(The FASEB Journal. 2005;19:331-341.)
© 2005 FASEB
Antibody phage display technologies with special reference to angiogenesis
Julia Smith*,1,
Roland E. Kontermann
,
Jim Embleton*,
and
Shant Kumar*,
* University of Manchester, Manchester, UK;
Vectron Therapeutics AG, Marburg, Germany; and
Christie Hospital NHS Trust, Manchester, UK
1Correspondence: University of Manchester, Stopford Building, Oxford Rd, Manchester, M13 9PT, UK. E-mail: Jusmith{at}fs1.scg.man.ac.uk
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ABSTRACT
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The presence of blood vessels is a prerequisite for normal development, tissue growth, and tissue repair. However, its abnormal occurrence or absence can also potentiate disease processes. Angiogenic therapies have been used to stimulate blood vessel growth in ischemic conditions such as severe end-stage peripheral vascular disease, ischemic heart disease and stroke and for inhibition of angiogenesis in tumors. The targeting and identification of novel endothelial cell (EC) markers that can ultimately be used in angiogenic strategies is an expanding field but is limited by the availability of reagents. For instance repeated injection of mouse monoclonal antibodies (Mabs) against angiogenic EC, can result in the production of autoantibodies. Therefore, these mouse Mabs cannot be used for therapeutic purposes. Phage display technology was employed in this context to select antibodies, proteins, and peptides against known or novel EC antigens. Furthermore, technologies have been developed that enable the specific targeting of epitopes on cells including the endothelium with high-affinity/ avidity antibodies. The focus for these antibody targeting strategies are markers that are unique or up-regulated on angiogenic EC including the vascular endothelial growth factor receptor (VEGFR) KDR, endoglin (CD105), and the extracellular domain B (ED-B) domain of fibronectin (FN). These markers are reviewed herein.—Smith, J., Kontermann, R. E., Embleton, J., Kumar, S. Antibody phage display technologies with special reference to angiogenesis.
Key Words: vasculogenesis • angiogenic therapy • vascular targeting • VEGF • CD105
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INTRODUCTION
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BLOOD VESSELS are a prerequisite for normal development, tissue growth and repair as they provide nutrients, remove waste products and are a means of transportation of cells to distant sites. Although the molecular mechanisms regulating blood vessel formation have received considerable attention, especially over the last decade, much remains to be determined.
Blood vessels form through one of two processes: vasculogenesis, where the blood vessels develop de novo via the assembly of endothelial cell precursors (angioblasts), or angiogenesis, when new blood vessels arise by sprouting from preexisting ones. Angiogenesis is tightly governed, limited by the metabolic demands of the tissue. In response to increases in mass, new capillaries arise from the vascular bed in embryogenesis, wound healing, the growth and repair of normal tissues and in disease states such as diabetic retinopathy, tumor growth and rheumatoid arthritis. Its occurrence can potentiate the disease process and many pathological conditions are characterized by over abundant or less frequently insufficient vascularization (1
, 2)
.
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ANGIOGENESIS
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Vast inroads have been made into research into anti-angiogenesis and currently there are over 60 anti-angiogenic/angiogenic drugs undergoing phase I, II or III clinical trials (3
4
5)
. These advances have occurred as a result of better understanding of the functions of growth factors, cytokines and extracellular matrix (ECM) molecules in blood vessel growth and maintenance and cloning of their genes. The ultimate target for both positive and negative regulators of blood vessel growth is the endothelium. Regulators of angiogenesis can act either directly on endothelial cells (EC) or indirectly after production from adjacent non-EC. A balance between the pro and anti-angiogenic factors, termed the "angiogenic switch" determines the activation status of EC (6)
. Once stimulated, EC synthesize proteases and ECM, creating a pathway on which they can migrate, thereby enabling sprouting of microvessels to occur.
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ANGIOGENIC AND ANTI-ANGIOGENIC STRATEGIES
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The development of therapeutic strategies has received impetus from promising studies in vitro and in vivo. Tumour targeting agents have traditionally focused on epitopes found on the tumor cell membranes. Recently the focus has switched to those expressed on endothelium as they are more readily accessible. Therapeutic targeting of angiogenic EC is an alternative strategy to control angiogenesis, as there is no barrier to the luminally expressed epitopes. Angiogenic therapies include stimulation of blood vessel growth in ischemic conditions such as severe end-stage peripheral vascular disease or ischemic heart disease and inhibition of angiogenesis in diabetes, rheumatoid arthritis or tumor progression.
A number of angiogenic strategies used to enhance blood vessel formation include topical application and injection of angiogenic proteins, injection of naked plasmids, viral vector gene therapy, cell-mediated gene transfer and stem cell directed therapy (reviewed in ref 7
). Some of these techniques have shown promise in animal models and others are being evaluated in early stage clinical trials in humans. For example, Steed (8)
reported that the topical application of recombinant human platelet-derived growth factor effectively and safely stimulated the healing of chronic diabetic lower extremity ulcers. Hughes et al. (9)
reviewed the therapeutic effects of basic fibroblast growth factor (FGF-1) and vascular endothelial cell growth factor (VEGF) in stimulating angiogenesis in ischemic heart muscle. They concluded that both growth factors may be useful for improving regional perfusion, but in combination with other additional therapies or growth factors (9)
. Yau et al. (10)
demonstrated that injection of VEGF transfected heart cells into rat cryoinjured myocardial tissue resulted in a 4-fold increase in VEGF165 expression in the scarred area lasting for at least 4 weeks (10)
. The authors concluded that transplantation of these modified heart cells was an excellent delivery system for myocardial gene therapy by producing the angiogenic factor in a localized and sustained pattern. Similarly injection of self bone marrow stem cells into rat ischemic heart arteries stimulated new blood vessel growth (11
, 12)
.
Anti-angiogenic therapies encompass injection of growth factors by themselves into tissues and inoculation of various combinations of molecules, such as growth factor/adhesion molecule-toxin conjugates, fixed whole cells, activated matrix molecules and their antagonists, and antibody/integrin antagonist conjugates. Anti-angiogenic targeting has many advantages; for example, the destruction of only a small number of EC in a segment of microvessel wall results in the death of thousands of dependent tumor cells, and there is limited drug resistance to most anti-angiogenic agents (13)
. Numerous growth factor-toxin or adhesion molecule-toxin conjugates have been described as coaguligands with the aim of preventing blood flow that restricts oxygen and nutrients supply to tissues (14
15
16)
. Tissue factor (TF) is the major initiating receptor for the blood coagulation cascade and is expressed on EC lining the tumor vasculature and on many types of tumor cells, but not normal vasculature. Ran et al. (16)
demonstrated that vascular cell adhesion molecule-1 (VCAM-1) covalently linked to tissue factor localized to the tumor vasculature when injected into mice bearing solid Hodgkin’s lymphomas, causing thrombosis and retarding tumor growth. VCAM-1 is also expressed in the heart and in lung vessels, and although the coaguligand was localized there, no thrombosis was observed. This was due to the absence of phosphatidylserine, which provides the coagulant surface onto which the complexes assemble, and suggested that this is a safety feature characteristic of coaguligands. The 
vß3 integrin has been localized to angiogenic vessels and its targeting with antibodies (Vitaxin) and antagonists (Cilengitide) results in blocking of integrin ligation, promotes apoptosis, and causes disruption of new blood vessel formation (17;
reviewed in ref 18
). Storgard et al. (19)
injected a cyclic peptide antagonist of the integrin into arthritic areas in rabbits and found there was a reduction in joint swelling, synovial infiltrate, and pannus formation and synovial angiogenesis was inhibited. Wei et al. (20)
described a novel immunotherapy to breach immune tolerance and elicit a response against angiogenic EC primarily in tumors. In a mouse model, fixed xenogeneic whole EC were injected near to the tumor site, which caused regression of the tumor and increased the survival of the tumor-bearing mice. The authors concluded that this strategy may induce an autoimmune response against the tumor EC as a cross-reaction.
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PROBLEMS WITH ANTI- ANGIOGENIC/ ANGIOGENIC THERAPY
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The progression of angiogenic strategies from cell culture and animal models to humans has yielded important findings, notably that blood vessels in tumors are abnormal, irregular in diameter, defective and leaky. Risk factors noted during human trials include the effects of the drug on the surrounding tissue. This can happen as a result of leakage of the injected growth factor protein or cDNA vector, leading to angiogenesis potentially promoting disease or conversely necrosis of the surrounding tissue, depending on the targeting agent (reviewed in ref 21
). Small-scale clinical trials may inadvertently misrepresent the nature of the applied therapy due to low patient numbers, so that selection of the appropriately large patient populations is important. For example, Losordo et al. (22)
reported improvements in five patients with cardiovascular disease treated with VEGF gene therapy. However, a larger study failed to confirm their findings (23)
. Finally, treatment with an angiogenic promoter or inhibitor requires coapplication of markers to monitor the effects of the drugs systemically. Animal models cannot predict any specific side effects in humans or long-term toxicity data. For instance, VEGF gene therapy in peripheral vascular disease enhanced vascularization of the area in patients, but caused edema of the lower leg (24)
. These problems, associated with the manipulation of angiogenesis, have been recognized (5
, 25
, 26)
.
Research is actively developing other agents capable of specifically targeting novel EC markers, which can ultimately be used in angiogenesis modulating strategies. However, in various diseased conditions it is equally important to image and quantify their blood vessels in order to evaluate the therapeutic effect of the therapy. Very often monoclonal antibodies (Mabs) have been used for this purpose, with fluorescently labeled markers, allowing visualization in tissue sections. However this is not a practicable proposition in patients and advances in technology are required to monitor changes. Mabs are being manipulated with the aim of modulating blood vessel development and hopefully overcoming some of the problems associated with current angiogenic strategies.
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MONOCLONAL ANTIBODIES
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Mabs are specialized targeting molecules that bind an epitope and can cause the destruction or immobilization of foreign bodies by recruiting complement and/or immune cells via their glycosylated Fc and regulate the turnover of Ig molecules in the circulation (27
, 28)
. There are at least 17 antibodies approved by the US Food and Drug Administration for clinical use in patients with various diseases, and a further 325 undergoing phase I and II clinical trials (29)
. This explosion in antibody use has arisen from the development of technologies enabling the conversion of existing mouse Mabs into mouse-human chimaeric antibodies or fully humanized Mabs (reviewed in ref 30
). Previously, Mabs were of rodent origin and their administration often elicited an immune response in patients, recognizing them as a foreign antigen. Mab-directed therapies have had limited success as monotherapy in human solid tumors (31)
. It is thought that the physical barriers, including elevated interstitial pressure, heterogeneous and reduced functional vasculature and the relatively large distances for Mabs to travel in the tumor interstitia, have contributed to limited tumor penetration. An alternative would be to target the vasculature. Proliferation of ECs in the normal adult is very low but the rate increases rapidly during spurts of angiogenesis. Therefore, markers associated with EC proliferation are potentially good candidates in anti-angiogenic therapy.
Mabs comprise a basic subunit of two 50 kDa heavy chains and two 25 kDa light chains linked by disulphide bonds to form a Y-shaped molecule (Fig. 1
). The antigen binding activity of a Mab is determined by the conformation of its amino acids in the complementarity determining regions (CDR). Three CDRs are present in the variable regions of both the light and heavy chains of the antibody. Immunoglobulin structure is conserved across different species and this has enabled the manipulation of Mabs by humanization or CDR grafting from one Mab to another.
Humanization can be achieved to differing degrees ranging from chimaeric antibodies, where human constant regions are spliced with rodent variable regions or fully humanized Mabs where the framework segment of the variable regions are humanized. Some of these engineered Mabs have their own problems. For example repeated use of a partially humanized Mabs used in rheumatoid arthritis patients caused human-anti-mouse antibody (HAMA) responses (32)
. The generation of human antibodies should reduce the risk of immunogenic reactions after their administration in patients.
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SINGLE CHAIN Fv, Fab, AND F(ab)2 ANTIBODIES
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To overcome problems associated with IgG molecules, such as Fc-mediated side effects and neutralizing immune responses, smaller recombinant antibody fragments carrying only antigen binding functions have been made and are termed scFvs, Fabs or F(ab)2 fragments (Fig. 1)
. The variable regions of the light and heavy chains (VL and VH) can be cloned and joined via a short spacer peptide to form a single chain Fv (scFv). A Fab comprises the variable regions with one constant region linked by disulphide bonds, and the F(ab)2 fragments are bivalent Fab molecules. Antibody fragments can be engineered to improve functional affinity and tissue retention by increasing the number of antigen binding sites: scFv can form diabodies, triabodies and tetrabodies; Fab can form dimers; all of which offer a number of advantages over Mabs. The large size of the intact Mab molecule limits its ability to diffuse from within the vasculature and prolongs its retention in the circulation, leading to bone marrow exposure and toxicity. In contrast the smaller scFv molecules quickly penetrate the vasculature and move into tissues, like solid tumors, and are rapidly eliminated from the circulation through the kidney (33)
. The in vivo half-life of scFvs can be extended if necessary for therapeutic purposes by conjugation to polyethylene glycol (PEG) (34)
. Furthermore, unlike antibody fragments interaction with the Fc receptors on cells in normal tissues can alter the biodistribution of the Mab. Conjugation of toxic drugs can affect the biodistribution, which would alter localization of the antibodies. To replace traditional use of Mabs, antibody fragments need to fulfil four important requirements. Primarily they must bind strongly and specifically to the target epitope, and have prolonged stability at body temperature for a long time as well as during storage and throughout preparatory incubation steps. The fragments to be of therapeutic value must be economical to bulk produce, and like human antibodies should not be immunogenic in patients.
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PHAGE DISPLAY
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Phage display technology allows selection of antibodies, proteins, and peptides against known or novel antigens. Bacteriophages (phage) are viruses that can infect bacterial cells and elicit the replication of their own DNA. In phage display M13 and Fd are the two most commonly used filamentous bacteriophages, comprising single stranded DNA that can infect E.coli through the male sex pilus. Phage DNA has a low transformation frequency and so phagemid vectors are now more commonly used in which phage and plasmid replication abilities are merged into the vector, enabling a 100-fold increase in transformation. The phage display library is a collection of independent clones, each carrying a different foreign DNA insert in the M13 or Fd genome or in the phagemid vector. The foreign gene sequence encoding an antibody, protein or peptide is spliced between genes encoding a phage signal peptide (or more usually a custom signal peptide in the case of phagemid vectors) and a portion of the coat protein, which ensures that the foreign protein is produced as a fusion with the coat protein. The expression of inserted genes is mediated through one of three structural coat proteins, gene 3 protein (pIII), gene 6 protein (pVI) or gene 8 protein (pVIII). Phagemids are designed to contain a gene encoding one of the coat proteins, predominantly pIII, with restriction sites to enable insertion of the foreign DNA sequences. The synthesized fusion coat protein is incorporated into the phage particles, that are released from the bacterial cell and the foreign protein or peptide is displayed on the phage surface (Fig. 2
). Epitopes of interest can be targeted with these foreign proteins expressed on the phage and those phage particles that bind to the epitope can be isolated, transduced back into the bacteria and the bacteria grown to expand the selected phage population.
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PHAGE ANTIBODY LIBRARIES
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The success of phage display selection depends primarily upon the quality of the original library used, as ligands that are not represented cannot be isolated. Phage display libraries contain repertoires of DNA encoding peptide, protein or antibody fragment sequences. Peptide libraries comprise millions of short variable amino acid sequences engineered into the coat protein, their construction and use has been reviewed extensively (35
36
37)
, and is only briefly mentioned here. Antibody fragment libraries can be constructed using Ig variable region genes derived from the B cells of an immunized donor (immune library), B cells of a nonimmunized donor (naive library), from a combination of germline V genes and synthetic oligonucleotide sequences encoding random CDRs (semi-synthetic library), or using fully synthetic sources (synthetic library). For the first option IgG genes from the spleen (i.e., B cells) of animals immunized with the target antigen are isolated (38)
. B cells can be harvested from patients who have potentially high levels of circulating antibodies for instance in patients with an autoimmune disorder or cancer (39
40
41)
. An immune B cell library is enriched in antigen specific antibodies that have been affinity matured by the hosts’ immune system. The advantage of using an immune phage display library is the selection of potentially high-affinity clones against the target antigen, with no additional benefit for selection against other nonimmunized molecules. The possible drawbacks are ethical constraints, where active immunization is not always possible or is ineffective. For instance, if the antigen does not elicit an immune response due to tolerance mechanisms or toxicity, then no enrichment is possible (reviewed in ref 42
).
Naive libraries are derived from the peripheral blood lymphocytes (43)
or lymphoid tissue samples of unimmunized donors. Germline V genes are reverse transcribed and amplified from B cell RNA and Ig heavy and light chain variable region (V) genes are randomly combined to encode the library (44
, 45)
. A naive library is not enriched for phages that bind specific antigens of interest. However, in principle a single naive library can be used to clone numerous antibodies against many different antigens including those previously found to be relatively toxic and nonimmunogenic, provided the initial library has a large enough repertoire to maximise the chance of isolating high-affinity antibodies. Semi-synthetic libraries are built artificially by the in vitro assembly of germline V sequences linked to randomized synthetic CDRs, where predominantly the CDR3 region of the heavy chain is altered and spliced with J regions to expand the library repertoire (46)
. The randomization of all the CDR regions has also been reported (47
, 48)
. A number of large semi-synthetic libraries available, one of the largest called the Griffin-1 library contains human scFv fragments (H Griffin, MRC, Cambridge, UK, unpublished data). This was constructed by recloning synthetic heavy and light chain variable regions from a 2-lox Fd phage library vector (49)
into the pHEN2 phagemid vector. In addition, several fully synthetic scFv libraries have been developed based on single or multiple variable domains used as scaffold and CDRs of varying composition and length (50
, 51)
.
The search for specific high-affinity antibody or peptide fragments has resulted in improvements in the construction of libraries, phage selection methodologies and fragment affinity maturation. Alterations in library construction have sought to improve their functional size, increase the repertoire of immune libraries and encode natural variation into the phage display system using in vivo technology (52
, 53)
. In general, the affinity of scFvs isolated from small naive libraries are lower than those isolated from larger libraries (44
, 49
, 54
, 55)
. The size of the library is not only important for the selection of high-affinity fragments; it also determines the success rate of selection of phage against a large repertoire of different antigens (54
, 55)
.
The use of immunized libraries improves the probability of successful selection of clones reacting with the antigen. For instance scFv with unusually high tumor specificity for human melanoma and breast cancer were isolated from a library constructed of V genes from donors with a high titer of autoantibodies (41)
. However, as there is strong bias in the diversity introduced because of the donors’ recent immune history, the library may not be sufficiently large enough to select high-affinity clones.
In vitro constructed libraries are based on the use of degenerate synthetic oligonucleotides forming the VH and VL regions, which have been randomly combined from a limited number of starting blocks. The encoded variation of these oligos does not necessarily resemble fully the variation found in naturally occurring immunoglobulin genes, as currently understanding of the immune repertoire is not complete. Söderlind et al. (56)
described a novel system of CDR implantation, which instead of combining one VH with one VL region comprising three CDR each that allows in vivo formed individual CDR segments to be combined randomly into a framework for phage display systems. More recently, a Fab library designed to facilitate variation in all six separate CDR regions has become available (BioInvent) (57)
that is thought to produce a library with greater variability than any of the previous ones.
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GENERAL METHODS FOR SELECTION OF PHAGE
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The ultimate aim of phage display is the selection of phage, which bind to the target antigen of interest with high affinity from a large excess of phage clones that do not bind or do so with lower affinity. This is achieved by multiple rounds of binding the phage to the target, washing to remove nonbound phage followed by elution and retrieval of specifically bound phage. Apart from the amount of antigen present, during selection other parameters have to be taken into account depending on the nature of the antigen.
Procedures for selecting against purified antigens rely upon the binding affinity of the phage for the antigen of interest relative to phage particles that do not bind, and require the antigen to be attached to a solid support (38
, 43
, 49)
.
Selection of phage antibody fragments has been achieved in vitro on nonpurified antigens attached to solid supports (58)
intact mammalian (59
60
61)
, and prokaryotic cells, native antigens in tissue section, nitrocellulose/PVDF membranes (after 1- or 2-dimensional electrophoresis of antigens, which cannot be readily purified) and in vivo (62)
(see also Fig. 3
for an overview of methods).
Antibody fragments can be raised against heterogeneous cell mixtures by fluorescence activated cell sorting (FACs) selection. The phage library is incubated with the cells and non binding phage is removed by washing. The cells of interest are labeled with a known fluorescence labeled Mab and cell sorting performed, sorted phages are eluted and amplified (63)
. Using biopanning against cells in suspension, non binding phage can be separated by differential centrifugation of the cell-phage complex through an organic phase. Bound phage is recovered in the cell pellet (60)
.
Phage display technology has been used to directly isolate peptides and antibody fragments that bind to antigens in vivo (62
, 64)
, or applying ex vivo selection protocols using isolated organs (65)
. In the in vivo selection methods phage are injected into the vasculature of live animals. Nonspecific peptides, which bind ubiquitously to EC in any organ, are diluted out by non-discriminate binding to the general vasculature and removed by perfusion, allowing phage that specifically bind to the endothelial cells of the organ of interest to be isolated after removal of the organ. Using this method, organ specific differences in the vascular tissue of a panel of markers have been identified (62
, 64)
. In vivo selection in humans has recently been reported (62)
but obviously the ethical constraints will seriously limit its use (64)
.
Success of the selection process can be assessed using one of three processes, panning, plaque lifts or capture lifts. After the desired phage have been selected and eluted if necessary they are transfected into bacteria where the phage particles are amplified and then purified. In summary selection procedures are highly flexible and can be tailored as required and have yielded antibody fragments against carbohydrate moieties and self-antibodies (59
, 66)
; two traditionally difficult areas in which to raise Mabs.
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AFFINITY AND STABILITY IMPROVEMENTS
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In general scFv have a lower avidity than a divalent IgG molecule. In addition, their small size and clearance rate result in problems with reliable tumor targeting and retention. The scFv molecule can be engineered to improve its retention by increasing its affinity to the target antigens. Various phage display strategies have been used to select for greater stability and include alteration of environmental conditions a) initially reducing the antibodies’ stability using protein engineering (67)
, b) selection of protease resistance as a property of native, thermodynamically stable proteins (68)
c) creating mutant scFv gene repertoires and the higher affinity binders can then be selected on antigen (69)
d) randomized mutagenesis of stable scFvs using DNA shuffling and oligonucleotide site-directed mutagenesis under selective pressure of temperature stress has produced antibodies with higher thermodynamic stability (70)
and e) temperature stress has been used in conjunction with agents such as guanidinium chloride to increase protein stability (58)
.
Although affinity selection allows large numbers of clones to be screened, it is potentially biased toward the enrichment of phage populations displaying multiple copies of the antigen binding site or rather than phage clones with binding sites of greater affinity. High-affinity binders are not always desirable. For instance, Lu and co-workers (71)
demonstrated that a high-affinity Fab antibody against KDR did not penetrate the tumors as well as a lower affinity Fab. The high-affinity Fab was restricted to the perivascular vessels of the tumor, impairing its anti-tumor activity.
One of the easiest methods of improving the binding of an scFv to its target antigen is to increase its functional affinity through the creation of a multimer. A number of multivalent scFv-based structures have been engineered (33)
. The multimers are formed following the inclusion of a short (5 amino acid chain) interdomain linkers in the scFv, that prevents intrachain pairing of the VH and VL domains, but allows interchain pairing to form diabodies, triabodies or tetrabodies (72)
.
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SELECTION OF ANTIBODIES FOR VASCULAR TARGETING AND ANTI-ANGIOGENIC STRATEGIES
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Different strategies have been applied to isolate vascular targeting antibodies from phage display libraries. As outlined above, the use of purified or recombinant expressed endothelial marker molecules allows for the selection of antibodies with defined specificities and thus is the method of choice if purified antigen is available. However, using this approach it is critical to identify those antibodies that also react with the antigen in the context of the endothelial cell membrane. This might require extensive screening or reselection on endothelial cells. Nevertheless, this approach has been successfully applied to isolate antibodies against various molecules expressed by endothelial cells or associated with the extracellular matrix.
Several studies described the isolation of antibody molecules against receptors or adhesion molecules expressed by endothelial cells. These include the selection of antibodies against VEGF receptor 2 or 3 by panning of murine or rabbit immune libraries or human naive libraries on soluble extracellular receptor fragments (71
, 73)
and the selection of scFv fragments directed against CD105 using semi-synthetic or fully synthetic human libraries (51
, 74)
; Recombinant antibodies were also selected against growth factors associated with angiogenesis, including scFv fragments directed against VEGF or the VEGF-VEGFR complex (75
, 76)
and angiopoietin-2 (Ang-2) (77)
.
Several studies have described the selection of antibodies directed against extracellular matrix proteins. Species-cross-reactive scFv fragments directed against fibroblast activation protein (FAP) were isolated from a murine immune library generated from a FAP–/– knockout mouse (78)
. FAP is a type-II membrane protein expressed on tumor stromal fibroblasts in > 90% of all carcinomas. It is a diagnostic marker and represents a potential target for therapeutic applications (79)
. In another approach, scFv fragments were selected against the ED-B domain of FN, which is inserted into FN by splicing and is found in the stroma of oncofetal and neoplastic tissues and blood vessels of tumors during angiogenesis (80)
. Applying a combinatorial mutagenesis approach, scFv fragments with improved affinity for ED-B were isolated from phage display libraries (81)
. Human scFv phage libraries were used to isolate high-affinity antibody fragments directed against tenascin-C (repeat C), an isoform generated by alternative splicing of nine fibronectin-like type III repeats and abundantly expressed in high-grade astrocytoma (80)
. Another study described the selection of antibodies against laminin. It is a component of the vascular basement membrane of tumor-associated vessels and is involved in tube formation (82)
.
One problem associated with phage display is that under some conditions it is possible to select specifically binding phage that interact with conformations of antigens other than the biologically active conformation (e.g., a denatured form of the antigen), so that the derived antibody fragment is unsuitable for its intended purpose. The selection of antibody or peptide fragments against the vascular endothelium poses these unique problems and various attempts been made to overcome them. Essentially even primary cultures of EC from any given tissue lose their landscape of antigens to a varying degree and these are not truly representative of the cell phenotype in vivo. Furthermore, expression of surface markers from different sources can alter following long-term culture and makes the use of EC cell lines a poor system in which to screen and select for antibody fragments.
Nevertheless, various groups reported the selection of novel antibodies selected on human endothelial cells, e.g., human umbilical vein endothelial cells (HUVEC) or human microvascular endothelial cells (HDMEC) (51
, 83)
. A detailed analysis of some of these antibodies demonstrated strong staining of the vasculature of a series of carcinomas, while none or only weak staining was seen for the vasculature of the corresponding normal tissues. Although the antigens recognized by these antibodies have not been identified, they may be useful for diagnosis as well as therapeutic applications. In addition to the use of primary endothelial cells, the isolation of tumor-derived endothelial cells (TEC) has been described that should be applicable for the selection of novel TEC-specific antibodies (84)
.
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DIRECTING FRAGMENTS AGAINST THE VASCULATURE
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Traditionally anti-angiogenesis therapies have targeted proliferating endothelial cells using drugs that can interfere with the normal cellular processes, via growth factors, protease inhibitors or altering endogenous inhibitor levels. Phage display peptide libraries have been used to screen the vasculature of both mice (85)
and humans (62)
confirming that there is widespread molecular heterogeneity in the vasculature of normal tissues such that peptides can be isolated to target the vasculature of specific organs (85)
. The presence of endothelial cell heterogeneity in tumors implies that antibodies could be sought and used for individual organs. The types of antigen targeted to date are widely expressed in all angiogenic tissues and include VEGF and VEGFR (71)
, CD105 (74
, 86)
, FN (81)
, vß3 (87)
, Ang-2 (77)
, laminin (82)
, FAP (79)
, and matrix metalloproteinases (88)
.
VEGF plays a central role in the angiogenic phenotype and the development of VEGF antagonists is a key player in anti-angiogenesis research. There are currently 10 different anti-VEGF compounds undergoing clinical trials and early analysis has produced some disappointments, for instance Avastin, a humanized VEGF neutralizing antibody, initially failed at stage III for the treatment of breast cancer (89)
, but had been approved in combination with intravenous 5-fluorouracil-based chemotherapy as a first-line treatment for patients with metastatic colorectal cancer. Various other recombinant anti-VEGF antibodies have been developed. Vitaliti and co-workers described an anti-VEGF scFv that blocks vascularization on the chorioallantoic membrane (CAM) and Lu and co-workers raised a Fab against the VEGF receptor KDR that blocks its interaction with the growth factor in vitro (71
, 75)
. Other studies described the use of 90Y-labeled nanoparticles targeted to the vasculature with anti-VEGFR antibodies for tumor therapy (90)
.
The early disappointments in clinical trials may in fact yield a valuable lesson, in that there is a strong possibility that the anti-VEGF therapy may cause tumor cells to preferentially select out tumor cell colonies that produce other growth factors such as FGF2 or TGFß and become apparently resistant to the anti-VEGF therapy. Furthermore, the murine tumor models employed to test agents tend to use fast-growing tumors that have active angiogenesis, whereas in humans tumors generally develop slowly with less angiogenesis and the levels of VEGF may not be the same (5)
.
Ang-2 is up-regulated by several angiogenic factors suggesting that Ang-2 may play a central role in growth factor-initiated angiogenesis. Thus, neutralization of Ang-2 should result in inhibition of angiogenesis. Indeed application of a neutralizing anti-Ang-2 scFv fragment, was able to inhibit VEGF-induced endothelial cell proliferation and migration in vitro (77)
.
Alphavß3-integrin is found on new blood vessels as well as the surface of many solid tumors. Vitaxin (MEDI-522), a humanized version of Mab LM609 directed against vß3-integrin, interferes with blood vessel formation by inducing apoptosis in proliferating endothelial cells (91)
. The antibody is currently in phase 2 clinical trials for the treatment of metastatic melanoma and prostate cancer. Vitaxin is being evaluated for the treatment of rheumatoid arthritis (92)
.
CD105 is highly expressed in angiogenic endothelial cells and has been recognized as a promising vascular target. CD105 is a 180 kDa homodimeric integral membrane glycoprotein composed of disulphide-linked 90-95kDa subunits. CD105 is a receptor for TGFß1 and TGFß3 expressed primarily in the EC of capillaries, arterioles, and venules, as well as on activated monocytes, some leukaemia cells and in the syncytiotrophoblast. It plays a pivotal role in angiogenesis (3
, 93)
. Mab CD105 binds strongly to tumor vasculature than to normal tissue, although variable CD105 expression in normal tissues has raised issues about its therapeutic applications due to potential side effects (94)
. A bispecific antibody single chain diabody that targets human CD105 and the adenovirus knob domain successfully mediated adenoviral transduction in endothelial cells in vitro (74)
. Korn and co-workers have used another bispecific antibody comprising the same CD105 scFv in combination with an scFv targeting the CD3- T cell receptor targeting (95)
. This antibody selectively targets the endothelial cells via CD105 and should be able to destroy the vascular bed by T cell mediated killing. Recently, Costello et al. showed that 99Tcm labeled anti-CD105 Mab (E9) specifically localized to the tumor mass in kidneys from patients with renal cell carcinoma in an ex vivo study (86)
. Mab E9 has been converted into a mouse scFv and shows specific binding to blood vessels in a variety of human tumors (Fig. 4
). Völkel and co-workers (96)
have raised additional scFv against CD105 protein (51)
. One of these scFvs was used to generate immunoliposomes for the targeting of encapsulated drugs to endothelial cells. Thus, CD105 may prove valuable in the development of therapeutic strategies to modulate angiogenesis.
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Figure 4. Top. perfusion of radiolabeled anti-CD105 Mab E9 in human kidney shows localization in tumor tissue (arrow) and Bottom. immunohistochemical staining of human breast tumor tissue section with mouse E9 scFv- an arrow points to one of the stained microvessels.
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It is known that the ED-B domain of FN specifically accumulates around neovascular structures (81)
. All of the work using phage display targeting toward the ED-B domain has centered on the scFv E1 and its mutant L19. The scFv L19 differs from the parent antibody by eight mutations introduced in the hypervariable loops and has a 760-fold improvement in affinity. This high-affinity scFv L19 was localized to aggressive tumors and significantly targets tumors better than E1 (61
, 81)
. Applications of these antibodies include radiotherapeutic use of radiohalogenated antibodies (97)
, photocoagulation of ocular angiogenesis with antibody-coupled photosensitizer (98)
, the enhancement of anti-tumor activities by targeted delivery of cytokines such as IL-2 and IL-12 (99
, 100)
the infarction of solid tumors using tissue factor-scFv fusion proteins (101)
, and the targeted delivery of drugs with immunoliposomes (102)
. Using a bispecific antibody comprising scFv L19 and mouse TNF, Borsi and co-workers (103)
demonstrated that the combination had a potent anti-tumor effect when compared with either component alone. This is a potentially exciting area and further in vivo studies are needed for its validation for clinical use.
Antibodies against FAP, a membrane protein expressed by tumor stroma fibroblasts, were successfully applied for retargeting cytotoxic T lymphocytes in vitro using a recombinant bispecific antibody molecule directed against FAP and the T cell coreceptor CD3 (104)
. In another study, a fusion protein consisting of the extracellular domain of tissue factor and an anti-FAP scFv was able to induce coagulation under physiological conditions (79)
, although as yet, no one has examined its therapeutic efficacy in animal models.
In conclusion phage display technology has already yielded peptides and antibodies that target specific regions of the vasculature. Selection of these molecules is being tailored to improve their affinity and their linkage with other cell receptors or growth factors may improve their potency. Phage display libraries are a dynamic resource containing a wealth of potentially exciting markers awaiting therapeutic exploitation.
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ACKNOWLEDGMENTS
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|---|
Support from the Wellcome Trust is gratefully acknowledged.
Received for publication September 10, 2004.
Accepted for publication October 15, 2004.
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REFERENCES
|
|---|
- Folkman, J. (1995) Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat. Med. 1,27-31[CrossRef][Medline]
- Ballara, S. C., Miotla, J. M., Paleolog, E. M. (1999) New vessels, new approaches: angiogenesis as a therapeutic target in musculoskeletal disorders. Int. J. Exp. Pathol. 80,235-250[CrossRef][Medline]
- Duff, S. E., Li, C., Garland, J. M., Kumar, S. (2003) CD105 is important for angiogenesis: evidence and potential applications. FASEB J. 17,984-992[Abstract/Free Full Text]
- Hudson, P. J., Souriau, C. (2003) Engineered antibodies. Nat. Med. 9,129-134[CrossRef][Medline]
- Cao, Y. (2004) Antiangiogenic cancer therapy. Semin. Cancer Biol. 14,139-145[CrossRef][Medline]
- Folkman, J., Hanahan, D. (1991) Switch to the angiogenic phenotype during tumorigenesis. Princess Takamatsu Symp. 22,339-347[Medline]
- Lei, Y., Haider, H. K., Shujia, J., Sim, E. S. (2004) Therapeutic angiogenesis. Devising new strategies based on past experiences. Basic Res. Cardiol. 99,121-132[CrossRef][Medline]
- Steed, D. L. (1995) Clinical evaluation of recombinant human platelet-derived growth factor for the treatment of lower extremity diabetic ulcers. Diabetic Ulcer Study Group. J. Vasc. Surg. 21,71-78[CrossRef][Medline]
- Hughes, G. C., Biswas, S. S., Yin, B., Coleman, R. E., DeGrado, T. R., Landolfo, C. K., Lowe, J. E., Annex, B. H., Landolfo, K. P. (2004) Therapeutic angiogenesis in chronically ischemic porcine myocardium: comparative effects of bFGF and VEGF. Ann. Thorac. Surg. 77,812-818[Abstract/Free Full Text]
- Yau, T. M., Li, G., Weisel, R. D., Reheman, A., Jia, Z. Q., Mickle, D. A., Li, R. K. (2004) Vascular endothelial growth factor transgene expression in cell-transplanted hearts. J. Thorac. Cardiovasc. Surg. 127,1180-1187[Abstract/Free Full Text]
- Kobayashi, T., Hamano, K., Li, T. S., Katoh, T., Kobayashi, S., Matsuzaki, M., Esato, K. (2000) Enhancement of angiogenesis by the implantation of self bone marrow cells in a rat ischemic heart model. J. Surg. Res. 89,189-195[CrossRef][Medline]
- Zwaginga, J. J., Doevendans, P. (2003) Stem cell-derived angiogenic/vasculogenic cells: possible therapies for tissue repair and tissue engineering. Clin. Exp. Pharmacol. Physiol. 30,900-908[CrossRef][Medline]
- Bredow, S., Lewin, M., Hofmann, B., Marecos, E., Weissleder, R. (2000) Imaging of tumour neovasculature by targeting the TGF-beta binding receptor endoglin. Eur. J. Cancer 36,675-681
- Huang, S. C., Yu, C. H., Huang, R. T., Hsu, K. F., Tsai, Y. C., Chou, C. Y. (1996) Intratumoral blood flow in uterine myoma correlated with a lower tumor size and volume, but not correlated with cell proliferation or angiogenesis. Obstet. Gynecol. 87,1019-1024[CrossRef][Medline]
- Molema, G., Griffioen, A. W. (1998) Rocking the foundations of solid tumor growth by attacking the tumor’s blood supply. Immunol. Today 19,392-394[CrossRef][Medline]
- Ran, S., Gao, B., Duffy, S., Watkins, L., Rote, N., Thorpe, P. E. (1998) Infarction of solid Hodgkin’s tumors in mice by antibody-directed targeting of tissue factor to tumor vasculature. Cancer Res. 58,4646-4653[Abstract/Free Full Text]
- Lode, H. N., Moehler, T., Xiang, R., Jonczyk, A., Gillies, S. D., Cheresh, D. A., Reisfeld, R. A. (1999) Synergy between an antiangiogenic integrin alphav antagonist and an antibody-cytokine fusion protein eradicates spontaneous tumor metastases. Proc. Natl. Acad. Sci. USA 96,1591-1596[Abstract/Free Full Text]
- Tucker, G. C. (2003) Alpha v integrin inhibitors and cancer therapy. Curr. Opin. Investig. Drugs 4,722-731[Medline]
- Storgard, C. M., Stupack, D. G., Jonczyk, A., Goodman, S. L., Fox, R. I., Cheresh, D. A. (1999) Decreased angiogenesis and arthritic disease in rabbits treated with an alphavbeta3 antagonist. J. Clin. Invest. 103,47-54[Medline]
- Wei, Y. Q., Wang, Q. R., Zhao, X., Yang, L., Tian, L., Lu, Y., Kang, B., Lu, C. J., Huang, M. J., Lou, Y. Y., et al (2000) Immunotherapy of tumors with xenogeneic endothelial cells as a vaccine. Nat. Med. 6,1160-1166[CrossRef][Medline]
- McDonald, D. M., Baluk, P. (2002) Significance of blood vessel leakiness in cancer. Cancer Res. 62,5381-5385[Abstract/Free Full Text]
- Losordo, D. W., Vale, P. R., Symes, J. F., Dunnington, C. H., Esakof, D. D., Maysky, M., Ashare, A. B., Lathi, K., Isner, J. M. (1998) Gene therapy for myocardial angiogenesis: initial clinical results with direct myocardial injection of phVEGF165 as sole therapy for myocardial ischemia. Circulation 98,2800-2804[Abstract/Free Full Text]
- Henry, T. D. (1999) Therapeutic angiogenesis. BMJ 318,1536-1539[Free Full Text]
- Isner, J. M., Pieczek, A., Schainfeld, R., Blair, R., Haley, L., Asahara, T., Rosenfield, K., Razvi, S., Walsh, K., Symes, J. F. (1996) Clinical evidence of angiogenesis after arterial gene transfer of phVEGF165 in patient with ischaemic limb. Lancet 348,370-374[CrossRef][Medline]
- Thompson, W. D., Li, W. W., Maragoudakis, M. (1999) The clinical manipulation of angiogenesis: pathology, side effects, surprises, and opportunities with novel human therapies. J. Pathol. 187,503-510[CrossRef][Medline]
- Simons, M., Bonow, R. O., Chronos, N. A., Cohen, D. J., Giordano, F. J., Hammond, H. K., Laham, R. J., Li, W., Pike, M., Sellke, F. W., et al (2000) Clinical trials in coronary angiogenesis: issues, problems, consensus: An expert panel summary. Circulation 102,E73-E86
- Kohler, G., Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (London) 256,495-497[CrossRef][Medline]
- Ghetie, V., Ward, E. S. (1997) FcRn: the MHC class I-related receptor that is more than an IgG transporter. Immunol. Today 18,592-598[CrossRef][Medline]
- Brekke, O. H., Loset, G. A. (2003) New technologies in therapeutic antibody development. Curr. Opin. Pharmacol. 3,544-550[CrossRef][Medline]
- Glennie, M. J., Johnson, P. W. (2000) Clinical trials of antibody therapy. Immunol. Today 21,403-410[CrossRef][Medline]
- Trail, P. A., Bianchi, A. B. (1999) Monoclonal antibody drug conjugates in the treatment of cancer. Curr. Opin. Immunol. 11,584-588[CrossRef][Medline]
- Matteson, E. L., Yocum, D. E., St Clair, E. W., Achkar, A. A., Thakor, M. S., Jacobs, M. R., Hays, A. E., Heitman, C. K., Johnston, J. M. (1995) Treatment of active refractory rheumatoid arthritis with humanized monoclonal antibody CAMPATH-1H administered by daily subcutaneous injection. Arthritis Rheum. 38,1187-1193[Medline]
- Adams, G. P., Schier, R. (1999) Generating improved single-chain Fv molecules for tumor targeting. J. Immunol. Methods 231,249-260[CrossRef][Medline]
- Lee, L. S., Conover, C., Shi, C., Whitlow, M., Filpula, D. (1999) Prolonged circulating lives of single-chain Fv proteins conjugated with polyethylene glycol: a comparison of conjugation chemistries and compounds. Bioconjug. Chem. 10,973-981[CrossRef][Medline]
- Zwick, M. B., Shen, J., Scott, J. K. (1998) Phage-displayed peptide libraries. Curr. Opin. Biotechnol. 9,427-436[CrossRef][Medline]
- Brown, K. C. (2000) New approaches for cell-specific targeting: identification of cell-selective peptides from combinatorial libraries. Curr. Opin. Chem. Biol. 4,16-21[CrossRef][Medline]
- Romanov, V. I. (2003) Phage display selection and evaluation of cancer drug targets. Curr. Cancer Drug Targets 3,119-129[CrossRef][Medline]
- Clackson, T., Hoogenboom, H. R., Griffiths, A. D., Winter, G. (1991) Making antibody fragments using phage display libraries. Nature (London) 352,624-628[CrossRef][Medline]
- Cai, X., Garen, A. (1997) Comparison of fusion phage libraries displaying VH or single-chain Fv antibody fragments derived from the antibody repertoire of a vaccinated melanoma patient as a source of melanoma-specific targeting molecules. Proc. Natl. Acad. Sci. USA 94,9261-9266[Abstract/Free Full Text]
- Hall, B. L., Boroughs, J., Kobrin, B. J. (1998) A novel tumor-specific human single-chain Fv selected from an active specific immunotherapy phage display library. Immunotechnology 4,127-140[CrossRef][Medline]
- Kupsch, J. M., Tidman, N. H., Kang, N. V., Truman, H., Hamilton, S., Patel, N., Newton Bishop, J. A., Leigh, I. M., Crowe, J. S. (1999) Isolation of human tumor-specific antibodies by selection of an antibody phage library on melanoma cells. Clin. Cancer Res. 5,925-931[Abstract/Free Full Text]
- Hoogenboom, H. R., De Bruine, A. P., Hufton, S. E., Hoet, R. M., Arends, J. W., Roovers, R. C. (1998) Antibody phage display technology and its applications. Immunotechnology 4,1-20[CrossRef][Medline]
- Marks, J. D., Hoogenboom, H. R., Bonnert, T. P., McCafferty, J., Griffiths, A. D., Winter, G. (1991) By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222,581-597[CrossRef][Medline]
- Winter, G., Griffiths, A. D., Hawkins, R. E., Hoogenboom, H. R. (1994) Making antibodies by phage display technology. Annu. Rev. Immunol. 12,433-455[Medline]
- Hoogenboom, H. R. (1997) Designing and optimizing library selection strategies for generating high-affinity antibodies. Trends Biotechnol. 15,62-70[CrossRef][Medline]
- Hoogenboom, H. R., Marks, J. D., Griffiths, A. D., Winter, G. (1992) Building antibodies from their genes. Immunol. Rev. 130,41-68[CrossRef][Medline]
- Garrard, L. J., Zhukovsky, E. A. (1992) Antibody expression in bacteriophage systems: the future of monoclonal antibodies?. Curr. Opin. Biotechnol. 3,474-480[CrossRef][Medline]
- Soderlind, E., Vergeles, M., Borrebaeck, C. A. (1995) Domain libraries: synthetic diversity for de novo design of antibody V-regions. Gene 160,269-272[CrossRef][Medline]
- Griffiths, A. D., Williams, S. C., Hartley, O., Tomlinson, I. M., Waterhouse, P., Crosby, W. L., Kontermann, R. E., Jones, P. T., Low, N. M., Allison, T. J. (1994) Isolation of high affinity human antibodies directly from large synthetic repertoires. EMBO J. 13,3245-3260[Medline]
- Knappik, A., Ge, L., Honegger, A., Pack, P., Fischer, M., Wellnhofer, G., Hoess, A., Wolle, J., Pluckthun, A., Virnekas, B. (2000) Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J. Mol. Biol. 296,57-86[CrossRef][Medline]
- Volkel, T., Muller, R., Kontermann, R. E. (2004) Isolation of endothelial cell-specific human antibodies from a novel fully synthetic scFv library. Biochem. Biophys. Res. Commun. 317,515-521[CrossRef][Medline]
- Holt, L. J., Bussow, K., Walter, G., Tomlinson, I. M. (2000) By-passing selection: direct screening for antibody-antigen interactions using protein arrays. Nucleic Acids Res. 28,E72
- Siegel, D. L. (2002) Recombinant monoclonal antibody technology. Transfus. Clin. Biol. 9,15-22[CrossRef][Medline]
- Vaughan, T. J., Williams, A. J., Pritchard, K., Osbourn, J. K., Pope, A. R., Earnshaw, J. C., McCafferty, J., Hodits, R. A., Wilton, J., Johnson, K. S. (1996) Human antibodies with subnanomolar affinities isolated from a large nonimmunized phage display library. Nat. Biotechnol. 14,309-314[CrossRef][Medline]
- Sheets, M. D., Amersdorfer, P., Finnern, R., Sargent, P., Lindquist, E., Schier, R., Hemingsen, G., Wong, C., Gerhart, J. C., Marks, J. D., et al (1998) Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens. Proc. Natl. Acad. Sci. USA 95,6157-6162[Abstract/Free Full Text]
- Soderlind, E., Ohlin, M., Carlsson, R. (1999) Complementarity-determining region (CDR) implantation: a theme of recombination. Immunotechnology 4,279-285[CrossRef][Medline]
- Carlsson, R., Soderlind, E. (2001) n-CoDeR concept: unique types of antibodies for diagnostic use and therapy. Expert Rev. Mol. Diagn. 1,102-108[CrossRef][Medline]
- Jung, S., Honegger, A., Pluckthun, A. (1999) Selection for improved protein stability by phage display. J. Mol. Biol. 294,163-180[CrossRef][Medline]
- Cai, X., Garen, A. (1995) Anti-melanoma antibodies from melanoma patients immunized with genetically modified autologous tumor cells: selection of specific antibodies from single-chain Fv fusion phage libraries. Proc. Natl. Acad. Sci. USA 92,6537-6541[Abstract/Free Full Text]
- Giordano, R. J., Cardo-Vila, M., Lahdenranta, J., Pasqualini, R., Arap, W. (2001) Biopanning and rapid analysis of selective interactive ligands. Nat. Med. 7,1249-1253[CrossRef][Medline]
- Viti, F., Giovannoni, L., Neri, D. (2002) Recombinant antibodies for the selective targeting of tumor neovasculature. Curr. Opin. Drug Discov. Dev. 5,204-213[Medline]
- Arap, W., Kolonin, M. G., Trepel, M., Lahdenranta, J., Cardo-Vila, M., Giordano, R. J., Mintz, P. J., Ardelt, P. U., Yao, V. J., Vidal, C. I., et al (2002) Steps toward mapping the human vasculature by phage display. Nat. Med. 8,121-127[CrossRef][Medline]
- Lekkerkerker, A., Logtenberg, T. (1999) Phage antibodies against human dendritic cell subpopulations obtained by flow cytometry-based selection on freshly isolated cells. J. Immunol. Methods 231,53-63[CrossRef][Medline]
- Rajotte, D., Arap, W., Hagedorn, M., Koivunen, E., Pasqualini, R., Ruoslahti, E. (1998) Molecular heterogeneity of the vascular endothelium revealed by in vivo phage display. J. Clin. Invest. 102,430-437[Medline]
- Maruta, F., Parker, A. L., Fisher, K. D., Murray, P. G., Kerr, D. J., Seymour, L. W. (2003) Use of a phage display library to identify oligopeptides binding to the lumenal surface of polarized endothelium by ex vivo perfusion of human umbilical veins. J. Drug Target. 11,53-59[CrossRef][Medline]
- Griffiths, A. D., Malmqvist, M., Marks, J. D., Bye, J. M., Embleton, M. J., McCafferty, J., Baier, M., Holliger, K. P., Gorick, B. D., Hughes-Jones, N. C. (1993) Human anti-self antibodies with high specificity from phage display libraries. EMBO J. 12,725-734[Medline]
- Ruan, B., Hoskins, J., Wang, L., Bryan, P. N. (1998) Stabilizing the subtilisin BPN’ pro-domain by phage display selection: how restrictive is the amino acid code for maximum protein stability?. Protein Sci. 7,2345-2353[Medline]
- Sieber, V., Pluckthun, A., Schmid, F. X. (1998) Selecting proteins with improved stability by a phage-based method. Nat. Biotechnol. 16,955-960[CrossRef][Medline]
- Adams, G. P. (1998) Improving the tumor specificity and retention of antibody-based molecules. In Vivo 12,11-21[Medline]
- Schier, R., McCall, A., Adams, G. P., Marshall, K. W., Merritt, H., Yim, M., Crawford, R. S., Weiner, L. M., Marks, C., Marks, J. D. (1996) 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. J. Mol. Biol. 263,551-567[CrossRef][Medline]
- Lu, D., Shen, J., Vil, M. D., Zhang, H., Jimenez, X., Bohlen, P., Witte, L., Zhu, Z. (2003) Tailoring in vitro selection for a picomolar affinity human antibody directed against vascular endothelial growth factor receptor 2 for enhanced neutralizing activity. J. Biol. Chem. 278,43496-43507[Abstract/Free Full Text]
- Atwell, J. L., Breheney, K. A., Lawrence, L. J., McCoy, A. J., Kortt, A. A., Hudson, P. J. (1999) scFv multimers of the anti-neuraminidase antibody NC10: length of the linker between VH and VL domains dictates precisely the transition between diabodies and triabodies. Protein Eng. 12,597-604[Abstract/Free Full Text]
- Persaud, K., Tille, J. C., Liu, M., Zhu, Z., Jimenez, X., Pereira, D. S., Miao, H. Q., Brennan, L. A., Witte, L., Pepper, M. S., et al (2004) Involvement of the VEGF receptor 3 in tubular morphogenesis demonstrated with a human anti-human VEGFR-3 monoclonal antibody that antagonizes receptor activation by VEGF-C. J. Cell Sci. 117,2745-2756[Abstract/Free Full Text]
- Nettelbeck, D. M., Miller, D. W., Jerome, V., Zuzarte, M., Watkins, S. J., Hawkins, R. E., Muller, R., Kontermann, R. E. (2001) Targeting of adenovirus to endothelial cells by a bispecific single-chain diabody directed against the adenovirus fiber knob domain and human endoglin (CD105). Mol. Ther. 3,882-891[CrossRef][Medline]
- Vitaliti, A., Wittmer, M., Steiner, R., Wyder, L., Neri, D., Klemenz, R. (2000) Inhibition of tumor angiogenesis by a single-chain antibody directed against vascular endothelial growth factor. Cancer Res. 60,4311-4314[Abstract/Free Full Text]
- Cooke, S. P., Boxer, G. M., Lawrence, L., Pedley, R. B., Spencer, D. I., Begent, R. H., Chester, K. A. (2001) A strategy for antitumor vascular therapy by targeting the vascular endothelial growth factor: receptor complex. Cancer Res. 61,3653-3659[Abstract/Free Full Text]
- Cai, M., Zhang, H., Hui, R. (2003) Single chain Fv antibody against angiopoietin-2 inhibits VEGF-induced endothelial cell proliferation and migration in vitro. Biochem. Biophys. Res. Commun. 309,946-951[CrossRef][Medline]
- Brocks, B., Garin-Chesa, P., Behrle, E., Park, J. E., Rettig, W. J., Pfizenmaier, K., Moosmayer, D. (2001) Species-crossreactive scFv against the tumor stroma marker "fibroblast activation protein" selected by phage display from an immunized FAP–/– knock-out mouse. Mol. Med. 7,461-469[Medline]
- Rippmann, J. F., Pfizenmaier, K., Mattes, R., Rettig, W. J., Moosmayer, D. (2000) Fusion of the tissue factor extracellular domain to a tumour stroma specific single-chain fragment variable antibody results in an antigen-specific coagulation-promoting molecule. Biochem. J. 349,805-812
- Carnemolla, B., Castellani, P., Ponassi, M., Borsi, L., Urbini, S., Nicolo, G., Dorcaratto, A., Viale, G., Winter, G., Neri, D., et al (1999) Identification of a glioblastoma-associated tenascin-C isoform by a high affinity recombinant antibody. Am. J. Pathol. 154,1345-1352[Abstract/Free Full Text]
- Viti, F., Tarli, L., Giovannoni, L., Zardi, L., Neri, D. (1999) Increased binding affinity and valence of recombinant antibody fragments lead to improved targeting of tumoral angiogenesis. Cancer Res. 59,347-352[Abstract/Free Full Text]
- Sanz, L., Kristensen, P., Blanco, B., Facteau, S., Russell, S. J., Winter, G., Alvarez-Vallina, L. (2002) Single-chain antibody-based gene therapy: inhibition of tumor growth by in situ production of phage-derived human antibody fragments blocking functionally active sites of cell-associated matrices. Gene Ther. 9,1049-1053[CrossRef][Medline]
- Mutuberria, R., Hoogenboom, H. R., van der, L. E., De Bruine, A. P., Roovers, R. C. (1999) Model systems to study the parameters determining the success of phage antibody selections on complex antigens. J. Immunol. Methods 231,65-81[CrossRef][Medline]
- Hannum, R. S., Ojeifo, J. O., Zwiebel, J. A., McLeskey, S. W. (2001) Isolation of tumor-derived endothelial cells. Microvasc. Res. 61,287-290[CrossRef][Medline]
- Essler, M., Ruoslahti, E. (2002) Molecular specialization of breast vasculature: a breast-homing phage-displayed peptide binds to aminopeptidase P in breast vasculature. Proc. Natl. Acad. Sci. USA 99,2252-2257[Abstract/Free Full Text]
- Costello, B., Li, C., Duff, S., Butterworth, D., Khan, A., Perkins, M., Owens, S., Al Mowallad, A. F., O’Dwyer, S., Kumar, S. (2004) Perfusion of 99Tcm-labeled CD105 Mab into kidneys from patients with renal carcinoma suggests that CD105 is a promising vascular target. Int. J. Cancer 109,436-441[CrossRef][Medline]
- Hood, J. D., Bednarski, M., Frausto, R., Guccione, S., Reisfeld, R. A., Xiang, R., Cheresh, D. A. (2002) Tumor regression by targeted gene delivery to the neovasculature. Science 296,2404-2407[Abstract/Free Full Text]
- Hu, J., Van den Steen, P. E., Houde, M., Ilenchuk, T. T., Opdenakker, G. (2004) Inhibitors of gelatinase B/matrix metalloproteinase-9 activity comparison of a peptidomimetic and polyhistidine with single-chain derivatives of a neutralizing monoclonal antibody. Biochem. Pharmacol. 67,1001-1009[CrossRef][Medline]
- Ferrara, N. (2002) Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications. Semin. Oncol. 29,10-14
- Li, L., Wartchow, C. A., Danthi, S. N., Shen, Z., Dechene, N., Pease, J., Choi, H. S., Doede, T., Chu, P., Ning, S., et al (2004) A novel antiangiogenesis therapy using an integrin antagonist or anti-Flk-1 antibody coated 90Y-labeled nanoparticles. Int. J. Radiat. Oncol. Biol. Phys. 58,1215-1227[Medline]
- Gutheil, J. C., Campbell, T. N., Pierce, P. R., Watkins, J. D., Huse, W. D., Bodkin, D. J., Cheresh, D. A. (2000) Targeted antiangiogenic therapy for cancer using Vitaxin: a humanized monoclonal antibody to the integrin alphavbeta3. Clin. Cancer Res. 6,3056-3061[Abstract/Free Full Text]
- Wilder, R. L. (2002) Integrin alpha V beta 3 as a target for treatment of rheumatoid arthritis and related rheumatic diseases. Ann. Rheum. Dis. 61(Suppl 2),ii96-ii99[Abstract/Free Full Text]
- Brekken, R. A., Li, C., Kumar, S. (2002) Strategies for vascular targeting in tumors. Int. J. Cancer 100,123-130[CrossRef][Medline]
- Balza, E., Castellani, P., Zijlstra, A., Neri, D., Zardi, L., Siri, A. (2001) Lack of specificity of endoglin expression for tumor blood vessels. Int. J. Cancer 94,579-585[CrossRef][Medline]
- Korn, T., Muller, R., Kontermann, R. E. (2004) Bispecific single-chain diabody-mediated killing of endoglin-positive endothelial cells by cytotoxic T lymphocytes. J. Immunother. 27,99-106
- Volkel, T., Holig, P., Merdan, T., Muller, R., Kontermann, R. E. (2004) Targeting of immunoliposomes to endothelial cells using a single-chain Fv fragment directed against human endoglin (CD105). Biochim. Biophys. Acta 1663,158-166[Medline]
- Demartis, S., Tarli, L., Borsi, L., Zardi, L., Neri, D. (2001) Selective targeting of tumour neovasculature by a radiohalogenated human antibody fragment specific for the ED-B domain of fibronectin. Eur. J. Nucleic Med. 28,534-539[CrossRef][Medline]
- Birchler, M., Viti, F., Zardi, L., Spiess, B., Neri, D. (1999) Selective targeting and photocoagulation of ocular angiogenesis mediated by a phage-derived human antibody fragment. Nat. Biotechnol. 17,984-988[CrossRef][Medline]
- Halin, C., Rondini, S., Nilsson, F., Berndt, A., Kosmehl, H., Zardi, L., Neri, D. (2002) Enhancement of the antitumor activity of interleukin-12 by targeted delivery to neovasculature. Nat. Biotechnol. 20,264-269[CrossRef][Medline]
- Carnemolla, B., Borsi, L., Balza, E., Castellani, P., Meazza, R., Berndt, A., Ferrini, S., Kosmehl, H., Neri, D., Zardi, L. (2002) Enhancement of the antitumor properties of interleukin-2 by its targeted delivery to the tumor blood vessel extracellular matrix. Blood 99,1659-1665[Abstract/Free Full Text]
- Nilsson, F., Kosmehl, H., Zardi, L., Neri, D. (2001) Targeted delivery of tissue factor to the ED-B domain of fibronectin, a marker of angiogenesis, mediates the infarction of solid tumors in mice. Cancer Res. 61,711-716[Abstract/Free Full Text]
- Marty, C., Odermatt, B., Schott, H., Neri, D., Ballmer-Hofer, K., Klemenz, R., Schwendener, R. A. (2002) Cytotoxic targeting of F9 teratocarcinoma tumours with anti-ED-B fibronectin scFv antibody modified liposomes. Br. J. Cancer 87,106-112[CrossRef][Medline]
- Borsi, L., Balza, E., Carnemolla, B., Sassi, F., Castellani, P., Berndt, A., Kosmehl, H., Biro, A., Siri, A., Orecchia, P., et al (2003) Selective targeted delivery of TNFalpha to tumor blood vessels. Blood 102,4384-4392[Abstract/Free Full Text]
- Wuest, T., Moosmayer, D., Pfizenmaier, K. (2001) Construction of a bispecific single chain antibody for recruitment of cytotoxic T cells to the tumour stroma associated antigen fibroblast activation protein. J. Biotechnol. 92,159-168[CrossRef][Medline]
- Hoogenboom, H. R., Henderikx, P., de Haard, H. (1998) Creating and engineering human antibodies for immunotherapy. Adv. Drug Deliv. Rev. 31,5-31[CrossRef][Medline]
- Hoogenboom, H. R. (2002) Overview of antibody phage-display technology and its applications. Methods Mol. Biol. 178,1-37[Medline]
- Azzazy, H. M., Highsmith, W. E., Jr (2002) Phage display technology: clinical applications and recent innovations. Clin. Biochem. 35,425-445[CrossRef][Medline]
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INTRODUCTION
