Troybodies and pepbodies.

All antibodies (Abs) with effector function are produced in mammalian cells, whereas bacterial production is restricted to smaller targeting fragments (scFv and Fab) without effector functions. In this project, we isolated different peptides that bind one of several Ab effector molecules. We have developed bacterial expression vectors for direct cloning of these peptides as fusions to scFv and Fab, and have obtained targeting fragments that also have the ability to bind Ab effector molecules. Some of these fusions (pepbodies) may also initiate Ab effector functions. We have also genetically inserted T-cell epitopes into Abs with specificity for antigen-presenting cell (APC) surface molecules to target the Ab-T-cell epitope fusions (Troybodies) to APCs. The approach is to exchange loops in Ig constant domains with single copies of well-defined T-cell epitopes. We have shown that a number of such T-cell epitopes are loaded on to MHC class II on APCs and are presented to specific T-cells. An increase in T-cell activation of up to four orders of magnitude is achieved compared with synthetic peptide. Our current goal is to identify all the loops in all Ig constant domains that may be loaded with T-cell epitopes to produce a multi-vaccine.

'Troy-bodies': antibodies as vector proteins for T cell epitopes.

A major objective in vaccine development is the design of reagents that give a strong, specific T cell response. 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-specificity and with T cell epitopes integrated in their C regions. We have made such antibodies with V regions specific for either IgD or MHC class II, and five different T cell epitopes have been tested. All epitopes could be introduced into loops of C domains without disrupting immunoglobulin (Ig) folding. Four have been tested in T cell activation studies, and all could be released and presented by APC. Furthermore, whether IgD- or MHC-specific, the molecules tested enhanced T cell stimulation compared to non-specific 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.

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 C(H)1 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 10(4) to 10(5) 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.

T cell recognition of the dominant I-A(k)-restricted hen egg lysozyme epitope: critical role for asparagine deamidation.

Type-B T cells raised against the immunodominant peptide in hen egg lysozyme (HEL(48-62)) do not respond to whole lysozyme, and this has been thought to indicate that peptide can bind to l-A(k) in different conformations. Here we demonstrate that such T cells recognize a deamidated form of the HEL peptide and not the native peptide. The sequence of the HEL epitope facilitates rapid and spontaneous deamidation when present as a free peptide or within a flexible domain. However, this deamidated epitope is not created within intact lysozyme, most likely because it resides in a highly structured part of the protein. These findings argue against the existence of multiple conformations of the same peptide-MHC complex and have important implications for the design of peptide-based vaccines. Furthermore, as the type-B T cells are known to selectively evade induction of tolerance when HEL is expressed as a transgene, these results suggest that recognition of posttranslationally modified self-antigen may play a role in autoimmunity.

"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 non-specific 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.

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.

Structural requirements for incorporation of J chain into human IgM and IgA.

J chain is associated with pentameric IgM and dimeric IgA via disulfide bonds involving the penultimate cysteine residue in the secretory tailpiece of the mu or the alpha heavy chain. We have investigated the structural basis for incorporation of J chain by analyzing several IgM mutants, IgA mutants and IgG/IgM hybrid molecules. IgM mutants with the mu secretory tailpiece replaced by the alpha secretory tailpiece and/or Cys414 replaced by serine incorporated J chain, although in reduced amounts correlating with reduced pentamer/polymer formation. In addition to pentamers, tetramers of IgMC414S contained J chain, while no J chain was associated with smaller polymers or hexamers of IgM. An IgA/IgM hybrid tailpiece abolished J chain incorporation to pentameric IgM. Analysis of IgG molecules that have added a secretory tailpiece and/or have IgM domain replacements showed that J chain incorporation depends on regions of the C(mu)4 domain in addition to the tailpiece. Features of the C(mu)3 domain other than Cys414 also play a role in efficient formation of pentamers and J chain incorporation, while the C(mu)2 domain is not specifically required. By analysis of two IgA mutants that formed larger polymers than IgAwt, we found J chain equally incorporated into dimers, trimers, tetramers and pentamers. Thus, the results show that J chain incorporation into IgA does not depend on the polymeric structure, while J chain incorporation into IgM is restricted to certain polymeric conformations.

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.

Effect of the IgM and IgA secretory tailpieces on polymerization and secretion of IgM and IgG.

Pentameric IgM and dimeric IgA both contain disulfide bonds between cysteines located in the secretory tailpieces of the heavy chains. To compare the influences of the mu and alpha tailpieces on the polymeric structure, we have replaced amino acids in the tailpiece of the human mu-chain with amino acids found in the corresponding positions in the human alpha tailpiece. We show that an IgM with an alpha tailpiece (IgM L561H, Y562V, L566V, S569A, D570E, T571V, and A572D) as well as IgM L561H, Y562V, and IgM A572D have a size distribution similar to that of wild-type IgM. However, one IgM mutant with a mu/alpha hybrid tailpiece (IgM L566V, S569A, D570E, T571V, and A572D) is secreted as a mixture of mainly hexamers, pentamers, tetramers, and dimers. The tetramers and dimers are specifically formed and secreted at the expense of pentamers and hexamers; no alterations in polymerization or secretion rates were observed. We have also incorporated the mu, alpha, and hybrid mu/alpha tailpieces to a human IgG3 or IgGL309C mutant. The IgG-tailpiece mutants are poorly secreted, but the secreted fractions contain multimeric molecules. Each of the mutants that contain both the L309C mutation and a secretory tailpiece forms mainly hexamers; however, small differences in polymer distribution exist for the different tailpieces. Comparison of the influence of different tailpieces on IgM and IgG polymeric structures suggests that the function of a specific tailpiece is dependent on other parts of the heavy chain, which can vary for different isotypes.

Effects of pneumoperitoneum on splanchnic hemodynamics: an experimental study in pigs.

OBJECTIVE: To study the effects on splanchnic haemodynamics of pneumoperitoneum induced by carbon dioxide insufflation. DESIGN: Controlled experimental study. ANIMALS: 11 Pigs weighing 19-30 kg. INTERVENTION: The animals were divided into a control group (n = 4) and a experimental group (n = 7). Experimental animals were subjected to stepwise increasing intra-abdominal pressure from 0 mm Hg to 25 mm Hg by carbon dioxide insufflation. MAIN OUTCOME MEASURES: Portal venous blood flow, portal venous blood pressure, portal/hepatic vascular resistance, and gastrointestinal vascular resistance. RESULTS: At 25 mm Hg portal venous blood flow was reduced (66% of baseline), and portal venous blood pressure and portal/hepatic vascular resistance were increased (360% and 650% of baseline, respectively). The increase in gastrointestinal vascular resistance was less pronounced. CONCLUSIONS: Increased intra-abdominal pressure caused significant changes in the splanchnic haemodynamics. The risk was greater if the intra-abdominal pressure exceeded 15 mm Hg.

Intestinal mucosal injury during porcine faecal peritonitis.

OBJECTIVE: To assess the incidence of intestinal mucosal injury during faecal peritonitis in pigs, the relation of such lesions to haemodynamic variables, intramucosal pH (pHi), and endothelial adherence of polymorphonuclear leucocytes (PMNs). DESIGN: Experimental laboratory study. SETTING: University department of surgery, Sweden. SUBJECTS: 57 Juvenile pigs. INTERVENTIONS: Pigs had faecal peritonitis induced (n = 39) or a sham procedure (n = 18). In addition, 15 animals were pretreated with the monoclonal CD18 receptor antibody IB4 before induction of peritonitis with the aim of preventing tissue accumulation of PMNs. MAIN OUTCOME MEASURES: Development of mucosal lesions and correlation with haemodynamic variables. RESULTS: 17/18 (94%) of control animals had normal mucosa. The incidence of mucosal lesions in animals with faecal peritonitis was 56%. Animals with severe mucosal injury (grade 4-5) had significantly lower mean arterial pressure, cardiac index, and pHi during the last hour of the experiment compared with animals without mucosal lesions. Pretreatment with IB4 did not prevent the development of intestinal mucosal injuries. Intramucosal pH decreased during sepsis and was not affected by IB4. CONCLUSIONS: Severe intestinal mucosal injury is associated with arterial hypotension, low cardiac index, and low pH. Neither the mucosal injury nor the reduction in pHi seen during porcine faecal peritonitis seemed to be leucocyte-related phenomena.

Relationship between plasma volume reduction and plasma electrolyte changes after prolonged bicycle exercise, passive heating and diuretic dehydration.

Plasma volume was decreased by prolonged bicycle exercise, by passive heating in warm water, by sauna dehydration, and by diuretically induced dehydration in eleven well trained subjects. Blood samples from an arm vein were taken before and after this pre-treatment, as well as after a subsequent standard exercise test (SET) on a bicycle ergometer (50%, 70% and 105% of max VO2; the SET with no pre-treatment was used as a control condition. The changes in plasma concentration of Na+, K+ and Cl- were not proportional to the calculated plasma volume changes. The Na+ and Cl- concentrations always increased in the plasma, while plasma potassium concentration was increased after prolonged exercise, but decreased after the other types of dehydrations. The standard exercise test produced a pronounced fall in total calculated plasma potassium and in K+ concentration measured 3-5 min after exercise in all types of experiments. In the standard exercise test the calculated water loss from the plasma volume was relatively large. It amounted to about 2/3 of the total water loss in the standard exercise test and was independent of the pre-treatments.