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H R Hoogenboom

Publications and source records attributed to H R Hoogenboom.

At least 37 records · Page 2Linked to original sources
PubMed1999

[Immunology in the medical practice. XXIV. Expression of antibodies on bacteriophages; possibilities for in vitro production of human antibodies for any desired application].

Although poly- and monoclonal antibodies are successfully applied in research, an expected clinical breakthrough of these reagents so far has not occurred. This can mainly be explained by the animal origin of antibodies, which may lead to a deleterious immune response upon therapeutic use in humans. Moreover, it has been technically demanding to alter the desired affinity, format and effector functions of existing antibodies. Currently, antibody phage-display technology, through construction of large and highly diverse antibody libraries, completely by-passing the immune system, enables the isolation of human antibodies, which can be engineered for every desired application.

Antibodies, Monoclonal↗
PubMed1999

CD3X anti-nitrophenyl bispecific diabodies: universal immunotherapeutic tools for retargeting T cells to tumors.

We developed a universal recombinant bispecific molecule (BiMol) that is capable of redirecting cytotoxic T cells to tumor cells via tagged anti-tumor ligands such as antibody fragments or cytokines. A recombinant bispecific diabody with binding specificities for the CD3 molecule on T cells as well as for the hapten nitrophenyl (NIP) was produced. This bispecific molecule is capable of redirecting cytotoxic T cells to kill a series of malignant cells, including B cell lymphoma, Hodgkin's lymphoma, and colon carcinoma via NIP-conjugated ligands to tumor-associated antigens. Cytotoxic activity of the diabody was found to be comparable to tetradoma-derived bispecific antibodies with similar specificities. Our findings demonstrate that universal CD3xanti-NIP diabodies could be used for T cell based cellular immunotherapy in a variety of human malignancies. Additionally, these bispecific molecules allow fast and economic testing of tumor-associated antigens on malignant cells for their potential use as immunotherapeutic target structures if corresponding hapten-conjugated antibodies or ligands are available.

Antibodies, Bispecific↗
PubMed1999

Design and expression of soluble CTLA-4 variable domain as a scaffold for the display of functional polypeptides.

We have designed and engineered the human cytotoxic T-lymphocyte associated protein-4 (CTLA-4) variable (V-like) domain to produce a human-based protein scaffold for peptide display. First, to test whether the CTLA-4 CDR-like loops were permissive to loop replacement/insertion we substituted either the CDR1 or CDR3 loop with somatostatin, a 14-residue intra-disulfide-linked neuropeptide. Upon expression as periplasmic-targeted proteins in Escherichia coli, molecules with superior solubility characteristics to the wild-type V-domain were produced. These mutations in CTLA-4 ablated binding to its natural ligands CD80 and CD86, whereas binding to a conformation-dependent anti-CTLA-4 monoclonal antibody showed that the V-domain framework remained correctly folded. Secondly, to develop a system for library selection, we displayed both wild-type and mutated CTLA-4 proteins on the surface of fd-bacteriophage as fusions with the geneIII protein. CTLA-4 displayed on phage bound specifically to immobilized CD80-Ig and CD86-Ig and in one-step panning enriched 5,000 to 2,600-fold respectively over wild-type phage. Bacteriophage displaying CTLA-4 with somatostatin in CDR3 (CTLA-4R-Som3) specifically bound somatostatin receptors on transfected CHO-K1 cells pre-incubated with 1 microg/ml tunicamycin to remove receptor glycosylation. Binding was specific, as 1 microM somatostatin successfully competed with CTLA-4R-Som3. CTLA-4R-Som3 also activated as well as binding preferentially to non-glycosylated receptor subtype Sst4. The ability to substitute CDR-like loops within CTLA-4 will enable design and construction of more complex libraries of single V-like domain binding molecules. Proteins 1999;36:217-227.

Abatacept↗
PubMed1999

A large non-immunized human Fab fragment phage library that permits rapid isolation and kinetic analysis of high affinity antibodies.

We report the design, construction, and use of the first very large non-immunized phage antibody library in Fab format, which allows the rapid isolation and affinity analysis of antigen-specific human antibody fragments. Individually cloned heavy and light chain variable region libraries were combined in an efficient two-step cloning procedure, permitting the cloning of a total of 3.7 x 10(10) independent Fab clones. The performance of the library was determined by the successful selection of on average 14 different Fabs against 6 antigens tested. These include tetanus toxoid, the hapten phenyl-oxazolone, the breast cancer-associated MUC1 antigen, and three highly related glycoprotein hormones: human chorionic gonadotropin, human luteinizing hormone, and human follicle-stimulating hormone. In the latter category, a panel of either homone-specific or cross-reactive antibodies were identified. The design of the library permits the monitoring of selections with polyclonal phage preparations and to carry out large scale screening of antibody off-rates with unpurified Fab fragments on BIAcore. Antibodies with off-rates in the order of 10(-2) to 10(-4) s-1 and affinities up to 2.7 nM were recovered. The kinetics of these phage antibodies are of the same order of magnitude as antibodies associated with a secondary immune response. This new phage antibody library is set to become a valuable source of antibodies to many different targets, and to play a vital role in target discovery and validation in the area of functional genomics.

Amino Acid Sequence↗
PubMed1999

A sensitive immunoassay for rat fatty acid translocase (CD36) using phage antibodies selected on cell transfectants: abundant presence of fatty acid translocase/CD36 in cardiac and red skeletal muscle and up-regulation in diabetes.

The rat membrane protein fatty acid translocase (FAT), which shows sequence similarity to human CD36 (a membrane protein supposedly involved in a variety of membrane processes), is implicated in the transport of long-chain fatty acids across cellular membranes. To set up an immunoassay for quantification of FAT in different tissues, we isolated a series of anti-FAT antibodies by panning a large naive phage antibody library on FAT-transfected H9c2 cells. All seven different phage antibody fragments isolated reacted specifically with FAT, and most likely recognize the same or closely located immunodominant sites on FAT, as a competitive monoclonal antibody (mAb) (CLB-IV7) completely blocked the binding of all these phage antibodies to cells. A sandwich ELISA was set up using mAb 131. 4 (directed against purified CD36 from human platelets) as capture antibody and phage antibodies and anti-phage sera as detector. With this ELISA (sensitivity 0.05 microgram/ml), the FAT content in isolated cardiomyocytes was found to be comparable with that of total heart ( approximately 3 mg/g of protein), while liver tissue and endothelial cells were below the detection limit (<0.1 mg of FAT/g of protein). During rat heart development, protein levels of FAT rose from 1.7+/-0.7 mg/g of protein on the day before birth to 3.6+/-0.4 mg/g of protein on day 70. Comparing control with streptozotocin-induced diabetic rats, a statistically significant (P<0.05) 2-4-fold increase of FAT was seen in heart (from 4.2+/-2.3 to 11.0+/-5.7 mg/g of protein), soleus (from 0.6+/-0.1 to 1.4+/-0.5 mg/g of protein) and extensor digitorum longus (EDL) muscle (from 0.3+/-0.1 to 1. 2+/-0.8 mg/g of protein). In addition, the FAT contents of each of these muscles were found to be of similar magnitude to the contents of cytoplasmic heart-type fatty-acid-binding protein in both diabetic rats and controls, supporting the suggested roles of these two proteins in cellular fatty acid metabolism. This is the first time phage display technology has been succesfully applied for direct selection, from a large naive antibody library, of antibodies that recognize selected membrane proteins in their natural context.

Animals↗
PubMed1999

Single-chain variable fragments selected on the 57-76 p21Ras neutralising epitope from phage antibody libraries recognise the parental protein.

Phage antibodies have been widely prospected as an alternative to the use of monoclonal antibodies prepared by traditional means. Many monoclonal antibodies prepared against peptides are able to recognise the native proteins from which they were derived. Here we show that the same is also true for phage antibodies. We have selected a number of single-chain variable fragments (scFv) from a large phage scFv library against a peptide from the switch region II of p21Ras. This peptide is known to reside in a mobile area of the native protein and is the epitope of a well characterised monoclonal antibody. Selected scFvs were able to recognise native p21Ras in both ELISA and Western blots, indicating that peptides are also likely to be very useful in selecting from phage antibody libraries.

Amino Acid Sequence↗
PubMed1999

A tandem repeat of MUC1 core protein induces a weak in vitro immune response in human B cells.

We have recently described an efficient method to study the human humoral immune response in vitro and to generate isotype-switched, antigen-specific human B cells, which has allowed us to produce high-affinity IgG antibodies against different peptides. In an attempt to study the in vitro immune response against self-antigens, such as tumour-associated antigens, this protocol was used to immunise resting human peripheral blood B cells with a peptide epitope from the human-adenocarcinoma-associated antigen, MUC1. After the two-step in vitro immunisation, the secondary immunised cultures were tested for MUC-1-specific antibodies by enzyme-linked immunosorbent assay (ELISA). Phage molecular libraries were subsequently constructed, using the variable parts of Ig genes derived from cells taken from ELISA-positive wells. The libraries were selected on the MUC1 core peptide. Antigen-specific Fab fragments, specific for the self antigen MUC1, were found in the library of secondary immunised IgG+ B cells and these antibodies were evaluated by BIAcore analysis. The specific Fab fragments exhibited an unusually rapid dissociation rate constant and the overall response frequency was lower, as compared to other antibodies generated by this protocol, which might be explained by the repetitive nature of the core peptide used for immunisation.

Amino Acid Sequence↗
PubMed1999

An anti-CD30 single-chain Fv selected by phage display and fused to Pseudomonas exotoxin A (Ki-4(scFv)-ETA') is a potent immunotoxin against a Hodgkin-derived cell line.

The human CD30 receptor is highly overexpressed on the surface of Hodgkin Reed-Sternberg cells and has been shown to be an excellent target for selective immunotherapy using monoclonal antibody-based agents such as immunotoxins. To construct a new recombinant immunotoxin for possible clinical use in patients with Hodgkin's lymphoma, we have chosen the murine anti-CD30 hybridoma Ki-4 to generate a high-affinity Ki-4 single-chain variable fragment (scFv). Hybridoma V-genes were polymerase chain reaction-amplified, assembled, cloned and expressed as a mini-library for display on filamentous phage. Functional Ki-4 scFv were obtained by selection of binding phage on the Hodgkin lymphoma-derived, CD30-expressing cell line L540Cy. The selected recombinant Ki-4 scFv was shown to specifically bind to an overlapping epitope on the CD30 antigen with binding kinetics similar to those of the original antibody. The Ki-4 scFv was subsequently fused to a deletion mutant of Pseudomonas exotoxin A (ETA'). The resulting immunotoxin Ki-4(scFv)-ETA' specifically binds to CD30+ L540Cy cells and inhibits the protein synthesis by 50% at a concentration (IC50) of 43 pM. This recombinant immunotoxin is a promising candidate for further clinical evaluation in patients with Hodgkin's lymphoma or other CD30+ malignancies.

Bacterial Toxins↗
PubMed1999

Selection-dominant and nonaccessible epitopes on cell-surface receptors revealed by cell-panning with a large phage antibody library.

To generate antibodies to defined cell-surface antigens, we used a large phage antibody fragment library to select on cell transfectants expressing one of three chosen receptors. First, in vitro panning procedures and phage antibody screening ELISAs were developed using whole live cells stably expressing the antigen of interest. When these methodologies were applied to Chinese hamster ovary (CHO) cells expressing one of the receptors for a neuropeptide, somatostatin, using either direct cell panning or a strategy of depletion or ligand-directed elution, many different pan-CHO-cell binders were selected, but none was receptor specific. However, when using direct panning on CHO-cells expressing the human membrane protein CD36, an extraordinary high frequency of antigen-specific phage antibodies was found. Panning on myoblasts expressing the rat homologue of CD36 revealed a similar selection dominance for anti-(CD36). Binding of all selected 20 different anti-(CD36) phage was surprisingly inhibited by one anti-(CD36) mAb CLB-IVC7, which recognizes a functional epitope that is also immunodominant in vivo. Similar inhibition was found for seven anti-(rat) CD36 that cross-reacted with human CD36. Our results show that, although cells can be used as antigen carriers to select and screen phage antibodies, the nature of the antigen target has a profound effect on the outcome of the selection.

Animals↗
PubMed1998

Protein LA, a novel hybrid protein with unique single-chain Fv antibody- and Fab-binding properties.

Existing Ig-binding proteins all suffer from limitations in their binding spectrum. In the pursuit of the ultimate, non-restricted, Ig-binding protein, we have constructed the hybrid protein LA, by fusing four of the Ig kappa light-chain-binding domains of peptostreptococcal protein L with four of the IgGFc- and Fab-binding regions of staphylococcal protein A. Ligand-blot experiments demonstrated that the L and the A components were both functional in the hybrid, as the protein was shown to bind purified kappa light chains and IgGFc. Protein LA bound human Ig of different classes and IgG from a wide range of mammalian species. IgG, IgM and IgA were purified from human serum and saliva by affinity chromatography on protein LA agarose. Similarly, single-chain Fv (scFv) antibodies carrying the kappa light-chain variable domain or expressing the V(H)III (variable domain of the heavy chain of Ig) determinant, were efficiently purified on immobilized protein LA. As judged by surface plasmon resonance (SPR), protein LA showed enhanced affinity for all tested ligands, including several scFv antibodies, compared with proteins L and A alone. SPR analysis also demonstrated that binding of a ligand to one of the components in protein LA did not affect the ability of the hybrid protein to interact simultaneously with a ligand for the other component. The antigen-binding capacity of a kappa-expressing scFv antibody was unaffected by the interaction with protein LA, whereas the binding of a V(H)III-expressing scFv antibody to its antigen was, unexpectedly, blocked by protein A and protein LA. Together, these data demonstrate that protein LA represents a highly versatile Ig-binding molecule.

Antibodies↗
PubMed1998

Human single-chain Fv antibodies to MUC1 core peptide selected from phage display libraries recognize unique epitopes and predominantly bind adenocarcinoma.

The tumor-associated antigen MUC1 is overexpressed and underglycosylated in human adenocarcinomas of diverse origins, such as breast, ovary, and colon. We isolated and describe five human single-chain (sc) Fv antibodies specific for the MUC1 variable number of tandem repeats region isolated by in vitro selection from a large naive phage antibody library containing over 6 x 10(9) different scFv antibodies. A synthetic biotinylated 100-mer peptide corresponding to five tandem repeats of the MUC1 peptide core was used for selection. Two of the antibodies were highly specific for MUC1 as judged by ELISA and flow cytometry. In immunohistochemistry, antibody clone 10A stained MUC1 in the cytoplasm and membrane of adenocarcinoma cells of breast and ovary, whereas in normal epithelium, only cytoplasmic or no staining was observed. With antibody clone 10B, staining was less pronounced and was not always membrane associated in adenocarcinoma. Determination of the fine specificity of 10A and 10B using a novel "indirect epitope fingerprinting" ELISA showed that both antibodies recognize unique epitopes that have not been described for hybridoma-derived anti-mucin antibodies of mouse origin. The selected human antibodies, like many of the murine MUC1 antibodies, recognize epitopes on the protein core of MUC1 that are abundantly present in the underglycosylated form of cell surface mucin on adenocarcinoma. The best human scFv, clone 10A, appears to distinguish normal cells from adenocarcinoma cells, which makes it an attractive candidate for use in antibody-based tumor targeting.

Adenocarcinoma↗
PubMed1998

Construction and characterization of a bispecific diabody for retargeting T cells to human carcinomas.

We describe the construction of a recombinant bispecific antibody fragment in the diabody format with specificity for both the well-established human pancarcinoma associated target antigen EGP2 (epithelial glycoprotein 2, also known as the CO17-1A antigen or KSA) and the CD3epsilon chain of human TCR/CD3 complex. The murine anti-EGP2 (MOC31) single chain variable fragment (scFv) and the humanized anti-CD3 (Ucht1v9) scFv were cast into a diabody format (designated Dia5v9) using a short 5 amino acid Gly-Ser linker between immunoglobulin heavy-chain and light-chain variable domains. Purification of the poly-histidine tagged Dia5v9 was achieved from extracts of protease deficient Escherichia coli by IMAC chromatography. The Dia5v9 diabody showed strong binding to both EGP2 and CD3 in transfected cells. The in vitro efficacy of Dia5v9 in mediating tumor cell lysis by interleukin-2 activated human T cells appeared to be similar to that of the hybrid-hybridoma-derived BsF(ab')2 Bis1 (anti-EGP2/anti-CD3) in a standard 4-hr 51Cr-release assay. This small and partially humanized recombinant bispecific antibody fragment may be valuable for T-cell-based immunotherapeutical treatment protocols, retargeting activated peripheral blood T lymphocytes to lyse various human carcinomas in vivo.

Animals↗
PubMed1998

Antibody phage display technology and its applications.

In recent years, the use of display vectors and in vitro selection technologies has transformed the way in which we generate ligands, such as antibodies and peptides, for a given target. Using this technology, we are now able to design repertoires of ligands from scratch and use the power of phage selection to select those ligands having the desired (biological) properties. With phage display, tailor-made antibodies may be synthesized and selected to acquire the desired affinity of binding and specificity for in vitro and in vivo diagnosis, or for immunotherapy of human disease. This review addresses recent progress in the construction of, and selection from phage antibody libraries, together with novel approaches for screening phage antibodies. As the quality of large naïve and synthetic antibody repertoires improves and libraries becomes more generally available, new and exciting applications are pioneered such as the identification of novel antigens using differential selection and the generation of receptor a(nta)gonists. A combination of the design and generation of millions to billions of different ligands, together with phage display for the isolation of binding ligands and with functional assays for identifying (and possibly selecting) bio-active ligands, will open even more challenging applications of this inspiring technology, and provide a powerful tool for drug and target discovery well into the next decade.

Animals↗
PubMed1998

High-affinity recombinant phage antibodies to the pan-carcinoma marker epithelial glycoprotein-2 for tumour targeting.

The tumour-associated antigen epithelial glycoprotein-2 (EGP-2) is a promising target for detection and treatment of a variety of human carcinomas. Antibodies to this antigen have been successfully used in patients for imaging of small-cell lung cancer and for adjuvant treatment of minimal residual disease of colon cancer. We describe here the isolation and complete characterization of high-affinity single-chain variable fragments (scFv) to the EGP-2 antigen. First, the binding kinetics of four murine whole antibodies directed to EGP-2 (17-1A, 323/A3, MOC-31 and MOC-161) were determined using surface plasmon resonance (SPR). The MOC-31 antibody has the lowest apparent off-rate, followed by MOC-161 and 323/A3. The V-genes of the two MOC hybridomas were cloned as scFv in a phage display vector and antigen-binding phage were selected by panning on recombinant antigen. The scFvs compete with the original hybridoma antibodies for binding to antigen and specifically bind to human carcinomas in immunohistochemistry. MOC-31 scFv has an off-rate which is better than those of the bivalent 17-1A and 323/A3 whole antibodies, providing it with an essential characteristic for tumour retention in vivo. The availability of these high-affinity anti-EGP-2 antibody fragments and of their encoding V-genes creates a variety of possibilities for their future use as tumour-targeting vehicles.

Animals↗
PubMed1998

Somatostatin displayed on filamentous phage as a receptor-specific agonist.

1. In search of methods to identify bio-active ligands specific for G protein-coupled receptors with seven transmembrane spanning regions, we have developed a filamentous phage-based selection and functional screening method. 2. First, methods for panning peptide phage on cells were established, using the hormone somatostatin as a model. Somatostatin was displayed on the surface of filamentous phage by cloning into phage(mid) vectors and fusion to either pIII or pVIII viral coat proteins. Peptide displaying phage bound to a polyclonal anti-somatostatin serum, and, more importantly, to several somatostatin receptor subtypes (Sst) expressed on transfected CHO-K1 cells, in a pattern which was dependent on the used display method. Binding was competed with somatostatin, with an IC50 in the nanomolar range. The phage were specifically enriched by panning on cells, establishing conditions for cell selections of phage libraries. 3. Binding of somatostatin displaying phage to sst2 on a reporter cell line, in which binding of natural ligand reduces secretion of alkaline phosphatase (via a cyclic AMP responsive element sensitive promoter), proved that the phage particles act as receptor-specific agonists. Less than 100 phage particles per cell were required for this activity, which is approximately 1000 fold less than soluble somatostatin, suggesting that phage binding interferes with normal receptor desensitization and/or recycling. 4. The combination of biopanning of phage libraries on cells with functional screening of phage particles for receptor triggering activity, may be used to select novel, bio-active ligands from phage libraries of random peptides, antibody fragments, or libraries based on the natural receptor ligand.

Amino Acid Sequence↗
PubMed1998

Absolute conservation of residue 6 of immunoglobulin heavy chain variable regions of class IIA is required for correct folding.

While studying the expression of single-chain antibodies (scFv) derived from several murine monoclonal antibodies, we found that residue 6 in Framework region 1 of the heavy chain variable domain plays a crucial role in antibody folding. Binding activity of three murine antibodies with a heavy chain variable region (VH) subgroup IIA was completely lost when at this position the wild-type residue glutamine (Q) was substituted by glutamate (E). Increased sensitivity towards trypsin digestion of soluble scFv suggested that the lack of binding activity was caused by incorrect folding of Q6E mutants. Grafting of the three additional class IA derived FR1 residues, based upon the comparison between both classes of VH sequences, on to the 'defect' subgroup IIA sequence, partially restored the antigen binding activity of the Q6E-containing scFv. Our results suggest that residue 6 of the heavy chain may be part of a folding nucleus, involving the first two beta-strands of Framework region 1. The evolutionary conservation of either glutamine or glutamate at position 6 in different antibody families may well indicate that within immunoglobulin VH domains, different family specific folding nuclei have evolved.

Amino Acid Sequence↗
PubMed1998

Selection of human anti-human immunodeficiency virus type 1 envelope single-chain antibodies from a peripheral blood cell-based phage repertoire.

Monoclonal antibodies play an important role in the development of diagnostic assays. Instead of using hybridoma technology to isolate human immunodeficiency virus type 1-specific antibodies, a phage-displayed antibody library was generated from a small number (10(7)) of peripheral blood lymphocytes from a seropositive donor. Two families of single-chain antibodies (scFvs) were selected by biopanning with the envelope precursor gp160. ELISA and competition in the BIAcore system revealed that one antibody family recognized a conformation-sensitive epitope within gp120, while the other antibody family was gp41-specific. The latter group had sequence similarity to antibodies recognizing the cluster III epitope of gp41. Binding of scFvs to gp160 could be inhibited with the donor's serum antibodies, indicating that antibodies with a similar specificity were circulating in the donor's blood. Competition experiments suggested that the epitope of the anti-gp41 antibodies was recognized by a broad range of patients' sera: 21 out of 22 sera from North American and all 20 sera from African seropositive patients inhibited binding of scFvs. In contrast, three sera from this panel did not react with the epitope of the anti-gp120 antibodies. These data indicate that, because of the conserved nature of its epitope, the anti-gp41 antibody will be suitable for diagnostic applications.

Amino Acid Sequence↗