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Use of a tag to enrich polypeptides libraries

Inactive Publication Date: 2005-07-21
BIOSITE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0007] The invention provides libraries of at least four chimeric antibodies. At least 50% of the antibodies in the library have specific affinity for the same target and no library member constitutes more than 25% of the library. In some libraries, the antibodies are Fab fragments. In some libraries, the antibodies are single-chain antibodies. In some libraries, the antibodies are intact a

Problems solved by technology

Although conventional display methods have achieved considerable success in isolating antibodies and other polypeptides with specific binding to selected targets, some inefficiencies and limitations remain.
In conventional methods, many library members bind nonspecifically to the target or the solid phase bearing the target and are amplified along with specifically bound library members causing poor efficiency at each round of affinity selection.
Not only can this waste time and effort in performing many rounds of affinity selection, but members bearing polypeptides having specific affinity are lost at each round.

Method used

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  • Use of a tag to enrich polypeptides libraries
  • Use of a tag to enrich polypeptides libraries
  • Use of a tag to enrich polypeptides libraries

Examples

Experimental program
Comparison scheme
Effect test

example 1

Immunization of Mice with Antigens and Purification of RNA from Mouse Spleens

[0146] Mice were immunized by the following method based on experience of the timing of spleen harvest for optimal recovery of mRNA coding for antibody. Two species of mice were used: Balb / c (Charles River Laboratories, Wilmington, Mass.) and A / J (Jackson Laboratories, Bar Harbor, Me.). Each of ten mice were immunized intraperitoneally with antigen using 50 μg protein in Freund's complete adjuvant on day 0, and day 28. Tests bleeds of mice were obtained through puncture of the retro-orbital sinus. If, by testing the titers, they were deemed high by ELISA using biotinylated antigen immobilized via streptavidin, the mice were boosted with 50 μg of protein on day 70, 71 and 72, with subsequent sacrifice and splenectomy on day 77. If titers of antibody were not deemed satisfactory, mice were boosted with 50 μg antigen on day 56 and a test bleed taken on day 63. If satisfactory titers were obtained, the animals...

example 2

Preparation of Complementary DNA (cDNA)

[0148] The total RNA purified as described above was used directly as template for cDNA. RNA (50 μg) was diluted to 100 μL with sterile water, and 10 μL-130 ng / μL oligo dT12 (synthesized on Applied Biosystems Model 392 DNA synthesizer at Biosite Diagnostics) was added. The sample was heated for 10 min at 70° C., then cooled on ice. 40 μL 5× first strand buffer was added (Gibco / BRL, Gaithersburg, Md.), 20 μL 0.1 M dithiothreitol (Gibco / BRL, Gaithersburg, Md.), 10 μL 20 mM deoxynucleoside triphosphates (dNTP's, Boehringer Mannheim, Indianapolis, Ind.), and 10 μL water on ice. The sample was then incubated at 37° C. for 2 min. 10 μL reverse transcriptase (Superscript™ II, Gibco / BRL, Gaithersburg, Md.) was added and incubation was continued at 37° C. for 1 hr. The cDNA products were used directly for polymerase chain reaction (PCR).

example 3

Amplification of cDNA by PCR

[0149] To amplify substantially all of the H and L chain genes using PCR, primers were chosen that corresponded to substantially all published sequences. Because the nucleotide sequences of the amino terminals of H and L contain considerable diversity, 33 oligonucleotides were synthesized to serve as 5′ primers for the H chains (FIG. 1), and 29 oligonucleotides were synthesized to serve as 5′ primers for the kappa L chains (FIG. 2). The 5′ primers were made according to the following criteria. First, the second and fourth amino acids of the L chain and the second amino acid of the heavy chain were conserved. Mismatches that changed the amino acid sequence of the antibody were allowed in any other position. Second, a 20 nucleotide sequence complementary to the M13 uracil template was synthesized on the 5′ side of each primer. This sequence is different between the H and L chain primers, corresponding to 20 nucleotides on the 3′ side of the pelB signal seq...

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Abstract

The invention is directed to production of chimeric antibodies using display screening methods. The invention is based in part on two related but self-sufficient improvements in conventional display methods. The first improvement provides methods of enriching conventional display libraries for members displaying more than one copy of a polypeptide prior to affinity screening of such libraries with a target of interest. These methods can achieve diverse populations in which the vast majority of members retaining full-length coding sequences encode polypeptides having specific affinity for the target. In a second aspect, the invention provides methods of subcloning nucleic acids encoding displayed polypeptides of enriched libraries from a display vector to an expression vector without the need for clonal isolation of individual members. These methods can be used to produce polyclonal libraries of chimeric antibodies for use, e.g., as diagnostic or therapeutic reagents.

Description

CROSS-REFERENCES TO RELATED APPLICATIONS [0001] The present application is a continuation-in-part of PCT 98 / 06704, filed, Apr. 3, 1998, which is a continuation-in-part of U.S. Ser. No. 08 / 835,159, filed Apr. 4, 1997 and U.S. Ser. No. 08 / 832,985, filed Apr. 4, 1997, all of which are incorporated by reference in their entirety for all purposes. Related application 60 / 044,292, filed Apr. 4, 1991 and 08 / 835,159, filed Apr. 4, 1997, are also incorporated by reference in their entirety for all purposes.BACKGROUND [0002] Over recent years, many publications have reported the use of phage-display technology to produce and screen libraries of polypeptides for binding to a selected target. See, e.g, Cwirla, et al., Proc. Natl. Acad. Sci. USA 87: 6378-6382 (1990); Devlin, et al., Science 249: 404-406 (1990), Scott & Smith, Science 249: 386-388 (1990); Ladner, et al., U.S. Pat. No. 5,571,698. A basic concept of phage display methods is the establishment of a physical association between DNA enc...

Claims

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Application Information

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IPC IPC(8): C07B61/00C07K14/00C07K16/00G01N33/53C07K16/12C12N15/09C12N15/10C12P21/02C12Q1/68C40B40/02G01N33/543
CPCC07K16/00C07K16/1282C07K2319/00G01N2400/50C40B40/02G01N33/54326G01N2333/33C12N15/1037
Inventor BUEEHLER, JOEVALKIRS, GUNARSGRAY, JEFF
Owner BIOSITE INC
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