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H R Hoogenboom

Publications and source records attributed to H R Hoogenboom.

At least 55 records · Page 3Linked to original sources
PubMed1998

Selection of recombinant, library-derived antibody fragments against p24 for human immunodeficiency virus type 1 diagnostics.

By application of combinatorial library technology, we generated the first recombinant antibody fragments directed against the major capsid protein p24 of human immunodeficiency virus type 1 (HIV-1). A library of single-chain Fv fragments (scFvs) was constructed by using the antibody variable-region (V) genes of B cells derived from the spleen of a viral lysate-immunized mouse. Antibodies were selected by panning or by enrichment with biotinylated antigen, yielding four different families of antibody fragments. The different types of scFvs were characterized by affinity measurements, by antigen recognition on Western blots, and by pepscan analysis. The epitope of one of the scFvs is located near the residues involved in CypA binding, thereby making it an attractive candidate for therapeutic applications. Comparison of the V gene sequence of this scFV with that of a previously described monoclonal antibody reactive against this immunodominant epitope revealed the usage of the identical combination of VH and Vkappa regions. Thus, this is one of the rare examples in which the original combination in a library-derived antibody fragment was retrieved. After appropriate affinity and format improvements, the best of our recombinant scFvs may form the basis for a sensitive p24 assay as a measure of viral load. In addition, anti-p24 scFvs could be expressed as intracellular antibodies (intrabodies) to aid in the treatment of HIV infections.

Amino Acid Sequence↗
PubMed1997

Targeting vectors for intracellular immunisation.

We define intracellular immunization as the inhibition or inactivation of the function of a molecule by the ectopic intracellular expression of antibody binding domains which recognise the molecule. Such recombinant antibodies can be directed to different compartments of eukaryotic cells by means of previously defined targeting signals, thus permiting the study of any molecule in any cellular compartment for which an antibody is available. For this purpose, we have created a set of vectors based on the VHExpress vector described [Persic, L., Roberts, A., Wilton, J., Cattaneo, A., Bradbury, A. and Hoogenboom, H.R. (1997) An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. Gene 187, 000-000], which has been modified to express scFvs (single chain fragments) linked to specific targeting signals. These permit the localisation of scFvs to different intracellular compartments: the endoplasmic reticulum (scFvE-er), the nucleus (scFvE-nuclear), the mitochondria (scFvE-mit), the cytoplasm (scFvE-cyto), and as secreted proteins (scFvE-sec). The function of these vectors has been assessed by the immunofluorescence of COS cells transiently transfected with constructs containing the alphaD11 scFv.

Amino Acid Sequence↗
PubMed1997

An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries.

Phage display is now an established method to select antibody fragments specific for a wide range of diverse antigens. In particular, isolation of human monoclonal antibodies has become a reality and for most purposes bacterial expression of the selected recombinant antibody fragments is sufficient. However, there are some cases where the expression of complete human immunoglobulin in mammalian cells is, if not essential, at least desirable. For this reason we have designed and constructed a set of mammalian expression vectors which permit facile and rapid cloning of antibody genes for both transient and stable expression in mammalian cells. Immunoglobulin genes may be cloned into these expression vectors as V regions or as Fabs for expression as either complete antibodies or as Fab fragments, using restriction sites which are rare in human V genes. All the important elements in the vectors--promoter, leader sequence, constant domains and selectable markers--are flanked by unique restriction sites, allowing simple substitution of elements. The vectors have been evaluated using the variable regions from the neutralizing anti-nerve growth factor (NGF) antibody, alphaD11, and the V regions from 2E10, a scFv selected from a scFv phagemid library.

Animals↗
PubMed1997

Designing and optimizing library selection strategies for generating high-affinity antibodies.

Since its invention at the beginning of the 1990s, antibody phage display has revolutionized the generation of monoclonal antibodies and their engineering. It is now possible to create antibodies binding to any chosen target antigen without the use of laboratory animals or hybridomas, in a system that completely by-passes the immune system. Making antibodies from single-pot phage libraries, and improving their affinity up to the picomolar range if necessary, has never appeared easier. In this review, a variety of phage library-based strategies for the isolation of high-affinity antibodies are presented.

Animals↗
PubMed1997

Cloning and expression of human V-genes derived from phage display libraries as fully assembled human anti-TNF alpha monoclonal antibodies.

BACKGROUND: With the advent of phage antibody libraries, access to completely human antibody fragments is feasible, either by direct selection from human antibody libraries, or by guided selection. After selection, Fabs and scFvs may need to be expressed as complete antibodies in mammalian cells for further characterisation, or if effector functions are required. OBJECTIVES: To rebuild and express the human anti-TNF alpha antibody Fab-P3A2 (isolated as a Fab fragment from phage display libraries by guided selection) as a fully assembled, functional human antibody (gamma-1, lambda) in Sp2/0 myeloma cells, and to perform preliminary characterisation studies of the secreted IgG1 molecule. A further objective was to investigate the kinetics of human antibody production and the stability of antibody secretion in transfectomas cultured in various media formulations. STUDY DESIGN: A tripartite strategy was employed for cloning heavy chain gene (VH)-P3 and light chain gene V lambda-A2-C lambda into mammalian cell expression vectors p alpha Lys-30 and p alpha Lys-17 respectively. The cell line P3A2.B5 was isolated after co-transfection of Sp2/0 mouse myelomas with the constructs, expanded and weaned into a protein free medium. Fully assembled Ig-P3A2 antibody was purified by Protein A affinity chromatography and characterised with respect to size of antibody chains, and affinity for human TNF alpha. Stability of secretion was investigated by extended serial sub-culture and analysis of P3A2.B5 sub-clones. Strategies of media enrichment were tested for any effect on antibody productivity by selected P3A2.B5 sub-clones. RESULTS: The cell line P3A2.B5 secreted an assembled, human antibody Ig-P3A2, with heavy and light chains of molecular weight 55 and 28 KD respectively. Equilibrium capture studies showed Ig-P3A2 to have a dissociation constant of approximately 1.5 x 10(-8) M. The mean specific productivity of the cell line increased from 1.2 pg/cell/day to 7.8 pg/cell/day by a combination of medium enrichment and serum reduction. Prolonged serial sub-culture of P3A2.B5 showed the cell line to be unstable with respect to antibody secretion. CONCLUSIONS: We have outlined a method for expression of human V genes as assembled antibodies in Sp2/0 myeloma cells. A cloning strategy for the stable expression of scFv or Fab genes isolated from phage display libraries as assembled human antibodies of the IgGl subclass in Sp2/0 myeloma cells has been described. For maximising specific productivity of antibody-producing cell lines, supplementation of culture media with glucose, glutamine and amino acids increases antibody yield significantly compared to that in conventional media, indicating the latter is stoichiometrically limiting for production purposes.

Animals↗
PubMed1996

Phage diabody repertoires for selection of large numbers of bispecific antibody fragments.

Methods for the generation of large numbers of different bispecific antibodies are presented. Cloning strategies are detailed to create repertoires of bispecific diabody molecules with variability on one or both of the antigen binding sites. This diabody format, when combined with the power of phage display technology, allows the generation and analysis of thousands of different bispecific molecules. Selection for binding presumably also selects for more stable diabodies. Phage diabody libraries enable screening or selection of the best combination bispecific molecule with regards to affinity of binding, epitope recognition and pairing before manufacture of the best candidate.

Animals↗
PubMed1994

On the interaction between single chain Fv antibodies and bacterial immunoglobulin-binding proteins.

Using four bacterial immunoglobulin-binding proteins, we have analyzed the binding characteristics of a panel of 34 human single chain Fv antibodies, expressed in E. coli and with known specificity and sequence. Several of the single chain Fv antibodies showed affinity for staphylococcal protein A and peptostreptococcal protein L, but not for the streptococcal proteins G or H. The affinity of the binding was higher for protein L (4.5 and 1.4 x 10(9) M-1) than for protein A (7.7 and 6.7 x 10(8) M-1), using the two single chain Fv antibodies displaying the strongest binding activity to these ligands. The binding was shown to be specific by Western blotting, and the single chain Fv antibodies could be purified from crude bacterial culture media by affinity chromatography on protein L- or A-Sepharose. Protein A, which has affinity for the VH domain of the scFv antibodies, was tested against scFv antibodies containing VH1, VH3, VH4 and VH5 domains, and its binding was restricted to approximately half of the scFv antibodies with a VH3 domain. Protein L, which has affinity for the VL domain, was tested against kappa 1, kappa 4, lambda 1, lambda 2 and lambda 3 domains, and it bound all kappa 1 domains, one lambda 2 and one lambda 3 domain. Comparison of the amino acid sequences of binding and non-binding VL domains demonstrated that amino acid residues crucial to the binding of protein L were distributed over a large area outside the hypervariable antigen-binding regions.

Amino Acid Sequence↗
PubMed1994

Antibody fragments from a 'single pot' phage display library as immunochemical reagents.

The display of repertoires of antibody fragments on the surface of filamentous bacteriophage offers a new way of making antibodies with predefined binding specificities. Here we explored the use of this technology to make immunochemical reagents to a range of antigens by selection from a repertoire of > 10(8) clones made in vitro from human V gene segments. From the same 'single pot' repertoire, phage were isolated with binding activities to each of 18 antigens, including the intracellular proteins p53, elongation factor EF-1 alpha, immunoglobulin binding protein, rhombotin-2 oncogene protein and sex determining region Y protein. Both phage and scFv fragments secreted from infected bacteria were used as monoclonal and polyclonal reagents in Western blots. Furthermore the monoclonal reagents were used for epitope mapping (a new epitope of p53 was identified) and for staining of cells. This shows that antibody reagents for research can be readily derived from 'single pot' phage display libraries.

Amino Acid Sequence↗
PubMed1994

Guiding the selection of human antibodies from phage display repertoires to a single epitope of an antigen.

We have developed a strategy for guiding the selection of human antibody fragments from phage display repertoires to a single epitope of an antigen, using rodent monoclonal antibodies as a template. Thus the heavy chain of a rodent antibody (MAb32) directed against human tumor necrosis factor alpha (TNF alpha) was cloned and paired as a template chain with a repertoire of human light chains for display as Fab fragments on filamentous phage. The phage were selected by binding to the antigen. The selected human light chains were in turn paired with a repertoire of human heavy chains displayed on phage, and the phage selected again. The isolated phage displaying human antibody fragments binding to TNF alpha also bound to a peptide comprising the N-terminal region of TNF alpha as with MAb32. One of the human Fab fragments was recloned for expression as a glycosylated human antibody in mammalian cells: Binding to TNF alpha could be competed with MAb32 or with anti-serum to the peptide, indicating the same epitope. The human antibody was found to have a binding affinity (Kd = 15 nM) similar to MAb32 (Kd = 26 nM). The process contrasts with existing means of "humanizing" rodent monoclonal antibodies in that the antibodies derived are completely human.

Amino Acid Sequence↗
PubMed1994

Making antibodies by phage display technology.

Antibody fragments of predetermined binding specificity have recently been constructed from repertoires of antibody V genes, bypassing hybridoma technology and even immunization. The V gene repertoires are harvested from populations of lymphocytes, or assembled in vitro, and cloned for display of associated heavy and light chain variable domains on the surface of filamentous bacteriophage. Rare phage are selected from the repertoire by binding to antigen; soluble antibody fragments are expressed from infected bacteria; and the affinity of binding of selected antibodies is improved by mutation. The process mimics immune selection, and antibodies with many different binding specificities have been isolated from the same phage repertoire. Thus human antibody fragments have been isolated with specificities against both foreign and self antigens, including haptens, carbohydrates, secreted and cell surface proteins, viral coat proteins, and intracellular antigens from the lumen of the endoplasmic reticulum and the nucleus. Such antibodies have potential as reagents for research and in therapy.

Animals↗
PubMed1993

Characterization of single-chain antibody (sFv)-toxin fusion proteins produced in vitro in rabbit reticulocyte lysate.

Chimeric proteins consisting of a fusion between binding-deficient mutants of diphtheria toxin (DT) or Pseudomonas exotoxin A (PE) and a single-chain antibody (E6 sFv) against the human transferrin receptor (TfnR) were expressed in a rabbit reticulocyte lysate system. Molecules utilizing PE40 (the carboxyl terminus 40 kDa of PE, lacking the binding domain) exhibited significant E6 sFv-mediated, cell type-specific cytotoxicity (IC50 1 x 10(-10) M) against a human erythroleukemia-derived cell line, K562. In contrast, a fusion protein between the same sFv and a DT mutant, DTM1 (containing two amino acid substitutions in the binding domain [S(508)F, S(525)F]) was not significantly cytotoxic, despite being enzymatically active. A tripartite protein in the form NH2-DTM1-E6 sFv-PE40-COOH exhibited cytotoxicity comparable to that of the PE40-sFv fusion (IC50 1 x 10(-10) M), suggesting that the deficit in activity of DTM1-sFv is not a function of misfolding of the sFv moiety or of a reduced ability to bind TfnR. In contrast to DTM1-E6 sFv, a fusion protein between a second DT mutant, CRM 107 [S(525)F], and the E6 sFv was specifically cytotoxic (IC50 1 x 10(-9) M), and toxicity could be blocked by addition of excess E6 antibody. The cell-free in vitro expression system we describe is rapid and may be used to express functional toxin-sFv fusion proteins. No protein refolding procedures are required, and the technique may be used to express proteins which, due to restrictions imposed on manipulation of toxin-encoding genes in Escherichia coli, could not be produced by more conventional methods.

ADP Ribose Transferases↗
PubMed1992

By-passing immunisation. Human antibodies from synthetic repertoires of germline VH gene segments rearranged in vitro.

By display of antibody repertoires on the surface of a filamentous bacteriophage and selection of the phage by binding to antigen, we can mimic immune selection. Recently, by tapping the repertoire of rearranged V-genes from the peripheral blood lymphocytes of unimmunised donors, we succeeded in making human antibody fragments with different specificities, including both haptens and proteins, from the same library of phage. Now we have built a repertoire of human VH genes from 49 human germline VH gene segments rearranged in vitro to create a synthetic third complementarity determining region (CDR) of five or eight residues. The rearranged VH genes were cloned with a human V lambda 3 light chain as single chain Fv fragments for phage display, and the library of phage panned by binding to each of two haptens, 2-phenyl-5-oxazolone (phOx) or 3-iodo-4-hydroxy-5-nitrophenyl-acetate (NIP) coupled to bovine serum albumin (BSA). Many different antibody fragments were isolated which bound specifically to hapten, some with affinities in the micromolar range. The in vitro "immune response" to the hapten NIP was dominated by the 9-1 segment (VH3 family), and that to phOx by the VH26 segment (VH3 family) with an invariant aromatic residue (Tyr, Phe, Trp) at residue 97 of CDR3. However, the isolation of phage against protein antigens proved more elusive, with a single phage binding to human tumour necrosis factor, and none to bovine serum albumin, turkey egg-white lysozyme or human thyroglobulin. Nevertheless, the work shows that human antibody fragments with specific binding activities can be made entirely in vitro.

Amino Acid Sequence↗
PubMed1992

Humanization of immunotoxins.

The construction and expression of a chimeric gene encoding a mouse/human antibody to the human transferrin receptor fused to the gene for angiogenin, a human homolog of pancreatic RNase, are described. F(ab')2-like antibody-enzyme fusions were prepared by linking the gene for human angiogenin to a chimeric anti-transferrin receptor heavy chain gene. The antibody-enzyme fusion gene was introduced into a transfectoma that secretes the chimeric light chain of the same antibody, and cell lines were cloned that synthesize and secrete the antibody-enzyme fusion protein of the expected size at a concentration of 1-5 ng/ml. Culture supernatants from clones secreting the fusion protein caused inhibition of growth and protein synthesis of K562 cells that express the human transferrin receptor but not toward a non-human-derived cell line that lacks this receptor. Whereas excess antibody to the same receptor did not itself inhibit protein synthesis, it was able to completely prevent the protein synthesis inhibition caused by the fusion protein. These results indicate that the cytotoxicity is due to a transferrin receptor-mediated mechanism involving the angiogenin portion of the fusion protein and demonstrate the feasibility of constructing recombinant antibody-RNase molecules capable of killing tumor cells bearing the transferrin receptor. The significance of the acquired cytotoxicity of a mouse/human chimeric antibody linked to a human protein may bear importantly in human therapeutic strategies that use mouse antibodies linked to toxins from plants or bacteria to target tumor cells. It is expected that the humanization of immunotoxins will lead to less toxicity and immunogenicity than currently available reagents.

Animals↗