/Kd.305.

7 (Schlueter et al., 1996).

The values reported in the Table 2 are from one representative experiment that has been repeated at least three times. Each data point used in determination of the Kd is the average of duplicate points. Hence, the affinity of soluble TCR analogs was measured in terms of their inhibition of 30.5.7 binding.

To determine the affinity of the soluble 2C TCR analogs, one has to first determine the Kd of 30.5.7 Fab fragments for peptide-loaded H-2 Ld molecules. This measurement was determined by direct saturation analysis of 30.5.7-FITC Fab binding to H-2 Ld molecules on the surface of RMA-S Ld cells. RMA-S cells were chosen since these cells express empty MHC molecules that can be readily loaded with specific peptides of interest (Catipovic et al Journal of Experimental Medicine 176:1611-1618 (1992); Townsend et al Nature 340:443-428 (1989). The affinity of 30.5.7 for H-2 Ld molecules is dependent on the peptide loaded into H-2 Ld (Table The affinity of the 30.5.7 for QL9 loaded H-2

L

d molecules is 12.2 nM while the affinities for p2Ca, pMCMV and SL9 loaded H-2 Ld molecules range between 4.8-6.4 nM. These small, peptide-dependent, differences in affinity are reproducible and variations in affinity were accounted for in the competitive binding assays. These values are in good agreement with the previously measured affinities of 1251-30.5.7 Fab for the same peptide/H-2 Ld complexes (8.8 to 16 nM (Schlueter et al., 1996)).

2C TCR/Ig inhibited binding of 30.5.7 Fab to H-2 Ld molecules loaded with either QL9 or p2Ca peptides but did not inhibit 30.5.7 Fab binding to pMCMV loaded H-2 Ld molecules (Figure The affinity of soluble divalent 2C TCR/Ig for QL9 loaded molecules is 13.3 nM (Figure 7 and Table As expected, the affinity of2C TCR/Ig for p2Ca loaded molecules, 90 nM, is lower than that for QL9 loaded H-2 Ld. Although a small amount of competitive inhibition was seen with SL9 loaded cells, the affinity of the soluble divalent 2C TCR/Ig chimeras for SL9 loaded molecules is too low to be accurately measured under the conditions tested (data not shown).

In all cases analyzed, the affinity of the soluble divalent 2C TCR/Ig was significantly higher than the affinity of the soluble monovalent 2C TCR for its cognate ligand (Figure 7 and Table The affinity of soluble divalent 2C TCR/Ig was 50-fold higher for QL9-loaded H-2 Ld and at least 20-fold higher for p2Ca-loaded H-2 Ld molecules than that of soluble monovalent 2C TCR for the same peptide/MHC complexes (Table 2).

Thus, the divalent nature of soluble 2C TCR/Ig chimeras significantly increased the affinity of the TCR analog for its cognate ligands. The finding that the chimeric molecules of the present invention demonstrate increased affinity for their specific ligands over what is seen for monovalent molecules was not an expected result. In fact, the chimeric CD4-IgG molecules disclosed in Capon et al. do not demonstrate improved target affinity, which is further evidence to the value and novelty of the compositions of the present invention.

TABLE 2 Measured Affinities of TCR analogs for peptide loaded RMA-SLd cells Peptide/MHC 30.5.7 Fab 2C TCR/Ig 2C-sm TCR complex Kd (nM) Kapp (nM) Kd (nM) Kapp (nM) Kd (nM) QL9 12.2 18.3 13.3 953.4 613.6 p2Ca 5.8 107.7 90.5 >20002 >20002 pMCMV 4.8 ndc' ndc' -3 3 'ndc no detectable competition with 30.5.7 Fab fragments 2 Competition was detected at the highest concentration of 2C-smTCR used, but an accurate measure of the Kd could not be determined.

3 not done.

Example 4: Binding specificity of soluble divalent TCR chimeras to peptide-loaded H-2 Ld molecules.

Based on the relatively high affinity of soluble divalent 2C TCR/Ig for peptide/MHC complexes, we postulated that these molecules might be useful in analysis of peptide/MHC complexes by direct flow cytometry based assays. To study peptide specificity of 2C TcR/Ig, we compared reactivity of 2C TcR/Ig with that of H-2 Ld reactive mAb, 30.5.7, in direct flow cytometry assays. Specific peptides (see Table 3 for sequences) were loaded into H-2 Ld molecules on RMA-S Ld cells. Peptides listed in Table 2 are a collection of H-2 Ld and H-2 Kb binding peptides used in analysis of the reactivity of the soluble divalent 2C TCR/Ig. Lysis and affinity data are summarized from their primary references (Corr et al., 1994; Huang et al., 1996; Solheim et al., 1993; 34 Sykulev et al., 1994a; Sykulev et al., 1994b; Tallquist et al., 1996; Udaka et al., 1996; Van Bleek and Nathanson, 1990).

TABLE 3 Peptides used in this study: Their reported effectiveness in 2C CTL assays and affinities of 2C TCR for peptide/MHC complexes.

peptide peptide MHC 2C-mediated Kd( a M) name sequence restriction lysis p2Ca LSPFPFDL H-2 L d 0.5-0.1 QL9 QLSPFPFDL H-2 Ld 0.066 SL9 LSPFPFDLL H-2 Ld 71 tum TQNHRALDL H-2 Ld na na pMCMV YPHFMPTNL H-2Ld nd gp 70 SPSYVYHQF H-2 Ld na na dEV-8 EQYKFYSV H-2 Kb unknown dEV-8 H-2 Kbm 3 unknown SIY SIYRYYGL H-2 Kb unknown SIY H-2 Kbm 3 unknown unknown pVSV RGYVYQGL H-2 Kb nd NP(52-59) na not available.

nd none detected. The affinity were below the sensitivity of the assay used.

The temperature-dependent reactivity of RMA-S Ld with 2C TCR/Ig was significantly different than the reactivity of RMA-S Ld with mAb 30.5.7. As expected (Solheim et al (1995) supra; Solheim et al (1993) supra), RMA-S Ld cells expressed more serologically reactive H-2 L d molecules recognized by mAb 30.5.7 on cells cultured at 27 0 C than when cells were cultured at 37C (Figure 8A); mean channel fluorescence (MCF) increased approximately 5 fold. Thus the epitope on H-2 Ld molecules recognized by mAb 30.5.7 can be stabilized by incubating cells at low temperatures. In contrast, RMA-S Ld cells expressed very low amounts of H-2 L d molecules recognized by 2C TcR/Ig on cells cultured at either 27 0 C or at 37 0 C (Figure 8, Panel This finding is consistent with the expected peptide-dependent reactivity of 2C TcR/Ig which should not recognize unloaded MHC even when conformationally stabilized by decreased temperature.

2C TcR/Ig reactivity also showed exquisite peptide specificity. As expected, all H-2 Ld-binding peptides stabilized expression of the epitope recognized by mAb 30.5.7 (Figure 8, Panels B-D and Figure Only H-2 L d molecules loaded with 2C reactive peptides, peptides p2Ca, QL9, and SL9 expressed peptide/H-2Ld epitopes that reacted with 2C TcR/Ig (Figure 8, Panels F-H and Figure MCF increased approximately 10-200 fold, from a MCF of 10 for either unloaded cells or cells loaded with an irrelevant H-2 L d binding peptide, to as high as 2200 for RMA-S Ld cells loaded with peptide QL9 (Figure The pattern of reactivity mimicked the known affinities of monovalent 2C TcR for peptide/H-2 L d complexes (see Table 3 for affinities). RMA-S L d cells loaded with peptide QL9, p2Ca, or SL9 had MCF values of 2200, 550, and 100, respectively, when stained with 2C TcR/Ig. Thus, soluble divalent 2C TcR/Ig chimeras reacted strongly with QL9/H-2 Ld complexes, modestly with p2Ca/H-2 L d complexes, and weakly with SL9/H-2 Ld complexes. The fact that 2C TcR/Ig bound to SL9-loaded H-2 L d molecules indicates, that even in a direct flow cytometry assay, soluble divalent 2C STcR/Ig chimeras could be used to detect specific peptide/MHC complexes that have affinities as weak as 71 mM for monovalent 2C TcR.

Example 5: Inhibition of In Vitro 2C T cell Mediated Lysis by Soluble Divalent 2C TcR/Ig Molecules Soluble divalent 2C TcR/Ig blocks 2C reactive T cell responses. Since soluble divalent 2C TcR/Ig interacts with high avidity with H-2 L d molecules loaded with the appropriate peptides in the flow cytometry assay, it was explored whether the reagent could effectively inhibit 2C T cells in vitro cytotoxicity CTL assays. This was analyzed using a cell line derived from 2C transgenic mice to lyse tumor target cells expressing H-2 L d CTL were tested in a routine 4 hour 51 Cr cytotoxicity assay. As targets for all the CTL assays, untransfected, MC57G, and Ld transfected, MC57G L d cells were used as targets. The percent specific lysis was determined as: 51 Cr cpm (experimental) -CPM (spontaneous)/cpm (maximum) -cpm (spontaneous). Standard errors were routinely less than 5% and spontaneous release was usually 10-15% of maximal release.

Using both untransfected MC57G and Ld transfected, MC57G L d we were able to establish a window ofH-2Ld specific lysis mediated by the 2C CTL line. To test the influence of 2C TcR/Ig, target cells were pretreated with either 2C TcR/Ig, or I-Ek/Ig and analyzed for lysis by the CTL cell line derived from 2C transgenic mice. Significant inhibition of lysis was seen at each effector to target cell ratio analyzed when cells were treated with 2C TcR/Ig (see Figure 10). While some non-specific inhibition was seen in the I-Ek/Ig treated target cells, significantly more inhibition was seen in the 2C TcR/Ig treated target cells.

In this assay, the target cells were normal tumor cells that load their cell surface MHC molecules with a variety of different endogenous peptides. Using these target cells, one does not need to specifically load H-2 Ld molecules with the p2Ca peptide since p2Ca or p2Ca-like peptides along with a large number of irrelevant peptides are endogenously loaded onto cellular MHC molecules. Inhibition of CTL-mediated lysis indicates that soluble divalent 2C TcR/Ig can effectively interact with the relevant peptides even within a milieu of a large number of irrelevant peptides. Thus, this approach could be used to search the universe of peptide/MHC complexes to identify only those complexes relevant Sto specific T cell responses of interest. In particular, these high avidity soluble analogs of I heterodimeric proteins may specifically be useful in identification of unknown tumor and autoimmune antigens.

Example 6: Binding of soluble divalent TCR chimeras to self restricted peptide/MHC complexes.

In addition to recognizing peptide/H-2 Ld ligands, two peptides, SIY and dEV-8, that sensitize either H-2 Kb or H-2 Kbm 3 targets for lysis by 2C CTL have also been defined (see Table 3 for sequences). To analyze the ability of 2C TcR/Ig to bind to these alternate 2C-reactive complexes, the binding of 2C TcR/Ig to peptide loaded transfected T2 cells was studied. Since T2 cells are derived from a human cell line, T2 cells do not naturally express H-2 K b as do RMA-S cells. Thus to study the binding of2C TcR/Ig to peptide-loaded H-2 K b or various H-2 Kbm mutant molecules, the T2 system was chosen since it is not complicated by the expression of MHC molecules from the parental cell line. Similar to RMA-S Ld cells, T2 cells also express empty MHC molecules that can be readily loaded with different peptides. For these studies peptide-loaded T2 cells transfected with: H-2 Kb, T2 Kb; H-2 Kbm 3 T2 Kbm 3 and H-2 Kbm i l T2 Kbm ll (Tallquist et al Journal of mmunology 155:2419-2426 (1995); Tallquist et al Journal of Experimental Medicine 184:1017-1026 (1996) were utilized.

Peptide SIY-loaded T2 Kb or T2 Kbm ll cells both expressed epitopes recognized by 2C TcR/Ig (Figure 11A,C). MCF of cells incubated with 2C TcR/Ig increased approximately 20 fold from 14 for pVSV loaded- to 276 for SIY loaded-T2 Kb and from 16 for pVSV loaded- to 250 for SIY loaded- T2 Kbml l SIY-loaded T2 Kbm 3 cells showed a much weaker but still significant interaction with 2C TcR/Ig (Figure 11B); compare SIY-loaded (solid lines; MCF, 36) to pVSV-loaded (dotted lines; MCF, 12) T2 Kbm 3 cells.

The 2C TcR/Ig binding data to SIY/MHC complexes was consistent with 2C CTL mediated lysis on various SIY/MHC targets (Figure 12). 2C CTL mediated efficient lysis i: of SIY loaded T2 Kb and T2 Kbm il cells (Figure 12B and data not shown, LD50-10ng/ml for SIY/ T2 Kb). 2C CTL mediated lysis of SIY loaded T2 Kbm 3 cells was significantly less efficient (Figure 12C, LD50-100ng/ml).

The binding of 2C TcR/Ig to dEV-8 loaded cells revealed a striking difference between the 20 affinity of 2C TcR/Ig for dEV-8/MHC complexes and the ability of that same peptide/MHC complex to mediate lysis by 2C CTL. As expected, dEV-8 loaded T2 Kb cells were neither lysed by 2C CTL (Figure 12B), nor were they recognized by 2C TcR/Ig in flow cytometry assays (Figure 12A). Interestingly, no significant binding of2C TcR/Ig could be found to dEV-8 loaded T2 Kbm 3 cells (Figure 12B). MCF of cells stained with 2C TcR/Ig was similar whether cells were loaded with either dEV-8 or a control H-2 Kb-binding peptide, pVSV (Figure 12; compare dotted to dashed lines). This is most surprising in that, consistent with previous reports (Tallquist et al (1996) supra, dEV-8 loaded T2 Kbm 3 cells were efficiently lysed by 2C CTL (Figure 12C). In fact, dEV-8 loaded T2 Kbm 3 cells were much better target cells (LD50 -0.5-1.Ong/ml), than SIY loaded T2 K bm 3 cells (LD50-1 00ng/ml), where a significant binding of 2C TcR/Ig was seen (Figure 12B). The efficiency of lysis by 2C CTL of dEV-8 loaded T2 Kbm 3 cells, was on the same order of magnitude as that ofp2Ca loaded T2 L d cells (Figure 12A,

I

38 which was also efficiently recognized in the 2C TcR/Ig binding assay (Figure A similar, although significantly less dramatic, lack of correlation between cytolysis and 2C TcR/Ig binding was seen for dEV-8 loaded T2 KbmlI cells. dEV-8 loaded T2 Kbm" 1 cells are relatively poor targets for 2C CTL (Tallquist et al (1996) supra) (data not shown), but were also not reactive with 2C TcR/Ig in flow cytometry assays (Figure 11C).

Example 7: Analysis of the effects of a-IFN on expression of endogenous 2C-specific peptide/MHC complexes.

The specificity and affinity of 2C TcR/Ig for peptide/MHC complexes suggested that one might be able to use this reagent to probe the influence of lymphokines on endogenous, cell surface, peptide/MHC complexes. To analyze this possibility and follow the expression of endogenous 2C-reactive peptide/H-2 L d complexes within a heterogeneous peptide/MHC environment, the influence of a-IFN on the H-2 Ld expressing murine cell line, RENCA was studied. RENCA cells were cultured in the presence of variable S. 15 amounts of A-IFN to induce up-regulation of naturally loaded peptide/MHC complexes.

2C TcR/Ig binding to RENCA cells increased as a function of A-IFN induction (Figure 13A-D, solid lines). The effect of a-IFN was dose dependent with a maximal 2-3 fold *..increase seen on cells treated with 10 units/ml of A-IFN. Since A-IFN is known to have a direct effect on class I expression (Figure 13E-H) (Hengel et al Journal of Virology 20 68:289-297 (1994)), it is necessary to normalize for any non-specific 2C TcR/Ig binding secondary to increased expression of H-2 L d This was accomplished by incubating RENCA cells with a control irrelevant H-2 Ld binding peptide, pMCMV. Since p2Ca is known to have a weak affinity for H-2 Ld (Sykulev et al Immunity 1:15-22 (1994a) exchange with a higher affinity H-2 Ld binding peptide like pMCMV (Sykulev et al (1994a) supra) should be very efficient. Therefore background reactivity of 2C TcR/Ig could be determined by the efficient displacement of endogenous p2Ca or p2Ca-like peptides by incubating the cells with saturating amounts of the control pMCMV peptide.

In all cases, 2C TcR/Ig binding could be blocked by prior incubation of cells with the control H-2 Ld binding, pMCMV (Figure 13A-D, dotted lines). Prior incubation of RENCA cells with a 2C specific peptide, QL9, induced a dramatic increase in 2C TcR/Ig binding (data not shown). The results of these experiments indicate that 2C TcR/Ig could be used as a sensitive probe to analyze cell surface expression of endogenous 2C-reactive 1. 1.

39 peptide/MHC complexes.

The effect of -IFN on 2C TcR/Ig reactivity was distinct from its effects on 30.5.7 reactivity. At all concentrations analyzed, 5-50 units/ml, 7' -IFN induced a 5-6 fold increase in serologically reactive H-2 Ld, as recognized by mAb 30.5.7 (Figure 13E-H).

MCF of unstimulated RENCA cells was 500, while the MCF of 7 -IFN stimulated cells was between 2666 and 3038. The maximal effect of 7 -IFN was seen at the lowest dose used, in the experiment presented, 5U/ml, and in other experiments was seen even at dose of r -IFN as low as 1 unit/ml (data not shown). Interestingly, the dose response curve of 7 -IFN on 2C TcR/Ig reactivity was shifted. r -IFN at 5U/ml had a relatively small but significant effect on 2C TcR/Ig reactivity. Maximal effects of A-IFN on 2C TcR/Ig reactivity required r -IFN treatment at 10 units/ml, approximately ten fold more than needed for maximal effects of r -IFN on 30.5.7 reactivity. These results indicate a differential effect of 7 -IFN on MHC heavy chain expression than that of -IFN on specific peptide antigen/MHC complex expression.

S 15 These results show that this approach is a general one for producing soluble divalent versions of heterodimeric proteins. Soluble divalent analogs of heterodimeric proteins of this invention are characterized as having high avidity for their targets.

The same way that this was done for a single murine class II MHC and a/ TcR, so to the same technology, generating soluble divalent heterodimeric proteins, can be used to develop other mammalian systems. These will include both rodent and human class II HLA molecules and a /3 and 6 T cell receptors.

The present invention may be embodied in forms other than those specifically disclosed above without departing from the spirit or essential characteristics of the invention. The particular embodiments of the invention described above, are therefore, to be considered as illustrative and not restrictive. All references and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The word 'comprising' and forms of the word 'comprising' as used in this description and in the claims does not limit the invention claimed to exclude any variance or additions.

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Claims (183)

1. An expression vector encoding a first fusion protein, wherein the first fusion protein comprises an immunoglobulin heavy chain comprising a variable region and (b) an extracellular domain of a first transmembrane polypeptide.

2. The expression vector of claim 1 wherein the immunoglobulin heavy chain is an IgG1 heavy chain.

3. The expression vector of claim 1 wherein the first transmembrane polypeptide is an MHC class 1 chain.

4. The expression vector of claim 1 wherein the first transmembrane polypeptide is a 10 TCRa chain.

5. The expression vector of claim 1 wherein the first transmembrane polypeptide is a TCRy chain.

6. The expression vector of claim 2 wherein the first transmembrane polypeptide is an MHC class II chain.

7. The expression vector of claim 2 wherein the first transmembrane polypeptide is a TCRa chain.

8. The expression vector of claim 2 wherein the first transmembrane polypeptide is a TCRy chain.

9. The expression vector of claim 1 wherein the first fusion protein further comprises a first peptide linker between the immunoglobulin heavy chain and the extracellular domain of the first transmembrane polypeptide. The expression vector of claim 9 wherein the first peptide linker is GLY-GLY- GLY-THR-SER-GLY (SEQ ID

11. An expression vector encoding a fusion protein, wherein the fusion protein comprises an IgGI heavy chain comprising a variable region, an extracellular domain of an MHC class IIP chain, and a peptide linker between the IgGI heavy chain and the extracellular domain of the MHC class IIP chain.

12. The expression vector of claim 11 wherein the peptide linker is GLY-GLY-GLY- THR-SER-GLY (SEQ ID NO:

13. An expression vector encoding a fusion protein, wherein the fusion protein 10 comprises an IgGI heavy chain comprising a variable region, an extracellular domain of a TCRa chain, and a peptide linker between the IgG heavy chain and the extracellular domain of the TCRa chain.

14. The expression vector of claim 13 wherein the peptide linker is GLY-GLY-GLY- THR-SER-GLY (SEQ ID

15. An expression vector encoding a fusion protein, wherein the fusion protein comprises an IgGI heavy chain comprising a variable region, an extracellular domain of a TCRy chain, and a peptide linker between the IgGI heavy chain and the extracellular domain of the TCRy chain.

16. The expression vector of claim 15 wherein the peptide linker is GLY-GLY-GLY- THR-SER-GLY (SEQ ID

17. The expression vector of claim 1 further encoding a second fusion protein, wherein the second fusion protein comprises an immunoglobulin light chain and an extracellular domain of a second transmembrane polypeptide.

18. The expression vector of claim 17 wherein the immunoglobulin light chain comprises a variable region.

19. The expression vector of claim 17 wherein the first transmembrane polypeptide is an MHC class IIP chain and the second transmembrane polypeptide is an MHC class IIa chain.

20. The expression vector of claim 17 wherein the first transmembrane polypeptide is a 10 TCRa chain and the second transmembrane polypeptide is a TCRP chain.

21. The expression vector of claim 17 wherein the first transmembrane polypeptide is a TCRy chain and the second transmembrane polypeptide is a TCR8 chain.

22. The expression vector of claim 17 wherein the immunoglobulin heavy chain is an IgGI chain.

23. The expression vector of claim 17 wherein the immunoglobulin light chain is an IgK chain.

24. The expression vector of claim 17 wherein the immunoglobulin light chain comprises a variable region. The expression vector of claim 17 wherein the first fusion protein comprises a first peptide linker between the immunoglobulin heavy chain and the extracellular domain of the first transmembrane polypeptide and wherein the second fusion protein comprises a second peptide linker between the immunoglobulin light chain and the extracellular 48 domain of the second transmembrane polypeptide.

26. The expression vector of claim 25 wherein the first peptide linker is GLY-GLY- GLY-THR-SER-GLY (SEQ ID NO:

27. The expression vector of claim 25 wherein the second peptide linker is GLY-SER- LEU-GLY-GLY-SER (SEQ ID NO: 11).

28. The expression vector of claim 25 wherein the first peptide linker is GLY-GLY- GLY-THR-SER-GLY (SEQ ID NO:10) and wherein the second peptide linker is GLY- SER-LEU-GLY-GLY-SER (SEQ ID NO: 11).

29. An expression vector encoding a fusion protein, wherein the fusion protein 10 comprises an immunoglobulin light chain comprising a variable region and an oo extracellular domain of a transmembrane polypeptide. The expression vector of claim 29 wherein the immunoglobulin light chain is an IgK chain.

31. The expression vector of claim 29 wherein the transmembrane polypeptide is an MHC class IIa chain.

32. The expression vector of claim 29 wherein the transmembrane polypeptide is a TCRP chain.

33. The expression vector of claim 29 wherein the transmembrane polypeptide is a chain.

34. The expression vector of claim 30 wherein the transmembrane polypeptide is an MHC class IIa chain. I L 49 The expression vector of claim 30 wherein the transmembrane polypeptide is a TCRp chain.

36. The expression vector of claim 30 wherein the transmembrane polypeptide is a TCR8 chain.

37. The expression vector of claim 29 wherein the fusion protein comprises a peptide linker between the immunoglobulin light chain and the transmembrane polypeptide.

38. The expression vector of claim 37 wherein the peptide linker is GLY-SER-LEU- GLY-GLY-SER (SEQ ID NO: 11).

39. An expression vector encoding a fusion protein comprising an IgK light chain i* 10 comprising a variable region, an extracellular domain of an MHC class IIa chain, and a peptide linker between the IgK light chain and the extracellular domain of the MHC class IIa chain.

40. The expression vector of claim 39 wherein the peptide linker is GLY-SER-LEU- GLY-GLY-SER (SEQ ID NO: 11).

41. An expression vector encoding a fusion protein comprising an IgK light chain comprising a variable region, an extracellular domain of a TCRP chain, and a peptide linker between the IgK light chain and the extracellular domain of the TCRp chain.

42. The expression vector of claim 41 wherein the peptide linker is GLY-SER-LEU- GLY-GLY-SER (SEQ ID NO: 11).

43. An expression vector encoding a fusion protein comprising an IgK light chain comprising a variable region, an extracellular domain of a TCR8 chain, and a peptide linker between the IgK light chain and the extracellular domain of the TCR6 chain.

44. The expression vector of claim 43 wherein the peptide linker is GLY-SER-LEU- GLY-GLY-SER (SEQ ID NO:11). An expression vector encoding a first and a second fusion protein, wherein the first fusion protein comprises an IgG1 heavy chain comprising a variable region, an extracellular domain of an MHC class IIP chain, and a first peptide linker between the IgG1 heavy chain and the extracellular domain of the MHC class IIp chain, and wherein the second fusion protein comprises an IgK light chain comprising a variable region, (e) an extracellular domain of an MHC class IIa chain, and a second peptide linker between the IgK chain and the extracellular domain of the MHC class IIa chain.

46. The expression vector of claim 45 wherein the first peptide linker is GLY-GLY- GLY-THR-SER-GLY (SEQ ID NO:10) and wherein the second peptide linker is GLY- SER-LEU-GLY-GLY-SER (SEQ ID NO:11).

47. An expression vector encoding a first and a second fusion protein, wherein the first fusion protein comprises an IgG1 heavy chain comprising a variable region, an 15 extracellular domain of a TCRa chain, and a first peptide linker between the IgG1 heavy chain and the extracellular domain of the TCRa chain, and wherein the second fusion protein comprises an IgK light chain comprising a variable region, an extracellular domain of a TCRP chain, and a second peptide linker between the IgK chain and the extracellular domain of the TCR3 chain.

48. The expression vector of claim 47 wherein the first peptide linker is GLY-GLY- GLY-THR-SER-GLY (SEQ ID NO:10) and wherein the second peptide linker is GLY- SER-LEU-GLY-GLY-SER (SEQ ID NO: 11).

49. An expression vector encoding a first and a second fusion protein, wherein the first fusion protein comprises an IgG1 heavy chain comprising a variable region, an extracellular domain of a TCRy chain, and a first peptide linker between the IgG1 heavy chain and the extracellular domain of the TCRy chain, and wherein the second fusion protein comprises an Igi light chain comprising a variable region, an extracellular domain of a TCR5 chain, and a second peptide linker between the IgK chain and the extracellular domain of the TCR8 chain. The expression vector of claim 49 wherein the first peptide linker is GLY-GLY- GLY-THR-SER-GLY (SEQ ID NO:10) and wherein the second peptide linker is GLY- SER-LEU-GLY-GLY-SER (SEQ ID NO:11).

51. A host cell comprising an expression vector encoding a first fusion protein, wherein the first fusion protein comprises an immunoglobulin heavy chain comprising a variable region and an extracellular domain of a first transmembrane polypeptide.

52. A host cell comprising an expression vector encoding a fusion protein, wherein the 15 fusion protein comprises an IgG1 heavy chain comprising a variable region, an extracellular domain of an MHC class IIP chain, and a peptide linker between the IgGI heavy chain and the extracellular domain of the MHC class IIP chain.

53. A host cell comprising an expression vector encoding a fusion protein, wherein the fusion protein comprises an IgG1 heavy chain comprising a variable region, an extracellular domain of a TCRa chain, and a peptide linker between the IgG1 heavy chain and the extracellular domain of the TCRa chain.

54. A host cell comprising an expression vector encoding a fusion protein, wherein the fusion protein comprises an IgG1 heavy chain comprising a variable region, an extracellular domain of a TCRy chain, and a peptide linker between the IgGI heavy chain and the extracellular domain of the TCRy chain.

55. A host cell comprising an expression vector encoding a first and a second fusion protein, wherein the first fusion protein comprises an immunoglobulin heavy chain comprising a variable region and an extracellular domain of a first transmembrane polypeptide, and wherein the second fusion protein comprises an immunoglobulin light chain and an extracellular domain of a second transmembrane polypeptide. 10 56. A host cell comprising an expression vector encoding a fusion protein, wherein the fusion protein comprises an immunoglobulin light chain comprising a variable region and an extracellular domain of a transmembrane polypeptide.

57. A host cell comprising an expression vector encoding a fusion protein comprising an IgK light chain comprising a variable region, an extracellular domain of an MHC 15 class IIa chain, and a peptide linker between the IgK light chain and the extracellular domain of the MHC class IIa chain. S58. A host cell comprising an expression vector encoding a fusion protein comprising an Igi light chain comprising a variable region, an extracellular domain of a TCR chain, and a peptide linker between the IgK light chain and the extracellular domain of the TCRP chain.

59. A host cell comprising an expression vector encoding a fusion protein comprising an IgK: light chain comprising a variable region, an extracellular domain of a TCR8 chain, and a peptide linker between the IgK: light chain and the extracellular domain of the TCR6 chain.

60. A host cell comprising an expression vector encoding a first and a second fusion protein, wherein the first fusion protein comprises an IgG1 heavy chain comprising a variable region, an extracellular domain of an MHC class II1P chain, and a first peptide linker between the IgG1 heavy chain and the extracellular domain of the MHC o~oo class 1I1P chain, and wherein the second fusion protein comprises an IgK light chain comprising a variable region, an extracellular domain of an MHC class Ilox chain, and a second peptide linker between the IgK chain and the extracellular domain of the MHC class Ilec chain. A host cell comprising an expression vector encoding a first and a second fusion protein, wherein the first fusion protein comprises an IgG1 heavy chain comprising a ooo o 15 variable region, an extracellular domain of a TCRot chain, and a first peptide linker 0000 between the IgG1 heavy chain and the extracellular domain of the TCRox chain, and wherein the second fusion protein comprises an IgK: light chain comprising a variable region, an extracellular domain of a TCR13 chain, and a second peptide linker between the IgK chain and the extracellular domain of the TCRP3 chain. 2 0 62. A host cell comprising an expression vector encoding a first and a second fusion protein, wherein the first fusion protein comprises an IgG1 heavy chain comprising a variable region, an extracellular domain of a TCRy' chain, and a first peptide linker between the IgG1 heavy chain and the extracellular domain of the TCR7, chain, and wherein the second fusion protein comprises an IgK light chain comprising a variable region, an extracellular domain of a TCR5 chain, and a second peptide linker between the IgK chain and the extracellular domain of the TCR6 chain.

63. A host cell comprising a first and a second expression vector, wherein the first expression vector encodes a fusion protein comprising an IgGI heavy chain comprising a variable region, an extracellular domain of an MHC class IIP chain, and a first peptide linker between the IgGI heavy chain and the extracellular domain of the MHC class IIp chain, and wherein the second expression vector encodes a fusion protein comprising an IgK light chain, an extracellular domain of an MHC class IIa chain, and a second peptide linker between the IgK light chain and the extracellular domain of :the MHC class IIa chain.

64. The host cell of claim 63 wherein the first peptide linker is GLY-GLY-GLY-THR- SER-GLY (SEQ ID NO:10) and wherein the second peptide linker is GLY-SER-LEU- GLY-GLY-SER (SEQ ID NO:11).

65. A host cell comprising a first and a second expression vector, wherein the first expression vector encodes a fusion protein comprising an IgGI heavy chain comprising a variable region, an extracellular domain of a TCRa chain, and a first peptide linker between the IgG1 heavy chain and the extracellular domain of the TCRa chain, and wherein the second expression vector encodes a fusion protein comprising an IgK light chain, an extracellular domain of a TCRP chain, and a second peptide linker between the IgK light chain and the extracellular domain of the TCRp chain.

66. The host cell of claim 65 wherein the first peptide linker is GLY-GLY-GLY-THR- SER-GLY (SEQ ID NO:10) and wherein the second peptide linker is GLY-SER-LEU- GLY-GLY-SER (SEQ ID NO: 11).

67. A host cell comprising a first and a second expression vector, wherein the first expression vector encodes a fusion protein comprising an IgGI heavy chain comprising a variable region, an extracellular domain of a TCRy chain, and a first peptide linker between the IgGI heavy chain and the extracellular domain of the TCRy chain, and wherein the second expression vector encodes a fusion protein comprising an IgK light chain, an extracellular domain of a TCR6 chain, and a second peptide linker S' 10 between the IgK light chain and the extracellular domain of the TCR5 chain.

68. The host cell of claim 67 wherein the first peptide linker is GLY-GLY-GLY-THR- SER-GLY (SEQ ID NO:10) and wherein the second peptide linker is GLY-SER-LEU- o SGLY-GLY-SER (SEQ ID NO: 11).

69. A method of making an expression vector encoding a fusion protein, comprising the steps of: inserting a first coding sequence for an extracellular domain of a transmembrane polypeptide into an expression vector; and inserting a second coding sequence for an immunoglobulin heavy chain comprising a variable region into the expression vector, wherein the first coding sequence is inserted 3' of the second coding sequence, such that the expression vector encodes a fusion protein comprising the immunoglobulin heavy chain and the extracellular domain of the transmembrane polypeptide. The method of claim 69 further comprising the step of expressing the fusion protein in a host cell.

71. The method of claim 69 wherein the immunoglobulin heavy chain is an IgG chain.

72. The method of claim 69 wherein the transmembrane polypeptide is an MHC class IIP chain.

73. The method of claim 69 wherein the transmembrane polypeptide is a TCRa chain.

74. The method of claim 69 wherein the transmembrane polypeptide is a TCRy chain.

75. The method of claim 71 wherein the transmembrane polypeptide is an MHC class IIp chain. 10 76. The method of claim 71 wherein the transmembrane polypeptide is a TCRa chain.

77. The method of claim 71 wherein the transmembrane polypeptide is a TCRy chain.

78. The method of claim 69 wherein the expression vector further comprises a third coding sequence for a peptide linker, wherein the third coding sequence is inserted between the first and second coding sequences, such that the fusion protein comprises the peptide linker between the immunoglobulin heavy chain and the extracellular domain of the transmembrane polypeptide.

79. The method of claim 78 wherein the linker is GLY-GLY-GLY-THR-SER-GLY (SEQ ID A method of making an expression vector encoding a fusion protein, comprising the steps of: inserting a first coding sequence for an IgG1 heavy chain comprising a variable region into an expression vector; inserting a second coding sequence for a peptide linker into the expression vector, wherein the second coding sequence is inserted 3' of the first coding sequence; and inserting a third coding sequence for an extracellular domain of an MHC class IIP chain into the expression vector, wherein the third coding sequence is inserted 3' of the second coding sequence, such that the expression vector encodes a fusion protein comprising the IgG1 heavy chain, the peptide linker, and the extracellular domain of the MHC class 11 chain, wherein the peptide linker is between the IgG1 heavy chain and the MHC class II1 chain.

81. The method of claim 80 wherein the peptide linker is GLY-GLY-GLY-THR-SER- GLY (SEQ ID

82. A method of making an expression vector encoding a fusion protein, comprising the steps of: inserting a first coding sequence an IgGI heavy chain comprising a variable region into an expression vector; inserting a second coding sequence for a peptide linker into the expression vector, wherein the second coding sequence is inserted 3' of the first coding sequence; and inserting a third coding sequence for an extracellular domain of a TCRa chain into the expression vector, wherein the third coding sequence is inserted 3' of the second coding sequence, such that the expression vector encodes a fusion protein comprising the IgG1 heavy chain, the peptide linker, and the extracellular domain of the TCRa chain, wherein the peptide linker is between the IgGI heavy chain and the TCRa chain.

83. The method of claim 82 wherein the peptide linker is GLY-GLY-GLY-THR-SER- GLY (SEQ ID

84. A method of making an expression vector encoding a fusion protein, comprising the steps of: inserting a first coding sequence an IgGI heavy chain comprising a variable region into an expression vector; inserting a second coding sequence for a peptide linker into the expression vector, wherein the second coding sequence is inserted 3' of the first coding sequence; and inserting a third coding sequence for an extracellular domain of a TCRy chain into the expression vector, wherein the third coding sequence is inserted 3' of the 10 second coding sequence, such that the expression vector encodes a fusion protein comprising the IgG1 heavy chain, the peptide linker, and the extracellular domain of the TCRy chain, wherein the peptide linker is between the IgGI heavy chain and the TCRy chain. The method of claim 84 wherein the peptide linker is GLY-GLY-GLY-THR-SER- GLY (SEQ ID

86. The method of claim 80 further comprising the step of expressing the fusion protein in a host cell.

87. The method of either of claims 82 or 84 further comprising the step of expressing the fusion protein in a host cell.

88. A method of making an expression vector encoding a fusion protein, comprising the steps of: inserting a first coding sequence for an extracellular domain of a transmembrane polypeptide into an expression vector; and inserting a second coding sequence for an immunoglobulin light chain comprising a variable region into the expression vector, wherein the first coding sequence is inserted 3' of the second coding sequence, such that the expression vector encodes a fusion protein comprising the immunoglobulin light chain and the extracellular domain of the transmembrane polypeptide.

89. The method of claim 88 further comprising the step of expressing the fusion protein in a host cell.

90. The method of claim 88 wherein the immunoglobulin light chain is an IgK chain. 10 91. The method of claim 88 wherein the transmembrane polypeptide is an MHC class IIH chain.

92. The method of claim 88 wherein the transmembrane polypeptide is a TCRP chain.

93. The method of claim 88 wherein the transmembrane polypeptide is a TCR5 chain.

94. The method of claim 90 wherein the transmembrane polypeptide is an MHC class 15 IIa chain. The method of claim 90 wherein the transmembrane polypeptide is a TCR chain. The method of claim 90 wherein the transmembrane polypeptide is a TCR3 chain.

96. The method of claim 90 wherein the transmembrane polypeptide is a TCR6 chain.

97. The method of claim 88 wherein the expression vector further comprises a third coding sequence for a peptide linker, wherein the third coding sequence is inserted between the first and second coding sequences, such that the fusion protein comprises the peptide linker between the immunoglobulin light chain and the extracellular domain of the transmembrane polypeptide.

98. The method of claim 97 wherein the linker is GLY-SER-LEU-GLY-GLY-SER (SEQ ID NO: 1).

99. A method of making an expression vector encoding a fusion protein, comprising the steps of: inserting a first coding sequence for an IgK light chain comprising a variable region into an expression vector; inserting a second coding sequence for a peptide linker into the expression vector, wherein the second coding sequence is inserted 3' of the first coding sequence; and inserting a third coding sequence for an extracellular domain of an MHC 10 class IIa chain into the expression vector, wherein the third coding sequence is inserted 3' of the second coding sequence, such that the expression vector encodes a fusion protein comprising the IgK light chain, the peptide linker, and the extracellular domain of the MHC class IIa chain, wherein the peptide linker is between the IgK light chain and the extracellular domain of the MHC class IIa chain.

100. The method of claim 99 wherein the linker is GLY-SER-LEU-GLY-GLY-SER (SEQ IDNO:11).

101. The method of claim 100 further comprising the step of expressing the fusion protein in a host cell.

102. A method of making an expression vector encoding a fusion protein, comprising the steps of: inserting a first coding sequence for an IgK light chain comprising a variable region into an expression vector; inserting a second coding sequence for a peptide linker into the expression vector, wherein the second coding sequence is inserted 3' of the first coding sequence; and inserting a third coding sequence for an extracellular domain of a TCRP chain into the expression vector, wherein the third coding sequence is inserted 3' of the second coding sequence, such that the expression vector encodes a fusion protein comprising the IgK light chain, the peptide linker, and the extracellular domain of the TCRp chain, wherein the peptide linker is between the IgK light chain and the extracellular domain of the TCRp chain.

103. The method of claim 102 wherein the linker is GLY-SER-LEU-GLY-GLY-SER (SEQ ID NO:11).

104. The method of claim 102 further comprising the step of expressing the fusion protein in a host cell.

105. A method of making an expression vector encoding a fusion protein, comprising the steps of: 15 inserting a first coding sequence for an IgK light chain comprising a variable region into an expression vector; inserting a second coding sequence for a peptide linker into the expression vector, wherein the second coding sequence is inserted 3' of the first coding sequence; and inserting a third coding sequence for an extracellular domain of a TCR8 chain into the expression vector, wherein the third coding sequence is inserted 3' of the second coding sequence, such that the expression vector encodes a fusion protein comprising the IgK light chain, the peptide linker, and the extracellular domain of the TCR5 chain, wherein the peptide linker is between the IgK light chain and the extracellular domain of the chain.

106. The method of claim 105 wherein the linker is GLY-SER-LEU-GLY-GLY-SER (SEQ ID NO:11).

107. The method of claim 105 further comprising the step of expressing the fusion protein in a host cell.

108. A method of making an expression vector encoding a first and a second fusion protein, comprising the steps of: inserting a first coding sequence for an extracellular domain of a first 10 transmembrane polypeptide into an expression vector; inserting a second coding sequence for an immunoglobulin heavy chain comprising a variable region into the expression vector, wherein the first coding sequence is inserted 3' to the second coding sequence, such that the expression vector encodes a first .fusion protein comprising the immunoglobulin heavy chain and the extracellular domain of the first transmembrane polypeptide; inserting a third coding sequence for an extracellular domain of a second transmembrane polypeptide into the expression vector; and inserting a fourth coding sequence for an immunoglobulin light chain into the expression vector, wherein the third coding sequence is inserted 3' to the fourth coding sequence, such that the expression vector encodes a second fusion protein comprising the immunoglobulin light chain and the extracellular domain of the second transmembrane polypeptide. 63

109. The method of claim 108 further comprising the step of expressing the fusion protein in a host cell.

110. The method of claim 108 wherein the immunoglobulin heavy chain is an IgGI chain.

111. The method of claim 108 wherein the immunoglobulin light chain is an IgK chain.

112. The method of claim 108 wherein the immunoglobulin light chain comprises a variable region.

113. The method of claim 108 wherein the first transmembrane polypeptide is an MHC class IIP chain and the second transmembrane polypeptide is an MHC class IIo chain. S 10 114. The method of claim 108 wherein the first transmembrane polypeptide is a TCRa chain and the second transmembrane polypeptide is a TCRp chain. 0

115. The method of claim 108 wherein the first transmembrane polypeptide is a TCRy chain and the second transmembrane polypeptide is a TCR8 chain.

116. The method of claim 108 wherein the expression vector further comprises a fifth 15 coding sequence for a first peptide linker and a sixth coding sequence for a second peptide linker, wherein the fifth coding sequence is inserted between the first and second coding sequences, such that the first fusion protein comprises the first peptide linker between the immunoglobulin heavy chain and the extracellular domain of the transmembrane polypeptide, and wherein the sixth coding sequence is inserted between the third and fourth coding sequences, such that the second fusion protein comprises the second peptide linker between the immunoglobulin light chain and the extracellular domain of the transmembrane polypeptide.

117. The method of claim 116 wherein the first peptide linker is GLY-GLY-GLY-THR- SER-GLY (SEQ ID NO:10) and wherein the second peptide linker is GLY-SER-LEU- GLY-GLY-SER (SEQ ID NO: 11).

118. The method of claim 116 further comprising the step of expressing the first and second fusion proteins in a host cell. 119 A polynucleotide encoding a first fusion protein, wherein the first fusion protein comprises an immunoglobulin heavy chain comprising a variable region and an extracellular domain of a first transmembrane polypeptide.

120. The polynucleotide of claim 119 wherein the immunoglobulin heavy chain is an 10 IgGI heavy chain.

121. The polynucleotide of claim 119 wherein the first transmembrane polypeptide is an MHC class II chain.

122. The polynucleotide of claim 119 wherein the first transmembrane polypeptide is a TCRao chain.

123. The polynucleotide of claim 119 wherein the first transmembrane polypeptide is a TCRy chain.

124. The polynucleotide of claim 120 wherein the first transmembrane polypeptide is an MHC class IIp chain.

125. The polynucleotide of claim 120 wherein the first transmembrane polypeptide is a TCRa chain.

126. The polynucleotide of claim 120 wherein the first transmembrane polypeptide is a TCRy chain.

127. The polynucleotide of claim 119 wherein the first fusion protein further comprises a first peptide linker between the immunoglobulin heavy chain and the extracellular domain of the first transmembrane polypeptide.

128. The polynucleotide of claim 127 wherein the first peptide linker is GLY-GLY- GLY-THR-SER-GLY (SEQ ID

129. A polynucleotide encoding a fusion protein, wherein the fusion protein comprises an IgG1 heavy chain comprising a variable region, an extracellular domain of an MHC class IIP chain, and a peptide linker between the IgG1 heavy chain and the extracellular domain of the MHC class 11 chain.

130. The polynucleotide of claim 129 wherein the peptide linker is GLY-GLY-GLY- THR-SER-GLY (SEQ ID

131. A polynucleotide encoding a fusion protein, wherein the fusion protein comprises an IgGI heavy chain comprising a variable region, an extracellular domain of a TCRa chain, and a peptide linker between the IgGI heavy chain and the extracellular domain of the TCRa chain.

132. The polynucleotide of claim 131 wherein the peptide linker is GLY-GLY-GLY- THR-SER-GLY (SEQ ID

133. A polynucleotide encoding a fusion protein, wherein the fusion protein comprises an IgG1 heavy chain comprising a variable region, an extracellular domain of a TCRy chain, and a peptide linker between the IgG1 heavy chain and the extracellular domain of the TCRy chain.

134. The polynucleotide of claim 133 wherein the peptide linker is GLY-GLY-GLY- THR-SER-GLY (SEQ ID

135. The polynucleotide of claim 119 further encoding a second fusion protein, wherein the second fusion protein comprises an immunoglobulin light chain and an extracellular domain of a second transmembrane polypeptide.

136. The polynucleotide of claim 135 wherein the immunoglobulin light chain comprises a variable region.

137. The polynucleotide of claim 135 wherein the first transmembrane polypeptide is an S. MHC class IIP chain and the second transmembrane polypeptide is an MHC class IIa chain. S 10 138. The polynucleotide of claim 135 wherein the first transmembrane polypeptide is a TCRa chain and the second transmembrane polypeptide is a TCRP chain.

139. The polynucleotide of claim 135 wherein the first transmembrane polypeptide is a TCRy chain and the second transmembrane polypeptide is a TCR8 chain.

140. The polynucleotide of claim 135 wherein the immunoglobulin heavy chain is an IgGI chain.

141. The polynucleotide of claim 135 wherein the immunoglobulin light chain is an IgKi chain.

142. The polynucleotide of claim 135 wherein the immunoglobulin light chain comprises a variable region.

143. The polynucleotide of claim 135 wherein the first fusion protein comprises a first peptide linker between the immunoglobulin heavy chain and the extracellular domain of the first transmembrane polypeptide and wherein the second fusion protein comprises a 67 second peptide linker between the immunoglobulin light chain and the extracellular domain of the second transmembrane polypeptide.

144. The polynucleotide of claim 143 wherein the first peptide linker is GLY-GLY- GLY-THR-SER-GLY (SEQ ID

145. The polynucleotide of claim 143 wherein the second peptide linker is GLY-SER- LEU-GLY-GLY-SER (SEQ ID NO: 11).

146. The polynucleotide of claim 143 wherein the first peptide linker is GLY-GLY- GLY-THR-SER-GLY (SEQ ID NO:10) and wherein the second peptide linker is GLY- SER-LEU-GLY-GLY-SER (SEQ ID NO:11).

147. A polynucleotide encoding a fusion protein, wherein the fusion protein comprises an immunoglobulin light chain comprising a variable region and an extracellular domain of a transmembrane polypeptide.

148. The polynucleotide of claim 147 wherein the immunoglobulin light chain is an IgK chain. 15 149. The polynucleotide of claim 147 wherein the transmembrane polypeptide is an MHC class IIa chain.

150. The polynucleotide of claim 147 wherein the transmembrane polypeptide is a TCRP chain.

151. The polynucleotide of claim 147 wherein the transmembrane polypeptide is a TCR5 chain.

152. The polynucleotide of claim 148 wherein the transmembrane polypeptide is an MHC class IIa chain. 68

153. The polynucleotide of claim 148 wherein the transmembrane polypeptide is a TCRp chain.

154. The polynucleotide of claim 148 wherein the transmembrane polypeptide is a TCR8 chain.

155. The polynucleotide of claim 147 wherein the fusion protein comprises a peptide linker between the immunoglobulin light chain and the transmembrane polypeptide.

156. The polynucleotide of claim 155 wherein the peptide linker is GLY-SER-LEU- GLY-GLY-SER (SEQ ID NO:11).

157. A polynucleotide encoding a fusion protein comprising an IgK light chain 10 comprising a variable region, an extracellular domain of an MHC class IIa chain, and a peptide linker between the IgK light chain and the extracellular domain of the MHC class IIa chain.

158. The polynucleotide of claim 157 wherein the peptide linker is GLY-SER-LEU- GLY-GLY-SER (SEQ ID NO: 11). 15 159. A polynucleotide encoding a fusion protein comprising an IgK light chain comprising a variable region, an extracellular domain of a TCRP chain, and a peptide linker between the IgrK light chain and the extracellular domain of the TCR3 chain.

160. The polynucleotide of claim 159 wherein the peptide linker is GLY-SER-LEU- GLY-GLY-SER (SEQ ID NO:11).

161. A polynucleotide encoding a fusion protein comprising an IgK light chain comprising a variable region, an extracellular domain of a TCR8 chain, and a peptide linker between the IgK light chain and the extracellular domain of the TCR5 chain. 69

162. The polynucleotide of claim 161 wherein the peptide linker is GLY-SER-LEU- GLY-GLY-SER (SEQ ID NO:11).

163. A polynucleotide encoding a first and a second fusion protein, wherein the first fusion protein comprises an IgG1 heavy chain comprising a variable region, an extracellular domain of an MHC class IIP chain, and a first peptide linker between the IgGI heavy chain and the extracellular domain of the MHC class IIP chain, and wherein the second fusion protein comprises an IgK light chain comprising a variable region, (e) an extracellular domain of an MHC class IIa chain, and a second peptide linker between the IgK chain and the extracellular domain of the MHC class IIa chain. S 10 164. The polynucleotide of claim 163 wherein the first peptide linker is GLY-GLY- GLY-THR-SER-GLY (SEQ ID NO:10) and wherein the second peptide linker is GLY- SER-LEU-GLY-GLY-SER (SEQ ID NO: 11).

165. A polynucleotide encoding a first and a second fusion protein, wherein the first fusion protein comprises an IgGI heavy chain comprising a variable region, an extracellular domain of a TCRa chain, and a first peptide linker between the IgG1 heavy chain and the extracellular domain of the TCRa chain, and wherein the second fusion protein comprises an IgK light chain comprising a variable region, an extracellular domain of a TCRP chain, and a second peptide linker between the IgK chain and the extracellular domain of the TCR3 chain.

166. The polynucleotide of claim 165 wherein the first peptide linker is GLY-GLY- GLY-THR-SER-GLY (SEQ ID NO:10) and wherein the second peptide linker is GLY- SER-LEU-GLY-GLY-SER (SEQ ID NO: 11).

167. A polynucleotide encoding a first and a second fusion protein, wherein the first fusion protein comprises an IgG1 heavy chain comprising a variable region, an extracellular domain of a TCRy chain, and a first peptide linker between the IgG1 heavy chain and the extracellular domain of the TCRy chain, and wherein the second fusion protein comprises an IgK light chain comprising a variable region, an extracellular domain of a TCR8 chain, and a second peptide linker between the IgK chain and the extracellular domain of the TCR5 chain.

168. The polynucleotide of claim 167 wherein the first peptide linker is GLY-GLY- GLY-THR-SER-GLY (SEQ ID NO:10) and wherein the second peptide linker is GLY- 10 SER-LEU-GLY-GLY-SER (SEQ ID NO: 11).

169. A method of making a first molecular complex comprising four fusion proteins, S* comprising the steps of: a expressing in a host cell a first fusion protein comprising an immunoglobulin heavy chain comprising a variable region and an extracellular domain of a first transmembrane polypeptide; and expressing in a host cell a second fusion protein comprising an immunoglobulin light chain and an extracellular domain of a second transmembrane polypeptide, whereby two first and two second fusion proteins associate to form a first molecular complex comprising at least four fusion proteins, wherein the first molecular complex comprises two ligand binding sites, each ligand binding site formed by the extracellular domains of the first and second transmembrane polypeptides, wherein the first molecular complex has an affinity for a cognate ligand which is increased relative to a second molecular complex consisting of the extracellular domain of the first transmembrane polypeptide and the extracellular domain of the second transmembrane polypeptide.

170. The method of claim 169 wherein the first and second fusion proteins are expressed from the same expression vector.

171. The method of claim 169 wherein the first and second fusion proteins are expressed from two different expression vectors.

172. The method of claim 169 wherein the first transmembrane polypeptide is an MHC class II3 chain and the second transmembrane polypeptide is an MHC class IIt chain.

173. The method of claim 169 wherein the first transmembrane polypeptide is a TCR3 chain and the second transmembrane polypeptide is a TCR3 chain.

174. The method of claim 169 wherein the first transmembrane polypeptide is a TCR8 chain and the second transmembrane polypeptide is a TCRp chain.

175. The method of claim 169 wherein the first fusion protein comprises a first peptide linker between the immunoglobulin heavy chain and the extracellular domain of the first S 15 transmembrane polypeptide and wherein the second fusion protein comprises a second peptide linker between the immunoglobulin light chain and the extracellular domain of the second transmembrane polypeptide.

176. The method of claim 175 wherein the first peptide linker is GLY-GLY-GLY-THR- SER-GLY (SEQ ID

177. The method of claim 175 wherein the second peptide linker is GLY-SER-LEU- GLY-GLY-SER (SEQ ID NO: 11).

178. The method of claim 179 wherein the immunoglobulin heavy chain is an IgGI heavy chain.

179. The method of claim 169 wherein the immunoglobulin light chain is an IgK light chain.

180. The method of claim 169 wherein the immunoglobulin light chain comprises a variable region.

181. A method of making a first molecular complex, comprising the steps of: expressing in a host cell a first fusion protein comprising an IgGI heavy chain comprising a variable region, an extracellular domain of an MHC class IIP chain, 0: and a peptide linker between the IgG1 heavy chain and the extracellular domain of the MHC class IIP chain; and expressing in a host cell a second fusion protein comprising an Igi light chain comprising a variable region, an extracellular domain of an MHC class IIa chain, and a peptide linker between the IgK light chain and the extracellular domain of the MHC class IIa chain, 15 whereby two first and two second fusion proteins associate to form a molecular complex comprising at least four fusion proteins, wherein the first molecular complex comprises two ligand binding sites, each ligand binding site formed by the extracellular domains of the first and second transmembrane polypeptides, wherein the first molecular complex has an affinity for a cognate ligand which is increased relative to a second molecular complex consisting of the extracellular domain of the first transmembrane polypeptide and the extracellular domain of the second transmembrane polypeptide.

182. The method of claim 181 wherein the first and second fusion proteins are expressed from the same expression vector.

183. The method of claim 181 wherein the first and second fusion proteins are expressed from two different expression vectors.

184. The method of claim 181 wherein the first peptide linker is GLY-GLY-GLY-THR- SER-GLY (SEQ ID NO:10) and wherein the second peptide linker is GLY-SER-LEU- GLY-GLY-SER (SEQ ID NO: 11).

185. A method of making a first molecular complex, comprising the steps of: expressing in a host cell a first fusion protein comprising an IgG1 heavy chain comprising a variable region, an extracellular domain of a TCRcc chain, and a peptide linker between the IgG1 heavy chain and the extracellular domain of the TCRoa chain; and expressing in a host cell a second fusion protein comprising an IgK light chain comprising a variable region, an extracellular domain of a TCR3 chain, and a o peptide linker between the IgK light chain and the extracellular domain of the TCR3 chain, 15 whereby two first and two second fusion proteins associate to form a molecular complex comprising at least four fusion proteins, wherein the first molecular complex comprises two ligand binding sites, each ligand binding site formed by the extracellular domains of the first and second transmembrane polypeptides, wherein the first molecular complex has an affinity for a cognate ligand which is increased relative to a second molecular complex consisting of the extracellular domain of the first transmembrane polypeptide and the extracellular domain of the second transmembrane polypeptide.

186. The method of claim 185 wherein the first and second fusion proteins are expressed from the same expression vector.

187. The method of claim 185 wherein the first and second fusion proteins are expressed from two different expression vectors.

188. The method of claim 185 wherein the first peptide linker is GLY-GLY-GLY-THR- SER-GLY (SEQ ID NO:10) and wherein the second peptide linker is GLY-SER-LEU- GLY-GLY-SER (SEQ ID NO: 11).

189. A method of making a first molecular complex, comprising the steps of: expressing in a host cell a first fusion protein comprising an IgG1 heavy chain comprising a variable region, an extracellular domain of a TCRy chain, and a peptide linker between the IgG1 heavy chain and the extracellular domain of the TCRy 10 chain; and expressing in a host cell a second fusion protein comprising an IgK light S chain comprising a variable region, an extracellular domain of a TCR8 chain, and a peptide linker between the IgK light chain and the extracellular domain of the TCR5 chain, whereby two first and two second fusion proteins associate to form a molecular complex 15 comprising at least four fusion proteins, wherein the first molecular complex comprises OS *S two ligand binding sites, each ligand binding site formed by the extracellular domains of the first and second transmembrane polypeptides, wherein the first molecular complex has an affinity for a cognate ligand which is increased relative to a second molecular complex consisting of the extracellular domain of the first transmembrane polypeptide and the extracellular domain of the second transmembrane polypeptide.

190. The method of claim 189 wherein the first and second fusion proteins are expressed from the same expression vector.

191. The method of claim 189 wherein the first and second fusion proteins are expressed from two different expression vectors.

192. The method of claim 189 wherein the first peptide linker is GLY-GLY-GLY-THR- SER-GLY (SEQ ID NO:10) and wherein the second peptide linker is GLY-SER-LEU- GLY-GLY-SER (SEQ ID NO:11).

193. A polynucleotide which encodes: a first fusion protein comprising an immunoglobulin heavy chain, wherein the immunoglobulin heavy chain comprises a variable region, and (ii) an extracellular domain of a first transmembrane polypeptide of a heterodimeric protein, wherein the immunoglobulin heavy chain is C-terminal to the extracellular domain of the first transmembrane polypeptide; and a second fusion protein comprising an immunoglobulin light chain and (ii) an extracellular domain of a second transmembrane polypeptide of the heterodimeric protein, wherein the immunoglobulin light chain is C-terminal to the extracellular portion of the second transmembrane polypeptide, 15 wherein the extracellular domain of the first transmembrane polypeptide and the extracellular domain of the second transmembrane polypeptide form a ligand binding site for a cognate ligand.

194. The polynucleotide of claim 193 wherein the polynucleotide further comprises a baculovirus expression vector.

195. The polynucleotide of claim 193 wherein the first transmembrane polypeptide is an MHC class 11 chain and wherein the second transmembrane polypeptide is an MHC class IIa chain.

196. The polynucleotide of claim 193 wherein the first transmembrane polypeptide is a TCRa chain and wherein the second transmembrane polypeptide is a TCRP chain.

197. The polynucleotide of claim 193 wherein the first transmembrane polypeptide is a TCRy chain and wherein the second transmembrane polypeptide is a TCR5 chain.

198. The polynucleotide of claim 193 wherein the immunoglobulin heavy chain is an IgG1 heavy chain.

199. The polynucleotide of claim 193 wherein the immunoglobulin light chain is an IgK light chain.

200. The polynucleotide of claim 193 wherein the first fusion protein comprises a first peptide linker between the immunoglobulin heavy chain and the extracellular domain of 10 the first transmembrane polypeptide and wherein the second fusion protein comprises a second peptide linker between the-immunoglobulin light chain and the extracellular domain of the second transmembrane polypeptide.

201. The polynucleotide of claim 200 wherein the first peptide linker is GLY-GLY- .:*GLY-THR-SER-GLY (SEQ ID

202. The polynucleotide of claim 200 herein the second peptide linker is GLY-SER- LEU-GLY-GLY-SER (SEQ ID NO:11).

203. A host cell comprising at least one polynucleotide encoding: a first fusion protein comprising an immunoglobulin heavy chain, wherein the immunoglobulin heavy chain comprises a variable region, and (ii) an extracellular domain of a first transmembrane polypeptide of a heterodimeric protein, wherein the immunoglobulin heavy chain is C-terminal to the extracellular domain of the first transmembrane polypeptide; and a second fusion protein comprising an immunoglobulin light chain and (ii) an extracellular domain of a second transmembrane polypeptide of the heterodimeric protein, wherein the immunoglobulin light chain is C-terminal to the extracellular portion of the second transmembrane polypeptide, wherein the extracellular domain of the first transmembrane polypeptide and the extracellular domain of the second transmembrane polypeptide form a ligand binding site.

204. The host cell of claim 203 wherein a first polynucleotide encodes the first fusion protein and wherein a second polynucleotide encodes the second fusion protein.

205. The host cell of claim 203, wherein the first and second fusion proteins form a first 10 molecular complex which has an affinity for a cognate ligand which is increased compared to a second molecular complex consisting of the extracellular domain of the first transmembrane polypeptide and the extracellular domain of the second transmembrane polypeptide. The Johns Hopkins University 30 April 2001 15 The Johns Hopkins University 30 April 2001 C