Elin Lunde

Antibodies engineered with IgD specificity efficiently deliver integrated T-cell epitopes for antigen presentation by B cells

We have developed a strategy for improving the stimulation of T cells during immune responses by constructing recombinant antibodies that enhance the delivery of antigen to antigen-presenting cells, such as B cells. These antibodies have variable regions specific for surface molecules on B cells, and a constant region with an inserted antigen. In vitro, such antibodies make B cells approximately 1000-fold more efficient at presenting antigen and stimulating specific T cells. In vivo, the antibodies turn B cells of the spleen into potent stimulators of T cells. This approach may be useful for the generation of new vaccines.

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.

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.

Immunoglobulin as a vehicle for foreign antigenic peptides immunogenic to T cells

Antibody (Ab) molecules may serve as targeting vehicles for delivery of foreign antigenic peptides to antigen presenting cells (APC). An attractive strategy is to substitute segments between beta-strands of immunoglobulin (Ig) constant (C)-region domains with antigenic peptides. For this to work, the mutant Ab must maintain its conformation so that it can be secreted from transfected cells. Furthermore, the antigenic peptides must be excised by the processing machinery of APC and loaded onto major histo-compatibility complex (MHC) class II molecules. To test this, we have introduced a peptide of eleven amino acids (a.a.) as either of three different loops in the first C-region domain of the heavy (H) chain (CH1) of human IgG3. When the resulting mutant H chain genes were expressed in a fibroblast cell line equipped with proper class II molecules, the H chains were retained intracellularly, probably due to the light (L) chain deficiency of the fibroblasts. Nevertheless, by the endogenous class II processing pathway, presentation of the epitope to CD4+ cells was observed for all three mutants. The presentation efficiency, however, depended on the position of the peptide in the H chain. This could be due to influence of flanking sequences, which differ in the three loop replacement mutants. When L chain-expressing Chinese hamster ovary (CHO) lambda cells were transfected with the same constructs, two out of the three mutant Ig were secreted. The mutants had the expected antigen specificity and were recognized by anti-IgG Ab. When added exogenously to dendritic cell APC, the mutant IgG3 were processed, and the liberated foreign epitopes presented to T cells. The results suggest that the loops connecting beta-strands in the Ig fold may be replaced by foreign peptides, which upon processing become stimulatory to CD4+ T cells. Combined with the well-known targeting function of antibodies, this principle may be useful for construction of a new generation of vaccines.

The cellular mechanism by which complementary Id + and anti-Id antibodies communicate: T cells integrated into idiotypic regulation

The V region antigenic determinants (idiotopes (Ids)) of antibodies (Abs) have been suggested to be involved in regulating the immune system. Certain diseases such as diabetes mellitus have recently been associated with a disequilibrium between Id+ and anti‐Id Abs. However, it is unknown how Abs carrying complementary idiotypes (that is, Id+ and anti‐Id Abs) regulate each other at the level of B and T cells. In this study, we show that B lymphoma cells genetically equipped with anti‐Id BCR V regions receive a signal when exposed to Id+Ig. Moreover, they become × 104 more efficient at presenting exogenous Id+ Ab to CD4+ T cells in vitro. Activated Id‐specific T cells in turn regulated the Id‐specific B lymphoma cells. Similar results were obtained in vivo in a surrogate model in which an Id‐peptide was incorporated genetically into the C‐region of a recombinant Ab that targeted IgD on B cells. The findings suggest that conventional T–B collaboration can explain communication between complementary Id+ and anti‐Id Ab at the cellular level. A model is suggested that integrates present and previous data on B‐cell regulation by Id‐specific T cells.

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.

Periplasmic expression of soluble single chain T cell receptors is rescued by the chaperone FkpA

BackgroundEfficient expression systems exist for antibody (Ab) molecules, which allow for characterization of large numbers of individual Ab variants. In contrast, such expression systems have been lacking for soluble T cell receptors (TCRs). Attempts to generate bacterial systems have generally resulted in low yields and material which is prone to aggregation and proteolysis. Here we present an optimized periplasmic bacterial expression system for soluble single chain (sc) TCRs.ResultsThe effect of 1) over-expression of the periplasmic chaperon FkpA, 2) culture conditions and 3) molecular design was investigated. Elevated levels of FkpA allowed periplasmic soluble scTCR expression, presumably by preventing premature aggregation and inclusion body formation. Periplasmic expression enables disulphide bond formation, which is a prerequisite for the scTCR to reach its correct fold. It also enables quick and easy recovery of correctly folded protein without the need for time-consuming downstream processing. Expression without IPTG induction further improved the periplasmic expression yield, while addition of sucrose to the growth medium showed little effect. Shaker flask yield of mg levels of active purified material was obtained. The Vαβ domain orientation was far superior to the Vβα domain orientation regarding monomeric yield of functionally folded molecules.ConclusionThe general expression regime presented here allows for rapid production of soluble scTCRs and is applicable for 1) high yield recovery sufficient for biophysical characterization and 2) high throughput screening of such molecules following molecular engineering.

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.

Induction of central T cell tolerance: Recombinant antibodies deliver peptides for deletion of antigen‐specific CD4+8+ thymocytes

In order to prevent or ameliorate autoimmune disease, it would be desirable to induce central tolerance to peripheral self‐antigens. We have investigated whether recombinant antibodies (Ab) that deliver T cell epitopes to antigen‐presenting cells (APC) in the thymus can be used to induce thymocyte deletion. Troybodies are recombinant Ab with V regions specific for APC surface molecules that have T cell epitopes genetically introduced in their C domains. When MHC class II‐specific Troybodies with the λ2315 T cell epitope were injected into λ2315‐specific TCR transgenic mice, a profound deletion of CD4+8+ thymocytes was observed. MHC class II‐specific Troybodies were 10–100‐fold more efficient than non‐targeting peptide Ab, and 500‐fold more efficient than synthetic peptide at inducing deletion. Similar findings were observed when MHC class II‐specific Troybodies with the OVA323–339 T cell epitope were injected into OVA‐specific TCR transgenic mice. Although deletion was transient after a single injection, newborn mice repeatedly injected with MHC class II‐specific Troybodies for 4 weeks, had reduced antigen‐specific T cells in peripheral lymphoid tissues and reduced T cell responses. These experiments suggest that Troybodies constructed to target specifically thymic APC could be useful tools for induction and maintenance of central T cell tolerance in autoimmune diseases.

Stabilizing mutations increase secretion of functional soluble TCR-Ig fusion proteins

BackgroundWhereas T cell receptors (TCRs) detect peptide/major histocompatibility complexes (pMHCs) with exquisite specificity, there are challenges regarding their expression and use as soluble detection molecules due to molecular instability. We have investigated strategies for the production of TCR-immunoglobulin (Ig) fusion proteins. Two different TCRs that are characteristic of a mouse model for idiotype (Id) dependent immune regulation were engineered. They are structurally unrelated with different variable (V), diversity (D) and joining (J) segments, but each share one V gene segment, either Vα or Vβ, with the well characterized murine TCR, 2C.ResultsSeveral TCR-Ig formats were assessed. In one, the TCR V domains were fused to Ig constant (C) regions. In others, the complete extracellular part of the TCR was fused either to a complete Ig or an Ig Fc region. All molecules were initially poorly secreted from eukaryotic cells, but replacement of unfavourable amino acids in the V regions improved secretion, as did the introduction of a disulfide bridge between the TCR C domains and the removal of an unpaired cysteine. A screening strategy for selection of mutations that stabilize the actual fusion molecules was developed and used successfully. Molecules that included the complete heterodimeric TCR, with a stabilizing disulfide bridge, were correctly folded as they bound TCR-specific antibodies (Abs) and detected pMHC on cells after specific peptide loading.ConclusionsWe show that fully functional TCR-Ig fusion proteins can be made in good yields following stabilizing engineering of TCR V and C region genes. This is important since TCR-Ig fusions will be important probes for the presence of specific pMHCs in vitro and in vivo. In the absence of further affinity maturation, the reagents will be very useful for the detection of kinetic stability of complexes of peptide and MHC.