There is still no general vaccine for prevention of disease caused by group-B meningococcal strai... more There is still no general vaccine for prevention of disease caused by group-B meningococcal strains. Meningococcal lipopolysaccharides (LPSs) have received attention as potential vaccine candidates, but concerns regarding their safety have been raised. Peptide mimics of LPS epitopes may represent safe alternatives to immunization with LPS. The monoclonal antibody (MoAb) 9-2-L3,7,9 [1] specific for Neisseria meningitidis LPS immunotype L3,7,9 is bactericidal and does not cross-react with human tissue. To explore the possibility of isolating peptide mimics of the epitope recognized by MoAb 9-2-L3,7,9, we have constructed two phage display libraries of six and nine random amino acids flanked by cysteines. Furthermore, we developed a system for the easy exchange of peptide-encoding sequences from the phage-display system to a hepatitis B core (HBc) expression system. Cyclic peptides that specifically bound MoAb 9-2-L3,7,9 at a site overlapping with the LPS-binding site were selected from both libraries. Three out of four tested peptides which reacted with MoAb 9-2-L3,7,9 were successfully presented as fusions to the immunodominant loop of HBc particles expressed in Escherichia coli. However, both peptide conjugates to keyhole limpet haemocyanin and HBc particle fusions failed to give an anti-LPS response in mice.
: The major histocompatibility class I-related neonatal Fc receptor, FcRn, salvages both IgG and ... more : The major histocompatibility class I-related neonatal Fc receptor, FcRn, salvages both IgG and albumin from degradation and thus contributes to maintain high serum levels of these proteins. Analbuminemia is a rare autosomal recessive disorder characterized by clinically observed allelic albumin variants that are absent or found in very low concentrations in the blood circulation. Such variants may have altered FcRn binding properties that affect their half-life, biodistribution and thereby transport ability. : We established an easy cloning, expression and purification strategy to obtain recombinant GST-tagged human serum albumin (HSA) variants for evaluation of pH dependent FcRn binding properties using an enzyme-linked immunosorbent assay (ELISA) and a real time surface plasmon resonance (SPR) biosensor system. : The strategy yielded purified GST-tagged albumin variants. A recombinant truncated HSA variant similar to a clinically observed splice mutant denoted Bartin, here abrogated HSA(Bartin), showed no detectable pH dependent FcRn binding compared to a fully functional albumin wild type variant, HSA(Wt), and a truncated HSA variant consisting of only the carboxy terminal domain III (HSA(DIII)). : The approach described can be used to rapidly screen clinically observed truncated or otherwise mutant or modified HSA variants regarding their pH dependent FcRn binding properties. Here, we demonstrate that a recombinant truncated HSA variant, HSA(Bartin), does not interact with FcRn, which gives a molecular explanation for the low serum levels. In addition, DIII of HSA alone was shown to retain its FcRn binding property.
Phage display has been instrumental for the success of antibody (Ab) technology. The aim of the p... more Phage display has been instrumental for the success of antibody (Ab) technology. The aim of the present study was to explore phage display of soluble T-cell receptors (TCRs). A library platform that supports engineering and selection of improved TCRs to be used as detection reagents for specific antigen presentation will be very useful. In such applications, high, equal and clone independent display levels are a prerequisite for 'fair' selection. Therefore, we explored how different pIII fusion formats and modes affected the display levels of two murine a/b TCRs. Both are derived from T-cell clones associated with the MOPC315 myeloma model. The results show that the design of the pIII fusion particle significantly affects the subsequent display levels. Furthermore, successful display may be obtained both in phagemid and phage versions. Importantly, improvement of poor display can be achieved by over-expressing the periplasmic chaperone FkpA.
We have developed a strategy for improving the stimulation of T cells during immune responses by ... more 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.
Recombinant antibodies are increasingly used for efficient delivery of T cell epitopes to antigen... more Recombinant antibodies are increasingly used for efficient delivery of T cell epitopes to antigenpresenting 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 b-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 g3 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.
Here we unravel the structural features of human IgM and IgA that govern their interaction with t... more 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.
We have constructed a fusion protein composed of TNF-α and the scFv region of mAb B1, an antibody... more We have constructed a fusion protein composed of TNF-α and the scFv region of mAb B1, an antibody that recognizes a cancer associated carbohydrate antigen (LeY). The fusion protein was expressed in E. coli and found in inclusion bodies.
Various native and hinge-modified forms of Ig with identical Ids were reacted with an anti-Id mAb... more 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, t...
In IgG molecules removal of N-linked glycan results in a conformational change that abolishes bin... more In IgG molecules removal of N-linked glycan results in a conformational change that abolishes binding to Fc gamma receptor proteins on the surface of leukocytes and clearance of abnormal target cells. We have developed a robust flow cytometric screening system for the isolation of aglycosylated Fc mutants exhibiting high affinity to desired Fc receptors despite the lack of glycosylation. A series of complex libraries (107-x109 clones) were generated by random or insertional mutagenesis of the Fc domain and screened for binding to purified Fc gamma receptors. Mutant aglycosylated Fcs that bind with high affinity and specificity were isolated and characterized in vitro. The aglycosylated Fc gamma receptor binding mutant proteins contain between 1-11 amino acid substitutions. We found that the mutations allowed highly selective binding of an aglycosylated, anti-Her2 (trastuzumab) to the FcRI receptor, with desired affinity indistinguishable from that of CHO-derived, glycosylated trastu...
Journal of immunology (Baltimore, Md. : 1950), Jan 22, 2015
Engineering of the constant Fc part of monoclonal human IgG1 (hIgG1) Abs is an approach to improv... more Engineering of the constant Fc part of monoclonal human IgG1 (hIgG1) Abs is an approach to improve effector functions and clinical efficacy of next-generation IgG1-based therapeutics. A main focus in such development is tailoring of in vivo half-life and transport properties by engineering the pH-dependent interaction between IgG and the neonatal Fc receptor (FcRn), as FcRn is the main homeostatic regulator of hIgG1 half-life. However, whether such engineering affects binding to other Fc-binding molecules, such as the classical FcγRs and complement factor C1q, has not been studied in detail. These effector molecules bind to IgG1 in the lower hinge-CH2 region, structurally distant from the binding site for FcRn at the CH2-CH3 elbow region. However, alterations of the structural composition of the Fc may have long-distance effects. Indeed, in this study we show that Fc engineering of hIgG1 for altered binding to FcRn also influences binding to both the classical FcγRs and complement f...
Idiotypes (Id) are antigenic determinants localized in variable (V) regions of Ig. Id-specific T ... more Idiotypes (Id) are antigenic determinants localized in variable (V) regions of Ig. Id-specific T and B cells (antibodies) play a role in immunotherapy of Id(+) tumors. However, vaccine strategies that enhance Id-specific responses are needed. Id(+) single-chain fragment variable (scFv) from multiple myelomas and B cell lymphomas were prepared in a fusion format that bivalently target surface molecules on antigen-presenting cells (APC). APC-specific targeting units were either scFv from APC-specific mAb (anti-MHC II, anti-CD40) or chemokines (MIP-1α, RANTES). Homodimeric Id-vaccines were injected intramuscularly or intradermally as plasmids in mice, combined with electroporation. (i) Transfected cells secreted plasmid-encoded Id(+) fusion proteins to extracellular fluid followed by binding of vaccine molecules to APC. (ii) Targeted vaccine molecules increased Id-specific B and T cell responses. (iii) Bivalency and xenogeneic sequences both contributed to enhanced responses. (iv) Targ...
The half-life of the two most abundant proteins in blood, immunoglobulin G (IgG) and serum albumi... more The half-life of the two most abundant proteins in blood, immunoglobulin G (IgG) and serum albumin, is extraordinary (approximately 19-23 days) compared to other circulating proteins. This phenomenon secures a broad biodistribution throughout the body of both molecules. The long half-life has made IgG the natural choice for engineering of antibody based therapeutics, while albumin is used as a fusion partner for or carrier of drugs. Remarkably, the half-life of these unrelated proteins has been shown prolonged by a receptor recycling pathway mediated by a common cell bound receptor named the neonatal Fc receptor (FcRn). This review summarizes our current understanding of FcRn function and discusses its relevance for development of new IgG and albumin based therapeutics and diagnostics.
We have developed a strategy for improving the stimulation of T cells during immune responses by ... more 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.
Antibody (Ab) molecules may serve as targeting vehicles for delivery of foreign antigenic peptide... more 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 endoge...
1) The influence of caffeine on growth and on the metabolism of thymidine was investigated in var... more 1) The influence of caffeine on growth and on the metabolism of thymidine was investigated in various E. coli strains. Caffeine caused filamentous growth in all strains investigated. The caffeine effect was reversible.
Background: Phage display is a platform for selection of specific binding molecules and this is a... more Background: Phage display is a platform for selection of specific binding molecules and this is a clear-cut motivation for increasing its performance. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII), or the minor coat protein III (pIII). Display on other coat proteins such as pVII allows for display of heterologous peptide sequences on the virions in addition to those displayed on pIII and pVIII. In addition, pVII display is an alternative to pIII or pVIII display.
Cells cultured from xeroderma pigmentosum (XP) patients are defective in excision repair of damag... more Cells cultured from xeroderma pigmentosum (XP) patients are defective in excision repair of damaged DNA specifically at the incision step. In Escherichia coli this step is mediated by the UvrA, UvrB and UvrC gene products. Our goal is to express each of these genes in XP cells, singly or in combination, and to determine the most suitable conditions for generating faithful E. coli Uvr protein copies in functional concentrations and properly localized for the eventual repair of damaged chromosomal DNA or DNA which is introduced exogenously.
There is still no general vaccine for prevention of disease caused by group-B meningococcal strai... more There is still no general vaccine for prevention of disease caused by group-B meningococcal strains. Meningococcal lipopolysaccharides (LPSs) have received attention as potential vaccine candidates, but concerns regarding their safety have been raised. Peptide mimics of LPS epitopes may represent safe alternatives to immunization with LPS. The monoclonal antibody (MoAb) 9-2-L3,7,9 [1] specific for Neisseria meningitidis LPS immunotype L3,7,9 is bactericidal and does not cross-react with human tissue. To explore the possibility of isolating peptide mimics of the epitope recognized by MoAb 9-2-L3,7,9, we have constructed two phage display libraries of six and nine random amino acids flanked by cysteines. Furthermore, we developed a system for the easy exchange of peptide-encoding sequences from the phage-display system to a hepatitis B core (HBc) expression system. Cyclic peptides that specifically bound MoAb 9-2-L3,7,9 at a site overlapping with the LPS-binding site were selected from both libraries. Three out of four tested peptides which reacted with MoAb 9-2-L3,7,9 were successfully presented as fusions to the immunodominant loop of HBc particles expressed in Escherichia coli. However, both peptide conjugates to keyhole limpet haemocyanin and HBc particle fusions failed to give an anti-LPS response in mice.
: The major histocompatibility class I-related neonatal Fc receptor, FcRn, salvages both IgG and ... more : The major histocompatibility class I-related neonatal Fc receptor, FcRn, salvages both IgG and albumin from degradation and thus contributes to maintain high serum levels of these proteins. Analbuminemia is a rare autosomal recessive disorder characterized by clinically observed allelic albumin variants that are absent or found in very low concentrations in the blood circulation. Such variants may have altered FcRn binding properties that affect their half-life, biodistribution and thereby transport ability. : We established an easy cloning, expression and purification strategy to obtain recombinant GST-tagged human serum albumin (HSA) variants for evaluation of pH dependent FcRn binding properties using an enzyme-linked immunosorbent assay (ELISA) and a real time surface plasmon resonance (SPR) biosensor system. : The strategy yielded purified GST-tagged albumin variants. A recombinant truncated HSA variant similar to a clinically observed splice mutant denoted Bartin, here abrogated HSA(Bartin), showed no detectable pH dependent FcRn binding compared to a fully functional albumin wild type variant, HSA(Wt), and a truncated HSA variant consisting of only the carboxy terminal domain III (HSA(DIII)). : The approach described can be used to rapidly screen clinically observed truncated or otherwise mutant or modified HSA variants regarding their pH dependent FcRn binding properties. Here, we demonstrate that a recombinant truncated HSA variant, HSA(Bartin), does not interact with FcRn, which gives a molecular explanation for the low serum levels. In addition, DIII of HSA alone was shown to retain its FcRn binding property.
Phage display has been instrumental for the success of antibody (Ab) technology. The aim of the p... more Phage display has been instrumental for the success of antibody (Ab) technology. The aim of the present study was to explore phage display of soluble T-cell receptors (TCRs). A library platform that supports engineering and selection of improved TCRs to be used as detection reagents for specific antigen presentation will be very useful. In such applications, high, equal and clone independent display levels are a prerequisite for 'fair' selection. Therefore, we explored how different pIII fusion formats and modes affected the display levels of two murine a/b TCRs. Both are derived from T-cell clones associated with the MOPC315 myeloma model. The results show that the design of the pIII fusion particle significantly affects the subsequent display levels. Furthermore, successful display may be obtained both in phagemid and phage versions. Importantly, improvement of poor display can be achieved by over-expressing the periplasmic chaperone FkpA.
We have developed a strategy for improving the stimulation of T cells during immune responses by ... more 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.
Recombinant antibodies are increasingly used for efficient delivery of T cell epitopes to antigen... more Recombinant antibodies are increasingly used for efficient delivery of T cell epitopes to antigenpresenting 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 b-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 g3 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.
Here we unravel the structural features of human IgM and IgA that govern their interaction with t... more 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.
We have constructed a fusion protein composed of TNF-α and the scFv region of mAb B1, an antibody... more We have constructed a fusion protein composed of TNF-α and the scFv region of mAb B1, an antibody that recognizes a cancer associated carbohydrate antigen (LeY). The fusion protein was expressed in E. coli and found in inclusion bodies.
Various native and hinge-modified forms of Ig with identical Ids were reacted with an anti-Id mAb... more 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, t...
In IgG molecules removal of N-linked glycan results in a conformational change that abolishes bin... more In IgG molecules removal of N-linked glycan results in a conformational change that abolishes binding to Fc gamma receptor proteins on the surface of leukocytes and clearance of abnormal target cells. We have developed a robust flow cytometric screening system for the isolation of aglycosylated Fc mutants exhibiting high affinity to desired Fc receptors despite the lack of glycosylation. A series of complex libraries (107-x109 clones) were generated by random or insertional mutagenesis of the Fc domain and screened for binding to purified Fc gamma receptors. Mutant aglycosylated Fcs that bind with high affinity and specificity were isolated and characterized in vitro. The aglycosylated Fc gamma receptor binding mutant proteins contain between 1-11 amino acid substitutions. We found that the mutations allowed highly selective binding of an aglycosylated, anti-Her2 (trastuzumab) to the FcRI receptor, with desired affinity indistinguishable from that of CHO-derived, glycosylated trastu...
Journal of immunology (Baltimore, Md. : 1950), Jan 22, 2015
Engineering of the constant Fc part of monoclonal human IgG1 (hIgG1) Abs is an approach to improv... more Engineering of the constant Fc part of monoclonal human IgG1 (hIgG1) Abs is an approach to improve effector functions and clinical efficacy of next-generation IgG1-based therapeutics. A main focus in such development is tailoring of in vivo half-life and transport properties by engineering the pH-dependent interaction between IgG and the neonatal Fc receptor (FcRn), as FcRn is the main homeostatic regulator of hIgG1 half-life. However, whether such engineering affects binding to other Fc-binding molecules, such as the classical FcγRs and complement factor C1q, has not been studied in detail. These effector molecules bind to IgG1 in the lower hinge-CH2 region, structurally distant from the binding site for FcRn at the CH2-CH3 elbow region. However, alterations of the structural composition of the Fc may have long-distance effects. Indeed, in this study we show that Fc engineering of hIgG1 for altered binding to FcRn also influences binding to both the classical FcγRs and complement f...
Idiotypes (Id) are antigenic determinants localized in variable (V) regions of Ig. Id-specific T ... more Idiotypes (Id) are antigenic determinants localized in variable (V) regions of Ig. Id-specific T and B cells (antibodies) play a role in immunotherapy of Id(+) tumors. However, vaccine strategies that enhance Id-specific responses are needed. Id(+) single-chain fragment variable (scFv) from multiple myelomas and B cell lymphomas were prepared in a fusion format that bivalently target surface molecules on antigen-presenting cells (APC). APC-specific targeting units were either scFv from APC-specific mAb (anti-MHC II, anti-CD40) or chemokines (MIP-1α, RANTES). Homodimeric Id-vaccines were injected intramuscularly or intradermally as plasmids in mice, combined with electroporation. (i) Transfected cells secreted plasmid-encoded Id(+) fusion proteins to extracellular fluid followed by binding of vaccine molecules to APC. (ii) Targeted vaccine molecules increased Id-specific B and T cell responses. (iii) Bivalency and xenogeneic sequences both contributed to enhanced responses. (iv) Targ...
The half-life of the two most abundant proteins in blood, immunoglobulin G (IgG) and serum albumi... more The half-life of the two most abundant proteins in blood, immunoglobulin G (IgG) and serum albumin, is extraordinary (approximately 19-23 days) compared to other circulating proteins. This phenomenon secures a broad biodistribution throughout the body of both molecules. The long half-life has made IgG the natural choice for engineering of antibody based therapeutics, while albumin is used as a fusion partner for or carrier of drugs. Remarkably, the half-life of these unrelated proteins has been shown prolonged by a receptor recycling pathway mediated by a common cell bound receptor named the neonatal Fc receptor (FcRn). This review summarizes our current understanding of FcRn function and discusses its relevance for development of new IgG and albumin based therapeutics and diagnostics.
We have developed a strategy for improving the stimulation of T cells during immune responses by ... more 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.
Antibody (Ab) molecules may serve as targeting vehicles for delivery of foreign antigenic peptide... more 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 endoge...
1) The influence of caffeine on growth and on the metabolism of thymidine was investigated in var... more 1) The influence of caffeine on growth and on the metabolism of thymidine was investigated in various E. coli strains. Caffeine caused filamentous growth in all strains investigated. The caffeine effect was reversible.
Background: Phage display is a platform for selection of specific binding molecules and this is a... more Background: Phage display is a platform for selection of specific binding molecules and this is a clear-cut motivation for increasing its performance. Polypeptides are normally displayed as fusions to the major coat protein VIII (pVIII), or the minor coat protein III (pIII). Display on other coat proteins such as pVII allows for display of heterologous peptide sequences on the virions in addition to those displayed on pIII and pVIII. In addition, pVII display is an alternative to pIII or pVIII display.
Cells cultured from xeroderma pigmentosum (XP) patients are defective in excision repair of damag... more Cells cultured from xeroderma pigmentosum (XP) patients are defective in excision repair of damaged DNA specifically at the incision step. In Escherichia coli this step is mediated by the UvrA, UvrB and UvrC gene products. Our goal is to express each of these genes in XP cells, singly or in combination, and to determine the most suitable conditions for generating faithful E. coli Uvr protein copies in functional concentrations and properly localized for the eventual repair of damaged chromosomal DNA or DNA which is introduced exogenously.
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Papers by Inger Sandlie