Selection of internalizing antibodies for drug delivery.
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Antibodies↗
Biomedical subjects
James D Marks
Publications and source records attributed to James D Marks.
Antibody affinity maturation by chain shuffling.
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Antibodies↗
Development of ligand-targeted liposomes for cancer therapy.
The continued evolution of targeted liposomal therapeutics has resulted in new agents with remarkable antitumour efficacy and relatively mild toxicity profiles. A careful selection of the ligand is necessary to reduce immunogenicity, retain extended circulation lifetimes, target tumour-specific cell surface epitopes, and induce internalisation and subsequent release of the therapeutic substance from the liposome. Methods for assembling targeted liposomes, including a novel micellar insertion technology, for incorporation of targeting molecules that efficiently transforms a non-targeted liposomal therapeutic to a targeted one, greatly assist the translation of targeted liposome technology into the clinic. Targeting strategies with liposomes directed at solid tumours and vascular targets are discussed. The authors believe the development of ligand-targeted liposomes is now in the advanced stage and offers unique and important advantages among other targeted therapies. Anti-HER2 immunoliposomal doxorubicin is awaiting Phase I clinical trials, the results of which should provide new insights into the promise of ligand-targeted liposomal therapies.
Animals↗
Epidermal growth factor receptor (EGFR)-targeted immunoliposomes mediate specific and efficient drug delivery to EGFR- and EGFRvIII-overexpressing tumor cells.
We hypothesized that immunoliposomes (ILs) that target epidermal growth factor receptor (EGFR) and/or its truncated variant EGFRvIII can be constructed to provide efficient intracellular drug delivery in tumor cells overexpressing these receptors. Monoclonal antibody fragments included Fab' fragments derived from C225, which binds both EGFR and EGFRvIII, or novel anti-EGFR scFv C10, which binds EGFR only. Monoclonal antibody fragments were covalently linked to liposomes containing various reporters or drugs. ILs were evaluated for specific binding, internalization, and cytotoxicity in EGFR/EGFRvIII-overexpressing cell lines in vitro. Flow cytometry and fluorescence microscopy showed that EGFR-targeted ILs, but not nontargeted liposomes or irrelevant ILs, were efficiently bound and internalized by EGFR-overexpressing cells, including glioma cells (U-87), carcinoma cells (A-431 and MDA-MB-468), and EGFRvIII stable transfectants (NR-6M). Furthermore, EGFR-targeted ILs did not bind to non-EGFR-overexpressing cells (MCF-7 and parental NR-6). ILs showed 3 orders of magnitude greater accumulation in NR-6-EGFRvIII stable transfectants versus parental NR-6 cells. Quantitative internalization studies indicated binding of EGFR-targeted ILs to target cells within 5 min, followed by intracellular accumulation beginning at 15 min; total uptake reached approximately 13,000 ILs/cell. ILs were used to deliver cytotoxic drugs doxorubicin, vinorelbine, or methotrexate to EGFR/EGFRvIII-overexpressing target cells in vitro. In each case, the IL agent was significantly more cytotoxic than the corresponding nontargeted liposomal drug in target cells, whereas it was equivalent in cells lacking EGFR/EGFRvIII overexpression. We conclude that EGFR-targeted ILs provide efficient and targeted delivery of anticancer drugs in cells overexpressing EGFR or EGFRvIII.
Adenocarcinoma↗
Predicting antigenic peptides suitable for the selection of phage antibodies.
Selection from phage antibody libraries can be considered to be an in vitro immune system in which the antibody response is reduced to the bare minimum of antigen recognition. Using selections of antibodies on peptides from a phage antibody library, we investigated what constitutes peptide antigenicity in the context of the antibody-protein binding site. We selected polyclonal antibodies in a high throughput format against 44% of 90 overlapping peptides derived from three different proteins. Of these, 33% of peptides (epitopic peptides) were able to select antibodies that recognized the protein from which the peptides were derived. Although no algorithm was able to predict all epitopic peptides, solvent accessibility was the best predictor in this cell-free antibody selection context. We subsequently applied solvent accessibility to successfully predict epitopic peptides from p53 and Znf217, and showed that such peptide selected single-chain antibodies were able to recognize soluble p53 in ELISA and Znf217 in a western blot. This is likely to have considerable utility in functional genomics and proteomics where it should be possible to select antibodies against gene products on the basis of deduced amino acid sequence in a high throughput fashion.
Amino Acid Sequence↗
Therapeutic efficacy of anti-ErbB2 immunoliposomes targeted by a phage antibody selected for cellular endocytosis.
Many targeted cancer therapies require endocytosis of the targeting molecule and delivery of the therapeutic agent to the interior of the tumor cell. To generate single chain Fv (scFv) antibodies capable of triggering receptor-mediated endocytosis, we previously developed a method to directly select phage antibodies for internalization by recovering infectious phage from the cytoplasm of the target cell. Using this methodology, we reported the selection of a panel of scFv that were internalized into breast cancer cells from a nonimmune phage library. For this work, an immunotherapeutic was generated from one of these scFv (F5), which bound to ErbB2 (HER2/neu). The F5 scFv was reengineered with a C-terminal cysteine, expressed at high levels in Escherichia coli, and coupled to sterically stabilized liposomes. F5 anti-ErbB2 immunoliposomes were immunoreactive as determined by surface plasmon resonance (SPR) and were avidly internalized by ErbB2-expressing tumor cell lines in proportion to the levels of ErbB2 expression. F5-scFv targeted liposomes containing doxorubicin had antitumor activity and produced significant reduction in tumor size in xenografted mice compared to nontargeted liposomes containing doxorubicin. This strategy should be applicable to generate immunotherapeutics for other malignancies by selecting phage antibodies for internalization into other tumor types and using the scFv to target liposomes or other nanoparticles.
Animals↗
Phage versus phagemid libraries for generation of human monoclonal antibodies.
Non-immune (naïve) phage antibody libraries have become an important source of antibodies for reagent, diagnostic, and therapeutic use. To date, reported naïve libraries have been constructed in phagemid vectors as fusions to pIII, yielding primarily single copy (monovalent) display of antibody fragments. For this work, we subcloned the single chain Fv (scFv) gene repertoire from a naïve phagemid antibody library into a true phage vector to create a multivalently displayed scFv phage library. Compared to monovalently displayed scFv, multivalent phage display resulted in improved efficiency of display as well as antibody selection. A greater number of antibodies were obtained and at earlier rounds of selection. Such increased efficiency allows the screening for binding antibodies after a single round of selection, greatly facilitating automation. Expression levels of antigen-binding scFv were also higher than from the phagemid library. In contrast, the affinities of scFv from the phage library were lower than from the phagemid library. This could be overcome by utilizing the scFv in a multivalent format, by affinity maturation, or by converting the library to monovalent display after the first round of selection.
Amino Acid Sequence↗
Generation and characterization of a protective monoclonal antibody to Pseudomonas aeruginosa PcrV.
Pseudomonas aeruginosa is a gram-negative pathogen causing life-threatening infections. Lung injury and the development of sepsis depend largely on the expression of type III secretion system (TTSS) virulence. TTSS functions as a molecular syringe to deliver toxins directly to the cytosol of cells, inhibit innate immune mechanisms, and prevent bacterial clearance. Polyclonal antibodies that bind to PcrV of P. aeruginosa inhibit the delivery of type III toxins and enhance the clearance of bacteria during acute lung infections. PcrV is a homologue of LcrV, a protective antigen in the Yersinia TTSS and an integral component of TTSS. In this study, a murine monoclonal antibody (MAb) to PcrV was generated: MAb 166, which is protective against P. aeruginosa when coinstilled with the bacterial inoculum or intraperitoneally transferred to mice. Fab fragments from MAb 166 prevent sepsis and death. The epitope bound by MAb 166 was mapped to the carboxyl-terminus of PcrV.
Animals↗
The intracellular antibody capture technology (IACT): towards a consensus sequence for intracellular antibodies.
We describe the application of an intracellular antibody capture technology (IACT) as a generic in vivo selection procedure for isolating intracellular antibodies or ICAbs. IACT was applied to the de novo selection of functional ICAbs against the microtubule-associated protein TAU, found in neurofibrillary lesions of Alzheimer's disease brains. A panel of 17 different ICAbs was created which bind TAU inside cells and the epitopes recognized by the selected ICAbs have been determined by an in vivo epitope mapping procedure. Finally, sequence analysis showed that the IACT-derived ICAbs are characterized by a common signature of conserved amino acid residues, suggesting that the IACT naturally selects a sort of "captured consensus sequence" for intracellular antibodies. The development of IACT, together with the possibility of scaling up in a high throughput and automated format, makes IACT a new enabling tool for target validation in functional genomics and global proteomics.
Alzheimer Disease↗
Genetic and immunological comparison of anti-botulinum type A antibodies from immune and non-immune human phage libraries.
Understanding the antibody response in botulinum intoxication is important for vaccine design and passive prophylaxis. To investigate this activity, we have studied the immune response to BoNT/A (botulinum neurotoxin serotype A) binding domain (HC) at the molecular level using phage display. The scFv antibodies were isolated from V-gene repertoires prepared from (a) human volunteer immunized with pentavalent botulinum toxoid and (b) non-immune human peripheral blood lymphocytes and spleenocytes. A large panel of serotype specific phage expressing botulinum binding scFv could be selected from both libraries. Epitope mapping of immune scFv binders towards BoNT/A HC revealed surprisingly a limited number of scFv recognizing conformational epitopes that corresponded to two distinct groups, clusters I and II. Only scFv from cluster I exhibited neutralizing activity in the mouse hemidiaphragm assay. Anti- BoNT/A HC clones derived from a non-immune library could be conveniently grouped into clusters III-XI and appeared to share no overlapping epitopes with cluster I or II. In addition they showed no neutralization of toxin at biologically significant concentrations. We therefore suggest that a vaccine based on the pentavalent botulinum toxoid directs the humoral immune response to a limited number of immunodominant epitopes exposed on the binding domain HC.
Antibodies, Bacterial↗
Towards proteome-wide production of monoclonal antibody by phage display.
Sequencing of the human genome reveals that there are approximately 30,000 genes that encode an even greater number of proteins which comprise the human proteome. Characterization of gene products at the genome-wide scale requires the development of high throughput methods to generate temporo-spatial information on each and every protein in the cell under normal and pathological conditions. Monoclonal antibodies are important reagents for these studies. We have developed a method to generate human monoclonal antibodies by selecting phage antibody libraries directly on antigen blotted onto poly(vinylidene fluoride) membranes. Cellular proteins are first separated by two dimensional (2D) gel electrophoresis, Western blotted onto poly(vinylidene fluoride) membranes, and used to select phage antibody libraries. Monoclonal antibodies can be generated against individual protein spots on a 2D gel. The antibodies are functional in Western blotting, ELISA, and immunohistochemistry. Automation of this process should allow high throughput production of monoclonal phage antibodies against cellular proteins as well as proteins that are uniquely expressed under pathological conditions.
Amino Acid Sequence↗
The cleavage site of C5 from man and animals as a common target for neutralizing human monoclonal antibodies: in vitro and in vivo studies.
The isolation of an anti-C5 single-chain fragment variable (scFv) antibody, TS-A12/22, from a human phage display library, is described. This antibody inhibits the activation of C5 and the assembly of the terminal complement complex implicated in cell and tissue damage. Using antibody-sensitized sheep erythrocytes and rabbit red cells as target cells in hemolytic assays, we found that TS-A12/22 inhibited the activation of C5 by the convertases of both classical and alternative pathways. Western blot analysis and competition experiments with synthetic peptides showed that TS-A12/22 reacted with the alpha chain of C5 and recognized the cleavage site of this complement component by the C5 convertase. As a result, the antibody prevented splitting of C5 and inhibited the generation of C5a and of the terminal complement complex. The identification of the TS-A12/22 recognition site as a conserved sequence in man, mouse, rat and rabbit enabled the demonstration of in vitro inhibition of complement activity in these species. The scFv TS-A12/22 was tested in a rat model of antigen-induced arthritis and proved to be effective in preventing influx of polymorphonuclear cells into the knee joint and C9 deposition on synovial tissue.
Animals↗
Phage-display antibody detection of Chlamydia trachomatis-associated antigens.
A phage-displayed human single-chain Fv antibody library (6.7x10(9) members) was used to select probes specific to components associated with the surface of Chlamydia trachomatis elementary bodies (EBs). Each of 15 antibodies was characterized by ELISA, dot-blot, immunoblot and immunocytochemistry, resulting in the identification of several new chlamydial components associated with the surface of EBs. In addition, six antibodies were specific for host-cell components associated with the surface of EBs. While phage display has been used effectively to produce specific antibodies for purified components, these data show that this technology is suitable for selection of specific probes from complex antigens such as the surface of a microbial pathogen.
Animals↗
Anti-HER2 immunoliposomes: enhanced efficacy attributable to targeted delivery.
PURPOSE: Anti-HER2 immunoliposomes combine the tumor-targeting of certain anti-HER2 monoclonal antibodies (MAbs) with the pharmacokinetic and drug delivery capabilities of sterically stabilized liposomes. We previously showed that anti-HER2 immunoliposomes bind efficiently to and internalize in HER2-overexpressing cells in vitro, resulting in intracellular drug delivery. EXPERIMENTAL DESIGN: Here we describe the pharmacokinetics and therapeutic efficacy of anti-HER2 immunoliposomes containing doxorubicin (dox) in a series of animal models. RESULTS: Immunoliposomes displayed long circulation that was identical to that of sterically stabilized liposomes in single- and multiple-dose studies in normal rats. Anti-HER2 immunoliposome-dox produced marked therapeutic results in four different HER2-overexpressing tumor xenograft models, including growth inhibition, regression, and cures. These results demonstrated that encapsulation of dox in anti-HER2 immunoliposomes greatly increased its therapeutic index, both by increasing antitumor efficacy and by reducing systemic toxicity. Immunoliposome-dox was significantly superior to all other treatment conditions tested, including free dox, liposomal dox, and anti-HER2 MAb (trastuzumab). When compared with liposomal dox in eight separate therapy studies in HER2-overexpressing models, immunoliposome delivery produced significantly superior antitumor efficacy in each study (P < 0.0001 to 0.04). Anti-HER2 immunoliposome-dox containing either recombinant human MAb HER2-Fab' or scFv C6.5 yielded comparable therapeutic efficacy. Cure rates for immunoliposome-dox reached 50% (11 of 21) with optimized immunoliposomes and Matrigel-free tumors and overall was 16% (18 of 115) versus no cures (0 of 124) with free dox or liposomal dox. Finally, anti-HER2 immunoliposome-dox was also superior to combinations consisting of free MAb plus free dox or free MAb plus liposomal dox. CONCLUSIONS: Anti-HER2 immunoliposomes produced enhanced antitumor efficacy via targeted delivery.
Animals↗
Preclinical manufacture of anti-HER2 liposome-inserting, scFv-PEG-lipid conjugate. 2. Conjugate micelle identity, purity, stability, and potency analysis.
Analytical methods optimized for micellar F5cys-MP-PEG(2000)-DPSE protein-lipopolymer conjugate are presented. The apparent micelle molecular weight, determined by size exclusion chromatography, ranged from 330 to 960 kDa. The F5cys antibody and conjugate melting points, determined by differential scanning calorimetry, were near 82 degrees C. Traditional methods for characterizing monodisperse protein species were inapplicable to conjugate analysis. The isoelectric point of F5cys (9.2) and the conjugate (8.9) were determined by capillary isoelectric focusing (cIEF) after addition of the zwitterionic detergent CHAPS to the buffer. Conjugate incubation with phospholipase B selectively removed DSPE lipid groups and dispersed the conjugate prior to separation by chromatographic methods. Alternatively, adding 2-propanol (29.4 vol %) and n-butanol (4.5 vol %) to buffers for salt-gradient cation exchange chromatography provided gentler, nonenzymatic dispersion, resulting in well-resolved peaks. This method was used to assess stability, identify contaminants, establish lot-to-lot comparability, and determine the average chromatographic purity (93%) for conjugate lots, described previously. The F5cys amino acid content was confirmed after conjugation. The expected conjugate avidity for immobilized HER-2/neu was measured by bimolecular interaction analysis (BIAcore). Mock therapeutic assemblies were made by conjugate insertion into preformed doxorubicin-encapsulating liposomes for antibody-directed uptake of doxorubicin by HER2-overexpressing cancer cells in vitro. Together these developed assays established that the manufacturing method as described in the first part of this study consistently produced F5cys-MP-PEG(2000)-DSPE having sufficient purity, stability, and functionality for use in preclinical toxicology investigations.
Calorimetry, Differential Scanning↗
Preclinical manufacture of an anti-HER2 scFv-PEG-DSPE, liposome-inserting conjugate. 1. Gram-scale production and purification.
A GMP-compliant process is described for producing F5cys-PEG-lipid conjugate. This material fuses with preformed, drug-loaded liposomes, to form "immunoliposomes" that bind to HER2/neu overexpressing carcinomas, stimulates drug internalization, and ideally improves the encapsulated drug's therapeutic index. The soluble, single-chain, variable region antibody fragment, designated F5cys, was produced in E. coli strain RV308 using high-density cultures. Affinity adsorption onto horizontally tumbled Streamline rProtein-A resin robustly recovered F5cys from high-pressure-disrupted, whole-cell homogenates. Two product-related impurity classes were identified: F5cys with mid-sequence discontinuities and F5cys with remnants of a pelB leader peptide. Low-pressure cation exchange chromatography, conducted at elevated pH under reducing conditions, enriched target F5cys relative to these impurities and prepared a C-terminal cysteine for conjugation. Site-directed conjugation, conducted at pH 5.9 +/- 0.1 with reaction monitoring and cysteine quenching, yielded F5cys-MP-PEG(2000)-DSPE. Low-pressure size exclusion chromatography separated spontaneously formed, high-molecular-weight conjugate micelles from low-molecular-weight impurities. When formulated at 1-2 mg/mL in 10 mM trisodium citrate, 10% sucrose (w/v), at pH 6.4 (HCl), the conjugate was stable when stored below -70 degrees C. Six scale-up lots were compared. The largest 40-L culture produced enough F5cys to manufacture 2,085 mg of conjugate, enough to support planned preclinical and future clinical trials. The conjugate was 93% pure, as measured by polyacrylamide gel electrophoresis. Impurities were primarily identified as product-related. Residual endotoxin, rProtein A, and genomic DNA, were at acceptable levels. This study successfully addressed a necessary step in the scale-up of immunoliposome-encapsulated therapeutics.
Amino Acid Sequence↗