Modern Antibody Technology: The Impact on Drug Development

Moroney, Simon E.; Plückthun, Andreas

doi:10.1002/9783527620982.ch48

Cited by 25 publications

(10 citation statements)

References 135 publications

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“…12 Therefore, the pools isolated after round six in this previous study were used as template for error-prone PCR. Several reactions were performed with different concentrations of the dNTP analogues dPTP (6-(deoxy-β-D-erythro-pentofuranosyl)-3,4-dihydro-8H-pyrimido- [4,5-c] [1,2]oxazine-7-one-5′-triphosphate) and 8-oxo-dGTP (8-oxo-2′-deoxyguanosine-5′-triphosphate) ranging from 2.5 μM to 20 μM each. The amplification was performed in a 50 μl reaction in the presence of 0.25 μM primers, one unit of Vent polymerase exo minus (NEB), 5% DMSO, 1.5 mM MgCl 2 and 200 μM dNTPs.…”

Section: Error-prone Pcrmentioning

confidence: 99%

“…Antibody engineering techniques have facilitated the generation of high affinity protein binders for targeted therapy. 1,2 The introduction of antibody-fragments and novel selection methods such as phage display or ribosome display has allowed the fast isolation of specific binding proteins from vast synthetic libraries in vitro. 3,4 Selections from such libraries have been shown to routinely yield specific binders, with the highest affinity binders typically being in the low nanomolar affinity-range.…”

Section: Introductionmentioning

confidence: 99%

“…Such evolved high-affinity antibodies have been used with great success in many applications. 2 An alternative, novel approach to developing high-affinity binding reagents takes advantage of another natural class of binding proteins, termed ankyrin repeat proteins. 8 Ankyrin repeat proteins are built from a single structural motif, which is assembled into a protein domain.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

A Designed Ankyrin Repeat Protein Evolved to Picomolar Affinity to Her2

Zahnd

Wyler

Schwenk

et al. 2007

Journal of Molecular Biology

219

195

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Section: Error-prone Pcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

A Designed Ankyrin Repeat Protein Evolved to Picomolar Affinity to Her2

Zahnd

Wyler

Schwenk

et al. 2007

Journal of Molecular Biology

219

195

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“…Antibodies have attracted attention as cancer therapeutics because of their ability to bind specifically to cell surface antigens that are selectively expressed on tumor cells [1]. One of these antigens, the 185-kDa phosphoglycoprotein human epidermal growth factor receptor 2 (HER2) (also referred to as HER-2, ERBB2, and p185HER-2), a member of the epidermal growth factor receptor family, has become one of the key targets for cancer therapeutics.…”

Section: Introductionmentioning

confidence: 99%

Antitumor activity and toxicity of anti-HER2 immunoRNase scFv 4D5-dibarnase in mice bearing human breast cancer xenografts

et al. 2009

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Ribonucleases (RNases) are a non-mutagenic alternative to harmful DNA-damaging anticancer drugs. Targeting of RNases with antibodies to surface antigens that are selectively expressed on tumor cells endows specificity to the cytotoxic actions of RNases. Barnase, a ribonuclease from Bacillus amyloliquefaciens, is a promising candidate for targeted delivery to cancer cells because of its insusceptibility to the ubiquitous cytoplasmic ribonuclease inhibitor, and its high stability and catalytic activity. Here, we characterized in vitro and in vivo an immunoRNase, scFv 4D5-dibarnase, which consists of two barnase molecules that are fused serially to the single-chain variable fragment (scFv) of humanized 4D5 antibody. The latter is directed against the extracellular domain of human epidermal growth factor receptor 2 (HER2), a cancer marker that is overexpressed in many human carcinomas. The scFv 4D5-dibarnase exerted a specific cytotoxic effect on HER2-overexpressing SKBR-3 and BT-474 human breast carcinoma cells (IC(50) = 4.1 and 2.4 nM, respectively) via induction of apoptosis. Ten doses of 0.7 mg/kg scFv 4D5-dibarnase to BALB/c nude mice that bore SKBR-3 human breast cancer xenografts resulted in a 76% reduction in tumor growth. A single injection of scFv 4D5-dibarnase at a total course dose of 7 mg/kg did not cause severe side effects in BALB/c nude or BDF1 mice. The cytotoxicity and selectivity of scFv 4D5-dibarnase merit consideration of this immunoRNase as a potent anticancer agent.

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“…S1), as well as in the form of other fusion proteins, all in soluble form and with the same high yield from E. coli. 4 High-affinity binders against a wide variety of targets and in particular HER2-specific DARPins have previously been selected from our DARPin libraries both by ribosome and phage display (23)(24)(25). From this latter work, we have isolated point mutants of a specific anti-HER2 DARPin (molecular weight, 14.5 kDa) showing affinities between 90 pmol/L and 270 nmol/L.…”

Section: Introductionmentioning

confidence: 99%

Efficient Tumor Targeting with High-Affinity Designed Ankyrin Repeat Proteins: Effects of Affinity and Molecular Size

Zahnd

Kawe²,

Stumpp³

et al. 2010

Cancer Research

223

209

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Slow-clearing, tumor-targeting proteins such as monoclonal antibodies typically exhibit high tumor accumulation but low tissue contrast, whereas intermediate-sized proteins such as scFvs show faster clearance but only moderate tumor accumulation. For both, tumor targeting does not seem to improve further above an optimal affinity. We show here that with very small high-affinity proteins such as designed ankyrin repeat proteins (DARPins), these limits can be overcome. We have systematically investigated the influence of molecular mass and affinity on tumor accumulation with DARPins with specificity for HER2 in SK-OV-3.ip nude mouse xenografts. DARPins with a mass of 14.5 kDa and affinities between 270 nmol/L and 90 pmol/L showed a strong correlation of tumor accumulation with affinity to HER2, with the highest affinity DARPin reaching 8% ID/g after 24 hours and 6.5% ID/g after 48 hours (tumor-to-blood ratio >60). Tumor autoradiographs showed good penetration throughout the tumor mass. Genetic fusion of two DARPins (30 kDa) resulted in significantly lower tumor accumulation, similar to values observed for scFvs, whereas valency had no influence on accumulation. PEGylation of the DARPins increased the circulation half-life, leading to higher tumor accumulation (13.4% ID/g after 24 hours) but lower tumor-to-blood ratios. Affinity was less important for tumor uptake of the PEGylated constructs. We conclude that two regimes exist for delivering high levels of drug to a tumor: small proteins with very high affinity, such as unmodified DARPins, and large proteins with extended half-life, such as PEGylated DARPins, in which the importance of affinity is less pronounced.

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Modern Antibody Technology: The Impact on Drug Development

Cited by 25 publications

References 135 publications

A Designed Ankyrin Repeat Protein Evolved to Picomolar Affinity to Her2

A Designed Ankyrin Repeat Protein Evolved to Picomolar Affinity to Her2

Antitumor activity and toxicity of anti-HER2 immunoRNase scFv 4D5-dibarnase in mice bearing human breast cancer xenografts

Efficient Tumor Targeting with High-Affinity Designed Ankyrin Repeat Proteins: Effects of Affinity and Molecular Size

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