Site-specific conjugation of single domain antibodies to liposomes enhances photosensitizer uptake and photodynamic therapy efficacy.

Photodynamic therapy for therapy-resistant cancers will greatly benefit from targeted delivery of tumor photosensitizing agents. In this study, a strategy for the site-specific conjugation of single domain antibodies onto liposomes containing the photosensitizer zinc phthalocyanine was developed and tested.

Crosstalk between epidermal growth factor receptor- and insulin-like growth factor-1 receptor signaling: implications for cancer therapy.

Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor-1 receptor (IGF-1R) can contribute to tumor development and -progression through their effects on cell proliferation, inhibition of apoptosis, angiogenesis, anchorage-independent growth and tumor-associated inflammation. EGFR-targeting monoclonal antibodies and small molecule tyrosine kinase inhibitors are currently in clinical use for the treatment of several types of cancer. However, primary and acquired resistance to these agents often occurs and thereby limits the clinical efficacy of mono-specific targeted therapy. Results from both in vitro and in vivo studies indicate that cross-talk between EGFR and IGF-1R can lead to acquired resistance against EGFR-targeted drugs. This review describes the interface between the EGFR and IGF-1R signaling networks and the implications of the extensive cross-talk between these two receptor systems for cancer therapy. EGFR and IGF-1R interact on multiple levels, either through a direct association between the two receptors, by mediating the availability of each others ligands, or indirectly, via common interaction partners such as G protein coupled receptors (GPCR) or downstream signaling molecules. This multi-layered cross-talk and its involvement in the induction of resistance to targeted therapies provide a clear rationale for dual targeting of EGFR and IGF-1R. We discuss several (potential) strategies to simultaneously inhibit EGFR and IGF-1R signaling as promising novel therapeutic approaches.

Evidence for a bias toward intracellular antigens in the local humoral anti-tumor immune response of a colorectal cancer patient revealed by phage display.

Many patients with colorectal carcinoma (CRC) mount a cellular as well as a humoral immune response to the tumor. To investigate the nature and specificity of the humoral immune response in a CRC patient, lymphocytes infiltrating the primary colorectal tumor and lymph nodes draining the tumor were used as antibody variable (V)-gene pools for the construction of phage antibody repertoires. These libraries were first validated by selection on the antigen tetanus toxoid and shown to contain antibodies that were probably derived from both naive and memory B cells. The repertoires were then screened for the presence of antibodies directed to CRC by selection on the cell line CaCo2. For comparison, the same selections were performed with a phage antibody repertoire made from B cells of healthy donors. Striking differences were observed in the panel of specificities selected from these different repertoires: although a large panel of antibodies reactive with patient-derived primary tumors was obtained from the immune repertoires, none of these discriminated between normal colonic epithelium and colon cancer and none were reactive with cell-surface antigens. However, selections using the non-immune library did result in numerous antibodies that recognized cell surface markers on CaCo2. These data suggest a bias in the local humoral immune response in this CRC patient, directed primarily toward intracellular epithelial-cell specific target antigens.

In vitro characterisation of a monovalent and bivalent form of a fully human anti Ep-CAM phage antibody.

Antibodies to tumour-associated antigens are increasingly being used as targeting vehicles for the visualisation and for therapy of human solid tumours. The epithelial cell adhesion molecule (Ep-CAM) is an antigen that is overexpressed on a variety of human solid tumours and constitutes an attractive target for immunotargeting. We set out to obtain fully human antibodies to this antigen by selecting from a large antibody repertoire displayed on bacteriophages. Two single-chain variable antibody fragments (scFv) were identified that specifically bound recombinant antigen in vitro. One of the selected antibodies (VEL-1) cross-reacted with extracellular matrix components in immunohistochemistry of colon carcinoma, whereas the other scFv (VEL-2) specifically recognised colon cancer cells. The latter antibody was further characterised with respect to epitope specificity and kinetics of antigen-binding. It showed no competition with the well-characterised anti Ep-CAM MOC-31 monoclonal antibody and had an off-rate of 5 x 10(-2) s-1. To obtain an antibody format more suitable for in vivo tumour targeting and to increase the apparent affinity through avidity, the genes of scFv VEL-2 were re-formatted by fusion to a human (gamma 1) hinge region and CH3 domain. This "minibody" was expressed in Escherichia coli, specifically bound the Ep-CAM antigen and showed a 20-fold reduced off-rate in surface plasmon resonance analysis. These results show that phage antibody selection, combined with antibody engineering, may result in fully human antibody molecules with promising characteristics for in vivo use in tumour targeting.

Identification of colon tumour-associated antigens by phage antibody selections on primary colorectal carcinoma.

Immunotargeting of solid tumours using antibodies has become a valuable tool for the detection of cancer metastases and the treatment of minimal residual disease. However, only few tumour antigens useful for targeting have been described to date. To identify cell-surface targets on colorectal carcinoma (CRC), we selected a large, human phage antibody repertoire on freshly isolated colon tumour cells. Two antibodies were identified that reacted with epithelial cell-restricted cell-surface antigens, whereas one clone preferentially reacted with stromal cells. These antigens are tumour-associated antigens, as shown by their uniform expression in tumours of different patients and of different differentiation stages and by their limited expression on normal tissues. The pattern of reactivity in immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA) suggests that these antigens are different from previously identified tumour-associated antigens (e.g. Ep-CAM or c-ERB-2). This phage antibody-based method may lead to the cloning of novel tumour antigens that are useful for the immunotargeting of solid tumours.

Design and application of diabodies, triabodies and tetrabodies for cancer targeting.

Multivalent recombinant antibody fragments provide high binding avidity and unique specificity to a wide range of target antigens and haptens. This review describes the design and expression of diabodies, triabodies and tetrabodies using examples of scFv molecules that target viruses (influenza neuraminidase) and cancer (Ep-CAM; epithelial cell adhesion molecule). We discuss the preferred choice of linker length between V-domains to direct the formation of either diabodies (60 kDa), triabodies (90 kDa) or tetrabodies (120 kDa), each with size, flexibility and valency suited to different applications for in vivo imaging and therapy. The increased binding valency of these scFv multimers results in high avidity (low off-rates). A particular advantage for tumour targeting is that molecules of 60-100 kDa have increased tumour penetration and fast clearance rates compared to the parent Ig (150 kDa). We highlight a number of cancer-targeting scFv multimers that have recently successfully undergone pre-clinical trials for in vivo stability and efficacy. We also review the design of multi-specific Fv modules suited to cross-link two or more different target antigens. These bi- and tri-specific multimers can be formed by association of different scFv molecules and, in the first examples, have been designed as cross-linking reagents for T-cell recruitment into tumours (immunotherapy), viral retargeting (gene therapy) and as red blood cell agglutination reagents (immunodiagnostics).

Human anti-CD30 recombinant antibodies by guided phage antibody selection using cell panning.

In various clinical studies, Hodgkin's patients have been treated with anti-CD30 immunotherapeutic agents and have shown promising responses. One of the problems that appeared from these studies is the development of an immune response against the nonhuman therapeutics, which limits repeated administration and reduces efficacy. We have set out to make a recombinant, human anti-CD30 single-chain variable fragment (scFv) antibody, which may serve as a targeting moiety with reduced immunogenicity and more rapid tumour penetration in similar clinical applications. Rather than selecting a naive phage antibody library on recombinant CD30 antigen, we used guided selection of a murine antibody in combination with panning on the CD30-positive cell line L540. The murine monoclonal antibody Ki-4 was chosen as starting antibody, because it inhibits the shedding of the extracellular part of the CD30 antigen. This makes the antibody better suited for CD30-targeting than most other anti-CD30 antibodies. We have previously isolated the murine Ki-4 scFv by selecting a mini-library of hybridoma-derived phage scFv-antibodies via panning on L540 cells. Here, we report that phage display technology was successfully used to obtain a human Ki-4 scFv version by guided selection. The murine variable heavy (VH) and light (VL) chain genes of the Ki-4 scFv were sequentially replaced by human V gene repertoires, while retaining only the major determinant for epitope-specificity: the heavy-chain complementarity determining region 3 (CDR3) of murine Ki-4. After two rounds of chain shuffling and selection by panning on L540 cells, a fully human anti-CD30 scFv was selected. It competes with the parental monoclonal antibody Ki-4 for binding to CD30, inhibits the shedding of the extracellular part of the CD30 receptor from L540 cells and is thus a promising candidate for the generation of anti-CD30 immunotherapeutics.

Guided selection of a pan carcinoma specific antibody reveals similar binding characteristics yet structural divergence between the original murine antibody and its human equivalent.

Antibody engineering provides an excellent tool for the generation of human immunotherapeutics for the targeted treatment of solid tumours. We have engineered and selected a completely human antibody to epithelial glycoprotein-2 (EGP-2), a transmembrane glycoprotein present on virtually all human simple epithelia and abundantly expressed on a variety of human carcinomas. We chose to use the procedure of "guided selection" to rebuild a high-affinity murine antibody into a human antibody, using two consecutive rounds of variable domain shuffling and phage library selection. As a starting antibody, the murine antibody MOC-31 was used. After the first round of guided selection, where the V(H) of MOC-31 was combined in Fab format with a human V(L)C(L) library, a small panel of human light chains was identified, originating from a segment of the VkappaIII family, whereas the MOC-31 V(L) is more homologous to the VkappaII family. Nevertheless, one of the chimaeric Fabs, C3, displayed an off-rate similar to MOC-31 scFv. Combining the V(L) of C3 with a human V(H) library, while retaining the V(H) CDR3 of MOC-31, clones were selected using human V(H) genes originating from the rarely used V(H)7 family. The best clone, 9E, shows over 13 amino acid mutations from the germline sequence, has an off-rate comparable to the original antibody and specifically binds to the "MOC-31"-epitope on EGP-2 in specificity and competition ELISA, FACS analysis and immunohistochemistry. In both V(L) and V(H) of antibody 9E, three germline mutations were found creating the MOC-31 homologue residue. Structural modelling of both murine and human antibodies reveals that one of the germline mutations, 53Y in V(H) CDR2, is likely to be involved in antigen binding. We conclude that, although they may bind the same epitope and have similar binding affinity to the antigen as the original murine antibody, human antibodies derived by guided selection unlike CDR-grafted antibodies, may retain only some of the original key elements of the binding site chemistry. The selected human anti-EGP-2 antibody will be a suitable reagent for tumour targeting.

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

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

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

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

Model systems to study the parameters determining the success of phage antibody selections on complex antigens.

Phage antibody display technology offers a powerful tool for the isolation of specific antibodies to defined target antigens. Most selection strategies described to date have relied on the availability of purified and often recombinant antigen, providing the possibility to perform selections on a well-defined antigen source. However, when the target antigen cannot be purified (e.g., an integral membrane protein), or if the antigen is unknown (e.g., when searching for novel markers on cells or tissues), panning of phage antibody libraries has to be performed on complex antigen sources such as cell surfaces or tissue sections, or even by in vivo selection methods. This provides a series of technical and experimental challenges. One focus of our research is to select antibodies directed to novel cancer-induced antigens expressed by tumours and by the tumour vasculature. To understand the parameters governing selection on complex antigen sources and to assess the efficiency of these phage library selections, we have set up two model selection systems in which both tumour cells and vascular endothelial cells serve as target "antigen". We describe a model based on phage antibodies directed to the tumour antigen epithelial glycoprotein-2, to compare phage antibody selections on a range of different antigen sources including purified and recombinant antigen, whole live cells, tissue cryosections and in vivo grown solid tumours. Secondly, we describe a model based on a phage antibody directed against the endothelial cell inducible adhesion molecule E-selectin. We compare selections on cultured cell monolayers with selections on cell suspensions immobilised on columns, to determine which selection approach is most suitable for the identification of novel tumour endothelial cell markers. Our data provide insight into the efficiency and thus potency of different selection strategies and show that there are very large differences in the recovery and enrichment of binding phage between the different methods tested. Our results further demonstrate the feasibility of phage antibody selections on whole, intact cells and show that these may sometimes compare favourably to selections on purified antigen. Selections on endothelial cells immobilised on columns compare favourably with selections on cell-monolayers; the most favourable conditions for both selection procedures are described. The implications of our data for phage antibody selections on these different complex antigen sources using either non-immune or immune phage antibody repertoires are discussed. The use of model systems such as the ones described here will help to determine optimal experimental conditions for phage library selections on complex antigens and aid in developing more powerful selection procedures for target discovery.

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

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

Antibody phage display technology and its applications.

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

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

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