Biomedical subjects

R Schier

Publications and source records attributed to R Schier.

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↗

PubMed2001

High affinity restricts the localization and tumor penetration of single-chain fv antibody molecules.

Antitumor monoclonal antibodies must bind to tumor antigens with high affinity to achieve durable tumor retention. This has spurred efforts to generate high affinity antibodies for use in cancer therapy. However, it has been hypothesized that very high affinity interactions between antibodies and tumor antigens may impair efficient tumor penetration of the monoclonal antibodies and thus diminish effective in vivo targeting (K. Fujimori et al., J. Nucl. Med., 31: 1191-1198, 1990). Here we show that intrinsic affinity properties regulate the quantitative delivery of antitumor single-chain Fv (scFv) molecules to solid tumors and the penetration of scFv from the vasculature into tumor masses. In biodistribution studies examining a series of radioiodinated scFv mutants with affinities ranging from 10(-7)-10(-11) M, quantitative tumor retention did not significantly increase with enhancements in affinity beyond 10(-9) M. Similar distribution patterns were observed when the scFv were evaluated in the absence of renal clearance in anephric mice, indicating that the rapid renal clearance of the scFv was not responsible for these observations. IHC and IF evaluations of tumor sections after the i.v. administration of scFv affinity mutants revealed that the lowest affinity molecule exhibited diffuse tumor staining whereas the highest affinity scFv was primarily retained in the perivascular regions of the tumor. These results indicate that antibody-based molecules with extremely high affinity have impaired tumor penetration properties that must be considered in the design of antibody-based cancer therapies.

Animals↗

PubMed2001

Increasing the affinity for tumor antigen enhances bispecific antibody cytotoxicity.

We tested the hypothesis that bispecific Abs (Bsab) with increased binding affinity for tumor Ags augment retargeted antitumor cytotoxicity. We report that an increase in the affinity of Bsab for the HER2/neu Ag correlates with an increase in the ability of the Bsab to promote retargeted cytotoxicity against HER2/neu-positive cell lines. A series of anti-HER2/neu extracellular domain-directed single-chain Fv fragments (scFv), ranging in affinity for HER2/neu from 10(-7) to 10(-11) M, were fused to the phage display-derived NM3E2 human scFV: NM3E2 associates with the extracellular domain of human FcgammaRIII (CD16). The resulting series of Bsab promoted cytotoxicity of SKOV3 human ovarian carcinoma cells overexpressing HER2/neu by human PBMC preparations containing CD16-positive NK cells. The affinity for HER2/neu clearly influenced the ability of the Bsab to promote cytotoxicity of (51)Cr-labeled SKOV3 cells. Lysis was 6.5% with an anti-HER2/neu K(D) = 1.7 x 10(-7) M, 14.5% with K(D) = 5.7 x 10(-9) M, and 21.3% with K(D) = 1.7 x 10(-10) M at 50:1 E:T ratios. These scFv-based Bsab did not cross-link receptors and induce leukocyte calcium mobilization in the absence of tumor cell engagement. Thus, these novel Bsab structures should not induce the dose-limiting cytokine release syndromes that have been observed in clinical trials with intact IgG BSAB: Additional manipulations in Bsab structure that improve selective tumor retention or facilitate the ability of Bsab to selectively cross-link tumor and effector cells at tumor sites should further improve the utility of this therapeutic strategy.

Adjuvants, Immunologic↗

PubMed1999

Generating improved single-chain Fv molecules for tumor targeting.

Due to their ease of isolation from phage display libraries and their ability to recognize conserved antigens, single-chain Fv (scFv) molecules are rapidly becoming commonplace. However, the monovalent nature of the scFv molecule often dictates, at best, transient interactions with target antigens when molecules with moderate to low affinity are employed. This, along with their rapid elimination from circulation, has limited the utility of scFv molecules for applications in the fields of cancer imaging and therapy. Recently, a number of strategies, including affinity maturation and modification of size and valence, have been evaluated for improving the in vivo efficacy of scFv molecules. In this review, we describe a number of these methods and discuss some of the characteristics that may belong to an optimal antibody-based targeting vehicle.

Animals↗

PubMed1999

Isolation and characterization of an anti-CD16 single-chain Fv fragment and construction of an anti-HER2/neu/anti-CD16 bispecific scFv that triggers CD16-dependent tumor cytolysis.

Bispecific antibody (bsAb)-based clinical trials of cancer have been conducted primarily using intact murine monoclonal antibody (mAb)-derived molecules. In some of these trials, toxicity resulting from the interactions of antibody Fc domains with cellular Fc receptors has limited the doses of antibody (Ab) that can be employed. Furthermore, human anti-mouse Ab responses prohibit multiple therapy courses. These factors have decreased the efficacy of the bsAb 2B1, which targets the extracellular domains (ECD) of the HER2/neu protooncogene product and the human FcgammaRIII (CD16). To address these obstacles, we have constructed and characterized a fully human gene-fused bsAb from single-chain Fv (scFv) molecules specific for HER2/neu and CD16. The human anti-CD16 scFv component, NM3E2, was isolated from a human scFv phage display library. As binding of NM3E2 to human neutrophil-associated CD16 decreased in the presence of plasma IgG, we have concluded that NM3E2 recognizes an epitope in the vicinity of the Fc binding pocket. Furthermore, the NM3E2 scFv was found by surface plasmon resonance-based epitope mapping to share an overlapping epitope with the Leu-11c mAb. The human anti-HER2/neu scFv component, C6.5, which was previously isolated from a human scFv phage display library, was employed as fusion partner for the creation of a bispecific scFv (bs-scFv). In the presence of the C6.5 x NM3E2 bs-scFv, peripheral blood lymphocytes promoted significant lysis of human SK-OV-3 ovarian cancer cells overexpressing HER2/neu. Biodistribution studies performed in SK-OV-3 tumor-bearing scid mice revealed that 1% ID/g of 125I-labeled C6.5 x NM3E2 bs-scFv was specifically retained in tumor at 23 h following injection. These results indicated that both scFv components of the bs-scFv retained their function in the fusion protein. This bsAb should overcome some of the problems associated with the 2B1 bsAb. C6.5 x NM3E2 bs-scFv offers promise as a platform for multifunctional binding proteins with potential clinical applications as a result of its human origin, lack of an Fc domain, ease of production, high level of in vitro tumor cell cytotoxicity and highly selective tumor targeting.

Animals↗

PubMed1998

Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens.

A large library of phage-displayed human single-chain Fv antibodies (scFv), containing 6.7 x 10(9) members, was generated by improving the steps of library construction. Fourteen different protein antigens were used to affinity select antibodies from this library. A panel of specific antibodies was isolated with each antigen, and each panel contained an average of 8.7 different scFv. Measurements of antibody-antigen interactions revealed several affinities below 1 nM, comparable to affinities observed during the secondary murine immune response. In particular, four different scFv recognizing the ErbB2 protein had affinities ranging from 220 pM to 4 nM. Antibodies derived from the library proved to be useful reagents for immunoassays. For example, antibodies generated to the Chlamydia trachomatis elementary bodies stained Chlamydia-infected cells, but not uninfected cells. These results demonstrate that phage antibody libraries are ideally suited for the rapid production of panels of high-affinity mAbs to a wide variety of protein antigens. Such libraries should prove especially useful for generating reagents to study the function of gene products identified by genome projects.

Animals↗

PubMed1998

Increased affinity leads to improved selective tumor delivery of single-chain Fv antibodies.

Mr 25,000 single-chain Fv (scFv) molecules are rapidly eliminated from the circulation of immunodeficient mice, yielding highly specific retention of small quantities of scFv in human tumor xenografts. We postulated that the specific retention of scFv in tumor could be enhanced by engineering significant increases in the affinity of the scFv for its target antigens. Affinity mutants of the human anti-HER2/neu (c-erbB-2) scFv C6.5 were generated by site-directed mutagenesis, which target the same antigenic epitope with a 320-fold range in affinity (3.2 x 10(-7) to 1.0 x 10(-9) M). In vitro, the Kd of each scFv correlated closely with the duration of its retention on the surface of human ovarian carcinoma SK-OV-3 cells overexpressing HER2/neu. In biodistribution studies performed in scid mice bearing established SK-OV-3 tumors, the degree and specificity of tumor localization increased significantly with increasing affinity. At 24 h after injection, tumor retention of the highest affinity scFv was 7-fold greater than that of a mutant with 320-fold lower affinity for HER2/neu. Because the rapid renal clearance of scFv may blunt the impact of improved affinity on tumor targeting, the distributions were also assayed in the absence of renal clearance (e.g., in mice rendered surgically anephric). In this model, the peak tumor retentions of the two higher affinity scFv approximated that reported previously for IgG targeting the same SK-OV-3 tumors in scid mice with intact kidneys. In contrast, the mutant with the lowest affinity for HER2/neu failed to accumulate in tumor, indicating the presence of an affinity threshold that must be exceeded for active in vivo tumor uptake. These results indicate that affinity can significantly impact the in vivo tumor-specific retention of scFv molecules.

Adenocarcinoma↗

PubMed1998

Prolonged in vivo tumour retention of a human diabody targeting the extracellular domain of human HER2/neu.

Single-chain Fv (scFv) molecules exhibit highly specific tumour-targeting properties in tumour-bearing mice. However, because of their smaller size and monovalent binding, the quantities of radiolabelled scFv retained in tumours limit their therapeutic applications. Diabodies are dimeric antibody-based molecules composed of two non-covalently associated scFv that bind to antigen in a divalent manner. In vitro, diabodies produced from the anti-HER2/neu (c-erbB-2) scFv C6.5 displayed approximately 40-fold greater affinity for HER2/neu by surface plasmon resonance biosensor measurements and significantly prolonged association with antigen on the surface of SK-OV-3 cells (t1/2 cell surface retention of > 5 h vs 5 min) compared with C6.5 scFv. In SK-OV-3 tumour-bearing scid mice, radioiodinated C6.5 diabody displayed a highly favourable balance of quantitative tumour retention and specificity. By as early as 4 h after i.v. administration, significantly more diabody was retained in tumour (10 %ID g(-1)) than in blood (6.7 %ID ml(-1)) or normal tissue (liver, 2.8 %ID g(-1); lung, 7.1 %ID g(-1); kidney, 5.2 %ID g(-1)). Over the next 20 h, the quantity present in blood and most tissues dropped approximately tenfold, while the tumour retained 6.5 %ID g(-1) or about two-thirds of its 4-h value. In contrast, the 24-h tumour retention of radioiodinated C6.5 scFv monomer was only 1 %ID g(-1). When diabody retentions were examined over the course of a 72-h study and cumulative area under the curve (AUC) values were determined, the resulting tumor-organ AUC ratios were found to be superior to those previously reported for other monovalent or divalent scFv molecules. In conclusion, the diabody format provides the C6.5 molecule with a distinct in vitro and in vivo targeting advantage and has promise as a delivery vehicle for therapeutic agents.

Animals↗

PubMed1996

Isolation of picomolar affinity anti-c-erbB-2 single-chain Fv by molecular evolution of the complementarity determining regions in the center of the antibody binding site.

We determined the extent to which additional binding energy could be achieved by diversifying the complementarity determining regions (CDRs) located in the center of the antibody combining site of C6.5, a human single-chain Fv (scFv) isolated from a non-immune phage library which binds the tumor antigen c-erbB-2. CDR3 of the light (V(L)) and heavy (V(H)) chain variable region of C6.5 were sequentially mutated, the mutant scFv displayed on phage, and higher affinity mutants selected on antigen. Mutation of V(L) CDR3 yielded a scFv (C6ML3-9) with a 16-fold lower Kd (1.0 x 10(-9) M) than C6.5. Due to its length of 20 amino acids, four V(H) CDR3 libraries of C6ML3-9 were constructed. The greatest increase in affinity from a single library was ninefold (Kd = 1.1 x 10(-10) M). Combination of mutations isolated from separate V(H) CDR3 libraries yielded additional ninefold decreases in Kd, resulting in a scFv with a 1230-fold increase in affinity from wild-type C6.5 (Kd = 1.3 x 10(-11) M). The increase in affinity, and its absolute value, are comparable to the largest values observed for antibody affinity maturation in vivo or in vitro and indicate that mutation of V(L) and V(H) CDR3 may be a particularly efficient means to increase antibody affinity. This result, combined with the location of amino acid conservation and substitution, suggests an overall strategy for in vitro antibody affinity maturation. In addition, the affinities and binding kinetics of the single-chain Fv provide reagents with potential tumor targeting abilities not previously available.

Alanine↗

PubMed1996

Identification of functional and structural amino-acid residues by parsimonious mutagenesis.

For in vitro evolution of protein function, we previously proposed using parsimonious mutagenesis (PM), a technique where mutagenic oligodeoxynucleotides (oligo) are designed to minimize coding sequence redundancy and limit the number of amino acid (aa) residues which do not retain parental structural features. For this work, PM was used to increase the affinity of C6.5, a human single-chain Fv (scFv) that binds the glycoprotein tumor antigen, c-erbB-2. A phage antibody library was created where 19 aa located in three of the heavy (H) and light (L) chain antigen-binding loops (L1, L3 and H2) were simultaneously mutated. After four rounds of selection, 50% of scFv had a lower dissociation rate constant (koff) than the parental scFv. The Kd of these scFv ranged from twofold (Kd=7.0 x 10(-9) M) to sixfold (Kd=2.4 x 10(-9) M) lower than the parental scFv (Kd=1.6 x 10(-8) M). In higher affinity scFv, substitutions occurred at 10/19 of the positions, with 21/28 substitutions occurring at only four positions, two in H2, and one each in L1 and L3. Only the wild type (wt) aa was observed at 9/19 aa. Based on a model of C6.5, seven of the nine conserved aa have a structural role in the variable domain, either in maintaining the main chain conformation of the loop, or in packing on the H-chain variable domain. Two of the conserved aa are solvent exposed, suggesting they may play a critical role in recognition. Thus, PM identified three types of aa: structural aa, functional aa which modulate affinity, and functional aa, which are critical for recognition. Since the sequence space was not completely sampled, higher affinity scFv could be produced by subjecting functional aa which modulate affinity to a higher rate of mutation. Furthermore, PM could prove useful for modifying function in other proteins that belong to structurally related families.

Amino Acid Sequence↗

PubMed1996

Isolation of high-affinity monomeric human anti-c-erbB-2 single chain Fv using affinity-driven selection.

The use of antibodies to target tumor antigens has had limited success, partially due to the large size of IgG molecules, difficulties in constructing smaller single chain Fv (scFv) antibody fragments, and immunogenicity of murine antibodies. These limitations can be overcome by selecting human scFv directly from non-immune or semi-synthetic phage antibody libraries; however, the affinities are typically too low for therapeutic application. For hapten antigens, higher-affinity scFv can be isolated from phage antibody libraries where the VH and VL genes of a binding scFv are replaced with repertoires of V genes (chain shuffling). The applicability of this approach to protein binding scFv is unknown. For this work, chain shuffling was used to increase the affinity of a non-immune human scFv, which binds the glycoprotein tumor antigen c-erbB-2 with an affinity of 1.6 x 10(-8) M. The affinity of the parental scFv was increased sixfold (Kd = 2.5 x 10(-9) M) by light-chain shuffling and fivefold (Kd = 3.1 x 10(-9) M) by heavy-chain shuffling, values comparable to those for antibodies against the same antigen produced by hybridomas. When selections were performed on antigen immobilized on polystyrene, spontaneously dimerizing scFv were isolated, the best of which had only a slightly lower Kd than wild type (Kd = 1.1 x 10(-8) M). These scFv dimerize on phage and are preferentially selected as a result of increased avidity. Compared to scFv which formed only monomer, dimerizing scFv had mutations located at the VH-VL interface, suggesting that VH-VL complementarity determines the extent of dimerization. Higher-affinity monomeric scFv were only obtained by selecting in solution using limiting concentrations of biotinylated antigen, followed by screening mutant scFv from bacterial periplasm by koff in a BIAcore. Using the proper selection and screening conditions, protein binding human scFv with affinities comparable to murine hybridomas can be produced without immunization.

Amino Acid Sequence↗

PubMed1996

Efficient in vitro affinity maturation of phage antibodies using BIAcore guided selections.

Selection of higher affinity mutant phage antibodies has proven less than straightforward due to sequence dependent differences in phage antibody expression, toxicity to Escherichia coli, and difficulty in eluting the highest affinity phage. These differences lead to selection for increased levels of expression or decreased toxicity rather than for higher affinity. In this work, we demonstrate how surface plasmon resonance as employed in the BIAcore can be used to increase the efficiency of phage antibody selections, yielding greater increments in affinity from a single library. A mutant phage antibody library was created by randomizing nine amino acids located in the V(L) CDR3 of C6.5, a human scFv which binds the tumor antigen c-erbB-2 with a Kd of 1.6 x 10-8 M. The library was subjected to five rounds of selection in solution using decreasing concentrations of biotinylated c-erbB-2. After each round of selection, polyclonal phage were prepared and the rate of binding to c-erbB-2 determined in a BIAcore under mass transport limited conditions. Determination of the rate of binding permitted calculation of the concentration, and hence percent, of binding phage present. Results were used to select the antigen concentration for the next round of selection. To determine the optimal eluent, polyclonal phage was injected in a BIAcore and eluted using one of five different solutions (10 mM HCl, 50 mM HCl, 100 mM HCl, 100 mM triethylamine, 2.6 M MgCl2). Differences were observed in eluent efficacy, which was reflected in significant differences in the affinities of phage antibodies isolated from the library after a round of selection using the different eluents. Use of the BIAcore to determine the optimal eluent and guide the antigen concentration used for selection yielded a C6.5 mutant with a 16 fold reduction in Kd (Kd = 1.0 x 10-9 M). This represents at least a twofold greater increment in affinity than previously obtained from a single library of phage antibodies which bind antigens.

Amino Acid Sequence↗

PubMed1995

In vitro and in vivo characterization of a human anti-c-erbB-2 single-chain Fv isolated from a filamentous phage antibody library.

BACKGROUND: Antibody-based reagents have failed to live up to their anticipated role as highly specific targeting agents for cancer therapy. Targeting with human single-chain Fv (sFv) molecules may overcome some of the limitations of murine IgG, but are difficult to produce with conventional hybridoma technology. Alternatively, phage display of antibody gene repertoires can be used to produce human sFv. OBJECTIVES: To isolate and characterize human single chain Fvs which bind to c-erbB-2, an oncogene product overexpressed by 30-50% of breast carcinomas and other adenocarcinomas. STUDY DESIGN: A non-immune human single-chain Fv phage antibody library was selected on human c-erbB extracellular domain and sFv characterized with respect to affinity, binding kinetics, and in vivo pharmacokinetics in tumor-bearing scid mice. RESULTS: A human single-chain Fv (C6.5) was isolated which binds specifically to c-erbB-2. C6.5 is entirely human in sequence, expresses at high level as native protein in E. coli, and is easily purified in high yield in two steps. C6.5 binds to immobilized c-erbB-2 extracellular domain with a Kd of 1.6 x 10(-8) M and to c-erbB-2 on SK-OV-3 cells with a Kd of 2.0 x 10(-8) M, an affinity that is similar to sFv produced against the same antigen from hybridomas. Biodistribution studies demonstrate 1.47% injected dose/g tumor 24 h after injection of 125I-C6.5 into scid mice bearing SK-OV-3 tumors. Tumor:normal organ ratios range from 8.9:1 for kidney to 283:1 for muscle. CONCLUSIONS: These results are the first in vivo biodistribution studies using an sFv isolated from a non-immune human repertoire and confirm the specificity of sFv produced in this manner. The use of phage display to produce C6.5 mutants with higher affinity and slower k(off) would permit rigorous evaluation of the role of antibody affinity and binding kinetics in tumor targeting, and could result in the production of a therapeutically useful targeting protein for radioimmunotherapy and other applications.

Amino Acid Sequence↗

PubMed1994

Neural networks.

Explore the source record for details and available documents.

Humans↗

PubMed1985

[Autotransfusion in aneurysm surgery].

In a group of selected 65 patients undergoing replacement of the thoraco-abdominal (TAA = 16) or abdominal (AAA = 49) aorta for large aneurysms intraoperative autotransfusion with the cell saver was used to salvage the patients' blood. The mean volumes of retransfused red blood cells were 1.21 (AAA) and 3.61 (TAA) respectively. Mean values for heparin, protein and hemolysis within the last sample of the autotransfused blood were 0.16 IU/ml, 1.24 g/l and 89.5 mumol/l respectively. T1/2 using chrom51 was 21 days. Our results confirm that intraoperative autotransfusion is a valuable and save adjunct in major vascular surgery.

Aorta, Abdominal↗