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

Schier, Robert; McCall, Adrian M.; Adams, Gregory P.; Marshall, Keith W.; Merritt, Hanne; Yim, Michael; Crawford, Robert S.; Weiner, Louis M.; Marks, Cara; Marks, James D.

doi:10.1006/jmbi.1996.0598

Cited by 350 publications

(232 citation statements)

References 50 publications

Supporting

Mentioning

224

Contrasting

Unclassified

Order By: Relevance

“…The generation of the anti-ErbB2 scFv antibodies has been described in detail. 37 The binding parameters of the anti-ErbB2 scFvs are summarized in Table 1.…”

Section: Cell Lines and Reagentsmentioning

confidence: 99%

CD28 cosignalling does not affect the activation threshold in a chimeric antigen receptor-redirected T-cell attack

2010

View full text Add to dashboard Cite

Adoptive immunotherapy of cancer using chimeric antigen receptor (CAR)-engineered T cells with redirected specificity showed efficacy in recent trials. In preclinical models, 'second-generation' CARs with CD28 costimulatory domain in addition to CD3z performed superior in redirecting T-cell effector functions and survival. Whereas CD28 costimulation sustains physiological T-cell receptor (TCR)-CD3 activation of naïve T cells, the impact of CD28 cosignalling on the threshold of CAR-mediated activation of pre-stimulated T cells without B7-CD28 recruitment remained unclear. Using CARs of different binding affinities, but same epitope specificity, we demonstrate that CD28 cosignalling neither lowered the antigen threshold nor the binding affinity for redirected T-cell activation. 'Affinity ceiling' above which increase in affinity does not increase T-cell activation was not altered. Accordingly, redirected tumor cell killing depended on the binding affinity but was likewise effective for CD3z and CD28-CD3z CARs. In contrast to CD3z, CD28-CD3z CAR-driven activation was not increased further by CD28-B7 engagement. However, CD28 cosignalling, which is required for interleukin-2 induction could not be replaced by high-affinity CD3z CAR binding or high-density antigen engagement. We conclude that CD28 CAR cosignalling does not alter the activation threshold but redirects T-cell effector functions.

show abstract

“…The generation of the anti-ErbB2 scFv antibodies has been described in detail. 37 The binding parameters of the anti-ErbB2 scFvs are summarized in Table 1.…”

Section: Cell Lines and Reagentsmentioning

confidence: 99%

CD28 cosignalling does not affect the activation threshold in a chimeric antigen receptor-redirected T-cell attack

2010

View full text Add to dashboard Cite

show abstract

“…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 uptake of radiolabelled antibody into tumours (reviewed by Goldenberg et al, 1990).…”

mentioning

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

View full text Add to dashboard Cite

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...

show abstract

“…The anti -ErbB2 ML39 ScFv gene sequence was removed from pSyn-1 ML39 20 and cloned into pAcGP67B baculovirus transfer vector at restriction sites NcoI and NotI to allow secretion of the ScFv and ScFv fusion proteins into the medium. A six -His tail was also engineered at the Cterminus of the peptide to facilitate purification.…”

Section: Cloning Expression Purification and Characterization Of Smentioning

confidence: 99%

“…Equal amount of proteins from each sample were used for luciferase analysis according to procedures recommended by the manufacturer. To confirm the specificity of ScFvmediated gene transfer, monoclonal antibody against ErbB2 ( Santa Cruz Biotechnology, Santa Cruz, CA ), or in some cases ScFv, 20 was preincubated with the cells for 10 minutes to block the ErbB2 receptor before transfection with DNA / vector complex was initiated.…”

Section: Analysis Of Transgene Expressionmentioning

confidence: 99%

Single-chain antibody-mediated gene delivery into ErbB2-positive human breast cancer cells

Stuckert

Bosch

et al. 2001

Cancer Gene Ther

View full text Add to dashboard Cite

Targeted gene transfer by nonviral vectors can be achieved through incorporation of specific ligand( s ) into the vectors. In this study, the effects of incorporation of an anti -ErbB2 single -chain antibody fragment ( ScFv ) into nonviral vectors for targeted gene delivery were investigated. The ML39 ScFv, selected from a human ScFv phage display library and affinity matured in vitro ( K d =1Â10 À 9 M ), was used as ligand specific for the extracellular domain of the tumor surface protein, ErbB2. Two approaches were taken: ( a ) development of a vector that is composed of a bifunctional fusion protein capable of binding DNA with the ErbB2 -specific ML39 ScFv at its N -terminus and a truncated form of human protamine at its C -terminus, and ( b ) formulation and evaluation of delivery vectors consisting of three independent components including ML39 ScFv, protamine, and cationic lipids. We demonstrate that fusion proteins comprised of the ML39 ScFv and a truncated form of protamine, denoted as ScFv -P -S, can selectively deliver exogenous DNA into ErbB2( + ) cells, with an 8 -to 10 -fold increase in expression levels of the luciferase reporter gene in ErbB2( + ) cells as compared to ErbB2( À ) cells. In addition, vectors formulated by appropriately mixing DNA, ScFv, protamine, and lipids in vitro could even more efficiently deliver the reporter gene into ErbB2( + ) cells with approximately 5 -fold increase in gene expression in ErbB2( + ) cell as compared to ErbB2( À ) cells. Expression and refolding of the ScFv fusion proteins, in addition to determination of optimal conditions for vector development using these approaches, are discussed.

show abstract

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

Cited by 350 publications

References 50 publications

CD28 cosignalling does not affect the activation threshold in a chimeric antigen receptor-redirected T-cell attack

CD28 cosignalling does not affect the activation threshold in a chimeric antigen receptor-redirected T-cell attack

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

Single-chain antibody-mediated gene delivery into ErbB2-positive human breast cancer cells

Contact Info

Product

Resources

About