WO1992015678A1 - Molecules d'adn dicistroniques produites par reaction en chaine de polymerase et servant a produire des anticorps - Google Patents

Molecules d'adn dicistroniques produites par reaction en chaine de polymerase et servant a produire des anticorps Download PDF

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Publication number
WO1992015678A1
WO1992015678A1 PCT/US1992/001475 US9201475W WO9215678A1 WO 1992015678 A1 WO1992015678 A1 WO 1992015678A1 US 9201475 W US9201475 W US 9201475W WO 9215678 A1 WO9215678 A1 WO 9215678A1
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εaid
primer
pcr
polypeptide
dna
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PCT/US1992/001475
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Joseph A. Sorge
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Stratagene
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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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins

Definitions

  • the present invention relates to a method for producing a library of dicistronic DNA molecules useful in expressing heterodimeric receptors, such as antibodies, T cell receptors and the like.
  • the expression of antibody libraries in bacteria has opened up new ways to uncover monoclonal antibody specificities.
  • the antigen binding domain of the antibody is composed of a heavy and a light chain. These chains are each encoded by separate genes.
  • both heavy and light chain coding sequences are typically coexpressed, which involves two cloning steps, one for the heavy chain and one for the light. This is generally accomplished by either inserting both heavy and light chain coding sequences into one vector, or by first making separate heavy and light chain libraries and recombining the genomes to make a combinatorial library encoding random combinations of the heavy and light sequences. In either case, the need to clone two separate DNA fragments is cumbersome and, therefore, a method that could fuse both heavy and light chain sequences together prior to vector ligation would be desirable.
  • the present invention contemplates a method of producing dicistronic DNA molecules each having upstream and downstream cistrons respectively coding for first and second polypeptides of a heterodimeric protein, such as a receptor.
  • the method comprises the following steps:
  • A Forming a first polymera ⁇ e chain reaction (PCR) admixture by combining, in a PCR buffer, first polypeptide-encoding genes and a first PCR primer pair defined by an outside first gene primer and an inside first gene primer.
  • the inside first gene primer has a 3 '-terminal priming portion and, preferably, a 5'- terminal non-priming portion.
  • the 3'-terminal priming portion comprises a nucleotide sequence homologous to a conserved portion of a first gene.
  • the inside gene primer has a 3'-terminal priming portion and, preferably, a 5'-terminal hybridizing portion complementary to a hybridizable portion of the 5'-terminal non-priming portion of the first inside gene primer.
  • the 3 '-terminal priming portion comprises a nucleotide sequence homologous to a conserved portion of a second polypeptide-coding gene.
  • the first and second inside primers when hybridized, form a duplex that codes for a double- stranded cistronic bridge that links the upstream and downstream cistrons.
  • One strand of the bridge codes for (i) at least one stop codon in the same reading frame as said upstream cistron, (ii) signals for the initiation of translation of the downstream in cistron.
  • signals include a riboso e binding site downstream from the stop codon, and at least one translation initiation codon in the same reading frame as the downstream cistron, the initiation codon being located downstream from the ribosome binding site.
  • D Subjecting the second PCR admixture to a plurality of PCR thermocycles to produce a plurality of second polypeptide-coding DNA ho ologs in double stranded form.
  • step (F) Hybridizing the separated strands of step (E) to form internally-primed duplexes.
  • each of the dicistronic DNA molecules produced contains a first polypeptide-coding sequence and a second polypeptide-coding sequence linked by the cistronic bridge.
  • the upstream cistron comprises one of the first polypeptide- or second polypeptide-coding DNA homologs.
  • the downstream cistron comprises the other of the first polypeptide- or second polypeptide-coding DNA homologs.
  • steps (A)-(D) are performed concurrently in one reaction vessel.
  • the polypeptide-encoding genes of steps (A) and (B) are present in respective repertoires of conserved genes.
  • the repertoires of steps (A) and (C) are usually formed by isolating mRNA from at least about 10 3 , preferably at least about 10 7 lymphocytes. It is preferred that the repertoire of first polypeptide genes comprises at least 10 5 different first polypeptide genes, and that the repertoire of second polypeptide genes comprises at least 10 5 different second polypeptide genes.
  • the method of the present invention can be used to operatively link for polyci ⁇ tronic expression any two genes.
  • this invention can be used to physically link two genes from a single cell, such as a B cell, T cell, and the like, and thereby take advantage of a native immune system's ability to select operative gene pairs from the immunological repertoire.
  • operative gene pairs i.e., a pair of genes encoding a heterodimeric receptor, from cells such as hybridomas, quadromas and the like, can be physically linked using the method of this invention.
  • the method further comprises step (H) wherein the dicistronic DNA molecules are PCR amplified by combining them with the outside first gene primer and the outside second gene primer to form a third PCR admixture.
  • the third PCR admixture is then subjected to a plurality of PCR thermocycles.
  • a repertoire of first and/or second polypeptide- encoding genes is used, an amplified library of dicistronic DNA molecules is produced.
  • the amplified products of step (H) are operatively linked for expression to a vector, preferably a phage vector.
  • a vector preferably a phage vector.
  • the steps for operatively linking the dicistronic DNA molecules to a vector and isolating a recombinant vector that expresses a desired heterodimeric receptor include the following:
  • the outside first gene primer hybridizes to a framework, leader or promoter region of a V H im unoglobulin gene
  • the outside second gene primer hybridizes to a J L , constant or framework region, of a V L immunoglobulin gene.
  • the 3'-terminal priming portion of the inside first gene primer hybridizes to a J H/ hinge, constant, or framework region of a V H immunoglobulin gene
  • the 3'- ter inal priming portion of the inside second gene primer hybridizes to a framework, leader or promoter region of a V L immunoglobulin gene.
  • a library of dicistronic DNA molecules comprising an upstream cistron and a downstream cistron, is produced by the following steps: (A) forming a poly erase chain reaction (PCR) admixture by combining, in a PCR buffer: (i) V H genes, (ii) V L genes,
  • a linking primer having a 3'- terminal priming portion, a cistronic bridge coding portion, and a 5'-terminal primer-template portion.
  • the 3 '-terminal priming portion has a nucleotide base sequence complementary to a portion of the primer extension product of one of the outside primers.
  • the 5'-terminal primer template portion has a nucleotide base sequence homologous to a portion of the primer extension product of the other of the outside primers.
  • the cistronic bridge coding portion is as previously described.
  • step (B) Subjecting the PCR admixture of step (A) to a plurality of PCR thermocycles.
  • the method further comprises steps (C)-(H) as follows:
  • (C) Subjecting the internally-primed duplexes to conditions for primer extension to produce dicistronic DNA molecules, each containing a V H -coding sequence and a V L -coding sequence linked by the cistronic bridge.
  • the upstream cistron comprises one of the V ⁇ - or " ⁇ -coding DNA homologs, and the downstream cistron comprising the other of the V H - V L - coding DNA homologs.
  • kits for producing a dicistronic DNA molecule as described herein are kits for producing a dicistronic DNA molecule as described herein.
  • the kit is an enclosure containing, in separate containers, an outside first polypeptide, preferably a V H , gene primer, an outside second polypeptide, preferably a V L , gene primer, and a linking primer defining a 3'-terminal priming portion, a cistronic bridge coding portion, and a 5'-terminal primer-template portion.
  • the 3'-terminal priming portion has a nucleotide base sequence complementary to a portion of the primer extension product of one of the outside primers.
  • the 5'-terminal primer-template portion encoding a nucleotide base sequence homologous to a portion of the primer extension product of the other of the outside primers.
  • the cistronic bridge coding portion is as previously described.
  • kits comprises an enclosure containing, in separate containers, an outside first polypeptide, preferably a V H , gene primer, an outside second polypeptide, preferably a v L , gene primer, an inside first polypeptide, preferably a V H , gene primer having a 3 '-terminal priming portion and a 5'-terminal non-priming portion.
  • the 3'-terminal priming portion comprises a nucleotide sequence homologous to a conserved portion of a V H gene.
  • the kit also contains an inside second polypeptide, preferably a V L , gene primer having a 3'- terminal priming portion and a 5'-terminal hybridizing portion complementary to the 5'-terminal non-priming portion of the first polypeptide gene primer, the 3'- terminal priming portion of which comprises a nucleotide sequence homologous to a conserved portion of a second polypeptide gene.
  • the first polypeptide inside and second polypeptide inside primers when hybridized, form a duplex that codes for a double- stranded DNA molecule containing the before described cistronic bridge for linking the upstream and downstream ci ⁇ trons.
  • Figure 1 illu ⁇ trate ⁇ the principal structural features of an immunoglobulin molecule.
  • the circled areas on the heavy and light chains represent the variable regions, (V H ) and (V L ) , a heterodimeric polypeptide containing a biologically active (ligand binding) portion of that region, and genes coding for the individual polypeptides, are produced by the methods of the present invention.
  • Figure 2 contain ⁇ three panels.
  • Panel 2A illustrate ⁇ variou ⁇ features of the heavy chain of human IgG (IgGl subclass) . Numbering is from the N- terminus on the left to the C-terminus on the right. Note the presence of four domains, each containing an intrachain di ⁇ ulfide bond (S-S) and spanning approximately 110 amino acid residues.
  • S-S intrachain di ⁇ ulfide bond
  • the symbol CHO stands for carbohydrate.
  • the V region of the heavy (H) chain (V H ) resembles V L in having three hypervariable complementarity determining regions (CDR'S) (not ⁇ hown) .
  • Panel 2B and 2C illustrate various features of a human kappa ( ) chain. Numbering is from the N- ter inus on the left to the C-terminus on the right. Note in Panel 2B the intrachain disulfide bond (S-S) spanning about the same number of amino acid residue ⁇ in the V L and C L domains. Panel 2C shows the locations of the CDRs in the V L domain. Segments out ⁇ ide the CDR are the framework segments (FR) .
  • S-S intrachain disulfide bond
  • Figure 3 illustrates a portion of the nucleotide base sequence of the 1661 base pair gene la B ⁇ equence from residue number 250 to re ⁇ idue number 651.
  • the position of the nucleotide base sequence is indicated by the numbers in the left margin of the figure.
  • the reading frame of the structural lamB gene is indicated by placement of the deduced amino acid residue sequence of the lambda receptor protein for which it codes below the nucleotide sequence such that the triple letter code for each amino acid residue is located directly below the three base ⁇ (codon) coding for each re ⁇ idue.
  • the re ⁇ idue sequence i ⁇ hown conventionally from left to right and in the direction of amino terminus to carboxy terminu ⁇ .
  • Figure 4 illu ⁇ trate ⁇ the strategy used to create immunoglobulin heavy and light chain PCR fusion products.
  • RNA and DNA are represented by dotted and solid lines, respectively.
  • Regions of the immunoglobulin heavy chain coding ⁇ trand area de ⁇ ignated V H , C H 1, C H 2, and C H 3 corre ⁇ pond to those functional regions in the protein.
  • the corresponding regions of the non-coding strand are designated by a prime (') following the symbol.
  • Regions V L and C L are similarly labelled for the light chain.
  • a region, X, unrelated to the natural immunoglobulin sequences is introduced into the fusion product by attaching X to the 5' end ⁇ of the C H 1' inside and V L in ⁇ ide primer ⁇ .
  • Figure 5 illu ⁇ trate ⁇ human fu ⁇ ion PCR in ⁇ ide primers.
  • the heavy chain C H 1' inside primer sequence is written 3* to 5* and the light chain V L inside primer sequence is written 5' to 3 ' . Note that it is not the primer strands that cros ⁇ -prime to create the fusion molecule, but the complementary PCR product strands.
  • Boxed nucleotides represent regions where the C H 1' primer hybridizes to the 3 ' end of C h l on human IgG heavy chain mRNA or where the V L primer hybridizes to the 5' end of V L framework-1 on human kappa light chain cDNA.
  • Underlined sequence ⁇ indicate the two stop codons.
  • the italicized amino acid and nucleotides indicate changes in sequence from the original pelB leader sequence.
  • the mouse fu ⁇ ion-PCR internal primers overlap in a similar manner.
  • Figure 6 illustrates the sequences of the synthetic DNAs inserted into Lambda ZAP to produce Lambda Zap II V H (ImmunoZAP H) (Panel A) and Lambda Zap V L (ImmunoZAP L) (Panel B) expression vectors.
  • the various features required for these vectors to express the V H and V L -coding DNA homologs include the Shine-Dalgarno ribosome binding ⁇ ite, a leader sequence to direct the expres ⁇ ed protein to the peripla ⁇ m a ⁇ de ⁇ cribed by Mouva et al. , J. Biol. Chem.
  • V H expression-vector sequence also contains a short nucleic acid sequence that codes for amino acids typically found in variable regions heavy chain (V H Backbone) .
  • V H Backbone is just upstream and in the proper reading as the V H DNA homologs that are operatively linked into the Xho I and Spe I restriction sites.
  • the V L DNA homologs are operatively linked into the V L sequence (Panel B) at the Sac I and Xba I restriction enzyme sites.
  • Figure 7 illustrates the major features of the bacterial expression vector Lambda Zap II V H (ImmunoZAP H) (V H - expression vector) .
  • the amino acids encoded by the synthetic DNA sequence from Figure 6A is shown at the top along with the T 3 polymerase promoter from Lambda Zap II.
  • the orientation of the insert in Lambda Zap II is as presented.
  • the V H DNA homologs were inserted into the phagemid that is produced by the in vivo excision protocol described by Short et al.. Nucleic Acids
  • V H DNA homologs were inserted into the Xho I and Spe I restriction enzyme ⁇ ite ⁇ .
  • the read through transcription produces the decapeptide epitope (tag) that is located just 3 ' of the cloning sites.
  • Figure 8 illustrates, in Panels 8A and 8B, the major features of the bacterial expression vector Lambda ZAP II Modified V H (Modified ImmunoZAP H) (V H - expre ⁇ ion vector) (IZ H) .
  • the amino acids encoded by the synthetic DNA sequence from Panel 8A is shown along with the T 3 polymerase promoter from Lambda ZAP II.
  • the orientation of the insert in Lambda ZAP II is as presented.
  • the insert was modified by the elimination of the Sac I site between the T 3 polymerase and Not I site and by the change of amino acids at the 5' end of the heavy chain rom QVKL to QVQL (a lysine residue was changed to a gluta ine residue) .
  • V H and V L DNA homologs were inserted into the Xho I and Xba I cloning sites of the phagemid as described in Figure 7 and shown in Panel 8B.
  • the modifications were made to create a fusion-PCR library from hybridoma RNA, to overcome decreased efficiency of secretion of positively charged amino acids in the amino terminus of the protein. Inouye et al., Proc. Natl. Acad. Sci.. USA. 85:7685-7689 (1988), and to make the V L Sac I cloning site a unique restriction site.
  • Figure 9 illustrates the major features of the bacterial expression vector Lambda Zap II V L (ImmunoZAP L) (V L expression vector) .
  • the amino acids encoded by the synthetic DNA sequence shown in Figure 6B is shown at the top along with the T 3 polymerase promoter from Lambda Zap II, The orientation of the insert in Lambda Zap II is as presented.
  • the V L DNA homologs are in ⁇ erted into the Sac I and Xba I cloning sites of the phagemid as described in Figure 7.
  • Figure 10 illustrates an ethidium bromide ⁇ tained agaro ⁇ e gel. After PCR amplification from human cloned DNA of heavy chain alone (HC) , light chain alone (LC) , and the heavy/light dicistronic DNA molecule (H/L) , DNA samples were electrophoresed. The expected size ⁇ of the HC, LC, and H/L products visualized on the gel were approximately 730, 690, and 1,390 base pair ⁇ , re ⁇ pectively.
  • Figure 11 illustrates an autoradiogram shoving ⁇ ignal ⁇ obtained from human phage clone ⁇ . Approximately 100 lambda phage were spotted onto E.
  • FIG 12 illustrates the major features of the bacterial expres ⁇ ion vector lambda ZAP H/L (ImmunoZAP H/L) (combined V H - and V L -expre ⁇ sion vector) .
  • the ImmunoZAP H/L vector is created from the heavy and light chain libraries by fusing the vectors at the Eco Rl site. DNA is purified from the light chain library and restriction digested with Mlu 1 and Eco Rl. This cleaves the DNA from the left arm of the vector into several pieces while leaving the right arm with the light chain inserts intact. DNA i ⁇ purified from the heavy chain librarie ⁇ and re ⁇ triction digested with Hind III and Eco Rl.
  • Nucleotide A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose) , a pho ⁇ phate, and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1' carbon of the pento ⁇ e) and that combination of ba ⁇ e and sugar is a nucleoside. When the nucleoside contains a phosphate group bonded to the 3 ' or 5* position of the pentose it is referred to as a nucleotide.
  • Base Pair (bp) A partnership of adenine (A) with thymine (T) , or of cytosine (C) with guanine (G) in a double stranded DNA molecule.
  • adenine A
  • C cytosine
  • G guanine
  • U uracil
  • Nucleic Acid A polymer of nucleotides, either single or double stranded.
  • Gene A nucleic acid whose nucleotide sequence codes for an RNA or polypeptide.
  • a gene can be either RNA or DNA.
  • Complementary Bases Nucleotides that normally pair up when DNA or RNA adopts a double stranded configuration.
  • Complementary Nucleotide Seguence A sequence of nucleotides in a single-stranded molecule of DNA or RNA that is sufficiently complementary to that on another single strand to specifically hybridize to it with consequent hydrogen bonding.
  • conserveed A nucleotide ⁇ equence i ⁇ conserved with respect to a preselected (reference) ⁇ equence if it non-randomly hybridizes to an exact complement of the preselected sequence.
  • Hybridization The pairing of substantially complementary nucleotide sequences (strand ⁇ of nucleic acid) to form a duplex or heteroduplex by the establishment of hydrogen bonds between complementary base pair ⁇ . It is a specific, i.e. non-random, interaction between two complementary polynucleotides that can be competitively inhibited.
  • Nucleotide Analog A purine or pyrimidine nucleotide that differs structurally from A, T, G, C, or U, but is sufficiently similar to substitute or the normal nucleotide in a nucleic acid molecule.
  • DNA Homolog I ⁇ a nucleic acid having a pre ⁇ elected conserved nucleotide sequence and a sequence coding for a receptor capable of binding a preselected ligand.
  • Receptor A receptor i ⁇ a molecule, ⁇ uch as a protein, glycoprotein and the like, that can specifically (non-randomly) bind to another molecule.
  • Antibody in its variou ⁇ grammatical form ⁇ i ⁇ u ⁇ ed herein to refer to immunoglobulin molecules and immunologically active portion ⁇ of immunoglobulin molecule ⁇ , i.e., molecule ⁇ that contain an antibody combining site or paratope.
  • Exemplary antibody molecules are intact immunoglobulin molecule ⁇ , ⁇ ub ⁇ tantially intact immunoglobulin molecules and portions of an immunoglobulin molecule, including those portions known in the art a ⁇ Fab, Fab 1 , F(ab') 2 and F(v) .
  • An antibody combining site is that structural portion of an antibody molecule comprised of a heavy and light chain variable and hypervariable regions that ⁇ pecifically bind ⁇ (immunoreacts with) an antigen.
  • the term immunoreact in it ⁇ variou ⁇ forms mean ⁇ specific binding between an antigenic determinant-containing molecule and a molecule containing an antibody combining site ⁇ uch a ⁇ a whole antibody molecule or a portion thereof.
  • the phra ⁇ e monoclonal antibody in its various grammatical forms refers to a population of antibody molecules that contain ⁇ only one species of antibody combining site capable of immunoreacting with a particular antigen.
  • a monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreact ⁇ .
  • a monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining ⁇ ite ⁇ , each immuno ⁇ pecific for a different antigen, e.g., a bi ⁇ pecific monoclonal antibody.
  • Up t eam In the direction oppo ⁇ ite to the direction of DNA tran ⁇ cription, and therefore going from 5* to 3' on the non-coding ⁇ trand, or 3' to 5' on the mRNA.
  • Downstream Further along a DNA sequence in the direction of sequence transcription or read out, that i ⁇ traveling in a 3'- to 5'-direction along the non-coding ⁇ trand of the DNA or 5•- to 3'- direction along the RITA tran ⁇ cript.
  • Ci ⁇ tron Sequence of nucleotides in a DNA molecule coding for an amino acid residue sequence.
  • Stop Codon Any of three codons that do not code for an amino acid, but in ⁇ tead cau ⁇ e s termination of protein ⁇ ynthesis. They are UAG, UAA and UGA. Al ⁇ o referred to as a nonsen ⁇ e or termination codon.
  • Leader Polypeptide A short length of amino acid sequence at the amino end of a protein, which carries or directs the protein through the inner membrane and so ensures its eventual secretion into the periplasmic space and perhaps beyond. The leader sequence peptide is commonly removed before the protein becomes active.
  • Reading Frame Particular sequence of contiguous nucleotide triplets (codons) employed in translation. The reading frame depends on the location of the translation initiation codon.
  • An inside primer is a polynucleotide that has a priming region located at the 3' terminu ⁇ of the primer which typically consists of 15 to 30 nucleotide ba ⁇ es.
  • the 3' terminal-priming portion i ⁇ capable of acting as a primer to catalyze nucleic acid ⁇ ynthesi ⁇ .
  • the 5'-terminal priming portion compri ⁇ e ⁇ a non-priming portion.
  • An outside primer comprises a 3'-terminal priming portion and a portion that may define an endonuclease re ⁇ triction ⁇ ite which i ⁇ typically located in a 5'-terminal non-priming portion of the outside primer.
  • the present invention contemplates a method of isolating from a repertoire of conserved genes a pair of genes coding for a dimeric receptor having a preselected activity.
  • the receptor will be a heterodimeric polypeptide capable of binding a ligand, such as an antibody molecule or immunologically active portion thereof, a cellular receptor, or a cellular adhesion protein coded for by one of the members of a family of conserved genes, i.e., genes containing a conserved nucleotide sequence of at least about 10 nucleotides in length.
  • Exemplary conserved gene families encoding different polypeptide claim ⁇ of a dimeric receptor are tho ⁇ e coding for immunoglobulins, major hi ⁇ tocompatibility complex antigen ⁇ of class I or II, lymphocyte receptors, integrins and the like.
  • a gene can be identified a ⁇ belonging to a repertoire of conserved genes using several methods. For example, an isolated gene may be used as a hybridization probe under low stringency conditions to detect other members of the repertoire of conserved genes present in genomic DNA using the methods described by Southern, J. Mol. Biol.. 98:503 (1975). If the gene used a ⁇ a hybridization probe hybridize ⁇ to multiple restriction endonuclease fragments of the genome, that gene is a member of a repertoire of conserved genes.
  • the immunoglobulins, or antibody molecule ⁇ are a large family of molecule ⁇ that include ⁇ everal types of molecules, ⁇ uch as IgD, IgG, IgA, IgM and IgE.
  • the antibody molecule is typically comprised of two heavy (H) and light (L) chains with both a variable (V) and constant (C) region present on each chain as shown in Figure 1.
  • Schematic diagrams of human IgG heavy chain and human kappa light chain are shown in Figure ⁇ 2A and 2B, re ⁇ pectively.
  • Several different region ⁇ of an immunoglobulin contain con ⁇ erved ⁇ e uence ⁇ useful for isolating an immunoglobulin repertoire.
  • the C region of the H chain defines the particular immunoglobulin type. Therefore the selection of conserved sequences a ⁇ defined herein from the C region of the H chain re ⁇ ult ⁇ in the preparation of a repertoire of immunoglobulin gene ⁇ having member ⁇ of the immunoglobulin type of the selected C region.
  • the V region of the H or L chain typically comprises four framework (FR) regions each containing relatively lower degree ⁇ of variability that include ⁇ length ⁇ of con ⁇ erved sequences.
  • FR1 and FR4 (J region) framework region ⁇ of the V H chain i ⁇ a preferred exemplary embodiment and is described herein in the Examples.
  • Framework regions are typically conserved acros ⁇ ⁇ everal or all immunoglobulin types and thus conserved sequences contained therein are particularly suited for preparing repertoires having several immunoglobulin types.
  • MHC major histocompatibility complex
  • Cla ⁇ s I MHC molecules are a polymorphic group of transplantation antigens representing a conserved family in which the antigen is comprised of a heavy chain and a non-MHC encoded light chain.
  • the heavy chain includes several regions, termed the N * Cl, C2, membrane and cytoplasmic region ⁇ .
  • Con ⁇ erved ⁇ equence ⁇ useful in the pre ⁇ ent invention are found primarily in the N, Cl and C2 region ⁇ and are identified a ⁇ continuou ⁇ ⁇ equence ⁇ of "invariant re ⁇ idue ⁇ " in Kabat et al. , supra.
  • Clas ⁇ II MHC molecule ⁇ comprise a conserved family of polymorphic antigens that participate in immune respon ⁇ ivene ⁇ s and are comprised of an alpha and a beta chain.
  • the genes coding for the alpha and beta chain each include several regions that contain conserved sequences suitable for producing MHC class
  • alpha or beta chain repertoires Exemplary conserved nucleotide sequences include those coding for amino acid residues 26-30 of the Al region, residue ⁇ 161-170 of the A2 region and residue ⁇ 195-206 of the membrane region, all of the alpha chain. Con ⁇ erved sequences are also present in the Bl, B2 and membrane region ⁇ of the beta chain at nucleotide sequences coding for amino acid residues 41-45, 150- 162 and 200-209, respectively.
  • Lymphocytes contain several families of proteins on their cell surface ⁇ including the T-cell receptor, Thy-1 antigen and numerou ⁇ T-cell surface antigens including the antigens defined by the monoclonal antibodies 0KT4 (leu3) , OKUT5/8 (leu2) , OKUT3, 0KUT1 (leul) , OKT 11 (leu5) 0KT6 and 0KT9. Paul, supra at pp. 458-479.
  • the T-cell receptor is a term used for a family of antigen binding molecules found on the surface of T-cell ⁇ .
  • the T-cell receptor a ⁇ a family exhibit ⁇ polymorphic binding specificity similar to immunoglobulins in its diversity.
  • the mature T-cell receptor i ⁇ comprised of alpha and beta chains each having a variable (V) and constant (C) region.
  • V variable
  • C constant
  • Exemplary con ⁇ erved ⁇ equence ⁇ include tho ⁇ e coding for amino acid re ⁇ idues 84-90 of alpha chain, amino acid re ⁇ idue ⁇ 107-115 of beta chain, and amino acid residues 91-95 and 111-116 of the gamma chain. Kabat et al., supra. p. 279.
  • Integrins are heterodimers co pri ⁇ ed of a beta and an alpha subunit.
  • Members of the integrin family include the cell surface glycoproteins platelet receptor GpIIb-IIIa, vitronectin, receptor (VnR) fibronectin receptor (FnR) and the leukocyte adhesion receptors LFA-1, Mac-1, Mo- 1 and 60.3. Rou ⁇ lahti et al., Science, 238:491-497 (1987) .
  • Nucleic acid and protein sequence data demonstrates regions of conserved sequences exist in the member ⁇ of the ⁇ e discipline ⁇ , particularly between the beta chain of GpIIb-IIIa VnR and FnR, and between the alpha ⁇ ubunit of VnR, Mac-1, LFA-1, FnR and GpIIb- IIIa. Suzuki et al., Proc. Natl. Acad. Sci. USA. 83:8614-8618, 1986? Ginsberg et al., J. Biol. Chem.. 262:5437-5440, 1987.
  • fusion PCR is used to generate two PCR-amplified DNA fragments, each of which have one of their ends modified by directed mispriming so that those ends share region ⁇ of complementarity, i.e., cohesive termini.
  • region ⁇ of complementarity i.e., cohesive termini.
  • PCR amplification methods are described in detail in U.S. Patent Nos. 4,683,192, 4,683,202, 4,800,159, and 4,965,188, and at least in several texts including "PCR Technology: Principles and Applications for DNA Amplification", H. Erlich, ed. , Stockton Press, New York (1989) ; and "PCR Protocols: A Guide to Methods and Applications", Inni ⁇ et al., ed ⁇ . , Academic Pre ⁇ , San Diego, California (1990). Cloning From Gene Repertoires
  • the following discussion illustrates the method of the present invention applied to isolating a pair of V H and V L genes from the immunoglobulin gene repertoire.
  • This discu ⁇ ion i ⁇ not to be taken a ⁇ limiting, but rather a ⁇ illustrating application of principles that can be used to operatively link and isolate a functionally similar pair of genes.
  • the illustrated method can be used with any family of conserved genes coding for functionally related dimeric receptors, whether obtained directly from a natural source, such naive or in vivo immunized cells, or from cells or one or more genes that have been treated or mutagenized in vitro.
  • the method combines the following elements:
  • fir ⁇ t and ⁇ econd PCR amplification products are produced u ⁇ ing re ⁇ pective fir ⁇ t and second PCR primer pairs.
  • the fir ⁇ t PCR primer pair comprises a first polypeptide outside primer and a first polypeptide inside primer.
  • the second PCR primer pair comprise ⁇ a ⁇ econd polypeptide out ⁇ ide primer and a ⁇ econd polypeptide in ⁇ ide primer.
  • the fir ⁇ t and second polypeptide inside primers contain complementary 5'- terminal sequence ⁇ that allow their DNA complements to hybridize and form an internally-primed duplex having 3'-overhanging termini.
  • the internally-primed duplex is then subjected to primer exten ⁇ ion reaction condition ⁇ to produce a double ⁇ tranded, dicistronic DNA having substantially blunt or blunt ends.
  • the dici ⁇ tronic DNA i ⁇ then PCR amplified u ⁇ ing the out ⁇ ide primer ⁇ a ⁇ a PCR primer pair.
  • a dici ⁇ tronic DNA molecule of this invention contains two amino acid residue-coding sequence ⁇ on the ⁇ ame ⁇ trand ⁇ eparated by at lea ⁇ t one ⁇ top codon and at least one signal sequence neces ⁇ ary for tran ⁇ lation of the down ⁇ tream ci ⁇ tron, ⁇ uch a ⁇ a translation initiation codon, ribosome binding site, and the like.
  • the upstream and downstream ci ⁇ trons of the dici ⁇ tronic DNA molecule are operatively linked by a ci ⁇ tronic bridge.
  • the ci ⁇ tronic bridge contain ⁇ the genetic element ⁇ necessary to terminate translation of the upstream ci ⁇ tron and initiate tran ⁇ lation of the downstream cistron.
  • the cistronic bridge coding strand preferably also encode ⁇ a ribosome binding site for the downstream ci ⁇ tron located downstream from the up ⁇ tream cistron's stop codon( ⁇ ).
  • the coding ⁇ trand of the cistronic bridge will al ⁇ o encode a leader polypeptide segment in the ⁇ ame tran ⁇ lational reading frame a ⁇ the down ⁇ tream cistron.
  • the nucleotide base sequence encoding the leader usually begins with an initiation codon located within an operative distance, i.e., i ⁇ operatively linked, to the ribosome binding site.
  • a receptor produced by the pre ⁇ ent invention a ⁇ umes a conformation having a binding site ⁇ pecific for, as evidenced by its ability to be competitively inhibited, a preselected or predetermined ligand such a ⁇ an antigen, enzymatic ⁇ ub ⁇ trate and the like.
  • a receptor of thi ⁇ invention i ⁇ a ligand binding heterodimeric polypeptide that form ⁇ an antigen binding ⁇ ite which specifically binds to a preselected antigen to form a complex having a sufficiently strong binding between the antigen and the binding site for the complex to be isolated.
  • affinity or avidity i ⁇ generally greater than 10 5 M "1 more u ⁇ ually greater than 10 6 M *1 and preferably greater than 10 8 M "1 .
  • a receptor of the subject invention binds a substrate and catalyzes the formation of a product from the substrate. While the topology of the ligand binding site of a catalytic receptor is probably more important for its preselected activity than its affinity (as ⁇ ociation con ⁇ tant or pKa) for the sub ⁇ trate, the subject catalytic receptors have an as ⁇ ociation constant for the preselected substrate generally greater than 10 3 M "1 , more usually greater than 10 5 M *1 or 10 6 M "1 and preferably greater than 10 7 M "1 .
  • the receptor produced by the subject invention i ⁇ heterodimeric and is therefore normally comprised of two different polypeptide chains, which together as ⁇ ume a conformation having a binding affinity, or a ⁇ sociation constant for the pre ⁇ elected ligand that i ⁇ different, preferably higher, than the affinity or association constant of either of the polypeptides alone, i.e., a ⁇ monomer ⁇ .
  • One or both of the different polypeptide chain ⁇ is derived from the variable region of the light and heavy chains of an immunoglobulin.
  • polypeptides comprising the light (V L ) and heavy (V H ) variable region ⁇ are employed together for binding the preselected ligand.
  • a receptor produced by the subject invention can be comprised of active monomer ⁇ V H and V L ligand binding polypeptide ⁇ produced by the present invention can be advantageously combined in the heterodimer to modulate the activity of either or to produce an activity unique to the heterodimer.
  • V H and V L The individual ligand polypeptides will be referred to as V H and V L and the heterodimer will be referred to as a F v .
  • a V H may contain in addition to the V H; ⁇ ubstantially all or a portion of the heavy chain constant region.
  • a V L may contain, in addition to the V L , ⁇ ubstantially all or a portion of the light chain constant region.
  • Fab can be advantageou ⁇ in some ⁇ ituation ⁇ becau ⁇ e the additional con ⁇ tant region ⁇ equence ⁇ contained in a Fab a ⁇ compared to a F v can ⁇ tabilize the V H and V L interaction. Such stabilization can cause the Fab to have higher affinity for antigen.
  • Fab i ⁇ more commonly used in the art and thus there are more commercial antibodie ⁇ available to ⁇ pecifically recognize a Fab in ⁇ creening procedure ⁇ .
  • the individual V H and V L polypeptide ⁇ can be produced in length ⁇ equal to or substantially equal to their naturally occurring length ⁇ . See Figure 2.
  • the v H and V L polypeptides will generally have fewer than 125 amino acid residue ⁇ , more u ⁇ ually fewer than about 120 amino acid residues, while normally having greater than 60 amino acid residue ⁇ , u ⁇ ually greater than about 95 amino acid re ⁇ idue ⁇ , more u ⁇ ually greater than about 100 amino acid residues.
  • the V H will be from about 110 to.about 125 amino acid re ⁇ idue ⁇ in length while V L will be from about 95 to about 115 amino acid re ⁇ idue ⁇ in length.
  • the amino acid re ⁇ idue ⁇ equence ⁇ will vary widely, depending upon the particular idiotype involved. U ⁇ ually, there will be at lea ⁇ t two cy ⁇ teines separated by from about 60 to 75 amino acid residues and joined by a disulfide bond.
  • the polypeptides produced by the subject invention will normally be substantial copies of idiotypes of the variable regions of the heavy and/or light chains of immunoglobulins, but in some situations a polypeptide may contain random mutations in amino acid residue sequences in order to advantageously improve the desired activity.
  • V H and V L polypeptides are desirable to provide for covalent cro ⁇ s linking of the V H and V L polypeptides, which can be accomplished by providing cy ⁇ teine resides at the carboxyl termini.
  • the polypeptide will normally be prepared free of the immunoglobulin constant regions, however a small portion of the J region may be included a ⁇ a result of the advantageou ⁇ selection of DNA synthe ⁇ i ⁇ primer ⁇ .
  • the D region will normally be included in the tran ⁇ cript of the V H .
  • the C terminu ⁇ region of the V H and V L polypeptide ⁇ will have a greater variety of ⁇ equence ⁇ than the N terminu ⁇ and, ba ⁇ ed on the pre ⁇ ent ⁇ trategy, can be further modified to permit a variation of the normally occurring V H and V L chains.
  • a synthetic polynucleotide can be employed to vary one or more amino acid in a hypervariable region.
  • a gene repertoire useful in practicing the present invention contains at least 10 3 , preferably at least 10 4 , more preferably at least 10 5 , and most preferably at least 10 7 different conserved genes.
  • V H and V L gene repertoires can be produced by isolating V H - and V L -coding mRNA from a heterogeneou ⁇ population of antibody producing cell ⁇ , i.e., B lymphocyte ⁇ (B cell ⁇ ) , preferably rearranged B cells such as those found in the circulation or spleen of a vertebrate.
  • B cell ⁇ B lymphocyte ⁇
  • Rearranged B cell ⁇ are those in which immunoglobulin gene tran ⁇ location, i.e., rearrangement, has occurred as evidenced by the presence in the cell of mRNA with the immunoglobulin gene V, D and J region transcript ⁇ adjacently located thereon.
  • the B cell ⁇ are collected in a 1-100 ml sample of blood which usually contains 10 6 B cells/ml.
  • bia ⁇ a repertoire for a pre ⁇ elected activity, such a ⁇ by using as a source of nucleic acid cells (source cells) from vertebrates in any one of variou ⁇ ⁇ tage ⁇ of age, health and immune re ⁇ pon ⁇ e.
  • source cells nucleic acid cells
  • repeated immunization of a healthy animal prior to collecting rearranged B cells results in obtaining a repertoire enriched for genetic material producing a receptor of high affinity.
  • collecting rearranged B cell ⁇ from a healthy animal whose immune system has not been recently challenged results in producing a repertoire that is not biased towards the production of high affinity V H and/or V L polypeptides.
  • the source cell ⁇ are obtained from a vertebrate, preferably a mammal, which has been immunized or partially immunized with an antigenic ligand (antigen) against which activity is sought, i.e., a preselected antigen.
  • the immunization can be carried out conventionally.
  • Antibody titer in the animal can be monitored to determine the stage of immunization desired, which stage correspond ⁇ to the amount of enrichment or biasing of the repertoire desired.
  • Partially immunized animals typically receive only one immunization and cells are collected from tho ⁇ e animal ⁇ ⁇ hortly after a re ⁇ ponse is detected.
  • Fully immunized animals display a peak titer, which i ⁇ achieved with one or more repeated injections of the antigen into the host mammal, normally at 2 to 3 week intervals. Usually three to five days after the last challenge, the spleen is removed and the genetic repertoire of the spleenocytes, about 90% of which are rearranged B cells, is isolated using standard procedures. See, Current Protocols in Molecular
  • V H and V L polypeptides can be derived from cells producing IgA, IgD, IgE, IgG or IgM, most preferably from IgM and IgG, producing cells.
  • the desired gene repertoire can be isolated from either genomic material containing the gene expres ⁇ ing the variable region or the me ⁇ senger RNA (mRNA) which represents a transcript of the variable region.
  • mRNA me ⁇ senger RNA
  • the difficulty in using the genomic DNA from other than non-rearranged B lymphocytes is in juxtaposing the sequences coding for the variable region, where the sequences are separated by introns.
  • the DNA fragment( ⁇ ) containing the proper exon ⁇ mu ⁇ t be i ⁇ olated, the introns excised, and the exons then spliced in the proper order and in the proper orientation.
  • the cells will be lysed under RNa ⁇ e inhibiting conditions.
  • the first step is to isolate the total cellular mRNA.
  • Poly A+ mRNA can then be selected by hybridization to an oligo-dT cellulose column.
  • the pre ⁇ ence of mRNA ⁇ coding for the heavy and/or light chain polypeptide ⁇ can then be assayed by hybridization with DNA single strand ⁇ of the appropriate genes.
  • the sequences coding for the constant portion of the V H and V L can be used as polynucleotide probes, which sequences can be obtained from available sources. See for example. Early and Hood, Genetic Engineering.
  • the preparation containing the total cellular mRNA i ⁇ first enriched for the presence of V H and/or V L coding mRNA. Enrichment is typically accomplished by subjecting the total mRNA preparation or partially purified mRNA product thereof to a primer extension reaction employing a polynucleotide synthesis primer of the present invention. Exemplary methods for producing V H and V L gene repertoires are de ⁇ cribed in PCT Application No.
  • isolated B cell ⁇ are immunized in vitro against a preselected antigen.
  • In vitro immunization i ⁇ defined a ⁇ the clonal expansion of epitope- ⁇ pecific B cells in culture, in respon ⁇ e to antigen stimulation.
  • the end re ⁇ ult i ⁇ to increa ⁇ e the frequency of antigen-specific B cell ⁇ in the immunoglobulin repertoire, and thereby decrease the number of clones in an expre ⁇ sion library that must be screened to identify a clone expres ⁇ ing an antibody of the de ⁇ ired specificity.
  • the advantage of in vitro immunization is that human monoclonal antibodies can be generated against a limitless number of therapeutically valuable antigen ⁇ , including toxic or weak immunogen ⁇ .
  • antibodie ⁇ ⁇ pecific for the polymorphic determinants of tumor-associated antigens, rheumatoid factors, and histocompatibility antigens can be produced, which can not be elicited in immunized animals.
  • it may be pos ⁇ ible to generate immune response ⁇ which are normally suppressed in vivo.
  • In vitro immunization can be used to give rise to either a primary or secondary immune respon ⁇ e.
  • a primary immune response resulting from first time exposure of a B cell to an antigen, results in clonal expansion of epitope-specific cells and the secretion of IgM antibodies with low to moderate apparent affinity constant ⁇ (lO ⁇ lO ⁇ '1 ) .
  • Primary immunization of human splenic and tonsillar lymphocytes in culture can be used to produce monoclonal antibodies against a variety of antigens, including cells, peptides, macromolecules, haptens, and tumor-associated antigens.
  • Memory B cell ⁇ from immunized donors can also be stimulated in culture to give rise to a secondary immune response characterized by clonal expansion and the production of high affinity antibodie ⁇ (>10 9 M "1 ) of the IgG i ⁇ otype, particularly again ⁇ t viral antigen ⁇ by clonally expanding sensitized lymphocytes derived from seropositive individuals.
  • peripheral blood lymphocytes are depleted of various cytolytic cells that appear to down-modulate antigen-specific B cell activation.
  • lyso ⁇ ome-rich ⁇ ubpopulation ⁇ natural killer cell ⁇ , cytotoxic and suppressor T cells, monocytes
  • the remaining cells including B cells, T helper cells, acces ⁇ ory cell ⁇
  • the lymphokine requirements for inducing antibody production in culture are satisfied by a culture supernatant from activated, irradiated T cells.
  • cell panning in addition to in vitro immunization, cell panning (immunoaffinity ab ⁇ orption) can be u ⁇ ed to further increa ⁇ e the frequency of antigen- ⁇ pecific B cell ⁇ .
  • Technique ⁇ for ⁇ electing B cell ⁇ ubpopulation ⁇ via ⁇ olid-pha ⁇ e antigen binding are well e ⁇ tablished.
  • Panning conditions can be optimized to selectively enrich for B cells which bind with high affinity to a , variety of antigens, including cell surface proteins. Panning can be used alone, or in combination with in vitro immunization to increase the frequency of antigen-specific cell ⁇ above the levels which can be obtained with either technique alone.
  • Immunoglobulin expression libraries constructed from enriched populations of B cells are biased in favor of antigen- specific antibody clone ⁇ , and thu ⁇ , enabling identification of clones with the desired specificities from smaller, less complex libraries.
  • Primers The term "polynucleotide" as used herein in reference to primers, probes and nucleic acid fragments or segments to be ⁇ ynthe ⁇ ized by primer extension is defined as a molecule comprised of two or more deoxyribonucleotides or ribonucleotide ⁇ , preferably more than 3. Its exact size will depend on many factors, which in turn depends on the ultimate conditions of use.
  • primer refers to a polynucleotide whether purified from a nucleic acid re ⁇ triction digest or produced synthetically, which is capable of acting as a point of initiation of nucleic acid synthe ⁇ i ⁇ when placed under conditions in which synthe ⁇ i ⁇ of a primer exten ⁇ ion product which is complementary to a nucleic acid strand is induced, i.e., in the presence of nucleotides and an agent for polymerization such as DNA polymerase, reverse transcripta ⁇ e and the like, and at a suitable temperature and pH.
  • the primer is preferably single stranded for maximum efficiency, but may alternatively be in double stranded form.
  • the primer is a polydeoxyribonucleotide.
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the agent ⁇ for polymerization.
  • the exact length ⁇ of the primer ⁇ will depend on may factor ⁇ , including temperature and the source of primer.
  • a polynucleotide primer typically contains 15 to 25 or more nucleotides, although it can contain fewer nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with template.
  • the primers used herein are selected to be "sub ⁇ tantially" complementary to the different strands of each specific sequence to be synthesized or amplified. This means that the primer mu ⁇ t be sufficiently complementary to non-randomly hybridize with its respective template strand. Therefore, the primer sequence may or may not reflect the exact sequence of the template.
  • a non- complementary nucleotide fragment can be attached to the 5' end of the primer, with the remainder of the primer sequence being ⁇ ub ⁇ tantially complementary to the ⁇ trand.
  • Such non-complementary fragment ⁇ typically code for an endonuclea ⁇ e re ⁇ triction site.
  • non-complementary base ⁇ or longer sequences can be intersper ⁇ ed into the primer, provided the primer ⁇ equence has ⁇ ufficient complementarily with the sequence of the ⁇ trand to be synthesized or amplified to non-randomly hybridize therewith and thereby form an extension product under polynucleotide synthe ⁇ izing condition ⁇ .
  • Primer ⁇ of the pre ⁇ ent invention may al ⁇ o contain a DNA-dependent RNA polymera ⁇ e promoter ⁇ equence or it ⁇ complement. See for example, Krieg et al., Nucleic Acid ⁇ Re ⁇ earch. 12:7057-70 (1984); Studier et al., J. Mol. Biol.. 189:113-130 (1986); and Molecular Cloning: A Laboratory Manual. Second Edition. Maniatis et al., eds. , Cold Spring Harbor, NY (1989).
  • the primer When a primer containing a DNA-dependent RNA polymerase promoter is used the primer is hybridized to the polynucleotide strand to be amplified and the ⁇ econd polynucleotide strand of the DNA-dependent RNA polymerase promoter is completed u ⁇ ing an inducing agent such as E. coli DNA polymerase I, or the Klenow fragment of E. coli DNA polymerase.
  • the starting polynucleotide is amplified by alternating between the production of an RNA polynucleotide and DNA polynucleotide.
  • Primers may also contain a template sequence or replication initiation site for a RNA-directed RNA polymerase.
  • Typical RNA-directed RNA polymerase include the QB replicase described by Lizardi et al., Biotechnology. 6:1197-1202 (1988).
  • RNA-directed polymerase ⁇ produce large number ⁇ of RNA ⁇ trand ⁇ from a small number of template RNA strand ⁇ that contain a template sequence or replication initiation site. These poly era ⁇ e ⁇ typically give a one million-fold amplification of the template ⁇ trand a ⁇ ha ⁇ been described by Kramer et al., J. Mol. Biol.. 89:719-736 (1974) .
  • the polynucleotide primer ⁇ can be prepared using any suitable method, such a ⁇ , for example, the phosphotriester or phosphodiester methods see Narang et al., Meth. Enzvmol.. 68:90, (1979); U.S. Patent No. 4,356,270; and Brown et al., Meth. Enzvmol.. 68:109, (1979) .
  • primers for Producing Gene depends on factors such a ⁇ the distance on the nucleic acid from the region coding for the desired receptor, its hybridization site on the nucleic acid relative to any second primer to be used, the number of genes in the repertoire it is to hybridize to, and the like.
  • Repertoires V H and V L gene repertoires can be separately prepared prior to their utilization in the present invention.
  • Repertoire preparation i ⁇ typically accomplished by primer extension, preferably by primer extension in a PCR format.
  • the nucleotide sequence of a primer is selected to hybridize with a plurality of immunoglobulin heavy chain genes at a site substantially adjacent to the V H -coding region so that a nucleotide sequence coding for a functional (capable of binding) polypeptide is obtained.
  • the primer To hybridize to a plurality of different V H -coding nucleic acid strands, the primer must be a substantial complement of a nucleotide sequence conserved among the different strands.
  • sites include nucleotide sequences in the constant region, any of the variable region framework region ⁇ , preferably the third framework region, leader region, promoter region, J region and the like.
  • V H -coding and V L -coding DNA homologs are to be produced by polymerase chain reaction (PCR) amplification
  • two primers i.e., a PCR primer pair
  • the first primer becomes part of the nonsen ⁇ e (minu ⁇ or complementary) ⁇ trand and hybridize ⁇ to a nucleotide sequence conserved among V H (plus or coding) strands within the repertoire.
  • first primers are therefore chosen to hybridize to (i.e. be complementary to) conserved regions within the J region, CHI region, hinge region, CH2 region, or CH3 region of immunoglobulin genes and the like.
  • first primers are chosen to hybridize with (i.e. be complementary to) a conserved region within the J region or constant region of immunoglobulin light chain genes and the like.
  • Second primers become part of the coding (plu ⁇ ) strand and hybridize to a nucleotide sequence conserved among minus ⁇ trand ⁇ .
  • ⁇ econd primer ⁇ are therefore cho ⁇ en to hybridize with a con ⁇ erved nucleotide ⁇ equence at the 5' end of the V H -coding immunoglobulin gene ⁇ uch a ⁇ in that area coding for the leader or first framework region.
  • the con ⁇ erved 5' nucleotide ⁇ equence of the ⁇ econd primer can be complementary to a sequence exogenously added u ⁇ ing terminal deoxynucleotidyl tran ⁇ fera ⁇ e a ⁇ described by Loh et al., Sci. Vol 243:217-220 (1989).
  • One or both of the fir ⁇ t and ⁇ econd primer ⁇ can contain a nucleotide sequence defining an endonuclease recognition ⁇ ite.
  • the ⁇ ite can be heterologou ⁇ to the immunoglobulin gene being amplified and typically appear ⁇ at or near the 5' end of the primer.
  • the pre ⁇ ent invention utilizes a set of polynucleotides that form in ⁇ ide primer ⁇ compri ⁇ ed of an up ⁇ tream inside primer and a down ⁇ tream in ⁇ ide primer.
  • Each of the in ⁇ ide primer ⁇ ha ⁇ a priming region located at the 3'-terminu ⁇ of the primer.
  • the priming region i ⁇ typically the 3'-mo ⁇ t (3 '-terminal) 15 to 30 nucleotide ba ⁇ e ⁇ .
  • each in ⁇ ide primer i ⁇ capable of acting as a primer to catalyze nucleic acid ⁇ ynthe ⁇ i ⁇ , i.e., initiate a primer exten ⁇ ion reaction off it ⁇ 3' terminu ⁇ .
  • One or both of the in ⁇ ide primer ⁇ i ⁇ further characterized by the pre ⁇ ence of a 5'-terminal (5'-mo ⁇ t) non-priming portion, i.e., a region that doe ⁇ not participate in hybridization to repertoire template.
  • each inside primer works in combination with an outside primer to amplify a target nucleic acid sequence.
  • the choice of PCR primer pairs for use in fu ⁇ ion PCR a ⁇ de ⁇ cribed herein is governed by the same consideration ⁇ a ⁇ previously discu ⁇ ed for choo ⁇ ing PCR primer pair ⁇ u ⁇ eful in producing gene repertoire ⁇ . That i ⁇ , the primer ⁇ have a nucleotide ⁇ equence that i ⁇ complementary to a ⁇ equence conserved in the repertoire.
  • Useful V L and V H inside priming ⁇ equence ⁇ are ⁇ hown in Table ⁇ 1 and 2, re ⁇ pectively, below.
  • Nucleotide sequences 1-10 are unique 5' primers for the amplification of kappa light chain variable region ⁇ .
  • Table 2 3' Priming Portion ⁇ of Variou ⁇ In ⁇ ide V H Primer ⁇
  • V H Unique 3' primer for amplification of V H including part of mouse gamma 1 first con ⁇ tant region and hinge region.
  • 3' primer for amplifying mou ⁇ e Fd including part of the mou ⁇ e IgG fir ⁇ t con ⁇ tant region and part of the hinge region.
  • 11 3' primer for amplifying human IgGl Fd including part of the human IgG first constant region and part of the hinge region including the two cy ⁇ teines which create the disulfide bridge for producing Fab'2 (the primer corresponds to Kabat numbers 2 1QQ to 247) .
  • a preferred set of in ⁇ ide primers used herein ha ⁇ primer ⁇ with complementary 5'-terminal non-priming region ⁇ , the complementary ⁇ trand ⁇ of which are capable of hybridizing to each other to form a duplex with 3' overhang ⁇ .
  • the duplex encode ⁇ all or part of a double stranded cistronic bridge.
  • the two in ⁇ ide primer ⁇ in combination encode both the plu ⁇ and minu ⁇ ⁇ trand ⁇ of all or part of the bridge.
  • one in ⁇ ide up ⁇ tream primer can have a sequence that forms a portion of the plu ⁇ strand of the bridge, and the other in ⁇ ide primer encode ⁇ the ⁇ equence, through complementarity, of the downstream portion of the plus strand.
  • the plus ⁇ trand of the ci ⁇ tronic bridge contain ⁇ , in the tran ⁇ lational reading frame and from an upstream position to a downstream position, sequence ⁇ coding for (i) at lea ⁇ t one ⁇ top codon, preferably two, in the same reading frame a ⁇ the up ⁇ tream ci ⁇ tron, (ii) a ribo ⁇ ome binding ⁇ ite, and (iii) a polypeptide leader, the tran ⁇ lation initiation codon of which i ⁇ in the same reading frame as the down ⁇ tream ci ⁇ tron.
  • the ribo ⁇ ome binding site includes an initiation codon (AUG) and a sequence 3-9 nucleotides long located 3 11 nucleotides upstream from the initiation codon [Shine et al.,
  • AGGAGGU which is called the Shine-Dalgarno (SD) sequence, is complementary to the 3' end of E. coli 16S mRNA.
  • Binding of the ribosome to mRNA and the sequence at the 3' end of the mRNA can be affected by several factors:
  • the complementary (overlapping) region of the inside primer ⁇ and the priming portion of the in ⁇ ide primer ⁇ have about the ⁇ ame denaturation temperature, Td.
  • a Td for the above-identified hybridizing region of about 45-55 ⁇ C, preferably about 50*C, i ⁇ preferred.
  • overlapping region ⁇ in the range of about 15 to 20 nucleotides works well in conjunction with priming regions in the range of 15-30 nucleotides.
  • the set of outside primers forms the termini of the dici ⁇ tronic DNA molecule.
  • the set of out ⁇ ide primer ⁇ compri ⁇ e ⁇ an up ⁇ tream out ⁇ ide primer and a own ⁇ tream out ⁇ ide primer.
  • the out ⁇ ide primer ⁇ each comprise a 3'-terminal priming portion, and preferably a portion that defines an endonuclease restriction site. When present, the re ⁇ triction site-defining portion is typically located in a 5'-terminal non- priming portion of the outside primer.
  • the restriction site defined by the up ⁇ tream out ⁇ ide primer i ⁇ typically cho ⁇ en to be one recognized by a restriction enzyme that doe ⁇ not recognize the re ⁇ triction ⁇ ite defined by the down ⁇ tream out ⁇ ide primer, the objective being to be able to produce a dici ⁇ tronic DNA having cohesive termini that are non- complementary to each other and thus allow directional insertion into a vector.
  • Nucleotide ⁇ equence ⁇ 21-28 are unique 5' primer ⁇ for the amplification of mouse V H gene ⁇ .
  • Nucleotide sequences 29-32 are unique 5' primers for amplification of nucleic acids coding for human variable regions.
  • V H and V L genes contained within a repertoire will depend, as is well known in the art, on the type, complexity, and purity of the nucleic acids making up the repertoire. Other factors include whether or not the genes are contained in one or a plurality of repertoires and whether or not they are to be amplified and/or mutagenized.
  • a library of dici ⁇ tronic DNA molecules containing upstream and down ⁇ tream ci ⁇ trons operatively linked by a cistronic bridge can be produced by the following steps: (a) Subjecting a repertoire of first polypeptide gene ⁇ (e.g., V H -coding gene ⁇ ), to PCR amplification u ⁇ ing fir ⁇ t outside and first inside primer ⁇ , i.e., a fir ⁇ t PCR primer pair, to form a first primary PCR product.
  • first polypeptide gene ⁇ e.g., V H -coding gene ⁇
  • PCR products to form internally (self) primed duplexes, i.e., duplexes having 3 '-hybridized and 5'- overhanging termini.
  • V H - and V L -coding gene repertoire ⁇ are compri ⁇ ed of polynucleotide coding ⁇ trand ⁇ , ⁇ uch a ⁇ mRNA and/or the sense strand of genomic DNA. If the repertoire i ⁇ in the form of double ⁇ tranded genomic DNA, it i ⁇ u ⁇ ually fir ⁇ t denatured, typically by melting, into ⁇ ingle ⁇ trand ⁇ .
  • a repertoire is ⁇ ubjected to a PCR reaction by treating (contacting) the repertoire with a PCR primer pair, each member of the pair having a pre ⁇ elected nucleotide ⁇ equence.
  • the PCR primer pair i ⁇ capable of initiating primer exten ⁇ ion reaction ⁇ by hybridizing to nucleotide ⁇ equences, preferably at least about 10 nucleotides in length and more preferably at lea ⁇ t about 20 nucleotide ⁇ in length, con ⁇ erved within the repertoire.
  • the fir ⁇ t primer of a PCR primer pair i ⁇ sometimes referred to herein as the "sense primer" because it hybridizes to the coding or sense stran of a nucleic acid.
  • the second primer of a PCR primer pair is sometime ⁇ referred to herein a ⁇ the "anti-sense primer" because it hybridizes to a non- coding or anti-sen ⁇ e ⁇ trand of a nucleic acid, i.e., a ⁇ trand complementary to a coding ⁇ trand.
  • the PCR reaction i ⁇ performed by mixing the PCR primer pair, preferably a predetermined amount thereof, with the nucleic acid ⁇ of the repertoire, preferably a predetermined amount thereof, in a PCR buffer to form a PCR reaction admixture.
  • the admixture i ⁇ maintained under polynucleotide synthe ⁇ izing condition ⁇ for a time period, which i ⁇ typically predetermined, sufficient for the formation of a PCR reaction product, thereby producing a plurality of different V ⁇ -coding and/or V L -coding DNA homologs.
  • a plurality of first primer and/or a plurality of second primers can be u ⁇ ed in each amplification, e.g., one ⁇ pecie ⁇ of fir ⁇ t primer can be paired with a number of different ⁇ econd primer ⁇ to form ⁇ everal different primer pair ⁇ .
  • an individual pair of fir ⁇ t and ⁇ econd primer ⁇ can be u ⁇ ed.
  • the amplification product ⁇ of amplification ⁇ u ⁇ ing the same or different combinations of first and second primers can be combined ' to increa ⁇ e the diver ⁇ ity of the gene library.
  • Methods for producing ⁇ uch complement ⁇ are well known in the art.
  • the PCR reaction is performed using any suitable method. Generally it occurs in a buffered aqueous solution, i.e., a PCR buffer, preferably at a pH of 7-9, most preferably about 8.
  • a molar excess (for genomic nucleic acid, usually about 10 6 :1 primer:tempiate) of the primer is admixed to the buffer containing the template ⁇ trand.
  • a large molar excess is preferred to improve the efficiency of the process.
  • the ratio of gene molecule ⁇ and their re ⁇ pective primer ⁇ i ⁇ a ⁇ follow ⁇ about l x l ⁇ 3 V H gene molecule ⁇ to about 1 x 10 8 out ⁇ ide V H primer molecule ⁇ , about 1 x 10 V H gene molecules, to about 1 x 10 7 in ⁇ ide V H gene primer molecule ⁇ , about 1 x 10 3 V L gene molecule ⁇ to about 1 x 10 8 out ⁇ ide V L gene primer molecules, about 1 x 10 4 V L gene molecule ⁇ to about 1 x 10 7 V L gene primer molecule ⁇ .
  • 10 A out ⁇ ide V H gene primer molecule ⁇ and 10 3 in ⁇ ide V H gene primer molecule ⁇ are u ⁇ ed for every V H gene molecule pre ⁇ ent in the PCR admixture.
  • 10 out ⁇ ide V L gene primer molecule ⁇ and 10 3 V L inside gene primer molecules are used for every V L gene molecule present in the PCR admixture.
  • there i ⁇ typically a 10 fold molar excess of outside primer to inside primer.
  • the gene repertoires are admixed with outside and inside primers, the out ⁇ ide primer ⁇ being pre ⁇ ent in exce ⁇ relative to the in ⁇ ide primer ⁇ .
  • the initial PCR thermocycle ⁇ produce intermediate products having complementary termini from each of the first and second gene repertoires. That is, the end of one strand from one primary PCR product is capable of hybridizing with the complementary end from the other primary PCR product.
  • the strands having the overlap at their 3' ends can act as primer ⁇ for one another, i.e., from an internally primed duplex, and be extended by the polymera ⁇ e to form the full length final product.
  • the final product i ⁇ then amplified by the set of out ⁇ ide primer ⁇ , which act a ⁇ a third PCR pair when the in ⁇ ide primer ⁇ have been exhau ⁇ ted, to form a secondary PCR product.
  • the molar ratio of outside primers to inside primers i ⁇ uch that the inside primers are effectively exhausted within about 2 to about 12, preferably about 5, 6 or 7 thermocycles.
  • the PCR buffer al ⁇ o contain ⁇ the deoxyribonucleotide tripho ⁇ phate ⁇ dATP, dCTP, dGTP, and dTTP and a polymera ⁇ e, typically thermo ⁇ table, all in adequate amount ⁇ for primer exten ⁇ ion
  • the resulting solution (PCR admixture) is heated to about 90*C - 100*C for about 1 to 10 minutes, preferably from 1 to 4 minutes. After thi ⁇ heating period the ⁇ olution i ⁇ allowed to cool to 54*C, which is preferable for primer hybridization.
  • the synthesi ⁇ reaction may occur at from room temperature up to a temperature above which the polymera ⁇ e (inducing agent) no longer function ⁇ efficiently.
  • An exemplary PCR buffer compri ⁇ e ⁇ the following: 50 mM KCI; 10 mM Tri ⁇ -HCl; pH 8.3; 1.5 mM MgCl 2 ; 0.001% (wt/vol) gelatin, 200 ⁇ M dATP; 200 ⁇ M dTTP; 200 ⁇ M dCTP; 200 ⁇ M dGTP; and 2.5 unit ⁇ Thermus aquaticu ⁇ DNA polymera ⁇ e I (U.S. Patent No. 4,889,818) per 100 microliter ⁇ of buffer.
  • the inducing agent may be any compound or ⁇ y ⁇ tem which will function to accomplish the synthesis of primer exten ⁇ ion product ⁇ , including enzymes.
  • Suitable enzymes for thi ⁇ purpo ⁇ e include, for example, E. coli DNA polymera ⁇ e I, Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, other available DNA polymera ⁇ e ⁇ , rever ⁇ e transcriptase, and other -enzymes, including heat- ⁇ table enzymes, which will facilitate combination of the nucleotides in the proper manner to form the primer extension product ⁇ which are complementary to each nucleic acid ⁇ trand.
  • the synthesis will be initiated at the 3' end of each primer and proceed in the 5' direction along the template strand, until synthesi ⁇ terminate ⁇ , producing molecules of different length ⁇ .
  • the inducing agent also may be a compound or sy ⁇ tem which will function to accomplish the synthesi ⁇ of RNA primer extension product ⁇ , including enzyme ⁇ .
  • the inducing agent may be a DNA-dependent RNA polymera ⁇ e such as T7 RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase. These polymera ⁇ e ⁇ produce a complementary RNA polynucleotide. The high turn over rate of the RNA polymerase amplifies the starting polynucleotide as ha ⁇ been de ⁇ cribed by Chamberlin et al., The Enzymes. ed. P. Boyer, PP. 87-108 ' , Academic Pres ⁇ , New York (1982) .
  • T7 RNA polymera ⁇ e i ⁇ that mutations can be introduced into the polynucleotide synthesi ⁇ by replacing a portion of cDNA with one or more mutagenic oligodeoxynucleotides (polynucleotides) and tran ⁇ cribing the partially- mi ⁇ matched template directly a ⁇ ha ⁇ been previou ⁇ ly described by Joyce et al., Nucleic Acid Research. 17:711-722 (1989). Amplification sy ⁇ tem ⁇ ba ⁇ ed on transcription have been described by Gingeras et al., in PCR Protocols. A Guide to Methods and Applications, pp 245-252, Academic Press, Inc., San Diego, CA (1990) .
  • the inducing agent i ⁇ a DNA-dependent RNA polymerase and therefore incorporates ribonucleotide tripho ⁇ phate ⁇
  • ⁇ ufficient amount ⁇ of ATP, CTP, GTP and UTP are admixed to the primer exten ⁇ ion reaction admixture and the re ⁇ ulting ⁇ olution i ⁇ treated a ⁇ de ⁇ cribed above.
  • the newly ⁇ ynthe ⁇ ized ⁇ trand and it ⁇ complementary nucleic acid ⁇ trand form a double- stranded molecule which can be used in the succeeding step ⁇ of the proce ⁇ .
  • the fir ⁇ t and/or second PCR reactions discussed above can advantageously be used to incorporate into the receptor a preselected epitope useful in immunologically detecting and/or isolating a receptor.
  • Thi ⁇ i ⁇ accompli ⁇ hed by utilizing a fir ⁇ t and/or second polynucleotide synthesis primer or expression vector to incorporate a predetermined amino acid residue ⁇ equence into the amino acid residue sequence of the receptor.
  • the dici ⁇ tronic DNA molecule ⁇ are typically further amplified. While the dici ⁇ tronic DNA molecule ⁇ can be amplified by classic techniques ⁇ uch a ⁇ incorporation into an autonomou ⁇ ly replicating vector, it is preferred to first amplify the molecules by subjecting them to a polymera ⁇ e chain reaction (PCR) prior to in ⁇ erting them into a vector.
  • PCR polymera ⁇ e chain reaction
  • the fir ⁇ t and second PCR reactions are performed in the same admixture that i ⁇ ⁇ ubject to a multiplicity of PCR thermocycle ⁇ where the out ⁇ ide primer ⁇ are in molar exce ⁇ .
  • PCR i ⁇ typically carried out by thermocycling i.e., repeatedly increasing and decreasing the temperature of a PCR reaction admixture within a temperature range whose lower limit is about 10*C to about 40 e C and whose upper limit is about 90"C to about 100*C.
  • the increasing and decreasing can be continuous, but i ⁇ preferably pha ⁇ ic with time periods of relative temperature ⁇ tability at each of temperature ⁇ favoring polynucleotide ⁇ ynthe ⁇ i ⁇ , denaturation and hybridization.
  • amplification reaction product ⁇ obtained from a plurality of different amplification ⁇ , each u ⁇ ing a plurality of different primer pair ⁇ , are then combined.
  • the pre ⁇ ent invention al ⁇ o contemplate ⁇ DNA homolog production via co- amplification (using two pairs of primer ⁇ ) , and - 54 - multiplex amplification (u ⁇ ing up to about 8, 9 or 10 primer pair ⁇ ) .
  • a diver ⁇ e library of dici ⁇ tronic DNA roolecule ⁇ having up ⁇ tream and down ⁇ tream ci ⁇ tron ⁇ can al ⁇ o be produced by combining, in a PCR buffer, double ⁇ tranded V H and V L repertoire ⁇ , V H and V L out ⁇ ide primer ⁇ , and an in ⁇ ide primer having a 3'-terminal priming portion, a ci ⁇ tronic bridge coding portion, and a 5'-terminal in ⁇ ide primer-template (primer- coding) portion.
  • the 3'-terminal priming portion ha ⁇ a nucleotide ba ⁇ e ⁇ equence complementary to a portion of the primer exten ⁇ ion product of one of the out ⁇ ide primer ⁇ .
  • the 5'-terminal primer-template portion ha ⁇ a nucleotide ba ⁇ e ⁇ equence homologou ⁇ (identical) to a portion of the primer exten ⁇ ion product of the other of the out ⁇ ide primer ⁇ . That i ⁇ , the linking primer ha ⁇ terminal ⁇ equence ⁇ homologou ⁇ to ⁇ equence ⁇ in both repertoire ⁇ .
  • the cistronic bridge coding portion codes for, either directly or through complementarily, at lea ⁇ t one ⁇ top codon in the same reading frame a ⁇ the up ⁇ tream cistron, a ribosome binding site located downstream from the ⁇ top codon, and a polypeptide leader haying a tran ⁇ lation initiation codon in the ⁇ ame reading frame a ⁇ the down ⁇ tream cistron, the initiation codon being located downstream from the ribosome binding ⁇ ite.
  • the dici ⁇ tronic DNA molecules containing operatively linked V H - and V L -coding DNA homolog ⁇ produced by PCR amplification are typically in double- stranded form and may have contiguou ⁇ or adjacent to each of their termini a nucleotide sequence defining an endonuclease restriction site. Digestion of the dicistronic DNA molecules having restriction ⁇ ite ⁇ at or near their termini with one or more appropriate endonuclea ⁇ e ⁇ results in the production of DNA molecule ⁇ having cohesive termini of predetermined specificity.
  • the present invention produces many non-naturally occurring antibodie ⁇ , i.e., combinations of V H and V L in a heterodimer.
  • the pre ⁇ ent invention also contemplates using fusion PCR to operatively link, and thereby recover, naturally occurring V H and V L combinations.
  • a fusion PCR method of the present invention is performed on repertoires comprising a plurality of substantially isolated cells containing genes coding for a heterodimeric receptor.
  • a plurality of PCR admixtures i ⁇ formed, each of which contain ⁇ (i) a ⁇ ample of substantially isolated B lymphocytes from a mammal producing antibody molecules against a preselected antigen, (ii) a PCR buffer, and (iv) either the previously described V H and V L PCR primer pair ⁇ or the set of outside V H and ⁇ PCR primer ⁇ in combination with the linking primer( ⁇ ) , al ⁇ o a ⁇ previously described.
  • the plurality of PCR admixtures i ⁇ then subjected to a multiplicity of PCR thermocycles a ⁇ de ⁇ cribed herein.
  • substantially isolated is meant a sample containing les ⁇ than about 100 target cell ⁇ , ⁇ uch a ⁇ B lymphocyte ⁇ , T cell ⁇ , and the like.
  • the plurality of PCR ad ixture ⁇ contain only about one cell.
  • the cell ⁇ are typically obtained from an individual mammal whose serum contains antibody molecules again ⁇ t the pre ⁇ elected antigen.
  • the collected cell ⁇ are typically seeded, usually at densities in the range of 0.5 to 100 cells per unit volume, into a plurality of individual PCR ves ⁇ el ⁇ , ⁇ uch a ⁇ microtiter plate well ⁇ and the like.
  • the plurality of PCR admixture ⁇ i ⁇ in the range of 800 to 1200, and preferably is about 1000, separate admixtures.
  • fewer cells are needed in each PCR admixture where the cell ⁇ are obtained from individual ⁇ expre ⁇ ing a high serum antibody titer against the preselected antigen.
  • B lymphocytes are obtained from an individual having a frequency of circulating B cells producing the antibody molecules of preselected ⁇ pecificity of 1/3000
  • each of about 800 to 1200 individual PCR admixture ⁇ need only contain about one B lymphocyte to re ⁇ ult in i ⁇ olation of the desired antibody.
  • the PCR proce ⁇ i ⁇ u ⁇ ed not only to produce a library of dici ⁇ tronic DNA molecules, but also to ' induce mutations within the library or to create diversity from a single parental clone and thereby provide a library having a greater heterogeneity.
  • the PCR proce ⁇ itself is inherently mutagenic due to a variety of factors well known in the art.
  • the PCR reaction admixture can be formed with different amounts of one or more of the nucleotides to be incorporated into the extension product. Under such conditions, the PCR reaction proceeds to produce nucleotide substitutions within the exten ⁇ ion product a ⁇ a result of the ⁇ carcity of a particular base.
  • nucleotides can be incorporated into the initial PCR reaction admixture in an amount to efficiently perform X number of cycles, and then cycling the admixture through a number of cycles in excess of X, such as, for instance, 2X.
  • mutations can be induced during the PCR reaction by incorporating into the reaction admixture nucleotide derivatives such as inosine, not normally found in the nucleic acid ⁇ of the repertoire being amplified. During subsequent in vivo amplification, the nucleotide derivative will be replaced with a sub ⁇ titute nucleotide thereby inducing a point mutation.
  • the dici ⁇ tronic DNA molecules produced by the above-described method can be operatively linked to a vector for amplification and/or expres ⁇ ion.
  • vector refers to a nucleic acid molecule capable of transporting between different genetic environments another nucleic acid to which it has been operatively linked.
  • One type of preferred vector i ⁇ an episome, i.e., a nucleic acid molecule capable of extra-chromo ⁇ omal replication.
  • Preferred vector ⁇ are tho ⁇ e capable of autonomou ⁇ replication and/or expre ⁇ ion of nucleic acid ⁇ to which they are linked.
  • Vector ⁇ capable of directing the expre ⁇ ion of gene ⁇ to which they are operatively linked are referred to herein a ⁇ "expre ⁇ ion vector ⁇ ".
  • V H - and V L - coding DNA homolog i ⁇ operatively linked depend ⁇ directly, a ⁇ i ⁇ well known in the art, on the functional propertie ⁇ desired, e.g., replication or protein expression, and the host cell to be transformed, these being limitation ⁇ inherent in the art of con ⁇ tructing recombinant DNA molecule ⁇ .
  • the vector utilized include ⁇ a prokaryotic replicon i.e., a DNA ⁇ equence having the ability to direct autonomou ⁇ replication and maintenance of the recombinant DNA molecule extra chromo ⁇ omally in a prokaryotic ho ⁇ t cell, ⁇ uch a ⁇ a bacterial ho ⁇ t cell, tran ⁇ formed therewith.
  • a prokaryotic replicon i.e., a DNA ⁇ equence having the ability to direct autonomou ⁇ replication and maintenance of the recombinant DNA molecule extra chromo ⁇ omally in a prokaryotic ho ⁇ t cell, ⁇ uch a ⁇ a bacterial ho ⁇ t cell, tran ⁇ formed therewith.
  • replicon ⁇ are well known in the art.
  • those embodiments that include a prokaryotic replicon also include a gene who ⁇ e expre ⁇ ion confer ⁇ a selective advantage, such as drug re ⁇ i ⁇ tance, to a bacterial ho ⁇
  • Those vectors that include a prokaryotic replicon can also include a prokaryotic promoter capable of directing the expres ⁇ ion (transcription and translation) of the V H - and V L -coding homologs in a bacterial ho ⁇ t cell, ⁇ uch a ⁇ E. coli tran ⁇ formed therewith.
  • a promoter i ⁇ an expre ⁇ ion control element formed by a DNA ⁇ eguence that permit ⁇ binding of RNA polymera ⁇ e and tran ⁇ cription to occur. Promoters contain two highly conserved regions, one located about 10 bp (-10 region on Priberow box) and the other about 35 bp (-35 region) upstream from the point at which transcription starts. These two region ⁇ typically determine promoter strength.
  • nucleotides that separate the conserved sequences is important for efficient promoter function. For example, 16 to 19 nucleotides typically separate the -10 and -35 region ⁇ , and changes in that spacing can change the efficiency of a promoter.
  • Promoter sequences compatible with bacterial ho ⁇ t ⁇ are typically provided in pla ⁇ mid vectors containing convenient restriction ⁇ ite ⁇ for in ⁇ ertion of a DNA ⁇ egment of the pre ⁇ ent invention. Typical of such vector plasmid ⁇ are pUC8, pUC9, pBR322, and pBR329 available from BioRad Laboratorie , (Richmond, CA) and pPL and pKK223 available from Pharmacia, (Piscataway, NJ) .
  • Promoters useful in this invention include Ptac ⁇ 1.1A, ⁇ 1.1B and ⁇ lO, which are recognized by T7 polymerase. See U.S. Patent No. 4,946,786.
  • Useful regulatable promoters include the E. coli lac promoter described in U.S. Patent No. 4,946,786 and the promoters for the temperature sensitive genes in U.S. Patent No. 4,806,471. See also U.S. Patent No. 4,711,845.
  • complementary cohesive termini can be engineered into the dici ⁇ tronic DNA molecule ⁇ during the primer extension reaction by use of an appropriately designed polynucleotide synthesis primer, a ⁇ previou ⁇ ly di ⁇ cu ⁇ ed.
  • the dici ⁇ tronic DNA molecule, and vector if nece ⁇ ary, i ⁇ cleaved with a re ⁇ triction endonuclea ⁇ e to produce termini complementary to tho ⁇ e of the vector.
  • the complementary cohe ⁇ ive termini of the vector and the dici ⁇ tronic DNA molecule are then operatively linked (ligated) to produce a unitary double stranded DNA molecule.
  • the present method produces a diverse population of double stranded DNA expression vectors wherein each vector expresses, under the control of a single promoter, one V H -coding DNA homolog and one V L - coding DNA homolog, the diversity of the population being the result of different V H - and V L -coding DNA - 60 - homolog combinations that occurs during the PCR reaction where both outside and both inside primers are present in effective amounts.
  • the vector ⁇ are linear double stranded DNA, such a ⁇ a Lambda Zap derived vector a ⁇ de ⁇ cribed herein.
  • the vector define ⁇ a nucleotide ⁇ eguence coding for a ribo ⁇ ome binding site and a leader, the sequence being located down ⁇ tream from a promoter and upstream from a sequence coding for a polypeptide leader.
  • the vector contains a selectable marker such that the presence of a dicistronic DNA molecule of thi ⁇ invention inserted into the vector, can be ⁇ elected.
  • selectable markers are well known to those skilled in the art. Examples of ⁇ uch marker ⁇ are antibiotic re ⁇ i ⁇ tance gene ⁇ , genetically ⁇ electable marker ⁇ , mutation suppres ⁇ ors ⁇ uch a ⁇ amber ⁇ uppre ⁇ or ⁇ and the like.
  • the ⁇ electable marker ⁇ are typically located up ⁇ tream of the promoter.
  • the resulting construct is then introduced into an appropriate host to provide amplification and/or expression of the V H - and V L -coding DNA homologs.
  • a functionally active heterodimeric receptor such as an F v .
  • Cellular ho ⁇ t ⁇ into which a V h - and V L -coding DNA homolog-containing con ⁇ truct ha ⁇ been introduced are referred to herein a ⁇ having been "tran ⁇ formed” or a ⁇ "tran ⁇ formant ⁇ ".
  • the ho ⁇ t cell can be either prokaryotic or eukaryotic.
  • Bacterial cell ⁇ are preferred prokaryotic host cell ⁇ for library screening, and typically are a strain of E. coli such as, for example, the E. coli strain DH5 available from Bethesda Re ⁇ earch Laboratorie ⁇ , Inc., Bethesda, MD.
  • Preferred eukaryotic ho ⁇ t cell ⁇ include yeast and mammalian cells
  • SUBS cell ⁇ preferably vertebrate cell ⁇ ⁇ uch a ⁇ tho ⁇ e from a mou ⁇ e, rat, monkey or human cell line.
  • transformation of prokaryotic host cell ⁇ see, for example, Cohen et al., Proc. Natl. Acad. Sci.. USA, 69:2110 (1972); and Maniatis et al., Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY (1982) .
  • retroviral vector ⁇ containing rDNAs ⁇ ee for example, Sorge et al., Mol. Cell. Biol.. 4:1730-1737 (1984); Graham et al., Virol.. 52:456 (1973); and Wigler et al., Proc. Natl. Acad. Sci.. USA, 76:1373-1376 (1979).
  • Successfully transformed cells i.e., cells containing a dicistronic DNA molecule operatively linked to a vector, can be identified by any ⁇ uitable well known technique for detecting the binding of a receptor to a ligand or the pre ⁇ ence of a polynucleotide coding for the receptor, preferably it ⁇ active ⁇ ite.
  • Preferred screening assays are those where the binding of ligand by the receptor produces a detectable signal, either directly or indirectly.
  • Such signals include, for example, the production of a complex, formation of a catalytic reaction product, the release or uptake of energy, and the like.
  • cells from a population subjected to transformation with a subject recombinant DNA can be cloned to produce monoclonal colonies.
  • Cell ⁇ form tho ⁇ e colonie ⁇ can be harve ⁇ ted, ly ⁇ ed and their DNA content examined for the pre ⁇ ence of the rDNA
  • the pre ⁇ ent invention include ⁇ a method for expre ⁇ ing a polypeptide on the outer surface of E. coli.
  • the surface expres ⁇ ion of a polypeptide provide ⁇ a particularly advantageou ⁇ technique for screening diverse libraries for a polypeptide, such a ⁇ a receptor, having a pre- ⁇ elected activity.
  • E. coli expre ⁇ ing a diver ⁇ e library of Fab fragment ⁇ on their ⁇ urface can be "panned" for tran ⁇ formant ⁇ carrying antibody activity against a specific antigen.
  • Any protein expressed on the cell surface of E. coli can provide the outer membrane spanning signal (surface expres ⁇ ion ⁇ ignal) for u ⁇ e in the pre ⁇ ent invention. More specifically, it has been di ⁇ covered that amino acid re ⁇ idue ⁇ 57-181 of mature lamB can act a ⁇ a signal for ⁇ urface expre ⁇ ion.
  • Such fu ⁇ ion polypeptides are represented by the formula, shown in the direction of amino- to carboxy-ter inu ⁇ : (Fl) NH 2 - B - Z - COOH ,
  • the heterologous polypeptide can itself be a fusion protein, and typically contains a periplasmic secretion ⁇ ignal ⁇ equence (polypeptide leader) , ⁇ uch as the pelB ⁇ ecretion ⁇ ignal, and the like. Thu ⁇ , a preferred fu ⁇ ion polypeptide is represented by the formula,
  • J is a ⁇ equence of amino acid re ⁇ idue ⁇ of from 6 to 350 residue ⁇ in length, and Z i ⁇ as described before in formula (Fl) .
  • J is from about 50 to about 150 amino acid re ⁇ idue ⁇ . More preferably, J i ⁇ a V H or V L a ⁇ de ⁇ cribed herein.
  • the pre ⁇ ent invention contemplate ⁇ .
  • the amino acid residue sequence of a protein or polypeptide is directly related via the genetic code to the deoxyribonucleic acid (DNA) sequence of the structural gene that codes for the protein.
  • DNA deoxyribonucleic acid
  • a structural gene for a fusion protein of this invention can be defined in terms of the amino acid residue sequence, i.e., protein or polypeptide, for which it
  • SUBSTITUTESHEET code ⁇ SUBSTITUTESHEET code ⁇ .
  • an important and well known feature of the genetic code is it ⁇ redundancy. That i ⁇ , for o ⁇ t of the amino acid ⁇ u ⁇ ed to make protein ⁇ , more than one coding nucleotide triplet (codon) can code for or de ⁇ ignate a particular amino acid re ⁇ idue. Therefore, a number of different nucleotide sequences may code for a particular amino acid residue sequence. Such nucleotide sequence ⁇ are con ⁇ idered functionally equivalent ⁇ ince they can result in the production of the same amino acid residue ⁇ equence in all organi ⁇ ms.
  • a methylated variant of a purine or pyrimidine may be incorporated into a given nucleotide sequence.
  • methylations do not affect the coding relation ⁇ hip in any way.
  • Recombinant DNA molecule ⁇ containing a nucleic acid sequence coding for a fusion polypeptide according to formula ⁇ (Fl) or (F2) are contemplated by thi ⁇ invention.
  • the pre ⁇ ent invention provide ⁇ for linking a nucleotide sequence coding for any polypeptide immunogen against which antibody production is desired to the outer membrane spanning ⁇ ignal (lamB) polypeptide and/or the ⁇ ecretion signal (pel B) polypeptide a ⁇ described herein.
  • the polypeptide immunogen i ⁇ a pathogen related immunogen and the conjugate ha ⁇ the capacity to induce the production of antibodie ⁇ that immunoreact with the pathogen when injected in an effective amount into an animal.
  • immunogen ⁇ of particular importance are derived from bacteria such a ⁇ B. pertu ⁇ i ⁇ . S. typho ⁇ a. S. paratyphoid A and B, C. diptheriae. C. tetani. C. botulinum. C. perfringen ⁇ , B. anthraci ⁇ . P, . pestis. P. multocida, V. cholerae, N. eningitide ⁇ . N. gonorrhea. H. influenzae. T. palladium, and the like; immunogen ⁇ derived from viru ⁇ e ⁇ ⁇ uch a ⁇ polio viru ⁇ , adenoviru ⁇ ,
  • the polypeptide immunogen i ⁇ a pathogen related immunogen that im unoreact ⁇ with, i.e., i ⁇ immunologically bound by, antibodie ⁇ induced by the pathogen. More preferably, the pathogen related immunogen i ⁇ capable of inducing an antibody response that provides protection against infection by the pathogen. Methods for determining the presence of both cros ⁇ -reactive and protective antibodie ⁇ are well known in the art.
  • Expres ⁇ ion Vector The pre ⁇ ent invention also contemplates various expression vectors useful in performing, inter alia, the ethod ⁇ of the present invention.
  • Each of the expres ⁇ ion vectors is a novel derivative of Lambda Zap. 1. Lambda Zap II
  • SUBSTITUTESHEET Lambda Zap II is prepared by replacing the -Lambda S gene of the vector Lambda Zap with the Lambda S gene from the Lambda gtlO vector, a ⁇ de ⁇ cribed in Example 7. 2.
  • Lambda ImmunoZAP H is prepared by replacing the -Lambda S gene of the vector Lambda Zap with the Lambda S gene from the Lambda gtlO vector, a ⁇ de ⁇ cribed in Example 7.
  • Lambda ImmunoZAP H i ⁇ prepared by inserting the synthetic DNA ⁇ equences illustrated in Figure 6A into the above-described Lambda Zap II vector.
  • the inserted nucleotide sequence advantageously provides a ribosome binding ⁇ ite
  • Modified Lambda ImmunoZAP H is prepared by inserting the modified synthetic DNA sequence ⁇ illu ⁇ trated in Figure 8A into the above-de ⁇ cribed Lambda ZAP II vector. The preparation of modified Lambda ImmunoZAP H and the detail ⁇ of the modification ⁇ are de ⁇ cribed in Example 8B. It ⁇ feature ⁇ are illu ⁇ trated in Figure 8A and 8B. 4. Lambda ImmunoZAP L
  • Lambda ImmunoZAP L i ⁇ prepared a ⁇ de ⁇ cribed in Example 9 by in ⁇ erting into Lambda Zap II the synthetic DNA sequence illustrated in Figure 6B. Important features of Lambda ImmunoZAP L are illu ⁇ trated in Figure 9.
  • the tran ⁇ formant ⁇ are u ⁇ eful, not
  • heterodimeric molecule ⁇ that assemble in the cell or in the periplasm, operatively linking the lamB outer membrane spanning ⁇ ignal sequence to the carboxy-terminus of one of the polypeptide chain ⁇ of the heterodimer, e.g., the heavy chain of a Fab, re ⁇ ult ⁇ in ⁇ urface expre ⁇ ion of the a ⁇ embled heterodimer.
  • a vaccine containing a transformant of thi ⁇ invention can be ea ⁇ ily prepared, lyophilized in the presence of appropriate inert, non-toxic carrier(s) (infra) in vials and stored at room temperature without loss of potency. No refrigeration or special storage equipment i ⁇ required.
  • the compo ⁇ ition of vaccine preparation ⁇ mu ⁇ t be known and con ⁇ i ⁇ tent.
  • Method ⁇ for the quality control of chemical component ⁇ are well establi ⁇ hed in the art and will not be di ⁇ cu ⁇ ed here.
  • Chemical purity in the vaccine preparation ⁇ i ⁇ defined a ⁇ freedom from toxic waste or cellular breakdown products and interfering or spurious immunogenic material.
  • the vaccines of the present invention can be administered to any warm-or cold-blooded animals susceptible to infection with pathogenic microorganisms. Human and non-human animals may benefit as hosts.
  • Administration can be parenteral, but preferably oral or intranasal, depending upon the natural route of infection.
  • the vaccine may be administered orally, by incorporation of the vaccine in feed or feed water.
  • the dosage administered may be dependent upon the age, health and weight of the recipient, kind of concurrent treatment if any, and nature of the organism.
  • a dosage of active ingredient will be from about l ⁇ ' to l ⁇ '° cells per application per host.
  • the preferred dose for intranasal administration would generally be about 10 6 organisms, su ⁇ pended in 0.05 to 0.1 ml of an immunologically inert carrier.
  • Peroral administration of a vaccine strain of, for example, Salmonella typhi developed according to the method de ⁇ cribed in this invention would probably require 10 6 to 10 8 organism ⁇ suspended in 1-2 mis of, for example, skim milk.
  • the vaccines can be employed in dosage forms such as capsule ⁇ , liquid solutions, su ⁇ pen ⁇ ion ⁇ , or elixirs, for oral administration, or sterile liquid for formulations such as solutions or suspensions for parenteral, intranasal or topical (e.g. wounds or burns) use.
  • An inert, immunologically acceptable carrier is preferably used, such as saline, phosphate buffered saline or skim milk.
  • compositions and Kits Many of the reagents de ⁇ cribed herein (e.g., nucleic acid ⁇ ⁇ uch a ⁇ primer ⁇ , vector ⁇ , and the like) have a number of form ⁇ , particularly variably protonated form ⁇ , and in equilibrium with each other. A ⁇ the ⁇ killed practitioner will under ⁇ tand, repre ⁇ entation herein of one form of a compound or reagent i ⁇ intended to include all form ⁇ thereof that are in equilibrium with each other.
  • the reagents de ⁇ cribed herein e.g., nucleic acid ⁇ ⁇ uch a ⁇ primer ⁇ , vector ⁇ , and the like
  • a ⁇ the ⁇ killed practitioner will under ⁇ tand, repre ⁇ entation herein of one form of a compound or reagent i ⁇ intended to include all form ⁇ thereof that are in equilibrium with each other.
  • the reagent ⁇ de ⁇ cribed herein can be packaged in kit form.
  • the term "package" refer ⁇ to a ⁇ olid matrix or material cu ⁇ tomarily utilized in a ⁇ y ⁇ tem and capable of holding within fixed limit ⁇ one or more of the reagent component ⁇ for u ⁇ e in a method of the pre ⁇ ent invention.
  • materials include glass and plastic (e.g., polyethylene, polypropylene and polycarbonate) bottles, vials, paper, plastic and plastic-foil laminated envelope ⁇ and the like.
  • a package can be a glas ⁇ vial used to contain the appropriate quantities of polynucleotide primer( ⁇ ) , vector ⁇ , re ⁇ triction enzyme( ⁇ ), DNA polymera ⁇ e, DNA liga ⁇ e, or a combination thereof.
  • An aliquot of each component sufficient to perform at least one PCR thermocycle will be provided in each container.
  • Kits useful for producing a template- complement or for amplification of a specific nucleic acid sequence u ⁇ ing a primer exten ⁇ ion reaction methodology al ⁇ o typically include, in separate containers within the kit, dNTP ⁇ where N is adenine, thymine, guanine and cytosine, and other like agent ⁇ for performing primer exten ⁇ ion reactions.
  • the reagent species of any system de ⁇ cribed herein can be provided in solution, a ⁇ a liquid di ⁇ per ⁇ ion or a ⁇ a ⁇ ubstantially dry powder, e.g., the pla ⁇ mid ⁇ may be provided in lyophilized form.
  • the kit i ⁇ an enclo ⁇ ure containing, in ⁇ eparate container ⁇ , an out ⁇ ide fir ⁇ t polypeptide, preferably a V H , gene primer, an out ⁇ ide ⁇ econd polypeptide, preferably a V L gene primer, and a linking primer defining a 3 » -terminal priming portion, a cistronic bridge coding portion, and a 5'-terminal primer template portion.
  • the 3 '-terminal priming portion has a nucleotide base sequence complementary to a portion of the primer extension product of one of the out ⁇ ide primers.
  • the 5'-terminal primer-template portion encoding a nucleotide base sequence homologou ⁇ to a portion of the primer exten ⁇ ion product of the other of the out ⁇ ide primer ⁇ .
  • the ci ⁇ tronic bridge coding portion i ⁇ a ⁇ previou ⁇ ly de ⁇ cribed.
  • kit compri ⁇ e ⁇ an enclo ⁇ ure containing, in separate containers, an out ⁇ ide fir ⁇ t polypeptide, preferably a V H , gene primer, an out ⁇ ide ⁇ econd polypeptide, preferably a V L , gene primer, an in ⁇ ide fir ⁇ t polypeptide, preferably a V H , gene primer having a 3'-terminal priming portion and a 5'-terminal non-priming portion.
  • the 3'-terminal priming portion compri ⁇ e ⁇ a nucleotide ⁇ equence homologou ⁇ to a con ⁇ erved portion of a V H gene.
  • the kit al ⁇ o contain ⁇ an in ⁇ ide ⁇ econd polypeptide, preferably a V L , gene primer having a 3'- terminal priming portion and a 5'-terminal hybridizing portion complementary to the 5•-terminal non-priming portion of the first polypeptide gene primer, the 3'- terminal priming portion of which comprises a nucleotide sequence homologous to a conserved portion of a second polypeptide gene.
  • the first polypeptide inside and second polypeptide inside primer ⁇ when hybridized, form a duplex that code ⁇ for a double- ⁇ tranded DNA molecule containing the before de ⁇ cribed ci ⁇ tronic bridge for linking the up ⁇ tream and down ⁇ tream ci ⁇ tron ⁇ .
  • Human heavy chain IgG and human kappa light chain are diagrammatically ⁇ ketched in Figure ⁇ 2A and 2B, respectively.
  • immunoglobulin heavy and light chain primers were designed to produce a region of homology between two polymera ⁇ e chain reaction (PCR) products. The complementary regions have been shown to hybridize predominantly under conditions where one set of primers ("inside primer pair”) is used in a limiting amount relative to the other set of primers ("outside primer pair").
  • the DNA polymerase After the 3' ends of the PCR products have hybridized, the DNA polymerase has been shown to extend the ends creating a fusion sequence carrying the unique sequences of both PCR fragments separated by one copy of region X cistronic bridge. A two-step cloning procedure is thus avoided.
  • an expression vector such as ImmunoZAP
  • SUBSTITUTESHEET The strategy used for producing immunoglobulin heavy and light chain PCR dici ⁇ tronic DNA i ⁇ ⁇ hown schematically in Figure 4. Regions of the immunoglobulin heavy chain coding strand are designated V H , C H 1, C H 2, and C H 3 corresponding to functional regions in the protein. The corresponding regions of the non-coding strand are designated by a prime (*) . Region ⁇ V L and C L are ⁇ imilarly labelled for the kappa light chain. Thi ⁇ procedure can al ⁇ o be performed u ⁇ ing lambda light chain specific regions.
  • a region, X, unrelated to the natural immunoglobulin sequence ⁇ , is introduced into the fusion product by attaching X to the 5' ends of both of the C H 1 » and V L in ⁇ ide primer ⁇ .
  • Overlapping oligonucleotide primer ⁇ u ⁇ ed in the fu ⁇ ion-PCR reaction ⁇ to produce dici ⁇ tronic DNA were de ⁇ igned to encode the following: amino acid ⁇ of 225 to 230 of the IgG heavy chain hinge region which are common to all human IgG i ⁇ otypes; an Spe "i re ⁇ triction ⁇ ite; two ⁇ top codon ⁇ ; a ribosome binding site; a peripla ⁇ mic (pelB) leader sequence (Better, et al., Science.
  • the overlapping regions of the human C H 1' in ⁇ ide and V L inside primers are illustrated in Figure 5.
  • the heavy chain downstream C H 1' inside primer sequence is written 3' to 5' and the light chain upstream V L inside primer sequence is written 5' to 3'.
  • Bold nucleotide ⁇ represent regions where the C H 1' in ⁇ ide primer hybridize ⁇ to the 3 ' end of C H 1 on human IgG heavy chain mRNA or where
  • the V L in ⁇ ide primer hybridizes to the 5' end of V L framework on human kappa light chain cDNA.
  • the amino acid and nucleotides in italics represent change ⁇ in sequence from the original pelB leader ⁇ equence.
  • the codon wa ⁇ changed from CTC to ATC re ⁇ ulting in a con ⁇ ervative amino acid change from a leucine to an i ⁇ oleucine a ⁇ ⁇ hown in Figure 5 and Table 7.
  • Hydrophobic amino acid ⁇ in the core region of peripla ⁇ mic leader ⁇ equence ⁇ have been ⁇ hown to be e ⁇ sential for correct proces ⁇ ing of the leader sequence and transport.
  • the additional dATP would then cau ⁇ e a mi ⁇ match between the overlapping PCR product ⁇ at the 3' terminu ⁇ and inhibit elongation by Taq DNA polymera ⁇ e. Sommer, et al. Nucl. Acid ⁇ Re ⁇ .. 17: 6749 (1989) . Therefore, the change to two dTTPs in this position of the oligonucleotide primers would allow proper ba ⁇ e pairing if up to two dATP ⁇ were added to the 3' terminu ⁇ of the heavy chain PCR product.
  • the kappa light chain PCR product wa ⁇ de ⁇ igned to terminate at a po ⁇ ition where two dTTPs occur 5' of the end of the product and did not require alterations of the nucleotide ⁇ equence.
  • Nucleotide ⁇ were changed in the kappa light chain primer encoding the pelB leader sequence without introducing amino acid changes in order to decrease the number of mismatche ⁇ between the primer and the leader ⁇ equence of the kappa light chain mRNA a ⁇ ⁇ hown in Figure 5 and Table 7.
  • Mullinax et al., supra. Briefly, the combinatorial library was prepared by the following approach. Volunteer donors, who had been previously immunized against tetanus but had not received booster* injections within the last year, received injection ⁇ on 2 con ⁇ ecutive days of 0.5 milliliters (ml) of alum- ab ⁇ orbed TT (40 microgram/ml (ug)/ml) (Connaught Laboratorie ⁇ , Swiftwater, Penn ⁇ ylvania) .
  • ml milliliters
  • alum- ab ⁇ orbed TT 40 microgram/ml (ug)/ml)
  • PBL ⁇ peripheral blood lymphocyte ⁇
  • Histopaque-1077 Sigma, St. Loui ⁇ , Missouri
  • centrifuging 400 x g for 30 minutes at 25 degrees Celsiu ⁇ (25'C).
  • I ⁇ olated PBL ⁇ were wa ⁇ hed twice with pho ⁇ phate buffered ⁇ aline (PBS) (150 mM sodium chloride and 150 mM sodium phosphate, pH 7.2 at 25'C) .
  • PBS pho ⁇ phate buffered ⁇ aline
  • RNA was then purified from the PBLs (10 6 B cell ⁇ per ml blood per 100 ml of blood) for an enriched ⁇ ource of B-cell mRNA coding for anti-TT IgG u ⁇ ing an RNA i ⁇ olation kit according to manufacturer' ⁇ in ⁇ truction ⁇ (Stratagene, La Jolla, California) and al ⁇ o de ⁇ cribed by Chomczyn ⁇ ki et al., Anal. Biochem.. 162:156-159 (1987).
  • the i ⁇ olated PBL ⁇ were homogenized in 10 ml of a denaturing ⁇ olution containing 4.0 M guanine isothiocyanate, 0.25 M sodium citrate at pH 7.0, and 0.1 M beta-mercaptoethanol.
  • the pelleted total cellular RNA wa ⁇ collected and di ⁇ olved in 3 ml of the denaturing solution described above. Three ml of isopropyl alcohol was added to the re-suspended total cellular RNA and inverted to mix. This solution was maintained at -20*C for at least 1 hour to precipitate the RNA. The ⁇ olution containing the precipitated RNA
  • RNA was prepared from the total cellular RNA using method ⁇ de ⁇ cribed in Molecular Cloning A Laboratory Manual. Maniatis et al., ed ⁇ ., Cold Spring Harbor, NY, (1982).
  • RNA i ⁇ olated from a PBL ⁇ prepared a ⁇ described above was re- ⁇ u ⁇ pended in one ml of IX ⁇ ample buffer (1 mM Tris-HCl, (Tris [hydroxylmethyl- aminomethane]) pH 7.5; 0.1 mM EDTA (disodium ethylene diamine tetra-acetic acid), 0.5 M NaCl) and maintained at 65*C for five minutes and then on ice for five more minutes.
  • IX ⁇ ample buffer 1 mM Tris-HCl, (Tris [hydroxylmethyl- aminomethane]) pH 7.5; 0.1 mM EDTA (disodium ethylene diamine tetra-acetic acid), 0.5 M NaCl
  • the mixture was then applied to an oligo-dT (Stratagene) column that was previously prepared by washing the oligo-dT with a solution containing 10 mM Tris-HCl, pH 7.5; 1 mM EDTA, 0.5 M NaCl.
  • the eluate was collected in a sterile polypropylene tube and reapplied to the same column after heating the eluate for five minute ⁇ at 65*C.
  • the oligo dT column wa ⁇ then washed with 0.4 ml of high ⁇ alt loading buffer con ⁇ i ⁇ ting of 10 mM Tri ⁇ -HCl at pH 7.5, 500 mM sodium chloride, and 1 mM EDTA.
  • the oligo dT column was then washed with 2 ml of 1 X low salt buffer consisting of 10 mM Tri ⁇ -HCl at pH 7.5, 100 mM sodium chloride, and 1 mM EDTA.
  • the mes ⁇ enger RNA wa ⁇ eluted from the oligo dT column with 0.6 ml of buffer consi ⁇ ting of 10 mM Tri ⁇ -HCl at pH 7.5, and 1 mM EDTA.
  • the messenger RNA was purified by extracting this solution with phenol/chloroform followed by a single extraction with 100% chloroform.
  • the messenger RNA isolated by the above process contains a plurality of different V H and V L coding polynucleotide ⁇ , i.e., greater than about 10 4 different V H - and V L -coding gene ⁇ .
  • 5 I ⁇ olated RNA wa ⁇ converted to cDNA by a primer exten ⁇ ion reaction with a fir ⁇ t-strand synthe ⁇ is kit according to manufacturer' ⁇ in ⁇ truction ⁇ (Stratagene) by u ⁇ ing an oligo (dT) primer for the light chain and a specific primer, C H 1', for the heavy chain.
  • Example 30 were then dige ⁇ ted with Sac I and Xba I and ligated into a modified Lambda Zap II vector as prepared in Example 9 to form a light chain ImmunoZap Library (ImmunoZAP L; Stratacyte, La Jolla, California) .
  • the PCR amplified heavy chain DNA was digested with Spe I
  • ImmunoZAP H with heavy chain inserts The left arm of the light chain library phage DNA was digested with Mlu I resulting in a right arm of ImmunoZAP with kappa light chain insert ⁇ . Both product ⁇ were then dige ⁇ ted with Eco Rl and ligated to create a combinatorial library that encoded human Fab fragment ⁇ including those ⁇ pecific for TT. Mullinax, et al., ⁇ upra.
  • Reactive plaque ⁇ were fir ⁇ t identified by binding to tetanu ⁇ toxoid a ⁇ de ⁇ cribed in Example 11. Bacteriophage from purified reactive plaques were then converted to the plasmid format by in vivo exci ⁇ ion with R408 helper phage (Stratagene) following method ⁇ de ⁇ cribed in Example 11 and familiar to one ⁇ killed in the art. Short, et al., Nucl. Acid ⁇ . Re ⁇ .. 16:7583- 7600 (1988) . The re ⁇ ulting purified pla ⁇ mid DNA encoding heavy and light chain wa ⁇ then u ⁇ ed in PCR reaction ⁇ a ⁇ de ⁇ cribed below in Example 3. b. Preparation of a V ⁇ - and V L -Coding
  • Purified population ⁇ of PBL ⁇ , other lymphocytes, and hybridoma ⁇ which express immunoglobulins including IgG, IgM, IgE, IgD, and IgA are u ⁇ ed a ⁇ ⁇ ource ⁇ for isolating mRNA encoding
  • Example 2a (Stratagene) as described in Example 2a.
  • the purified RNA is then converted to cDNA with a first- ⁇ trand ⁇ ynthe ⁇ is kit as de ⁇ cribed in Example 2a.
  • the resultant cDNA i ⁇ then u ⁇ ed a ⁇ a template in PCR amplication reaction ⁇ a ⁇ described below in Example 3 for the production of dicistronic molecule ⁇ expre ⁇ sing heavy and light chains.
  • Populations of cell ⁇ de ⁇ cribed above can be i ⁇ olated from other mammalian sources such as mouse or rabbit. Both mRNA and rearranged DNA can be i ⁇ olated a ⁇ de ⁇ cribed above and u ⁇ ed a ⁇ template ⁇ in PCR amplification reaction ⁇ .
  • cDNA ⁇ ynthe ⁇ ized from mRNA i ⁇ olated from a mou ⁇ e anti-human fibronectin hybridoma (ATCC, CRL-1606) wa ⁇ u ⁇ ed a ⁇ a preferred template for the production of dicistronic molecule ⁇ expressing heavy and light chain.
  • V H -Coding Repertoire From Rearranged DNA Rearranged DNA isolated from PBLs, other lymphocyte ⁇ , and hybridoma ⁇ which expre ⁇ s immunoglobulin ⁇ can be u ⁇ ed to prepare a V H -coding repertoire.
  • the amplification procedure for preparing a V ⁇ -coding repertoire u ⁇ ing rearranged DNA i ⁇ performed a ⁇ described in Example 3.
  • SUBST which bind tetanu ⁇ toxoid (TT) , wa ⁇ u ⁇ ed a ⁇ a template for preparing a V H -coding double ⁇ tranded DNA homolog.
  • Human heavy chain containing both the V H and C H 1 coding region and de ⁇ ignated a ⁇ Fd, wa ⁇ amplified in a PCR reaction.
  • the amplification wa ⁇ performed in a 100 ul reaction containing 5 nanogram ⁇ (ng) of the cloned DNA in PCR buffer con ⁇ i ⁇ ting of the following: 10 mM .Tri ⁇ -HCl, pH 8.3; 50 mM KCI, 1.5 mM MgCl 2 ; 0.001% (w/v) gelatin; 200 mM of each dNTP; 200 nanomolar (nM) of each primer; and 2.5 unit ⁇ of Taq DNA polymera ⁇ e.
  • the human V H outside primer and C H 1' inside primer were used a ⁇ a PCR primer pair for amplification of the heavy chain (Table 7 and Figure 4) .
  • the reaction mixture wa ⁇ overlaid with mineral oil and ⁇ ubjected to 40 cycle ⁇ of amplification.
  • Each amplification cycle (thermocycle) involved denaturation at 94 * C for 1.5 minute ⁇ , annealing at 54 ' C for 2.5 minute ⁇ and polynucleotide ⁇ ynthe ⁇ is by primer extension (elongation) at 72*C for 3.0 minutes followed by a return to the denaturation temperature.
  • the resultant amplified V H -coding DNA homolog containing sample ⁇ were then gel purified, extracted twice with phenol/chloroform, once with chloroform followed by ethanol precipitation and were ⁇ tored at -70*C in 10 mM Tri ⁇ -HCl, pH 7.5, and 1 mM EDTA.
  • the PCR purified product ⁇ were electrophore ⁇ ed in an agaro ⁇ e gel.
  • the expected size of the heavy chain was approximately 730 base pairs as shown in Figure 10.
  • the V H -coding double stranded DNA homolog ⁇ were then u ⁇ ed in subsequent PCR amplification reaction ⁇ with V L -coding counterparts prepared below for the production of dicistronic DNA molecules having V H and V L cistronic portions as illustrated in Example 4.
  • Cloned DNA prepared in Example 2 from a combinatorial library that encodes human Fab fragments which bind tetanus toxoid (TT) , wa ⁇ u ⁇ ed as a template for preparing a V L -coding double ⁇ tranded DNA homolog.
  • Human light chain containing the entire coding region of kappa light chain (V L and C L ) , wa ⁇ amplified u ⁇ ing the same PCR conditions de ⁇ cribed for human heavy chain with the exception that a human V L in ⁇ ide primer and C L * out ⁇ ide primer were u ⁇ ed a ⁇ the PCR primer pair (Table 7 and Figure 4) .
  • the re ⁇ ultant V L -coding double ⁇ tranded DNA homolog wa ⁇ gel purified and stored as described above.
  • the PCR purified products were electrophore ⁇ ed in an agaro ⁇ e gel.
  • the expected ⁇ ize of the light chain wa ⁇ approximately 690 ba ⁇ e pair ⁇ a ⁇ shown in Figure 10.
  • the V L -coding double stranded DNA homologs were then used in subsequent PCR amplification reactions with V H -coding counterparts prepared above for the production of dicistronic DNA molecules a ⁇ illu ⁇ trated in Example 4.
  • V ⁇ - and V-coding double ⁇ tranded DNA homolog ⁇ prepare in Example 3a and 3b, re ⁇ pectively, were admixed together and denatured at 95*C for 5 minute ⁇ to ⁇ eparate the strands of each homolog.
  • the denatured V H -and V L -coding DNA strands in the admixture were then annealed at 54*C for 5 minutes to form a V H - and V L -coding duplex DNA molecule hybridized at the 3' ends at region X of each original
  • the hybridized recombinant V H - and V L - coding DNA molecule (internally primed duplex) was subjected to primer extension and then amplified with only the V H and C L ' primer ⁇ following the PCR reaction procedure described in Example 3a.
  • the PCR reaction product ⁇ were gel electrophore ⁇ ed to verify the pre ⁇ ence of the re ⁇ ultant V H -and V L -coding dici ⁇ tronic DNA molecule ⁇ .
  • the expected ⁇ ize of the dici ⁇ tronic molecule wa ⁇ about 1390 base pairs and i ⁇ ⁇ hown in Figure 10.
  • Mouse hybridoma heavy and light chain cDNA prepared in Example 2b was amplified in a single PCR reaction using the reaction conditions given above with an exce ⁇ of the out ⁇ ide primers (200 nM concentration of both the mou ⁇ e V H primer and C L '
  • Another approach to producing a library of dici ⁇ tronic DNA molecule ⁇ i ⁇ to u ⁇ e a single internal primer in ⁇ tead of u ⁇ ing two ⁇ eparately internal primer ⁇ The proce ⁇ of creating a dici ⁇ tronic molecule compri ⁇ ing an up ⁇ tream V H ci ⁇ tron and a down ⁇ tream V L ci ⁇ tron i ⁇ to combine in a PCR buffer the following: a repertoire of V H gene ⁇ con ⁇ i ⁇ ting of at least 10 5 different genes; a repertoire of V L genes consisting of at least 10 4 different genes; an outside V H primer; an outside V L primer; and a polynucleotide strand having a 3'-terminal priming portion, a ci ⁇ tronic bridge coding portion, and a 5'-terminal primer-template portion.
  • the PCR reaction is performed as described in Example 2a.
  • the 3'-terminal priming portion of a polynucleotide strand has a nucleotide base sequence homologous to a portion of the primer exten ⁇ ion product of one of the outside primers.
  • the 5'-terminal priming portion encodes a nucleotide base sequence homologous to a portion of the primer extension product of the other outside primer.
  • the cistronic bridge coding portion encodes at least one ⁇ top codon in the same reading frame a ⁇ the up ⁇ tream cistron, a ribo ⁇ ome binding site downstream from the ⁇ top codon and a polypeptide leader (pelB) having a
  • the resultant single step internally primed dici ⁇ tronic DNA molecule can then be ligated into modified ImmunoZAP H for con ⁇ truction of an expre ⁇ ion vector a ⁇ de ⁇ cribed in Example 10.
  • the vector Lambda ZapTM II (Stratagene) i ⁇ a derivative of the original Lambda Zap (ATCC # 40,298) that maintains all of the characteristic ⁇ of the original Lambda Zap including 6 unique cloning ⁇ ite ⁇ , fu ⁇ ion protein expre ⁇ ion, and the ability to rapidly exci ⁇ e the in ⁇ ert in the form of a phagemid (Blue ⁇ cript SK-) , but lack ⁇ the SAM 100 mutation, allowing growth on many Non-Sup F strains, including XLl-Blue.
  • the Lambda Zap II was constructed as de ⁇ cribed in Short et al., Nucleic Acids Re ⁇ ..
  • Bacteriophage lambda was selected a ⁇ the expression vector for three reasons. First, in vitro packaging of phage DNA is the most efficient method of reintroducing DNA into host cells. Second,
  • leader ⁇ equence directing the expre ⁇ ed protein to the peripla ⁇ mic ⁇ pace provided a polynucleotide ⁇ equence that coded for a known epitope (epitope tag) and also provided a polynucleotide that coded for a ⁇ pacer protein between the V H -coding DNA homolog and the polynucleotide coding for the epitope tag.
  • the individual ⁇ ingle- ⁇ tranded polynucleotide ⁇ (N T -N ⁇ ) are ⁇ hown in Table 9 below.
  • Polynucleotide ⁇ 2, 3, 9-4', 11, 10-5', 6, 7 and 8 were kina ⁇ ed by adding 1 ⁇ l of each polynucleotide (0.1 ug/ ⁇ l) and 20 unit ⁇ of T 4 polynucleotide kina ⁇ e to a ⁇ olution containing 70 mM Tri ⁇ -HCl at pH 7.6, 10 mM MgCl 2 , 5 mM DTT, 10 mM beta mercaptoethanol, 500 ug/ml of BSA. The solution was maintained at 37*C for 30 minutes and the reaction stopped by maintaining the ⁇ olution at 65*C for 10 minute ⁇ .
  • the individual polynucleotides were covalently linked to each other to ⁇ tabilize the ⁇ ynthetic DNA in ⁇ ert by adding 40 ⁇ l of the above reaction to a solution containing 50 mM Tris-HCl, pH 7.5, 7 mM MgCl 2 , 1 mM DTT, 1 mM ATP and 10 units of T4 DNA ligase. This solution was maintained at 37*C for 30 minutes and then the T4 DNA ligase was inactivated by maintaining
  • the packaged ligation mixture was plated on XLl-blue cells (Stratagene) .
  • Individual Lambda Zap II plaques were cored and the insert ⁇ exci ⁇ ed according to the in. vivo excision protocol provided by the manufacturer (Stratagene) .
  • SUBSTITUTESHEET vector wa ⁇ performed as described above with the following modifications: elimination of the Sac I ⁇ ite between the T 3 polymerase and Not I site ⁇ and changing the nucleotide base residue sequence from AAA to CAG which resulted in an amino acid residue change from lysine to glutamine a ⁇ shown in Figure 8A and 8B.
  • the individual single-stranded polynucleotide ⁇ (N-i, 4 , N and N 7 ) , which were modified from their counterparts li ⁇ ted in Table 9, are li ⁇ ted in Table 10 below.
  • the modifications also improved the efficiency of secretion of positively changed amino acid ⁇ in the amino terminu ⁇ of the expre ⁇ ed protein. Inouye et al., Proc. Natl. Acad. Sci. USA. 85:7685-7689 (1988).
  • the individual single-stranded polynucleotides (N ⁇ Ne) are shown in Table 9 above.
  • Polynucleotides N2, N3, N4, N6, N7 and N8 were kina ⁇ ed by adding 1 ⁇ l of each polynucleotide and 20 unit ⁇ of T* polynucleotide kina ⁇ e to a ⁇ olution containing 70 mM Tri ⁇ -HCl, pH 7.6, 10 mM MgCl 2 , 5 mM DDT, 10 mM 2ME, 500 microgram ⁇ per ml of BSA.
  • the ⁇ olution wa ⁇ maintained at 37*C for 30 minutes and the reaction stopped by maintaining the solution at 65 ' C for 10 minute ⁇ .
  • the two end polynucleotide ⁇ 20 ng of polynucleotide ⁇ Nl and polynucleotide ⁇ N5 were added to the above kina ⁇ ing reaction ⁇ olution together with 1/10 volume of a solution containing 20 mM Tris-HCl, pH 7.4, 2 mM MgCl 2 and 50 mM NaCl.
  • This solution wa ⁇ heated to 70*C for 5 minute ⁇ and allowed to cool to room temperature, approximately 25*C, over 1.5 hour ⁇ in a 500 ml beaker of water.
  • all the polynucleotide ⁇ annealed to form the double ⁇ tranded synthetic DNA insert.
  • SUBSTITUTESHEET polynucleotide ⁇ were covalently linked to each other to ⁇ tabilize the ⁇ ynthetic DNA insert with adding 40 ⁇ l of the above reaction to a solution containing 50 ⁇ l Tri ⁇ -HCl, pH 7.5, 7 mM MgCl 2 , 1 mM DTT, 1 mM ATP and 10 unit ⁇ of T4 DNA liga ⁇ e.
  • Thi ⁇ ⁇ olution wa ⁇ maintained at 37'C for 30 minute ⁇ and then the T4 DNA liga ⁇ e wa ⁇ inactivated by maintaining the ⁇ olution at 65*C for 10 minute ⁇ .
  • the end polynucleotide ⁇ were kina ⁇ ed by mixing 52 ⁇ l of the above reaction, 4 ⁇ l of a solution recontaining 10 mM ATP and 5 units of T4 polynucleotide kinase. This solution was maintained at 37*C for 30 minutes and then the T4 polynucleotide kinase was inactivated by maintaining the ⁇ olution at 65'C for 10 minute ⁇ .
  • the ligation mixture was packaged according to the manufacture's instruction ⁇ u ⁇ ing Gigapack II Gold packing extract and the packaged ligation mixture wa ⁇ plated on XL1- Blue cell ⁇ a ⁇ de ⁇ cribed in Example 8a.
  • Individual lambda Zap II plaques were cored and the insert ⁇ excised according to the in vivo excision protocol as de ⁇ cribed in Example 8a.
  • Thi ⁇ in vivo exci ⁇ ion protocol convert ⁇ the cloned in ⁇ ert from the Lambda Zap II vector into a phagemid vector to allow ea ⁇ y manipulation and sequencing and also produces the phagemid version of the V L expression vectors.
  • the phagemid was produced, as detailed above, by the in vivo exci ⁇ ion proce ⁇ from the Lambda Zap V L expre ⁇ ion vector ( Figure 9) .
  • V H -V L -coding (V HL ) dicistronic DNA molecule ⁇ PCR amplified product ⁇ (human or mou ⁇ e) prepared in Example ⁇ 4, 5 and 6 (50 mM NaCl, 25 M Tri ⁇ -HCl, pH 7.7, 10 mM MgCl 2 , 10 M ⁇ - mercaptoethanol, 100 ug/ml BSA, at 37 ' C were digested with restriction enzymes Xho I and Xba I at a concentration of 60 unit ⁇ of enzyme per ug of DNA, and purified on a 1% agaro ⁇ e gel.
  • V HL dici ⁇ tronic molecule ⁇ E. coli were infected to yield approximately 100 plaque ⁇ per plate.
  • Replica filter lift ⁇ of the plaque ⁇ on an agar plate were produced by overlaying a nitrocellulo ⁇ e filter that had been ⁇ oaked in 10 mM i ⁇ opropyl beta-dithiogalactopyranoside on each plate with transfer for 15 hours at 23*C.
  • the filter ⁇ were screened with rabbit anti-human heavy and light chain antibodies followed by goat anti-rabbit antibody coupled to alkaline pho ⁇ phata ⁇ e (Cappel Laboratorie ⁇ , Malvern, Penn ⁇ ylvania) . The detection of immunoreactive product confirmed the presence and expre ⁇ ion of V HL antibody fragment ⁇ .
  • plaque ⁇ were plated and protein ⁇ expressed a ⁇ de ⁇ cribed above.
  • Replica filter ⁇ were incubated with 0.2 nM 125 I-tetanus toxoid and washed. Positive plaques were identified by autoradiography and isolated. The frequency of positive clones in the library was equivalent to (number of positive clones)/[number of plaques screened) X (fraction of plaques expres ⁇ ing V HL ) .
  • Mou ⁇ e antibody-producing plaque ⁇ prepared in Example 7 were ⁇ creened for antibody expres ⁇ ion with rabbit anti-mouse heavy and light chain antibody (Cappel Laboratories) as described above.
  • SUBSTITUTESHEET 500 ul of a buffer containing 50 mM Tri ⁇ -HCl, pH 7.5, 100 mM NaCl, 10 mM MgS0 4 , and 0.01% (w/v) gelatin and 20 ul of chloroform.
  • Double ⁇ tranded DNA wa ⁇ prepared from the phagemid containing cell ⁇ according to the methods described by Holme ⁇ et al., Anal. Bioche .. 114:193, (1981). Clone ⁇ were fir ⁇ t ⁇ creened for DNA in ⁇ ert ⁇ by restriction digests with Xho 1 and Xba 1. The detection of 1390 ba ⁇ e pair fragment on an agaro ⁇ e gel confirmed the pre ⁇ ence of a V KL dici ⁇ tronic molecule insert. b. Sequencing of Plasmid ⁇ from Expre ⁇ ion Library
  • SUB PCR amplification can, therefore, be u ⁇ ed to fu ⁇ e ⁇ eguence ⁇ re ⁇ pon ⁇ ible for encoding ⁇ ubunit ⁇ of a heterodimeric protein together into a ⁇ ingle DNA fragment that can then direct the expre ⁇ ion of both subunits from one expression vector.
  • the source of nucleic acid template comes from hybridoma mRNA, there is only one heavy and light chain sequence to choose from, and thu ⁇ the heavy:light pair i ⁇ a "natural" pair.
  • the PCR fu ⁇ ion reaction to form a dici ⁇ tronic DNA molecule can randomly pair heavy and light chain ⁇ from different cell ⁇ , producing a combinatorial library.
  • a ⁇ mall fraction of the clone ⁇ contain the original heavy and light chain pair ⁇ .
  • Thi ⁇ may not be a problem if the de ⁇ ired natural pair i ⁇ well repre ⁇ ented in the original B-cell population, a ⁇ i ⁇ the ca ⁇ e with hyperimmunized donor ⁇ .
  • one wi ⁇ hes to find a naturally occurring rare ⁇ pecificity in a combinatorial library one may have to ⁇ creen an large number of clone ⁇ .
  • the fu ⁇ ion method pre ⁇ ented here may offer a ⁇ olution to the random combinatorial problem. If one begin ⁇ with a very dilute population of B-cells (po ⁇ ibly in a medium that limit ⁇ diffu ⁇ ion) , it may be po ⁇ ible for the dici ⁇ tronic event to occur between naturally paired heavy and light chain sequences before significant mixing between B-cell RNA occur ⁇ . Thu ⁇ , the fu ⁇ ed heavy and light chain ⁇ eguence ⁇ would be the original pair ⁇ , and the re ⁇ ulting library would expre ⁇ predominantly the naturally occurring antibody ⁇ pecificitie ⁇ . Such a library would be highly preferable when rare natural ⁇ pecificitie ⁇ are sought.
  • SUBSTITUTESHEET Another advantage to thi ⁇ method i ⁇ that only one vector and one cloning ⁇ tep are nece ⁇ ary. Thi ⁇ ⁇ ave ⁇ a ⁇ ubstantial amount of time, resources, and effort. Moreover, the ease of the single PCR reaction greatly simplified the proces ⁇ of going from B-cell RNA to an E. coli library, making thi ⁇ approach a noteworthy alternative to ⁇ tandard hybridoma technology.
  • PCR primer ⁇ are u ⁇ ed to produce a DNA ⁇ egment encoding the ⁇ urface expre ⁇ ion ⁇ ignal amino acid re ⁇ idue sequence of lamB, (i.e., residue positions 51-184 as shown in Figure 3) :
  • the primers are mixed pairvi ⁇ e with genomic DNA u ⁇ ed from E. coli having the lamB gene a ⁇ template.
  • the amplified DNA segment i ⁇ purified by preparative agarose gel electrophoresi ⁇ , dige ⁇ ted with Spe I and Xba I restriction endonuclease ⁇ , and
  • the immunoZAP vector (H/L) is created from the heavy and light chain libraries, prepared in Example ⁇ 8 and 9, respectively, by fusing the vector ⁇ at the Eco Rl ⁇ ite a ⁇ follow ⁇ .
  • DNA i ⁇ purified from the light chain library and restriction digested with Mlu 1 and Eco Rl. This cleaves the DNA from the left arm of the vector into several pieces while leaving the right arm with the light chain inserts intact.
  • DNA is purified from the heavy chain libraries and re ⁇ triction digested with Hind III and Eco Rl. This cleaves the DNA from the right arm of the vector into several pieces while leaving the left arm with the heavy chain inserts intact.
  • the intact left arm of the heavy chain vector containing the heavy chain in ⁇ ert ⁇ and right arm of the light chain vector containing the light chain in ⁇ erts are then mixed and ligated at the common Eco Rl re ⁇ triction site.
  • the re ⁇ ultant ImmunoZAP H/L vector is shown in Figure 12.
  • the ligations and packaging are a ⁇ de ⁇ cribed in Example 2 to create the ImmunoZAP H/L library.
  • a DNA segment coding for a preselected polypeptide, such as a V H , can then be ligated into the lamB-modified ImmunoZap H expression vector at position between, and is the same reading frame with, the pelB leader and the lamB signal sequences.
  • the vector thus produced expresses the preselected polypeptide as a double-fusion protein, i.e., having pelB leader and lamB surface expression signal polypeptide segments operatively linked to the preselected polypeptides amino- and carboxy-ter ini,
  • TyrPheAsp GluProLysSerCy ⁇ AspLysThrHi ⁇ ThrSerTyrPheTyr A ⁇ pValProA ⁇ pTyrGlySerLy ⁇ SerSerPheTyrPheA ⁇ p
  • SUBSTITUTESHEET 7 3' heavy chain C H 1 overlapping primer for in ⁇ ert C. 8 5' lamB overlapping primer for in ⁇ ert D. 9 3' heavy chain C H 1 overlapping primer for in ⁇ ert D. 103 • lamB overlapping primer with 5' light chain primer.
  • the inserts between the heavy chain and lamB sequences are made using the PCR-fusion procedure for producing dicistronic DNA as prepared in Examples 2 and 3 with the following exceptions.
  • the light chain and lamB sequences are fused together u ⁇ ing the out ⁇ ide primer ⁇ and limiting amount ⁇ of the in ⁇ ide primer ⁇ (Table 13) .
  • the re ⁇ ultant PCR product ⁇ are gel purified u ⁇ ing Gene Clean (BIO 101) a ⁇ described in Example 10 before PCR- fu ⁇ ing it to the heavy chain u ⁇ ing only out ⁇ ide primer ⁇ (Table 13) .
  • the re ⁇ ultant PCR-fu ⁇ ion product con ⁇ ists of V H -in ⁇ ert A, B, C or D-lamB-light chain.
  • the region in ⁇ erted by the PCR primer ⁇ between the lamB and light chain create ⁇ the same dici ⁇ tronic bridge previou ⁇ ly in ⁇ erted between the heavy and light chain DNA ⁇ .
  • Thi ⁇ product is ligated with the modified ImmunoZAP H vector restriction digested with the enzymes Xho I and Xba I as prepared in Example 10. After insertion, the dici ⁇ tronic e ⁇ age encoded by the DNA allows expres ⁇ ion of the heavy chain and lamB a ⁇ a fu ⁇ ion protein and the light chain a ⁇ a separate protein.
  • Blood wa ⁇ collected from healthy donor ⁇ and PBL ⁇ were i ⁇ olated a ⁇ de ⁇ cribed in Example 2.
  • I ⁇ olated PBLs were then fractionated into T and non-T cell ⁇ by AET-SRBC (2-aminoethylthiouronium bromide- ⁇ heep red blood cell) ro ⁇ etting according to the procedure de ⁇ cribed by Callard. Callard et al., Eur. J. Immunol.. 11, 206 (1981). Briefly, the isolated PBLs were treated with a 1% suspen ⁇ ion of AET-modified ⁇ heep red blood cell ⁇ . The ro ⁇ ette wa ⁇ purified over a Ficoll gradient and the red blood cell ⁇ removed by hypotonic ly ⁇ i ⁇ .
  • the procedure for preparing the T cell replacing factor, ⁇ -PWM-T was performed as de ⁇ cribed by Daniel ⁇ on. Danielson et al., Immunol.. 61:51-55 (1987).
  • the T cell ⁇ were activated by treatment with 10 ug of pokeweed itogen (PWM)/ml (Sigma) for 24 hour ⁇ at 37*C.
  • PWM pokeweed itogen
  • SUBSTITUTESHEET activation of T cells result ⁇ in ⁇ ecretion of gamma interferon, interleukin-2 (IL-2) and various undefined B cell growth factor ⁇ into the medium.
  • Growth factor containing supernatant from the PWM-treated T cells hereinafter designated s-PWM-T, was collected and added to lymphocyte cell cultures prepared below.
  • the cell ⁇ were incubated at room temperature for 40 minute ⁇ and then wa ⁇ hed three time ⁇ in RPMI-1640 containing 2% heat-inactivated human serum. Cell recovery after treatment with Leu-OMe ranged from 30- 90%. The treatment with Leu-OMe was performed to effect the removal of a Leu-OMe-sensitive ⁇ ubpopulation leaving a population of cell ⁇ that re ⁇ pond to T-cell dependent antigen stimulation in vitro.
  • Leu-OMe-treated PBLs were immunized in vitro with either keyhole limpet hemocyanin (KLH) (Sigma) or tetanus toxoid (TT) (Example 2).
  • KLH keyhole limpet hemocyanin
  • TT tetanus toxoid
  • the Leu-OMe- treated T cells were first suspended in supplemented RPMI-1640, containing 50 uM beta- ercaptoethanol, 10% heat-inactivated human AB serum, 30% (v/v) s-PWM-T, and antigen (1-lOOOng/ml) .
  • the cell ⁇ were maintained in heat-inactivated fetal bovine ⁇ erum in ⁇ tead of human AB ⁇ erum.
  • the antigen- treated Leu-OMe-treated PBL ⁇ were then plated at a concentration of 2 X 10 6 cells/ml in a 4-ml (six-well plates) or 30-ml (75-cm 2 flask) and maintained at 37 * C in 5% C0 2 for three days.
  • the cell ⁇ were pelleted and wa ⁇ hed one time with RPMI-1640 supplemented medium prepared above lacking antigen to effect the removal of antigen.
  • the washed antigen-treated cells were resu ⁇ pended in fresh medium containing s-PWM-T, but lacking antigen.
  • the cells were thereafter cultured for three to four more days for a total maintenance period of six to seven days, at which time the level ⁇ of antigen- ⁇ pecific antibody and/or the number of antigen- ⁇ pecific antibody ⁇ ecreting cell ⁇ were determined by ELISA and ELISPOT a ⁇ ay ⁇ , re ⁇ pectively.
  • SUBSTITUTESHEET ug/ml-final concentration (Boehringer Mannheim, Indianapolis, Indiana) wa ⁇ diluted in 50 mM PBS, pH 7.5, containing 1.5 M sodium chloride and 0.1% Tween 20 and 100 ul of diluted AP antiglobulin conjugate were then added to each well and maintained at room temperature for one hour or at 4'C overnight. The well ⁇ were then rin ⁇ ed three time ⁇ with PBS 0.05% Tween 20.
  • ELISPOT assay ⁇ are performed a ⁇ de ⁇ cribed by Czerkinsky. Czerkinsky et al., J. Immunol. Method ⁇ , 65:190-121 (1983). For mea ⁇ uring the number of antigen-specific antibody-secreting cells in the in vitro immunized PBL cultures ELISPOT was performed. For this as ⁇ ay, 3.5 centimeter diameter poly ⁇ tyrene petri di ⁇ he ⁇ (Falcon, Oxnard, California) were filled with 1.5 ml of either KLH or TT antigen at a concentration of 1 ug/ml. Borrebaeck et al., ⁇ upra.
  • the plate ⁇ were washed as described for the ELISA assay.
  • the antigen-coated plates were then blocked with 0.2% gelatin at 37*C.
  • Lymphocytes (10 5 to 10 6 ) were added to each dish and allowed to incubate undisturbed overnight at 37*C.
  • the cells were removed and the plates were washed twice with cold PBS and then maintained for 10 minutes with cold 10 mM EDTA- PBS.
  • the plates were then rinsed three times with PBS containing 0.5% Tween-20.
  • the frequency of antigen- ⁇ pecific B cell ⁇ wa ⁇ determined a ⁇ (number of antigen-specific antibody ⁇ ecreting cell ⁇ )/(number of B cell ⁇ added per plate).
  • the total number of lymphocyte ⁇ wa ⁇ determined by trypan exclu ⁇ ion. The re ⁇ ult ⁇ of the ⁇ e a ⁇ ay ⁇ are de ⁇ cribed below.
  • SUBSTITUTESHEET Depletion of antigen-specific B cell ⁇ wa ⁇ demonstrated by culturing the non-adherent cell ⁇ in the pre ⁇ ence of ⁇ -PWM-T, as described above, for 6 days. The number of antigen- ⁇ pecific antibody producing cell ⁇ was then determined by the ELISPOT as ⁇ ay. The number of B cell ⁇ which adhere under the condition ⁇ de ⁇ cribed above was determined using two different methods. An enriched population of B cell ⁇ wa ⁇ obtained by ro ⁇ etting with AET-treated ⁇ heep red blood cell ⁇ . The non-ro ⁇ etting cell ⁇ were then panned on autologou ⁇ plasma-coated petri dishes, and the non- adherent lymphocytes (B cell ⁇ ) recovered.
  • the B cell ⁇ were labelled overnight with 35 S-methionine, panned a ⁇ de ⁇ cribed above, and the percent radioactivity adhering to the di ⁇ hes determined.
  • the number of purified cell ⁇ which adhered wa ⁇ determined micro ⁇ copically u ⁇ ing an ocular grid. The re ⁇ ult ⁇ of the experiment ⁇ are de ⁇ cribed below. 4) Panning In Vitro Immunized Cell ⁇
  • KLH and tetanus toxoid (TT) a ⁇ model antigen ⁇ in the above-de ⁇ cribed procedure re ⁇ ulted in a 2-3 fold increa ⁇ e in the frequency of both TT- and KLH- ⁇ pecific B cell ⁇ .
  • the frequency of KLH- ⁇ pecific B cell ⁇ wa ⁇ con ⁇ iderably influenced by
  • Peripheral blood lymphocyte ⁇ from unboosted donors were panned on TT- and gelatin-coated petri dishes and the number of TT-specific B cells in the non-adherent cell population determined.
  • 100% and 90% of the anti-TT antibody secreting cells, respectively were depleted when panned on TT plates, while only 28% and 8% were depleted when panned on gelatin (not shown) .
  • SUBST - Ill - condition ⁇ ranged from 1.5 to 10% and wa ⁇ determined either by labeling the cell ⁇ with 35 S-methionine (expt. 3) or by examining the adherent cell ⁇ micro ⁇ copically with an ocular grid (expt. 4).
  • the ⁇ e preliminary re ⁇ ult ⁇ indicate that a ⁇ ingle cycle of cell panning can be u ⁇ ed to increa ⁇ e the frequency of antigen- ⁇ pecific B cell ⁇ by at lea ⁇ t 9-fold, and po ⁇ ibly a ⁇ high a ⁇ 67-fold. It should be possible to further deplete B cells which bind non- ⁇ pecifically or with low affinity to antigen by performing ⁇ equential i ⁇ olation ⁇ or by altering the epitope density of the solid matrix.
  • a ⁇ Table 17 indicate ⁇ , panning at day ⁇ 6 and 7 (peak of antibody production) is inefficient, possibly due to either down-modulation of surface IgM receptors or interference by secreted anti-KLH antibody. To recover the greatest enrichment antigen-specific B cells, panning should be performed at day 5 to ' ensure maximal clonal expansion.
  • the ⁇ e ⁇ tudie ⁇ have demon ⁇ trated, with model antigen ⁇ , that in vitro immunization or cell panning can be u ⁇ ed to increa ⁇ e the frequency of antigen- ⁇ pecific B cell ⁇ by at lea ⁇ t 10-fold.
  • Preliminary re ⁇ ult ⁇ indicate that the two technique ⁇ can be combined to give ri ⁇ e to frequencie ⁇ which are comparable to tho ⁇ e of the lymphocyte population u ⁇ ed to construct the TT-specific library (10 "3 ) .
  • the ⁇ e technique ⁇ may obviate the requirement for .in vivo immunization, thereby eliminating one of the major ob ⁇ tacle ⁇ to the routine production of human monoclonal antibodie ⁇ .

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Abstract

Procédé de production de molécules d'ADN dicistroniques présentant chacune des cistrons amont et aval codant respectivement les premier et deuxième polypeptides d'un récepteur hétérodimérique. L'invention se rapporte également à des trousses contenant, dans des récipients séparés, les amorces et/ou les vecteurs revendiqués dans des quantités suffisantes pour produire et/ou exprimer les molécules d'ADN dicistroniques.
PCT/US1992/001475 1991-03-01 1992-02-27 Molecules d'adn dicistroniques produites par reaction en chaine de polymerase et servant a produire des anticorps WO1992015678A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0744958A1 (fr) * 1994-01-31 1996-12-04 Trustees Of Boston University Banques d'anticorps polyclonaux
WO1999064624A2 (fr) * 1998-06-12 1999-12-16 Central Manchester Healthcare Nhs Trust Acides nucleiques
US6395475B1 (en) 1993-05-18 2002-05-28 Florida State University Semiautomated method for finger-printing bacterial DNA
EP1516929A2 (fr) * 2003-09-18 2005-03-23 Symphogen A/S Procédé de liaison de séquences nucléotidiques d'intérêt
WO2010136598A1 (fr) 2009-05-29 2010-12-02 Morphosys Ag Collection et ses procédés d'utilisation
WO2012066129A1 (fr) 2010-11-19 2012-05-24 Morphosys Ag Collection et méthodes pour l'utiliser
US8283294B2 (en) 2007-03-01 2012-10-09 Symphogen A/S Method for cloning cognate antibodies

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DE69233750D1 (de) * 1991-04-10 2009-01-02 Scripps Research Inst Bibliotheken heterodimerer Rezeptoren mittels Phagemiden

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WO1990014430A1 (fr) * 1989-05-16 1990-11-29 Scripps Clinic And Research Foundation Nouveau procede d'exploitation du repertoire immunologique

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WO1990014430A1 (fr) * 1989-05-16 1990-11-29 Scripps Clinic And Research Foundation Nouveau procede d'exploitation du repertoire immunologique

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Title
FASEB JOURNAL. vol. 5, no. 6, 19 March 1991, BETHESDA, MD US; A-1717, ABSTR. 7820 R. L. MULLINAX ET AL.: 'ANTIBODY EXPRESSION LIBRARIES IN E. COLI: SIMPLIFIED CONSTRUCTION USING PCR-MEDIATED GENE FUSION' *
GENE. vol. 77, 1989, AMSTERDAM NL pages 61 - 68; R. M. HORTON ET AL.: 'Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension' *

Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6395475B1 (en) 1993-05-18 2002-05-28 Florida State University Semiautomated method for finger-printing bacterial DNA
EP0744958A1 (fr) * 1994-01-31 1996-12-04 Trustees Of Boston University Banques d'anticorps polyclonaux
EP0744958A4 (fr) * 1994-01-31 1997-07-30 Univ Boston Banques d'anticorps polyclonaux
US5789208A (en) * 1994-01-31 1998-08-04 The Trustees Of Boston University Polyclonal antibody libraries
US6335163B1 (en) 1994-01-31 2002-01-01 The Trustees Of Boston University Polyclonal antibody libraries
EP1231268A2 (fr) * 1994-01-31 2002-08-14 Trustees Of Boston University Banques d'anticorps polyclonaux
EP1231268A3 (fr) * 1994-01-31 2002-08-21 Trustees Of Boston University Banques d'anticorps polyclonaux
WO1999064624A2 (fr) * 1998-06-12 1999-12-16 Central Manchester Healthcare Nhs Trust Acides nucleiques
WO1999064624A3 (fr) * 1998-06-12 2000-09-14 Central Manchester Healthcare Acides nucleiques
EP1516929A3 (fr) * 2003-09-18 2006-06-07 Symphogen A/S Procede de liaison de sequences nucleotidiques d'interet
WO2005042774A3 (fr) * 2003-09-18 2005-06-09 Symphogen As Procede de liaison de sequences specifiques
EP1516929A2 (fr) * 2003-09-18 2005-03-23 Symphogen A/S Procédé de liaison de séquences nucléotidiques d'intérêt
EP1921144A3 (fr) * 2003-09-18 2009-12-30 Symphogen A/S Procedé de liaison de sequences nucleotidiques d'interet
US7749697B2 (en) 2003-09-18 2010-07-06 Symphogen A/S Method for linking sequences of interest
WO2005042774A2 (fr) * 2003-09-18 2005-05-12 Symphogen A/S Procede de liaison de sequences specifiques
US8283294B2 (en) 2007-03-01 2012-10-09 Symphogen A/S Method for cloning cognate antibodies
US8685896B2 (en) 2009-05-29 2014-04-01 Morphosys Ag Collection and methods for its use
WO2010136598A1 (fr) 2009-05-29 2010-12-02 Morphosys Ag Collection et ses procédés d'utilisation
US9624293B2 (en) 2009-05-29 2017-04-18 Morphosys Ag Collection and methods for its use
US10647757B2 (en) 2009-05-29 2020-05-12 Morphosys Ag Collection and methods for its use
WO2012066129A1 (fr) 2010-11-19 2012-05-24 Morphosys Ag Collection et méthodes pour l'utiliser
US8367586B2 (en) 2010-11-19 2013-02-05 Morphosys Ag Collection and methods for its use
US8728981B2 (en) 2010-11-19 2014-05-20 Morphosys Ag Collection and methods for its use
US9541559B2 (en) 2010-11-19 2017-01-10 Morphosys Ag Collection and methods for its use

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