US20040029113A1 - Novel methods of constructing libraries of genetic packages that collectively display the members of a diverse family of peptides, polypeptides or proteins - Google Patents

Novel methods of constructing libraries of genetic packages that collectively display the members of a diverse family of peptides, polypeptides or proteins Download PDF

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US20040029113A1
US20040029113A1 US09/837,306 US83730601A US2004029113A1 US 20040029113 A1 US20040029113 A1 US 20040029113A1 US 83730601 A US83730601 A US 83730601A US 2004029113 A1 US2004029113 A1 US 2004029113A1
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nucleic acid
stranded
oligonucleotide
tct
cleavage
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Robert Ladner
Edward Cohen
Horacio Nastri
Kristin Rookey
Rene Hoet
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Dyax Corp
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Dyax Corp
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Priority to US10/045,674 priority patent/US8288322B2/en
Priority to CA2747868A priority patent/CA2747868A1/en
Priority to PCT/US2002/012405 priority patent/WO2002083872A2/en
Priority to AU2002307422A priority patent/AU2002307422B2/en
Priority to DK02762148.1T priority patent/DK1578903T4/da
Priority to PT02762148T priority patent/PT1578903E/pt
Priority to EP02762148.1A priority patent/EP1578903B2/en
Priority to ES02762148.1T priority patent/ES2375952T5/es
Priority to AT02762148T priority patent/ATE534735T2/de
Priority to CA2458462A priority patent/CA2458462C/en
Publication of US20040029113A1 publication Critical patent/US20040029113A1/en
Priority to US11/365,556 priority patent/US9382535B2/en
Assigned to PAUL ROYALTY FUND HOLDINGS II reassignment PAUL ROYALTY FUND HOLDINGS II SECURITY AGREEMENT Assignors: DYAX CORP.
Assigned to DYAX CORP., A DELAWARE CORPORATION reassignment DYAX CORP., A DELAWARE CORPORATION RELEASE BY SECURED PARTY (SEE DOCUMENT FOR DETAILS). Assignors: PAUL ROYALTY FUND HOLDINGS II
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Priority to AU2009200092A priority patent/AU2009200092B2/en
Assigned to COWEN HEALTHCARE ROYALTY PARTNERS, L.P. reassignment COWEN HEALTHCARE ROYALTY PARTNERS, L.P. SECURITY AGREEMENT Assignors: DYAX CORP., A DELAWARE CORPORATION
Priority to CY20121100019T priority patent/CY1112220T1/el
Priority to US13/464,047 priority patent/US8901045B2/en
Assigned to DYAX CORP., A DELAWARE CORPORATION reassignment DYAX CORP., A DELAWARE CORPORATION RELEASE BY SECURED PARTY (SEE DOCUMENT FOR DETAILS). Assignors: HEALTHCARE ROYALTY PARTNERS, L.P. F/K/A COWEN HEALTHCARE ROYALTY PARTNERS, L.P.
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Priority to US14/557,171 priority patent/US9683028B2/en
Assigned to DYAX CORP. reassignment DYAX CORP. RELEASE BY SECURED PARTY (SEE DOCUMENT FOR DETAILS). Assignors: LFRP INVESTORS, L.P.
Priority to US15/612,938 priority patent/US10829541B2/en
Priority to US16/984,655 priority patent/US20210087256A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1093—General methods of preparing gene libraries, not provided for in other subgroups
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors

Definitions

  • DNAs that encode members of the families to be displayed must be amplified before they are cloned and used to display the desired member on the surface of a genetic package. Such amplification typically makes use of forward and backward primers.
  • Such primers can be complementary to sequences native to the DNA to be amplified or complementary to oligonucleotides attached at the 5′ or 3′ ends of that DNA.
  • Primers that are complementary to sequences native to the DNA to be amplified are disadvantaged in that they bias the members of the families to be displayed. Only those members that contain a sequence in the native DNA that is substantially complementary to the primer will be amplified. Those that do not will be absent from the family. For those members that are amplified, any diversity within the primer region will be suppressed.
  • the primer that is used is at the 5′ end of the V H region of an antibody gene. It anneals to a sequence region in the native DNA that is said to be “sufficiently well conserved” within a single species. Such primer will bias the members amplified to those having this “conserved” region. Any diversity within this region is extinguished.
  • CDRs Complementary Determining Regions
  • FRs Framework Regions
  • the DNAs must be cleaved to produce appropriate ends for ligation to a vector. Such cleavage is generally effected using restriction endonuclease recognition sites carried on the primers.
  • restriction endonuclease recognition sites carried on the primers.
  • the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.
  • the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.
  • This method involves ligating the individual single-stranded DNA members of the family to a partially duplex DNA complex. The method comprises the steps of:
  • FIG. 1 is a schematic of various methods that may be employed to amplify VH genes without using primers specific for VH sequences.
  • FIG. 2 is a schematic of various methods that may be employed to amplify VL genes without using VL sequences.
  • FIG. 3 depicts gel analysis of cleaved kappa DNA from Example 2.
  • FIG. 4 depicts gel analysis of cleaved kappa DNA from Example 2.
  • FIG. 5 depicts gel analysis of amplified kappa DNA from Example 2.
  • FIG. 6 depicts gel purified amplified kappa DNA from Example 2.
  • Sense strand The upper strand of ds DNA as usually written. In the sense strand, 5′-ATG-3′ codes for Met.
  • Antisense strand The lower strand of ds DNA as usually written. In the antisense strand, 3′-TAC-5′ would correspond to a Met codon in the sense strand.
  • Forward primer A “forward” primer is complementary to a part of the sense strand and primes for synthesis of a new antisense- strand molecule. “Forward primer” and “lower-strand primer” are equivalent.
  • Backward primer A “backward” primer is complementary to a part of the antisense strand and primes for synthesis of a new sense- strand molecule. “Backward primer” and “top-strand primer” are equivalent. Bases: Bases are specified either by their position in a vector or gene as their position within a gene by codon and base. For example, “89.1” is the first base of codon 89, 89.2 is the second base of codon 89. Sv Streptavidin Ap Ampicillin ap R A gene conferring ampicillin resistance. RE Restriction endonuclease URE Universal restriction endonuclease Functionally Two sequences are sufficiently complementary complementary so as to anneal under the chosen conditions.
  • Ab Antibody an immunoglobin.
  • the term also covers any protein having a binding domain which is homologous to an immunoglobin binding domain.
  • antibodies within this definition are, inter alia, immunoglobin isotypes and the Fab, F(ab 1 ) 2 , scfv, Fv, dAb and Fd fragments.
  • Fab Two chain molecule comprising an Ab light chain and part of a heavy-chain.
  • scFv A single-chain Ab comprising either VH::linker::VL or VL::linker::VH w.t. Wild type HC Heavy chain LC Light chain
  • VK A variable domain of a Kappa light chain.
  • VH A variable domain of a heavy chain.
  • VL A variable domain of a lambda light chain.
  • nucleic acid sequences that are useful in the methods of this invention may be naturally occurring, synthetic or a combination thereof. They may be mRNA, DNA or cDNA.
  • nucleic acids encode antibodies. Most preferably, they encode Fabs.
  • nucleic acids useful in this invention may be naturally diverse, synthetic diversity may be introduced into those naturally diverse members, or the diversity may be entirely synthetic.
  • synthetic diversity can be introduced into one or more CDRs of antibody genes.
  • Synthetic diversity may be created, for example, through the use of TRIM technology (U.S. Pat. No. 5,869,644).
  • TRIM technology allows control over exactly which amino-acid types are allowed at variegated positions and in what proportions.
  • codons to be diversified are synthesized using mixtures of trinucleotides. This allows any set of amino acid types to be included in any proportion.
  • Another alternative that may be used to generate diversified DNA is mixed oligonucleotide synthesis.
  • TRIM technology one could allow Ala and Trp.
  • mixed oligonucleotide synthesis a mixture that included Ala and Trp would also necessarily include Ser and Gly.
  • the amino-acid types allowed at the variegated positions are picked with reference to the structure of antibodies, or other peptides, polypeptides or proteins of the family, the observed diversity in germline genes, the observed somatic mutations frequently observed, and the desired areas and types of variegation.
  • the nucleic acid sequences for at least one CDR or other region of the peptides, polypeptides or proteins of the family are cDNAs produced by reverse transcription from mRNA. More preferably, the mRNAs are obtained from peripheral blood cells, bone marrow cells, spleen cells or lymph node cells (such as B-lymphocytes or plasma cells) that express members of naturally diverse sets of related genes. More preferable, the mRNAs encode a diverse family of antibodies. Most preferably, the mRNAs are obtained from patients suffering from at least one autoimmune disorder or cancer. Preferably, mRNAs containing a high diversity of autoimmune diseases, such as systemic lupus erythematosus, systemic sclerosis, rheumatoid arthritis, antiphospholipid syndrome and vasculitis are used.
  • autoimmune diseases such as systemic lupus erythematosus, systemic sclerosis, rheumatoid arthritis, antiphospholipid syndrome and vasculitis are used
  • the cDNAs are produced from the mRNAs using reverse transcription.
  • the mRNAs are separated from the cell and degraded using standard methods, such that only the full length (i.e., capped) mRNAs remain. The cap is then removed and reverse transcription used to produce the cDNAs.
  • the reverse transcription of the first (antisense) strand can be done in any manner with any suitable primer. See, e.g., H J de Haard et al., Journal of Biological Chemistry, 274(26):18218-30 (1999).
  • primers that are complementary to the constant regions of antibody genes may be used. Those primers are useful because they do not generate bias toward subclasses of antibodies.
  • poly-dT primers may be used (and may be preferred for the heavy-chain genes).
  • sequences complementary to the primer may be attached to the termini of the antisense strand.
  • the reverse transcriptase primer may be biotinylated, thus allowing the cDNA product to be immobilized on streptavidin (Sv) beads. Immobilization can also be effected using a primer labeled at the 5′ end with one of a) free amine group, b) thiol, c) carboxylic acid, or d) another group not found in DNA that can react to form a strong bond to a known partner on an insoluble medium. If, for example, a free amine (preferably primary amine) is provided at the 5′ end of a DNA primer, this amine can be reacted with carboxylic acid groups on a polymer bead using standard amide-forming chemistry. If such preferred immobilization is used during reverse transcription, the top strand RNA is degraded using well-known enzymes, such as a combination of RNAseH and RNAseA, either before or after immobilization.
  • Sv streptavidin
  • nucleic acid sequences useful in the methods of this invention are generally amplified before being used to display the peptides, polypeptides or proteins that they encode.
  • the single-stranded DNAs Prior to amplification, the single-stranded DNAs may be cleaved using either of the methods described before. Alternatively, the single-stranded DNAs may be amplified and then cleaved using one of those methods.
  • any of the well known methods for amplifying nucleic acid sequences may be used for such amplification. Methods that maximize, and do not bias, diversity are preferred.
  • the present invention preferably utilizes primers in the constant regions of the heavy and light chain genes and primers to a synthetic sequence that are attached at the 5′ end of the sense strand Priming at such synthetic sequence avoids the use of sequences within the variable regions of the antibody genes.
  • Those variable region priming sites generate bias against V genes that are either of rare subclasses or that have been mutated at the priming sites. This bias is partly due to suppression of diversity within the primer region and partly due to lack of priming when many mutations are present in the region complementary to the primer.
  • the methods disclosed in this invention have the advantage of not biasing the population of amplified antibody genes for particular V gene types.
  • the synthetic sequences may be attached to the 5′ end of the DNA strand by various methods well known for ligating DNA sequences together. RT CapExtention is one preferred method.
  • RT CapExtention derived from Smart PCR (TM)
  • TM Smart PCR
  • UDP-GGG upper-strand primer
  • reverse transcriptases are used so that the reverse complement of the upper-strand primer is attached to the lower strand.
  • the upper strand or lower strand primer may be also biotinylated or labeled at the 5′ end with one of a) free amino group, b) thiol, c) carboxylic acid and d) another group not found in DNA that can react to form a strong bond to a known partner as an insoluble medium. These can then be used to immobilize the labeled strand after amplification.
  • the immobilized DNA can be either single or double-stranded.
  • FIG. 1 shows a schematic of the amplification of VH genes.
  • FIG. 1 Panel A shows a primer specific to the poly-dT region of the 3′ UTR priming synthesis of the first, lower strand. Primers that bind in the constant region are also suitable.
  • Panel B shows the lower strand extended at its 3′ end by three Cs that are not complementary to the mRNA.
  • Panel C shows the result of annealing a synthetic top-strand primer ending in three GGGs that hybridize to the 3′ terminal CCCs and extending the reverse transcription extending the lower strand by the reverse complement of the synthetic primer sequence.
  • Panel D shows the result of PCR amplification using a 5′ biotinylated synthetic top-strand primer that replicates the 5′ end of the synthetic primer of panel C and a bottom-strand primer complementary to part of the constant domain.
  • Panel E shows immobilized double-stranded (ds) cDNA obtained by using a 5′-biotinylated top-strand primer.
  • FIG. 2 shows a similar schematic for amplification of VL genes.
  • Panel A shows a primer specific to the constant region at or near the 3′ end priming synthesis of the first, lower strand. Primers that bind in the poly-dT region are also suitable.
  • Panel B shows the lower strand extended at its 3′ end by three Cs that are not complementary to the mRNA.
  • Panel C shows the result of annealing a synthetic top-strand primer ending in three GGGs that hybridize to the 3′ terminal CCCs and extending the reverse transcription extending the lower strand by the reverse complement of the synthetic primer sequence.
  • Panel D shows the result of PCR amplification using a 5′ biotinylated synthetic top-strand primer that replicates the 5′ end of the synthetic primer of panel C and a bottom-strand primer complementary to part of the constant domain.
  • the bottom-strand primer also contains a useful restriction endonuclease site, such as AscI.
  • Panel E shows immobilized ds cDNA obtained by using a 5′-biotinylated top-strand primer.
  • each V gene consists of a 5′ untranslated region (UTR) and a secretion signal, followed by the variable region, followed by a constant region, followed by a 3′ untranslated region (which typically ends in poly-A).
  • An initial primer for reverse transcription may be complementary to the constant region or to the poly A segment of the 3′-UTR.
  • a primer of 15 T is preferred.
  • Reverse transcriptases attach several C residues to the 3′ end of the newly synthesized DNA. RT CapExtention exploits this feature.
  • the reverse transcription reaction is first run with only a lower-strand primer. After about 1 hour, a primer ending in GGG (USP-GGG) and more RTase are added.
  • the DNAs of this invention are rendered single-stranded.
  • the strands can be separated by using a biotinylated primer, capturing the biotinylated product on streptavidin beads, denaturing the DNA, and washing away the complementary strand.
  • a biotinylated primer capturing the biotinylated product on streptavidin beads
  • denaturing the DNA denaturing the DNA
  • washing away the complementary strand Depending on which end of the captured DNA is wanted, one will choose to immobilize either the upper (sense) strand or the lower (antisense) strand.
  • any 5′ untranslated regions and mammalian signal sequences must be removed and replaced, in frame, by a suitable signal sequence that functions in the display host.
  • parts of the variable domains in antibody genes may be removed and replaced by synthetic segments containing synthetic diversity. The diversity of other gene families may likewise be expanded with synthetic diversity.
  • the first method comprises the steps of:
  • the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.
  • short oligonucleotides are annealed to the single-stranded DNA so that restriction endonuclease recognition sites formed within the now locally double-stranded regions of the DNA can be cleaved.
  • a recognition site that occurs at the same position in a substantial fraction of the single-stranded DNAs is identical.
  • Table 195 depicts the DNA sequences of the FR3 regions of the 51 known human VH germline genes.
  • the genes contain restriction endonuclease recognition sites shown in Table 200. Restriction endonucleases that cleave a large fraction of germline genes at the same site are preferred over endonucleases that cut at a variety of sites.
  • An enzyme that cleaves downstream in FR3 is also more preferable because it captures fewer mutations in the framework. This may be advantageous is some cases. However, it is well known that framework mutations exist and confer and enhance antibody binding.
  • the present invention by choice of appropriate restriction site, allows all or part of FR3 diversity to be captured. Hence, the method also allows extensive diversity to be captured.
  • restriction endonucleases that are active between about 45° and about 75° C. are used.
  • enzymes that are active above 50° C., and more preferably active about 55° C., are used. Such temperatures maintain the nucleic acid sequence to be cleaved in substantially single-stranded form.
  • Enzymes shown in Table 200 that cut many of the heavy chain FR3 germline genes at a single position include: MaeIII(24@4), Tsp45I(21@4), HphI(44@5), BsaJT(23@65), AluI(23@47), BlpI(21@48), DdeI(29@58), BglTI(10@61), MslI(44@72), BsiEI(23@74), EaeI(23@74), EagI(23@74), HaeIII(25@75), Bst4CI(51@86), HpyCH41II(51@86), HinfI(38@2), MlyI(18@2), PleI(18@2), MnlI(31@67), HpyCH4V(21@44), BsmAI(16@11), BpmI(19@12), XmnI(12@30), and SacI(11@51). (The notation used means, for
  • restriction endonucleases are: Bst4CI (or TaaI or HpyCH41II), BlpI, HpyCH4V, and MslI. Because ACNGT (the restriction endonuclease recognition site for Bst4CI, TaaI, and HpyCH41II) is found at a consistent site in all the human FR3 germline genes, one of those enzymes is the most preferred for capture of heavy chain CDR3 diversity. BlpI and HpyCH4V are complementary.
  • BlpI cuts most members of the VH1 and VH4 families while HpyCH4V cuts most members of the VH3, VH5, VH6, and VH7 families.
  • Neither enzyme cuts VH2s, but this is a very small family, containing only three members. Thus, these enzymes may also be used in preferred embodiments of the methods of this invention.
  • restriction endonucleases HpyCH4III, Bst4CI, and TaaI all recognize 5′-ACnGT-3′ and cut upper strand DNA after n and lower strand DNA before the base complementary to n. This is the most preferred restriction endonuclease recognition site for this method on human heavy chains because it is found in all germline genes. Furthermore, the restriction endonuclease recognition region (ACnGT) matches the second and third bases of a tyrosine codon (tay) and the following cysteine codon (tgy) as shown in Table 206. These codons are highly conserved, especially the cysteine in mature antibody genes.
  • Table 250 E shows the distinct oligonucleotides of length 22 (except the last one which is of length 20) bases.
  • Table 255 C shows the analysis of 1617 actual heavy chain antibody genes. Of these, 1511 have the site and match one of the candidate oligonucleotides to within 4 mismatches. Eight oligonucleotides account for most of the matches and are given in Table 250 F.1. The 8 oligonucleotides are very similar so that it is likely that satisfactory cleavage will be achieved with only one oligonucleotide (such as H43.77.97.1-02#1) by adjusting temperature, pH, salinity, and the like.
  • One or two oligonucleotides may likewise suffice whenever the germline gene sequences differ very little and especially if they differ very little close to the restriction endonuclease recognition region to be cleaved.
  • Table 255 D shows a repeat analysis of 1617 actual heavy chain antibody genes using only the 8 chosen oligonucleotides. This shows that 1463 of the sequences match at least one of the oligonucleotides to within 4 mismatches and have the site as expected. Only 7 sequences have a second HpyCH4III restriction endonuclease recognition region in this region.
  • the germline genes for human heavy chain FR1 are shown in Table 217.
  • Table 220 shows the restriction endonuclease recognition sites found in human germline genes FR1s.
  • the preferred sites are BsgI(GTGCAG;39@4), BsoFI(GCngc;43@6,11@9,2@3,1@12), TseI (Gcwgc;43@6,11@9,2@3,1@12), MspA1I(CMGckg;46@7,2@1), PvuII(CAGctg;46@7,2@1), AluI(AGct;48@82@2), DdeI (Ctnag;22@52,9@48), HphI(tcacc;22@80), BssKI(Nccngg;35@39,2@40), BsaJI(Ccnngg;32@40,2@41), BstNI(CCwgg;33@40), ScrFI (CCn
  • MspAI and PvuII have 46 sites at 7-12 and 2 at 1-6. To avoid cleavage at both sites, oligonucleotides are used that do not fully cover the site at 1-6. Thus, the DNA will not be cleaved at that site. We have shown that DNA that extends 3, 4, or 5 bases beyond a PvuII-site can be cleaved efficiently.
  • FIG. 300 shows the human kappa FR1 germline genes and Table 302 shows restriction endonuclease recognition sites that are found in a substantial number of human kappa FR1 germline genes at consistent locations.
  • BsmAI and PflFI are the most preferred enzymes. BsmAI sites are found at base 18 in 35 of 40 germline genes. PflFI sites are found in 35 of 40 germline genes at base 12.
  • Another example of choosing an appropriate restriction endonuclease recognition site involves cleavage in FR1 of the human lambda light chain.
  • Table 400 shows the 31 known human lambda FR1 germline gene sequences.
  • Table 405 shows restriction endonuclease recognition sites found in human lambda FR1 germline genes. HinfI and DdeI are the most preferred restriction endonucleases for cutting human lambda chains in FR1.
  • oligonucleotides are prepared so as to functionally complement, alone or in combination, the chosen recognition site.
  • the oligonucleotides also include sequences that flank the recognition site in the majority of the amplified genes. This flanking region allows the sequence to anneal to the single-stranded DNA sufficiently to allow cleavage by the restriction endonuclease specific for the site chosen.
  • the actual length and sequence of the oligonucleotide depends on the recognition site and the conditions to be used for contacting and cleavage. The length must be sufficient so that the oligonucleotide is functionally complementary to the single-stranded DNA over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and solely at the desired location.
  • the oligonucleotides of this preferred method of the invention are about 17 to about 30 nucleotides in length. Below about 17 bases, annealing is too weak and above 30 bases there can be a loss of specificity. A preferred length is 18 to 24 bases.
  • Oligonucleotides of this length need not be identical complements of the germline genes. Rather, a few mismatches taken may be tolerated. Preferably, however, no more than 1-3 mismatches are allowed. Such mismatches do not adversely affect annealing of the oligonucleotide to the single-stranded DNA. Hence, the two DNAs are said to be functionally complementary.
  • the second method to manipulate the amplified single-stranded DNAs of this invention for cloning comprises the steps of:
  • the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.
  • UREs are partially double-stranded oligonucleotides.
  • the single-stranded portion or overlap of the URE consists of a DNA adapter that is functionally complementary to the sequence to be cleaved in the single-stranded DNA.
  • the double-stranded portion consists of a type II-S restriction endonuclease recognition site.
  • the URE method of this invention is specific and precise and can tolerate some (e.g., 1-3) mismatches in the complementary regions, i.e., it is functionally complementary to that region. Further, conditions under which the URE is used can be adjusted so that most of the genes that are amplified can be cut, reducing bias in the library produced from those genes.
  • the sequence of the single-stranded DNA adapter or overlap portion of the URE typically consists of about 14-22 bases. However, longer or shorter adapters may be used. The size depends on the ability of the adapter to associate with its functional complement in the single-stranded DNA and the temperature used for contacting the URE and the single-stranded DNA at the temperature used for cleaving the DNA with the type II-S enzyme.
  • the adapter must be functionally complementary to the single-stranded DNA ver a large enough region to allow the two strands to associate such that the cleavage may occur at the chosen temperature and at the desired location. We prefer singe-stranded or overlap portions of 14-17 bases in length, and more preferably 18-20 bases in length.
  • the site chosen for cleavage using the URE is preferably one that is substantially conserved in the family of amplified DNAs. As compared to the first cleavage method of this invention, these sites do not need to be endonuclease recognition sites. However, like the first method, the sites chosen can be synthetic rather than existing in the native DNA. Such sites may be chosen by references to the sequences of known antibodies or other families of genes. For example, the sequences of many germline genes are reported at http://www.mrc-cpe.cam.ac.uk/imt-doc/restricted/ok.html. For example, one preferred site occurs near the end of FR3—codon 89 through the second base of codon 93. CDR3 begins at codon 95.
  • sequences of 79 human heavy-chain genes are also available at http://www.ncbi.nlm.nih.gov/entre2/nucleotide.html. This site can be used to identify appropriate sequences for URE cleavage according to the methods of this invention. See, e.g., Table 8B.
  • sequences are identified using these sites or other available sequence information. These sequences together are present in a substantial fraction of the amplified DNAs. For example, multiple sequences could be used to allow for known diversity in germline genes or for frequent somatic mutations. Synthetic degenerate sequences could also be used. Preferably, a sequence(s) that occurs in at least 65% of genes examined with no more than 2-3 mismatches is chosen.
  • URE single-stranded adapters or overlaps are then made to be complementary to the chosen regions.
  • Conditions for using the UREs are determined empirically. These conditions should allow cleavage of DNA that contains the functionally complementary sequences with no more than 2 or 3 mismatches but that do not allow cleavage of DNA lacking such sequences.
  • the double-stranded portion of the URE includes a Type II-S endonuclease recognition site.
  • Any Type II-S enzyme that is active at a temperature necessary to maintain the single-stranded DNA substantially in that form and to allow the single-stranded DNA adapter portion of the URE to anneal long enough to the single-stranded DNA to permit cleavage at the desired site may be used.
  • Type II-S enzymes for use in the URE methods of this invention provide asymmetrical cleavage of the single-stranded DNA. Among these are the enzymes listed in Table 800. The most preferred Type II-S enzyme is FokI.
  • An activator may be used to activate part of the FokI enzyme to dimerize without causing cleavage. Examples of appropriate activators are shown in Table 510.
  • the cleavage reaction is performed at a temperature between 45°-75° C., preferably above 50° C. and most preferably above 55° C.
  • the UREs used in the prior art contained a 14-base single-stranded segment, a 10-base stem (containing a FokI site), followed by the palindrome of the 10-base stem. While such UREs may be used in the methods of this invention, the preferred UREs of this invention also include a segment of three to eight bases (a loop) between the FokI restriction endonuclease recognition site containing segments.
  • the stem (containing the FokI site) and its palindrome are also longer than 10 bases. Preferably, they are 10-14 bases in length. Examples of these “lollipop” URE adapters are shown in Table 5.
  • a URE to cleave an single-stranded DNA involves the FR3 region of human heavy chain.
  • Each has a 20-base adaptor sequence to complement the germline gene, a ten-base stem segment containing a FokI site, a five base loop, and the reverse complement of the first stem segment.
  • FIG. 512 shows an analysis of 182 full-length human kappa chains for matching by the four 19-base probe sequences shown. Ninety-six percent of the sequences match one of the probes with 2 or fewer mismatches.
  • the URE adapters shown in Table 512 are for cleavage of the sense strand of kappa chains.
  • the adaptor sequences are the reverse complement of the germline gene sequences.
  • the URE consists of a ten-base stem, a five base loop, the reverse complement of the stem and the complementation sequence.
  • the loop shown here is TTGTT, but other sequences could be used. Its function is to interrupt the palindrome of the stems so that formation of a lollypop monomer is favored over dimerization.
  • Table 512 also shows where the sense strand is cleaved.
  • Table 515 shows analysis of 128 human lambda light chains for matching the four 19-base probes shown. With three or fewer mismatches, 88 of 128 (69%) of the chains match one of the probes. Table 515 also shows URE adapters corresponding to these probes. Annealing these adapters to upper-strand ssDNA of lambda chains and treatment with FokI in the presence of FOKIact at a temperature at or above 45° C. will lead to specific and precise cleavage of the chains.
  • the conditions under which the short oligonucleotide sequences of the first method and the UREs of the second method are contacted with the single-stranded DNAs may be empirically determined.
  • the conditions must be such that the single-stranded DNA remains in substantially single-stranded form. More particularly, the conditions must be such that the single-stranded DNA does not form loops that may interfere with its association with the oligonucleotide sequence or the URE or that may themselves provide sites for cleavage by the chosen restriction endonuclease.
  • first method and UREs can be adjusted by controlling the concentrations of the URE adapters/oligonucleotides and substrate DNA, the temperature, the pH, the concentration of metal ions, the ionic strength, the concentration of chaotropes (such as urea and formamide), the concentration of the restriction endonuclease(e.g., FokI), and the time of the digestion.
  • concentrations of the URE adapters/oligonucleotides and substrate DNA the temperature, the pH, the concentration of metal ions, the ionic strength, the concentration of chaotropes (such as urea and formamide), the concentration of the restriction endonuclease(e.g., FokI), and the time of the digestion.
  • synthetic oligonucleotides having: 1) target germline gene sequences, 2) mutated target gene sequences, or 3) somewhat related non-target sequences. The goal is to cleave most of the target sequences and minimal amounts of non-targets.
  • the single-stranded DNA is maintained in substantially that form using a temperature between 45° C. to 75° C. More preferably, a temperature between 50° C. and 60° C., most preferably between 55° C. and 60° C., is used. These temperatures are employed both when contacting the DNA with the oligonucleotide or URE and when cleaving the DNA using the methods of this invention.
  • the two cleavage methods of this invention have several advantages.
  • the first method allows the individual members of the family of single-stranded DNAs to be cleaved solely at one substantially conserved endonuclease recognition site.
  • the method also does not require an endonuclease recognition site to be built in to the reverse transcription or amplification primers. Any native or synthetic site in the family can be used.
  • the second method has both of these advantages.
  • the URE method allows the single-stranded DNAs to be cleaved at positions where no endonuclease recognition site naturally occurs or has been synthetically constructed.
  • both cleavage methods permit the use of 5′ and 3′ primers so as to maximize diversity and then cleavage to remove unwanted or deleterious sequences before cloning and display.
  • the DNA is prepared for cloning. This is done by using a partially duplexed synthetic DNA adapter, whose terminal sequence is based on the specific cleavage site at which the amplified DNA has been cleaved.
  • the synthetic DNA is designed such that when it is ligated to the cleaved single-stranded DNA, it allows that DNA to be expressed in the correct reading frame so as to display the desired peptide, polypeptide or protein on the surface of the genetic package.
  • the double-stranded portion of the adapter comprises the sequence of several codons that encode the amino acid sequence characteristic of the family of peptides, polypeptides or proteins up to the cleavage site.
  • the amino acids of the 3-23 framework are preferably used to provide the sequences required for expression of the cleaved DNA.
  • the double-stranded portion of the adapter is about 12 to 100 bases in length. More preferably, about 20 to 100 bases are used.
  • the double-standard region of the adapter also preferably contains at least one endonuclease recognition site useful for cloning the DNA into a suitable display vector (or a recipient vector used to archive the diversity).
  • This endonuclease restriction site may be native to the germline gene sequences used to extend the DNA sequence. It may be also constructed using degenerate sequences to the native germline gene sequences. Or, it may be wholly synthetic.
  • the single-stranded portion of the adapter is complementary to the region of the cleavage in the single-stranded DNA.
  • the overlap can be from about 2 bases up to about 15 bases. The longer the overlap, the more efficient the ligation is likely to be. A preferred length for the overlap is 7 to 10. This allows some mismatches in the region so that diversity in this region may be captured.
  • the single-stranded region or overlap of the partially duplexed adapter is advantageous because it allows DNA cleaved at the chosen site, but not other fragments to be captured. Such fragments would contaminate the library with genes encoding sequences that will not fold into proper antibodies and are likely to be non-specifically sticky.
  • One illustration of the use of a partially duplexed adaptor in the methods of this invention involves ligating such adaptor to a human FR3 region that has been cleaved, as described above, at 5′-ACnGT-3′ using HpyCH4III, Bst4CI or TaaI.
  • Table 250 F.2 shows the bottom strand of the double-stranded portion of the adaptor for ligation to the cleaved bottom-strand DNA. Since the HpyCH4III-Site is so far to the right (as shown in Table 206), a sequence that includes the AflII-site as well as the XbaI site can be added. This bottom strand portion of the partially-duplexed adaptor, H43.XAExt, incorporates both XbaI and AflII-sites.
  • the top strand of the double-stranded portion of the adaptor has neither site (due to planned mismatches in the segments opposite the XbaI and AflII-Sites of H43.XAExt), but will anneal very tightly to H43.XAExt.
  • H43AExt contains only the AflII-site and is to be used with the top strands H43.ABr1 and H43.ABr2 (which have intentional alterations to destroy the AflII-site).
  • the desired, captured DNA can be PCR amplified again, if desired, using in the preferred embodiment a primer to the downstream constant region of the antibody gene and a primer to part of the double-standard region of the adapter.
  • the primers may also carry restriction endonuclease sites for use in cloning the amplified DNA.
  • the composite DNA is cleaved at chosen 5′ and 3′ endonuclease recognition sites.
  • the cleavage sites useful for cloning depend on the phage or phagemid into which the cassette will be inserted and the available sites in the antibody genes.
  • Table 1 provides restriction endonuclease data for 75 human light chains.
  • Table 2 shows corresponding data for 79 human heavy chains in each Table, the endonucleases are ordered by increasing frequency of cutting.
  • Nch is the number of chains cut by the enzyme and Ns is the number of sites (some chains have more than one site).
  • SfiI, NotI, AflII, ApaLI, and AscI are very suitable.
  • SfiI and NotI are preferably used in pCES1 to insert the heavy-chain display segment.
  • ApaLI and AscI are preferably used in pCES1 to insert the light-chain display segment.
  • BstEII-sites occur in 97% of germ-line JH genes. In rearranged V genes, only 54/79 (68%) of heavy-chain genes contain a BstEII-Site and 7/61 of these contain two sites. Thus, 47/79 (59%) contain a single BstEII-Site.
  • An alternative to using BstEII is to cleave via UREs at the end of JH and ligate to a synthetic oligonucleotide that encodes part of CH1.
  • One example of preparing a family of DNA sequences using the methods of this invention involves capturing human CDR 3 diversity. As described above, mRNAs from various autoimmune patients is reverse transcribed into lower strand cDNA. After the top strand RNA is degraded, the lower strand is immobilized and a short oligonucleotide used to cleave the cDNA upstream of CDR3. A partially duplexed synthetic DNA adapter is then annealed to the DNA and the DNA is amplified using a primer to the adapter and a primer to the constant region (after FR4).
  • the DNA is then cleaved using BstEII (in FR4) and a restriction endonuclease appropriate to the partially double-stranded adapter (e.g., Xba I and AflII (in FR3)).
  • the DNA is then ligated into a synthetic VH skeleton such as 3-23.
  • One example of preparing a single-stranded DNA that was cleaved using the URE method involves the human Kappa chain.
  • the cleavage site in the sense strand of this chain is depicted in Table 512.
  • the oligonucleotide kapextURE is annealed to the oligonucleotides (kaBR01UR, kaBR02UR, kaBR03UR, and kaBR04UR) to form a partially duplex DNA.
  • This DNA is then ligated to the cleaved soluble kappa chains.
  • the ligation product is then amplified using primers kapextUREPCR and CKForeAsc (which inserts a AscI site after the end of C kappa). This product is then cleaved with ApaLI and AscI and ligated to similarly cut recipient vector.
  • Another example involves the cleavage illustrated in Table 515. After cleavage, an extender (ON_LamEx133) and four bridge oligonucleotides (ON_LamB1-133, ON_LamB2-133, ON_LamB3-133, and ON_LamB4-133) are annealed to form a partially duplex DNA. That DNA is ligated to the cleaved lambda-chain sense strands. After ligation, the DNA is amplified with ON_Lam133PCR and a forward primer specific to the lambda constant domain, such as CL2ForeAsc or CL7ForeAsc (Table 130).
  • ON_LamEx133 an extender
  • four bridge oligonucleotides ON_LamB1-133, ON_LamB2-133, ON_LamB3-133, and ON_LamB4-133
  • the preferred process of this invention is to provide recipient vectors having sites that allow cloning of either light or heavy chains. Such vectors are well known and widely used in the art.
  • a preferred phage display vector in accordance with this invention is phage MALIA3. This displays in gene III. The sequence of the phage MALIA3 is shown in Table 120A (annotated) and Table 120B (condensed).
  • the DNA encoding the selected regions of the light or heavy chains can be transferred to the vectors using endonucleases that cut either light or heavy chains only very rarely.
  • light chains may be captured with ApaLI and AscI.
  • Heavy-chain genes are preferably cloned into a recipient vector having SfiI, NcoI, XbaI, AflII, BstEII, ApaI, and NotI sites.
  • the light chains are preferably moved into the library as ApaLI-AscI fragments.
  • the heavy chains are preferably moved into the library as SfiI-NotI fragments.
  • the display is had on the surface of a derivative of M13 phage.
  • the most preferred vector contains all the genes of M13, an antibiotic resistance gene, and the display cassette.
  • the preferred vector is provided with restriction sites that allow introduction and excision of members of the diverse family of genes, as cassettes.
  • the preferred vector is stable against rearrangement under the growth conditions used to amplify phage.
  • the diversity captured by the methods of the present invention may be displayed in a phagemid vector (e.g., pCES1) that displays the peptide, polypeptide or protein on the III protein.
  • a phagemid vector e.g., pCES1
  • Such vectors may also be used to store the diversity for subsequent display using other vectors or phage.
  • the mode of display may be through a short linker to three possible anchor domains.
  • One anchor domain being the final portion of M13 III (“IIIstump”)
  • a second anchor being the full length III mature protein
  • the third being the M13 VIII mature protein.
  • the IIIstump fragment contains enough of M13 III to assemble into phage but not the domains involved in mediating infectivity. Because the w.t. III and VIII proteins are present, the phage is unlikely to delete the antibody genes and phage that do delete these segments receive only a very small growth advantage.
  • the DNA encodes the w.t. AA sequence, but differs from the w.t. DNA sequence to a very high extent. This will greatly reduce the potential for homologous recombination between the display anchor and the w.t. gene that is also present.
  • the present invention uses a complete phage carrying an antibiotic-resistance gene (such as an ampicillin-resistance gene) and the display cassette. Because the w.t. iii and viii genes are present, the w.t. proteins are also present.
  • the display cassette is transcribed from a regulatable promoter (e.g., P LacZ )
  • a regulatable promoter allows control of the ratio of the fusion display gene to the corresponding w.t. coat protein. This ratio determines the average number of copies of the display fusion per phage (or phagemid) particle.
  • Another aspect of the invention is a method of displaying peptides, polypeptides or proteins (and particularly Fabs) on filamentous phage.
  • this method displays FABs and comprises:
  • P req is a regulatable promoter
  • RBS1 is a first ribosome binding site
  • SS1 is a signal sequence operable in the host strain
  • VL is a member of a diverse set of light-chain variable regions
  • CL is a light-chain constant region
  • stop is one or more stop codons
  • RBS2 is a second ribosome binding site
  • SS2 is a second signal sequence operable in the host strain
  • VH is a member of a diverse set of heavy-chain variable regions
  • CH1 is an antibody heavy-chain first constant domain
  • linker is a sequence of amino acids of one to about 50 residues
  • anchor is a protein that will assemble into the filamentous phage particle and stop is a second example of one or more stop codons
  • anchor is a protein that will assemble into the filamentous phage particle and stop is a second example of one or more stop codons
  • the DNA encoding the anchor protein in the above preferred cassette should be designed to encode the same (or a closely related) amino acid sequence as is found in one of the coat proteins of the phage, but with a distinct DNA sequence. This is to prevent unwanted homologous recombination with the w.t. gene.
  • the cassette should be placed in the intergenic region. The positioning and orientation of the display cassette can influence the behavior of the phage.
  • a transcription terminator may be placed after the second stop of the display cassette above (e.g., Trp). This will reduce interaction between the display cassette and other genes in the phage antibody display vector (PADV).
  • PADV phage antibody display vector
  • the phage or phagemid can display proteins other than Fab, by replacing the Fab portions indicated above, with other protein genes.
  • Various hosts can be used for growth of the display phage or phagemids of this invention. Such hosts are well known in the art. In the preferred embodiment, where Fabs are being displayed, the preferred host should grow at 30° C. and be RecA ⁇ (to reduce unwanted genetic recombination) and EndA ⁇ (to make recovery of RF DNA easier). It is also preferred that the host strain be easily transformed by electroporation.
  • XL1-Blue MRF′ satisfies most of these preferences, but does not grow well at 30° C. XL1-Blue MRF′ does grow slowly at 38° C. and thus is an acceptable host. TG-1 is also an acceptable host although it is RecA + and EndA + . XL1-Blue MRF′ is more preferred for the intermediate host used to accumulate diversity prior to final construction of the library.
  • the libraries of this invention may be screened using well known and conventionally used techniques.
  • the selected peptides, polypeptides or proteins may then be used to treat disease.
  • the peptides, polypeptides or proteins for use in therapy or in pharmaceutical compositions are produced by isolating the DNA encoding the desired peptide, polypeptide or protein from the member of the library selected. That DNA is then used in conventional methods to produce the peptide, polypeptides or protein it encodes in appropriate host cells, preferably mammalian host cells, e.g., CHO cells. After isolation, the peptide, polypeptide or protein is used alone or with pharmaceutically acceptable compositions in therapy to treat disease.
  • a repertoire of human-kappa chain mRNAs was prepared by treating total or poly(A+) RNA isolated from a collection of patients having various autoimmune diseases with calf intestinal phosphatase to remove the 5′-phosphate from all molecules that have them, such as ribosomal RNA, fragmented mRNA, tRNA and genomic DNA. Full length mRNA (containing a protective 7-methyl cap structure) is unaffected. The RNA is then treated with tobacco acid pyrophosphatase to remove the cap structure from full length mRNAs leaving a 5′-monophosphate group.
  • the short oligonucleotides shown in Table 525 were added in 40 fold molar excess in 100 ⁇ L of NEB buffer 2 (50 ⁇ M NaCl, 10 mM Tris-HCl, 10 mM MgCl 2 , 1 mM dithiothreitol pH 7.9) to the dry beads. The mixture was incubated at 95° C. for 5 minutes then cooled down to 55° C. over 30 minutes. Excess oligonucleotide was washed away with 2 washes of NEB buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl 2 , 1 mM dithiothreitol pH 7.9).
  • NEB BsmAI
  • a partially double-stranded adaptor was prepared using the oligonucleotide shown in Table 525.
  • the adaptor was added to the single-stranded DNA in 100 fold molar excess along with 1000 units of T4 DNA ligase (NEB) and incubated overnight at 16° C.
  • the excess oligonucleotide was removed with a Qiagen PCR purification column.
  • the ligated material was amplified by PCR using the primers kapPCRt1 and kapfor shown in Table 525 for 10 cycles with the program shown in Table 530.
  • the soluble PCR product was run on a gel and showed a band of approximately 700 n, as expected (FIGS. 5 and 6).
  • the DNA was cleaved with enzymes ApaLI and AscI, gel purified, and ligated to similarly cleaved vector pCES1.
  • the presence of the correct size insert was checked by PCR in several clones as shown in FIG. 15.
  • Table 500 shows the DNA sequence of a kappa light chain captured by this procedure.
  • Table 501 shows a second sequence captured by this procedure.
  • CDR 1 and 2 diversity were constructed in the 3-23 VH framework in a two step process: first, a vector containing the 3-23 VH framework was constructed, and then, a synthetic CDR 1 and 2 was assembled and cloned into this vector.
  • oligos and two PCR primers (long oligonucleotides: TOPFRLA, BOTFR1B, BOTFR2, BOTFR3, F06, BOTFR4, ON-vgC1, and ON-vgC2 and primers: SFPRMET and BOTPCRPRIM, shown in Table 600) that overlap were designed based on the Genebank sequence of V323 VH.
  • the design incorporated at least one useful restriction site in each framework region, as shown in Table 600.
  • the segments that were synthesized are shown as bold, the overlapping regions are underscored, and the PCR priming regions at each end are underscored.
  • PCR Polymerase Chain Reaction
  • the PCR mixture contained 200 uM dNTPs, 2.5 mM MgCl 2 , 0.02U Pfu TurboT DNA Polymerase, 1U Qiagen HotStart Taq DNA Polymerase, and 1 ⁇ Qiagen PCR buffer.
  • the PCR program consisted of 10 cycles of 94° C. for 30s, 55° C. for 30s, and 72° C. for 30s.
  • the assembled V3-23 DNA sequence was then amplified, using 2.5 ul of a 10-fold dilution from the initial PCR in 100 ul PCR reaction.
  • the PCR reaction contained 200 uM dNTPs, 2.5 mM MgCl 2 , 0.02U Pfu TurboTM DNA Polymerase, 1U Qiagen HotStart Taq DNA Polymerase, 1 ⁇ Qiagen PCR Buffer and 2 outside primers (SFPRMET and BOTPCRPRIM) at a concentration of 1 uM.
  • the PCR program consisted of 23 cycles at 94° C. for 30 s, 55° C. for 30 s, and 72° C. for 60 s.
  • V3-23 VH DNA sequence was digested and cloned into pCES1 (phagemid vector) using the SfiI and BstEII restriction endonuclease sites (All restriction enzymes mentioned herein were supplied by New England BioLabs, Beverly, Mass. and used as per manufacturer's instructions).
  • Stuffer sequences (shown in Table 610 and Table 620) were introduced into pCES1 to replace CDR1/CDR2 sequences (900 bases between BspEI and XbaI RE sites) and CDR3 sequences (358 bases between AflII and BstEII), prior to cloning the CDR1/CDR2 diversity.
  • the new vector is pCES5 and its sequence is given in Table 620. Having stuffers in place of the CDRs avoids the risk that a parental sequence would be over-represented in the library.
  • the CDR1-2 stuffer contains restriction sites for BglII, Bsu36I, BclI, XcmI, MluI, PvuII, HpaI, and HincII, the underscored sites being unique within the vector pCES5.
  • the stuffer that replaces CDR3 contains the unique restriction endonuclease site RsrII.
  • the stuffer sequences are fragments from the penicillase gene of E. coli.
  • oligonucleotides ON-vgCl, ON_Br12, ON_CD2Xba, and ON-vgC2, shown in Table 600 and Table 630
  • a mix of these 4 oligos was combined at a final concentration of 2.5 uM in a 40 ul PCR reaction.
  • Two of the 4 oligos contained variegated sequences positioned at the CDR1 and the CDR2.
  • the PCR mixture contained 200 uM dNTPs, 2.5U Pwo DNA Polymerase (Roche), and 1 ⁇ Pwo PCR buffer with 2 mM MgSO 4 .
  • the PCR program consisted of 10 cycles at 94° C. for 30 s, 60° C. for 30 s, and 72° C. for 60 s.
  • This assembled CDR1/2 DNA sequence was amplified, using 2.5 ul of the mixture in 100 ul PCR reaction.
  • the PCR reaction contained 200 uM dNTPs, 2.5U Pwo DNA Polymerase, 1 ⁇ Pwo PCR Buffer with 2 mM MgSO 4 and 2 outside primers at a concentration of 1 uM.
  • the PCR program consisted of 10 cycles at 94° C. for 30 s, 60° C. for 30 s, and 72° C. for 60 s. These variegated sequences were digested and cloned into the V3-23 framework in place of the CDR1/2 stuffer.
  • Id is the number of the adapter.
  • VH5 cag gtc acc atc tca gcc gac aag tcc atc agc acc gcc tac ctg cag tgg agc agc ctg aag gcc tcg gac acc gcc atg tat tac tgt gcg aga ca !
  • BlpF3HC6-1 matches 4-30.1, not 6-1.
  • D.2 Segment of synthetic 3 - 23 gene into which captured CDR3 is to be cloned ! BlpI ! XbaI... . ... ... !D323* cgCttcacTaag TCT AGA gac aaC tcT aag aaT acT ctC taC Ttg caG atg aac ! ! AflII... ! a gC TTA AG G D.3 Extender and Bridges !
  • Bridges (BlpF3Brl) 5′-cgCttcacTcag tcT aga gaT aaC AGT aaA aaT acT TtG- tac Ttg caG Ctg a
  • lower strand is cut here ! !
  • BlpF3Ext is the reverse complement of: ! 5′-cgCttcacTcag tcT aga gaT aaC AGT aaA aaT acT TtG taC Ttg caG Ctg a-3′ !
  • XbaI and AflII Sites in bridges are bunged (H43.XABr1) 5′-ggtgtagtga-
  • H43.XABr2 5′-ggtgtagtga-
  • H43.ABr1 5′-ggtgtagtga-
  • H43.ABr2 5′-ggtgtagtga-
  • H43.AExt 5′- ATAgTAgAcTgcAgTgTccTcAgccTTAAgcTgTTTcAcTAcAcc-3′ !(H43.AExt) is the reverse complement of 5′-ggtgtagtga- ′
  • VHBA881 5′-cgCttcacTaag-
  • VHBB881 5′-cgCttcacTaag-
  • pV323 is same as pCES1, except as noted. ! BstEII sites may occur in light chains; not likely to be unique in final ! vector. ! ! 143 144 145 146 147 148 149 150 151 152 ! A S T K G P S V F P 2769 gcc tcc acc aaG GGC CCa tcg GTC TTC ccc ! Bsp120I. BbsI...(2/2) ! ApaI.... ! ! 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 !
  • MscI 6664 act ggt gaa tct gec aat gta aat aat cca ttt cag acg att gag cgt 6712 caa aat gta ggt att tcc atg agc gtt ttt cct gtt gca atg gct ggc 6760 ggt aat att gtt ctg gat att acc agc aag gcc gat agt ttg agt tct 6808 tct act cag gca agt gat gtt att act aat caa aga agt att gct aca 6856 acg gtt aat ttg cgt gat gga cag act ctt tta ctc ggt ggt
  • End IV 7060 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc 7120 gcccgctcct ttcgctttct tccgttcctt tctcgccacg ttcGCCGGCt ttccccgtca !
  • NgoMI 7180 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc 7240 caaaaactt gatttgggtg atggttCACG TAGTGggcca tcgccctgat agacggtttt !
  • DraIII 7300 tcgccctttG ACGTTGGAGT Ccacgttcttt taatagtgga ctcttgttcc aaactggaac !
  • DrdI 7360 aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcgga 7420 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgaaa 7480 ctctctcagg gccaggcggt gaagggcaat CAGCTGttgc cCGTCact ggtgaaaga ! PvuII. BsmBI. 7540 aaaaccaccc tGGATCC AAGCTT !
  • cag tct gcc ctg act cag cct cGc tcA gTg tcc ggg tct cct gga cag tca gtc acc atc tcc tgc ! 2e cag tct gcc ctg act cag cct Gcc tcc gTg tcT ggg tct cct gga cag tcG Atc acc atc tcc tgc !
  • cag tct gcc ctg act cag cct ccc tcc gTg tcc ggg tct cct gga cag tca gtc acc atc tcc tgc ! 2d cag tct gcc ctg act cag cct Gcc tcc gTg tcT ggg tct cct gga cag tcG Atc acc atc tcc tgc ! 2b2 !
  • 3a tcT tCt gag ctg act cag GAC ccT GcT gtg tcT gtg Gcc TTG gga cag aca gTc agG atc acA tgc ! 3l tcc tat gTg ctg act cag cca ccc tca gtg tcA gtg Gcc cca gga Aag acG gcc agG atT acc tgT !
  • 4c cAg cct gtg ctg act caA TcA TcC tct gcC tct gcT tCC ctg gga Tcc tcg Gtc aag ctc acc tgc ! 4a cAg cTt gtg ctg act caA TcG ccC tct gcC tct gcc tCC ctg gga gcc tcg Gtc aag ctc acc tgc ! 4b !
  • VHS881-1.1 5′-gctgtgtat
  • VHS881-1.2 5′-gccgtgtat
  • VHS881-2.1 5′-gccgtatat
  • VHS881-4.1 5′-gccgtgtat
  • VHS881-9.1 5′-gccatgtat
  • VHEx881 is the reverse complement of the ON below ! [RC] 5′-cgCttcacTaag- ! Scab........ ! Synthetic 3-23 as in Table 206 !
  • VHBA881 5′-cgCttcacTaag-
  • VHBB881 5′-cgCttcacTaag- TCT
  • VH881PCR 5′

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US09/837,306 US20040029113A1 (en) 2000-04-17 2001-04-17 Novel methods of constructing libraries of genetic packages that collectively display the members of a diverse family of peptides, polypeptides or proteins
US10/045,674 US8288322B2 (en) 2000-04-17 2001-10-25 Methods of constructing libraries comprising displayed and/or expressed members of a diverse family of peptides, polypeptides or proteins and the novel libraries
AT02762148T ATE534735T2 (de) 2001-04-17 2002-04-17 Neue verfahren zum aufbau von präsentierten und/oder exprimierten mitgliedern einer weitläufigen familie von peptiden, polypeptiden oder proteinen umfassenden bibliotheken sowie die neuen bibliotheken
CA2458462A CA2458462C (en) 2001-04-17 2002-04-17 Novel methods of constructing libraries comprising displayed and/or expressed members of a diverse family of peptides, polypeptides or proteins and the novel libraries
CA2747868A CA2747868A1 (en) 2001-04-17 2002-04-17 Novel methods of constructing libraries comprising displayed and/or expressed members of a diverse family of peptides, polypeptides or proteins and the novel libraries
AU2002307422A AU2002307422B2 (en) 2001-04-17 2002-04-17 Novel methods of constructing libraries comprising displayed and/or expressed members of a diverse family of peptides, polypeptides or proteins and the novel libraries
DK02762148.1T DK1578903T4 (da) 2001-04-17 2002-04-17 Hidtil ukendte fremgangsmåder til fremstilling af biblioteker omfattende viste og/eller udtrykte medlemmer af diverse familier af peptider, polypeptider eller proteiner og hidtil ukendte biblioteker
PT02762148T PT1578903E (pt) 2001-04-17 2002-04-17 Novos métodos de construção de bibliotecas compreendendo membros apresentados e/ou expressos de uma família diversa de péptidos, polipéptidos ou proteínas e novas bibliotecas
EP02762148.1A EP1578903B2 (en) 2001-04-17 2002-04-17 Novel methods of constructing libraries comprising displayed and/or expressed members of a diverse family of peptides, polypeptides or proteins and the novel libraries
ES02762148.1T ES2375952T5 (es) 2001-04-17 2002-04-17 Nuevos métodos para construir bibliotecas que comprenden miembros presentados y/o expresados de una familia diversa de péptidos, polipéptidos o proteínas y las nuevas bibliotecas
PCT/US2002/012405 WO2002083872A2 (en) 2001-04-17 2002-04-17 Methods and products of cleaving ssdna using oligonucleotides to form restriction sites
US11/365,556 US9382535B2 (en) 2000-04-17 2006-02-28 Methods of constructing libraries of genetic packages that collectively display the members of a diverse family of peptides, polypeptides or proteins
AU2009200092A AU2009200092B2 (en) 2001-04-17 2009-01-09 Novel Methods of Constructing Libraries Comprising Displayed and/or Expressed Members of a Diverse Family of Peptides, Polypeptides or Proteins and the Novel Libraries
CY20121100019T CY1112220T1 (el) 2001-04-17 2012-01-05 Νεες μεθοδοι δημιουργιας βιβλιοθηκων, οι οποιες περιεχουν παρουσιαζομενα και/ή εκφραζομενα μελη μιας ποικιλομορφης οικογενειας πεπτιδιων, πολυπεπτιδιων ή πρωτεϊνων και οι νεες βιβλιοθηκες
US13/464,047 US8901045B2 (en) 2000-04-17 2012-05-04 Methods of constructing libraries comprising displayed and/or expressed members of a diverse family of peptides, polypeptides or proteins and the novel libraries
US14/557,171 US9683028B2 (en) 2000-04-17 2014-12-01 Methods of constructing libraries comprising displayed and/or expressed members of a diverse family of peptides, polypeptides or proteins and the novel libraries
US15/612,938 US10829541B2 (en) 2000-04-17 2017-06-02 Methods of constructing libraries comprising displayed and/or expressed members of a diverse family of peptides, polypeptides or proteins and the novel libraries
US16/984,655 US20210087256A1 (en) 2000-04-17 2020-08-04 Novel methods of constructing libraries comprising displayed and/or expressed members of a diverse family of peptides, polypeptides or proteins and the novel libraries

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AU2001253589B2 (en) 2007-08-16
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JP5149476B2 (ja) 2013-02-20
WO2001079481A2 (en) 2001-10-25
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