Clinical evidence of efficient tumor targetting based on single–chain Fv antibody selected from a combinatorial library

Begent, R. H. J.; Verhaar, Marianne; Chester, Kerry; Casey, Joanne L.; Green, A.J.; Napier, Mark; Hope‐Stone, Laura; Cushen, Noreen; Keep, P. A.; Johnson, C J; Hawkins, Robert E.; Hilson, A. J.W.; Robson, L

doi:10.1038/nm0996-979

Cited by 231 publications

(136 citation statements)

References 28 publications

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“…The use of diabodies would have some advantages over the use of other formats of bispecific Abs, including the lack of an Fc portion, thus avoiding the killing of FcR-positive bystander cells (39) and their small size for better penetration of tumors (40). The anti-CEA Ab used in this study has shown excellent tumor localization in colon carcinoma patients in an scFv format (41) and in colon-carcinoma xenografts in nude mice, both as a bispecific dAb ␣CEA ϫ ␣CD3 (data not shown) as well as a bivalent ␣CEA dAb (42). Tumor-specific dAb-based molecules were secreted at high levels and were able to efficiently activate unstimulated human peripheral blood T cells to proliferate and eliminate CEA-expressing tumor cells.…”

Section: Discussionmentioning

confidence: 91%

Induction of Human T Lymphocyte Cytotoxicity and Inhibition of Tumor Growth by Tumor-Specific Diabody-Based Molecules Secreted from Gene-Modified Bystander Cells

Blanco

Holliger

Vile

et al. 2003

The Journal of Immunology

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Infiltrating T cells are found in many malignancies, but they appear to be mostly anergic and do not attack the tumor, presumably because of the absence of activation and/or costimulatory signals. We describe a strategy for cellular antitumor immunotherapy by the in situ production of soluble bifunctional Ab-based molecules that activate and retarget T cells to the tumor. We genetically modified cells to simultaneously secrete two bifunctional molecules, a bispecific diabody directed against the carcinoembryonic Ag (CEA) and the CD3ε chain of the TCR (αCEA × αCD3), and a fusion protein comprising the extracellular portion of B7-1 fused to a bivalent anti-CEA diabody (B7-αCEA). Together, αCEA × αCD3 and B7-αCEA proved potent at inducing the activation, proliferation, and survival of primary human T cells. When producer cells were cocultured with primary T cells and CEA+ cancer cells, αCEA × αCD3 and B7-αCEA acted in combination to activate and retarget T cell cytotoxicity and completely abrogate tumor growth in the coculture. Furthermore, the introduction of just a few such producer cells at the tumor site efficiently inhibited the growth of established human colon carcinoma xenografts. Despite a cumbersome generation process, the use of autologous gene-modified producer cells opens the way for a new diabody-based gene therapy strategy of cancer.

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Section: Discussionmentioning

confidence: 91%

Induction of Human T Lymphocyte Cytotoxicity and Inhibition of Tumor Growth by Tumor-Specific Diabody-Based Molecules Secreted from Gene-Modified Bystander Cells

Blanco

Holliger

Vile

et al. 2003

The Journal of Immunology

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“…The MFE-23 scFv in pUC119 30 was recloned into the pET-20b vector as an Nco I/Not I fragment and the protein expressed in BL21(DE3) cells. Cultures of 500 ml of 2TY media containing 100 lg/ml ampicillin were grown at 30°C and induced at an optical density (600 nm) of 0.7 by adding IPTG to a final concentration of 1 mM.…”

Section: Cell Culturementioning

confidence: 99%

Targeted photodynamic therapy with multiply‐loaded recombinant antibody fragments

Bhatti

Yahioglu

Milgrom

et al. 2007

Intl Journal of Cancer

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Current photodynamic therapy (PDT) of cancer is limited by inefficiencies involved in specifically targeting photosensitizers to tumors. Although antibodies are being explored as targeting vehicles, they present significant challenges, particularly in terms of pharmacokinetics and drug-coupling. We describe here a novel and effective system to covalently attach multiple photosensitizer molecules (both preclinical, pyropheophorbide-a and clinically approved, verteporfin photosensitizers) to single-chain Fvs. Further, we demonstrate that not only do the resulting photoimmunoconjugates retain photophysical functionality, they are more potent than either free photosensitizer, effectively killing tumor cells in vitro and in vivo. For example, treatment of human breast cancer xenografts with a photoimmunoconjugate comprising an anti-HER-2 scFv linked to 8-10 molecules of pyropheophorbide-a leads to significant tumor regression. These results give an insight into the important features that make scFvs good carriers for PDT drugs and provide proof of concept of our unique approach to targeted photodynamic therapy (tPDT). This promises to significantly improve on current photodynamic therapies for the treatment of cancer. ' 2007 Wiley-Liss, Inc.Key words: photodynamic therapy; single chain Fv; pyropheophorbide-a; verteporfin Photodynamic therapy (PDT) is a minimally invasive procedure used in a range of conditions where superficially localized lesions such as age-related macular degeneration (AMD) or tumors need to be treated. 1 PDT is typically a 2-step process that involves the administration of a photosensitizer (PS) leading to marginal accumulation in the tumor. Following this, the PS is activated by exposure to light of an appropriate wavelength. This ultimately leads to the conversion of molecular oxygen into reactive oxygen species (ROS), primarily singlet oxygen, leading to tumor cell death via irreversible damage to cellular components such as proteins, lipids and DNA. 2 Current clinical use of PDT achieves efficacies similar to conventional therapies but with lower morbidity, simplicity of use and improved functional and cosmetic outcome. 3,4 PDT has mainly been used where conventional approaches have failed or were unsuitable. These include premalignant dysplastic lesions and noninvasive cancers, which are commonly found in the mucosa of the aerodigestive 5 and urinary tracts. 6 Success in treating these types of cancers has been achieved using Photofrin 1 , 7 Levulan 18 and Foscan 1 . 9 The most successful application of PDT has been for wet age-related macular degeneration (AMD) for which the photosensitizer verteporfin (Visudyne 1 ) has been used to destroy ocular neovasculature. 10 PDT has also had great successes in dermatology because of its impressive cosmetic outcome. Methyl 5-aminolaevulinate (Metvix 1 ) has been used to treat actinic keratosis with up to 90% cure. 11 Although PSs accumulate in cancer cells, the tumor specificity ratios are low and their inherent hydrophobicity, causes them to persist...

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“…Control scFv were as follows: a human scFv FITC-E2, specific for the hapten fluorescein (Vaughan et al, 1996) was Table 1 Affinity-matured scFv derivatives of CEA6 scFv Origin included as a negative control, while a murine CEA-specific scFv, MFE23, kindly provided by Dr K Chester, Royal Free Hospital, London, UK, was used as a positive control. MFE23 has previously been shown to localize to CEA-expressing tumours in mice and to metastatic liver deposits of colorectal tumours in humans (Chester et al, 1994;Verhaar et al, 1995;Begent et al, 1996). lodination was carried out using a modification of the lodoGen method (Pimm and Gribben, 1993).…”

Section: Radioiodinationmentioning

confidence: 99%

Antigen specificity and tumour targeting efficiency of a human carcinoembryonic antigen-specific scFv and affinity-matured derivatives

Jackson

Bacon²,

Pedley³

et al. 1998

Br J Cancer

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Summary We have examined the biological properties of CEA6, a human carcinoembryonic antigen (CEA)-specific single-chain Fv (scFv) isolated by phage display, and five related clones derived by affinity maturation and selected for improved off-rate (Kff). All clones bind strongly and specifically to CEA-positive human tumours by immunocytochemistry and show negligible cross-reactivity with normal colon. Flow cytometry of scFv on human liver cells indicates a shift in fine epitope specificity resulting from mutagenesis. All monomeric scFv have been radioiodinated, retaining effectively full binding activity. A single intravenous injection into nude mice bearing human colon tumour xenografts confirms tumour targeting in all cases. As reported in other studies, the kidney is the main route of elimination of scFv at early time points. Tumour binding of the parental antibody CEA6 consistently gives the highest tumour-blood ratios at 24 h (mean 16:1). Clone T06D11, which has a sevenfold reduced K0ff relative to CEA6, showed no difference in tumour uptake at 24 h but persisted at the tumour site for longer than CEA6. This study demonstrates a possible correlation between binding affinity and tumour residence time when examined in this model. Keywords: human scFv; carcinoembryonic antigen; affinity maturation Advances in recombinant antibody technology have allowed many of the problems encountered in antibody targeting of tumours to be overcome (Huston et al, 1993). Poor tumour penetrance of whole immunoglobulin has been addressed through the use of smaller fragments derived both enzymatically and by recombinant methods (Yokota et al, 1992;Hu et al, 1996;Rowlinson-Busza et al, 1996). Further advantages of fragments are their rapid extravasation and pharmacokinetic clearance, which are clearly contributing factors in their improved performance as imaging agents (Colcher et al, 1990;Milenic et al, 1991;Verhaar et al, 1995). Moreover, the immunogenicity of rodent monoclonal antibodies (mAbs) has been reduced either by humanization strategies or by de novo isolation of antibody fragments from combinatorial libraries of human V genes (reviewed by Johnson and Chiswell, 1993). Rapid expression in bacteria has greatly assisted the study of improved engineered antibody fragments and has avoided timeconsuming and expensive mammalian cell expression systems.The isolation and characterization of scFv from large phage display libraries has demonstrated several instances where the dissociation constant of the molecule, and more specifically the off-rate (Kodf), is the main predictive factor in performance in in vitro bioassays (Thompson et al, 1996;Schier et al, 1996; Roberts et al, in preparation). The relationship between binding kinetics and tumour targeting efficiency is rather more complicated, mainly because so many additional factors (tumour size, antigen density and rate of turnover, epitope, size and charge of antibody, dose, isotope and labelling chemistry used, presence of circulating antigen) are known to influence the...

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Clinical evidence of efficient tumor targetting based on single–chain Fv antibody selected from a combinatorial library

Cited by 231 publications

References 28 publications

Induction of Human T Lymphocyte Cytotoxicity and Inhibition of Tumor Growth by Tumor-Specific Diabody-Based Molecules Secreted from Gene-Modified Bystander Cells

Induction of Human T Lymphocyte Cytotoxicity and Inhibition of Tumor Growth by Tumor-Specific Diabody-Based Molecules Secreted from Gene-Modified Bystander Cells

Targeted photodynamic therapy with multiply‐loaded recombinant antibody fragments

Antigen specificity and tumour targeting efficiency of a human carcinoembryonic antigen-specific scFv and affinity-matured derivatives

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