EP1214352A2 - Bibliotheque amelioree d'affichage de phages de fragments de vh humain et procedes de production des memes - Google Patents

Bibliotheque amelioree d'affichage de phages de fragments de vh humain et procedes de production des memes

Info

Publication number
EP1214352A2
EP1214352A2 EP00960243A EP00960243A EP1214352A2 EP 1214352 A2 EP1214352 A2 EP 1214352A2 EP 00960243 A EP00960243 A EP 00960243A EP 00960243 A EP00960243 A EP 00960243A EP 1214352 A2 EP1214352 A2 EP 1214352A2
Authority
EP
European Patent Office
Prior art keywords
library
parental
amino acids
ofthe
binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00960243A
Other languages
German (de)
English (en)
Inventor
Howard Kaplan
Joycelyn Entwistle
Jamshid Tanha
Saran Narang
Michael Dan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Viventia Bio Inc
Original Assignee
Viventia Biotech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CA 2282179 external-priority patent/CA2282179A1/fr
Application filed by Viventia Biotech Inc filed Critical Viventia Biotech Inc
Publication of EP1214352A2 publication Critical patent/EP1214352A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/02—Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display

Definitions

  • the present invention relates to combinatorial libraries including phage display libraries which display binding fragments having preferred characteristics of solubility.
  • the present invention also describes methods of producing phage libraries in which the phage population displays binding fragments having characteristics which are biased towards characteristics of the wild type or parental binding fragment.
  • U.S. Patent No. 5,702,892 discloses a phage display library constructed in an M13 derived expression vector, in which recombinant phage ofthe library contain a polynucleotide encoding a fusion protein which comprises a phage coat protein and an immunoglobulin heavy chain binding-fragment.
  • the heavy-chain binding- fragment spans from a position upstream of CDR1 to a position downstream of CDR3.
  • '892 describes that the DNA sequence encoding the CDR3 region and/or the CDR1 region may be randomly varied so that the population of phage expresses a series of potential heavy chain binding domains for panning against the target ligand.
  • U.S. Patent No. 5,759,808 discloses a phage display library comprising a population of phage based on random variation of a cDNA sequence obtained from lymphocytes of camelids previously immunized with target antigens.
  • Camelid heavy chain antibodies occur naturally, in a composition of about 45%, as heavy chain dimers.
  • Heavy chain antibodies specific for a target antigen may be generated by immunizing a member ofthe camelid species with the target antigen (see Lauwereys et al. (1998) The EMBO J. 17, 3512-3520).
  • camelid heavy chain antibodies are naturally more hydrophilic at amino acid residues at locations 44, 45 and 47 (Kabat numbering system), in FR2, which corresponds to the surface where they normally contact the V L domain.
  • Another salient feature of a camelid V H is that it generally has a comparatively longer CDR3 with a high incidence of cysteines and thus may form, via paired cysteines in CDR1 and CDR3, exposed loops, which are more amenable to binding into cavities such as the active site of enzymes and antibodies (Desmyter et al. (1996) Nat. Struct. Biol. Vol. 3, No. 9, p. 803).
  • the present invention discloses advances in the technology related to creating libraries containing immunoglobulin-like proteins that specifically bind target ligands eg. antigens.
  • the invention is directed to a population of variants of at least one parental ligand-binding molecule, wherein said parental ligand-binding molecule comprises an immunoglobulin V H binding fragment comprising, at least in substantial part, at least the framework (FR) regions ofthe immunoglobulin V H fragment depicted in one of Figures 1 or 2 and wherein said variants comprise at least in substantial part, the FR regions ofthe immunoglobulin V H fragment depicted in one of Figures 1 or 2 and differ from said parental ligand-binding molecule in amino acid residues constituting at least part of at least one of the CDRs of said parental ligand-binding molecule.
  • said population of variants is constituted by one or more combinatorial libraries of such variants, for example, protein arrays, phage display libraries, ribosome display libraries etc.
  • variants may (though not necessarily) form part of another structure or molecule, for example in the case of phage display, part ofthe coat protein ofthe phage. Accordingly, the term variant is used broadly to refer to variants ofthe essential molecule (a ligand-binding molecule) when forming part of another structure or molecule (eg. as in phage display or ribosome display) or when independent of any such combination, eg. in the case of protein arrays whose members maynot be associated with individual supporting structures/molecules.
  • the essential molecule a ligand-binding molecule
  • the invention is directed to a ligand-binding molecule which has been identified as binding to a target ligand by screening a combinatorial library ofthe invention for one or more ligand-binding molecules which specifically recognize said target ligand.
  • the invention is also directed more generally to any specific such ligand-binding molecule which is derived from such combinatorial library ofthe invention. It is understood herein that such specific ligand-binding molecule may be directly obtained from such a library or may be indirectly derived, for example, through the course of further antibody engineering or other modification steps (eg. creating fragments, derivatives, a secondary library, etc) using a ligand-binding molecule directly or indirectly obtained from such library. It also understood that the invention excludes known ligand-binding molecules.
  • the target ligand is a cancer antigen.
  • Figures 2, 3 and 4 depict preferred variations, more fully described below, on the preferred immunoglobulin V H binding fragment and/or nucleic acid construct (depicted in Figure 1.
  • Figure 1 describes a wild-type parental immunoglobulin V H binding fragment derived from human monoclonal antibody BT32/A6 (hereinafter referred to as "A6") partially described in U.S Patent No. 5,639,863 (hereinafter referred to as the '863 patent). It has now been found that A6 has preferred solubility and other characteristics which lend themselves well to the creation of libraries, including na ⁇ ve libraries, of various types of human immunoglobulin fragments including scFvs, Fds, Fabs etc., as more fully described below.
  • At least a substantial part ofthe framework (FR) regions ofthe A6 V H fragment depicted in Figure 1, alone or in combination with features of its CDR3, provide a useful departure point, in the form of a parental ligand- binding molecule, for the randomization or partial randomization of amino acid residues which tend to play a predominant role in ligand-binding, namely the CDR regions ofthe heavy chain and particularly the CDR3 ofthe heavy chain.
  • the nucleic acid changes (removal ofthe recombination site) relative to A6 wild-type Figure lreflected in Figure 14 may be incorporated into Figures 1 or 2 to create preferred Figures 3 and 4, respectively.
  • the combinatorial library ofthe invention may be generated by phage display. Accordingly, in a preferred embodiment of one aspect ofthe invention, the invention is directed to a phage display library displaying a plurality of different variants of a parental ligand-binding molecule, wherein said parental ligand-binding molecule comprises an immunoglobulin V H binding fragment comprising, at least in substantial part, at least the FR regions ofthe immunoglobulin V H fragment depicted in one of Figures 1 or 2 and wherein said variants are encoded by nucleic acid sequences which vary from the nucleic acid sequence encoding said parental ligand-binding molecule in a sub-sequence (at least one) encoding at least part of one of the CDRs of said parental ligand-binding molecule, preferably the CDR3, whereby said plurality of variants comprise at least, in substantial part, the FR regions ofthe immunoglobulin V H fragment depicted in one of Figures 1 or 2 and are differentiated, at least
  • the A6-based parental ligand molecule comprises (and therefore preserves within members ofthe library), in substantial part (subject to at least partial randomization of selected regions of one of the CDRs, preferably the CDR3, to create binding diversity within the library), one or more of the CDR regions ofthe A6 V H fragment, particularly the CDR3.
  • at least the length of the wild-type V H CDR3 (23 amino acids) and preferably also elements of its amino acid composition is preserved or at least partially preserved (approximately 16-23 amino acids and more particularly 18 to 23 amino acids).
  • the CDR3 may also be lengthened by approximately 1 to 10 residues.
  • the library may optionally have representation of binding molecules having CDR3s of varying lengths.
  • the parental ligand-binding molecule is a dAb fragment. It is known that a dAb molecule , due to the removal of its light chain partner, tends to, in most, if not all cases, aggregate, in varying degrees due to the "sticky" nature ofthe V L interface. This stickyness is attributable, at least in part, to the hydrophobic nature ofthe V H residues at this interface. This stickyness results in substantial dimer and/or multimer formation which may reduce, on the whole, the solubility characteristics of members ofthe library.
  • A6 V H amino acid residues at the VL interface are substituted by residues which tend to minimize aggregate formation, for example, hydrophilic amino acids, and preferably one or more ofthe substitutions reflected in Figure 2, relative to Figure 1.
  • substitutions are not fixed within the entire population ofthe library, but are introduced by randomizing or partially randomizing various A6 V H amino acid residues, particularly including FR residues, among the residues at the interface, (see for example, Padlan et al "Anatomy of the Antibody Molecule” Molecular Immunology Vol. 31, pi 69- 217, Table 25 for itemization and related discussion of these residues).
  • FR regions other than, or in addition to, modifications to the V L interface (FR2) may be modified by at least partial randomization, for example, one or both of FR1 (one or more of residues 4 to 21) and FR4 (one or more of residues lOOo to 113) to improve, on the whole, the solubility characteristics of members ofthe library (for example, biasing at least some and preferably all of one or both of these sets of residues (at least 70% or more), preferably 90% in favour ofthe parental amino acid constitution to achieve 10% randomization).
  • FR1 one or more of residues 4 to 21
  • FR4 one or more of residues lOOo to 113
  • the present invention provides a heterogeneous population of genetic packages (eg. phage) having a genetically determined outer surface protein, wherein the genetic packages collectively display a plurality of different, preferably human, (ie. having substantial identity, preferably at least 80% homology to human framework and other conserved regions) V H ligand-binding fragments, each genetic package including a nucleic acid construct coding for a fusion protein which comprises at least a portion ofthe outer surface protein and a variant of at least one soluble parental ligand-binding fragment preferably derived from or having a substantial part of the FR regions ofthe amino acid sequence identified in one of Figures 1 or 2 (or a sequence at least 80%, preferably 85 to 100%, more preferably 90-100%, homologous (% identity) thereto), wherein the variant V H ligand-binding fragments preferably span from a position upstream of an immunoglobulin heavy chain CDR1 to a position downstream of CDR3 (preferably including substantially all of
  • the parental V H binding-fragment naturally has a long CDR3 that is amenable to forming exposed loops for binding into cavities.
  • the parental V H ligand-binding fragment is built on a human framework or is adapted from or adaptable to a human framework.
  • the preferred binding region of the variants (corresponding to the randomized or partially randomized part ofthe CDR3) is located in carboxy terminal region ofthe CDR3.
  • a substantial part ofthe amino acid sequence identified in Figure 1, preferably including at least part ofthe CDR3, supplies the preferred amino acid constitution ofthe various preferred parental ligand-binding molecules, such that a population of variant heavy chain ligand-binding molecules built on this framework of amino acids are on the whole better adapted to be or better capable of being expressed as soluble proteins.
  • FIG. 1 is a sequence diagram showing a parental V H ligand-binding molecule (A6) according to the invention.
  • FIG. 2 is a sequence diagram showing another parental V H ligand-binding molecule (A6.1) according to the invention, additionally showing modified nucleic acid bases corresponding to amino acids 24 and 25, for introducing the Nhel site. Introduction of this Nhel site does not alter the amino acid constitution of A6.1.
  • Figure 3 is a sequence diagram showing the A6 V H ligand-binding molecule (encoded by A6- chi(-)) according to the invention, in which the nucleic acid residues co ⁇ esponding to amino acids 3 to 16 of A6 wild-type have been modified to remove a putative recombination site, leaving the amino acid constitution of A6 unchanged.
  • Figure 4 is a sequence diagram showing the A6.1 V H ligand-binding molecule (encoded by A6.1-chi (-)) according to the invention, in which nucleic acids co ⁇ esponding to amino acids 3 to 16 of A6.1 have been modified to remove a putative recombination site, leaving the amino acid constitution of A6.1 unchanged. The altered nucleic acid residues corresponding to Nhel are also shown.
  • Figure 5 is a facsimile of an SDS-PAGE showing high expression of human A6.1 dAb in E. Coli.
  • Figure 6 shows size exclusion chromatograms of molecular weight markers (A), dAb (B) and A6.1 dAb obtained by gel filtration obtained using a Superdex 75 column. The masses of the markers were 2,000, 67, 43, 25 and 14 kDa.
  • Figure 7 is a size exclusion chromatogram of A6.1 dAb following IMAC purification showing molecular weights associated with the peaks.
  • Figure 8 is an NMR 2-D spectra showing the molecular configuration of two embodiments of an A6.1 based dAb.
  • the two 15N-1H HSQC spectra are: a: R3A10(Cys-), the spectrum was acquired at 308 K; b: M2R2-1, the spectrum was acquired at 298 K.
  • Figure 9 is diagrammatic representation of the amino acid substitutions in parental ligand- binding molecule A6.1 and A6.1C relative to wild-type A6.
  • FIG. 10 is a sequence diagram showing a parental V H ligand-binding molecule designated A6.1C.
  • Figure 11 is a graphic representation ofthe binding characteristics of A6.1C library binders to 3B1 and control BS A.
  • Figure 12 is a sensogram overlay showing the binding characteristics of a potential V H binding fragment generated against anti-FLAG antibody (M2) using a phage display library ofthe invention.
  • Figure 13 is a diagrammatic representation of vector, SJFI, used to create the vector into which the library is cloned.
  • Figure 14 is a listing ofthe nucleotide and amino acid sequence of A6 V H after introduction ofthe Nhel site and removal ofthe putative recombination site at amino acid residues 3 to 16.
  • Figure 15 is a schematic representation of steps taken to remove the putative recombination site ofthe 5 end ofthe A6 V H gene.
  • SEQ. ID. NO. 1 co ⁇ esponds to the nucleic acid sequence shown in Figure 1.
  • SEQ. ID. NO. 2 co ⁇ esponds to the amino acid sequence shown in Figure 1.
  • SEQ. ID. NO. 3 co ⁇ esponds to the amino acid sequence of CDR1 shown in Figure 1.
  • SEQ. ID. NO. 4 co ⁇ esponds to the amino acid sequence of CDR2 shown in Figure 1.
  • SEQ. ID. NO. 5 co ⁇ esponds to the amino acid sequence of CDR3 shown in Figure 1.
  • SEQ. ID. NOS. 6-11 and 25 co ⁇ espond to the nucleotide sequences of primers disclosed herein.
  • SEQ. ID. NOS. 12-24 correspond to the amino acid sequences of CDR3 variants disclosed herein at Table 2.
  • the invention is directed to a population of genetic packages having a genetically determined outer surface protein including genetic packages which collectively display a plurality of different ligand-binding molecules in association with the outer surface protein, each package including a nucleic acid construct coding for a fusion protein which is at least a portion ofthe outer surface protein and a variant of at least one soluble parental ligand-binding molecule derived from or having the amino acid sequence identified in Figure 1 (or a sequence preferably at least 80% homologous in the framework and conserved regions thereof), wherein at least part ofthe construct, preferably including at least part ofthe CDR3 identified in Figure 1, encodes or is biased in favor of encoding, the amino acid constitution ofthe parental ligand binding fragment such that the plurality of different ligand-binding domains are on the whole better adapted to be or better capable of being expressed as soluble proteins.
  • the variant V H ligand-binding molecules are preferably characterized by a CDR3 having 16 to 33 amino acids.
  • the replicable genetic package is a recombinant phage and the heterogeneous population of replicable genetic packages collectively constitute a phage display library.
  • the parental ligand-binding molecule is a V H binding fragment, and the plurality of variant ligand-binding fragments are expressed in the absence of light chains.
  • the parental ligand-binding-molecule is a natural occurring antibody or fragment thereof, having a natural human V interface.
  • the V L interface is engineered to avoid hydrophobic amino acids.
  • the V L interface is engineered for amino acids, which form weak interactions.
  • the parental ligand binding molecule has a camelid type V L interface.
  • at least one ofthe V L interface amino acids are randomized or partially randomized in the construction ofthe library.
  • the potential V H binding fragments include the entire FRl through to FR4 regions, although it is to be understood that partial deletions, for example, within CDR2, are contemplated to be within the scope ofthe invention.
  • CDR3s of a variety of different lengths from 16 to 33 amino acids are predominantly represented among the potential V H binding fragments.
  • CDR3s of a variety of different lengths, from 18 to 28 amino acids, or from 20 to 25, or from 18 to 23, amino acids are predominantly represented in the library.
  • the parental V H ligand-binding fragment is built on a human framework and preferably is the parental V H ligand-binding fragment identified in Figure 1 which has a CDR3 of 23 amino acids in length.
  • the invention encompasses a phage display library which is constructed using a parental V H ligand-binding molecule derived from a human parental V H ligand-binding fragment, or is built on any framework which is at least 80% (preferably 85%, more preferably 90 to 95%) homologous to the framework and other conserved regions of a fully human V H chain.
  • the invention also contemplates that the parental V H binding-fragment, though not human, is adapted (eg. humanized) or adaptable (eg. to be adapted after selection of prefe ⁇ ed binders) to a human framework.
  • the invention also contemplates the random, biased or fixed occu ⁇ ence of features disclosed in the camelid literature, for example pairable cysteines in CDRl and CDR3 (optional) and/or the substitution of hydrophilic amino acids at least one of positions 44, 45, and 47 and preferably also positions 93 and 94 (Kabat numbering system).
  • the parental ligand-binding molecule is a V H fragment derived from a human IgM heavy chain, and preferably comprises FRl through FR4 ofthe V H chain.
  • a partial sequence ofthe preferred antibody BT32/A6 (A6) is disclosed in U.S. Patent No. 5,639,863, incorporated herein by reference. The entire sequence is supplied now in Figure 1.
  • Figure 1 the CDR regions are demarcated.
  • the amino acid residue numbers in Figure 1 and throughout the disclosure refer to the Kabat numbering system (Kabat et al. 1991, Sequences of Proteins of Immunological Interest, publication No. 91-3242, U.S. Public Health Services, NIH, Bethesda MD) except in the sequence listings and where explicitly stated or otherwise implied.
  • Figure 1 co ⁇ esponds to SEQ. ID. NO. 1 (nucleic acid) and SEQ. ID. NO. 2 (amino acid).
  • Figure 1 demarcates and labels regions CDRl (corresponding to SEQ. ID. NO. 3), CDR2 (SEQ. ID. NO. 4) and CDR3 (SEQ. ID. NO. 5).
  • prefe ⁇ ed solubility characteristics for creating dAb libraries.
  • prefe ⁇ ed solubility characteristics refers to at least one ofthe several, often co ⁇ elated, characteristics including good yield, expression as a soluble product (as opposed to inclusion bodies) within the periplasm ofthe host organism, eg. Escherichia. Coli. and a reduced tendency to dimerize and other aggregate formation.
  • polypeptide polypeptide
  • peptide protein
  • protein protein
  • combinatorial library is used herein to refer to a set of molecules, typically belonging to a defined (na ⁇ owly or broadly) class comprising a substantial number of potentially useful variants, wherein the variations in the molecule represent a complete or partial set of permutations or combinations of at least some constituent elements of a reference molecule, which is typically a template or "parental” molecule, or simply the class itself.
  • the constituent elements are amino acids and nucleic acid bases, respectively.
  • the phrase "in substantial part” refers to variations relative to a referenced molecule which do not significantly impair the "functionality" of that molecule.
  • functionality refers primarily to the solubility and binding characteristics ofthe molecule.
  • Such variations ie. the referenced molecule in substantial part
  • a substantial part ofthe framework would preferably refer to at least 80% identity of the amino acid residues and more preferably an 85 to 100% identity, and even more preferably at least a 90% identity ofthe amino acid residues.
  • ligand-binding fragment is used broadly to define the whole or any part of an antibody that is capable of specifically binding to any ligand, in the broadest sense of the term ligand.
  • An A6-based human heavy domain ligand-binding-fragment is well suited for the development of a combinatorial library (optionally a phage display library) that is used to generate soluble binding fragments that are useful for human diagnosis and therapy (due to limited HAMA response).
  • phage display libraries are used to selectively generate molecular probes that specifically interact with a ligand, including without limitation, natural and synthetic molecules and macromolecules and can be used in vitro (i.e., a diagnostic) and in vivo (i.e., a diagnostic and/or therapeutic) as indicators, inhibitors and immunological agents.
  • the types of natural and synthetic molecules and macromolecules include but are not limited to: antibodies and fragments thereof; enzymes; cell receptors; proteins, polypeptides, peptides; polynucleotides, oligonucleotides; carbohydrates such as polysaccharides, oligosaccharides, saccharides; lipids; organic-based and inorganic-based molecules such as antibiotics, steroids, hormones, pesticides, herbicides, dyes, polymers.
  • A6.1 V H has preferred solubility characteristics.
  • This SDS-PAGE shows a particularly heavy band showing strong expression in E. Coli of an A6.1 dAb, designated R3A10.
  • R3A10 was expressed as a soluble V H in E. Coli. Yields as high as 55 mg/L of bacterial culture were obtained by IMAC chromatography of periplasmic extracts. The single domain product was shown to be highly pure and homogeneous by SDS-PAGE ( Figure 5).
  • the protein yields of many dAbs from the A6.1 library were above 5 mg per liter of bacterial culture in shaker flasks. Some had yields more than 10 mg and one over 50 mg.
  • the solubility ofthe wild type and the camelized versions were very high as shown by NMR studies.
  • R3A10 and M2R2-l(Cys " ) for example, were soluble in mM concentrations over extended periods of time allowing good quality NMR data collection.
  • a NMR structure of a human VH camelized in this manner has been described (Reichmann, J Mol, Biol) but in order to reduce aggregation and achieve sufficient solubility CHAPS detergent had to be added to the sample during NMR data collection.
  • the A6.1 dAb molecules described here were completely free of aggregated material in the absence of detergent.
  • V H heavy chain variable domain
  • Single domain antibodies derived from these antibodies are highly soluble and the structural basis of solubility has been partially elucidated.
  • conserved human/murine interface residues such as Val37, Gly44, Leu45 and Trp47 are generally replaced in heavy chain antibodies by tyrosine or phenylalanine, glutamate, arginine or cysteine, and glycine, respectively.
  • These mutations increase the hydrophilicity ofthe V L interface either by non-polar to polar substitutions or, in a more subtle way, by inducing local conformational changes (Desmyter, Transue, et al., 1996) (Spinelli, Frenken, et al.,).
  • the disulfide linkage imparts rigidity on the CDR3 loop which extends out ofthe combining site and penetrates deep into the active site of lysozyme (Desmyter, Transue, et al., 1996).
  • a second feature is the longer average length ofthe V H H CDR3, relative to human or murine V H s (Muyldermans, Atarhouch, et al., 1994).
  • a longer CDR3, which is a feature of A6, increases the antigen binding surface and, to some extent, compensates for the absence ofthe antigen binding surface provided by the V L in conventional antibodies (Desmyter, Transue, et al., 1996).
  • a third feature is the absence ofthe CDR3 salt linkage that is typically present in conventional antibodies and formed by arginine or lysine residues at position 94 and aspartate at position 101 (Desmyter, Transue, et al., 1996) (Muyldermans, Atarhouch, et al., 1994) (Spinelli, Frenken, et al., 1996) (Davies & Riechmann, 1996) (Chothia & Lesk, 1987) (Morea, Tramontano, et al., 1998).
  • dAbs are an attractive alternative to scFvs because of their much smaller size and the fact that they demonstrate affinities comparable to those demonstrated by scFvs (Ward, Gussow, et al., 1989) (Spinelli, Frenken, et al., 1996) (Lauwereys, Arbabi, et al., 1998) (Davies & Riechmann, 1995) (Arbabi, Desmyter, et al., 1997) (Reiter, Schuck, et al., 1999). Smaller size is an advantage in applications requiring tissue penetration and rapid blood clearance. Smaller molecules also offer a tremendous advantage in terms of structural studies (Davies & Riechmann, 1994) (Constantine, Goldfarb, et al., 1992 (Constantine, Goldfarb, et al, 1993).
  • Phage antibody library construction is much simpler and more efficient if single domain antibodies are used instead of Fabs or single chain Fvs. Randomization can be introduced at a much higher percentage of CDR positions without exceeding practical library size. The problem of shuffling original V L -V H pairings is also avoided.
  • Camelid phage dAb libraries constructed from the V H H repertoire of camels immunized with target antigens have performed well (Arbabi, Desmyter, et al., 1997) (Lauwereys, Arbabi, et al., 1998) (Decanniere, Desmyter, et al., 1999). However, construction of libraries from immunized camels presents obvious problems.
  • the parental ligand-binding fragment has amino acid substitutions at V L interface which reduce the tendency to aggregation attributable to the "stickyness" of the V H dAb at this interface.
  • the parental ligand-binding fragment has a long CDR3 similar to some camelid antibodies.
  • an A6 dAb based library is prefe ⁇ ed, because A6 has an unusually long CDR3 of 23 amino acids.
  • the library preserves the entire length of this CDR3 and at least one of positions 44, 45 and 47 are altered, preferably 44 or 45 to camelid type residues.
  • the CDR3 was randomized and cysteine residues were introduced at positions 33 and lOOe in the expectation that the residues would form the CDR1-CDR3 disulphide bridge present in the camel antibody Cab-Lys3 (Desmyter, Transue, et al. 1996).
  • the library was evaluated by panning against an IgG that binds a peptide of known sequence. Procedures for the construction and testing of this library is described in examples 5 through 13 and below as follows.
  • A6 dAb is expressed surprisingly well as a soluble product in E. coli with a yield of approximately 10 mg per liter of bacterial culture .
  • Mass spectrometry has confirmed that the product has the expected molecular weight.
  • size exclusion chromatography ofthe product reveals three components that are thought to co ⁇ espond to monomer, dimer and higher oligomer on the basis of their elution volumes ( Figure 6A and B).
  • the monomer peak elutes unusually late suggesting that the dAb is interacting non-specifically with the gel matrix possibly through its exposed hydrophobic V L interface.
  • the dAb was further modified by introducing Val93 Ala and Lys94Ala mutations in FR3 and an Ala33Cys mutation in CDRl .
  • a prefe ⁇ ed library having the itemized substitutions at positions 44, 45, 47 93 and 94 is refe ⁇ ed to as "A6.1” Libraries with Ala 33 Cys and Cys at position 1 OOe (see discussion below) are termed "A6.1C". These substitutions are diagrammatically illustrated in Figure 9 and the sequences for A6.1 and A6.1C are shown in Figures 2 and 10, respectively.
  • positions 93 and 94 are predominantly occupied by Ala residues and Cys is frequently found at position 33 (Muyldermans, Atarhouch, et al., 1994) (Vu, Ghahroudi, et al., 1997).
  • the library was constructed by randomizing 19 amino acids in CDR3, leaving the last three residues, PhelOO, AsplOl, and Tyrl02, unchanged (Davies & Riechmann, 1995).
  • the degenerate oligonucleotide used for randomization was designed so as to always introduce Cys at position lOOe.
  • the library was initially placed in a phagemid vector. Following transformation the size ofthe library was determined to be 2.1 x 10 . Of 80 randomly picked clones analyzed by PCR all but one had the dAb insert. In addition, all twenty that were sequenced were unique, demonstrating the diversity ofthe library. To convert the display format from monovalent to multivalent, the library was sub-cloned into a phage vector (MacKenzie and To, (1998). Following transformation, the size ofthe library was determined to be 6.6 x 10 7 . Therefore, on a random basis each member ofthe original phagemid library is represented 3 times.
  • the library was panned, in both formats, against 3B1 scFv, which is specific for a bacterial carbohydrate (Deng, MacKenzie, et al., 1994).
  • the phagemid vector format panning failed to enrich for binders and PCR analysis of clones selected at different stages of the panning process revealed almost universal deletion ofthe dAb inserts.
  • the phage vector seven different dAbs that bound to 3B1 were identified. As shown in Figure 11, these dAbs bound to the target antigen, 3B1, in ELISA experiments and showed no detectable binding to the control BSA.
  • the consensus sequence was present at the extreme C-terminal end of CDR3 (see Table 1 and Table 2 below).
  • the library was also panned against M2 IgG, an antibody which was raised against the FLAG peptide DYKDDDDK (Knappik & Pluckthun, 1994). More recent studies showed that M2 recognizes the consensus sequence XYKXXD and prefers epitopes with aspartate at the first position (Miceli, Degraaf, et al., 1994). Twenty- four different dAbs with the FLAG consensus sequence were identified the sequencing of clones randomly selected after 3 rounds of panning (Table 2 below).
  • M2R2-l a M2R2-l
  • b M2R2-2 c M2R2-4
  • d M2R2-5 e M2R2-9
  • f M2R2-10 g M2R2-13; h M2R2-14; i M2R2-15; j M2R2-18; k M2R3-4; ⁇ RS-IS.
  • A6VH displays a number of features that makes it a desirable template for camelized library construction.
  • BT32/A6 offers the option for introduction of a CDR1-CDR3 disulphide bridge. Formation ofthe disulphide bond was confirmed for several sequences and introduction ofthe two cysteines did not have a negative impact ofthe yield of soluble product. For M-2 binders the presence of the disulfide imposed a constraint that prevented optimal interaction with M-2. In other instances, however, the presence ofthe bridge would probably be advantageous since this is a common feature of heavy chain antibodies. Construction and pooling of two libraries, one with and one without the bridge, would appear to be advantageous.
  • the preferential use ofthe C-terminus as an antigen contact region is in sharp contrast to an anti- lysozyme dAb where all the antigen contacting residues of CDR3 are located at its N- terminal half (Desmyter, Transue, et al., 1996),
  • the dAb are displayed 3-5 copies and therefore there is potential for avidity which increases the likelihood of isolating weak binders (Nissim, Hoogenboom, et al., 1994).
  • the randomized positions in A6 and A6.1 libraries are preferably at positions lOOi to lOOn as indicated by the data demonstrating binding in the C-terminal regionof the CDR3 loop.
  • CDRl positions could be identified for limited randomization. Libraries containing shorter and partially randomized CDR3 could be constructed and pooled to further increase diversity.
  • Figure 15 is a schematic representation ofthe steps taken to remove the recombination site at the 5' end ofthe A6VH gene.
  • plasmid pSJF-A6VH as template and 1 & 3 and 2 & 4 primer pairs, two overlapping fragments were constructed by PCR. From these, a larger construct (Fgmtl) was assembled by splice overlap extension (SOE) and further amplified by PCR using primers 1 and 2.
  • SOE splice overlap extension
  • the present inventors have also found a method of enhancing the probability that the binding fragments displayed in the library have characteristics which approximate the desired solubility characteristics found in the wild type binding fragment.
  • nucleotides ofthe variable region are added in a step-wise addition and by selecting a nucleotide ratio which is biased in favor of producing amino acids which reflect the DNA of the parental or wild type species.
  • a method for biasing a library in favor of obtaining selected percentages of wild type amino acid residues is achieved by creating residue substitutions by using different spiking levels ofthe various dNTPs as described below.
  • the randomization of amino acids is often achieved by DNA synthesis.
  • a primer is annealed next to DNA encoding for the variable region, and nucleotides are randomly added to synthesize randomized variable regions.
  • nucleotide ratio of 1 : 1 : 1 : 1 , which generates a totally random variable region.
  • the likelihood of achieving affinity or other desirable traits found in the wild type as follows.
  • a dNTP ratio which is biased in favor of producing amino acids which reflect the DNA of the parental (wild type) species.
  • Table 3 charts particular amino acid residues or sequences of residues and prefe ⁇ ed types of amino acid substitutions according to various examples ofthe invention to be defined hereafter.
  • the selection of amino acids for randomization or partial randomization is based on adopting one or more of a variety of approaches including one of more ofthe following:
  • phrases such as at least approximately 10%, or approximately 10-100% are intended to specify a preference for each ofthe unit percentages between about 7 and 100% that are practically achievable by oligonucleotide primer design and PCR amplification described herein below, as well as other well known PCR techniques and techniques of Controlled Mutation described in the art, and routine variations of such techniques.
  • phrases such as at least 80% are intended to specify a preference for each ofthe unit percentages between 80% and 100%. It is to be understood that biasing of a percentage less than 100% implies unless otherwise implied or stated that the remaining percentage is fully randomized.
  • 90% biasing in favor of wild-type amino acids at a given amino acid position is to be approximated by controlling the percentage amounts of each ofthe three relevant nucleotides (so that, for example, the product ofthe probabilities of occurrence ofthe three desired nucleotides in sequence in the growing chain is 90%) so as to supply 90% of co ⁇ ect coding triplet(s) and a total of 10% of random coding triplets, having regard to the degeneracy of the genetic code (for example if two different coding triplets result in a given amino acid, then the sum ofthe probabilities of achieving those two triplets will have to equal 90%).
  • the library produced may be a pooled library in which several libraries each having varying degrees of biasing to wild-type, for example, 60%, 50%, 40% and 30%, are pooled together to obtain the both desired variability and similarity.
  • the prefe ⁇ ed parental binding-fragment may be engineered to maximize the desired characteristic (e.g. solubility, intermolecular interaction) and then made the subject of libraries with varying degrees of biasing.
  • the library could be biased to be rich in amino acids, which are highly soluble.
  • both arms (halves) ofthe prefe ⁇ ed longer loop forming CDR3s may be biased to amino acids that are favored for intermolecular interaction, preferably charged amino acids, so as to provide a method of generating, in addition to loop size, varying loop structures.
  • This bias may be systematically introduced or systematically reduced by randomization, in cooperating pooled libraries having varying degrees of biasing.
  • CDR3s of a variety of different lengths from 16 to 33 amino acids are predominantly represented among the variant V H ligand-binding fragments.
  • CDR3s of a variety of different lengths, from 18 to 25 amino acids, or, from 18 to 23 amino acids are predominantly represented in the library.
  • the term "predominant" ordinarily implies a majority representation of the specified long CDR3 variant V H ligand-binding fragments, the invention also contemplates an even less substantial representation, especially within a reasonably large size library (>10 7 ).
  • the specified long CDR3 variant V H ligand-binding fragments have a majority representation within the library and more preferably an even greater or exclusive representation.
  • the parental V H ligand-bindingmolecule is reduced in size and the parental V H ligand-binding molecule is optionally modified by deleting a portion ofthe CDR2.
  • CDR3s ofthe same length as that of the parental V H ligand-binding molecule are predominantly or exclusively represented in the variant V H ligand-binding fragments.
  • the CDR3 region is specifically retained along with human sequence elements of other regions that confer favorable characteristics solubility, to create a phage display library having favorable characteristics of solubility, preferably when compared with variant V H ligand-binding fragments that have fully randomized hypervariable regions (particularly CDR3).
  • the present inventors have found that favorable solubility characteristics of a parental V H ligand-binding molecule can be maintained in the population of variant V H ligand-binding fragments in the course of randomizing the hypervariable regions by biasing all or selected amino acids residues to wild-type and/or biasing in favor of amino acids residues that favor certain or a variety of types of intermolecular interaction.
  • variant V H ligand-binding fragments having relatively long CDR3s of varying lengths are produced by randomly or partially randomly inserting varying numbers of nucleotide triplets in any part of a randomized portion ofthe parental V H framework. Primers ofthe desired length and nucleotide composition are synthesized followed by PCR amplification. Desired randomization can be achieved by biasing nucleotide composition ofthe primer.
  • the production of displays of long CDR3 variant binders may also be accomplished by pooling several libraries of variantV ⁇ ligand-binding fragments having randomized or partially randomized CDR3s of different respective uniform lengths. These strategies are not mutually exclusive.
  • Binasing biased in favor of and related forms of these terms are generally intended to refer to weighting in the course of introducing variation in the parental ligand-binding molecule.
  • “Homologous” or “homology” as used herein refers to "identity” or “similarity” as used in the art, meaning relationships between two or more polynucleotide or amino acid sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Both identity and similarity can be readily calculated (Lesk, A. M., ed., Computational Molecular Biology, Oxford University Press, New York, 1988; Smith, D. W., ed., Biocomputing: Informatics and Genome Projects, Academic Press, New York, 1993; Griffin, A. M., and Griffin, H.
  • Methods commonly employed to determine identity or similarity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D. (1988, SIAMJ. Applied Math., 48: 1073). Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al.(1984), Nucleic Acids Research 12(1): 387), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al.(1990), J. Molec. Biol. 215: 403).
  • Percent homology or “% homologous” or related terms include both ofthe following interpretations / methods of calculation: 1) an approximate percentage ofthe sequence referenced in terms of the number of common residues (e.g. 80% of 11 is understood to be an approximation insofar as application ofthe percentage does not yield a unit number of residues, in which case both the immediately higher number and immediately lower unit numbers, 9 and 8 respectively, are deemed to be covered); 2) the percentage of binding fragments theoretically achievable that have the full wild-type sequence, which is calculated as a product ofthe probabilities that the wild-type amino acid will occur at a given amino acid position.
  • Consed regions refer to those which are commonly found in at least other antibodies of the same type or in at least the same species of mammal.
  • Wild-type refers to the parental binding-fragment, which may be a variant ofthe natural or to the native A6 V H parental ligand-binding fragment, depending on the context.
  • Step-wise refers to the addition of, for example, nucleic acids, in a manner such that the quantity of nucleic acids added at each step is rigorously control, usually one nucleic acid at a time.
  • “Camelid type” refers specifically to one or more features ofthe camelid V L interface.
  • Soluble includes the generally ascribed meaning in the art and without limitation includes (based on solubility co ⁇ elated phenomena) the relative amounts of naturally- folded recombinant protein released from the cell.
  • Percent biasing or “% of binding fragments” refers to biasing on an individual amino acid basis (though other techniques to accomplish the same effect might apparent to those skilled in the art).
  • the specification that wild-type amino acids occur at a specified position or series of positions in, for example, at least approximately 50% of potential binding fragments is intended to mean both that 50% biasing is sought at a given such position or that a total of 50% ofthe correct nucleotide triplets are represented.
  • Suitable parental binding-fragments include any known in the art and include the group consisting of an scFv, Fab, V H , Fd, Fabc, F(ab') , F(ab) derived from A6.
  • Nucleotide sequences can be isolated, amplified, and processed by standard recombinant techniques. Standard technique in the art include digestion with restriction nucleases, and amplification by polymerase chain reaction (PCR), or a suitable combination thereof. PCR technology is described in U.S. Patent Nos. 4,683,195; 4,800,159; 4,754,065; and 4,683,202, as well as PCR: The Polymerase Chain Reaction, Mullis et al., eds., Birkauswer Press, Boston (1994).
  • oligonucleotide primers consisting of specified amino acid residues (one or more) of the wild-type A6 molecule (e.g. CDR3 residues), mixing these in appropriate concentrations with a completely randomized (e.g. CDR3) oligonucleotide primer and subjecting the mixture of oligonucleotide primers to PCR.
  • CDR3 residues a completely randomized oligonucleotide primer and subjecting the mixture of oligonucleotide primers to PCR.
  • Polynucleotides comprising a desired sequence can be inserted into a suitable vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification.
  • Polynucleotides can be introduced into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, f-mating or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell by standard methods. See, e.g., Sambrook et al. (1989). RNA can also be obtained from transformed host cell, or it can be obtained directly from the DNA by using a DNA-dependent RNA polymerase.
  • Suitable cloning and expression vectors include any known in the art, e.g., those for use in bacterial, mammalian, yeast and insect expression systems. Specific vectors and suitable host cells are known in the art and are not described in detail herein. See e.g. Gacesa and Ramji, Vectors, John Wiley & Sons (1994). Phage display techniques are generally described or referenced in some of the preceding general references, as well as in U.S. Patent Nos. 4,593,002; 5,403,484; 5,837,500; 5,571,698; 5,750,373; 5,821,047; 5,223,409 and 5,702,892. "Phage Display of Peptides and Proteins", (Kay, Brian K.
  • DNA encoding millions of variants of a parental binding-fragment can be batch- cloned into the phage genome as a fusion to the gene encoding one ofthe phage coat proteins (pill, pVI or pVIII).
  • the coat protein fusion will be inco ⁇ orated into new phage particles that are assembled in the bacterium.
  • Expression ofthe fusion product and its subsequent inco ⁇ oration into the mature phage coat results in the ligand being presented on the phage surface, while its genetic material resides within the phage particle. This connection between ligand genotype and phenotype allows the enrichment of specific phage, e.g. using selection on immobilized target.
  • Phage that display a relevant ligand will be retained, while non-adherent phage will be washed away. Bound phage can be recovered from the surface, reinfected into bacteria and re-grown for further enrichment, and eventually for analysis of binding.
  • the success of ligand phage display hinges on the combination of this display and enrichment method, with the synthesis of large combinatorial repertoires on phage.
  • phage While the use of phage is described as an embodiment for the production of libraries for displaying, and selecting particular binding fragments, it is to be understood that and suitable genetic package may be used for the production of libraries ofthe invention.
  • suitable genetic packages include cells, spores and viruses (see US Patent No. 5,571,698), or any other suitable replicable genetic packages.
  • cell based approaches Another popular method of presenting a library is the two-hybrid system (Feilds and Stemglanz, 1994, Trends in Genetics 10:286-292).
  • Ribosome display is a well documented technique that may be useful for generating libraries. This entirely in vitro method allows for libraries with a diversity of >10 12 .
  • a peptide is displayed on the surface of a ribosome that is translating it.
  • a library of mRNA molecules (we could start with A6) is translated in vitro translation system to the 3' end, such that the ribosome does not fall off.
  • the protein emerges from the ribosome in such a way that it can fold, but does not fall off. In some instances, there is an additional folding step in an oxiding environment (important for proteins with disulfide bonds).
  • the whole complex of folded protein, ribosome and mRNA, which is stable for several days, can then be panned against a ligand that is recognized by the translated protein.
  • the translated protein could be an antibody and the ligand is its antigen.
  • the mRNA can then be amplified by reverse transcription and PCR. This technique has been used to successfully generate scFv antibody fragments with high affinity for their target. Reference is made to Hanes,J., Jermutus,L., Weber-Bornhauser,S., Bosshard,H.R. & Pluckthun,A. Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries. Proc. Natl. Acad. Sci.
  • A6 V H gene was used as a PCR template to amplify a shorter internal fragment employing the primers A6V H / ⁇ heI- 5'(TGTTCAGCTAGCGGATTC)3' and A6V H /BstEII- 5 '(TGAGGAGACGGTGACCGTTGTCCCTTGGCCCCAGATATCAAA)3 ' .
  • PCR polymerase chain reaction
  • the amplified product was purified using QIAquick PCR Purification kitTM (QIAGEN, Mississauga, ON), digested with Nhel and BstEll restriction endonucleases and subsequently ligated to the NAeI/__tetEII-restricted pSJFl-10A12 vector derived from pUC 8 (Narang et al., 1987) to replace a portion ofthe existing A6 V H gene.
  • QIAquick PCR Purification kitTM QIAGEN, Mississauga, ON
  • the pUC 8 plasmid (Vie ⁇ a and Messing, 1982; Messing, 1983) was modified by inserting the OmpA signal sequence and the His -carboxy tail between the ⁇ coRI and Hindlll restriction sites ofthe pUC 8 polylinker region, using oligonucleotide primers and PCR (Narang et al., 1987).
  • Electro-competent E.coli TGI cells were prepared (Tung and Chow, 1995) and an aliquot of the ligated product was used to transform the cells. Transformation was carried out using the BIO-RAD Gene PulserTM (Bio-Rad Laboratories, Mississauga), ON according to the manufacturer's instructions and the clone harbouring the mutated A6 dAb gene was confirmed by sequencing (Sanger, F. et al., 1977) using the AmpliTaq DNA Polymerase FS kit and 373A DNA Sequencer Stretch (PE Applied Biosystems, Mississauga, ON). All the cloning steps were performed as previously described (Sambrook et al. 1989). The resulting vector is termed pSJFl-A6VH.NjeI.
  • A6 dAb library construction involved a series of sequential PCR experiments.
  • the PCR mixture contained 200 M each ofthe four d ⁇ TPs, 0.2 pmol/ 1 each ofthe two primers, IX buffer (Perkin Elmer), and 0.05 units/ 1 of AmpliTaq D ⁇ A polymerase (Perkin Elmer). (The former primer also introduces Apa ⁇ l site at the 5' end ofthe PCR product.)
  • the amplified fragment from step (1) was purified by QIAquick Gel Extraction kitTM (QIAGE ⁇ ) and subsequently used as the template in a second PCR reaction using the primers A6VH._4/. ⁇ /I and A6VH.RndmCDR3.F - 5' (GCCCCAGATATCAAA20 [((A/C) ⁇ )]TTTCACACAGTAATA)3 ⁇ At the protein level the second primer results in the randomization ofthe first 20 residues in CDR3.
  • the PCR mixture was identical to above except that the concentration ofthe primers was increased to 0.5 pmol/1 to ensure that sufficient amounts of oligonucleotide primers and dNTPs were provided for the generation of a large randomized library.
  • Example 4 Addition of a Notl restriction site, ligation to the phage vector and library construction.
  • amplified fragments were purified as above and used as templates in a third round of PCR employing 2 pmol/ul each ofthe two primers A6VH/ApaK (described above) and A6VH.NotI.EXT.F - 5 ' (CGATTCTGCGGCCGCTGAGGAGACGGTG ACCGTT-
  • the latter primer inco ⁇ orates the Notl site (underlined) at the 3' end ofthe amplified products.
  • the amplified fragments were purified using QIAquick PCR Purification kitTM (QIAGE ⁇ ), digested with ApaR and Notl, and ligated to ⁇ /. ⁇ /I/Notl-digested fd-tet phage vector (McAfferty et al., 1990; Zacher et al., 1980).
  • the ligated product was desalted using QIAquick PCR Purification kitTM (QIAGE ⁇ ).
  • the SOC medium per L: bacto-tryptone, 20 g; bacto-yeast extract, 5 g; ⁇ aCl, 0.5 g; glucose, 3.6 g
  • a small aliquot ofthe electroporated cells were serially diluted in exponentially growing E. coli strain TGI cells.
  • Two hundred ⁇ l ofthe diluted cells were mixed with 3ml of 50°C top agar and immediately poured onto 2xYT (per L: bacto-tryptone, 16 g; bacto-yeast extract, 10 g; ⁇ aCl, 5 g) plates pre-warmed to 37°C.
  • the recombinant phage vectors 1.5 ⁇ g, were mixed with 40 ⁇ l of competent E. coli strain TGI and the cells were transformed by electroporation. Following transformation, 1 ml of SOC medium was added to each electroporation mixture (45 ml in total).
  • the mixture was divided into three equal aliquots, each of which were added to tubes containing 3 ml of top agar at 50°C, vortexed immediately, poured onto pre-warmed 2xYT agar plates, and incubated at 37°C overnight.
  • Five ml of sterile PBS per L: NaCl, 8 g; KC1, 0.2 g; Na 2 HPO , 1.44 g; KH PO 4 , 0.24 g; pH 7.4 was added to the plates and the phage particles were eluted by gently shaking the plates at 4°C for 3 hr.
  • the phage-containing PBS supernatant was collected, the plates rinsed with an additional 5 ml of PBS and the two supernatants were pooled. The supernatants were centrifuged at 6000g for 15 min at 4°C, the cleared supernatant decanted and the phage were purified as described by Ha ⁇ ison et al. (1996). The phage pellet was dissolved in 20 ml of sterile PBS, divided into 100 ⁇ l aliquots and stored in liquid nitrogen.
  • A6 dAb was constructed from the heavy chain variable domain (VH) ofthe A6, an anti-tumor
  • dAb gene was amplified by polymerase chain reaction (PCR) using the primers:
  • A6VH. 5'(TATGAAGACACCAGGCCGAGGTCCAGCTGCAGGAG)3' which contain the BamHl and Bbsl sites (underlined).
  • PCR was performed in a total volume of 50 1 containing 200 M each ofthe four dNTPs, 100 pmol each ofthe two primers, 5 1 10X buffer (NEB), and 2 units of Vent DNA polymerase (NEB).
  • the amplified product was purified using QIAquick PCR Purification kitTM (QIAGEN), digested with BamHl and Bbsl restriction endonucleases, and subsequently ligated to the expression vector pSJF2 (Simon J. Foote, personal communication). Transformation was performed as described in Example 1.
  • the vector containing A6 dAb gene (pSJF2-A6dAb) was used as template to amplify two overlapping 5' and 3' fragments.
  • the 5' fragment was amplified using the RP primer 5'(GCGGATAACAATTTCACACAGGAA)3' and A ⁇ .ldAb analogue .bk primer 5'(AGCCTGGCGGACCCAGTGCATAGCATAGCTACTGAAGGTGAATCCGCTAGCTG AACAGGAGAGTCT)3'.
  • the 3' fragment was amplified using the FP primer 5'(CCAGGGTTTTCCCAGTCACGAC)3' and the mutagenic primer A6.1 dAb analogue fw, 5'(TGGGTCCGCCAGGCTCCAGGGAAGGAACGTGAAGGTGTTTCAGCTATTAGT)3'. (At the protein level the b old codons in the mutagenic primer introduce Glu, Arg, and Gly at positions 44, 45, and 47, respectively).
  • the two fragments were gel purified using the QIAquick Gel Extraction kitTM (QIAGEN), and a larger construct was assembled from the 5' and 3' fragments by performing splice overlap extension (Clackson, et al., 1991).
  • the amplified product was gel purified, digested with EcoRI and Hindl ⁇ l, purified again, and ligated to the EcoRl/Hindlll restricted pSJF2-A6dAb. An aliquot ofthe ligated product was used to transform the TGI cells and the clone harboring the A6.1 dAb was identified by sequencing.
  • the camelized dAb gene was used as the template in PCR to amplify a shorter fragment.
  • PCR was performed as described above using the two mutagenic primers A6VH.33C,
  • the second primer results in the randomization of 19 residues in CDR3 and introduces a cysteine at position lOOe.
  • the amplified fragments were used as the templates in a third round of PCR employing the primers A6VH.33C and A ⁇ VH.BstEII,
  • PCR was performed as above using 0.5 pmol of template and 100 pmol of each ofthe two primers.
  • the amplified fragments were purified, digested with Nhel and BstEll, and ligated to NAel/i-StEII-treated pSJF6-A6dAb phagemid (Simon J. Foote, personal comunication).
  • the ligated product was desalted using QIAquick PCR Purification kitTM (QIAG ⁇ ) and used to transform E.
  • coli strain XL 1 -Blue Various dilutions ofthe transformation mixture was spread on LB/ampicillin plates and incubated overnight. In the morning, the number of ampicillin resistant colonies were used to calculate the size ofthe library. Following this, single colonies were suspended in PCR mixture, and the DNA inserts were amplified. The size ofthe amplified product, determined by agarose gel electrophoresis, was used to determine the fraction ofthe library with full-size dAb insert. Diversity of the library was determined by sequencing 20 dAb genes from the library. Growth of the library was performed as described (Harrison et al., 1996).
  • 5YCATGACCACAGTGCACAGGAGGTCCAGCTGCAGGAGTC)3' and A6VH.NotI 5'.CGATTCTGCGGCCGCTGAGGAGACGGTGACCGTTG)3' were used in PCR to amplify the dAb genes.
  • the primers are complimentary to the 5' and 3' ends ofthe dAb genes and inco ⁇ orate Apall and Notl restriction sites (underlined sequences) at the end ofthe amplified genes.
  • the amplified products were purified, cut sequentially with Apall and Notl restriction endonucleases, purified again, and ligated to the _4/> ⁇ /I/NotI-treated fd-tet phage vector. Following this, 1.5 g of the desalted ligated product was mixed with 40 1 of competent E. coli strain TGI and the cells were transformed by electroporation. Transformation, library phage amplification and purification and library size determination were performed as in Example 4.
  • Example 8 Library size determination. To determine the size ofthe library, immediately following the transformation and after the addition ofthe SOC medium an small aliquot ofthe electroporated cells were serially diluted in exponentially growing TGI cells. Two hundred 1 of the diluted cells was mixed with 3 ml of 50 C agarose top and immediately poured onto 2xYT plates pre-warmed to 37 C. Plates were incubated overnight at 37°C and the number of plaques were used to determine the size ofthe library.
  • Panning was performed using the Nunc-Immuno MaxiSo ⁇ TM 8-well strips (Nunc). Briefly, the wells were coated overnight by adding 150 1 of lOO g/ m l antigen in PBS. In the morning, they were rinsed three times with PBS and subsequently blocked with 400 I P B S- 2% (w/v) skim milk (2% MPBS) at 37 ° C for 2 hr. The wells were rinsed as above and 10 12
  • transducing units phage in 2% MPBS were added.
  • the mixture was incubated at room temperature for 1.5 hr after which the unbound phage in the supernatant was removed.
  • the wells were rinsed 10 times with PBS-0.1% (v/v) Tween 20 and then 10 times with PBS to remove the detergent.
  • the bound phage was eluted by adding freshly prepared 200 1 1 00 mM triethylamine, pipetting the content ofthe well up and down several times and incubating the mixture at room temperature for 10 min.
  • the eluted phage was transfered to a tube containing 100 1 1 M Tris-HCl, pH 7.4 and vortexed to neutralize triethylamine.
  • 10 ml exponentially growing TGI culture was infected with 150 1 e luted phage by incubating the mixture at 37 ° C for 30 min. Serial dilutions ofthe infected cells were used to
  • the titer ofthe eluted phage was determined as described in the previous section. The remaining of the infected cells were spun down and then resuspend in 900 1 2 x YT. The cells were mixed in 300 1 aliquots with 3 ml agarose top and the phage propagated on the plates overnight at 37 ° C. In the morning the phage was purified, the titer was determined, and a total of 10 1 '
  • transducing units phage were used for further rounds of selectio
  • the culture was shaken for another 60 hr, the periplasmic fraction was extracted by osmotic shock method (Anand et al., 1991), and the presence of dAb in the extract was detected by Western blotting (MacKenzie 1994).
  • the periplasmic fraction was dialyzed extensively in 10 mM HEPES (N-[2-hydroxyethyl]piperazine-N - [2-ethanesulfonic acid]) buffer pH 7.0, 500 mM NaCl.
  • the presence ofthe dAb C-terminal His tag allowed a one step protein purification by immobilized metal affinity chromatography using HiTrap ChelatingTM column (Phamacia).
  • the 5-ml column was charged with Ni 2+ by applying 30 ml of a 5 mg/ml NiCl .6H O solution and subsequently washed with 15 ml deionized water. Purification was carried out as described (MacKenzie, 1994) except that the starting buffer was 10 mM HEPES buffer, 10 mM imidazole, 500 mM NaCl, pH 7.0, and the bound protein was eluted with a 10-500 mM imidazole gradient. The purityof the protein was determined by SDS- PAGE (Laemmli). To detect the presence of dimer/multimer dAb in the protein preparation, gel filtration chromatography was performed using Superdex75 (Pharmacia) as described (Deng et al., 1995).
  • Alkylation reactions were performed using iodoacetic acid. Briefly, 5x vol. cold acetone were added to 200 g of dAb solution and the contents were mixed, followed by centrifugation in a micro fuge at maximum speed at 4°C for 10 min. The pellet was dissolved in 500 1 of 6 M guanidinium hydrochloride and 55 1 of 1 M Tris buffer, pH 8.0 were added. Subsequently, a 25 molar excess of DTT, relative to Cys residues, was added and the mixture was incubated at room temperature for 30 min. To this, a 2.2 molar excess, relative to DTT, of freshly-made iodoacetic was added and the reaction was incubated as described above.
  • the alkylated product was concentrated and dissolved in 50 1 of distilled water using Ultrafree-MC 10,000 NMWL filter unit according to the manufacturer's instructions (Millipore, Nepean, ON, Canada). Control experiments were identical except that DTT was replaced with water. The MWs ofthe iodoacetic acid-treated dAbs were determined by mass spectroscopy.
  • Binding studies were performed using BIACORE Upgrade (Biacore Inc., Piscataway, NJ) as described (J ⁇ nsson et al., 1991). Approximately 14, 000 RU of anti-FLAG M2 IgG or control IgG were immobilized on CM5 sensor chips by amine coupling. Single-domain antibodies were passed over the sensor chips surfaces in 10 mM HEPES buffer, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% P-20 (Biacore Inc.) at 25°C and at a flow rate of 5 ⁇ l/min.
  • dAbs were incubated with DTT prior to injection and the above buffer was supplemented with appropriate amount of DTT. Surfaces were regenerated with 10 mM HCI. Sensorgram data were analyzed using the BIAevaluation 3.0 software package (Biacore Inc.).
  • Nunc-Immuno MaxiSo ⁇ TM plates (Nunc) were coated overnight at 4°C with 150 1 o f 10 g/ml of 3B1 scFv or BSA in PBS. The contents were removed and the plates were tapped on a paper towel to remove any liquid remaining in the wells. The wells were blocked by adding 300 ⁇ l of 2% MPBS and incubating for 2 hr at 37°C. The contents ofthe wells were
  • Isotopically labeled proteins were prepared from cells grown on 15 N- and/or 13 C-enriched media (Bio-Express, Cambridge Isotopes Laboratory, Andover, MA). Briefly, six ml of LB containinglOO ug/ml ampicilin was inoculated with a single recombinant colony and
  • periplasmic fraction was extracted by osmotic shock method (Anand, Dubuc, et al.. 1991) and the presence of dAb in the extract was detected by Western blotting (MacKenzie, Sharma, et al.. 1994).
  • the periplasmic fraction was dialyzed extensively in 10 mM HEPES buffer, pH 7.0, 500 mM NaCl.
  • the presence of a C-terminal His 5 tag allowed a one step protein purification by IMAC using HiTrap ChelatingTM column (Amersham Phamacia Biotech, Baie d'Urfe, QC, Canada).
  • the 5-ml column was charged with Ni 2+ by applying 30 ml of a 5 mg/ml NiCl 2 .6H O solution and subsequently washed with 15 ml deionized water.
  • Purification was carried out as described previously (MacKenzie, Sharma, et al.. 1994) except that the starting buffer was 10 mM HEPES buffer, 10 mM imidazole, 500 mM NaCl, pH 7.0, and the bound protein was eluted with a 10-500 mM imidazole gradient. The purity ofthe protein was determined by SDS-PAGE (Laemmli 1970).
  • NMR samples were prepared by concentration and extensive buffer exchanging on a YM10 membrane (Amicon).
  • the final buffer contained 10 mM sodium phosphate, 150 mM NaCl, and 0.2 mM EDTA at pH 6.8.
  • the final protein concentration ofthe NMR samples was ⁇ 1 mM.
  • NMR experiments were performed at 298 K and 308 K on a Bruker Avance800 spectrometer equipped with pulse field gradient accessories.
  • 2D 15 N- ⁇ HSQC Bodenhausen and Ruben, 1980 was acquired using solvent suppression via the WATERGATE method implemented through the 3-9-19 pulse train (Piotto et al., 1992; Sklenar et al., 1993).
  • Triple-resonance experiments (Slatter et al..
  • HNCACB CBCA(CO)NH
  • HNCA HNCA
  • HN(CO)CA HNCO
  • HBHA(CO)NH 15 N-edited 3D NOESY- HSQC
  • 3D TOCSY-HSQC 15 N-edited 3D NOESY- HSQC
  • the NMR data were processed using NMRPipe/NMRDraw (Delaglio et al.., 1995) and analyzed by the use of NMRView (Johnson and Blevins, 1994).
  • the phage display A6-based dAb library was panned against the anti-FLAG M2 monoclonal antibody as described by the New England Biolabs (Beverly, MA) (NEB Technical Bulletin (1998): Ph.D.O Phage Display Peptide Library Kits; Knappik and Pluckthun, 1994); the FLAG peptide epitope recognized by the M2 monoclonal antibody is (X)YKXXD where the first position has a preference for aspartic acid (Miceli et al., 1994).
  • the consensus sequence should occur at a frequency of 4x10 "4 .
  • the FLAG peptide epitope should be represented by approximately 5x10 2 independent clones.
  • Oligonucleotides comprising randomly mutated CDR3 regions were prepared on an Applied Biosystems 394 DNA synthesizer as described above.
  • the anti-codon formula [(A/C)NN] is used resulting in a reduction in possible codon usage from 64 to 32 and reduces the number of possible stop codons. Position one, therefore, comprises only A and C in the synthetic reaction mixture.
  • the dNTP mixture comprise 25% each of A,G,C and T.
  • the 3' oligonucleotide randomizing primer was designed such that the last 15 nucleotides of framework 3 and the first 17 nucleotides of framework 4 were kept constant for hybridization.
  • the primers are adapted by reducing n to 15-23 in the above primer formulae whilst keeping the flanking nucleotides constant.
  • the primers would be adapted by increasing n to 24-33 in the above primer formula while keeping the flanking nucleotides constant.
  • Example 17 Selective Randomization Biasing for 50% Homology to Parental Tyrosine
  • the following example would be used.
  • tyrosine which is encoded by TAC or TAT (antisense strand GTA or ATA) the nucleotides would be spiked as follows for the antisense strand.
  • First anticodon nucleotide position 80% of A and 20% of C is added to the dNTP solution, and G and T are not added to reduce codon degeneracy.
  • Second anticodon nucleotide position 80% T and approximately 6.67% of C, 6.67 of
  • approximately 51% ofthe chains ofthe library will contain a wild-type A6 tyrosine in that specified position.
  • nucleotide spiking levels would be as follows:
  • First anticodon nucleotide position 50% A and 50% C.
  • Second anticodon nucleotide position 35.35% C, 35.35% G, 14.65% A and 14.65% T
  • Third anticodon nucleotide position 35.35% A, 35.35% T, 14.65% C and 14.65% G.
  • the following example can be used.
  • tyrosine which is encoded by TAC or TAT (antisense strand GTA or ATA) the nucleotides would be spiked as follows for the anti sense strand.
  • Second anticodon nucleotide position 47% T and approximately 17.67 % of C, 17.67 of A and 17.67% of G.
  • First anticodon nucleotide position 97% of A and 3% of C is added, G and T are not added to reduce codon degeneracy. For this reason, only A and C are used in the first anticodon position for all 20 naturally occurring amino acids.
  • Second antcodon nucleotide position 97% T and approximately 1 % of C, 1% of A and l% of G.
  • approximately 90% ofthe chains ofthe library will contain a wild-type A6 tyrosine in that specified position.
  • the PCR protocol consisted of an initial denaturation step at 94°C for 3 min followed by 30 cycles of 94°C for 30 sec, 55°C for 30 sec, 72°C for 1 min and a final extension step at 72°C for 10 min.
  • the two fragments were gel purified using the QIAquick Gel ExtractionTM kit (QIAGEN), and a larger construct was assembled from the 5' and 3' fragments by performing splice overlap extension (SOE). Briefly, the reaction vial containing both 5' and 3' fragments, 200 ⁇ M each ofthe four dNTPs, 5 ⁇ l 10X buffer (NEB), and 2 units of Vent DNA
  • NEB polymerase
  • the amplified product was purified (QIAquick PCR PurificationTM kit) and

Abstract

L'invention concerne des bibliothèques d'affichage de phages dans lesquelles la population de phages recombinants présente plusieurs fragments de liaison potentiels possédant des caractéristiques préférées de solubilité et/ou d'interaction intermoléculaire. L'invention concerne également des procédés de dépolarisation de bibliothèques d'affichage, afin de produire des variants, qui approchent au plus près des caractéristiques préférées du fragment de liaison parent.
EP00960243A 1999-09-07 2000-09-07 Bibliotheque amelioree d'affichage de phages de fragments de vh humain et procedes de production des memes Withdrawn EP1214352A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
CA2282179 1999-09-07
CA 2282179 CA2282179A1 (fr) 1999-09-07 1999-09-07 Presentoir de librairie a phage ameliore
US16354699P 1999-11-04 1999-11-04
US163546P 1999-11-04
PCT/CA2000/001027 WO2001018058A2 (fr) 1999-09-07 2000-09-07 Bibliotheque amelioree d'affichage de phages de fragments de vh humain et procedes de production des memes

Publications (1)

Publication Number Publication Date
EP1214352A2 true EP1214352A2 (fr) 2002-06-19

Family

ID=25681199

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00960243A Withdrawn EP1214352A2 (fr) 1999-09-07 2000-09-07 Bibliotheque amelioree d'affichage de phages de fragments de vh humain et procedes de production des memes

Country Status (3)

Country Link
EP (1) EP1214352A2 (fr)
AU (1) AU7262700A (fr)
WO (1) WO2001018058A2 (fr)

Families Citing this family (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7858559B2 (en) 2000-11-17 2010-12-28 University Of Rochester In vitro methods of producing and identifying immunoglobulin molecules in eukaryotic cells
WO2002051870A2 (fr) * 2000-12-22 2002-07-04 GRAD, Carole Legal Representative of KAPLAN, Howard Bibliotheques d'affichage de phages de fragments vh humains
AU2002359913A1 (en) * 2001-12-20 2003-07-09 Agricultural Research Council Semi-synthetic antibody fragment combinatorial library based on avian immunoglobulin genes
US20090005257A1 (en) * 2003-05-14 2009-01-01 Jespers Laurent S Process for Recovering Polypeptides that Unfold Reversibly from a Polypeptide Repertoire
DE602006018578D1 (de) 2005-01-28 2011-01-13 Childrens Medical Center Diagnose- und prognoseverfahren von blasenkrebs.
AU2007217861A1 (en) 2006-02-17 2007-08-30 Children's Medical Center Corporation Free NGAL as a biomarker for cancer
EP2010917B1 (fr) 2006-04-10 2012-06-27 Trustees of Boston University Biomarqueur de mélanome et procédés d'utilisation
EP3666284A1 (fr) 2007-06-22 2020-06-17 Children's Medical Center, Corp. Procédés et utilisations d'un fragment de saposine a
EP2382466B1 (fr) 2008-12-30 2016-03-09 Children's Medical Center Corporation Procédé de prédiction d'une appendicite aiguë
WO2011069093A1 (fr) 2009-12-04 2011-06-09 Boston Biomedical Research Institute, Inc. Détection et dénombrement de cellules souches spécifiques d'un tissu et utilisations associées
AU2010339794B2 (en) 2009-12-17 2016-09-22 Children's Medical Center Corporation Saposin-A derived peptides and uses thereof
EP4306123A3 (fr) 2011-12-22 2024-04-17 Children's Medical Center Corporation Peptides dérivés de la saposine-a et leurs utilisations
CN103215255B (zh) * 2012-01-19 2015-06-17 深圳华大基因科技有限公司 用于扩增免疫球蛋白轻链cdr3序列的引物集及其用途
EP3165535B1 (fr) * 2012-08-22 2019-05-15 Mogam Biotechnology Research Institute Procédé de criblage et de fabrication de domaines variables d'immunoglobulines super-stables et applications associées

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5639863A (en) * 1994-06-21 1997-06-17 Dan; Michael D. Human monoclonal antibodies specific to cell cycle independent glioma surface antigen
IL127127A0 (en) * 1998-11-18 1999-09-22 Peptor Ltd Small functional units of antibody heavy chain variable regions

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0118058A3 *

Also Published As

Publication number Publication date
WO2001018058A2 (fr) 2001-03-15
WO2001018058A3 (fr) 2001-10-04
AU7262700A (en) 2001-04-10

Similar Documents

Publication Publication Date Title
US8372954B2 (en) Phage display libraries of human VH fragments
US8257705B2 (en) Single-domain antigen-binding antibody fragments derived from llama antibodies
US9062305B2 (en) Generation of human de novo pIX phage display libraries
US7196187B2 (en) Method for producing antibody fragments
EP2242843B1 (fr) Procédés et matériaux pour mutagenèse ciblée
US20090286282A1 (en) Method for Producing Antibody Fragments
US20040202995A1 (en) Nucleic acids, proteins, and screening methods
de Haard et al. Creating and engineering human antibodies for immunotherapy
JP2010029198A (ja) 蛋白質／（ポリ）ペプチドライブラリー
WO2001018058A2 (fr) Bibliotheque amelioree d'affichage de phages de fragments de vh humain et procedes de production des memes
JP2021531822A (ja) 抗体の開発可能性が最大化された抗体ライブラリー
Crameri pJuFo: a phage surface display system for cloning genes based on protein-ligand interaction
US20110118149A1 (en) Synthetic Polypeptide Libraries and Methods for Generating Naturally Diversified Polypeptide Variants
CA2399809A1 (fr) Domaine de proteines combinatoire
Tanha et al. Phage Display Libraries of Human Vh Fragments
CA2384388A1 (fr) Bibliotheque amelioree d'affichage de phages de fragments de vh humain et procedes de production des memes
JP2019510507A (ja) 合成抗体ライブラリーを作製する方法、前記ライブラリー、及びその適用
Plückthun Studying Protein Structure and Function by Directed Evolution: Examples with Engineered Antibodies
CA2282179A1 (fr) Presentoir de librairie a phage ameliore
Pliickthun STUDYING PROTEIN STRUCTURE AND FUNCTION BY DI CTED EVOLUTION
Plückthun Examples with Engineered Antibodies

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20020408

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

RIN1 Information on inventor provided before grant (corrected)

Inventor name: TANHA, JAMSHID

Inventor name: ENTWISTLE, JOYCELYN

Inventor name: NARANG, SARAN

Inventor name: DAN, MICHAEL

Inventor name: KAPLAN, HOWARD

17Q First examination report despatched

Effective date: 20030812

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20040224