Isolation of High-affinity Monomeric Human Anti-c-erbB-2 Single chain Fv Using Affinity-driven Selection

Schier, Robert; Bye, Jacqueline M.; Apell, Gerald; McCall, Adrian M.; Adams, Gregory P.; Malmqvist, Magnus; Weiner, Louis M.; Marks, James D.

doi:10.1006/jmbi.1996.0004

Cited by 287 publications

(189 citation statements)

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“…Chief among these advantages is the facile means whereby anti-oncoprotein scFvs may be derived. Such methods have included genetic techniques of deriving cDNA directly from hybridomas [21][22][23][24][25]34 as well as biopanning of phage scFv 33,35 libraries and others. 38 These methods have allowed direct isolation of scFvs against virtually any subcellular target protein.…”

Section: Discussionmentioning

confidence: 99%

“…In this regard, various techniques have been used to improve the affinity of scFvs for cognate targets. [33][34][35] Such efforts have largely been endeavored in the context of the comparison of recombinant immunotoxins, whereby the affinity of the antibody clearly correlated with tumor targeting and with the overall therapeutic efficacy. In our studies, increasing the binding affinity of the scFv did not translate into an improvement in intrabody-mediated tumor cell cytotoxicity induction.…”

Section: Discussionmentioning

confidence: 99%

“…In this regard, screening of a phage display library and sitedirected mutagenesis has been used to obtain an antierbB-2 scFv such that this new derivative anti-erbB-2 scFv, C6.5, possesses an affinity for its target that is 1000 times greater than that for e23. [33][34][35] We thus used this scFv to determine the degree to which affinity differential predicated utility for intrabody knockout approaches. Our results demonstrated that affinity enhancement of the parent scFv does not necessarily predict an improvement in intrabody-mediated antineoplastic effects.…”

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confidence: 99%

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Antineoplastic effect of anti-erbB-2 intrabody is not correlated with scFv affinity for its target

et al. 2000

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Intracellular single-chain antibodies (scFvs) have emerged as a powerful method to knock out expression of oncoproteins. We have demonstrated previously that scFvs directed against a variety of molecular targets induce specific toxicity in tumor cells. Recently, the utility of an anti-erbB-2 scFv has predicated its evaluation in a phase I gene therapy clinical trial. The utility of scFv as an intrabody is closely linked to its interaction with a target, limiting the contribution of the latter to the neoplastic phenotype. In this study, we sought to determine whether improvement in the affinity of the scFv for its cognate target could improve the efficiency of intrabody-mediated oncoprotein knockout. We compared in erbB-2-positive and -negative tumor cells the function of plasmids encoding a newly developed C6.5 anti-erbB-2 scFv, which has a 1000-fold higher affinity, with our original e23 anti-erbB-2 scFv. Intracellular scFv expression, target binding, and tumor cell cytotoxicity were found to be similar in all conditions tested, including dose-response studies with limiting dilutions of the scFv. On this basis, we have concluded that the antineoplastic effect of anti-erbB-2 intrabody is not correlated with scFv affinity for its target.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Antineoplastic effect of anti-erbB-2 intrabody is not correlated with scFv affinity for its target

et al. 2000

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“…A single-chain Fv antibody against the extracellular domain of c-erbB-2 has previously been isolated by panning a combinatorial antibody library [19,32] constructed from peripheral blood mononuclear cells from a healthy individual. Clearly, antibodies isolated through this process are unlikely to reflect a naturally occurring immune response.…”

Section: Discussionmentioning

confidence: 99%

Isolation of human anti-c-erbB-2 Fabs from a lymph node-derived phage display library

Clark

Hawkins²,

Papaioannou

et al. 1997

Clinical and Experimental Immunology

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SUMMARYAn immunoglobulin phage display library constructed from a tumour-associated pericolic lymph node was panned against the extracellular domain of the oncoprotein c-erbB-2. Sixteen independent clones were confirmed as positive binders based on ELISA analysis of soluble Fabs. Nucleotide sequencing demonstrated that the V H region of 12 clones belonged to four different V gene families, and the clones demonstrated varying degrees of somatic mutation compared with germ-line sequences. Fab fragments were examined for cross-reactivity by ELISA and shown to be negative against a panel of irrelevant self and non-self antigens, including bovine serum albumin (BSA), mouse immunoglobulin, tetanus toxoid, heregulin-PE40-FLAG and insulin. Reactivity of Fabs in vitro was verified by immunocytochemistry, which showed binding to the c-erbB-2 over-expressing breast cancer cell line SKBR3 but not to the lowexpressing cell line MDA-MB-231. We conclude that a single lymph node library of moderate diversity (2 × 10 7 k light chain and g heavy chain clones), when derived from an individual whose colorectal tumour over-expressed c-erbB-2, can be successfully panned to isolate a number of unique Fabs specific for this antigen. The nature of the anti-c-erbB-2 Fabs recovered from this library suggests that they may have resulted from a humoral immune response in the individual, and that in vivo antibody responses to tumour-associated antigens may be exploited in vitro for the production of tumour-specific recombinant antibodies.

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“…It can be calculated that for efficient tumour retention, off-rates in the region of 10-5 S-l or better are required (Schier et al, 1996), yielding a half-life of bound complexes of 19 h or longer. As the MOC-3 1 antibody V-genes were cloned in a phagemid vector allowing expression on phage, it is possible to affinity mature the antibody fragments (and in particular the offrate), for example by chain shuffling or by random mutagenesis of the genes (for review, see Winter et al, 1994).…”

Section: Discussionmentioning

confidence: 99%

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

Roovers

Henderikx²,

Helfrich³

et al. 1998

Br J Cancer

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Summary 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 singlechain 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-1 61) 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.

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Isolation of High-affinity Monomeric Human Anti-c-erbB-2 Single chain Fv Using Affinity-driven Selection

Cited by 287 publications

References 0 publications

Antineoplastic effect of anti-erbB-2 intrabody is not correlated with scFv affinity for its target

Antineoplastic effect of anti-erbB-2 intrabody is not correlated with scFv affinity for its target

Isolation of human anti-c-erbB-2 Fabs from a lymph node-derived phage display library

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

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