WO2001092295A2 - Ligands for cd21 and compositions thereof for modulating immune responses - Google Patents

Abstract

Ligands for the CD21 molecule corresponding to the CD21 contacting amino acid residies 36-39 and 160-167 of the C3d molecule are provided. These ligands have the ability to bind CD21 and stimulate the CD21/CD19 complex and may be polypeptides, peptides or mimetics. The ligands coupled to antigens modulate the immune response of hosts immunized therewith. Site-specific mutated analogs of C3d are provided that have enhanced binding to CD21.

Description

TITLE OF THE INVENTION

LIGANDS FOR CD21 AND COMPOSITIONS THEREOF FOR MODULATING IMMUNE RESPONSES

FIELD OF THE INVENTION The present invention relates to the field of immunology and is particularly concerned with ligands for the CD21 molecule and compositions comprising such ligands and antigens for modulation of immune responses.

BACKGROUND OF THE INVENTION There are many reasons why the immune system does not mount a substantial immune response when an antigen is encountered for the first time. Thus, the threshold for activating unprimed T cells is higher and B cells express unmutated low affinity receptors. Other portions of the immune system capable of recognizing infectious agents in the preimmune host have evolved to augment the primary immune response. Complement is a component of normal plasma that augments opsonization of bacteria by antibodies and allows some antibodies to kill bacteria. The complement system involves a large number of different plasma proteins. One of these is directly activated by bound antibody to trigger a cascade of reactions each of which results in the activation of the next complement component. Some activated complement proteins bind covalently to the bacteria opsonizing them for engulfment by phagocytes bearing complement receptors while small fragments of complement proteins recruit phagocytes to the site of complement activation. In addition, the terminal complement components damage bacteria by generating pores in bacterial membranes. C3 is the most important protein in the complement system. Its primary activation product C3b binds to target immune complexes or foreign substances and targets them for destruction or clearance. The complement system generates C3b either by the "classical pathway" or the "alternative pathway". C3b is generated from its precursor C3 by the proteolytic enzymes termed "C3 convertase". A summary of the complement system is shown in Figure 1. The generation of the C3 convertase results in the rapid cleavage of many molecules of C3 to produce C3b, which bind covalently to antigen surfaces. This cleavage of C3 and the binding of large numbers of C3b molecules to the surface of the pathogen is the pivotal event in complement activation. The bound C3b is the major opsonin of the complement system, it binds to complement receptors on phagocytes and facilitates engulfment of the pathogen. The covalent incorporation of a C3b subunit into either the classical or alternative pathway C3 convertase enables the enzyme to cleave C5 by facilitating the binding of C5 to the complex. Cleavage of C5 initiates assembly of the membrane attack complex. C5a and C3a mediate local inflammatory responses recruiting fluid, cells and proteins to the site of infection. Binding of C3b also initiates the alternative pathway thereby amplifying complement activation.

Thus, the most important action of complement is to facilitate the uptake and destruction of pathogens by phagocytic cells. This occurs by specific recognition of bound complement components by complement receptors (CR) on phagocytes. CR1 is a receptor for C3b and is expressed on both macrophages and polymorphonuclear leukocytes.

More recently, there has been an accumulating body of evidence indicating that C3 also participates in the regulation of the adaptive immune response (References 1 and 2 - Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are nearby incorporated by reference into the present disclosure).

The majority of soluble antigens do not contain multiple identical epitopes and cannot activate B-cells by cross-linking their receptors. This problem is of particular relevance in the development of defined subunit vaccines. The B-cell response to most antigens involves interactions with T-cells, which recognize antigen presented on the surface of B-cells and deliver signals which activate the B-cell. Following activation, the amplification of the antibody response involves further cell-to-cell interaction that occurs in the lymphoid follicles and involves follicular dendritic cells resulting in cross-linking of receptor molecules.

However, even activated B-cells whose surface immunoglobulin molecules are cross-linked make very limited amounts of antibody in the absence of other signals. Follicular dendritic cells are believed to increase the sensitivity of B-cells to antigen through a molecule CD23. This molecule is expressed on the follicular dendritic cells surface and binds to a B-cell surface complex known as a co-receptor. The B-cell co-receptor is a complex of three proteins. The first of these is the receptor CD21 (CR2) that binds activated complement components and also binds the CD23 molecule. The second component, TAPA-1, is a serine protease protein that spans the membrane four times and the third component is CD 19. The co-receptor is triggered by the binding of ligand which ultimately generates intracellular signals that augment the signal transduced via the B-cell receptor itself, (Fig. 1).

C3 contains a thioester that mediates attachment to other biological molecules. Activation of C3 to the CRl ligand C3b by the "C3 convertase" of the classical or alternative pathway makes the thioester accessible to attack by weak nucleophiles such as the -OH of carbohydrates. The C3b glycoconjugate interacts with CRl on B cells, macrophages, and follicular dendritic cells (FDCs). CRl is a co-factor for plasma protease, factor I, that processes C3b to iC3b and C3dg. It is these proteolytic fragments of C3d that then bind CD21 and mediate the immune enhancing effect of C3.

Thus, the regulatory action is mediated by the interaction of C3dg, an activation product of C3, with its receptor CD21 , the latter being primarily present on the membranes of B cells and follicular cells. Several in vivo studies have pointed out the importance of this interaction in the modulation of the antibody- mediated humoral response. For example, the administration to mice of a blocking anti-mouse CD21 mAb (Ref 3), or of a soluble form of CD21 (Ref. 4), at the time of the primary immunization with sub-optimal doses of T-dependent antigens diminished the primary response and abolished the secondary response and isotype switching. Knockout mice generated by disruption of the Cr2 gene displayed an impaired primary response, virtually no secondary response and greatly reduced number and size of germinal centers upon immunization with T- dependent antigen (Ref 5, 6). Since CRl and CD21 are differential splice products of the Cr2 gene in the mouse (Ref 7), the phenotype of the knockout reflects losing both types of receptors. Nevertheless, in view of the similarity of the immune response profile of the CR1/CR2 knockout mice to the mice in which binding of ligand to CD21 was blocked by a CD21 -specific mAb, or by a competing soluble form of CD21, it suggests that minimally interaction between iC3b/C3dg-coated antigen and CD21 is required for a normal immune response. The phenotype of the Cr2 gene knockout is in fact very similar to that observed in C3- and C4-deficient mice (Ref 8) and to that of CD19-deficient mice (Ref 9, 10). The mechanism through which CD21 is thought to participate in a B cell signaling event involves its association with a pre-existing signal transduction complex, namely the CD19/CR2/TAPA-1 complex (Ref 11). CD19 is considered to be the key molecule in the signaling process, but its ligand has not been identified. Thus, a model has been proposed that bridges the B cell receptor (BCR) to CD 19 via the dual recognition of a C3dg-coated antigen by CD21 and by the antibody component of the BCR complex (Ref 2). In support of this model, it has been found that there was about a 10-fold decrease in the threshold concentration of anti-BCR required for B cell activation in vitro when antibodies that simultaneously cross-linked the CD19/CD21 complex were also present (Ref 12).

Thus the covalent attachment of activated complement C3 (C3d) to antigen links the innate and adaptive arms of the immune system by targeting antigen to follicular dendritic cells and B-cells via specific receptor CD21 and CD35. The enhancing role for complement by either co-ligating CD21/CD19 co- receptor and BCR or trapping antigen on FDC, requires the CD21 ligand C3d to become attached to antigen, hi International Patent Application WO 96/17625, published June 13, 1996 by Fearon et al, and in Reference 13 there is described the enhancement of humoral responses by coupling C3d to the C-terminus of lysozyme. The modulation of the immune response described is an increase or a decrease in the level of antibody response to immunogen administration. Thus, the immune response is enhanced by coupling a plurality of C3d molecules to the immunogen and reduced by coupling only one C3d molecule to the immunogen. In this application the preferred ligand is described as C3d, however derivatives or fragments thereof are also proposed such as a portion of C3d that retains the ability to bind CD21 and stimulate B-cells through the CD21/CD19 complex. 01 00785

CD21 is a member of the regulators of complement activation (RCA) family (Ref 14) and depending on splice site utilization, its extracellular region consists of 15 or 16 short consensus repeat (SCR) domains. The binding sites for its ligands, C3dg and iC3b, have been localized to SCR domains 1 and 2 (Ref 15) and peptide segments 10-LNGRIS-15 of SCR-1, 84-GSTPYRHGDSVTFA-97 of SCR-2 (Ref 16), and 63-EYFNKYS-69, located in between SCRs 1 and 2 (Ref 17) have been suggested to be important for iC3b/C3dg binding. The three- dimensional structure of CR2 has not been determined but a model of CR2 SCR-1 and SCR-2 has been produced based on the NMR structure of SCRs 15-16 of factor H (Ref 18). In this model the peptide segments considered important for iC3b/C3dg binding are largely surface exposed and with some rotation about the interdomain segment, the 10-15 and 84-97 peptide segments can be configured to form a contiguous patch (Ref 16). An electrostatic surface potential rendition of the modeled CD21 domains indicated that the basic residues within these latter two segments were part of two prominent electropositive patches (Ref 19).

The identification of a site (s) within C3d that mediates the interaction with CD21 has been the subject of some controversy, as has been the issue of whether there are supplementary sites in iC3b in addition to the site (s) in C3d. In 1985, Lambris et al, based largely on synthetic peptide mimetic studies (Ref 20) proposed that a C3d segment having the sequence 1199-EDPGKQLYNVEA-1210 (mature C3 numbering) played a major role in the interaction with CD21. Further studies from this same group suggested that in addition to the C3d contacts, there were additional contacts for CD21 in the C3c portion of iC3b (Ref 21). However, extensive mutagenesis of the 1199-1210 segment of human C3 led to minimal effects on the binding of either iC3b or C3dg to CD21, suggesting no more than a minor role for this segment of C3d in mediating binding to CD21 (Ref 22). Furthermore, there is little difference between the respective CD21 binding activities of iC3b and C3dg.

Due to the importance of CD21 (CR2) when associated with CD19 in the development and maintenance of immune responses, including the generation of memory B lymphocytes, the identification of ligands for the CD21 molecule (other than C3d) is an important problem unsolved in the art. Furthermore, 0785

because of the previous proposed CD21 -binding site on C3 by Lambris et al (1985) (Ref 20), the need to search for such ligands had not even been previously appreciated.

By site-directed mutagenesis of C3d, amino acid residues crucial for the interaction with CD21 were identified and ligands for the CD21 molecule provided. These ligands have particular utility in compositions comprising at least one of the ligands and at least one antigen attached thereto for modulating the immune response of a host immunized with the composition.

SUMMARY OF THE INVENTION The present invention is directed towards the provision of ligands for the

CD21 molecule and their use in immunogenic compositions.

In accordance with one aspect of the invention there is provided a ligand for the CD21 molecule corresponding to the CD21 contacting amino acid residues 36-39 and 160-167 of the C3d molecule (the C3d numbering system as per Ref 19), the molecule having the ability to bind CD21 and stimulate B-cells through the CD21/CD19 complex wherein the molecule is not C3d. In one embodiment of the present invention the ligand may be a fragment of C3d and it may be a peptide or it may be a mimetic. In a further embodiment of the present invention there is provided a composition comprising at least one ligand as provided herein and at least one antigen attached thereto. The antigen may be a protein, a polypeptide or a peptide, and the at least one antigen and the at least one ligand may comprise a fusion protein. In particular embodiments, the at least one antigen may be an antigen derived from or corresponding to a pathogen or it may be at least part of an antibody molecule such as a variable region of an antibody. In a further aspect of the invention there is provided a ligand for the CD21 molecule that has an enhanced binding activity to CD21 compared to the corresponding ligand of the wild-type C3d molecule. The ligand with enhanced binding activity may be a fragment of C3d, an anolog thereof, a peptide, or a mimetic. In a further aspect of the invention there is provided an analog of C3d that has an enhanced binding activity to CD21 compared to a wild-type C3d molecule. 01 00785

The analog having the enhanced binding activity may have at least one amino acid in the wild-type C3d molecule deleted or replaced. In a particular embodiment the at least one amino acid may comprise amino acid lysine at position 162 and may be replaced by alanine. The ligands provided herein and the compositions comprising at least one antigen and at least one ligand are useful for the modulation of the immune response and are useful as antigens in immunogenic compositions, therapeutics diagnostic reagents and in the generation of diagnostic agents. The compositions may be provided as immunogenic compositions and may be formulated as vaccines for in vivo administration to a host, including a human, to confer protection against disease caused by a pathogen from which, for example, the antigen has been derived. The compositions can also be used as cancer therapeutics.

The invention also extends to nucleic acid molecules enconding the ligands and compositions as provided herein, expression vectors comprising such nucleic acid molecules, futher comprising regulatory sequences for expression and host cells containing the nucleic acid molecules and expression vectors as provided herein. The invention further provides methods for making the ligands and compositions as provided herein comprising culturing host cells containing the nucleic acid molecules and expression vectors and isolating the compositions. The invention also extends to a method of altering the immunogenicity of an antigen comprising coupling the antigen with the ligands of the present invention.

The invention also extends to methods of generating immune responses in a host including a human comprising administering thereto an immuno-effective amount of the immunogenic compositions provided herein.

The invention also includes the use of ligands and compositions as described herein in the manufacture of a medicament for modulating the immune response of a host. The present invention will be further understood from the following description with reference to the drawings, in which: BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic of the main components and effector actions of complement generated by the classical and alternative pathways and the interaction of C3d-antigen complexes with CD21 (CR2) of the B-cell co-receptor complex;

Figure 2 is a graphical representation showing the ability of purified iC3b and C3dg (and C4 as a negative control) to inhibit rosette formation between iC3b-coated red cells and CR2-bearing Raji cells;

Figure 3 illustrates a space filling model of the concave surface of C3d showing the respective locations of the seventeen amino acids mutated;

Figure 4 is an SDS-PAGE and autofluorograph demonstrating the mutant C3b molecules deposited on the C3 convertase-bearing red cells were convertible to iC3b by factors H and I;

Figure 5 is a graphical representation of the rosette formation between CR2-bearing Raji cells and sheep red cells bearing various amounts of recombinant iC3b;

Figure 6 is a bar graph showing a summary of the CR2 binding activities of all of the mutants engineered for this study as determined from the CR2- dependent rosette assay; Figure 7 is a graphical representation of the flow cytometric measurement of the binding of soluble CR2 to sheep red cells bearing various amounts of recombinant iC3b;

Figure 8 is a graphical representation showing the ability of purified C3d peptides to inhibit rosette formation between iC3b-coated red cells and CD21- bearing Raji cells;

Figure 9 is a graphical representation showing the ability of C3d cyclic peptides (Segment 33-40) to inhibit rosette formation; and

Figure 10 is a graphical representation showing the ability of C3D cyclic peptides (Segment 160-168) to inhibit rosette formation. DETAILED DESCRIPTION OF THE INVENTION

The involvement of complement in adaptive humoral immunity is dependant upon the interaction between B cell and FDC-resident complement 0785

receptor 2 (CD21 or CR2) and its antigen-associated ligands iC3b and C3dg. The location of a CD21 (CR2) binding site within C3d has not been firmly established in the art. The present invention provides an identification of this binding site novel ligands for the CD21 molecule are also provided. The structure of human C3d has been published (Ref 19) and reveals the molecule to be a highly helical alpha-alpha barrel presenting two distinct surfaces, a convex surface bearing the thioester residues and, at the opposite end a concave surface. Furthermore, the rim of the cavity on the concave surface is surrounded by acidic residues that form a V-shaped entity providing^" a surface with a prominent negatively-charged electrostatic potential.

In the present invention, the involvement of the acidic pocket of C3d in mediating interaction with CD21 was dissected by alanine scanning mutugenesis of charged and non-charged residues of the acidic pocket. The analogs were then assessed for their ability to bind to CD21. This study identified two clusters of C3d residues that are involved in interaction with CD21. Based on this information, novel ligands for CD21 were prepared.

The mutation of C3d residues that have surface exposed side chains lying within or just adjacent to the boundaries of the electrostatic surface potential- defined negatively charged pocket was performed within the context of the intact human C3 molecule. Following transient expression in COS-1 cells of the C3 analog cDNAs, the culture supernatants served as a source of recombinant analog C3. The analogs were then deposited onto C3 convertase-bearring sheep red cells (EAC42). The deposited C3b molecules were then converted to iC3b by treatment of the cells with excess H and I and these EAC423bi cells were then used in rosetting assays with Riji cells. These cells bear CD21 on their surfaces but have no other complement receptors.

The location of the amino acid residues in C3d selected for mutation are shown in Figure 3 and the 28 C3b analogs that where constructed are shown in Table 1. The numbering system used refers to the C3d residue number since this allows reference to the C3d structure, hi addition to the 17 single residue mutants constructed, acidic residues within each of the 36-39 and 160-167 segments were mutated in several combinations. In two cases (analogs D36N/E37Q/E39Q and T A01/00785

10

D163N) the effects of isosteric amide substitutions were also examined and Arg49 was replaced by both Ala and the more isosteric residue Met. With the exception of the R49A analog, the expression levels of all of the C3 analogs fell within the range (250-450 ng/ml) observed for wild-type recombinant C3 in the numerous transfections carried out. The most isosteric R49M analog was expressed at normal levels and could be evaluated for its role in mediating CD21 binding. The isosteric amide substitution analog D163N was secreted at wild-type levels but was only poorly deposited on the C3 convertase-bearing red cells. In contrast the D163A analog was normal in all respects. Since it is known that there is a binding site for factor H within C3d (Ref

23, 24) and further that red cells on which the deposited C3b was not converted to iC3b would show very much impaired ability to form CD21 -dependent rosettes (Ref 22), it was important to establish that none of the mutations that we introduced prevented the factor H and I mediated conversion of the cell-associated C3b to iC3b. To examine this issue, culture supernatants from transfected COS-1 cells that had been metabolically labeled with ³⁵S-Met/Cys were used to build the red cells bearing the various C3b analog molecules. These cells were then treated with factors H and I to convert the deposited C3b to iC3b. Following hypotonic lysis and recovery of the membrane ghosts bearing ester-linked adducts of S- labeled iC3b (or C3b if the conversion did not work), hydroxylamine was added to break the ester linkages and the chain structure of the released S-labeled protein was assessed by SDS-PAGE autofluorography. The results of this work are shown in Figure 4. As a reference point, the hydroxylamine-released chains of the wild-type protein are shown both before and after treatment with factors H and I (right side of upper panel). Whereas the former shows predominantly bands migrating at the positions of α'-chain and β-chain, the latter shows the chains characteristic of iC3b, namely β, α'-67, and a doublet of α'-43/α'-40, where the doublet represents the respective products of the first and second factor I mediated cleavages. It has long been assumed, and now has been directly demonstrated, that it is the first factor I mediated cleavage which correlates with the functional status of the molecule (Ref 25). It can further be seen in Figure 4 that all of the analogs examined displayed wild-type-like behavior with respect to extent of deposition and conversion to their respective iC3b forms.

Rosetting of Wild-Type and Mutant iCSh-coated Red Cells to CR2-bearing Raji Cells Aliquots of C3 convertase-bearing red cells were treated with various amounts of recombinant C3-containing supernatants in order to create sets of cells with various amounts of iC3b on their surfaces. Within an experiment, the relative amount of iC3b deposited on each group of red cells was determined by measuring the binding of I-labelled anti-C3c to a fixed number of red cells. Figure 5 presents the rosette formation dose response curves for the various alanine substitution mutants engineered for this study. Each panel represents a separate experiment in which the dose response curve for the wild-type molecule acted as an internal reference point. The solid line near the bottom of each panel represents the background rosetting observed in control experiments employing EAC42 cells. The presence in the assay of the CR2-specific, functional site- blocking monoclonal antibody OKB-7 yielded a similar level of background rosetting when EAC423bi cells bearing high levels of wild-type iC3b were employed as the source of CD21 ligand. These data are representative of minimally two, and often three, repeat experiments for every mutant examined in this study. The data were also plotted using a semi-logarithmic scale for the x- axis, which denoted the relative amount of iC3b ligand per red cell. The horizontal displacement of the psuedo-linear part of a given dose response curve from that of the wild-type curve allows the estimation of the extent of the defect or enhancement in CD21 -binding introduced by any given mutation. Figure 6 summarizes the results of all the rosette experiments in a more convenient bar graph format.

The D36/E37/E39 cluster lines one edge of the negatively charged depression visible in the electrostatic surface potential rendering of the C3d molecule (see Fig. 3 in Ref. 19), D163 and E166 line the opposite edge, and the carboxylate side chain of El 60 extends down into the depression. Dealing first with the D36/E37/E39 cluster (Fig. 4 panels A, B and Fig. 5), it can be seen that whereas mutation of D36 on its own is without effect, both the E37A and E39A mutant molecules show about a 2-fold defect in CD21 binding activity in the rosette assay. The combination E37A/E39A mutant molecule was more than 4- fold compromised in CD21-binding activity and mutation of D36A in combination with E37A somewhat magnified the defect observed over that for E37A alone. Although the combination mutant D36A/E39A showed a similar CD21-binding defect as did the single E39A mutant, the D36A/E37A/E39A triple mutation, which would effectively remove the negatively charged rim from that side of the depression, yields a molecule that displays only about 10% of the wild- type CD21 -binding activity. To further test the importance of the negative charges per se, and at the same time address the issue of side chain stereochemistry that becomes more of a factor when analyzing the results of a triple mutation, the isosteric amide version of this mutant was engineered. Like its alanine-substituted counterpart, the D36N/E37Q/E39Q mutant was also found to be essentially inactive in mediating CD21 binding. Finally alanine substitutions were made at Glu 42, an acidic residue which protrudes up at the extreme edge of this surface of the molecule, and Asp 292, which extends the negative surface at the other edge of this rim of acidic residues (Fig. 3). In each case the resultant analog molecules retained between 80-90% of wild-type CR2 binding activity (Fig. 5E,F and Fig. 6). Collectively, these data indicate that the side chains of the D36/E37/E39 cluster make important ionic interactions with CD21 and the contacts with E37 and E39 appear to be the most important within this grouping of acidic residues.

The opposite rim of the depression encompasses residues 160-167 of the primary sequence (Fig. 3) and also within this surface patch, towards the edge of the cavity rim, is the hydrophobic side chain of 1164 and protruding away from the rim, but in between acidic residues D163 and E166, is the basic side chain of K162 (Fig. 3). This region is extremely important for the interaction between C3d and CD21 as even single alanine substitutions at D163, E166 and 1164 were sufficient to decrease CD21 binding levels to less than 5% of wild-type for D163 and 1164 and to about 10% of wild-type for E166 (Fig. 5 C,E and Fig. 6). D163 was also mutated to its isosteric amide, and although the deposition efficiency onto the C3 convertase-bearing cells was lower for the D163N analog protein than for any of the other analogs examined, by using more concentrated culture supernatants, sufficient deposition was achieved to ascertain that it was the negative charge per se at the D 163 position that was required to mediate binding to CD21 (Fig. 6). The mutation to alanine of D160, which lies at the bottom of the cavity, had a somewhat lesser effect, but still reduced CD21 binding 4-fold relative to wild-type, hi contrast, El 67, which relative to the cavity is the most distally located acidic residue in this segment, could be mutated to alanine with minimal effect on CD21 -binding activity. The K162A analog displayed a 2-fold enhancement in CD21 binding activity. One interpretation of this result is that the positive side chain of K162 partially inhibits the binding to this region of C3d of the electropositive surface contributed by CD21, so its removal results in increased binding of CD21. In keeping with this interpretation, in contrast to the almost complete absence of CD21-binding activity displayed by the D163A mutant, the double mutant K162AJD163A retains much of the CD21 -binding activity of the wild-type protein (Fig. 5D and Fig. 6). It would seem that in order to overcome the charge repulsion caused by K162, contact with acidic residues D163, El 66, and perhaps El 60 is necessary. However, in the absence of the K162 positive charge, the ionic contribution of at least D163 to the binding interface with CD21 becomes dispensable. "When mutations to the acidic residues in the 160-167 segment were carried out in combination, the molecules were generally defective in CD21 binding (Fig. 5D, Fig. 6). For example, the residual binding of the E160A mutant is abrogated in the double mutant analog E160A/D163A and the combined mutation of D163A and E166A, both of which were largely inactive as single mutants, does not result in the acquisition of any substantial new CD21 binding activity. The one exception to this general rule is that the combination of El 66 A, which was largely inactive as a single mutant, with El 67 A, which as a single mutant retained a near wild-type level of activity, yielded a molecule retaining approximately 30% of wild-type CD21-binding activity. It is possible that in the absence of the mutual charge repulsion between the carboxylates of D163, E166 and E167, the side chain of D163 may reorient so as to partially accommodate the loss of the ionic contribution of El 66 to the binding energy. Panels E through F of Figure 5 show the rosette assay results for six additional single residue mutants examined in this study (V97A, N98A, R49M, K251A, K291A and Y201A). It can be seen that these mutations were found to have relatively minor effects on CD21 binding activity, the largest effect being seen for the K251A mutant which retains about 60% of wild-type activity. Thus, CD21 primarily bridges between contacts in the D36/D37/D39 cluster and the E160/D163/I164/E166 cluster. Binding of Soluble CD21 to Wild-Type and Mutant iC3b-coated Red Cells:

Since the rosette assay is highly dependent on avidity effects, i.e. there are many potential points of contact between the ligand-bearing red cell and the receptor-bearing Raji cell, it is possible that this assay would miss the effects of mutations that bring about relatively minor changes to the intrinsic affinity governing the interaction between individual iC3b and CD21 molecules. This may be especially true for some of the single residue mutations examined in the previous section. Although not completely devoid of avidity effects, an assay measuring the binding of a bivalent soluble form of CD21 to iC3b-coated red cells should be less prone to such avidity "masking" effects since the ligand-receptor contacts are maximally bivalent. Accordingly, red cells bearing various amounts of wild-type or single-residue-mutant iC3b were prepared and incubated with a fixed concentration of soluble CD21 in the form of a (CD21)2-IgG fusion protein (4,22). Binding of the soluble CD21 to the cells was revealed by an FITC- conjugated anti-mouse IgG in conjunction with flow cytometric analysis. The relative amount of iC3b ligand per cell was determined, as before, usmg I- labeled anti-C3c and background fluorescence levels were determined using EAC42 cells. The results of such assays employing all of the single mutants engineered for this study are shown in Figure 7. In general the results were concordant with the rosette assay. In no case did mutants which showed severe defects in the rosette assay not also show severe defects in the soluble CD21 binding assay, however, as expected, the effects of some mutations were more severe in the soluble CD21 binding assay. For example, whereas the D36A and E42A mutations were without effect in both assays, near complete CD21 -binding impairment was seen in mutants E37A and E39A (Fig. 7A), this contrasting with the 2-fold defect seen in the rosette assay for these latter two mutants. The data in Figure 6B independently confirm the enhancing effect of the K162A mutation noted in the rosette assay. Additionally, the E160A mutation on its own is sufficient to completely abrogate CD21 -binding activity measured by this assay, whereas this mutant showed about 25% residual activity in the rosette assay. The small decreases in CD21 binding detected by the rosette assay for mutants D292A and K251A are confirmed and mutation of V97, a non-polar residue lying immediately adjacent to the critical 1164, gives rise to an approximately 2-fold defect in the soluble CD21 binding assay (Fig. 7D). The results conclusively show the involvement of two predominantly acidic surface patches in providing crucial contacts for the interaction with the C3d-binding domains of CD21 (i.e. CD21 SCR domains 1 and 2). Of the two acidic clusters, E37/E39 and E160/D163/E166, which are located on opposite sides of the depression on the concave surface of C3d, the latter appears to be the more important based on the fact that single mutations within it were sufficient to almost completely abrogate CD21 binding. The contribution of the hydrophobic side chain of the adjacent residue 1164 also appears to be vital. Inhibition of the iC3b-CD21 Interaction by Linear Peptide Mimetics

As a first step toward developing peptide-based mimetics of the CD21- contacting residues of C3d, linear peptides corresponding to the two clusters identified in the mutagenesis experiments were synthesized. Besides the wild- type sequences corresponding to C3d residues 36-42 and 160-167, two mutant versions of the latter segment were also synthesized. One of these peptides contained the K162A mutation, which in the context of iC3b enhanced binding to CR2, whereas the other had the three acidic residues of this segment replaced by their isosteric amides. A C4-derived peptide corresponding to C4 residues 740- 756 (not C4d derived) was available as an additional negative control. Shown in Figure 8 are the results of an experiment in which variable concentrations of the various peptides (100-300 μM range)were assessed for their abilities to inhibit rosette formation between wild-type iC3b-coated red cells and Raji cell CD21. It can be seen that K162A mutant derivative of the C3d 160-167 segment peptide, and to a lesser extent the 36-42 segment peptide, were able to inhibit rosette formation, presumably reflecting the CD21 -binding abilities of these two linear peptides and their abilities to out-compete the authentic iC3b ligand. As expected from the mutagenesis data, the isosteric amide derivative of the 160-167 peptide behaved like the negative control C4-derived peptide. The wild-type 160-167 peptide was not inhibitory in this assay, and emphasized the importance of the enhancing effect of the K162A mutation noted in the site-directed mutagenesis experiments. Inhibition of the iC3b-CD21 Interaction by Cyclic Peptide Mimetics of C3d

The relatively high concentrations of peptide required to compete with the authentic ligand in the above experiment, as well as the inability of the wild-type 160-167 segment peptide to act as a competitive inhibitor, are observations consistent with linear peptides often being poor mimetics of the parent protein because they have too much conformational freedom. By contrast, there have been several examples in the literature of cyclic peptides, i.e. disulfide bond- constrained peptides, displaying submicromolar binding affinities for their receptor/acceptor molecules (Ref. 31-35). Thus, it is clearly apparent to one skilled in the art that second generation conformationally-constrained peptides of the following forms are expected to show improved binding ability to CD21:

GG³⁶DCETEQ⁴⁰GCGGGSOrn(Bb)CONH₂, CG³⁶DETE³⁹CGGGSOrn(Bb)CONH₂,

GG¹⁶⁰ESCDISEE¹⁶⁷CGGGSOrn(Bb)CONH₂, GC¹⁶⁰ESADICEE¹⁶⁷GGGSOrn(Bb)CONH₂.

These peptides have designed into them a GGG spacer group and an ornithine- benzoylbenzoate photo-crosslinking group which enables them to be decorated onto antigen of interest. In an immunization procedure, such peptide-decorated antigens are designed to simultaneously engage both the B cell receptor and the CD21/CD19 co-receptor signaling complex for providing a molecular adjuvant effect.

In one aspect of the invention, the structure of C3d in the vicinity of the 33-40 and 160-168 segment peptides was analysed for residues at which it would be possible to insert a pair of cysteines such that when they formed a disulfide bond, the peptide segment would adopt a structure resembling that in the native C3d molecule. For the 33-40 segment, the "virtual mutagenesis" experiment suggested that if L35 was replaced by cysteine, it would be able to form a disulfide bond with a cysteine that replaced either W41 or Q40, without requiring any movement on the part of the rest of the peptide segment. Since the 160-168 segment contains within it C165, a residue which in native C3d is involved in formation of a disulfide bond with C108, we decided to use C165 as one of the sulfhydryl donor residues. In this case, the only residue at which the structure predicted that substitution of an apposing cysteine would have the desired effect was at A161. Accordingly, the synthesis of the six cyclic peptides shown below was commissioned from the Alberta Peptide Institute, Edmonton, AB.

I. NH2-Gly-33His-Tyr-Cys-Asp-Glu-Thr-Glu-Gln-Cys-CONH2

II. NH2-Gly-33His-Tyr-Cys-Asp-Glu-Thr-Glu-Cys-Gln-CONH2 in. NH2-Gly-33His-Tyr-Cys-Asp-Gln-Thr-Gln-Cys-Gln-COOH

TV. NH2-Gly-160Glu-Cys-Ala-Asp-Ile-Cys-Glu-Glu-Gln-CONH2

V. NH2-Gly- 160Glu-Cys-Lys- Asp-Ile-Cys-Glu-Glu-Gln-CONH2

VI. NH2-Gly-160Gln-Cys-Lys-Asn-Ala-Cys-Glu-Glu-Gln-CONH2 Peptides I and II are the two variant cyclic peptide mimetics referred to above for the 33-40 segment of residues. Peptide El is a control in which isosteric amide substitutions have been introduced at residues E37 and E39, these substitutions being equivalent to ones which diminished the CD21 binding activity of iC3b. Peptides IV and V are two cyclic peptides corresponding to the 160-168 segment. The only difference between them is that peptide IV contains the K162A mutation that was found to be enhancing in the context of the authentic CD21 ligands iC3b and C3dg. Peptide VI contains three substitutions, E160Q, D163N and I164A, each of which, in the context of the authentic ligand, greatly impaired CD21 binding activity. Reverse phase HPLC chromatography and mass spectroscopy analysis performed by the manufacturer indicated that all the peptides were approximately 95% pure and of the expected molecular mass for a cyclic monomer. These cyclic peptides were used in a rosette inhibition assay similar to that described for the linear peptides. As can be seen in Fig. 9 A, and Fig. 10A, cyclic peptides I, IV and V showed significant inhibitory activity, suggesting that they were able to compete with red cell-associated iC3b for binding to the Raji cell CD21. Peptide V was clearly the most potent as at 200 μM it was capable of almost completely inhibiting rosette formation. This degree of inhibition was significantly greater than that achieved by comparable concentrations of any of the linear peptides employed in the earlier experiments (Fig. 8). Also in contrast to the results with the linear peptides, the wild-type Kl 62 -containing peptide was in this case more effective than the K162A substitution mutant. Neither of the two peptides designed to be specificity controls (i.e. cyclic peptides III and VI) showed any inhibitory activity in the rosette assay.

Interestingly, only one of the two 33-40 segment mimetic cyclic peptides (I) showed substantial inhibitory activity. This could either be because the position of the cysteine in cyclic peptide E did not provide for a native-like conformation, or because disruption of the native sequence by the insertion of a cysteine between E39 and Q40 ablated the CD21 binding capacity of the peptide. To distinguish between these possibilities, but also to test whether the CD21 binding capacity of peptides IV and V were dependent on the constrained conformation of the cyclic peptides, the peptides were reduced (5 mM DTT, lhr, 22°C, pH 8) and alkylated (20 mM iodoacetamide, 0.5 hr, 22°C, pH 8) prior to their use in the rosette inhibition assay. The results of these experiments are presented in Figures 9B and 10B for peptides I, II, III and IV, V, VI, respectively. It can be seen in Fig. 9B that reduction and alkylation have little effect on the CD21 binding activity of peptide I. Moreover, peptide E does not acquire any new inhibitory activity following reduction and alkylation. These results suggest that the activity of peptide I is not dependent upon any conformational constraints imparted by the disulfide bond and further, that it is the disruption of the native sequence between E39 and Q40 that likely accounts for the inactivity of peptide E in the rosette inhibition assay. By contrast, as can be seen in Fig. 10B, the CD21 binding activities of peptides IV and V are completely dependent upon the conformational constraints imposed by the disulfide bond, as both became completely inactive in the rosette inhibition assay following reduction and alkylation. As reagent controls for the reduction and alkylation experiment, it was found that the addition of DTT, followed by iodoacetamide, to a buffer control did not alter the % rosettes formed from that seen in the absence of peptide inhibitor (Fig. 9B). Additionally, treatment of C3d with iodoacetamide and DTT under the same conditions, but in reverse order, did not alter its rosette inhibitory capacity. Thus, the effects of reduction and alkylation on the behavior of the peptides is not due to non-specific effects of the reagents on the rosette assay.

Cumulatively, the peptide inhibition data show that it is possible to create peptide mimetics of C3d that can specifically bind to CD21 and that, in the case of the 160-168 segment, the introduction of a conformation-constraining disulfide bond considerably augments the binding affinity relative to that observed with similar linear peptides. It is clearly apparent to one skilled in the art that other cysteine placements within this target region, may further increase the intrinsic affinity down into the low micromolar range.

Other permutations of the peptide mimetic approach designed for improving binding to CD21 include linking together linear or cyclic peptides corresponding to each of the 36-42 and 160-167 segments using a flexible (GGGGS)₂ spacer segment. Such a spacer roughly bridges the distance of these two segments in the C3d molecule and are designed to allow independent CD21 subsite engagement for a resulting increase in affinity.

Another approach that takes advantage of avidity effects involves synthesizing the various peptides as multidentate polymers using the so-called MAPs approach (multiple antigen peptide system) in which a multivalent branched core of amino groups on a carbon skeleton (e.g. tetravalent or octavalent) is used as the acceptor for peptide elongation (Ref. 36). Such octavalent peptide mimetics of regions in CD21's SCR1 and SCR2 that interacted with red cell-bound iC3b were about 100-fold more effective than there monomeric counterparts (Ref 37). Such MAPs-based peptides could also be conjugated to antigen of interest by having the first amino acid added be the ornithine benzoyl-benzoic acid derivative. EXAMPLES The above disclosure generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for purposes of illustration and are not intended to limit the scope of the invention. Changes in form and substitution of equivalents are contemplated as circumstances may suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation. Example 1: This Example illustrates the site-directed mutagenesis and expression of recombinant C3.

All mutants were generated by an overlap-extension PCR method using the proofreading enzyme Vent polymerase (Biolabs) (Ref 26) with a modified version (pSV-C3(LC)) of the expression plasmid pSV-C3 containing the full- length cDNA of human C3 (Ref 27). pSV-C3 was modified in order to introduce an Agel restriction site at nucleotide 3713 and to delete a BglR site at nucleotide 4220 of the C3 cDNA sequence. In neither case did the base substitutions alter the amino acids coded for. As delimited by the sense and anti-sense flanking primers used for the overlap-extension PCR, the target region for mutagenesis spanned nucleotides coding for amino acid residues 925 through 1371 of C3 (mature C3 numbering), which includes within it the nucleotides encoding the entire C3d fragment (residues 980 to 1281). The overlap-extension PCR fragments of mutants located upstream of the Age! site were restricted with Sail and Agel to produce a 670 bp fragment. For mutants located downstream of the Agel site the overlap-extension fragments were restricted with Agel and BglR to produce a 507 bp fragment. The restricted fragments were then exchanged with the corresponding segments in wild-type pSV-C3(LC). The mutations were confirmed by strand denaturation dideoxy DNA sequencing using a T7 polymerase sequencing kit (Amersham Pharmacia Biotech Inc., Canada). Wild-type or mutant plasmid DNAs (5 μg) were transfected into COS-1 cells by our previously described modification of the DEAE-dextran method (Ref 28, 29) in 100 mm plates. Culture supernatants (about 8 ml) were harvested after five days, dialyzed against VBS and concentrated about 5-fold using Biomax concentrators (Millipore Corporation, Bedford, MA). Recombinant C3 protein concentration in the concentrated supernatants was determined by a C3-specific ELISA, using plates coated with 10 μg/ml of rabbit anti-C3c antibody (Sigma, St. Louis, MO), a goat anti-C3 sandwiching antibody (Quidel, San Diego, CA), and finally an alkaline phosphatase-conjugated anti-goat IgG for detection. Purified human C3 was used to obtain a standard curve.

Metabolic labeling of transfected COS-1 cells was performed 48h post- transfection as described previously (Ref 22). Example 2:

This Example illustrates the preparation of iC3b-coated erythrocytes. The preparation of sheep erythrocytes bearing varying amounts of wild- type or mutant iC3b was done via minor modifications of our previously described procedure (Ref 22). Briefly, 0.25 ml of EAC4b2a (10⁹ cells/ml) in SGVB-E were incubated with increasing amount of concentrated tiansfection supernatants (from 0.175 ml to 1.5 ml at a concentration of 1-1.5 μg/ml) for 2 h at 37°C. The resulting EAC423b were converted to EAC423bi cells by incubating with 4 μg of factor H and 0.4 μg of factor I for 3 h at 37°C, followed by washing.

The relative number of iC3b molecules/cell was determined by evaluating the binding of 2.5 μg of ¹²⁵I-labeled anti-C3 antibody to 50 μl containing 5 x 10⁷ EAC423bi cells as previously described (Ref 26). The non-specific binding component was determined by incubating the ¹²⁵I-labeled anti-C3c with an equal number of EAC4b2a cells.

The conversion of red cell-bound recombinant C3b to iC3b was monitored in experiments using metabolically labeled culture supernatants as the source of recombinant C3 for building the EAC423bi cells. As described in detail in an earlier study (Ref 30), membranes of the EAC423bi cells were solubilized in 0.75%o SDS, treated with 1 M hydroxylamine to break the ester linkages between the ³⁵S-iC3b and red cell surface molecules and then the released labeled molecules were analyzed by SDS-PAGE autoflurography under reducing conditions. Example 3

This Example illustrates the Rosetting of iC3b-coated erythrocytes to CD21 -bearing Raji cells.

Raji cells (5 x 10⁴) were incubated with 4 x 10⁶ EAC423bi in 50 μl of the low ionic strength buffer, SGVB, for 30 min at 37°C with gentle rotation. Cells were fixed with 0.2% glutaraldehyde for 5 min before quenching with 20 mM Tris-HCl, pH 8.2. Rosette formation was evaluated in duplicate by assessing the percentage of Raji cells bearing four or more erythrocytes. Binding specificity was determined by incubating Raji cells with EAC42 and also by pre-incubating Raji cells with 4 μg/ml of OKB7, an IgG2b functional site blocking anti-human CD21 mAb, (provided by Dr. P. Rao, Ortho Diagnostics Systems, Raritan, NJ) before the addition of iC3b-coated erythrocytes. The IgG2b anti-CR3 mAb OKMI (provided by Dr. W. Reed, University of North Carolina, Chapel Hill, NC) was used as negative control of Ab binding. Rosette inhibition by purified iC3b and C3dg was performed as above for the OKB7 blocking mAb experiments except that instead of OKB7, the Raji cells were pre-incubated with various concentration of purified iC3b or C3dg before the addition of EAC423bi cells made with sufficient purified C3 to yield approximately 80 to 90% rosette formation in the absence of inhibitor (Fig. 2). C4 was used as a negative control in these experiments. Example 4

This example illustrates the Binding of soluble CR2 to iC3b-coated erythrocytes.

Culture supernatants from J558L-pSNRCR2 cells were dialyzed against DVB. The amount of secreted (CR2)₂-IgGl was determined by ELISA, using plates coated with a rabbit antibody to mouse IgG (Jackson ImmunoResearch, West Grove, PA) and a sandwiching rabbit antibody to mouse IgG that is conjugated to alkaline phosphatase (Jackson ImmunoResearch,). Purified mouse IgG (Sigma) was used to obtain a standard curve. For the binding assay, 50 μl of culture supernatant, containing 4 μg/ml of

(CR2)₂-IgGl, were incubated overnight at 4°C with 5 x 10⁶ iC3b-coated erythrocytes. Binding specificity was established by pre-incubating (CR2)₂-IgGl with 40 μg/ml of OKB7 or OKMI at 37°C for 1 h. Erythrocytes were then washed with DGVB and binding was detected by using a fluorescein-conjugated donkey antibody to mouse IgG (Jackson ImmunoResearch). After 1 h of incubation at 4°C, cells were washed and resuspended in 1 ml DGVB. Cell-bound fluorescence intensity was determined using a FACSCalibur flow cytometer (Becton Dickinson) and analyzed as a function of cell number using the Cell Quest software package (Becton Dickinson). Example 5 This example illustrates the ability of C3d peptides to inhibit rosette formation between iC3b-coated erythrocytes.

The assay was performed essentially as described in example 3. The only difference was that the purified iC3b and C3dg was replaced by the following C3d peptides: 160-167 NH₂-GEAKDISEEGGGGS(LysBb)CONH₂

160-167/K162A NH₂-GEAADISEEGGGGS(LysBb)CONH₂

160-167/QNQ NH₂-GQAKNISQEGGGGS(LysBb)CONH₂

36-42 NH₂-RGDETEQAEGGGGS(LysBb)CONH₂

C4 740-756 NHa-EILQEEDLIDEDDJRVRCCONHz (negative control)

All peptides have free amino groups at their N-termini, but carboxyamide C-termini. The GGGGS(LysBb) segment represents a spacer arm and a photoactive benzoylbenzene group which is present to enable photo-crosslinking to a protein molecule. The results shown in Figure 8 indicate that the K162A peptide, and to a lesser extent the 36-42 peptide, were able to inhibit the rosette formation indicating the ability of these peptides to bind to the CD21 molecules and out-compete the iC3b coated erythrocytes.

SUMMARY OF DISCLOSURE In summary of this disclosure, the present invention provides ligands for the CD21 molecule and complexes comprising the ligands and antigens for modulation of the immune response. Analogs of C3d are provided that have an enhanced ability to bind to CD21. The disclosure further provides methods of making and using the ligands, complexes and analogs including in their use in immunization. Modifications are possible within the scope of this invention.

a Low expression refers to less than 100 ng/ml of recombinant protein in culture supernatants, insufficient to achieve the normal dose-response range of deposited iC3b required in the rosette assay.

Low deposition refers to a defective ability of recombinant proteins expressed at normal levels to deposit on EAC42. REFERENCES Carroll, M.C. 1998. The role of complement and complement receptors in induction and regulation of immunity. Ann. Rev. Immunol. 16:545.

Fearon, D.T., and R.H. Carter. 1995. The CD19/CR2/TAPA-1 complex of B lymphocytes: linking natural to acquired immunity. Annu. Rev. Immunol. 13: 127.

Heyman, B., E.J. Wiersma, and T. Kinoshita. 1990. In vivo inhibition of the antibody response by complement receptor-specific monoclonal antibody. J. Exp. Med. 172:665.

Hebell, T., J.M. Ahearn, and Fearon, D.T. 1991. Suppression of the immune response by a soluble complement receptor of B lymphocytes. Science 254:102.

Ahearn, J.M., M.B. Fischer, D. Croix, S. Georg, M. Ma, J. Xia, X. Zhou, R.G. Howard, T.L. Rothstein, and M.C. Carroll M.C. 1996. Disruption of the CR2 locus results in a reduction in B-la cells and an impaired response to T- dependent antigen. Immunity 4:251.

Croix, D.A., J.M. Aheam, A.M. Rosengard, S. Han, G. Kelsoe, M. Ma, and M.C. Carroll. 1996. Antibody response to T-dependent antigen requires B cell expression of complement receptors. J. Exp. Med. 183:1857.

Kurtz, C.B., E. O'Toole, S.M. Christensen, and J.H. Weis. 1990. The murine complement receptor gene family. IV. Alternative splicing of Cr2 gene transcripts predicts two distinct gene products that share homologous domains with both human CR2 and CRl. J. Immunol. 144:3581.

Fischer, M.B., M. Ma, S. Georg, X. Zhou, J. Xia, O. Finco, S. Han, G. Kelsoe, R.G. Howard, T.l. Rothstein, E. Kremmer, F.S. Rosen, and M.C. Carroll. 1996. Regulation of the B cell response to T-dependent antigens by classical complement pathway. J. Immunol. 157:549.

Rickert, R.C., K. Rajewsky, and J. Roes. 1995. Impairment of T-cell- dependent B-cells responses and B-l cell development in CD19-deficient mice. Nature 376:352.

Engel, P., L.-J. Zhou, D.C. Ord, S. Sato, B. Koller, and T.F. Tedder. 1995. Abnormal B lymphocyte development, activation and differentiation in mice that lack or overexpress the CD 19 signal transduction molecule. Immunity 3:39. Matsumoto, A.K., J. Kopicky-Burd, R.H. Carter, D.A. Tuveson, T.F. Tedder, and D.T. Fearon. 1991. Intersection of the complement and immune systems: a signal transduction complex of the B-lymphocyte-containing complement receptor type 2 and CD19. J. Exp. Med. 173: 55.

Carter, R.H., and D.T. Fearon. 1992. Lowering the threshold for antigen receptor stimulation of B lymphocytes. Science 256^'.' 105. Demρsey, P.W., M.E.D. Allison, S. Akkaraju, C.C. Goodnow, and D.T. Fearon. 1996. C3d of complement as a molecular adjuvant: bridging innate and acquired immunity. Science 271 : 348.

Hourcade, D., V.M. Holers, and J.P. Atkinson. 1989. The regulators of complement activation (RCA) gene cluster. Advances in Immunology 45: 381.

Lowell, C.A., L.B. Klickstein, R.H. Carter, J.A. Mitchell, D.T. Fearon, and J.M. Ahearn. 1989. Mapping of the Epstein-Barr virus and the C3dg binding sites to a common domain on complement receptor type 2. J. Exp. Med. 170: 1931.

Molina, H., S.J. Perkins, J. Guthridge, J. Gorka, T. Kinoshita, and V.M. Holers. 1995. Characterization of a complement receptor 2 (CR2, CD21) ligand binding site for C3: an initial model of ligand interaction with two linked short consensus repeat modules. J. Immunol. 154: 5426.

Prodinger, W.M., M.G. Schwendinger, J. Schoch, M. Kochle, C. Larcher, and M.P. Dierich. 1998. Characterization of C3dg binding to a recess formed between short consensus repeats 1 and 2 of complement receptor type 2 (CR2; CD21). J. Immunol. 161: 4604.

Barlow, P.N., A. Steinkasserer, D.G. Norman, B. Kiefler, A.P. Wiles, R.B. Sim, and J.D. Campbell. 1993. Solution structure of a pair of complement modules by nuclear magnetic resonance. J. Mol. Biol. 232: 268.

Nagar, B., R.G. Jones, R.J. Diefenbach, D.E. Isenman, and J.M. Rini. 1998. X- ray crystal structure of C3d: a C3 fragment and ligand for complement receptor 2. Science 280: 1277. Lambris, J.D., V.S. Ganu, S. Hirani, and H.J. Mϋller-Eberhard. 1985. Mapping of the C3d-receptor (CR2)-binding site and a neoantigenic site in the C3d domain of the third component of complement. Proc. Natl. Acad. Sci. USA 82: 4235.

Esparza, I., J.D. Becherer, J. Alsenz, A. De la Hera, Z. Lao, CD. Tsoukas, and J.D. Lambris. 1991. Evidence for multiple sites of interaction in C3 for complement receptor type 2 (C3d/EBV receptor, CD21). Eur. J. Immunol. 21: 2829.

Diefenbach, R.J., and D.E. Isenman. 1995. Mutation of residues in the C3dg region of human complement component C3 corresponding to a proposed binding site for complement receptor type 2 (CR2, CD21) does not abolish binding of iC3b or C3dg to CR2. J. Immunol. 154: 2303.

Lambris, J. D., D. Avila, and H.J. Mϋller-Eberhard. 1988. A discontinuous Factor H Binding Site in the Third Component of Complement as Delineated by Synthetic Peptides. J. Biol. Chem. 263:12147

Hellwage, J., T.S. Jokiranta, V. Koistinen, O. Vaarala, S. Meri, and P.F. Zipfel. 1999. Functional properties of complement factor H-related proteins FHR-3 and FHR-4: binding of the C3d region of C3b and differential regulation by heparin. FEBS Lett .462:345.

Sahu. A., S.N. Isaacs, A.M. Soulika, and J.D. Lambris. 1998. Interaction of vaccinia virus complement control protein with human complement proteins: factor I-mediated degradation of C3b to iC3bl inactivates the alternative complement pathway. J. Immunol. 160:5596.

Ho, S.N., H.D. Hunt, R.M. Horton, J.K. Pullen, and L.R. Pease. 1989. Site- directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51.

Taniguchi-Sidle.A., and D.E. Isenman 1992. Mutagenesis of the Arg-Gly- Asp triplet in human complement component C3 does not abolish binding of iC3b to the leukocyte integrin complement receptor type El (CR3, CDl lb/CD18). J. Biol. Chem. 267:635.

Oprian D.D., Molday R.S., Kaufinan R J., Khorana H.G. 1987. Expression of a synthetic bovine rhodopsin gene in monkey kidney cells. Proc. Natl. Acad. Sci. USA 84: 8874.

Ebanks, R., A. Jaikaran, M.C. Carroll, M.J. Anderson, R.D. Campbell, and D.E. Isenman. 1992. A single arginine to tryptophan interchange at -chain residue 458 of human complement component C4 accounts for the defect in the classical pathway C5 convertase activity of allotype C4A6: implications for the location of a C5 binding site in C4. J. Immunol. 148: 2803.

Taniguchi-Sidle, A. and Isenman, D.E. 1994. Interactions of human complement component C3 with factor B and complement receptors type 1 (CRl, CD35) and type 3 (CR3, CDllb/CD18) involve an acidic sequence at the N-terminus of C3 '-chain. J. Immunol. 153:5285. O'Neil, K.T., Hoess, R.H., Jackson, S.A., Ramachandan, N.S., Mousa, S.A. and DeGrado, W.F. (1992). Identification of novel peptide antagonists for gpEb/EIa from a conformationally constrained phage peptide library. Proteins: Struc. Func. Gen. 14:509.

McConnell, S.J., Kendall, M.L.,Reilly, T.M. and Hoess, R.H. (1994). Constrained peptide libraries as a tool for finding mimotopes. Gene 151:115.

Giebel, L.B., Cass, R.T., Milligan, D.L., Young, D.C., Arze, R. and Johnson, C.R. (1995). Screening of cyclic peptide phage libraries identifies ligands that bind strepavidin with high affinities. Biochemistry 34: 15430.

Sahu, A., Kay, B.K. and Lambris, J.D. (1996). inhibition of human complement by a C3 -binding peptide isolated from a phage-displayed random peptide library. J. hnmunol. 157:884.

Baglia, F.A., Jameson, B.A. and Walsh, P.N. (1993). Identification and characterization of a binding site for factor XEa in the Apple 4 domain of coagulation factor XL J. Biol. Chem. 268:3838.

Tam, J.P. (1988). Synthetic peptide vaccine design: Synthesis and properties of a high density multiple antigenic peptide system. Proc. Natl. Acad. Sci. USA 85:5409.

Molina, H., Perkins, S.J., Guthridge, J., Gorka, J., Kinoshita, T. and Holers, V.M. (1995). Characterization of a complement receptor 2 (CR2, CD21) ligand binding site for C3: An initial model of ligand interaction with two linked short consensus repeat modules. J. Immunol. 154:5426.

Claims

What we claim is:

A ligand for the CD21 molecule, said ligand comprising CD21 contacting amino acid residues 36-39 and 160-167 of the C3d molecule, wherein said ligand has the ability to bind CD21 and stimulate B cells through the

CD21/CD19 complex and wherein said ligand is other than naturally occurring C3d.

The ligand of claim 1, wherein said ligand is a fragment of C3d.

The ligand of claim 1, wherein said ligand is a peptide.

The ligand of claim 1, wherein said ligand is a mimetic.

The ligand of claim 1, wherein said ligand is a cyclic peptide mimetic.

A non-naturally occurring ligand for the CD21 molecule, wherein said ligand demonstrates an enhanced binding affinity for CD21 as compared to the binding affinity of a wild-type C3d molecule.

7. The ligand of claim 6, wherein said ligand is a fragment of C3d.

8. The ligand of claim 6, wherein said ligand is a peptide.

9. The ligand of claim 6, wherein said ligand is a mimetic.

10. The ligand of claim 6, wherein said ligand is a cyclic peptide mimetic.

11. An analog of C3d, said analog having an enhanced binding affinity for

CD21 as compared to the binding affinity a wild-type C3d molecule.

12. The analog of claim 11, wherein said analog has an amino acid sequence corresponding to the amino acid sequence of wild-type C3d in which at least one amino acid has been inserted, deleted or replaced.

13. The analog of claim 12, wherein at least one amino acid within the acidic pocket of C3d is replaced.

14. The analog of claim 13, wherein the at least one amino acid is Lysine-162. 15. The analog of claim 11, wherein said ligand is a cyclic peptide mimetic. 16. The analog of claim 15, wherein said mimetic comprises a disulfide bond.

17. The analog of claim 15, wherein at least one amino acid has been replaced by cysteine.

18. The analog of claim 15, wherein said analog is selected from the group consisting of: a) NH2-Gly-33His-Tyr-Cys-Asp-Glu-Thr-Glu-Gιn-Cys-CONH2 b) NH2-Gly-33His-Tyr-Cys-Asp-Glu-Thr-Glu-Cys-Gln-CONH2 c) NH2-Gly-160Glu-Cys-Ala-Asp-Ile-Cys-Glu-Glu-Gln-CONH2 d) NH2-Gly-160Glu-Cys-Lys-Asp-Ile-Cys-Glu-Glu-Gln-CONH2 e) NH2-Gly-160Gln-Cys-Lys-Asn-Ala-Cys-Glu-Glu-Gln-CONH2

19. A composition comprising; a) a first component selected from the group consisting of a ligand as defined in any one of claims 1-10, an analog as defined in any one of claims 11-18 and mixtures thereof; and b) a second component comprising at least one entity capable of binding to a B-cell receptor.

20. The composition of claim 19 wherein said entity is an antigen.

21. The composition of claim 20, wherein the antigen is a protein, a polypeptide or a peptide.

22. The composition of claim 20, wherein the antigen is an antigen from a pathogen.

23. The composition of claim 19, wherein the entity is at least part of an antibody molecule.

24. The composition of claim 24, wherein the at least part of an antibody is a variable region of an antibody.

25. The composition of any one of claims 19 to 24, wherein said first component and said second component are provided as a fusion protein.

26. A nucleic acid molecule encoding the ligand any one of claims 1 to 10.

27. A nucleic acid molecule encoding the analog of any one of claims 11-18.

28. A nucleic acid molecule encoding the fusion protein of claim 25.

29. An expression vector comprising the nucleic acid molecule of any one of claims 26-28.

30. A host cell comprising the expression vector of claim 29. A method of altering the immunogenicity of an antigen, said method comprising coupling said antigen to a ligand as defined in any one of claims 1 to 10 or to an analog as defined in claims 11-18. A method of inducing or enhancing an immune response to an antigen in a host, said method comprising administering the composition of any one of claims 19 to 25 to the host. The method of claim 31 or 32, wherein the antigen is an antigen derived from a pathogen. The method of claim 33, wherein said immune response protects the host against disease caused by the pathogen. Use, of a composition as defined in any one of claims 19 to 25, in the manufacture of a medicament for modulating the immune response of a host. Use according to claim 35 wherein said medicament is a vaccine. Use according to claim 35 wherein said medicament is a tumor vaccine. Use of the composition, as defined in any one of claims 19 to 25, to modulate an immune response in a host.