Discovery of Human Antibodies to Cell Surface Antigens by Capture Lift Screening of Phage-Expressed Antibody Libraries

Watkins, Jeffry D.; Beuerlein, Gregory; Wu, Herren; McFadden, Paul R.; Pancook, James D.; Huse, William D.

doi:10.1006/abio.1997.2523

Cited by 20 publications

(13 citation statements)

References 36 publications

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“…Vitaxin variants were screened initially by a modified plaque lift approach, termed capture lift (22), in which the nitrocellulose was precoated with goat anti-human kappa antibody and blocked with BSA before application to the phage-infected bacterial lawn. After the capture of phage-expressed Vitaxin variant Fabs, filters were incubated with 1.0 g͞ml biotinylated ␣ v ␤ 3 for 3 h at 4°C, washed four times, incubated with 2.3 g͞ml NeutrAvidin-alkaline phosphatase (Pierce) for 15 min at 25°C, and washed four times.…”

Section: Methodsmentioning

confidence: 99%

“…To permit the efficient screening of the initial libraries a highly sensitive plaque lift assay, termed capture lift (22), was used. Phage-expressed Vitaxin variants were selectively captured on nitrocellulose filters coated with goat antihuman kappa chain antibody, probed with biotinylated ␣ v ␤ 3 , and detected with NeutrAvidin-alkaline phosphatase.…”

Section: Methodsmentioning

confidence: 99%

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Stepwisein vitroaffinity maturation of Vitaxin, an α_vβ₃-specific humanized mAb

Beuerlein

Nie

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

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138

106

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A protein engineering strategy based on efficient and focused mutagenesis implemented by codon-based mutagenesis was developed. Vitaxin, a humanized version of the antiangiogenic antibody LM609 directed against a conformational epitope of the ␣ v ␤ 3 integrin complex, was used as a model system. Specifically, focused mutagenesis was used in a stepwise fashion to rapidly improve the affinity of the antigen binding fragment by greater than 90-fold. In the complete absence of structural information about the Vitaxin-

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Stepwisein vitroaffinity maturation of Vitaxin, an α_vβ₃-specific humanized mAb

Beuerlein

Nie

et al. 1998

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

138

106

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“…Compared to the higher throughput plaque and capture lift assays, 43,53 this method is not restricted to interactions on filter membranes. Moreover, there is no need for a master plate to pick up the positive clones after screening because the bound phages can be recovered after detection and used to reinfect bacterial cells for subsequent phagemid preparation and sequencing.…”

Section: Discussionmentioning

confidence: 99%

Rescue and In Situ Selection and Evaluation (RISE): A Method for High-Throughput Panning of Phage Display Libraries

Vanhercke¹,

Ampè²,

Tirry³

et al. 2005

SLAS Discovery

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Phage display has proven to be an invaluable instrument in the search for proteins and peptides with optimized or novel functions. The amplification and selection of phage libraries typically involve several operations and handling large bacterial cultures during each round. Purification of the assembled phage particles after rescue adds to the labor and time demand. The authors therefore devised a method, termed rescue and in situ selection and evaluation (RISE), which combines all steps from rescue to binding in a single microwell. To test this concept, wells were precoated with different antibodies, which allowed newly formed phage particles to be captured directly in situ during overnight rescue. Following 6 washing steps, the retained phages could be easily detected in an enzyme-linked immunosorbent assay (ELISA), thus eliminating the need for purification or concentration of the viral particles. As a consequence, RISE enables a rapid characterization of phagedisplayed proteins. In addition, this method allowed for the selective enrichment of phages displaying a hemagglutinin (HA) epitope tag, spiked in a 10 4 -fold excess of wild-type background. Because the combination of phage rescue, selection, or evaluation in a single microwell is amenable to automation, RISE may boost the high-throughput screening of smaller sized phage display libraries. (Journal of Biomolecular Screening 2005:108-117)

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“…The filters were incubated with 5 g/ml MRK-16 in 5% nonfat powdered milk, 0.2% Tween 20, 0.01% anti-foam A emulsion, and 0.01% thimerosal in PBS and washed three times with PBS containing 0.1% Tween 20 (PBS-T). Reactive plaques were identified colorimetrically following incubation with goat anti-murine IgG-alkaline phosphatase conjugate (25).…”

Section: Methodsmentioning

confidence: 99%

“…The relative importance of specific framework residues varies between different mAbs and consequently, identifying important residues and determining the optimal amino acid at those positions has proven difficult. Previously, we described an approach for the humanization of antibodies that uses phage expression of combinatorial antibody framework libraries and CDR grafting coupled with identification of the most active humanized variant by rapid screening methods (23)(24)(25). Phage expression of combinatorial framework libraries takes advantage of the efficiency of bacterial cloning systems and, when coupled with the appropriate assays, increases the likelihood of identifying fully active humanized variants.…”

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confidence: 99%

Use of a Peptide Mimotope to Guide the Humanization of MRK-16, an Anti-P-glycoprotein Monoclonal Antibody

Tang¹,

Beuerlein²,

Pecht³

et al. 1999

Journal of Biological Chemistry

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A mimotope-guided strategy for engineering antibodies directed against orphan targets or antigens that are difficult to purify was developed and used to humanize the murine MRK-16 monoclonal antibody (mAb). MRK-16 recognizes a conformational epitope of a 170-kDa membrane protein, termed P-glycoprotein (P-gp). Elevated expression of P-gp on tumor cells is associated with resistance to cytotoxic drugs, a major obstacle in chemotherapy. Murine MRK-16 was used to enrich and screen a phage-displayed peptide library to identify reactive mimotopes. One peptide, termed ALR1, was enriched to a greater extent than others and subsequently was expressed as a fusion protein with glutathione Stransferase. ALR1 fusion protein bound MRK-16 specifically and inhibited binding of MRK-16 to cells expressing elevated levels of P-gp. To humanize MRK-16, the murine complementarity determining regions were grafted onto homologous human heavy and light chain variable region frameworks. Framework residues that differed between the murine MRK-16 and the homologous human templates were analyzed and subsequently, five framework positions potentially important for maintaining the specificity and affinity of MRK-16 were identified. A combinatorial library consisting of 32 variants encoding all possible combinations of murine and human residues at the five differing framework positions was expressed in a phage system. In the absence of purified P-gp, ALR1 fusion protein was used as surrogate antigen to screen the antibody library to identify the framework combination that most preserved the binding activity of the mAb. On the basis of the initial screening against the mimotope four antibody variants were selected for further characterization. The binding affinity of these variants for the ALR1 fusion protein correlated with their binding to cells expressing elevated levels of P-gp. Thus, peptide mimotopes which can be identified for virtually any antibody including those that recognize conformational or carbohydrate epitopes, can serve as antigen templates for antibody engineering.The widespread success of murine hybridoma technology has resulted in the discovery of numerous well characterized monoclonal antibodies (mAbs) 1 with unique specificities. Many of these mAbs display tremendous therapeutic potential both as vehicles for targeting cytotoxic agents (reviewed in Ref. 1) and as function blocking molecules (2-7). However, murine mAbs are generally recognized as foreign antigens by the human immune system preventing the administration of multiple doses (8, 9). As a result, there has been considerable effort devoted to circumventing the immunogenicity of murine mAbs, including the development of methods for discovering human antibodies. For example, human lymphocytes have been stimulated in vitro (10, 11), phage-expressed human antibody libraries have been synthesized (reviewed in Ref. 12), and transgenic mice expressing human Ig genes have been created (13). Although human mAbs have been discovered by these approaches there remains a need ...

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Discovery of Human Antibodies to Cell Surface Antigens by Capture Lift Screening of Phage-Expressed Antibody Libraries

Cited by 20 publications

References 36 publications

Stepwisein vitroaffinity maturation of Vitaxin, an α_vβ₃-specific humanized mAb

Stepwisein vitroaffinity maturation of Vitaxin, an α_vβ₃-specific humanized mAb

Rescue and In Situ Selection and Evaluation (RISE): A Method for High-Throughput Panning of Phage Display Libraries

Use of a Peptide Mimotope to Guide the Humanization of MRK-16, an Anti-P-glycoprotein Monoclonal Antibody

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Discovery of Human Antibodies to Cell Surface Antigens by Capture Lift Screening of Phage-Expressed Antibody Libraries

Cited by 20 publications

References 36 publications

Stepwisein vitroaffinity maturation of Vitaxin, an αvβ3-specific humanized mAb

Stepwisein vitroaffinity maturation of Vitaxin, an αvβ3-specific humanized mAb

Rescue and In Situ Selection and Evaluation (RISE): A Method for High-Throughput Panning of Phage Display Libraries

Use of a Peptide Mimotope to Guide the Humanization of MRK-16, an Anti-P-glycoprotein Monoclonal Antibody

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Stepwisein vitroaffinity maturation of Vitaxin, an α_vβ₃-specific humanized mAb

Stepwisein vitroaffinity maturation of Vitaxin, an α_vβ₃-specific humanized mAb