Biomedical subjects

T Kretzschmar

Publications and source records attributed to T Kretzschmar.

15 recordsLinked to original sources

PubMed2001

High-throughput generation and engineering of recombinant human antibodies.

The first version of the Human Combinatorial Antibody Library (HuCAL) is a single-chain Fv-based phage display library (HuCAL-scFv) with 2x10(9) members optimised for high-throughput generation and targeted engineering of human antibodies. 61% of the library genes code for functional scFv as judged by sequencing. We show here that since HuCAL-scFv antibodies are expressed in high levels in Escherichia coli, automated panning and screening in miniaturised settings (96- and 384-well format) have now become feasible. Additionally, the unique modular design of HuCAL-genes and -vectors allows the distinctly facilitated conversion of scFv into Fab, miniantibody and immunoglobulin formats, and the fusion with a variety of effector functions and tags not only convenient for therapeutic applications but also for high-throughput purification and detection. Thus, the HuCAL principle enables the rapid and high-throughput development of human antibodies by optimisation strategies proven useful in classical low molecular weight drug development. We demonstrate in this report that HuCAL is a very convenient source of human antibodies for various applications.

Animals↗

PubMed2000

Bypassing hybridoma technology: HLA-C reactive human single-chain antibody fragments (scFv) derived from a synthetic phage display library (HuCAL) and their potential to discriminate HLA class I specificities.

The generation of discriminative, monospecific anti-HLA antibodies used to be a difficult endeavor. Phage display technology, using single-chain antibody fragments (scFv) offers a powerful alternative obtaining target-specific, genetically stable reagents. Most of scFv obtained to date have been enriched by panning phage libraries to solid-phase coupled antigens. In the present study, HLA-C-specific scFv were isolated using a synthetic phage library in combination with a Cw*0602 overexpressing cell line. ScFv from this procedure precipitated HLA-Cw*0602 heavy chains from whole cell lysates. Flow cytometry analysis revealed that scFv stained HLA-Cw*0602-positive cells, but not cells expressing HLA alleles Cw*0302, Cw*0802, A*0201, B*2705, or Gm1*01011, indicating the specificity of scFv. Similarly they showed an ability to discriminate Cw*0602-positive from Cw*0602-negative peripheral blood lymphocytes (PBL). The results of our study demonstrate the feasibility to genetically engineer single-chain HLA-class I-specific antibodies, by phage display technology. This approach might be a valuable tool to develop a broad range of novel monospecific antibodies against HLA-class I specificities.

Alleles↗

PubMed1998

Scoring functions for computational algorithms applicable to the design of spiked oligonucleotides.

Protein engineering by inserting stretches of random DNA sequences into target genes in combination with adequate screening or selection methods is a versatile technique to elucidate and improve protein functions. Established compounds for generating semi-random DNA sequences are spiked oligonucleotides which are synthesised by interspersing wild type (wt) nucleotides of the target sequence with certain amounts of other nucleotides. Directed spiking strategies reduce the complexity of a library to a manageable format compared with completely random libraries. Computational algorithms render feasible the calculation of appropriate nucleotide mixtures to encode specified amino acid subpopulations. The crucial element in the ranking of spiked codons generated during an iterative algorithm is the scoring function. In this report three scoring functions are analysed: the sum-of-square-differences function s, a modified cubic function c, and a scoring function m derived from maximum likelihood considerations. The impact of these scoring functions on calculated amino acid distributions is demonstrated by an example of mutagenising a domain surrounding the active site serine of subtilisin-like proteases. At default weight settings of one for each amino acid, the new scoring function m is superior to functions s and c in finding matches to a given amino acid population.

Algorithms↗

PubMed1996

High-level expression in insect cells and purification of secreted monomeric single-chain Fv antibodies.

We have constructed a recombinant baculovirus encoding an anti-(phenyl-oxazolone) single-chain Fv antibody (anti-phOx-scFv) fused to the baculovirus GP67 secretion signal sequence, 6 liters of Sf9 insect cells were infected with this virus at a multiplicity of infection of one and cultured in a bioreactor for 72 h. The dialyzed supernatant was subjected to cation exchange chromatography at pH 6.0 followed by size exclusion chromatography on a Sephadex G100 superfine matrix. This rapid protocol resulted in the isolation of monomeric scFv with a purity of greater than 98%. The final yield was 32 mg/l (10(9) cells/l). Partial amino-terminal sequencing revealed that the GP67 signal sequence was completely removed upon secretion. The dissociation constant of the scFv monomers is about 1 x 10(-4) M. By competitive ELISA scFv dimers yielded a half maximum inhibitory concentration of 3.4 x 10(-7 M which matches the earlier measured Kd for the anti-phOx-scFv (3.2-5.3 x 10-7 M. Marks et al. (1991) J. Mol. Biol. 222, 581-597: Marks et al. (1992) Bio/Technology 10, 779-783). This method is readily scaled up for the preparation of scFv antibodies in high yield and purity obviating any affinity chromatography and/or refolding steps by exploitation of insect cell expression as an efficient alternative to E. coli expression.

Animals↗

PubMed1995

Evaluation of antibodies fused to minor coat protein III and major coat protein VIII of bacteriophage M13.

A gene coding for an anti-(2-phenyl-5-oxazolone) single-chain Fv antibody (Ab) fragment (anti-phOx scFv) was cloned in-frame into phagemid vectors upstream from genes encoding (i) the wild-type (wt) minor coat protein (cp) III of the filamentous bacteriophage M13 of Escherichia coli, (ii) a truncated version of cpIII (amino acid (aa) positions 198-406), (iii) the wt major cpVIII, or (iv) a hybrid of interleukin-1 beta (IL-1 beta; aa 10-152) and wt cpVIII. Recombinant (re-) phage obtained by phagemid rescue were examined for the efficiency of displaying these various anti-phOx scFv::cp hybrids with commercially available anti-M13 enzyme-linked immunosorbent assays (ELISA), by immunoblotting with an anti-c-myc Ab, and by selection experiments. We found that the highest ELISA signals were obtained with the cpIII constructs and also that more immunoreactive material was detected by blotting than with Ab::cpVIII fusions. Consequently, more scFv::cpIII than scFv::cpVIII phage could be recovered in micropanning experiments with the antigen phOx as target.

Amino Acid Sequence↗

PubMed1995

Selection procedures for nonmatured phage antibodies: a quantitative comparison and optimization strategies.

Libraries of peptides and proteins can be displayed on the surface of filamentous bacteriophage. The efficient capturing of phage recognizing a defined target molecule remains a serious obstacle, in particular when the phage are present at a low frequency or have a reduced affinity like nonmatured phage antibodies and when the availability of target molecules is limited. We present theoretical considerations and experimental data which allowed us to substantially improve microselection under these conditions. We used a model phage displaying an anti-(2-phenyl-5-oxazolone) single-chain Fv antibody fragment. Following standard protocols and aiming at a low nonspecific binding, only 3.6 x 10(-3)% of the phage input could be recovered from a single round of selection performed in the wells of a microtiter plate. Our results explain why this often employed panning in wells is not efficient, especially with high-molecular-weight target molecules. We devised a procedure which increased the probability of microselection by a factor of 34. An alternative capturing method using immunotubes with a new protocol decreased the amount of required work by a factor of 30. In the case of a nonlimited supply of target molecules, column-affinity chromatography is recommended.

Antibodies, Viral↗

PubMed1993

Chronic myelogenous leukemia-derived basophilic granulocytes express a functional active receptor for the anaphylatoxin C3a.

The receptor for the inflammatory peptide C3a has scarcely been examined on human cells. This work demonstrates that human tumor-derived basophilic granulocytes express C3a receptors, and presents parts of the hitherto unknown C3a-signal transduction. When incubated with IL-3, these cells specifically liberated histamine on C3a stimulation. Independent from IL-3, 240,000 +/- 100,000 receptors per cell with a Kd of 5.6 +/- 0.9 nM were determined. [Ca2+]i increased from 120 +/- 35 nM to 300 +/- 80 nM after a C3a challenge, as measured by digital imaging fluorescence microscopy, and rested at its basal level in the presence of C3a-desArg, the immediate catabolic product of C3a in vivo. This [Ca2+]i increase could be completely desensitized homologously by C3a as well as inhibited by up to 75% by pertussis toxin. Thus, tumor-derived basophils are suitable for cloning of the human C3a receptor.

Anaphylatoxins↗

PubMed1993

Cyclic disulfide analogues of the complement component C3a. Synthesis and conformational investigations.

The flexible C-terminal region of the anaphylatoxic peptide C3a was reported to contain the receptor binding site. To elucidate the receptor binding conformation of the C-terminus, as well as to examine a synthetic approach to potential C3a-antagonists, 26 cyclic disulfide bridged C3a analogues were synthesized. Solid phase peptide synthesis was performed on different polymeric supports by individual peptide synthesis, with Fmoc strategy, and simultaneous multiple peptide synthesis, using Boc and Fmoc strategies. Both strategies gave open-chain peptides in comparable yields. Syntheses using the Boc strategy employed the HF-labile 4(methoxy)benzyl group (Mob) for beta-thiol protection of cysteine; in contrast, the TFA-stable protecting groups, acetamidomethyl (Acm) and trityl (Trt), were chosen for syntheses employing Fmoc strategy. Ring closure reactions by iodine oxidation were carried out starting from protected (Acm/Acm, Trt/Acm) or unprotected dithiols. The resulting cyclic C3a analogues were characterized by HPLC, amino acid analysis, and FAB-MS. Conformational investigations using CD spectroscopy and theoretical structural investigations by means of molecular dynamics calculations revealed that slight variations in sequence result in pronounced conformational consequences. The potential of cyclic C3a analogues to activate or to desensitize guinea pig platelets, a standard test system for biological activities of anaphylatoxic peptides like C3a, revealed relatively low activities for cyclic peptides (< 0.1% C3a activity). N-terminal acylation with cationic, arginine-rich sequences like YRRGR- led to amplified biological effects. Three of the synthesized peptides, namely CAALCLAR (P1), YRRGRCGGLCLAR (P5) and YRRGRAhxCGGLCLAR (P8), point in the direction of C3a antagonists.

Amino Acid Sequence↗

PubMed1992

C3a receptor on dibutyryl-cAMP-differentiated U937 cells and human neutrophils: the human C3a receptor characterized by functional responses and 125I-C3a binding.

The anaphylatoxic peptide C3a is part of a basic immunological defense mechanism, the complement system. Research on the human C3a receptor and signal transduction is hampered by the lack of a suitable human cell or cell line. We screened tumor cell lines and human blood cells for a C3a-dependent increase in cytosolic Ca2+ ([Ca2+]i) and analyzed this reaction in a fura-2/AM fluorescence assay for cells in suspension. U937 cells, when differentiated with dibutyryl-cAMP (Bt2cAMP), and purified human neutrophils reacted in a dose-dependent fashion to C3a and a C3a analogue synthetic peptide. We found complete homologous desensitization of this response and no heterologous desensitization to human C5a. Pertussis toxin totally blocked the increase in [Ca2+]i, indicating the possible involvement of a G-protein. Single-cell analysis by digital imaging fluorescence microscopy indicated that neutrophilic granulocytes responded to C3a. In binding studies with Bt2cAMP-differentiated U937 cells and human granulocytes, the 125I-C3a binding was displaced by C3a, yielding one class of C3a binding sites with dissociation constants (Kd) in the low nanomolar range. We identified myo-inositol 1,4,5-trisphosphate (IP3) as the second messenger possibly causing the [Ca2+]i increase and the release of N-acetyl-beta-D-glucosaminidase as one secretory cell response. By functional and binding studies we demonstrated the expression of the C3a receptor on Bt2-cAMP-differentiated U937 cells and human neutrophils and characterized parts of the C3a signal pathway. Our data support a physiological concept in which C3a might be more important than presently thought.

Acetylglucosaminidase↗

PubMed1992

A recombinant hybrid anaphylatoxin with dual C3a/C5a activity.

By site-directed mutagenesis of a human complement factor C5a cDNA clone, we have designed a hybrid anaphylatoxin in which three amino acid residues in the C-terminal sequence of human C5a were exchanged to create the native C-terminal human C3a (hC3a) sequence Leu-Gly-Leu-Ala-Arg. This hybrid anaphylatoxin rC5a-(1-69)-LGLAR exhibited true C3a and C5a activity when tested in the guinea pig ileum contraction assay. Quantitative measurements of ATP release from guinea pig platelets revealed about 1% intrinsic C3a activity for this hybrid, while the C5a activity was essentially unchanged. Competitive binding assays confirmed that the rC5a-(1-69)-LGLAR mutant was able to displace radioiodinated rhC5a with a KI of approx. 40 nM and hC3a with a KI of approx. 3.7 microM from guinea pig platelets. Since the C-termini of both human C3a and C5a anaphylatoxins are known to interact with their respective receptors, we conclude that the same peptidic sequence, LGLAR, is able to bind to and activate two different receptors, the C3a receptor as well as the C5a receptor. This clone provides a novel tool for the identification of further receptor-binding residues in both anaphylatoxins, since any mutants may be tested for altered C3a and C5a activity simultaneously.

Adenosine Triphosphate↗

PubMed1992

Synthetic peptides as antagonists of the anaphylatoxin C3a.

Peptide compounds resembling the receptor-binding C-terminal domain of the anaphylatoxic peptide C3a were synthesized to examine two kinds of C3a antagonism: (a) specific desensitization of C3a-sensitive cells and (b) competitive binding to the C3a receptor. We used guinea-pig platelets, which express a C3a receptor and specifically release ATP upon stimulation, to evaluate the actions of the C3a analogues. The ATP liberation can be inhibited by pretreatment (i.e. desensitization) of the guinea-pig platelets with substimulatory concentrations of C3a or its analogues. Compared to C3a, several peptides were found with at least a tenfold greater difference between the required concentrations for C3a-specific half-maximal desensitization (DD50) and half-maximal platelet activation (ED50). The most potent compounds were YAAALKLAR and Fmoc-EAALKLAR (Fmoc: 9-fluorenylmethoxycarbonyl) with an ED50/DD50 of 140 +/- 28 and 80 +/- 17, respectively (mean +/- standard deviation). The ED50/DD50 of human C3a was found to be only 6 +/- 2. Some C3a derivatives were also tested in competitive binding studies for their ability to compete with C3a for receptor sites on guinea-pig platelets. Three of them were considered partial antagonists [YRRGRCGGLCLAR, YRRGRXCGGLCLAR and YRRGRXCGALCLAR (X = 6-aminohexanoyl)] because their Ki were smaller than their ED50 (Ki/ED50 = 0.6 +/- 0.3, 0.5 +/- 0.1 and 0.4 +/- 0.2, respectively). Interestingly, the last two compounds also had ED50/DD50 values greater than 60. Common to all three peptides are N-terminal arginine-rich sequences and intramolecular disulfide bridges which introduce conformational constraint.

Amino Acid Sequence↗

PubMed1992

Human C5a anaphylatoxin: gene cloning and expression in Escherichia coli.

A gene coding for the human anaphylatoxin C5a was cloned and expressed in Escherichia coli. A combination of reverse transcription of mRNA of the U937 cell line with subsequent preparative polymerase chain reaction was employed to obtain the gene. The sequence was cloned into the plasmid vector pKK 233-2 behind an ATG initiation codon under the control of a trc promotor. After purification by ion exchange chromatography and reversed phase FPLC a mixture of predominantly non-glycosylated recombinant human C5a with a beta-mercaptoethanol adduct at cysteine 27 and the N-methionyl derivative was obtained which was homogeneous on silver-stained gels, immunoreactive with C5a-specific monoclonal antibodies and functionally active in releasing myeloperoxidase from human granulocytes and ATP from guinea pig platelets. The final yield was about 0.4-0.8 mg purified recombinant C5a per liter bacterial culture.

Amino Acid Sequence↗

PubMed1992

Functional activities of synthetic anaphylatoxic peptides in widely used biological assays.

A comparison study was carried out between the modern ATP release assay (ARA) with guinea-pig platelets and the traditional guinea-pig ileum contraction assay (ICA). The biological activities of the anaphylatoxin C3a and synthetic C3a analogue peptides were determined in both assays. In dose-response curves with C3a, a human C3a peptide with the last 21 amino acids of the C terminus (C3a 56-77) and a peptide with 13 amino acids which was acylated N-terminal with the aromatic fluorenylmethoxycarbonyl group and an aminohexanoyl group (Fmoc-Ahx YRRGRAAALGLAR) were tested. The ARA turned out to be 100 times more sensitive than the ICA. In contrast to previous reports the 21 amino acid long C3a analogue peptide did not exhibit full C3a activity but only 7% (ARA) or 12% (ICA). The potentiation of biological activity in the ARA by coupling non-peptide acyl-residues N terminal to peptide C3a analogues could be confirmed with Fmoc-Ahx-YRRGRAAALGLAR in the ICA. In addition, the tri-peptide Fmoc-Ahx-LAR displayed C3a specific activity in the ICA demonstrated by desensitization experiments.

Adenosine Triphosphate↗

PubMed1991

Functional expression of a human C5a receptor clone in Xenopus oocytes requires additional RNA.

cRNA from a PCR-generated C5aR clone was prepared by in vitro transcription and microinjected into Xenopus laevis oocytes. Ligand-induced whole cell current could be detected after co-injection of cRNA for the C5aR with total RNA of the unstimulated U937 cell line, but not with either of the components injected alone. These data clearly demonstrate an absolute requirement of the C5aR for an additional human factor to become functionally expressed in Xenopus oocytes.

Animals↗

PubMed1991

Characterization of the C5a receptor on guinea pig platelets.

Guinea pig (gp) platelets react to nanomolar doses of the complement-derived anaphylatoxin C5a with a shape change, aggregation and release of biogenic amines and nucleotides from their granules. We have investigated the specific receptor for C5a on gp platelets which mediates these biological effects. Competitive binding studies with 125I-labeled guinea pig C5a (125I-gpC5a) revealed approx. 4000 binding sites/cell with Kd = 6 x 10(-9) M. The more than 60-fold higher biological activity (ATP-release from gp platelets) of gpC5a versus recombinant human C5a (rhuC5a) and the different binding behavior of gpC5a and rhuC5a point to a species restriction in the gp platelet system. Cross-linking of 125I-gpC5a to gp platelets (250 microM DSS) and analysis by SDS-PAGE under reducing conditions resulted in labeling of a single band with a molecular mass of 32 kDa (ligand-receptor complex). Because of these characteristics, the C5a receptor on gp platelets clearly differs from all previously described C5a receptors.

Adenosine Triphosphate↗