EP1444344A1 - Nouveaux vecteurs de phage 88 - Google Patents

Nouveaux vecteurs de phage 88

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Publication number
EP1444344A1
EP1444344A1 EP02744148A EP02744148A EP1444344A1 EP 1444344 A1 EP1444344 A1 EP 1444344A1 EP 02744148 A EP02744148 A EP 02744148A EP 02744148 A EP02744148 A EP 02744148A EP 1444344 A1 EP1444344 A1 EP 1444344A1
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EP
European Patent Office
Prior art keywords
phage
gene
vector
polypeptide
site
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02744148A
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German (de)
English (en)
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EP1444344A4 (fr
Inventor
Katherine S. Bowdish
Shana Barbas-Frederickson
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Alexion Pharmaceuticals Inc
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Alexion Pharmaceuticals Inc
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Publication of EP1444344A1 publication Critical patent/EP1444344A1/fr
Publication of EP1444344A4 publication Critical patent/EP1444344A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • 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

Definitions

  • This disclosure relates to phage vectors useful for generating phage display libraries.
  • this disclosure relates to vectors useful for display of antibodies on phage particles.
  • Filamentous bacteriophage consist of a circular, single-stranded DNA molecule surrounded by a cylinder of coat proteins. There are about 2,700 molecules of the major coat protein pVIII that envelope the phage. At one end of the phage particle, there are five copies each of gene III and VI proteins (pill and pVI) that are involved in host-cell binding and in the termination of the assembly process. The other end contains five copies each of pVII and pIX that are required for the initiation of assembly and for maintenance of virion stability. In recent years, vectors have been developed that allow the display of foreign peptides on the surface of a filamentous phage particle.
  • chimeric proteins By insertion of specific oligonucleotides or entire protein coding regions into genes encoding specific phage capsid proteins, chimeric proteins can be produced which are able to be assembled into phage particles. This results in the display of the foreign protein or peptide on the surface of the phage particle.
  • the display of peptides and proteins on the surface of bacteriophage represents a powerful methodology for selection of rare members in a complex library and for carrying out molecular evolution in the laboratory. The ability to construct libraries of enormous molecular diversity and to select for molecules with predetermined properties has made this technology applicable to a wide range of problems.
  • phage display of natural peptides including, mapping epitopes of monoclonal and polyclonal antibodies and generating immunogens
  • phage display of random peptides including mapping epitopes of monoclonal and polyclonal antibodies, identifying peptide ligands, and mapping substrate sites for proteases and kinases
  • phage display of protein and protein domains including directed evolution of proteins, isolation of antibodies, and cDNA expression screening.
  • phage display has been to construct combinatorial peptide libraries.
  • Synthetic ohgonucleotides fixed in length but with unspecified codons, can be cloned as fusions to genes III or VIII of phage where they are expressed as a plurality of peptide apsid fusion proteins.
  • the libraries often referred to as random peptide libraries, can then be tested for binding to target molecules of interest. This is most often done using a form of affinity selection known as "biopanning" or simply “panning”.
  • Phagemid III amp R pHENl III Sfil-S-Notl amp R pComb3 III pComb ⁇ VIII pCANTAB 5E III Sfil-SNotl amp p8V5 VIII BstXI-S-BstXl amp" ⁇ SurfZap III Notl-S-Spel amp R
  • phage and phagemid vectors have been constructed and utilized for phage display. Each of the existing vectors has its advantages and disadvantages.
  • vectors that fuse a gene of interest whose protein product is to be displayed to gene VIII have been categorized as either type 8, type 8+8 or type 88.
  • Type 8 vectors are phage vectors where all copies of gene VIII are fused to a gene of interest for display. With approximately 2700 copies of pVIII on the surface of the phage particle, there is little tolerance for large inserts to be displayed on the phage surface. In addition, strong avidity effects due to multivalent display reduce selective pressure for high affinity that is commonly desired and may be taken into account.
  • the 8+8 vectors are phagemid vectors.
  • helper phage are required to package the phagemid genome into a phagemid particle that is extruded out of the cell.
  • the gene of interest is fused to a copy of gene VIII on the plasmid, while the helper phage retains a wildtype, unfused copy of gene VIII.
  • the coat of the phagemid particle is made up of both wildtype and pVITI fusion proteins leading to more stability and a loss of some avidity effects.
  • helper phage and phagemid viral particles will have fusion proteins on the surface leading to a loss of the corresponding genetic information from helper phage particles that inadvertently display selected proteins.
  • the type 88 vectors are phage vectors where both a fused and unfused copy of gene VIII are present on the phage vector.
  • the phage vector system is less complex in that helper phage are not required. Additionally, there is no loss of selected clones that result from inadvertent display on the helper phage surface.
  • the presently known 88 vectors are derivatives of fd-tet, where an insert conferring tetracycline resistance was introduced at a convenient restriction site. Unfortunately, the insert disrupts the minus strand origin of replication, leading to a defect in minus strand synthesis. As a result, these vectors have a very low intracellular RF copy number, making vector production for cloning as well as library amplification difficult.
  • the size of the insert conferring tetracycline resistance is approximately 2.6 kb.
  • This large insert in addition to insertions into the phage for protein of interest display (including promoter, ribosomal binding sites, signal sequences, st ⁇ ffer fragments in the case of the cloning vectors, and antibody genes in the case of antibody display) yield a large phage genome that is not packaged as efficiently as smaller phage genomes.
  • the fd-tet vector has served as the starting point of construction of a variety of phage vectors including the fUSE vectors of G. Smith (Scott and Smith, Science, Vol.
  • phage vectors useful for generating phage display libraries.
  • the novel vectors described herein are produced as the result of modification of a phage genome at an artificially created cloning site not employed in previous phage vector constructions.
  • a phage genome is engineered in accordance with this disclosure to include a restriction site at one of two different positions.
  • a restriction site is inserted into the phage genome between the end of gene IV and the MOS hairpin which serves as a phage packaging signal for newly synthesized single strands of phage DNA.
  • a restriction site is inserted into the phage genome after the MOS hairpin and prior to the minus strand origin.
  • the vector is engineered to be a "88" vector by inserting at the new restriction site a nucleotide sequence encoding at least a functional domain of pVIII and at least a first cloning site for receiving a gene encoding a polypeptide to be displayed.
  • the 88 vector is engineered to cause display of a dimeric (e.g., heterodimeric) species by inserting first cloning site for receiving first gene encoding a polypeptide to be displayed and a second cloning site for receiving a second gene encoding a polypeptide capable of dimerizing with the polypeptide to be displayed, thereby resulting in display of a dimeric polypeptide or protein.
  • the first and second cloning sites can be inserted with the nucleotide sequence encoding at least a functional domain of pVIII as part of a single cassette referred to herein as a display cassette.
  • the novel vectors are engineered to produce phage particles that display antibodies.
  • a first gene encoding an antibody heavy chain Fd is inserted adjacent the nucleotide sequence encoding at least a functional domain of pVIII to produce a pVIII fused with a heavy chain Fd.
  • a second gene encoding an antibody light chain is also inserted into the vector.
  • Fig. 1 is a flow chart illustrating the strategy for making a vector based on modification of the fl genome between gene IV and the MOS hair pin;
  • Fig. 2 is a flow chart illustrating the strategy for making a vector based on modification of the fl genome between the MOS hairpin and the minus strand origin;
  • Fig. 3 is a map of the vector produced in Example 1 ;
  • Fig 4a is the sequence (Seq. ID No. 2) of cassette la used in Example 1 ;
  • Fig. 4b is the sequence (Seq. ID No. 7) of cassette 2 used in Examples 1 and 2;
  • Fig. 4c is the sequence (Seq. ID No. 12) of cassette 3 used in Examples 1 and 2;
  • Fig. 4d is the sequence (Seq. ID No. 22) of an alternative display cassette useful in making an 88 vector in accordance with this disclosure;
  • Fig. 5a is a map of the pAX131 vector;
  • Figs. 5b-e show the sequence (Seq. ID No.13) of the pAX131 vector
  • Fig. 6 is the sequence (Seq. ID No. 14) of the synthetic gVIII portion of cassette 3.
  • Fig. 7 shows the alignment of the oligos for preparation of the synthetic gVIII;
  • Fig. 8 shows a map of the vector pAX131-gVIII
  • Fig. 9 is the sequence (Seq. ID No. 23) of the final inserted construct resulting from the insertion of cassettes la, 2 and 3 in Example 1;
  • Fig. 10 is a map of the vector produced in Example 2;
  • Fig. 11 is the sequence (Seq. ID No. 25) for cassette lb used in Example 2; and
  • Fig. 12 is the sequence (Seq. ID No. 30) of the final construct resulting from the insertion of cassettes lb, 2 and 3 as described in Example 2.
  • Fig. 12 is the sequence (Seq. ID No. 30) of the final construct resulting from the insertion of cassettes lb, 2 and 3 as described in Example 2.
  • DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The novel vectors described herein are prepared by modifying a phage genome.
  • phage genomes e.g., Ml 3, fd, etc.
  • pVIII a truncated version or fragment thereof is contemplated (unless the context indicates otherwise) provided the display function of the protein is maintained.
  • the present vectors are the result of modification of the fl genome between gene IV and the hairpin which serves as a packaging signal (MOS).
  • MOS packaging signal
  • the phage genome is engineered to contain a novel restriction site at this location.
  • at least a first cloning site and a nucleotide sequence encoding at least a functional domain of pVIII are inserted at the newly formed restriction site.
  • a first gene encoding a polypeptide to be displayed can be inserted at the first cloning site.
  • the vector effectively encodes a fusion protein of pVIII and a polypeptide to be displayed by the phage particle.
  • Any polypeptide that can be displayed by phage can be fused to pVIII.
  • Non-limiting examples of polypeptides that can be displayed include naturally occurring and synthetic enzymes, hormones, antibodies, antigens, toxins and cytokines. For a nonlimiting list of proteins and protein domains that can be displayed, see Phage Display of Peptides and Proteins, A Laboratory Manual, Kay et al., ed., Academic Press, 1996.
  • a second cloning site is also inserted at the novel restriction site.
  • the second cloning site is adapted to receive a second gene that encodes a polypeptide that can dimerize with the polypeptide fused to pVIII.
  • display of a dimeric species e.g., a heterodimeric species
  • the second gene can be eliminated.
  • the polypeptide fused to pVIII is an antibody heavy chain Fd and the modification to the fl genome also involves inserting a site for cloning into the vector a second gene encoding an antibody light chain.
  • the vector can be used to make phage particles that display antibody libraries.
  • Figure 1 is a flow chart showing the steps involved in a particularly useful method for producing a phage vector capable of generating phage display of polypeptides (e.g., libraries of antibodies) in accordance with this disclosure.
  • a restriction site is introduced into the into the fl genome between the end of gene IV and the hairpin which serves as a packaging signal (MOS).
  • the restriction site can be any known restriction site. Suitable restriction sites for insertion include, but are not limited to Nhe I, Hind III, Nco I, Xma I, Bgl II, Bst I, Pvu I, etc.
  • restriction site selected for insertion is present in the native genome, it may be desirable to remove or disable the native restriction site to avoid unwanted digestion during further processing.
  • the restriction site can be inserted using any technique known to those skilled in the art. In a particularly useful embodiment, overlap PCR is used to generate a restriction fragment containing the desired restriction site. This fragment is then cloned into the phage genome at suitable sites.
  • the replicative form (RF) DNA is opened by digestion and a first cassette containing a terminator and multiple cloning sites is added. Depending on the particular restriction site inserted in the first step, specific methods for opening the RF DNA are known to and readily selected by those skilled in the art.
  • the first cassette is engineered to include overhangs which, when combined with the ends of the DNA fo ⁇ ned by the digestive opening thereof at the inserted restriction site will create a hybrid site that will no longer be recognized as the inserted restriction site.
  • subsequent cloning steps advantageously occur at the cloning sites within the first cassette.
  • one of the cloning sites within the first cassette can be the same as the restriction site inserted in the first step to decrease the number of different enzymes employed in the process.
  • suitable cassettes can be created using overlapping ohgonucleotides ("oligos") in a PCR fill in reaction.
  • cassettes can be created using long complementary oligos which can form a double stranded DNA cassette.
  • the oligos are mixed in a 1 :1 ratio, heat denatured and slowly cooled to allow the duplexed cassette to form.
  • Other suitable techniques for creating cassettes will be evident to those skilled in the art.
  • the process shown in Figure 1 involves again opening the RF DNA at one of the cloning sites within the first cassette and inserting a second cassette that includes a promoter.
  • a promoter recognized by a host cell can be employed. Suitable promoters include, but are not limited to, ara, lac and trc promoters.
  • the promoter drives expression of other sequences inserted into the vector, such as, for example expression of the pVIII fusion protein and any polypeptides intended to dimerize therewith.
  • the display cassette contains at least a nucleotide sequence encoding at least a functional domain of pVIII and a first cloning site adapted to receive a gene encoding a polypeptide to be displayed.
  • the nucleotide sequence encoding at least a functional domain of pVIII can be natural or synthetic.
  • the display cassette contains a synthetic gene VIII to avoid having identical native gene VIII sequences at two different locations within the vector.
  • the nucleotide sequence can encode a truncated pVIII provided the display function of the protein is maintained.
  • the display cassette contains at least a first cloning site for receiving a first gene encoding a polypeptide to be displayed.
  • the cloning site is a region of the nucleic acid between two restriction sites, typically with a nonessential region of nucleotide sequence (commonly refe ⁇ ed to as a "stuffer" sequence) positioned therebetween.
  • the first cloning site is defined by Xhol and Spel restriction sites adjacent to the synthetic gene VIII.
  • a suppressible stop codon could be positioned between the first gene and the nucleotide sequence encoding at least a functional domain of pVIII such that fusion display is obtained in a suppressing host (as long as the first gene is inserted in-frame) and a secreted protein without pVIII is obtained in a non-suppressing host.
  • the display cassette optionally also contains a second cloning region for receiving a second gene encoding a polypeptide that can dimerize with the polypeptide to be displayed.
  • the second gene preferably encodes an antibody light chain.
  • the second cloning site is a region of the vector between two restriction sites, typically with a stuffer positioned therebetween. In the flow chart of Fig. 1, the second cloning site is defined by Sad and Xbal restriction sites.
  • the present vectors are the result of modification of the fl genome between the hairpin which serves as a packaging signal (MOS) and the minus strand origin.
  • the vector After engineering a novel restriction site at this location, the vector has inserted at this site at least a nucleotide sequence encoding a pVIII and a cloning site for receiving a first gene encoding a polypeptide to be fused to pVIII and thus displayed by the phage particle. Suitable polypeptides to be displayed are those described above in connection with the previous embodiment.
  • a cloning site for receiving a second gene is also inserted at this site.
  • the second gene preferably encodes a polypeptide that can dimerize with the polypeptide fused to pVIII. In this manner, display of a dimeric species (e.g., a heterodimeric species) can be achieved. Where monomeric display of a single polypeptide or protein is intended, the second gene can be eliminated.
  • the polypeptide fused to pVIII is a heavy chain Fd and the modification to the fl genome also involves inserting a site for cloning into the vector a second gene encoding an antibody light chain.
  • the vector can be used to make phage particles that display antibody libraries.
  • the first step involves introducing a restriction site into the fl genome between the hairpin which serves as a packaging signal (MOS) and the minus strand origin.
  • the restriction site can be any known restriction site.
  • Suitable restriction sites for insertion include Nhe I, Hind III, Nco I, Xma I, Bgl II, Bst I, Pvu I, etc. It should be understood that if a restriction site selected for insertion is present in the native genome, it may be desirable to remove or disable the native restriction site to avoid unwanted digestion during further processing.
  • the restriction site can be inserted using any teclmique known to those skilled in the art. Tn a particularly useful embodiment, overlap PCR is used to generate a restriction fragment containing the desired restriction site. This fragment is then cloned into the phage genome at suitable sites.
  • the replicative form (RF) DNA is opened by digestion and a first cassette containing multiple cloning sites and a terminator is added.
  • a first cassette containing multiple cloning sites and a terminator is added.
  • specific methods for opening the RF DNA will be known to and readily selected by those skilled in the art.
  • the first cassette is engineered to include overhangs which, when ligated with the ends of the DNA formed by digestion at the inserted restriction site will create a hybrid site that will no longer be recognized as the inserted restriction site. In this manner, subsequent cloning steps advantageously occur at the cloning sites within the first cassette.
  • one of the cloning sites within the first cassette can be the same as the restriction site inserted in the first step to decrease the number of different enzymes employed in the process.
  • the process shown in Figure 2 involves again opening the RF DNA at one of the cloning sites within the first cassette and inserting a second cassette that includes a promoter.
  • a promoter recognized by the host cell can be employed. Suitable promoters include, but are not limited to, ara, lac and trc promoters.
  • the display cassette contains a synthetic gVIII and at least a first cloning site for receiving a first gene that encodes a polypeptide to be displayed, such as, for example, an antibody heavy chain Fd.
  • the display cassette optionally contains a second cloning region for receiving a second gene, such as, for example, a gene encoding an antibody light chain.
  • the phage vector produced by the process illustrated in Figure 2 will be a modified fl genome that contains, after the MOS hairpin but before the minus strand origin, a promoter, an antibody cloning region for receiving a gene encoding an antibody light chain, an antibody cloning region for receiving a gene encoding an antibody heavy chain Fd to be displayed, a synthetic gene VIII and a terminator.
  • a selectable marker can be added to the present vectors.
  • suitable markers include tetracycline or kanamycin resistance.
  • the vectors described herein can be transformed into a host cell using known techniques (e.g., electroporation) and amplified.
  • the vectors described herein can also be digested and have a first gene and optionally a second gene ligated therein in accordance with this disclosure.
  • the vector so engineered can be transformed into a host cell using known techniques and amplified or to effect expression of polypeptides and/or proteins encoded thereby to produce phage particles displaying single polypeptides or dimeric species.
  • known techniques e.g., electroporation
  • the vector so engineered can be transformed into a host cell using known techniques and amplified or to effect expression of polypeptides and/or proteins encoded thereby to produce phage particles displaying single polypeptides or dimeric species.
  • a novel vector was prepared by using the phage fl genome as the starling material.
  • a unique Nhe I restriction site was introduced into the fl genome (GenBank accession
  • Plaque assays were performed by allowing dilutions of phage 205-13.1-1 to infect a bacterial host, then the mixture was plated in top agar onto an LB-agar plate. The plates were incubated overnight to allow a bacterial lawn to form. Circular areas of slower bacterial growth are the result of phage infection and were visualized on the plate.
  • the modified fl phage 205-13.1-1 was digested with the restriction endonuclease Nhe I and cassette 1 a (Seq. ID No. 2) (see Figure 4a), which contains a terminator (Krebber, A., Bu ⁇ nester, ⁇ ., and Pluckthun, A., Gene (1996) 178, pp71-4) and multiple cloning sites was ligated into that position.
  • Cassette la was created by making use of long complimentary oligos which formed the double-stranded DNA cassette. The two oligos were mixed together at a 1 :1 molar ratio, heat denatured and slowly cooled to allow the duplexed insert to form.
  • the annealing of the oligos was such that single stranded DNA overhangs were present at each end (underlined). These overhangs are compatible with the Nhe I overhangs remaining after Nhe I digestion of the vector. However, the ends do not regenerate functional Nhe I sites after annealing.
  • the oligos used for this construction method were: Cas. la-F2: 5 ' CTAGAGTACCCGATAAAAGCGGCTTCCTGAC AGGAGGCCGTTTTGTTTTGCAGC CCACCTGCTAGCATGAATTCGTGGTACCT 3 ' (Seq. ID No. 5) Cas. la-B2:
  • the RF DNA of 205-63.1 was digested with Nhe I and Eco RI and cassette 2, (Seq. ID No. 7, see Fig.4b) which contains the trc promotor, was added (see for example Invitrogen's pTrcHis A promotor sequence).
  • Cassette 2 was generated by making use of long complementary oligos which formed the double stranded DNA cassette. The two oligos were mixed together at a 1 :1 molar ratio, heat denatured and slowly cooled to allow the duplexed insert to form. The annealing of the oligos was such that single stranded DNA overhangs were present at each end, which were compatible with the Nhel/ EcoR I digested vector.
  • the oligos used for construction were: Cas. 2-F2
  • Cassette 3 (Seq. ID No. 12, see Fig. 4c) is the display cassette and contains the first and second cloning regions and a synthetic gene VIII from the vector pAX131-gene VIII (205-91, see below).
  • PAX131 is a phagemid vector prepared by modifying Bluescript II.
  • Fig. 5a is a map of pAX131.
  • Figs. 5b-e show the nucleic acid sequence (Seq. ID No. 13) for pAX131.
  • the preparation of pAX131 is described more fully in commonly owned pending application entitled PHAGEMID VECTORS filed on even date herewith under Express Mail Label No. EL820507456US (U.S. Provisional Application Serial No. 60/287,355, filed April 27, 2001), the disclosure of which is incorporated herein in its entirety by this reference.
  • the cloning region of pAX131 was constructed using an overlapping oligo approach (synthetically generated region).
  • the area of interest includes a ribosomal binding site followed by an optimized (for E. coli expression) ompA leader sequence, an Sfi I, then Sac I and Xba I cloning sites for antibody light chains, another ribosomal binding sequence, an optimized pel B leader sequence, Xho 1 and Spe I heavy chain cloning sites followed by a downstream Sfi I.
  • the portion o pAX131 which was replaced in the creation of cassette 3 includes the sequence for a gene TIT of fl phage. See Figure 5a.
  • a synthetic gene VIII was generated with a nucleotide sequence optimized for bacterial expression (Seq. ID No. 14, see Fig. 6).
  • the gene was assembled using overlapping phosphorylated ohgonucleotides ligated together and cloned into a PCR script vector.
  • the assembled gene was cut from this vector using the flanking Spe I and Not I sites and cloned into pAX131 at the same sites.
  • the sequences of the overlapping oligos were:
  • G8-1f 5'CTAGT GGC CAG GCC GGC Ctg GCT GAA GGC GAC GAC CCG
  • GCT AAA GCT GCT TTC GAC TCC CTG CAG
  • GCT TCC GCT ACC GAA TAC ATC
  • G8-2f 5'GCT TGG GCT ATG GTG GTG GTG ATC GTG GGC GCT ACC ATC GGC ATC AAA CTG TTC AAA AAA TTC ACC TCC AAA GCT TCC taa GGT ACC GC
  • G8-3b 5'GGC CGC GGT ACC tta GGA AGC TTT GGA GGT GAA TTT TTT
  • G8-4b 5' AGC GCC CAC GAT CAC CAC CAC CAT AGC CCA AGC GTA GCC GAT GTA TTC GGT AGC GGA AGC CTG 3' (Seq. ID No.18)
  • G8-5b 5' CAG GGA GTC GAA AGC AGC TTT AGC CGG GTC GTC GCC TTC
  • Figure 7 shows the alignment of the oligos for the preparation of the synthetic gVIII. All oligo sequences are shown in the sense orientation, meaning reverse oligos G8-3b, G8-4b, and G8-5b are shown in Figure 7 as their reverse complement in order to see the alignment with the forward oligos G8-l f and G8-2f Construction of the synthetic gene was actually done by contract with Aptagen.
  • the resulting vector 205-91 was digested by restriction enzymes EcoR I and Kpn I to create the display cassette (Seq. ID No. 12, see Fig. 4c). This display cassette was then inserted into Eco RI and Kpn I digested 205-87 to create the final vector, 228-49.14.
  • Verification of the final construct includes sequence analysis of the resulting RF DNA and phage plaque size as described above. Additionally, a tetanus toxoid control antibody was cloned into the phage using the Sac I/Xba I sites for the light chain and Xho 1/ Spe I sites for the heavy chain Fd to create 241-15.29. Western blots of phage virion preps of 241-15.29 indicated that the Fab was expressed as a fusion protein with the synthetic gene VIII and incorporated into virions.
  • a test panning experiment will also be performed to ensure that the Fab-fusion is presented on the phage surface and available for antigen selection.
  • a phage mixture at a ratio of 1 specific phage/antibody into 10 6 or more non-specific phage/antibody was used as the starting sample. Following 3 to 4 rounds of panning, the specific antibody was selected and therefore present at a much higher ratio than the starting ratio. Solid phase panning was also performed
  • plaques by adding 5 mis media to each large petri dish and scrapping the top agar into 50 ml conical tubes. Agar debris was removed by centrifugation. Phage stock was used directly but can be concentrated by PEG precipitation if necessay : 4% PEG 8000 + 0.5M NaCl on ice for
  • MOS hairpin and the minus origin See Fig. 10).
  • overlap PCR was used to generate the restriction site between the MOS hairpin and the minus origin (see
  • 205-13.2-1 was digested with Nhe 1.
  • Cassette lb was created for insertion into 205- 13.2-1 by making use of long complementary oligos which form the double stranded DNA cassette.
  • the two ohgos were mixed together at a 1 : 1 molar ratio, heat denatured and slowly cooled to allow the duplexed insert to form.
  • the annealing of the oligos was such that ends of single stranded DNA overhangs were present at each end, which were complementary to the Nhe I digested vector.
  • Oligos used for this method were: Cas.lb-F2: 5' CT AGA GCT AGC at GAA TTC st GGT ACCgta ccc gat aaa age ggc ttc ctg aca gga ggc cgt ttt gtt ttg cag ccc ace tT 3' (Seq. ID No.
  • Cas.lb-B2 5' CTA GAa ggt ggg ctg caa aac aaa acg gcc tec tgt cag gaa gcc get ttt ate ggg tac GGT ACC ac GAA TTC at GCT AGC T 3' (Seq. ID No. 29)
  • the overlapping ends are underlined, the Nhe I, Eco RI, and Kpn I sites are double underlined. The sequence is such that the ends do not regenerate a functional Nhe I site. Insertion of Cassette lb into 205-13.2-1 generated 205-83.1. This was digested with
  • Nhe I and Eco RI and cassette 2 (Seq. ID No. 7, see Fig. 4b) was generated and added as described above in Example 1 to create construct 205-93.12. This was verified by sequence analysis, digested with Eco RI and Kpn I, and cassette 3 was inserted as described above in Example 1 to create the final construct 228-88.5. This was verified by sequence analysis and analysis of plaque size, which again did not appear to be significantly affected.
  • the present novel vector can be used in connection with the production and screening of libraries made in accordance with conventional phage display technologies. Both natural and synthetic antibody repertoires have been generated as phage displayed libraries. Natural antibodies can be cloned from B-cell mRNA isolated from peripheral blood lymphocytes, bone ma ⁇ ow, spleen, or other lymphatic tissue of a human or non-human donor. Donors with an immune response to the antigen(s) of interest can be used to create immune antibody libraries. Alternatively, non-immune libraries may be generated from donors by isolating naive antibody B cell genes. PCR using antibody specific primers on the 1 st strand cDNA allows the isolation of light chain and heavy chain antibody fragments which can then be cloned into the display vector.
  • Synthetic antibodies or antibody libraries can be made up in part or entirely with regions of synthetically derived sequence.
  • Library diversity can be engineered within variable regions, particularly within CDRs, through the use of degenerate ohgonucleotides.
  • a single Fab gene may be modified at the heavy chain CDR3 position to contain random nucleotide sequences.
  • the random sequence can be introduced into the heavy chain gene using an oligonucleotide which contains the degenerate coding region in an overlap PCR approach.
  • degenerate oligo cassettes can be cloned into restriction sites that flank the CDR(s) to create diversity.
  • the resulting library generated by such approaches can then be cloned into a display vector in accordance with this disclosure.
  • phage particles Upon introduction of the display into bacteria, phage particles will be generated that have antibody displayed on the surface.
  • the resulting collection of phage-displayed antibodies can be selected for those with the ability to bind to the antigen of interest using techniques known to those skilled in the art.
  • Antibodies identified by this system can be used therapeutically, as diagnostic reagents, or as research tools.

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Abstract

Cette invention permet de modifier phonétiquement un génome de phage pour qu'il contienne un nouveau site de restriction dans l'une ou l'autre de deux positions différentes. Dans un premier mode de réalisation de cette invention, un site de restriction est inséré dans le génome du phage entre l'extrémité du gène IV et la boucle en épingle à cheveux MOS qui sert de signal de conditionnement de phage pour les brins simples nouvellement synthétisés de l'ADN du phage. Dans un second mode de réalisation de cette invention, un site de restriction est inséré dans le génome du phage après la boucle en épingle à cheveux MOS et avant l'origine du brin négatif. Une fois que le génome du phage est modifié de façon à contenir le nouveau site de restriction, le vecteur peut être modifié génétiquement en vecteur « 88 » par insertion dans le nouveau site de restriction d'une séquence nucléotidique codant au moins un domaine fonctionnel de la protéine pVIII et au moins un premier site de clonage destiné à recevoir un gène codant un polypeptide à exprimer en surface et, éventuellement, un second site de clonage destiné à recevoir un second gène codant un polypeptide capable de dimérisation avec le polypeptide à exprimer. Dans des modes de réalisation particulièrement utiles, ces nouveaux vecteurs sont modifiés génétiquement de façon à produire des particules de phage qui expriment des anticorps.
EP02744148A 2001-04-27 2002-04-29 Nouveaux vecteurs de phage 88 Withdrawn EP1444344A4 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US28723901P 2001-04-27 2001-04-27
US287239P 2001-04-27
PCT/US2002/014971 WO2003093471A1 (fr) 2001-04-27 2002-04-29 Nouveaux vecteurs de phage 88

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EP1444344A1 true EP1444344A1 (fr) 2004-08-11
EP1444344A4 EP1444344A4 (fr) 2005-08-24

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AU6235294A (en) * 1993-02-02 1994-08-29 Scripps Research Institute, The Methods for producing polypeptide binding sites
GB9313509D0 (en) * 1993-06-30 1993-08-11 Medical Res Council Chemisynthetic libraries
US5702892A (en) * 1995-05-09 1997-12-30 The United States Of America As Represented By The Department Of Health And Human Services Phage-display of immunoglobulin heavy chain libraries
AU2002305246B2 (en) * 2001-04-27 2006-06-08 Alexion Pharmaceuticals, Inc. Phagemid vectors

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PINI A ET AL: "PHAGE DISPLAY OF ANTIBODY FRAGMENTS" CURRENT PROTEIN AND PEPTIDE SCIENCE, BENTHAM SCIENCE PULBISHERS, NL, vol. 1, no. 2, September 2000 (2000-09), pages 155-169, XP001084580 ISSN: 1389-2037 *
See also references of WO03093471A1 *

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