Efficient one-step direct labelling of recombinant antibodies with technetium-99m

Liberatore, M.; Neri, Dario; Neri, Giovanni; Pini, Alessandro; Iurilli, Anna Paola; Ponzo, Fabio; Spampinato, Giuseppe; Padula, Fabrizio; Pala, Andrea Norcini; Colella, A. Centi

doi:10.1007/bf00801622

Cited by 17 publications

(9 citation statements)

References 10 publications

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“…Labelling of scFv with Technetium-99m at single specific sites remote from the binding domains has been successful for imaging applications (Liberatore et al, 1995;Verhaar et al, 1996) but is less sensitive than iodine. However, one disadvantage of iodination is that it modifies tyrosine residues including those in the antigen binding site.…”

Section: Discussionmentioning

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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Section: Discussionmentioning

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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“…The Cys thiol group, along with 3 amides in the peptide backbone, creates a site-specific N 3 S chelator for labeling with 99m Tc, resulting in a controlled, homogeneous product. This approach has been applied for the labeling of single-chain Fv fragments (23)(24)(25). We previously evaluated the labeling of Affibody molecules with CGG and CGGG sequences incorporated at the N terminus of Affibody molecule Z HER2:342 (26).…”

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confidence: 99%

Targeting of HER2-Expressing Tumors with a Site-Specifically ^99mTc-Labeled Recombinant Affibody Molecule, Z_HER2:2395, with C-Terminally Engineered Cysteine

Ahlgren¹,

Wållberg²,

Tran³

et al. 2009

J Nucl Med

102

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The detection of human epidermal growth factor receptor type 2 (HER2) expression in malignant tumors provides important information influencing patient management. Radionuclide in vivo imaging of HER2 may permit the detection of HER2 in both primary tumors and metastases by a single noninvasive procedure. Small (7 kDa) high-affinity anti-HER2 Affibody molecules may be suitable tracers for SPECT visualization of HER2-expressing tumors. The use of generator-produced 99m Tc as a label would facilitate the prompt translation of anti-HER2 Affibody molecules into use in clinics. Methods: A C-terminal cysteine was introduced into the Affibody molecule Z HER2:342 to enable sitespecific labeling with 99m Tc. Two recombinant variants, His 6 -Z HER2:342 -Cys (dissociation constant [K D ], 29 pM) and Z HER2:2395 -Cys, lacking a His tag (K D , 27 pM), were labeled with 99m Tc in yields exceeding 90%. The binding specificity and the cellular processing of Affibody molecules were studied in vitro. Biodistribution and g-camera imaging studies were performed in mice bearing HER2-expressing xenografts. Results: 99m TcHis 6 -Z HER2:342 -Cys was capable of targeting HER2-expressing SKOV-3 xenografts in SCID mice, but the liver radioactivity uptake was high. A series of comparative biodistribution experiments indicated that the presence of the His tag caused elevated accumulation in the liver. 99m Tc-Z HER2:2395 -Cys, not containing a His tag, showed low uptake in the liver and high and specific uptake in HER2-expressing xenografts. Four hours after injection, the radioactivity uptake values (percentage of injected activity per gram of tissue [%IA/g]) were 6.9 6 2.5 (mean 6 SD) %IA/g in LS174T xenografts (moderate level of HER2 expression) and 15 6 3 %IA/g in SKOV-3 xenografts (high level of HER2 expression). The corresponding tumor-to-blood ratios were 88 6 24 and 121 6 24, respectively. Both LS174T and SKOV-3 xenografts were clearly visualized with a clinical g-camera 1 h after injection of 99m Tc-Z HER2:2395 -Cys. Conclusion: The Affibody molecule 99m Tc-Z HER2:2395 -Cys is a promising tracer for SPECT visualization of HER2-expressing tumors.

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“…Mercaptoacetyl‐containing SN 3 ‐chelators can be site‐specifically introduced into peptides by employing chemical synthesis . When using recombinantly produced proteins, a site‐specific chelator can be introduced by the engineering of a unique cysteine into the peptide sequence . Experience with Affibody molecules carrying different peptide‐based chelators labeled with 99m Tc has furthermore shown that the residualizing properties of 99m Tc are influenced by the side‐chains of the amino acids participating in the coordination of 99m Tc, as further described in Section .…”

Section: Radionuclides For Labeling Of Peptides and Proteins For Molementioning

confidence: 99%

Design and evaluation of radiolabeled tracers for tumor imaging

Wållberg

Ståhl

2013

Biotech and App Biochem

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The growing understanding of tumor biology and the identification of tumor-specific genetic and molecular alterations, such as the overexpression of membrane receptors and other proteins, allows for personalization of patient management using targeted therapies. However, this puts stringent demands on the diagnostic tools used to identify patients who are likely to respond to a particular treatment. Radionuclide molecular imaging is a promising noninvasive method to visualize and characterize the expression of such targets. A number of different proteins, from full-length antibodies and their derivatives to small scaffold proteins and peptide receptor-ligands, have been applied to molecular imaging, each demonstrating strengths and weaknesses. Here, we discuss the concept of molecular targeting and, in particular, molecular imaging of cancer-associated targets. Additionally, we describe important biotechnological considerations and desired features when designing and developing tracers for radionuclide molecular imaging.

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Efficient one-step direct labelling of recombinant antibodies with technetium-99m

Cited by 17 publications

References 10 publications

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

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

Targeting of HER2-Expressing Tumors with a Site-Specifically ^99mTc-Labeled Recombinant Affibody Molecule, Z_HER2:2395, with C-Terminally Engineered Cysteine

Design and evaluation of radiolabeled tracers for tumor imaging

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Efficient one-step direct labelling of recombinant antibodies with technetium-99m

Cited by 17 publications

References 10 publications

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

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

Targeting of HER2-Expressing Tumors with a Site-Specifically 99mTc-Labeled Recombinant Affibody Molecule, ZHER2:2395, with C-Terminally Engineered Cysteine

Design and evaluation of radiolabeled tracers for tumor imaging

Contact Info

Product

Resources

About

Targeting of HER2-Expressing Tumors with a Site-Specifically ^99mTc-Labeled Recombinant Affibody Molecule, Z_HER2:2395, with C-Terminally Engineered Cysteine