Biomedical subjects

I Sandlie

Publications and source records attributed to I Sandlie.

At least 19 recordsLinked to original sources

PubMed2002

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.

Amino Acid Sequence↗

PubMed2001

'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.

Animals↗

PubMed2001

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.

Amino Acid Sequence↗

PubMed2001

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.

Amino Acid Sequence↗

PubMed2001

Recombinant chimeric OKT3 scFv IgM antibodies mediate immune suppression while reducing T cell activation in vitro.

OKT3, a mouse anti-human CD3 monoclonal antibody (mAb), is a potent immunosuppressive agent used in clinical transplantation to treat allograft rejection. Two major drawbacks of this therapy are the systemic release of several cytokines due to cross-linking mediated by the mAb between T cells and FcgammaR-bearing cells and the human anti-mouse antibody (HAMA) response. To overcome these side effects, three chimeric OKT3 single chain variable fragment (scFv) IgM antibodies, scOKT3-gamma DeltaIgM wt, scOKT3-gamma DeltaIgM C575S and scOKT3-gamma DeltaIgM VAEVD, were generated. They consist of the light and heavy variable binding domains of OKT3 mAb as well as the CH3 and CH4 domains of different human IgM variants linked with a human IgG3 hinge region to provide more flexibility and stability. Like the native IgM, scOKT3-gamma DeltaIgM antibodies are able to form polymeric structures, which lead to an increase in binding affinity and immunosuppressive potential compared with the parental OKT3 mAb. However, independently of their polymerization, all scOKT3-gamma DeltaIgM constructs do not induce any significant T cell proliferation or cytokine release (IL-2, TNF-alpha and IFN-gamma) in in vitro assays, while their CD3-modulating properties are retained. These results suggest that the use of scOKT3-gamma DeltaIgM antibodies may offer significant advantages over the OKT3 mAb in improving clinical immunosuppressive treatment.

Adult↗

PubMed2001

"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.

Animals↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed2000

Lysine 322 in the human IgG3 C(H)2 domain is crucial for antibody dependent complement activation.

The classical complement activation cascade of the immune system is initiated by multivalent binding of its first component, C1q, to the Fc region of immunoglobulins in immune complexes. The C1q binding site on mouse IgG2b has been shown to contain the amino acids Glu 318, Lys 320 and Lys 322 in the C(H)2 domain (Duncan, A.R., Winter, G.,1988. The binding site for C1q on IgG. Nature 322 738-740). Identical or closely related motifs are found on all IgGs in all species, and the binding site has therefore been thought to be universal. However, the results from another study indicate that the site is different in human IgG1 molecules (Morgan, A., Jones, N.D., Nesbitt, A.M., et al., 1995. The N-terminal end of the C(H)2 domain of chimeric human IgG1 anti-HLA-DR is necessary for C1q, Fc gamma RI and Fc gamma RIII binding. Immunology 86 319-324). To determine the site(s) responsible for complement activation in anti-NIP-mouse/human IgG3 antibodies, we have mutated amino acids Lys 276, Tyr 278, Asp 280, Glu 318, Lys 320 and Lys 322 in two beta-strands in the C(H)2 domains of human IgG3. In addition, we mutated the Glu 333, which resides in close proximity to the postulated C1q-binding site of mouse IgG2b, as well as Leu 235 in the lower hinge region. All mutants were tested in Antibody Dependent Complement Mediated Lysis (ADCML)(4) assays, where the antigen concentration on target cells was varied and human serum was complement source. Only the mutants that lacked the positively charged side chain of lysine in position 322 showed strong reduction in ADCML, particularly at low antigen density on target cells. Alanine scanning of positions 318 and 320 did not affect ADCML, contrary to what was observed for mouse IgG2b. Neither did a leucine to glutamic acid mutation in position 235 have the effect that has been reported for human IgG1. These results suggest that the complement binding site on human IgG3 molecules is different from that found on mouse IgG2b, and possibly on human IgG1 as well. Thus the contact site may not be conserved.

Binding Sites↗

PubMed2000

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.

Humans↗

PubMed1999

Polymerization of IgA and IgM: roles of Cys309/Cys414 and the secretory tailpiece.

We have investigated how the secretory tailpiece (tp), Cys414 and the amino acids flanking Cys414 or Cys309 are involved in regulating the different polymerization of IgM and IgA to pentamers and dimers/monomers, respectively. Whereas changing the tp of IgM to that of IgA has little effect on IgM polymerization, introducing the mu tp to IgA leads to the formation of larger than wild-type IgA polymers, including pentamers and hexamer. This shows that the secretory tp can differentially regulate polymerization depending on the heavy chain context. Cys414, which is engaged in intermonomeric disulfide bonds in IgM, is not crucial for the difference in IgM and IgA polymerization; IgM with a C414S mutation forms more large polymers than IgA. Also, IgA with IgM-like mutations in the five amino acids flanking Cys309, which is homologous to Cys414, oligomerize similarly as IgA wild type. Thus, IgA appears to have an inherent tendency to form monomers and dimers that is partially regulated by the tp, while the Cys309 region has only a minor effect. We also show that complement activation by IgM is sensitive to alterations in the polymeric structure, while IgA is inactive in classical complement activation even for polymers such as pentamers and hexamers.

Amino Acid Sequence↗

PubMed1999

Recombinant expression of polymeric IgA: incorporation of J chain and secretory component of human origin.

Mucosal J (joining) chain-expressing IgA immunocytes produce dimeric IgA that is actively transported by the epithelial polymeric Ig receptor (pIgR) to exocrine secretions. Release of secretory IgA (SIgA) occurs by cleavage of the covalently linked pIgR ectodomain, also known as bound secretory component. We have identified the human J-chain cDNA sequence through database screening, and isolated it from B cells for recombinant expression. Co-expression of this cDNA with an alpha heavy chain and a lambda light chain in Chinese hamster ovary (CHO) cells resulted in a mixture of recombinant monomeric and dimeric IgA in culture supernatants. This dimeric IgA was transported by the pIgR-mediated mechanism in vitro. Furthermore, expression of the human pIgR ectodomain together with the dimeric IgA, resulted in production of complete SIgA by the CHO cells. These results demonstrated that co-expression of the necessary polypeptide components allows a single mammalian cell to produce SIgA. Development of production systems for human antigen-specific recombinant SIgA may be important for applications in passive mucosal vaccination.

Amino Acid Sequence↗

PubMed1999

Complement-mediated lysis of cultured osteosarcoma cell lines using chimeric mouse/human TP-1 IgG1 and IgG3 antibodies.

Osteosarcoma is the commonest malignant tumour of the bones. The presence of micrometastases at the time of primary diagnosis is associated with poor prognosis. Despite developments in surgery and aggressive chemotherapy, about 50% of the patients still succumb to the disease. Thus, there is a need to develop alternative treatment modalities. One such strategy is to use antibodies with improved effector functions. The two monoclonal antibodies, TP-1 and TP-3, recognize a tumour-associated antigen on human osteosarcoma cells. In the present study, we describe the cloning of the TP-1 variable genes, and the production of complete chimeric mouse/human monoclonal antibodies. Constructs containing the constant genes from human IgG1, IgG3 or a mutant IgG3 with a shortened hinge region, called m15, were expressed in the mouse myeloma cell line, NS0. The m15 mutant has been shown to be very potent in triggering complement-mediated lysis. Our goal was to investigate whether this mutant could overcome the complement protection on human osteosarcoma cells, which is generally present on all human cells. We found that the target cells expressed several membrane-bound complement inhibitors, and that masking of these inhibitors rendered the cells sensitive to lysis. The m15 mutant exhibited greater lytic activity than both IgG3 and IgG1, although it could not cause extensive killing of the target cells alone.

Animals↗

PubMed1999

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.

Animals↗

PubMed1998

Comparisons of the ability of human IgG3 hinge mutants, IgM, IgE, and IgA2, to form small immune complexes: a role for flexibility and geometry.

Various native and hinge-modified forms of Ig with identical Ids were reacted with an anti-Id mAb, and the resultant immune complexes were analyzed by negative stain immunoelectron microscopy. Complexes were scored for their geometry (linear versus ring complexes) and size (dimer, trimer, etc.). Ring dimers are the thermodynamically most favorable configuration, unless inhibited by steric and/or flexibility constraints. We found ring dimerization to correlate with the length of the upper, but not middle or lower, hinge. In contrast, the geometry and size of complexes of those molecules lacking formal hinges were unpredictable. A hingeless IgG mutant and native IgE readily formed ring dimers. Remarkably, monomeric IgM formed more ring dimers than any of the other Igs tested, including IgG3. We also tagged the Fab arms and measured the mean Fab-Fab angles and the degree of angular variation for each type of Ig. Surprisingly, IgM proved the most flexible by this assay. In hinged Igs, there was a correlation between length of the upper hinge and Fab-Fab flexibility. In contrast, we found no correlation between the mean Fab-Fab angle in uncomplexed Igs and their ability to dimerize with anti-Id mAb. These data suggest that the physicochemical methods typically used to evaluate molecular flexibility are often of low predictive value when tested in a functional assay.

Amino Acid Sequence↗

PubMed1998

The influence of the hinge region length in binding of human IgG to human Fcgamma receptors.

Interactions between human IgG with human FcgammaRI and FcgammaRIIa (R131) were studied to investigate the role of the hinge region of IgG3 and IgG1 in the binding of the antibodies to FcgammaR. It was found that a hinge deletion mutant of IgG3 (IgG3 m15) was reduced in its ability to bind to FcgammaRI and FcgammaRIIa but was more potent at activating ADCC by activated lymphocytes (FcgammaRIIIa-mediated), compared to the wild-type version of IgG3. The human IgG1 allotype G1m(a,z) was more efficient at binding to FcgammaRI than the two IgG3 antibodies tested. The IgG1 and IgG3 wild type antibodies were better able to bind to FcgammaRII than the hinge deletion mutant version of IgG3. The data suggest a role for the hinge region in influencing FcgammaR mediated effector functions in IgG3.

Amino Acid Sequence↗

PubMed1998

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.

Amino Acid Sequence↗

PubMed1998

Activation of complement by human IgG1 and human IgG3 antibodies against the human leucocyte antigen CD52.

Activation of the complement cascade by immunoglobulin G (IgG) plays a major role in the host defense against pathogens. Using recombinant human antibodies specific for the leucocyte antigen CD52, different allotypes of human IgG1 subclass were compared for their ability to activate human complement. In addition the roles of the different length hinge regions of IgG1 and IgG3 were investigated. It was found that the naturally occurring allotypes G1m(a,z) and G1m(f), and one artificially created isoallotype, G1m(null), did not significantly differ in their overall ability to cause cell lysis. However, some differences in binding of individual components of the classical activation pathway were detected. More of the complement component C1s seemed to be associated with the allotype G1m(f), although this did not result in an overall improvement in lytic potency. In this system the wild-type IgG3 was found to be less effective in complement lysis than IgG1. By shortening the hinge region of IgG3 to resemble that of an IgG1 antibody, increased complement binding was observed compared with that of wild-type IgG3 and the IgG1 allotypes. The overall lytic potency of the antibody was also improved compared with wild type IgG3 and it was also slightly more effective than the IgG1 allotypes.

Antigens, CD↗

PubMed1997

Versatile vectors for transient and stable expression of recombinant antibody molecules in mammalian cells.

We have developed new cassette expression vectors for the cloning of any intact V-region gene followed by any C-region gene. Both the heavy-and light chain vectors harbor a strong hCMV promoter, restriction site cassettes for cloning of both V- and C-region genes, transcription termination signals, fl-ori for single stranded DNA (ssDNA) synthesis, selection marker for Neomycin and SV40 ori for transient expression. The vectors accept VH and VL chain genes obtained by RT-PCR. Reamplification of the V genes is then performed with a new set of primers which are designed specifically for each individual V gene. Cloning into the vectors is aided by restriction sites located just outside the V-gene coding region, thus keeping the V-genes intact. The vectors also contain cloning sites for the exchange of genomic C-genes so that the resulting Ig genes may code for complete antibodies, antibody fragments or fusion proteins. A simple subcloning step permits the expression of both heavy and light chain genes from one single vector, thus avoiding co-transfection of the two vectors. The usefulness of the vectors was confirmed by construction of mouse-human chimeric antibodies. The V-genes were derived from a hybridoma cell line, TP-3, and was combined with human C kappa, C gamma 3 and C gamma 1 genes as well as with CH1 gamma 3. High yields of recombinant antibody products in NSO cells were obtained. Transient expression was also demonstrated.

Animals↗