Ingunn B. Rasmussen

Identification of residues in the Cmu4 domain of polymeric IgM essential for interaction with Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1).

The binding of nonspecific human IgM to the surface of infected erythrocytes is important in rosetting, a major virulence factor in the pathogenesis of severe malaria due to Plasmodium falciparum, and IgM binding has also been implicated in placental malaria. Herein we have identified the IgM-binding parasite ligand from a virulent P. falciparum strain as PfEMP1 (TM284var1 variant), and localized the region within this PfEMP1 variant that binds IgM (DBL4β domain). We have used this parasite IgM-binding protein to investigate the interaction with human IgM. Interaction studies with domain-swapped Abs, IgM mutants, and anti-IgM mAbs showed that PfEMP1 binds to the Fc portion of the human IgM H chain and requires the IgM Cμ4 domain. Polymerization of IgM was shown to be crucial for the interaction because PfEMP1 binding did not occur with mutant monomeric IgM molecules. These results with PfEMP1 protein have physiological relevance because infected erythrocytes from strain TM284 and four other IgM-binding P. falciparum strains showed analogous results to those seen with the DBL4β domain. Detailed investigation of the PfEMP1 binding site on IgM showed that some of the critical amino acids in the IgM Cμ4 domain are equivalent to those regions of IgG and IgA recognized by Fc-binding proteins from bacteria, suggesting that this region of Ig molecules may be of major functional significance in host-microbe interactions. We have therefore shown that PfEMP1 is an Fc-binding protein of malaria parasites specific for polymeric human IgM, and that it shows functional similarities with Fc-binding proteins from pathogenic bacteria.

Efficient Delivery of T Cell Epitopes to APC by Use of MHC Class II-Specific Troybodies

A major objective in vaccine development is the design of reagents that give strong, specific T cell responses. We have constructed a series of rAb with specificity for MHC class II (I-E). Each has one of four different class II-restricted T cell epitopes genetically introduced into the first C domain of the H chain. These four epitopes are: 91–101 λ2315, which is presented by I-Ed; 110–120 hemagglutinin (I-Ed); 323–339 OVA (I-Ad); and 46–61 hen egg lysozyme (I-Ak). We denote such APC-specific, epitope-containing Ab “Troybodies.” When mixed with APC, all four class II-specific Troybodies were ∼1,000 times more efficient at inducing specific T cell activation in vitro compared with nontargeting peptide Ab. Furthermore, they were 1,000–10,000 times more efficient than synthetic peptide or native protein. Conventional intracellular processing of the Troybodies was required to load the epitopes onto MHC class II. Different types of professional APC, such as purified B cells, dendritic cells, and macrophages, were equally efficient at processing and presenting the Troybodies. In vivo, class II-specific Troybodies were at least 100 times more efficient at targeting APC and activating TCR-transgenic T cells than were the nontargeting peptide Ab. Furthermore, they were 100–100,000 times more efficient than synthetic peptide or native protein. The study shows that class II-specific Troybodies can deliver a variety of T cell epitopes to professional APC for efficient presentation, in vitro as well as in vivo. Thus, Troybodies may be useful as tools in vaccine development.

Structural requirements for the interaction of human IgM and IgA with the human Fcα/μ receptor

Here we unravel the structural features of human IgM and IgA that govern their interaction with the human Fcα/μ receptor (hFcα/μR). Ligand polymerization status was crucial for the interaction, because hFcα/μR binding did not occur with monomeric Ab of either class. hFcα/μR bound IgM with an affinity in the nanomolar range, whereas the affinity for dimeric IgA (dIgA) was tenfold lower. Panels of mutant IgM and dIgA were used to identify regions critical for hFcα/μR binding. IgM binding required contributions from both Cμ3 and Cμ4 Fc domains, whereas for dIgA, an exposed loop in the Cα3 domain was crucial. This loop, comprising residues Pro440–Phe443, lies at the Fc domain interface and has been implicated in the binding of host receptors FcαRI and polymeric Ig receptor (pIgR), as well as IgA‐binding proteins produced by certain pathogenic bacteria. Substitutions within the Pro440–Phe443 loop resulted in loss of hFcα/μR binding. Furthermore, secretory component (SC, the extracellular portion of pIgR) and bacterial IgA‐binding proteins were shown to inhibit the dIgA–hFcα/μR interaction. Therefore, we have identified a motif in the IgA–Fc inter‐domain region critical for hFcα/μR interaction, and highlighted the multi‐functional nature of a key site for protein–protein interaction at the IgA Fc domain interface.

The principle of delivery of T cell epitopes to antigen-presenting cells applied to peptides from influenza virus, ovalbumin, and hen egg lysozyme: Implications for peptide vaccination

Targeting of antigens to antigen-presenting cells (APCs) increases CD4+ T cell activation, and this observation can be exploited in the development of new vaccines. We have chosen an antigen-targeting approach in which we make recombinant antibodies (Abs) with T cell epitopes in their constant region and APC-specific variable regions. Three commonly used model epitopes, amino acids 110–120 of hemagglutinin, 323–339 of ovalbumin, and 46–61 of hen egg lysozyme, were introduced as loops in the CH1 domain of human IgG3. For all three epitopes, we show that the recombinant molecules are secreted from transfected cells. The epitopes are presented to specific T cells, and targeting to IgD on B cells in vitro enhances the presentation efficiency by 104 to 105 compared with the free peptide. After i.v. injection, the epitopes targeted to IgD are presented by splenic APCs to activate specific T cells, whereas little or no activation could be detected without targeting, even after the amount of antigen injected was increased 100-fold or more. Because a wide variety of T cell epitopes, in terms of both length and secondary structure, can be tolerated in loops in constant domains of Abs, the Ab constant region seems to have the intrinsic stability that is needed for this fusion molecule strategy. It might thus be possible to load the Ab with several different epitopes in loops in different domains and thereby make a targeted multisubunit vaccine.

IgM secretory tailpiece drives multimerisation of bivalent scFv fragments in eukaryotic cells

BACKGROUND The monoclonal antibody (mAb) TP-3 binds selectively to human and canine osteosarcoma (OS) cells and is therefore a potential candidate for use as a targeting agent in radioimmunoimaging and therapy of OS metastases. However, intact murine mAbs have several drawbacks such as large size, delayed blood clearance and high immunogenicity, all of which can be overcome by genetic engineering. OBJECTIVES To construct and express bivalent and multivalent TP-3 scFv fragments from the mammalian expression vector, pLNO. This vector has unique restriction sites for simple cassette cloning of any individual variable (V) and constant (C) genes and has previously been used for expression of intact chimeric TP-3 mAbs and Fab fragments. Furthermore, it is also suitable for expression of any modified V region, such as a scFv fragment, fused to any modified C region or to non-immunoglobulin protein sequences. STUDY DESIGN Six different constructs were made; three scFv-CH3 fragments that differed in the design of linker between the scFv fragment and the IgG CH3 domain. These constructs were also made with the IgM secretory tailpiece (microtp) attached to the C terminus. RESULTS All constructs were secreted as bivalent antibody fragments with a molecular weight of about 100 kDa. A band corresponding to a dimer appeared in all the supernatants from TP-3 scFv-CH3 producing cells, whether microtp was present or not, whereas higher orders of multimers were not seen. However, pulse chase analyses of the cells revealed that a small fraction of higher order polymers was formed from genes including the fragment encoding microtp and that microtp conferred retention both to monomers and intermediate polymers. The recombinant TP-3 antibody fragments were shown to bind human OS cells. CONCLUSION Recombinant mAb fragments can be designed and cloned into the mammalian expression vector, pLNO. This vector is flexible in the sense that the genes encoding such fragments can be expressed from either cDNA or from genomic DNA. A microtp attached to the CH3 domain in these fragments was sufficient to drive polymerization, however inefficiently and intracellular retention of both monomers and intermediate polymers was observed.

Recombinant antibodies as carrier proteins for sub-unit vaccines: influence of mode of fusion on protein production and T-cell activation

A major objective in development of vaccines is the design of sub-unit vaccines with the ability to induce strong T-cell responses. For this purpose, T-cell epitopes have been genetically inserted into various carrier proteins. Ig molecules may be especially useful as vehicles for delivery of CD4(+) T-cell epitopes to antigen presenting cells (APC). We have previously replaced loop structures between beta-strands in the C(H)1 domain of human IgG3 with a defined 11 amino acids long, MHC class II-restricted T-cell epitope. In this report we have added the same T-cell epitope into loops in the C(H)1 domain of mouse IgG2b. The following major points can be made: (1) Loops can accommodate an elongation of at least 11 amino acids without disruption of the overall Ig structure and secretion. (2) The recombinant Ig molecules are processed by spleen APC and the epitopes that are released are presented to T-cells. (3) Site of integration influences efficiency of processing and presentation. (4) Elongation of two neighbouring loops reduces Ig secretion. Taken together, our present results indicate that IgG C(H)1 domains may be engineered to carry T-cell epitopes in loop structures between beta-strands, but not all loops may be equally suitable for this purpose.

Processing of an Antigenic Sequence from IgG Constant Domains for Presentation by MHC Class II

Targeting of T cell epitopes to APC enhances T cell responses. We used an APC-specific Ab (anti-IgD) and substituted either of 18 loops connecting β strands in human IgG constant H (CH) domains with a characterized T cell peptide epitope. All Ab-epitope fusion molecules were secreted from producing cells except IgG-loop 2(BC)CH1, and comparing levels, a hierarchy appeared with fusions involving CH2≥CH1>CH3. Within each domain, fusion at loop 6(FG) showed best secretion, while low secretion correlated with the substitution of native loops that contain conserved amino acids buried within the folded molecule. Comparing the APC-specific rAb molecules for their ability to induce T cell activation in vitro, the six mutants with epitope in CH2 were the most effective, with loop 4CH2 ranking on top. The CH1 mutants were more resistant to processing, and the loop 6CH1 mutant only induced detectable activation. The efficiency of the CH3 mutants varied, with loop 6CH3 being the least effective and equal to loop 6 CH1. Considering both rAb secretion level and T cell activation efficiency, a total of eight loops may carry T cell epitopes to APC for processing and presentation to T cells, namely, all in CH2 in addition to loop 6 in CH1 and CH3. Comparing loop 4CH2 with loop 6CH1 mutants after injection of Ab in BALB/c mice, the former was by far the most efficient and induced specific T cell activation at concentrations at least 100-fold lower than loop 6CH1.

Recombinant antibodies for delivery of antigen : a single loop between β-strands in the constant region can accommodate long, complex and tandem T cell epitopes

Recombinant antibodies are increasingly used for efficient delivery of T cell epitopes to antigen-presenting cells (APCs), both for vaccination purposes and for immune modulation. We have previously shown that recombinant antibodies can accommodate single T cell epitopes inserted into loops between beta-strands in constant (C) domains. Such recombinant antibodies have in addition been equipped with variable regions that target APCs for increased delivery of C region T cell epitopes. We here show that loop 6 (loop FG) in C(H)1 of human gamma 3 can be exchanged with (i) long T cell epitopes up to 37 amino acids, (ii) epitopes with complex secondary structure such as gluten epitopes with a type II polyproline helical confirmation and (iii) two tandemly linked T cell epitopes. T cell responses increased with T cell epitope elongation, presumably due to a positive influence of flanking residues. Recombinant antibodies targeted to either CD14 on monocytes or HLA-DP on monocytes and dendritic cells gave similar results and were 2-4 logs more efficient at stimulating human T cells than were non-targeted controls. Thus, single loops in C regions of recombinant antibodies seem versatile and may be used for delivery of lengthy, complex and multiple T cell epitopes to human APCs.

CD40/APC-specific antibodies with three T-cell epitopes loaded in the constant domains induce CD4+ T-cell responses.

CD4+ T lymphocytes play a central role in the orchestration and maintenance of the adaptive immune response. Targeting of antigen to antigen presenting cells (APCs) increases peptide loading of major histocompatibility complex (MHC) class II molecules and CD4+ T-cell activation. APCs have been targeted by APC-specific recombinant antibodies (rAbs) with single T-cell epitopes integrated in the constant region of the heavy chain (C(H)). However, the strategy may be improved if several T-cell epitopes could be delivered simultaneously by one rAb. We here demonstrate that a single rAb can be loaded with multiple identical or different T-cell epitopes, integrated as loops between β-strands in C(H) domains. One epitope was inserted in C(H)1, while two were placed in C(H)2 of IgG. T-cell proliferation assays showed that all three peptides were excised from loops and presented on MHC class II to T-cells. Induction of T-cell activation by each epitope in the multi-peptide rAb was as good, or even better, than that elicited by corresponding single-peptide rAbs. Furthermore, following DNA vaccination of mice with plasmids that encode CD40-specific rAbs loaded with either one or three peptides, T-cell responses were induced. Thus, integration of multiple epitopes in C(H) region loops of APC-specific rAbs is feasible and may be utilized in design of multi-vaccines.

“Troy-bodies”: Recombinant Antibodies that Target T Cell Epitopes to Antigen Presenting Cells

Targeting of antigens to antigen presenting cells (APC) results in enhanced antigen presentation and T cell activation. In this paper, we describe a novel targeting reagent denoted “Troy-bodies”, namely recombinant antibodies with APC-specific V regions and C regions with integrated T cell epitopes. We have made such antibodies with V regions specific for either IgD or MHC class II, and four different T cell epitopes have been tested. All four epitopes could be introduced into loops of C domains without disrupting Ig folding, and they could be released and presented by APC. Furthermore, whether IgD-or MHC-specific, the molecules enhanced T cell stimulation compared to nonspecific control antibodies in vitro as well as in vivo. Using this technology, specific reagents can be designed that target selected antigenic peptides to an APC of choice. Troy-bodies may therefore be useful for manipulation of immune responses, and in particular for vaccination purposes.