WO1995027045A1 - The isolation and production of catalytic antibodies using phage technology - Google Patents

Abstract

Disclosed and claimed are methods for producing catalytic antibodies, including human catalytic antibodies, from bacteriophage. The methods require the cloning, selection, screening, and isolation of catalytic antibodies. Also disclosed and claimed are the products themselves, the catalytic antibodies, made from the phage technology. In addition, catalytic antibodies produced from the phage technology and useful in prodrug activation are disclosed and claimed. And finally, the invention also understands the production of catalytic antibodies to phosphonates.

Description

  
 



   THE ISOLATION AND PRODUCTION OF
 CATALYTIC ANTIBODIES USING PHAGE TECHNOLOGY
REFERENCE TO RELATED APPLICATIONS
 This application is a continuation-in-part of International Application WO 92/01047 (PCT/GB91/01134) having an International filing date of July 10,1991, a publication date of
January 23,1992, and wherein the U. S. is a designated State, said International Application
WO 92/01047 (PCT/GB91/01134) being hereby incorporated by reference.



  FIELD OF THE INVENTION
 The present invention relates to the isolation and production of catalytic antibodies displayed on bacteriophage and, more particularly, the isolation and production of human catalytic antibodies. This invention also relates to the isolation and production of catalytic antibodies for use in prodrug activation. This invention further relates to production of catalytic antibodies that bind to transitional state analogs.



  BACKGROUND OF THE INVENTION
 Monoclonal antibodies are traditionally made by establishing an immortal mammalian cell line which is derived from a single immunoglobulin producing cell secreting one form of a biologically functional antibody molecule with a particular specificity. Because the antibodysecreting mammalian cell line is immortal, the characteristics of the antibody are reproducible from batch to batch. The key proprieties of monoclonal antibodies are their specificity for a particular antigen and the reproducibility with which they can be manufactured.



   Structurally, the simplest antibody (IgG) comprises four polypeptide chains, two heavy (H) chains and two light (L) chains connected by disulfide bonds. The light chains exist in two distinct forms called K (klPPa) and. (lambda). Each chain has a constant region (C) and a variable region (V). Each chain is organized into a series of domains. The light chains have
 two domains, corresponding to the C region and the other to the V Region. The heavy chains  have four domains, one corresponding to the V region and three domains (l, 2, and 3) in the C region. The antibody has two arms (each arm being a Fab region), each of which has a VL and a VIl region associated with each other.

   It is this pair of V regions (VL and VH) that differ from one antibody'o to another (owing to amino acid sequence variations), and which together are responsible for recognizing the antigen and providing an antigen binding site (ABS). In even more detail, each V region is made up from three complementarily determining regions (CDR) separated by four framework regions (FR). The CDRs are the most variable part of the variable regions, and they perform the critical antigen binding function. The CDR regions are derived from many potential germ line sequences via a complex process involving recombination, mutation, and selection.



   It has been shown that the function of binding antigens can be performed by fragments of a whole antibody. Example binding fragments are (i) the Fab fragment consisting of the
VL, VII, CL and CHI domains; (ii) the Fd fragment consisting of the VH and Ctll domains; (iii) the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (iv) the dAb fragment (Ward et al., Nature 341 (1989): 544-546) which consists of a Vil domain; (v) isolated CDR regions; and (vi) F (ab') 2 fragments, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region.



   Although the two domains of the Fv fragment are coded for by separate genes, it has proved possible to make a synthetic linker that enables them to be made as a single protein chain (known as single chain Fv (scFv); Bird et al., Science 242 (1988): 423-426; Huston et al., Proc. Natl. Acad. Sci., USA 85 (1988): 5879-5883); by recombinant methods. These scFv fragments were assemble from genes from cnonoclonals that had been previously isolated. In our earlier application, WO 92/01047, we described a process to assemble scFv fragments from VIS and VL domains that were not part of a previously isolated antibody.



   Although moncclonal antibodies, their fragments and derivatives have been enormously advantageous, there are nevertheless a number of limitations associated with them. First, the
 therapeutic applications of monoclonal antibodies produced by human immortal cell lines holds great promise for the treatment of a wide range of diseases (Lennox, Clinical Applications of  
Monoclonal Antibodies, British Medical Bulletin 1984). Unfortunately, immortal antibodyproducing human cell lines are very difficult to establish and they give low yields of antibody (approximately 1 gg/ml). In contrast, equivalent rodent cell lines yield high amounts of antibody (approximately 100 Ag/ml). However, the repeated administration of t oese foreign rodent proteins to humans can lead to harmful hypersensitivity reactions.

   As a result, these rodent-derived monoclonal antibodies have limited therapeutic use.



   Second, a key aspect in the isolation of monoclonal antibodies is how many different clones of antibody producing cells with different specificities, can be practically established and sampled compared to how many theoretically need to be sampled in order to isolate a cell producing antibody with the desired specificity characteristics (Milstein, Royal Soc. Croonian
Lecture, Proc. R. Soc. London B. 239 (1990): 1-16). For example, the number of different specificities expressed at any one time by lymphocytes of the marine immune system is thought to be approximately 1 () 7 and this is only a small proportion of the potential repertoire of specificities. However, during the isolation of a typical antibody producing cell with a desired specificity, the investigator is only able to sample 1 () 3 to 104 individual specificities.

   The problem is worse in the human, where one has approximately 1012 lymphocyte specificities with the limitation on sampling of 103 or 104 remaining.



   This problem has been alleviated to some extent in laboratory animals by the use of immunization regimes. Thus, where one wants to produce monoclonal antibodies having a specificity against a particular epitope, an animal is immunized with an immunogen expressing
 that epitope. The animal will then mount an immune response against the immunogen and
 there will be a proliferation of lymphocytes which have specificity against the epitope. Owing
 to this proliferation of lymphocytes with the desired specificity, it becomes easier to detect them
 in the sampling procedure. However, this approach is not successful in all cases as a suitable
 immunogen may not be available. Furthermore, where one wants to produce human
 monoclonal antibodies (e. g., for therapeutic administration), such an approach is not practically
 or ethically feasible.  



   In our earlier application, WO 92/01047, we described methods of constructing a bacteriophage that expresses and displays at its surface a large biologically functional binding molecule (e. g., antibody fragments, enzymes, and receptors) and which remains intact and infectious. We called the structure which comi : ; ses a virus particle and a binding molecule displayed at the viral surface a"package". Where the binding molecule is an antibody, an antibody derivative or fragment, or a domain that is homologous to an immunoglobulin domain, we called the package a"phage antibody" (pAb). However, except where the context demande otherwise, where the term phage antibody is used generally, it was also interpreted as referring to any package comprising a virus particle and a biologically functional binding molecule displayed at the viral surface.

   Since the original filing of WO 92/01047, a number of examples of functional antibody and other protein domains expressed on the surface of bacteriophage have been reported in both the literature and additional patent applications.



   This simple substitution of immortalized cells with bacterial cells as the"factory", considerably simplifies procedures for preparing large amounts of binding molecules expressed on the surface of the bacteriophage. Furthermore, the use of polymerase chain reaction (PCR) amplification (Saiki et al., Science 239 (1988): 487-491) to isolate antibody producing sequences from cells (e. g., hybridomas and B cells) has great potential for speeding up the timescale under which binding specificities can be isolated. Phage antibody expression libraries can be easily generated by cloning the amplified VH and VL genes directly into bacteriophage expression vectors. Furthermore, a bacteriophage based recombinant production system allows scope for producing tailor-made antibodies and fragments thereof.

   For example, it is possible to produce chimeric molecules with new combinations of binding and effector functions, humanized antibodies (e. g., murine variable regions combined with human constant domains or murine-antibody CDRs grafted onto a human FR) and novel antigen
 binding molecules. The key advantage of the phage based system being the ability to directly
 screen the recombinant antibodies directly for the desired binding specificities.



   In creating recombinant VII and VL phage libraries several problems need to be
 aciciressed. For example, in a mouse there are approximately 10 possible H chains and 10  possible L chains. Therefore, there are 1014 possible combinations of H and L chains, and to test for anything like this number of combinations, one would have to create and screen a library of about 1014 clones. This had not previously been a practical possibility.



  PC'r/GB92/00883 and PCT/GB92/01755 applications, which are herein incorporated by reference, disclose a number of approaches which ameliorate this problem. Each of these applications is a continuation-in-part of our International Application WO 92/01047.



   In addition, a number of molecular biological techniques which have previously been developed for engineering of antibody active sites can be applied in combination with the phage antibody library approaches described previously. These techniques include site-directed mutagenesis of residues within a CDR, replacement of all or portions of CDR (s) with random amino acid sequence, CDR shuffling in which a CDR region is essentially replaced with a library of CDR regions. The use of pAbs may also allow the construction of entirely synthetic antibodies. Furthermore, antibodies may be made which have some synthetic sequences, for example, CDRs, and some naturally derived sequences (see for example PCT/BG92/06372).



  For example, V-gene repertoires could be made in vitro by combining un-rearranged V genes, with D and J segments. Libraries of pAbs could then be selected by binding to antigen, hypermutated in vitro in the antigen-binding loops or V domain framework regions, and subjected to further rounds of selection and mutagenesis.
 pAbs have a range of applications in selecting antibody genes encoding antigen binding activities. One particularly exciting area of application is in the development of antibodies with catalytic properties (catalytic antibodies). Catalytic antibodies have been described in U. S.



  Patent Nos. 4,888,281 to Schochetman et al.; 4,963,355 to Kim et al.; and to
Schochetman et al., all hereby incorporated by reference. As disclosed therein, catalytic antibodies combine the catalytic abilities of enzymes with the binding capabilities of antibcxlies.



   All catalytic antibodies described to date have been generated using monoclonal
 antibody technology. The details of that process are well known to those of ordinary skill in
 the art. A typical methodology first involves immunizing mice with an appropriate antigen.



  The antigen may be the desired reactant; the desired reactant bound to a peptide or other carrier  molecule; a reaction intermediate or an analog of the reactant; or the product or a reaction intermediate.



   "Analog"as the term is used here can encompass isomers, homologs, transition state analogs or other compounds sufficiently resembling the reactant in terms of chel ! al stmcture such that an antibody raised against the analog may participate in an immunological reaction with the reactant but will not necessarily catalyze a reaction of the analog.



   Although a number of different types of antibody catalysts have been developed with this technology, the time required to establish and then screen the hybridomas for the desired specificity is of considerable importance.



   If the desired specificity is sufficiently rare, it may be impractical or impossible to sample enough hybridomas cell lines to recover the desired specificity.



   Additionally, there is currently no suitable hybridoma based technology for generating entirely human catalytic antibodies.



   The methods of the invention can also be used to effect a cleavage that leads to the activation of some biological function.



   Such reactions include the cleavage of peptide bonds, but may also include ester bonds or glycosidic bonds or other types of bonds.



   One example of the cleavage of a biomolecule which leads to the activation of a biological function is the treatment of insulin-dependent diabetes. Patients self-administer insulin by injection. Prior attempts to develop a formulation of insulin whose release into the circulation mimics the pharmacokinetics of the release of natural pancreatic insulin have not proved successful. Insulin exists in the pancreas in a pro-form, proinsulin, whose activity is many orders of magnitude lower than insulin itself. An antibody protease specific for the peptide bond that leads to conversion of proinsulin to insulin can be designed so that its kinetic characteristics allow release of insulin in vivo after an injection of proinsulin plus antibody protease. This is an example of prodrug activation where the drug in this instance is a natural protein hormone.

   Prodrugs may include many therapeutically active molecules which lead to the activation or deactivation of a biological function. The pro-form may either take advantage  
 of a natural modification of the drug or any suitable synthetic modification thereof. Suitable
 drug derivatives with low activity (therapeutically beneficial or toxic), which, on modification
 with a suitable catalytic antibody, are converted to an active form. A particular example of this
 process is given in PCT/US89/01951 filed May 1989, which is hereby incorporated by
 reference.



   SUMMARY OF THE INVENTION
 One object of the present invention is to provide a method for producing and isolating a
 catalytic antibody displayed on phage which is capable of catalytically increasing the rate of a
 chemical reaction.



   Another object of the present invention is to produce human catalytic antibodies by one
 of several different methods outlined hereafter.



   A further object of the present invention is to isolate phage displaying antibody epitopes
 that bind to transition state analogs, phosphonates, and RT3.



   Still another object of the present invention is to produce a catalytic antibody from
 antibodies (either mouse or human-derived) that bind transition state analogs, phosphonates,
 and RT3 by one of several different methods including mutagenesis, chain shuffling, and CDR
 shuffling or various combinations of these procedures.



   And finally, yet another object of the present invention is to produce catalytic antibodies
 for use in prodrug activation.



   Accordingly, me present invention provides a method for producing catalytic antibodies
 displayed on phage comprising the steps of :
 (a) generating a gene library of antibody-derived domains;
 (b) inserting coding for said domains into a phage expression vector; and (c) isolating said catalytic antibodies.



   The invention further provides a method for isolating catalytic antibodies displayed on
 phage comprising the following steps:  
 (a) preparing an antigen;
 (b) immunizing with said antigen;
 (c) generating a library of VH and VL domains from said immunized animal;
 (d) cloning said VIS and VL domains into a phage expression vector to generate
 phage display antibodies;
 (e) selecting phage display antibodies which bind specifically to said antigen;
 (f) screening said selected phage display antibodies for catalytic activity to
 substrate; and
 (g) isolating said catalytic antibodies.



   The invention further provides a method for isolating catalytic human antibodies displayed on phage comprising the following steps:
 (a) preparing an antigen;
 (b) generating a library of VH and VL domains;
 (c) cloning said VI-I and VL domains into a phage expression vector to generate
 phage display antibodies;
 (d) selecting phage display antibodies which bind specifically to said antigen;
 (e) screening said selected phage display antibodies for catalytic activity to
 substrate; and
 (f) isolating said catalytic antibodies.



   The invention further provides a method for producing catalytic antibodies displayed on phage through chain shuffling comprising the following steps:
 (a) combining a library of VL genes with VH genes to form a chain shuffled
 library;
 (b) cloning the shuffled chain;
 (c) expressing said catalytic antibody on phage;
 (d) selecting against an antigen; and  
 (e) screening for catalytic activity.



   The invention further provides a method for producing catalytic antibodies displayed on phage through CDR shuffling comprising the following steps:
 (a) isolating VL and VH genes;
 (b) isolating a library of CDR regions;
 (c) recombining said VL and VI-I genes with said library of CDR regions to
 produce a CDR shuffled library; and
 (d) cloning the CDR shuffled library;
 (e) expressing said CDR shuffled library on phage;
 (f) selecting against an antigen; and
 (g) screening for catalytic activity.



   The invention further provides a method for producing catalytic antibodies displayed on phage through imprinting comprising the following steps:
 (a) selecting a set of antibodies;
 (b) isolating a set of VH and a set of VL genes from said antibodies;
 (c) combining said set of VH with a library of VL and combining said set of VL
 with a library of VH to form two combination libraries;
 (d) cloning said combination libraries;
 (e) expressing said libraries on phage;
 (f) selecting against an antigen;
 (g) isolating selected libraries of VH and VL genes;
 (h) combining said libraries of VH and VL genes;
 (i) cloning said combined libraries;
 (j) expressing said combined libraries on phage;
 (k) reselecting against an antigen; and
 (1) screening for catalytic activity.  



   The invention further provides a method for enhancing the rate of cleavage or formation of a specific bond within a molecule in vivo which comprises introducing into an animal an effective amount of a phage-derived catalytic antibody.



   The invention further provides a method for in vivo activation of a prodrug comprising:
 (a) introducing a prodrug into a patient, said prodrug having a chemical bond
 therein which upon cleavage releases the active form of said drug; and
 (b) introducing into said patient an effective amount of a phage-derived catalytic
 antibody capable of cleaving said bond in said prodnig.



  BRIEF DESCRIPTION OF THE DRAWINGS
 The present invention as defined in the claims can be better understood with reference to the text and to the drawings.



   FIGS. 1-4 show the reaction schemes for the synthesis of Compounds 7 (RT3 hapten), 8,12, and 15, respectively, and intermediates thereof.



   FIG. 5 shows the plasmid construct resulting from the insertion of His-6 oligo into pHEN-OX16 to give pHEN-OX1611isl l.



   FIG. 6 shows an SDS polyacrylanide gel starved with coomassie are:
 (a) total periplasmic proteins from 1 ml of cells.



   (b) unbound fraction from I ml of cells, after addition of binding matrix.



   (c) fraction bound and eluted from matrix, equivalent to 1 ml of cells.



   (d-f) are purified fractions equivalent to 5 mls of cells.



   (d) pOX16-his-11 antibody fragment eluted with PBS+1M Nacl, 250 mNi
 imidazole.



   (e) pOX16-his-11 antibody fragment eluted with PBS, 250 mM imidazole.  
 pSCFv4his-6 antibody fragment eluted with PBS+1M NaCl, 250 mM
 imidazole.



   FIG. 7 shows pCANTAB vectors encoding C terminal his-6 peptides.



   FIG. 8 shows the competitive assay results for selected mouse RT3 phage antibodies
 with haptens (RT3) portions of the haptens (RT3A and RT3B) or portions of the product (Prod
 A and Prod B).



   FIG. 9 shows the genetic sequence of light chain pattern A and light chain pattern C
 from mouse-derived RT3 phage antibodies.



   FIG. 10 shows the alignment of the mouse germiine to the genetic sequence of light
 chain pattersn B, D, and I from mouse-derived RT3 phage antibodies.



   FIG. 11 shows the comparison of genetic sequences of light chain patterns A, B, C, D,
 and I from mouse-derived RT3 phage antibodies.



   FIG. 12 shows the genetic sequence of heavy chain pattern A from mouse-derived RT3
 phage antibodies.



   FIG. 13 shows the alignment of the mouse germline to the genetic sequence of heavy
 chain patterns B and D from mouse-derived R'13 phage antibodies.



  FIG 14 shows the comparison of genetic sequences of heavy chain patterns A, B, C,
 D, and I from mouse-derived RT3 phage antibodies.



   FIG. 15A shows an HPLC chromatogram of a catalytic assay of IMAC  pure scFv from clone 18.



   FIG. 15B shows an HPLC chromatogram of a catalytic assay + RT3 hapten of IMAC pure scFv from clone 18
 FIG. 15C shows an HPLC chromatogram of a catalytic RT3 assay blank at pli 9.0
 FIG. 16 shows an HPLC chromatogram of a catalytic assay of HIC pure scFv from clone 18.



   FIG. 17A shows an HPLC chromatogram of a catalytic assay of IMAC pure scFv from clone 83.



   FIG. 17B shows an HPLC chromatogram of a catalytic assay + RT3 hapten of IMAC pure scFv from clone 83
 FIG. 18 shows an HPLC chromatogram of a catalytic assay of HIC pure scFv from clone 83
 FIG. 19A shows binding pattern of clones to RT3 obtained after 3 rounds of panning of a naive human-derived phage antibody library.



   FIG. 19B shows binding pattern of clones to RT3 after 4 rounds of panning of a naive human-derived phage antibody library.



   FIG. 20 shows genetic sequences of heavy and light chains of RT3 specific phage
 antibodies selected from a naive human phage antibody library.  



   FIG. 21 shows the general scheme for VH and VL chain shuffling.



   FIG. 22 shows RT3-BSA ELISA assay with polyclonal phage derived from human shufflid libraries after PANO, PAN1, and PAN2.



   FIG. 23A shows inhibitoin of phage antibody bidning to RT3-BSA by left hand portion of RT3 hapten (RT3A) or substrate (Product A).



   FIG. 23B shows inhibition of phage antibody binding to RT3-BSA by right hand portion of RT3 hapten (RT3B) or substrate (Product B).



   FIG. 24A shows yield of plrige elutecl with IZT3, RT3A, RT3B, TEA and PBS from
ELISA wells coated with 0.3 tg of RT3-BSA.



   FIG. 24B shows yield of phage eluted with RT3, RT3A, RT3B, and TEA from ELISA wells coated with 15 ytg of RT3-BSA.



   In order that the invention herein described may be more fully understood, the following detailed description is set forth. This description, while exemplary of the present invention, is not to be construed as specifically limiting the invention and such variations which would be within the purview of one skilled in this art are to be considered to fall withill the scope of this invention.



  DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
 For the sake of convenience and ready reference, the following definitions will be used
 in describing the instant invention.



   Analog encompasses isomers, homologs, transition states or other compounds  sufficiently resembling the reactant in terms of chemical structure such that an antibody raised against an analog may participate in an immunological reaction with the reactant but will not necessarily catalyze a reaction of the analog.



   Antibody describes an immunoglobulin whether natural or partly or wholly synthetically produced. The term also covers any protein having a binding domain which is homologous to an immunoglobulin binding domain. These proteins can be derived from natural sources, or partly or wholly synthetically produced. Example antibodies are the immunoglobulin isotopes and the Fab, F (abl) 2, scFv, fV, dAb, Fd fragments.



   An antibody-derived domain refers to a sequence derived from an antibody molecule.



   Antigen is a substance, frequently a protein, that can stimulate an animal organism to produce antibodies and that can combine with the antibodies these produced.



   A domain is a part of a protein that is folded within itself and independently of other parts of the same protein and independently of a complementary binding member.



   Homologs indicate polypeptides having the same or conserved residues at a corresponding position in their primary, secondary, or tertiary structure. The term a ! so extends to two or more nucleotide sequences encoding the homologous polypeptides. Examples of homologous peptides are the immunoglobulin isotopes.



   Isolating refers to the separation of a specific phage from the library.



   Library is a collection of oligo or polynucleotides, e. g., DNA sequences within clones.



   A naive library is a phage display library of immunoglobulin sequences derived from an animal which has not been immunized with the following: the reactant; the reactant bound to a peptide or other carrier, a reaction intermediate; an analog of the reactant; an analog of the product in which the antibody so generated is capable of binding to the reactant or a reaction intermediate; and an analog of a reaction intermediate or transition state.



   A package describes a repliable genetic display package in which the particle is
 displaying a member of a sbp at its surface. The package may be a bacteriophage which
 displays an antigen binding domain at its surface. This type of package has been called a phage
 antibody (pAb).  



   A phage vector is a vector derived by modification of a phage genome, containing an origin of replication for a bacteriophage, but not one for a plasmid.



   A phagemid vector is a vector derived by modification of a plasmid genome, containing an origin of replication for a bacteriophage as well as the plasmid origin of replication.



   A vector is a DNA molecule, capable of replication in a host organism, into which a gene is inserted to construct a recombinant DNA molecule.



  An Overview Of The Method
 The invention describes methods to generate and isolate phage particles which express on their surface an antibody with catalytic properties. In the practice of the invention the antibody domains encoding the catalytic functionality can be prepared from either specifically immunized or non-immunized animal or human sources as defined below. Additionally, the invention describes methods of generating or improving binding and/or catalytic function by one of several methodologies including but not limited to chain shuffling, CDR grafting, and mutagenesis.

   In a further embodiment of the invention, a method is disclosed for converting a catalytic antibody encoded entirely by mouse-derived VI-I and VL domains into one encode by human-derived VH and VL domains
 The first step in generating antibodies with specific catalytic function requires, but is
 not necessarily limited to, a chemical hapten ten (e. g., transition state analog (TSA) that is related
 to, but distinct from the substrate of the reaction to be catalyzed). The structure, synthesis, and
 use of said TSA (s) as a means to generate antibodies with catalytic function has been described
 in U. S. Patent No. 4,196,265 issued Avril 1,1980, which is hereby incorporated by
 reference. As described in the prior art, the traditional route for producing and isolating
 catalytic antibodies has been through a monoclonal antibody approach (hybridoma technology).



   The present invention utilizes a phosphonate transition state analog as either an
 immunogen or immobilized on a solid phase to allow generation and selection of antibodies
 which bind said TSA and which may have catalytic properties (see FIG. 1). Unlike previous
 inventions in which catalytic antibodies are produced via hybridoma methods, the unique  embodiment of the invention is the ability to express antibody domains, including those with catalytic function, on the surface of a bacteriophage. Methods for generating libraries of phage with the potential for displaying catalytic antibody domains on their surface are described in detail below.



  1. Production Of Binding and/or Catalytic Phage Antibodies From An Immunized Source
 The TSA is bound to a carrier molecule or peptide and immunized into BALB/C mice.



  After an appropriate amount of time spleens are removed from the mice and mRNA isolated from the cells. The RNA serves as a starting source material for amplifying immunoglobulin variable domains for cloning into bacteriophage expression vector and subsequent expression on the surface of bacteriophage particle. In the embodiment of this invention the variable domains are typically linked by a short peptide to produce a scFv as described in the background to the present invention. It should be noted that alternative phage expression vectors could be used for expression of the antibody as Fab. Techniques for creating said phage antibody libraries have been described previously and the details of the process are well known to those skilled in the art. (see, e. g., WO 92/01047; McCafferty et al., Nature (1990): 552-564.; Hoogenboom et al., Nucl. Acids. Res. 

   (1991): 4133-4137; Marks et al., J.



  Mol. Biol. (1991) : 581-597).



   Phage antibodies which specifically bind and recognize the TSA are isolated f 



   Phage antibodies that bind and are immobilized on the solid phase surface can be removed by one of several methods including:
 a) Non-specific elution by using buffers of either low (acidic) or high (basic) pH.
 b) Specific elution with free hapten such as the original phosphonate TSA or
 substrate or product of the reaction
 c) Specific elution with portions of the TSA, or substrate or product.



   In addition to specific elution of phage antibodies bound to the TSA, it may be desirable to control the binding of the phage antibodies during the initial panning or affinity chromatography step. One method would be to use competitive inhibition in which the phage antibodies are first preincubated with reactants, reaction products or portions of the TSA (see
Example 9). The purpose of such"preselection"would be to eliminate from the population of binding antibodies those least likely to be catalytic. In the context of the present example, those eliminated would be phage antibodies that do not substantially bind the phosphonate portion of
 the TSA.

   The type of preselection of the phage antibody library would need to be determined
 experimentally, but ultimately could lead to methods to enrich within the population of TSA
 binding phage antibodies the proportion of catalytic over non-catalytic phage. A greater degree
 of flexibility could be exerted if such procedures were carried out in ELISA wells. Thus,
 following a particular procedure, the eluate could be collected and the whole plate carried
 through a detection procedure. Based on the results, the eluate from specific wells could be
 selected for further analysis/pannings.



   Following elution of phage antibodies by any or all of the above methods, phage are
 collected and can be subjected to additional pannings (2,3,4,5, etc.) simply by collecting the
 eluted phage from the previous panning and reincubating on TSA solid phase. Since, a certain
 percentage of phage that do not specifically bind to the TSA are carried through each panning
 step (i. e., non-specific binders), pools of phage clones or individually isolated phage clones
 are typically rescreened for binding to the TSA by a solid phase ELISA assay. The ELISA
 assay can be done with antibody expressed on the bacteriophage surface or expressed in a  soluble form as described below.

   Other formats of the ELISA assay, for example competitive inhibition with free hapten, substrate or product or various halves or portions of said hapten, substrate or product can be employed to further characterize the binding specificity of the pools of phage clones or individual binding clones.



   Individual phage antibody clones or pools of clones which have the appropriate binding specificity are then assayed for catalytic activity. Assay for catalysis is most conveniently done using soluble antibody and methods for producing soluble antibody from a phage antibody expressing E. coli clone have been described previously (Marks et al, J. Mol. Biol. (1991): 581597). A criteria for attributing catalytic activity to the antibody active site is rigorous purification of the antibody away from contaminating proteins or enzymes. In this invention, purification of the soluble antibody is facilitated by incorporation of specific peptides at the 3' carboxyl terminus of the expressed antibody.

   Examples of such peptides currently used and as reported in the prior art include:
 a) histidine peptide-allows purification of antibody on metal immobilized on a   
 column matrix (IMAC, Hochuli et al., Bio/Technology (1988):1321
   
 1325).
 b) myc peptide-allows purification on a column matrix on which antibody that
 binds specifically to the myc peptide has been immobilized (Clackson et al.



   Nature (1991): 624-628).



   Previous vectors utilized for display of antibody fragments contained only the myc peptide (see FIG. 5). The vectors described and disclosed in this application represent the first example of incorporation of the histidine peptide in tandem with the myc peptide. This represents an improvement over the previous art because it allows purification of the soluble antibody using two uniquely different formats and purification conditions.



   Additional purification of antibody can be achieved by utilizing specific properties unique to the antibody of interest such as hydrophobicity, charge, and size. Purification is
 affected by any of a number of standard protein purification techniques as described previously
 (Deutscher, Methods in Enzvmolocv. Vol. 182, Guide to Protein Purification (1990).  



   Antibodies isolated by phage antibody techniques described above can be screened for the ability to catalyze the desired reaction by a number of methods well known in the art. In its simples form screening is accomplished by incubating antibody and reactant (substrate) under appropriate conditions and measuring the formation of product by any of a number of means such as spectrophotometric methods or high pressure liquid chromatography.



  2. Production Of Binding and/or Catalytic Phage Antibodies From A Non-imnunized
Source
 In this embodiment of the invention source material for generating a phage antibody library is from a non-immunized animal or mammal such as human. Non-immunized in this example means not specifically immunized with a specific reactant (either bound to a carrier protein or as free reactant), reaction intermediate, analog of a reactant or expected products of a particular reaction. As demonstrated in the prior art, low and moderate affinity human antibodies have been generated to specific antigens using phage antibody libraries generated from a non-immunized source. Such an approach provides a method for generating animal or human antibodies that bind to a TSA simply by panning the naive animal or human derived phage antibody library on TSA as described above.

   Phage clones which specifically bind and recognize the TSA can then be assayed for the desired catalytic function as described. Such an approach provides a method to isolate directly an entirely human derived catalytic antibody.



  3. Production Of Binding and/or Catalytic Phage Antibodies By Chain-Shuffling
 A chain-shuffling approach for generating phage antibody libraries takes advantage of the promiscuity of binding between VH and VK pairs. In this embodiment of the invention, the VH or VK domain from one, several or many different phage antibody clones is recombined with a library of VK or VH domains. The phage clones and libraries of VH and
VK domains can be obtained from an immunized source as described in Section 1 above or an
 non-immunized source as described in Section 2 above. In addition, the phage clones chosen
 for chain shuffling can be, but is not necessarily limited to, those that have previously been  selected for binding to a particular TSA. Following the chain shuffling procedure the recombined chains (i. e., shuffled chains) are cloned back into the phage antibody expression vector.

   The expressed phage antibody library is repanned on the TSA and individual binding clones screened for catalytic actity as earlier described.



  4. Production of Binding and/or Catalytic Phage Antibodies by CDR Shuffling
 It is well known that antibody specificity and antigen binding affinity are specified by the six CDR's encoded by the VH and VL domains. It follows then that altering any or all of the CDR's from a given antibody will have dramatic effects on the binding properties of that antibody. CDR shuffling as it relates to phage antibodies describes a process for replacing a region encoding a CDR or CDR's within a VH or VL domain with a library of CDR or CDR's.



  As with the chain shuffling approach described in Section 3 above, the VH or VK domain used for CDR shuffling can be from one, several or many different phage antibody clones.



  The phage clones and libraries of CDR regions used for shuffling can be obtained from either immunized or non-immunized sources. Following CDR shuffling the recombinant VH and
VL domains are recloned into the phage antibody expression vector. The expressed phage antibodies are repannned against the TSA and individual binding clones assayed for catalytic activity as described above.



  5. Production of Binding and/or Catalytic Phage Antibodies by Mutagenesis
 As described above for CDR shuffling binding specificity of an antibody can be altered by changing amino acids encoded within CDRs. CDR mutagenesis for the purposes of this invention can be defined as:
 a) site-directed in which one or a few specific amino acids within a particular CDR
 are mutagenized. This process normully results in alteration of the wild-type
 amino sequence to several different amino acids dependent upon the nucleotide
 sequence of the region being mutagenized and the sequence of the mutagenic
 primer.  
 b) random mutagenesis in which some or all of the amino acids within a CDR or
 CDRs is replaced with a random nucleotide sequence such that the wild type
 sequence is replaced by all possible combinations of amino acids.



   A number of different methodologies for both site-directed and random mutagenesis have been described in the literature and are well known to those in the art. As with the other methodologies, the phage antibody clone or clones chosen for mutagenesis could be, but is not limited to, ones already selected for binding to the TSA. In addition, the chosen binding clones could be ones isolated from phage antibody libraries derived from either immunized or nonimmunized animal or human sources.



   Recent successes in modelling antigen binding sites augurs well for de novo design.



  The approach is especially attractive for making, catalytic antibodies, particularly for small substrates. Here side chains or binding sites for prosthetic groups might be introduced, not only to bind selectively to the transition state of the substrate, but also to participate directly in bond making and breaking.



  6. Derivation of Human Catalytic Antibodies by"imprinting"
 The process of"imprinting"involves using an existing antibody with desired binding characteristics, to derive new antibodies, with similar characteristics. This is done by recombining original antibody chains, or parts thereof, with a library of complementary parts.



  When new antibody elements are found, which complement the original antibody binding characteristics, these are recombined with a library which replaces the original antibody binding characteristics, these are recombined With a library which replaces the original antibody part, to give an entirely new antibody which mimics the binding of the original antibody (PCT/GB/ 92/01755).



   For example, an imprinting approach would have value for humanizing a mouse
 catalytic antibody isolated using phage antibody technology as described in Section 1 above.



   Once isolated, the mouse encoded VH or VL domain of the catalytic antibody could be
 recombined with a library of the complimentary human derived VL or VH domains. The half  mouse-half human phage antibodies are then reselected for binding to the TSA used originally to generate the catalytic activity. The remaining mouse VH or VL domain of the selected binders is now replaced with a library of the complimentary human derived VL or VH domains. The resulting phage antibodies which now are encoded entirely by human derived sequences entirely are again reselected for binding to the TSA. Inaividual clones selected for binding to me TSA are then assayed as described above for catalytic activity.



   Having now generally described this invention, the following examples are included for purposes of illustration and are not intended as any form of limitation.



  Example 1.1
Synthesis of RT3 Phosphonate Transition State Analog Hapten
 The reaction scheme for the synthesis of the RT3 phosphonate transition state analog is shown in Figure 1. a2-Chloroisodurene (1) by lillogen exchange using sodium iodide in acetone gave a2-Iodoisodurene (2). Michaelis-Arbusov reaction with trimethylphosphite yielded the dimethoxy phosphonate compound (3). Activation by heating with phosphores pentachloride and subsequent reaction with Methy 4-Hydroxyphenylacetate (4) afforded compound (5). Demethylation using thiophenol and triethylamine produced compound (6)
 which under strong basic conditions of lithium hydroxide was fully deprotected to the derised product (7).



   More specifically, a2-Iodoisodurene (Compound 2) was prepared as follows:
 a2-Chloroisodurene (Compound 1) (2.28 g) was dissolved in acetone (45 ml) and NaI
 (2.25 g) was added. The reaction mixture was stirred vigorously at 75'C in the dark for 16
 hours. The reaction mixture was concentrated, redissolved in ethyl acetate (100 ml), washed
 with water and concentrated to a solid. The solid was redissolved in ethyl acetate (5 ml) and
 purified by flash chromatography using 100 g of silica and eluting with hexane to give a2  
Iodiosodurene (2) (2.717 g). This was confirmed by spectroscopy IH NMR (CDC13) 8 2.25 (s, 3H), 2.33 (s, 6H), 4.48 (s, 2H), 6.85 (s, 2H).



  Preparation of Compound 3
 Freshly distilled Trimethoxyphosphite (5 ml) and a2-Iodoisodurene (2) (0.765 g) were heated together at 110 C for 16 hours. Te reaction mixture was concentrated to a small volume and purified by flash chromatography using 50 g of silica and eluting with ethyl acetate-hexane (2: 8 volume by volume) to give compound 3 (0.550 g). This was confirmed by spectroscopy-1H NMR (CDC13) 8 2.25 and 2.27 (2s, 3H), 2.40 (Is, 6H), 3.25 (d, 2H), 3.65 (d, 6H), 6.88 (s, 2H).



  Methyl 4-Iydroxyphenylacetate (Compound 4)
 4-Hydroxyphenylacetic acid (1.75 g) was dissolved in methanol (30 ml) and 10 M aqueous hydrochloric acid (0.15 ml) was added and heated to reflux for 15 hours. After cooling to room temperature, triethylamine (1 ml) was added, the mixture concentrated, purified by flash chromatography using silica gel (30 g) and eluted with ethyl acetate-hexane (2: 8 volume by volume) to give Methyl 4-Hydroxyphenylacetate (4) (1.693 g). This was confirmed by spectroscopy-IH NMR (CDCI3) 8 3.58 (s, 2H), 3.73 (s, 3H), 6.75 (d, 2H), 7.10 (d, 2H).



  Preparation of Compound 5
 Compound 3 (0.243 g) was dissolved in dry chloroform (3 ml) and a solution of
 phosphores pentachloride (0.240 g) in dry chloroform (3 ml) added followed by heating at
 60* C for 3 hours. The reaction mixture was concentrated to an oil which was left to stir under
 high vacuum for 16 hours. The resultant phosphorochloridate was redissloved in dry
 dichloromethane (3 m ) and added to a solution of Metliyl 4-Flydroxyplienylacetate (4) (0.150  g) and 4-Dimethylaminopyridine (0.146 g) in dry dicliloromethane (3 ml) at 0 C. The reaction mixture was stirred at room temperature for 16 hours. Saturated aqueous ammonium chloride (15 ml) was added and products were extracted into dichloromethane (40 ml).

   The organic layer was washed with water (10 ml), dried over anhydrous MgS04, filtered and concentrated then purified by flash chromatography using silica (30 g) and eluting with ethyl acetate-hexane (4: 6 volume by volume) to give compound 5 (0.20 g). This was confirmed by spectroscopy
IH NMR (CDCI3) 8 2.27 and 2.30 (2s, 31-1), 2.40 and 2.42 (2s, 6H), 3.40 (d, 2H), 3.58 (s, 2H), 3.67 (d, 3H), 3.69 (s, 3H), 6.88 (s, 211), 7.05 (d, 2H), 7.20 (d, 2H).



  Preparation of Compound 6
 Compound 5 (0.19 g) was dissolved in dioxane (1.5 ml). Tliiophenol (0.575 g) and triethylamine (0.70 ml) were added and the reaction mixture was stirred for 16 hours. The mixture was concentrated, redissolved in water (30 ml) and washed with dichloromethane (5 x 25 ml). The aqueous layer was adjusted to pH 1 with aqueous HCI and extracted with ethyl acetate (5 x 30 ml). The organic layers were combined, dried over anhydrous MgS04, filtered and concentrated to give compound 6 (0.176 g). This was confirmed by spectroscopy-IH
NMR (CDC13) 8 2.15 (bs, 3H), 2.28 (bs, 6H), 3.13 (d, 2H) 3.40 (bs, 2H), 3.60 (bs, 3H), 6.68 (bs, 2H), 6.85 (bs, 2H), 7.00 (bs, 2H).



  Preparation of Compound 7
 Compound 6 (0.087 g) was treated with a solution of lithium hydroxide monohydrate
 (0.025 g) in methanol (1.4 ml) and water (0.30 ml) with vigorous stirring for 50 hours. The reaction mixture was concentrated to a third of its volume, water (10 ml) was added and the
 aqueous layer was washed with dichloromethane (3 x 10 ml). The aqueous layer was adjusted
 to pH 1 with concentrated HCI and was extracted with ethyl acetate (7 x 20 mL). Organic
 layers were combined, dried over anhydrous MgS04, filtered and concentrated to give  compound 7 (0.063 g). This was confirmed by spectroscopy- lI NMR (d6 DMSO) 8 2.170 and 2.175 (2s, 3H), 2.30 (s, 6H), 3.20 (d, 2H), 3.50 (s, 2H), 6.80 (s, 2H), 7.00 (d, 2H), 7.20 (d, 2H).



  Ex uz le 1.2
Synthesis of Left Hand Portion (Compound 8) of RT3! osphonate Transition State Analog (RT3A)(see FIG. 2)
Preparation of Compound 8 from the demethylation of compound 3 using thiophenol and triethylamine.



   Compound 3 (0.122 g) was dissolved in dioxane (1.5 ml) and with stirring a solution of thiophenol (0.55 g) in dioxane (1.5 ml) was added. Triethylamine (0. 70 ml) is then added and the solution is stirred for 24 hours at room temperature. Reaction mixture was trallsferre to a separating funnel, water (50 ml) was added, the aqueous layer was adjusted to pH 7 with aqueous HC1 and then it was washed with dichioromethane (5 x 50 ml). The aclueous luyer was acidifie to pH 1 with 1 M aqueous IICI and extracted with ethyl acetate (2 x 75 ml). The organic layers were combined and washed with water (5 ml), dried over anhydrous MgS04, filtered and concentrated. Purification using silica (l ) g) and eiuting with methanotdichloromethane (8: 92 volume by volume-15: 85 volume by volume) gave compound 8 (0.055 g).

   This was confirmed by spectroscopy-1 NMR (d6 DMSO) 8 2.10 (bs, 3t-1), 2.28 (bs, 6H), 3.2 (d, 2H), 3.30 (d, 3fi), 6.85 (s, 2H).



  Example 1.3
Synthesis of Right Iland Portion (Compound 12) of R'1'3 Pluosphonate'1'ransition State
Analog (RT3B) (see FIG. 3)
 Preparation of Dibenzylmethylphosphorate (Compound 9)  
 Sodium hydride (60% dispersion in mineral oil) (0.16 g) was washed with dry hexane (2 x 10 ml). To the decanted solid, dry THE (5 ml) was added and the stirred suspension was cooled to 0  C. A solution of Dibenzyl phosphite (1.048 g) in dry THF (5 ml) was added and the mixture warmed to room tempera. ! : 2e. After 30 minutes, methyl iodide (0.32 ml) was added and the reaction mixture stirred for 2 hours. The reaction mixture was concentrated, redissolved in ethyl acetate (75 ml), washed with saturated aqueous ammonium chloride solution (50 ml) and water (10 ml).

   The organic layer was dried over anhydrous MgS04, filtered, concentrated then purified by flash chromatography using silica (10 g) and eluting with ethyl acetate-hexane (1: 1 volume by volume) which gave Dibenzylmethylphosphonate (9) (0.750 g). This was confirmed by spectroscopy * NMR (CDC13) 6 1.48 (d, 3H), 5.00 (m, 4H), 7.40 (s, 10H).



  Preparation of Benzylmethylphosphoric Acid (Compound 10)
 Dibenzylmethylphosphonate (9) (0.277 g) was dissolved in dioxane (1 ml) and water (0.5 ml). Aqueous 2 M LiOI-l (1 ml) was added and the mixture was vigorously stirred for 48 hours. Water (25 ml) was added and the aqueous layer was washed with ethyl acetate (25 ml).



  The aqueous layer was acidified to pH 1 with concentrated HCI and extracted with ethyl acetate
 (2 x 35 ml). Organic layers were combined, dried over anhydrous MgS04, filtered and
 concentrated to give Benzylmethylphosphoric acid (10) (0.183 g). This was confirmed by
 spectroscopy-1H NMR (CDC13) 8 1.53 (d, 31-1), 5.08 (d, 21-1), 7.40 (m, 511), 11.90 (s, IH).



   Preparation of Compound 11
 Benzynnethylphosphoric acid (10) (0.118 g) was dissolved in thionyl chloride (1 ml)
 and stirred for 4 hours. The reaction mixture was concentrated to dryness and was) eft under
 high vacuum for 16 hours. This was redissolved in dry dichloromethane (1 ml) and DMP (1.5
 ml). With stirring Methy ! 4-Hydroxyphenyl acetate (4) (0.083 g) and triethylamine (0.170  mL) was added. After 16 hours, saturated a (lueous ammonium chloride (30 ml) was added and the mixture was extracted with ethyl acetate (2 x 50 ml). Organic extracts were combined, dried over anhydrous MgSO4, filtered and concentrated. Purification was achieved using preparative tic plates (1 mm) and using ethyl acetate-hexane (4: 6 volume by volume) as the solvent to give compound (11) (0.068 g).

   This was confirmed by spectroscopy-l l I NMR (CDC13) 8 1.67 (d, 311), 3.60 (s, 2H), 3.73 (s, 311), 5.15 (m, 211), 7.13 (d, 2H), 7.25 (d, 2H), 7.40 (s, 5H).



  Preparation of Compound 12
 Methylation ofdibenzyt phosphite using methyt iodide gave Dibenzyl methyl pluyolphonate (9) which on lithium hyclroxicle hydrolysis afforded the phosploric acid 10.



  Activation with thioiiyl chloride and subsequent reaction with Methy 4-Hydroxyphenylacetate (4) produced compound 11. Final product 12 was obtained by the catalytic hydrogenation of 11.



   Compound 11 (0.060 g) was dissolved in ethyl acetate (10 mi) and 10% palladium on charcoal (0.03 g) was added. The mixture was stirred under an atmosphere of hydrogen for 3 hours. It was then filtered through a bed of celite and ethyl acetate (2 x 10 ml) added to wash products from the celite. All washings and Ciltrates were combined and concentrated to give compound 12 (0.038 g). This was confirmed by spectroscopy"NMR (CDCI3) 6 1.48 (vbs, 3H), 3.60 (bs, 2H), 3.68 (bs, 31-1), 7.15 (m, 4H), 8.20 (vbs, 1H).



  Exalllple 1.4
Synthesis of RT3 Substrate (see FIG. 4)
 Preparation of 4-Hydroxyphenylacetamide (Compound 13)
 Methyl 4-Hydroxyphenylacetate (4) (0.83 g) was dissolved in saturated methanolic
 ammonia (30 ml) and place (l in a thick wa ! ! ed tube with Teflon screw cap. The solution was  stirred in this sealed tube at room temperature for 72 hours. Reaction mixture was concentrated and redissolved in methanol-chloroform (2: 8 volume by volume, 75 ml). No crystallization occurred so the solution was concentrated to half its volume and hexane was added with heating. Cooling to 0* C gave crystals of 4-Hydroxyphenylacetamide (13) (0.524 g). This was confirmed by spectroscopy- l NMR (d6 DMSO + CF3C02D) 5 3.23 (s, 211), 6.63 (d, 2H), 7.00 (d, 2H).



  Preparation of Compound 15
 The amide 13 was prepared by the arnmono ! ysis of the methyl ester 4. Activation of mesitylacetic acid with thionyl chloride and subsequent reaction with amide 13 gave the final compound 15.



   Mesitylacetic acid (0.10 g) was dissolved in thionyl chloride (1 ml) and stirred for 4 hours. The reaction mixture was concentrated to dryness and placed under high vacuum for 16 hours. The resultant mesitylacetylcllloride (14) was dissolved in dry dichloromethane (1 ml) and added to a solution of 4-1-lydroxyphenylacelanlide (13) (0.076 g) and triethylamine (0.077 mL) in dry DMF (1 ml). The reaction mixture was stirred for 90 minutes then concentrated, redissolved in ethyl acetate (30 ml) and washed with saturated aqueous sodium bicarbonate (25 ml) and water (5 cul). The organic layer was dried over anhydrous MgS04, filtered and concentrated. Purification was acheived by preparative silica tlc (I mm) using ethyl acetate as solvent to give compound 15 (0.055 g).

   This was confirmed by spectroscopy- H NMR (d6
DMSO + CF3C02D) 8 2.13 (s, 3H), 2.23 (s, 611), 3.35 (s, 21-1), 3.83 (s, 3H), 6.80 (s, 2FI), 6.93 (d, 2H), 7.23 (d, 2fil).



  Exarnpie 2
Hapten Conjugations
 RT3 hapten, 4- (carboxymethyl) phenyl-(2, 4,6-trimethylphenyl)-methyl phosphonate   (compound 7, Figure 1), was conjugated to bovine serum albumin (BSA) and keyhole limpe hemocyanin (KLH), via the free carboxyl group on the hapten.



   5.4 mg of RT3 was dissolved in phosphate buffer at 37'C, and then mixed with 6 mg,
EDC, l-ethyl-3- (3-dimethy ! aminopropyt) car ! x : diimide-HC !, and N-hydroxysulfosucccinimide (S-NIIS), at a molar ratio of 1: 2: 2, respectively. 10 mg of BSA was dissolved in water and then added to the hapten. The molar ratio of hapten to BSA was 100: 1, using a BSA molecular weight of 64,000. The mixture was stirred at room temperature for 3 hours and then dialyzed against 2 changes of phosphate buffered saline (PBS) at 4  C over 2 days.



   The RT3-KLH conjugate was made in a similar manner to RT3-BSA except that the pli of the hapten, EDC, S-NHS mixture was adjusted to 6.0 with Na011 before the addition of the
KLI-I. The hapten to protein ratio was 100: 1 using a protein molecular weight of 64,000. The reaction mixture was stirred for 2 hours at room temperature, and then dialyzed against PBS at 4'C over 2 days.



   After the dialysis, the protein concentrations were determined by the micro-bicinchonic acid assay using BSA as the protein standard (Pierce, Rockford, Illinois).



  Example 3
Immunizations And mRNA Isolation
 BALB/c female mice, 14-weeks-old, were injected intraperitoneally with 50 ig of RT3
KLI-I emulsified in complete Freund's adjuvant. The mice were boosted with 10 ig of RT3
 KLH emulsified in incomplete Freund's adjuvant at weeks 4 and 7. The mice were sacrifice
 three days after the last injection and the spleen removed and used as a source of mRNA. Tie
 immune response after the second injection was measured by ELISA. The titer of the
 antiserum against RT3-BSA was 1: 100,000.



   Preparation of mRNA-mRNA was isolated from 1 () 5 mg of spleen obtained from a
 mouse immunized with RT3-KLH as described above. mRNA was purified using a FastTrack
 mRNA isolation kit (Invitrogen Corp, San Diego, CA.) following manufacturers instructions.



  The mRNA yield was 5.4 ug as determined spectrophotometrically using the following  formula:
 mRNA = (A26o) (0.04 ug/ul) D where D is the dilution factor
Example 4
Materials and Methods for Construction Of Phage Display Libraries
 Protocols used in the following procedures were described in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989).



   Restriction digestion, analysis of restriction enzyme digestion products on agarose gels, purification of DNA using phenol/chloroform, preparation of 2xTY medium and plates, preparation of tetracycline and ampicillin stock solutions, PAGE of proteins, Preparation of phosphate buffered saline, preparation of plasmid DNA by alkaline lysis, cesium chloride purification of plasmid DNA.



   All enzymes were supplied by New England Biolabs (Beverly, MA) and were used according to manufacturer's instructions unless otherwise stated.



   Ligations were done using an Amersham (Arlington Heights, IL) ligation kdt. DNA purifications using glass milk (Bio 101, La Jolla, CA) or magic minipreps or magic PCR preps (Promega, Madison, WI) were done according to manufacturers conditions.



   Preparation of competent cells and transformation were done according to the method described in the Bio-Rad (Hercules, CA) electro-transformadon protocols.



   The following are described in McCafferty et al., 1992, Patent No. W092/01047: preparation of phage, phagemid particles, single stranded DNA, expression of soluble single chain Fv antibodies, the procedures for panning and ELISA, analysis of diversity by PCR and
BstNl digestion.



   DNA was transformed into competent TG1 cells (genotype: K 12d (lac-pro), sup E, thi, hsdD5/F'traD36, pro A+B+, Lac Iq, lac ZdM15) or HB2151 cells (genotype: K12d (lac-pro), thi/F'pro A+B+, Lac IqZ dM15).



   The mouse PCR primers, the vector pCANTAB 3 and pCANTAB 5, and and-M13 antibodies are available from Pharmacia (Piscataway, NJ) (Cat. No. 27-9400-01,27-9401-01,  27-9402-01 respectively).



  Examnle 4.1
Preparation Of Veia ors Facilitating RIpici/Mtiltiple Isolations Of Soluble Single Chain Fv (scFv) Antibodies Using"Irnmobilized Metal Affinity Cchromatography Procedure" (IMAC)
 In screening for catalytic antibodies, it would be advantageous to have a means of readily purifying/concentrating bacterially expressed antibodies from piiagemid vectors.'1'he following changes were incorporated into the phagemid vectors pHEN, pCANTAB (see
McCafferty et al., (1992) Patent Application WO 92/01047, Hoogenboom H. R. et al., Nucl.



  Acid Res. 19, (1991): 4133-4137, Pharmacia product literature Cat. No. 27-9401-01):
 i) sequences encoding six histidine residues at the C terminus of the antibody
 were introduced.
 ii) sequences encoding a myc tag peptide at the C terminus of the antibody were
 included for sensitive detection/alternative purification of SCFv's. By
 incorporating these changes, a very simple and rapid procedure for
 concentrating and purifying bacterially expressed antibodies has been
 developed.

 

   Two pairs of oligonucleotides were synthesised to generate the double stranded inserts
 shown below. These have 5'overhangs compatible le with the Noti site and so can be clones
 into this site in plIEN, pCANTAB, regenerating the Nos 1 site at the 5'end as shown below.



   Ibis-6 1/2
 ala ala his his his his his his amb 5'G GCC GCA CAT CAT CAT CAC CAT CAC TA 3'
 3'CGT GTA GTA GTA GTG GTA GTG ATC CGG 5'
 Hies-6314
 ala ala his his his his his his gly
 5'G GCC GCA CAT CAT CAT CAC CAT CAC GG 3'
 3'CGT GTA GTA GTA GTG GTA GTG CCC CGG 5'
 (amb= amber codon)  
 His-6 3/4 was cloned into pHEN-OX16 which consists of the high affinity oxazalone binding clone described in Clackson et al., Nature 352 (1991): 624-628, cloned into the
Pstl/Notl site of pHENl. This construct will give rise to a product consisting of [aOX antibody-his-6-myc tag-amber codon-gene3 which can be detected with the 9E10 antibody 9 (the cell line producing 9E10 antibody 9 is available from ATCC, Rockville, MD, CRL1729 designated MYC1-9E10.2). The new construct is called pOX16his-11 and is shown in Figure 5.



   This clone was used to work out the"immobilized metal affinity chromatography procedure" (IMAC) purification regime described below. An additional construct, was made by inserting His-6 1/2 into the clone scFv4, which consists of the lysozome binding D1. 3 scFv antibody cloned into pCANTAB3. This construct will give rise to a product consisting of (D1. 3 antibody-his-6-amber codon-gene 3) which can be detected with anti-Dl. 3 antiserum.



   All cloning manipulations were carried out in TG1 and the correct clones introduced into the non-suppressor strain HB2151 for expression as single chain Fvs.



   All volumes are for an initial culture volume of 50 mis and all bacterial growth was at 30  C in the host-HB2151. E. coli cells carrying the plasmid of interest were grown to 0.7-1.0
O. D./ml in 2xTY medium supplemented with 2% glucose, 1001lglml ampicillin. The culture was centrifuged in a 50 ml Falcon tube at 3500 rpm for 10 minutes at room temperature, resuspended in 2xTY/100Ag1ml ampicillin/1mM IPTG and grown for 3 hours. The culture was centrifuged in a 50 ml Falcon tube at 3500 rpm for 15 minutes at a temperature of 4  C and is resuspended in 1 ml of cold buffer A (PBS/1M NaCl/lmM EDTA) and left on ice for 15 minutes.

   The sample was cenifuged 2x10 minutes, the supernatant carrying the periplasmic contents collected and MgCl2 added to 1-2 mE
 400 gl of a 1: 1 slurry of Ni-NTA agarose: buffer 1 (Qiagen, Chatsworth, CA) which had been pre-equilibrated with buffer A was added to the periplasmic preparation and incubated for 10 minutes on an inverting platform at room temperature. The mixture was centrifuged at low speed on a microfuge for 10-15 seconds and the pellet resuspended in 1 ml of buffer A.  



  This process was repeated another 2 times before resuspending in 100 pi of either PBS or buffer A carrying 250 mM imidazole. After 10 minutes the supernatant was collected and the pellet re-extracted with another 100 Wl of the same buffer and pooled.



   Results are shown in FIG. 6, in lanes marked with the capital letters A, B, C, D, E and
F. Lane A, FIG. 6 shows abundant accumulation of scFv in the periplasm after 3 hours induction. As expected scFv was only found in the culture supernatant after overnight incubation (data not shown). Isolation of antibody from the periplasm not only has the advantage that it can be prepared after a shorter induction, with the potential for a better quality product, but also the initial centrifugation step itself effectively concentrates the antibody, when working from the periplasm. Lanes B and C show that the antibody fragment is efficiently bound and recovered after incubating the Ni-NTA matrix with periplasmic extract (see Lane A) and eluting bound scFv with Buffer A/250 mM Imidazole as described above.

   Lanes D and E shows that elution can be carried out in PBS/250 mM imidazole, without added NaCl. This may be a more useful buffer for subsequent use of the antibody. Lane F shows that the clone scFv4his-6 produces an antibody fragment which can be recovered in the same way.



   This procedure is a very simple means of concentrating/purifying antibodies which will facilitate the preparation of multiple samples simultaneously as required for screening for catalysis.



   Vector forms of the above construct were prepared by cleavage with Notl and Bam Hl to isolate DNA extending from the Notl cloning site through to the BamHl site in the middle of gene III. This was used to replace the equivalent Notl/BamHl site within PCANTAB 3 and pCANTAB5 to give the vectors PCANTAB 3 his-6 and pCANTAB5 his-6. This transfers the myc tag and the his-6 tag to the new backbone (Figure 7).



  Example 4.2
Preparation Of A Phage Library Derived From Mice Immunized With RT3
 Sequences of all primers used for the construction, PCR and sequence analysis of mouse derived phage display libraries are shown below:  
SEQUENCE OF VK PRIMARY PRIMERS.



  VKA BACK 5'GAT GTT TTG ATG ACC CAA ACT CCA 3'
VKB BACK 5'GAT ATT GTG A1'A ACC CAG GAZ GAZA 3'
VKC BACK 5'GAC ATT GTG CTA/G ACC CAG TCT CCA 3'
VKD BACK 5'GAC ATC CAG ATG ACN CAG TCr CCA 3'
VKE BACK 5'CAATT GTT CI'C ACC CAG TCT'CCA 3'
VKF BACK 5'GAA AAT GTG CTC ACC CAG TCI'CCA 3'
MJK I FONX 5'CCG TTT GAT TTC CAG CTT GGT GCC 3'
MJK2FONX 5'CCG TTT TAT TTC CAG CTT GGT CCC 3'
MJK4FONX 5'CCG TTT TAT TTC CAA CIT TGT CCC 3'
MJK5FONX 5'CCG TTT CAG CTC CAG CUT GC'I'CCC 3'
SEQUENCE OF VI ! PRIMARY PRIMERS.



  VH I FOR-2 5'TGA GGA GAC GGT GAC CGT GG1'CCC TTG GCC CC 3'
Vl i I BACK 5'AGG TSM ARC TGC AGS AGT CWG G 3'
SEQUENCE OF VK LINKER PRIMERS.



  VKALINKFOR
TGG AGT TTG GGT CAT CAA AAC ATC CGA TCC GCC ACC GCC AGA GCC
VKBLINKFOR
TTC ATC CrG GGT TAT CAC AAT ATC CGA TCC GCC ACC GCC AGA GCC
VKCLINKFOR
TGG AGA CrG GGT T/CAG CAC AAT GTC CGA TCC GCC ACC GCC AGA GCC
VKDLINKFOR
TGG AGA CTG XGT CAT CTG GAT GTC CGA TCC GCC ACC GCC AGA GCC
VKELINKFOR
TGG AGA CTG GGT GAG AAC AAT TTG CGA TCC GCC ACC GCC AGA GCC
 VKFLINKFOR
TGG AGA CTG GGT GAG CAC ATT TTC CGA TCC GCC ACC GCC AGA CCC
 SEQUENCE OF VH LINKER PRIMER.



   LINK BACK 5'GGG ACC ACG GTC ACC GTC TCC TCA 3'
 PULLTIIROUGH PRIMERS
 HBKAPA10 5'CAT GAC CAC AGT GCA CAG GTS MAR CTG CAG SAG TCW GG 3'
 JKINOTIO 5'GAG TCA TTC TGC GGC CGC CCG TTT GAT TTC CAG CTT GGT GCC 3'
 JK2NOT10 5'GAG TCA TTC TGC GGC CGC CCG TTT TAT TTC CAG CTT GGT CCC 3'
 JK4NOT10 5'GAG TCA TTC TGC GGC CGC CCG TTT TAT TTC CAA CTT TGT CCC 3'
 JK5NOT10 5'GAG TCA TTC TGC GGC CGC CCG TTT CAG CTC CAG CTT GGT CCC 3'
 PCR SCREEN PRIMERS
 KSJ28 5'GTC ATT GTC GGC GCA ACT ATC GGT ATC 3'
 FDTSEQ I 5'GTC GTC TTT CCA GAC GTT AGT 3'
 Spleen mRNA was used from a mouse immunized with RT3-KLH (see Example 3)
 and cDNA was prepared using random hexamers (Pharmacia, Piscataway, N. J.) as primers.



   PCR reaction conditions are essentially as in McCafferty et al., Patent application WO
 92/01047, using Taq polymerase according to manufacturers conditions.  



   The primary heavy chain product (VH) was made using the primer VH1FOR-2 and
VH1BACK. The primary light chain PCR product (VL) was made in 5 separate reactions using an equimolar amount of the 4 MJKFONX primers with one of the 5 VKBACK primers (VKABACK, VKCBACK, VKDBACK, VKEBACK, VKFBACK). PCR conditions for
VL's were 25 cycles of 94* C for 1 minute, 55'C for 1 minute and 72'C for 2 minutes, followed by a 72'C incubation for 10 minutes. For the VH's 60'C was used as the hybridization temperature rather than 55'C as this gave better results.



   Linker fragments were prepared using the template pscFvNQ 11 (McCafferty, J. et al.,
WO 92/01047) with the primer LINKBACK with 5 separate reactions each containing one of primers VKALINKFOR, VKCLINKFOR, VKDLINKFOR, VKELINKFOR,
VKFLINKFOR.



   Primary products were gel purified and linked together in 5 separate linkage reactions using linker fragments complementary with the 3'end ofVH and with the 5'end of the various
VL's. Linkage was done in a"mock"PCR reaction using the three fragments and no added primers. The linkage was carried out in duplicate in a 25 til volume with approximately 10 ng of each fragment present. This linkage was taken through 25 temperature cycles of 94'C for 1 minute, 60* C for 2 minutes and 72* C for 2 minutes followed by a 72* C incubation for 10 minutes. 25 Rl of assembly reaction was run on a gel and after de-staining the assembled product was just visible on the gel (data not shown)
 The material for cloning was prepared in a secondary PCR reaction using primers which introduce cloning sites (VIIIBACKAPAIO and a mix of JKINOT10, JK2NOTl (),
JK4NOT10, JK5NOT10).

   A small amount of product from the linkage reaction was used as template (1 il into a 50 tl PCR reaction). PCR conditions were 25 cycles of 94* C for 1 minute, 55* C for 1 minute and 72* C for 2 minutes followed by a 72'C incubation for 10 minutes.



   The secondary PCR product was cut with the enzymes ApaLl/Notl, gel purified,
 cloned into the ApaLl and Notl sites of pCANTAB3his-6 and transformed into
 electrocompetentTGl cells. (Transformation efficiencies were 5xl () S/ (ig forpUC19 and  1x106-K) 7/p. g for ligated vector). A library of 1.2 x 106 clones was generated and 18/20 clones were found to have insert. Analysis by PCR and BstNl digestion indicate that these are all different.



  Exampie 4.3
Panning The Mouse Anti-RT3 Library Against RT3-BSA
 The panning procedure was essentially as described in Marks, J. D. et al.,
Biotechnology 10 (1992): 779-783. The RT3 hapten (compound 7, figure 1) was conjugated to
BSA as described in example 2. Nunc (Kamstrup, Denmark) immunosorb tubes were coated with 1 mi of RT3-BSA at 20 mg/ml. The tubes were blocked to the top with 4 mls PBS/2% milk powder for 2 hours at 37* C and 0.8-1.0 ml of concentrated phage (equivalent to 10-50 mls of culture supernatant) was used for binding. Tubes were not inverted. Binding of phage and washing was done using MOPS buffered saline (MBS which is 50 mM MOPS pH7.4,
 150mMNaCI).



   Washing was done ten times with MBS/0.1% Tween 20 and ten times with MBS.



   Bound phage were eluted using 800 ml of 100 mM triethylamine, neutralized with 400 mi of
 1M Tris pH 7.4 and infected, into exponentially growing TG l-tr cells (T phage resistant TG 1
 cells). The cells infected with the eluate were plated onto large (22x22cm) TY plates
 supplemented with 2% glucose/100 mg/ml ampicillin. Bacterial stocks were prepared next
 day, liquid cultures were inoculated from them and rescued with M 13 helper phage and the
 panning procedure was repeated a second time with the concentrated phage.



   The panning process was repeated and concentrated phage was used in a polyclonal
 ELISA. No signal was achieved from the unpanned library but increasing signal was achieved
 through successive pannings (not shown). The numbers of phage eluting after PAN I, PAN2
 and PAN3 increased each time as expected and 2200 x 106 infectious phage
 respectively).



   Eluted phage from PAN1 and PAN2 was introduced into 1IB2151 cells (a
 non-suppressor line producing soluble SCFv). Individual colonies were picked into 96 well  plates containing TY medium with 100 mg/ml ampicillin supplemented with 2% glucose (TY/G/A) and grown for 4-16 hours (stock plate). These cultures were used to inocule a second 96 well plate containing TY/A and 0.1 % glucose. This plate was incubated for 2-4 hours at 30 C before inducing by adding IPTG to 1 mM and growing overnight Next day culture supematants were added to ELISA plates previously coated with 2 mg/ml Rr,-BSA and blocked with 2% milk powder. Binding was carried out in 1X MBS/2% milk powder and binding was detected using the mouse 9E10 antibody followed by goat anti-mouse-peroxidase (Sigma, SL Louis, Missouri).

   The 9E10 antibody used to detect the myc tag peptide is available from the ATCC, Rockville, Maryland (CRL1729, Name given is MYC1-9E10 2).



   Screening for binding from PAN1 using RT3-BSA as antigen and MBS buffer throughout the procedure identified 47 positives from 364 clones. In a similar way, 115/184 positives were identified from PAN2. The diversity of the clones was analyzed by BstNI digestion of PCR amplified single chain DNA insert from each clone as described in Example 4.0.



   The results are summarized in Table 1 on a group-by-group basis as shown below.



  17 of 48 binders analysed from PAN1 (35%) had pattern A. A total of 78 binding clones from 115 from PAN 2 (68%) had PCR pattern A. (22 of these were restreaked and analyzed further and these are presented in the Figure 1 and Table 1). Pattern B was found in 2 of 48 (4.1%) clones from PAN1 and 24 of 115 (21 %) from PAN2.2 of 48 from PAN1 had pattern C (4.1 %), while 3 of 115 from PAN 2 (2.6%) had this pattern. Pattern D was found in 3 of 48 clones from PAN1 (6.2%) and 3 of 115 from PAN 2 (2.6%). Thus, the proportion of positives from each group appears to alter from PAN1 to PAN2. This could result in the loss of potentially catalytic clones after several rounds of panning if selection is based solely on strength of binding to RT3.  



  Table 1. Grouping Of Mouse RT3 Binders According To PCR Pattern
EMI38.1     


<tb>  <SEP> PCR <SEP> Pattern <SEP> Sample <SEP> No.
<tb>



   <SEP> A <SEP> PAN1-3,4,6,8,9,14,17,18,24,25,27,30,35,36,45,46,47.
<tb>



  PAN2-60,62,63,64,65,66,67,70,72,74,77,78,79,84,85,86, <SEP> i
<tb>  <SEP> 87.88.91.92.96.97.
<tb>



   <SEP> B <SEP> PAN1-12,20.
<tb>



   <SEP> PAN2-49,50,51,52,53,54,55,56,57,58,59,69,82,90,93,94,
<tb>  <SEP> 102,103,104,105,106.
<tb>



   <SEP> C <SEP> PAN1-5,48
<tb>  <SEP> PAN2-75,76,80.
<tb>



   <SEP> D <SEP> PAN1-10,26,43.
<tb>



   <SEP> PAN2-68,83,89.
<tb>  <SEP> a <SEP> PAN1-2,13.
<tb>  <SEP> e <SEP> PAN1-11,19.
<tb>  <SEP> q <SEP> PAN1-7,15.
<tb>  <SEP> i <SEP> (small <SEP> PAN1-23,38,44.
<tb>  <SEP> insert)
<tb>  <SEP> j <SEP> (small <SEP> PAN1-31,40,42.
<tb>  <SEP> insert)
<tb>  <SEP> E <SEP> PAN2-71,73
<tb>  <SEP> 10 <SEP> unique <SEP> b-21, <SEP> d-41, <SEP> f-1, <SEP> g-33, <SEP> h-16, <SEP> k-28, <SEP> 1-39, <SEP> n-34, <SEP> p-32, <SEP> G-22.
<tb>  <SEP> patterns
<tb>  <SEP> from <SEP> PAN1
<tb>  <SEP> 4 <SEP> unique <SEP> F-61, <SEP> H-81,1-95, <SEP> J-98.
<tb>  <SEP> patterns
<tb>  <SEP> from <SEP> PAN2
<tb> 
 At least 15 other patterns were found in PAN1 with many appearing only once. Many other patterns present in PAN1 were not identified in PAN2. In addition, some patterns  appeared in PAN2 which had not been identified in PAN1.

   This argues that there is much greater diversity in the library than is indicated by the PCR pattern groups which we have identified.



  Example 4.4
Binding Analysis of Selected Clones from Mouse RUs Phage Antibody Library
 Several mouse RT3 phage antibody clones isolated from PAN 2 (see Table 1, Example 4.3) were characterized further in terms of binding specificity. Analysis was done using a competitive inhibition format, in which the antibody is first reacted with free hapten or product or portions of hapten and product, prior to addition to RT3-BSA coated wells as described in detail below.



   The clones selected for analysis and their corresponding PCR pattern (see Table 1,
Example 4.3) were: 50 (PCR B), 64 (PCR A), 68 (PCR D), 71 (PCR E), 80 (PCR C), 84 (PCR A), 95 (PCR I), 96 (PCR A), and 97 (PCR A). Soluble scFv was purified from 50 ml cultures of each clone using the IMAC protocol described in Example 4.1, except bound antibody was eluted with 50 mM EDTA, 0.5 M NaCl. The scFv concentration for each clone was estimated from silver stained SDS polyacrylamide gels by running a portion of the eluted protein on a gel containing appropriate scFv concentration standards. The scFv protein was diluted to 4 ug/ml and then serially diluted 1: 2 across 11 wells of RT3-BSA coated ELISA plate. The concentration of scFv giving 50% of the maximum ELISA signal was determined from the titration.

   This concentration of scFv was used for a subsequent competitve inhibition assay described below.



   A competitive inhibition ELISA assay was performed by incubating scFV (at a concentration as determined by titration above) with 100 uM of each of the following compounds: RT3 hapten (Compound 7, Figure 1), left hand portion of RT3 hapten (designated
RT3A, Compound 8, Figure 2), right hand portion of RT3 hapten (designated RT3B,
Compound 12, Figure 3), The left and right hand portions of the expected products from the esteriolytic cleavage of substrate (Compound 15, Figure 4) designated Product A   (Mesitylacetic acid, Figure 4) and Product B (Compound 13, Figure 4). The scFv was preincubated with the the inhibitor compound in tubes at room temperature for 1 hour prior to addition to RT3-BSA coated ELISA wells. The results of the assay are shown in Figure 8.



  The OD4i5 nm readings have been normalized to a value of 1, which represents the ELISA signal seen for the corresponding scFv in the absence of added competitive inhibitor. The results show all of the clones with the exception of 68 and 71 are inhibited in their binding to
RT3-BSA with free RT3 hapten. The clones which did not show inhibition with free RT3 were shown to have cross reactivity with BSA. Clones 64,84,96,97 and 50 also show binding inhibition with RT3A andto a lesser extent with Product A. No inhibition is seen for any of the clones tested with RT3B or Product B.



  Example 4.5
Sequencing Of Mouse Anti-RT3 SCFv (s)
 Although a large proportion of clones from PAN1 and PAN2 fall into the PCR A pattern group, it was not clear whether clones in this group were identical or diverse, and so a number of these clones were sequenced. Furthermore, in an attempt to determine whether the same heavy and light chains were being used within other major pattern groups, some representative clones from the other major groups were sequenced.



   Single stranded DNA was prepared from those clones which are emboldened in Table 1 and sequencing was carried out using the Sequenase kit (USB, Cleveland, OH). Sequence alignments to Genbank germline sequences and between clones were done using the "MacVectorlM"aBI, New Haven, Connecticut) program. Since the sequences at the 5'and 3' ends were encoded and enforced by PCR primers, these were"removed"for aliments. In the presentation of light chain sequences, the primer encoded sequences are not shown but the primers which were used are indicated in the right hand column. For the heavy chains, since the 5'primer is a single but degenerate primer, the sequence introduced by this primer is shown in each case. For comparison, the actual heavy chain primer sequence is shown at the 5'and 3'ends of each clone.

   The sequence of one clone is presented on the top line and the  differences from this sequence are indicated for the other clones. All PCR A mutations shown, which give rise to amino acid, changes were re-checked on the sequencing gels. For the heavy chains of pattern A, all unique changes were re-checked on the sequencing gel, and changes which occured in a number of clones were checked on at least one of the clones carrying that change.



  Light Chain Sequences Of Mouse RU Binders
 As shown in FIG. 9, eight different light chains have been used with the 15 different clones from pattern A. The chain associated with clones mR6 and mR8, differs from the germline V gene by a single silent nucleotide change. The chain used in mR9, mRl8 and mR27 differs from mR6 and mR8 by an additional single silent mutation. Thus, these 5 clones share the same protein sequence as the germline. Clones mR9 and mR27 have used different primers to derive the same sequence, indicating that they are independant isolates of this same sequence.



   Clones mR3 and mR25 are identical in the sequence which has been amplified but have also used different primers from each other. The sequence which mR3 and mR25 share in common, differs from mR6 and mR8 by 2 silent nucleotide changes and 2 changes resulting in 2 amino acid changes in FR3 and CDR3. Most changes have occurred in the light chain shared by clones 14,30,36,84, and 96. In these and in all the others light chains of this group, most amino acid changes are clustered in FR3, CDR3, and FR4.



   One can envisage the basic germline clone represented in mR6/8 or mR9/25, changing
S to N in CDR2 and then changing in 3 different ways to give the clones represented by 4,97 and 14 (+4 others). Similarly, there may have been a change of Y to F in CDR 3 from the same starting point, giving rise to mR3 and mR25. A third series of changes may have given rise to mR24.



   The light chain associated with pattern C (clone mR80) is also shown in FIG. 9 aligned with the germline sequence used in pattern A clones. The pattern C light chain appears to be a more highiy mutated form, derived from the same germline as used in pattern A.  



   Clones representing the other PCR patterns appear to use different ermline derived sequences. The relationship of these other clones to their nearest germline is shown in FIG.



  10. In pattern B (50,69), 2 nucleotide changes from germline give rise to 1 amino acid change. In pattern D (10,43, and 68, and 83), 8 nucleotide changes from germline give rise ta 5 amino acid changes. In pattern I (95), 2 nucleotide changes from germline give rise to I amino acid change.



   FIG. 11 shows the relationship of the different light chain sequences to that of pattern
A (for mR6,8). There is a great deal of difference between them. For patterns D and I, only the protein sequence is shown, since the nucleotide sequences have many differences. The latter two groups have longer CDR2s than the others.



  Heavy Chain Sequences Of Mouse RT3 Binders
 Analysis of the heavy chain sequences associated with PCR pattern A, reveals that, as for the light chain, they are all closely related but in most cases are different from each other (Figure 12). The alignment to aermline is less clear in these samples. The closest germline belongs to sub-group VH-II, but there are numerous differences from this germline, in the isolated clones. In addition, there appears to be a greater number of amino acid changes between clones. As expected the changes are clustered in the CDR (s).



   The heavy chains of pattern B (FIG. 13), align to a different germline and again shows numerous changes from this. All 4 clones in this group appear to be identical. Thus it appears that the clones sequenced from pattern B are multiple isolates of the same antibody. The clones represented by pattern D are all identical to each other and, excluding the sequence of CDR3, differ from the closest germline by 4 amino acids (FIG. 13).



  * The alignment of all the different heavy chain patterns with that of pattern B is shown in
FIG. 14. The heavy chains of pattern C (80) and pattern I (95) are closely related to that of pattern B. Pattern C differs by 4 amino acids. Pattern I differs by one silent mutation and one amino acid change. Interestingly, the heavy chain associated with patterns B, C, and I has a
CDR of only 3 amino acids.  



   The amino acid change in mR95 appears to introduce an amber codon. This would introduce an amino acid at this position when the suppressor line TG1 is used in the preparation of phage, but would be expected to act as a stop codon, in the non-suppressor line
HB2151 used in the screening of soluble antibodies.



  Diversity Of Clones In Pattern A
 Table 2 collates the information derived from sequencing the clones in PCR pattern A.



  Each different light chain sequence in the group is given a label ai-aviii. Each different heavy chain sequence in the group is given a label Ai-Ax.  
TABLE 2. Chain usage of mouse RT3 binders-PCR pattern A)
 LIGHT CHAINS
EMI44.1     


<tb> HE. <SEP> r, <SEP> VY <SEP> CHAINS <SEP> ai <SEP> I <SEP> aii <SEP> aiii <SEP> aiv <SEP> av <SEP> avi <SEP> avii <SEP> aviii
<tb> Ai <SEP> 14, <SEP> 30, <SEP> 36,
<tb>  <SEP> 84, <SEP> 96
<tb> Ail <SEP> 3
<tb> Aiii <SEP> 4
<tb> Aiv <SEP> 24
<tb> Au <SEP> 9
<tb> Avi6,8
<tb> Avii <SEP> 64
<tb> Aviii <SEP> 97
<tb> Aix <SEP> 27
<tb> Ax <SEP> 25
<tb> 
 Clones (PAN1) 84,96 (PAN) 2 are identical and probably represent duplicate isolates of the same initial clone. Clones 6 and 8 are also identical to each other.



  Otherwise, every clone is different There are two cases where the same light chain has been used with two different heavy chains (aii in mR 9/mR27 and aiii in mR3 and mR25). As described earlier, the light chains in each pairing used different primers. Apart from the duplicate isolates, there are no cases here, of the same heavy chain being used in different clones.



   These PCR and sequencing experiments suggest that there is indeed a great diversity in  the mouse library both at the gross scale, as judged by PCR analysis, and at a more subtle level, as judged by sequencing.



  Example 5. 1
Screening of scFv Molecules for Catalytic Activity 1. Initial Selection
 An early screen protocol was used to rapidly select a subset of potentially catalytic scFv
 molecules from the large number of scFv fragments that had been selected on the basis of hapten affinity.
 a) Immobilization: The scFv fragments that bound to hapten in an ELISA assay were selected for screening to detect catalytic activity. A 96-well Millititer GV filtration plate (Millipore) was pre-wetted and washed in PBS containing 0.05% Tween-20. Suspensions of scFv fragments immobilized on anti-mvc antibody Protein A agarose (vide infra, also see
Example 6.1) were each transferred to separate wells in the 96 well filter plate. Residual supernatant was removed by aspiration through the filter plate.

   The immobilized scFv fragments were washed in the wells by filtration at 4  C with PBS/Tween (5 x 200 1L), PBS (3 x 200 AL), and 25 mM HEPES, pH 7.0,140 mM NaCl, 0.01% NaN3 (3 x 200 I1L).
 b) Incubation of scFv and substrate: To immobilized washed antibody was added 200 pL of approximately 50 LM substrate (1) in 25 mM Hepes, pH mM NaCI, 0.01%
NaN3 was added. Following incubation at room temperature (approximately 22* C) for approximately 24 hours after which substrate solution (but not beads) was withdrawn and frozen (-20 C) until analyzed by high performance liquid chromatography (HPLC). The same 96 well plate, still containing immobilized scFv, was washed with 4 x 200 IlLlwell with 10 mM Tris, pH 9.0,140 mM NaCl, 0.01% NaN3. Again, 200 WL of 35-50 gM of RT3 substrate (Compound 15, FIG. 4) was added, this time in the pH 9.0 buffer described above.



  The scFv fragments were incubated with compound 1 for 3 hours and, as at pH 7.0, substrate solution was withdrawn frozen for later analysis of product formation.
 c) Analysis of Reaction Mixtures for Product Formation: To reduce the number of  samples, pools of generally two or three reaction mixtures (50 StL of each reaction mixture) were subjected to HPLC analysis. Mixtures (100 or 150 pL) were centrifuged in an Eppendorf centrifuge to prevent any carry-over of the agarose onto the HPLC system. Samples were then injected onto a Waters HPLC system equipped with a Vydac C-18 analytical reverse phase column. Components of the eluent were separated using a linear gradient over 30 minutes from 0.1% TFA in water to 0.1% TFA in acetonitrile. Product was detected and quantitated specrrophotometrically using a Waters spectral detection system, typically set at 215 or 270 nm.



   Early screen analysis of 46 hapten-binding scFv fragments was carried out at pH 7.0 and 9.0 to detect catalytic activity. HPLC analysis was carried out and peak areas determined for those sample pools which gave a peak at the expected retention time for the product. The results are summarized in Table 3 below. HPLC analyses of the reaction mixtures (as pools of 2 or 3) indicated that at pH 7.0, one pool of three samples and one pool of two samples appeared to have substantial product formation. At pH 9.0 a number of pools showed product peak areas above background. Three pools of three samples showed large product peaks with peak areas greater than 0.6 and these were scored as being positive. Thus, early screen assays narrowed the number of potentially catalytic scFv fragments from 46 to 5 at pH 7.0 and from 46 to 9 at pH 9.0.

   Three of the candidates at pH 7.0 were the same scFv fragments as three of the candidates at pH 9.0.  



   TABLE 3
HPLC Assay Results of Early Screen Phage Antibody Pools at pH 7.0 and 9.0
 ph 7.0
EMI47.1     


<tb> SAMPLE <SEP> PEAK <SEP> AREA
<tb> BLANK. <SEP> 18
<tb> 1,2,3
<tb> 4,5,6
<tb> 8,11,12
<tb> 14,16,17
<tb> 18,19,20 <SEP> 0.52
<tb> 21,24,25
<tb> 27,28,29
<tb> 30,31,32
<tb> 33,34,35
<tb> 36,37,38
<tb> 39,40,41
<tb> 42,44,45 <SEP> 0.455
<tb> 46,47,48 <SEP> 0.476 <SEP> 
<tb> 50,65,76 <SEP> 0.559
<tb> 83,97 <SEP> 0.571
<tb>   ph 9.0
EMI48.1     


SAMPLE <SEP> PEAK <SEP> AREA
<tb> BLANK
<tb> 1,2,3 <SEP> 0.572 <SEP> 
<tb> 4,5,6
<tb> 8,11,12 <SEP> 0.674
<tb> 14,16,17 <SEP> 0.581
<tb> 18,19,20 <SEP> 0.79
<tb> 21,24,25 <SEP> 0.522
<tb> 27,28,29 <SEP> 0.504
<tb> 30,31,32 <SEP> 0.627
<tb> 33,34,35 <SEP> 0.496
<tb> 36,37,38 <SEP> 0.492
<tb> 39,40,41 <SEP> 0.492
<tb> 42,44,45 <SEP> 0.470
<tb> 46,47,48 <SEP> 0.471
<tb>  <SEP> 0.511
<tb> 83, 

  97 <SEP> 0.407
<tb> 
Individual clones from each of the active pools identified as above were reassayed for catalytic  activity. Since the scFv remains bound to the anti-Myc agarose the same material used for the pool assays was reused for the assay of the individual clones. The results of the catalytic assays are shown in Table 4 below. The catalytic assay identified 6 clones: 11,12,18,19,30, and 83, which gave a product peak on HPLC. Clone 83 was active at pH 7.0, but not pH 9.0. Clones 18 and 19 were active at botii pH 7.0 and 9.0. Clones 11,12, and 30 were active only at pH 9.0.



   TABLE 4
 HPLC Assay Results of Individual Page Ab Clones at pH 7.0 and 9.0
 ph 7.0
EMI49.1     


SAMPLE <SEP> PEAK <SEP> AREA <SEP> | ph 9.0
EMI50.1     


SAMPLE <SEP> PEAK <SEP> AREA
<tb>  <SEP> 8 <SEP> 0
<tb>  <SEP> 11 <SEP> 1.5
<tb>  <SEP> 12 <SEP> 1.21
<tb>  <SEP> 18 <SEP> 1.09
<tb>  <SEP> 19
<tb>  <SEP> 30 <SEP> 0.96
<tb>  <SEP> 31 <SEP> 1
<tb>  <SEP> 32
<tb> 
Example 5.2
 1. Secondary Screening for Catalytic Activity
 To further examine the scFv fragments for catalytic activity, the potentially-catalytic proteins identified in the early screen described above were individually grown and purified.



  Purification of the scFv was acheived as described in Example 6.1 using either IMAC or affinity chromotagraphy on anti-Myc-Protein A agarose. Assays were performed in the same buffer systems and pH values as in the early screen but the antibodies were tested individually and they were not immobilized but free in solution.



   From these secondary assays, two scFv molecules, designated 18 and 83, catalytic activity was found. Clone 18 appeared to be active at pH 9.0 but not at pH 7.0 while clone 83 appeared to be active at pH 7.0 but not at pH 9.0 or 5.0. Both activities were significantly inhibited by hapten (2) when 10 LiM antibody was assayed with 40 tam substrate and 30 pLM  hapten. These two clones were selected as candidates for large scale purifcation of scFv as described below. The results of these assays are presented in Example 6.4.



  Example 6.1
Large Scale Purification of scFv from Catalytic rnRrD Phage Antibody Clones 18 and 83
 Preparation of Periplasmic Lysates-E. coli HB2151 clones expressing soluble anti
RT3 scFv were grown overnight in 2XYT containing 2% Glucose and 100 ug/ml ampicillin.



  Overnight cultures were used to inoculate 500 ml of 2XYT at a starting OD600 of 0.1 and cultures were shaken at 28  C for 3 to 5 hours until OD600 of 1.2 to 1.8. IPTG was added to 1 rnNI final concentration and shaking incubation was continued at 25'C for 2.5 hours. Cells were pelleted at 4, OOOXG for 10 minutes and pellets were resuspended in 6 ml of periplasmic lysate buffer (10 mM phosphate buffer, 1 M NaCl, 1 mM EDTA, pH 7.5. Following incubation on ice for 30 minutes, lysates were centrifuged at 6,000XG to remove cellular debris. PMSF was added to the cleared lysate at a final concentration of 5 llg/ml and lysate was stored on ice until purification as described below.



  Immobilized Metal Affinity Chromotagraphy (IMAC)
 MgCl2 was added to the periplasmic lysate to 1 mM final concentration and lysate was passed through a 1 ml bed volume Ni+2 charged sepharose column (Probond Metal binding
Resin. Invitrogen Corp., San Diego, CA.) washed and equilibrated with 10 mM phosphate buffer, l M NaCL Column was washed with 10 bed volumes of 10 mM phosphate, 1 M NaCl, pH 7.5 and bound scFv was eluted with 50 mM EDTA, 0.5 M NaCI. Column eluate was concentrated and dialyzed into 7 mM phosphate buffer, 0.15 M NaCl, pH 8.0 using a
Centricon 10 microconcentrator (Amicon, Beverly, MA) following manufacturers instructions.



  (see Mol. Cell. Biol. 5 (1985): 3610-3616). For some preperations of scFv concentration of the IMAC eluate was not required.



   Anti-myc Peptide Affinity Purification-Monoclonal antibody 9E10 that recognizes a 13 amino acid peptide tag at the C-terminus of the scFV was cross-linked to Protein A agarose  using an Affinica Antibody Orientation Kit (Schleicher and Schull, Keene, NH) following manufacturers instructions. A 1 ml bed volume column was prewashed with 0.23 M Glycine, 03 M NaCl, pH 2.5 and reequilabrated with 10 mM Phosphate buffer, 0.5M NaCl, pH 7.5.



  Periplasmic lysates were diluted with an equal volume of 10 mM phosphate buffer, pH7.5 and then passed through column. Column was washed with 10 bed volumes of 10 mM phosphate buffer, 0.5M NaCl and bound scFv was eluted with 0.23M glycine, pH2.5,0.3M NaCl. For some preperations the column eluate was dialyzed and concentrated using a Centricon 10 microconcentrator as described above.



  Example 6.2
Purification Of ScFv Fragments from Phage Antibody Catalytic Clones 18 and 83 By
Hydrophobic Interaction Chromatography
 Following IMAC or anti-myc peptide Protein A agarose purification of scFv derived from lysates of E. coli clones 18 and 83 (see Example 6.1), further purification of the scFv was accomplished on an alkyl superose 5/5 column attached to an FPLC system (Pharmacia).



   Chromatography was performed using a linear reverse gradient of (NH4) 2SO4 in 0. 1 M
Na phosphate pH 7.0. This was formed from two buffers:
Buffer A: 2M (NH4) 2SO4 in 0.1 M Na phosphate pH 7.0
Buffer B: 0.1 M Na phosphate pH 7.0
Gradient conditions were:
 0-3 ml: 10% Buffer B
 3-39 ml: Linear gradient, 10%-70% Buffer B 39-42 ml: Linear gradient, 70%-100% Buffer B 42-44 ml: 100% Buffer B 44-46 ml: Linear gradient, 100%-10% Buffer B 46-49 ml: 10% Buffer B  
 Samples were adjusted to a final concentration of 1.8 M (NH4) 2S04 by the addition of 0.783 vols of a saturated solution of (NN4) 2SO4 in 0.1 M Na phosphate (4.1 M (NH4) 2SO4) and dilution with an appropriate volume of 10% Buffer B: 90% Buffer A to increase the volume to a value suitable for injection onto the column. Fractions were collected and the peak (s) corresponding to ScFv identified by SDS PAGE as described below.

   These were pooled, concentrated and used in assays for binding or catalytic activity as appropriate.



   Elution of protein from the column was monitored by OD280 and plotted automatically. A typical chromatogram for IMAC pure scFv from clone 18 shows the bulk of the protein elutes in two distinct peaks. Fractions corresponding to each of the peaks were pooled as indicated.



  Peak 1 consisits of a broad shoulder eluting at 17.25 to 21.90% Buffer B (Pool 1) followed by a sharp peak at 24.2% Buffer B (Pool 2). Peak 2 is a sharp peak eluting at 48.10% buffer B (Pool 3). Purification of the scFv was monitored by silver stained SDS/PAGE with the following results. Load material for the HIC column (IMAC pure scFv + (NH4) 2SO4) showed  > 90% of the protein was scFv. At least four additional bands were also visible.



  Analysis of Pool 1 and Pool 2 obtained following HIC showed the majority of the scFv that was loaded was distributed equally in these pools and no substantial purification of the scFv was acheived. Pool 3 contained a small amount of scFv and an additional low molecular weight band.



   The chromatogram for clone 83 shows the bulk of the scFv protein elutes in a single sharp peak at 54.7% Buffer B. A second minor peak of protein elutes during the final wash with 100% Buffer B. Fractions corresponding to these peaks were pooled and, analyzed by
SDS/PAGE followed by silver staining. The column load material for HIC (IMAC purified material + (NH4) 2SO4) contained  > 90% scFv. After HIC the majority of the scFv is recovered in the single main peak eluting at 4.7% Buffer B. As with clone 18, no substantial purification of the scFv was acheived by HIC. A small amount of scFv is found in the late eludng minor peak.  



  Example 6.3
Binding Assays of IMAC and HIC Pure scFv from Phage Antibody Catalytic Clones 18 and 83
 Fractions or pools of fractions from the purification protocols described above (see Example 6.1 or 6.2) were analyzed for RT3 binding activity using an RU-BISA solid phase ELISA assay essentially as described in example 4.4. The fraction or fraction pools were first diluted 1: 5 or 1: 10 in PBS/Tween-20 and then serially diluted 1: 2 with PBS/Tween-20 across 11 wells of the RT3-BSA coated ELISA plate. The titer which gave the 50% maximal ELISA signal was determined for each sample analyzed. By multiplying this titer by the volume of the pool or fraction analyzed, an estimate of the number of binding units in each sample could be determined.

   This analysis showed that for clone 83 even though majority of the scFv loaded on the HIC column was recovered it had less than 10% of the binding activity compared to the column load. This result suggests that HIC may be unsuitable for purification of the scFv, since it may result in perturbations of the scFv protein structure resulting in loss of binding and presumably catalytic activity.



  Example 6.4
Catalytic Assays of IMAC and HIC Purified scFv from Phage Antibody Clones 18 and 83
 For clone 18 a typical catalytic assay was set up as follows: 50 plu of scFv was added to 145 pi of RT3 substrate (Compound 15, FIG. 4) and 5 jll of water or for some assays 5ul of RT3 hapten. A blank consisting of 50 111 of water and 147 il of RT3 substrate was set up to monitor the background hydrolysis of the RT3 substrate. The reaction was allowed to proceed for 6 hours after which samples were frozen at-20'C to stop the reaction. Samples were analyzed by HPLC as described in example 5.1. The amount of scFv added to an assay typically ranged from 1 to 5 ug and protein was buffered in 10 mM Hepes, 150 mM NaCl, pH 7.3. The typical RT3 substrate concentration was 50 uM buffered in 25 mM Tris-Cl, pH 9.0, 140 mM NacL and 0.01% NaN3.  



   An HPLC profile showing a typical positive catalytic assay result for IMAC pure 18 scFv is shown in Figure 15A. Figure 15B shows the HPLC profile of the same assay but done in the presence of RT3 hapten (Compound 5, FIG. 1). Finally, Figure 15C shows the
HPLC profile of the blank (no added scFv).



   In a similar manner assays of the fraction pools obtained following HIC purification of 18 scFv (see Example 6.2) were also performed. A typical result for the assay of the main scFv-containing pool (Pool 2 as described in Example 6.2 above) is shown in Figure 16.



  Similar results were obtained from assays of Pool 1 and Pool 3 (data not shown).



   Assay of IMAC or HIC purified scFv from clone 83 was done in a similar manner as described for clone 18 with the following exceptions. Substrate was buffered in 25 mM
HEPES, pH 7.0,140 mM NaCl and 0.01% NaN3. Due to the lower background hydrolysis of RT3 substrate at neutral pH, reactions were typically run for 24 hours. An HPLC profile of a positive catalytic assay result for IMAC pure 83 scFv is shown in Figure 17A. The same assay repeated in the presence of RT3 hapten is shown in Figure 17B. A blank was also analyzed and gives a profile similar to the that in Figure 17B (data not shown). A catalytic assay of the main scFv containing pool obtained following HIC (see Example 6.2) is shown in
Figure 18.



   It should be noted that the retention times of the expected product peak as well as nonproduct related peaks varied from run to run on the HPLC. The reason for this variation is not known. Test runs of the RT3 product (Compound 13, FIG. 4) alone on HPLC and monitoring at 215 nM showed a distinct peak profile was produced. This 215 nM profile was used as an internal control to accurately determine the position of the product peak on the 270 nM profile for each HPLC run.



   Conclusions from the results of the catalytic assays performed as described above are as follows. IMAC pure scFv from both clone 18 and 83 is able to hydrolyze the RT3 substrate and produces a product peak that elutes from the HPLC column at the correct retention time.



  In the presence of RT3 hapten, catalysis is completely abrogated presumably due to the much tighter binding of the RU hapten in the antibody pocket compared to the RT3 substrate. This  
 is further evidence that the scFv is responsible for catalysis since it is unlikely that natural
 esterase exists which is capable of specifically recognizing and binding the RT3 substrate or
 hapten with high affinity.



   Following HIC purification of scFv for either clone 18 or 83 no catalytic activity was
 observed in the scFv containing fractions. Loss of activity could possibly be due to instability
 of the scFv resulting in qnfolding or aggregation. Instability of the scFv for clone 83 was
 clearly demonstrated by the loss of binding in the assays performed on HIC purified scFv as described in Example 6. 3
Example 7.1
Isolation Of Binders To The Transition State Analogue, REND From A Naive Human Library
 It has been demonstrated that immunization schemes can be by-passed and that low and moderate affinity human antibodies (Kds down to 86 nM) can be isolated directly from human antibody libraries derived from non-immunized sources (Marks et al., J. Mol. Biol. 222 (1991): 581-597).

   This approach could provide a starting clone (or clones) which could be improved by a number of approaches as described by example below (for related examples see
Marks et al., BioTechnolovv 10 (1992): 779-783). These approaches could, therefore, lead to the isolation of entirely human catalytic antibodies which could prove extremely valuable, particularly in the area of therapeutic catalytic antibodies.



   The non-immunized human library described in Marks et al., J. Mol.. Biol. 222 (1991): 581-597, was panned against RT3-BSA, coated onto tubes as described for the immunized mouse library except 100-200 Ag/ml RT3-BSA coating concentration was used.



  The progress of the purification schemes was monitored by ELISA (s) using polyclonal phage.



  Polyclonal phage derived from 2 rounds of panning against RT3-BSA (RT3BSA: 2) gives a signal which is visible after overnight incubation with substrate. Polyclonal phage derived from 3 rounds of panning against RT3-BSA (RT3BSA: 3) gives a strong signal on ELISA (1 O. D. in 5 minutes). No binding to BSA was observed and binding to RT3-BSA was inhibited by pre-incubation with 10-100 Ag/ml of soluble, unconjugated RT3 suggesting that it was specific  for RT3 (not shown).



   Individual clones derived from 3 and 4 rounds of panning were examined for binding using methods as described in example 4. 3. Before beginning to screen on a large scale, the mmimum ELISA coating concentration was determined. Reducing antigen concentration from 100 Wg/ml tO 1.6 Ag/ml (100 pLUwell) causes only a 30% reduction in signal (data not shown) 2 llg/ml concentration was, therefore, used in all subsequent ELISA screenings
 144 clones derived from 3 rounds of panning were examined by ELISA assay for specific binding to RT3-BSA using 100 il of culture supernatant from IPTG induced HB2151 clones. As shown in FIG. 19A, 40 clones gave strong ELISA signals (0.7 to 2.5 O. D. after overnight incubation with substrate) and 27 gave moderate signals (0.2-0.5 O. D. after overnight incubation with substrate).

   FIG. 19B shows the results for 48 clones derived from 4 rounds of panning against RT3-BSA. From this 39 clones showed strong ELISA signal (0.72.5 O. D.) and 1 moderate ELISA signal (0.34 O. D.).



   PCR analysis and Bst N1 digestion revealed that all the the good binders examined from both rounds of panning shared a common PCR pattern and all but one of the weaker binders examined shared a common PCR pattern which is different to that of the high binders.



  One additional pattern was found associated with one of the weak binders. It is interesting to see enrichment with successive rounds of panning of the clone associated with high ELISA signal relative to that for moderate signal, as previously described by Clackson et al., Nature 352 (1991): 624-628.



  Example 7.2
Sequence Analysis Of Human RT3 Binders
 Sequencing was carried out on various members of each PCR pattern group as shown below:
PCR1= RT3: 63,80.



  PCR2= RT3: 47,54
PCR3= RT3: 61  nucleotide and deduced amino acid sequences of each group FIG. 20.



  Example 8. t
Chain Shuffling Of Human RT3 Binders
 The scheme used for chain shuffling is shown in FIG. 21. All of the scFv clones in the human or mouse libraries share certain common sequences including the plasmid sequences upstream of the heavy chain, the linker sequences between the heavy and light chains and gene 3 sequences downstre. im of the light chain. Primers were selected/synthesized from these areas to provide a general means of ampifying cloned heavy or light chain V regions. Thus,
PCR using the primers LMB3 and PCRFILINK will give rise to a heavy chain product while the primers FDTSEQ1 and LINKPCRL will give rise to a light chain product. LINKPCRL and PCRHLINK are complementary and so provide a means of linking the products.

   In this way, the separate heavy or light chains from each clone can be linked to a whole population of complementary chains derived from the initial library. The linked product acts as a template for a secondary PCR using the primers LMB3 and FDTSEQ1 and the secondary product is digested with Sfil and Notl for cloning. The primers were chosen to enable a change in
 fragment size to be observed following each digestion step. In addition, the efficiency of
 digestion is probably improved by having a relatively large overhang upstream of the restriction
 site.



   Method For Chain Shuffling
 The following primers are used:
 FDTSEQ1 5'GTCGTC1TTCCAGACGTTAGT 3
 LMB3 5'CAGGAAACAGCrArGAC 3
 PCRHLINK 5'ACC GCC AGA GCC ACC TCC GCC 3'
 LINKPCRL 5'GGCGGA GGTGGCTCrGGCGGT 3  
Primary heavy and light chain PCR products are prepared in the following reactions:
 HEAVY LIGHT
LMB3 primer FDTSEQ1 primer (lOpmotes/m !) 2.5 t1 (lOpmoles/ml) 2.5 l
PCRHLINK nrimer LINKPCRL primer (10pmoles/ml) 2.5 l (10pmoles/ml) 2.5111 10X PCR 10X PCR reaction buffer 5. OyI reaction buffer 5.0 l 5mM each dNTP's 2.5111 5mMeachdNTP's 2.5p11
Taq polmerase Taq polymerase (5U/ml) 0.3 l (5U/ml) 0.3Kl1 water 37 l water 371il
 PCR conditions are 25 cycles of 94  C I minute, 60  C I minute, 72  C 2 minutes with a final 1 () minutes at 72* C. For isolated clones temp ! ate can be most simply provided as a toothpick innoculum from a bacterial colony.

   For library material, DNA was prepared from a frozen bacterial stock and 2-10 ng added to the reaction. Primary PCR products were purified on agarose gels and purified using 5 zip of Geneclean "glass milk" (Bio 101, La Jolla, CA) with two e ! utions in water of 10 tir each.



   Assembly is carried out as follows: purified heavy 2.5 Ktl (20-50 ng) purified light 2.5 il (20-50 ng) 10X reaction buffer 2 1 5mM each dNTP (s) 1.0 Kt1
Taq polymerase 0.2 1 water 37  l
 PCR conditions are 25 cycles of 94  C 1 minute, 65  C for 4 minutes with a final 10 minutes at 72* C.



   For secondary PCRs, 1 pi of the linked material was used as template. The reaction was set-up as follows: linked PCR product 1 1
LMB3 primer (10pmoles/ml) 2.5 gl
FDTSEQ1 primer (lOpmoles/ml) 2.5 1
 I OX PCR  reaction buffer 5.0 zip 5mM each dNTP (s) 2.5 Rl
Taq polmerase (5U/ml) 0.3 pll water 37 p
 PCR conditions are 25 cycles of 94 C 1 minute, 60  C 1 minute, 72'C 2 minutes with a final 10 minutes at 72'C (5)il can easily be seen on a gel).



   The secondary product was extracted with phenol: chloroform and precipitated with ethanol, to remove Taq polymerase. The PCR product was digested overnight at 50  C with
Sfil according to manufacturers instructions. Next day 1/1Oth volume of 1M NaCl was added to give a final concentration of 150 mM Nad and Triton-Xl00 added to a final concentration of 0.01% before digesting with Nos 1 for 3 hours at 37  C. The digest was treated with phenol: chloroform, precipitated, dissolved in l12O and purified by running on a 1.5% agarose gel and purified with"Geneclean" (Bio 101, I, a Jolla, CA). The DNA was eluted into a final volume of 10-15 Ill and cloned into the Sf i I/Not I site of pCANTAB5 his-6.



   Plasmid DNA of pCANTAB5 his-6 was prepared by the alkaline lysis method and was purified by cesium chloride centrifugation. The purified DNA was digested at a DNA concentration of 100 ig/nil with Sfil according to manufacturers instructions (50* C for Sfil,
 overnight) followed by a 3 hour digestion with Not 1. The digestion product was loaded on
 directly on to a Chromaspin 1000 column (Clontech, Pa) o A) to, CA) to remove the stuffer
 fragment and spun for 3 minutes at 2200 rpm in a bench top centrifuge. The DNA was then
 phenol: chloroform extracted and dissolved at 100 pg/riii for use.



   Ligations are carried out using an Amersham (Arlington Heights, IL) ligation kit as
 follows:
 Vector DNA I Kll (100 ng)
 insert DNA 2 it (10-50 nu)
 1 () mMMgCl,
 200 mM Tris pH7.4 3 Vil
 buffer A 24 pl
 buffer B 6 Kll  
 Incubate for 30-60 minutes at 16  C. For library preparation, 5 times the volumes shown above were used. The ligation product was concentrated and purified using Geneclean and eluted into a volume of 10-15 Fll of water. This was introduced into electrocompetent T phage resistant TGI cells using a Bio-Rad (Heicutes, CA) eiectroporator, according to manufacturers instructions.



  Three clones, hRT3-1, hRT3-47, and hRT3-61 isolated after four rounds of panning of the naive human library (see Example 5.1) were used as templates for the chain shuffling protcol described above. As described in Example 5.2, sequence analysis showed each of the three clones were unique from each other in terms of VH and VL gene usage. Six different libraries were prepared. In each case, the name of the library refers to the fixed chaill an (l the clone number from which it was derived. Thus, H47 is a library with a fixed heavy chain from
RT3: 47 combined with a library of human light chains.



   The library sizes obtained were as follows:
 TABLE 3
 Library Size (X 1 () 6) Proportion with insert
 H1 7.8 9/10
 H47 6.2 8/10
 H61 6.8 9/10
 L1 9.6 9/10
 LA7 9.8 9/1 ()
 L61 8.4 8/10
 PCR using the primers FDTSEQ1 and LMB3 was carried out on 10 colonies from each library to determine the proportion with insert. The results are shown in the table above. In addition the PCR products were digested with BstN l to determine the diversity. It should be
 remembered, however, that approximately 2/3 of the sequence (of any given clone) in the chain
 shuffled library, is now fixed and that different members of the same V gene family may give
 the same"BstNl signature". Despite this, none of the library members had the pattern
 associated with the original clone.

   In some cases, patterns were found in duplicate within
 some libraries and one pattern may have appeared 3 times in the III library.  



  Example 8.2
Panning Human Chain Shuffled Library
 Phage particles were rescued from the libraries as described in Marks et. al.,
Biotechnologv 10 (1992): 779-783. Phage from pairs of libraries derived from the same starting clone were pooled and panned against RT3-BSA (e. g., III and LI). Panning and rescue were done essentially as described in Marks et al., J. Mol. Biol. 222 (1991): 581-597). except Nunc (Kamstrup, Denmark) immunosorb tubes were coated overnight with 1 ml of
RT3-BSA at 20 tg/ml. Coating and blocking were done in phosphate buffered saline (PBS) as before. The equivalent of 20 mis of phage was used in a final volume of 800 Kll of MOPS buffered saline (MBS) with 2% dried milk powder. Washing, elution, infection, and rescue with Ml3 helper phage were as described above.



   "Polyclonal"phage derived from either the unpanned libraries (PANO), from the first round of panning (PAN1), or from 2 rounds of panning (PAN2) were used in an ELISA to determine the progress of the panning process for each pair of libraries.



   As shown in FIG. 18, no signal was observed from PANO samples. Low level signal is observed in PAN1 samples derived from RT3: 1 and RT3: 47 and there is a marked improvement in PAN2 samples. With libraries derived from RT3: 61, ELISA signal is still relatively low after two rounds of panning. The ELISA results are mirrored when the eluate from each round of panning is quantitated as shown in Table 4. Increasing numbers of phage are eluted from the second panning. The numbers of phage yielded from PAN1 and PAN2 on the H61/L61 libraries is lower titan the corresponding yield from HI/LI and H47/L47 libraries.



   TABLE 4
 Yield of phage from pannings (XI 06)
 shuffled shuffled shuffled
 RT3: 1 RT3: 47 RT3: 61 eluate of PAN1 1.4 2.9 0.34 eluate of PAN2 1300 160 () 20 ()  input phage approximately 2-10 X 1012
 Table 5 shows the proportion of positives derived from panning the reshuffled human binders. For reshuffled RT3: 1 and RT3: 47, even after one round of panning the majority scored positive. Since polyclonal phage from reshuffled RT3: 61 was negative after PAN1, and positive after PAN2, individual colonies were only analyzed from PAN2 from this library.



   Table 5 Proportion Of Positives From Reshuffled Human Libraries
EMI63.1     


<tb>  <SEP> PROPORTION <SEP> NUMBERS
<tb> POPULATION <SEP> POSITIVE <SEP> RESTREAKED
<tb> RT3: <SEP> 1 <SEP> Reshuffle
<tb> PAN1 <SEP> 39/44 <SEP> 28
<tb> PAN2 <SEP> 42/44 <SEP> 15
<tb> RT3: <SEP> 47 <SEP> Reshuffle
<tb> PAN1 <SEP> 29/44 <SEP> 25
<tb> PAN2 <SEP> 35/48 <SEP> 28
<tb> RT3: <SEP> 61 <SEP> Reshuffle
<tb> PAN2 <SEP> 44/96 <SEP> 37
<tb> 
 Positive clones were restreaked and retested for RT3 and BSA binding. All reshuffled human libraries gave rise to a high proportion of binders after 1-2 rounds of panning. These have been grouped by PCR/BstNI digestion (using FTDSEQ1 and LMB3) into 9 PCR pattern groups for the RT3: 1 reshuffled library, 4 PCR pattern groups for the RT3: 47 reshuffled library and 8 PCR pattern groups for the RT3: 61 reshuffled library as shown in Table 6 below.



   There is a strong possibility that a heavy chain shuffled, with a library of light chains, will pull out different light chains, which are related to each other, and so the potential for PCR pattern diversity is reduced. Conversely, it is likely that a degree of diversity will be found by sequencing, even within a given PCR grouping.  



   Table 6. Grouping Of Human RT3 Binders According To PCR
A. RESHUFFLED CLONES ARISING FROM HU RT3: 1
EMI64.1     


<tb> PCR <SEP> Pattem <SEP> Sample <SEP> No.
<tb>



   <SEP> PAN1-1,5,6,9, <SEP> 10, <SEP> 12,21,27.
<tb>  <SEP> PAN2-31,34,35,36, <SEP> 38, <SEP> 41,42,43. <SEP> 
<tb>



   <SEP> B <SEP> PAN <SEP> 1-2, <SEP> 3,4,8,11,13,22,24 <SEP> [25].
<tb>



   <SEP> PAN2-30,39.
<tb>



  C <SEP> PAN1- <SEP> (7, <SEP> 14, <SEP> [15], <SEP> 19, <SEP> 23.
<tb>



   <SEP> PAN2-29,33.
<tb>



   <SEP> D <SEP> PAN1-17, <SEP> [18], <SEP> [26].
<tb>



   <SEP> PAN2- <SEP> (44.
<tb>



  E <SEP> PAN2-32,40.
<tb>



  F <SEP> PAN <SEP> 1- <SEP> 20.
<tb>



  G <SEP> PAN1-28.
<tb>



   <SEP> H <SEP> PAN1-16.
<tb>



   <SEP> I <SEP> PAN1-37.
<tb> 



  3/All samples from RT3: 1 were negative for BSA binding apart from a very low level in sa  
B. RESHUFFLED CLONE ARISING FROM HU RT3: 47
EMI65.1     


<tb> PCR <SEP> Pattem <SEP> SamDle <SEP> No.
<tb>



   <SEP> F <SEP> PAN1-2,4,12,17,20,21,22.
<tb>



   <SEP> PAN2-4, <SEP> 33,40,41,42,44,48,
<tb>  <SEP> 49,51, <SEP> 52,53. <SEP> 
<tb>



  B <SEP> PAN1-9, <SEP> 10.
<tb>



  PAN1-1.
<tb>



   <SEP> PAN2-37.
<tb>



  D <SEP> PAN1-3, <SEP> 26.
<tb>



  Pattern <SEP> Negative <SEP> on <SEP> re-screening
<tb> Unknown <SEP> 19,23,24,25.
<tb>



   <SEP> 35,36,38,39,43,45,46,47,50. <SEP> 
<tb>



   <SEP> Binder <SEP> to <SEP> BSA
<tb>  <SEP> PAN1-11
<tb>   
C. RESHUFFLED CLONE ARISING FROM HU RT3: 61
EMI66.1     


<tb> PCR <SEP> Pattern <SEP> Sample <SEP> No.
<tb>



   <SEP> A <SEP> PAN2-2, <SEP> 8,10,11,13,14,15,17,18,20,21,23,24,
<tb>  <SEP> 25,26,28,29,32,34,35.
<tb>



  B <SEP> PAN2-16,30,33.
<tb>



  C <SEP> PAN2-4.
<tb>



  D <SEP> PAN2-6.
<tb>



   <SEP> E <SEP> PAN2-12.
<tb>



   <SEP> F <SEP> PAN2-9.
<tb>



   <SEP> G <SEP> PAN2-22.
<tb>



   <SEP> PAN2-31.
<tb>



  Pattern <SEP> Negative <SEP> on <SEP> re-screening.
<tb>



  Unknown <SEP> PAN2-1,3,5,27,36,37.
<tb>



   <SEP> Positive <SEP> on <SEP> re-screening.
<tb>



   <SEP> PAN2-7.
<tb>



   <SEP> Binder <SEP> to <SEP> BSA.
<tb>



   <SEP> PAN2-19.
<tb> 



  Foomote: 1/Samples are all labelled hu (original clone number) : reshuffled clone number e. g. hu 47: 12 human clone derived from chain shuffling clone RT3: 47 from the first panning and numbering derivative 12. For presentation here, only the clone number is given.



  The use of the same letters for pattern groups derived from different starting clones, is not meant to imply that they are the same.



  3/Samples in rackets had been scored positive first time round but the clones picked after restreaking did not come up positive. This is either due to mixed colonies in the original were variable expression from different preparation or initial false positives. Sequencing was carried out on a number of clones derived from huRT3: 47 and these are emboldened in Section
B.

 

   In an attempt to determine which antibody chain was derived from the original human clone, separate PCR of the heavy and light V genes was carried out on individual clones (using either FDTSEQ1 or LMB3 in conjunction with primers located in the sequence encoding the  flexible linker peptide between the chains (PCRHLINK and LINKPCRL). Pairs of clones from each PCR group were analyzed (underlined in Table 6). This result indicates that all heavy chains, with the exception of hu61: 16 and hu61: 33 (pattern B) had the same heavy chain as the original isolate. These two clones now have a heavy chain pattern similar to RT3 : 47 and a light chain pattern which may be similar to the parent clone, RT3: 61.

   Reshuffled clones which had been described as having the same PCR pattern (from PCR/digest of whole SCFv, run on 3% gel) now show subtle differences (from PCR/digest of in triethylamine, it was found that the overnight ELISA signal following elution, went from 0.289 to 0.019. (Using 2.5 X 1011 polyclonal phage/well derived from one round of panning the shuffled human RU, 47 library (47PAN1 phage). Titration of the eluate showed that 7. 5X 107 infectious phage were collected, i. e., 0.03% of input. This compares favorably with elution from Nunc immunosorb tubes where 1.6X 109 infectious phage were yielded from an input of 1X 1013 for the same sample, i. e., 0.016% (see Table 3). By this type of approach, a range of specific elution procedures could be compared and the most suitable samples infected into E. coli for further work.



   The minimal transition state analogues, equivalent to the left and right hand of the RT3 molecule, will be referred to as RT3a (Compound 8, FIG. 2) and RT3b (Compound 12, FIG.



  3). The left and right hand products of substrate cleavage will be referred to as product A (Mesitylacetic acid, FIG. 4) and product B (Compound 13, FIG. 4). In order to determine the optimal concentrations of the various components required for panning/elution of the original mouse library, a dilution series was prepared for RT3, RT3a, RT3b, and both reaction products (product A and product B). These were pre-incubated with 100 111 of 10x polyclonal phage derived from one round of panning the mouse RT3 library (usin--triethvlamine elurion).



   The results of this analysis are presented in FIG. 23A and 23B. The most effective inhibition occurs with RT3 itself. It is clear that binding of this selected phage population is inhibited to a far greater extent by the left hand TSA and product (FIG. 23A) than by the right hand portions (FIG. 23B). Indeed, it is not at all clear if any inhibition occurs at the concentrations tested with right hand TSA or product. Furthermore, it appears that there is greater inhibition by the left hand TSA than the left hand product
 Specific elution was attempted using the original unpanned mouse library. This was carried out by binding 200 fll of lOx phage concentrate to ELISA wells coated with 150) lof2 p. ml RT3-BSA and blocked with 200 pLI of 2% Marvel.

   Phage were allowed to bind for 1 hour and were eluted by adding 200 pli ouf the following :  0.05 p-NI, 0.5 p1M or 5 AM RT3 5 WM, 50, ulNl or 500 WM RT3a 5 pM, 50iM or 500 WM RT3b 100 mM triethylamine
 Phage derived from two 15 minute lutions were pooled and reintroduced into TG1 or
HB2151 cells. FIG. 24A plots the yield of phage under each set of conditions. Triethylamine gives approximately 104 phage from an input equivalent to 2 ml of culture supernatant. This is in line with the successful panning described in the previous report, which has given rise to all the mouse clones described earlier. In that experiment, approximately 105 phage were derived from an input, equivalent to 20 ml of culture supernatant. Elution with RT3 and RT3a gives rise to a greater number of phage than triethylamine.

   RT3b gives a level of elution equivalent to or just greater than that achieved in a"buffer only"control.



   This experiment was repeated, but a higher coating concentration of RT3-BSA was used (100 fig/ml), and volumes were adjusted to ensure that coating and blocking volumes exceeded the volume of input phage (to prevent any background problems associated with non-specific sticking of phage up the side of the well). In this experiment (Figure 24B), the overall yield of eluted phage in all samples was reduced from before.



   Polyclonal phage and soluble antibody was prepared from the various populations and tested in ELISA. Positive signals were achieved with soluble and phage ELISA, from sample derived by elution with 500LiM Rna where. Individual colonies from this population were screened and 9f7/144 of the clones were found to be positive.



   Panning was also carried out in the original immunosorb tubes (Nunc). Elution was carried out using either 100 mM triethylamine or 500 pM Rna. Binding was carried out in the presence or absence of product A. (50,500,5000 1M). The results are summarized below:  
ELUTION REGIME CONCENTRATION OF COMPETING PRODUCT YIELD ff iQ6)
 1/100 rmM triethylamine 0 AM product a 52* 2/500 AM RT3a 0 1M product a 42 2 3/500 1M RT3a 50 llM product a 0.59 4/500 $1M RT3a 500 pM product a 0.85 5/500 AM RT3a 5000 pLM product a 47
 In this experiment, the yield of phage by triethylamine and RT3a elution (42-52 x106) is higher than previous experiments. The yield of phage from RT3a elution is reduced by 70 fold when 50 lit product A is present during binding (suggesting binding is specific).

   There is a similar reduction with 500 RM product A, but when 5000 1M product A is used, the yield returns to 47 x 106. (This may be an effect of DMS, used to dissolve the product, which was present at 7.2% in this particular sample). Thus, it is possible to elute phage from either 96 well plates or immunosorb tubes using hapten elution with minimal TSA molecules.



  Furthermore, the binding profile may be altered by competing with reaction products, thereby tailoring the binding profile of the eluted population according to the desired requirements.



  Example 10
CDR Shuffling Of Human RT3 Binders
 The scheme used for shuffling CDR fragments is a modification of the chain shuffling scheme described in Example 8.1.



   The primers VHCDR3BACK and REV VHCDR3BACK are complementary to each other and to a conserved sequence in the framework region of human VH genes immediately upstream of CDR3. A population of DNA fragments which inclues both CDR1 and CDR2 of the heavy chain from the library described by Marks et al., (1991) can be amplified using REV
VHCDR3BACK (see below for sequence) and LBM3 (described in Example 4.2) and the remainder of the scFV from the chosen clone can be amplified using VHCDR3BACK (see below for sequence) and FDTSEQ1 (described in Example 4.2). This permits the linkage of a population of CDRs 1 and 2 with the remaining portion of a single clone by a two-fragment  assembly reaction.



   Similarly, a library of DNA fragments containing the CDR3 region of the heavy chain may be amplified using VHCDR3BACK and the linker located primer, PCRHLINK (see example 4.2). The remaining potion of the heavy chain from the chosen clone was amplified with REV VHCDR3BACK and LMB3 and the light chain was amplified with LINKPCRL (see example 4.2) and FDTSEQ1. Thus, a population of CDR3 fragments may be introduced into a single clone by two sequential two-fragment assembly reactions: the first invovling assembly of CDR 1 and 2 from the clone with the population of CDR3s. This is followed by a secondary PCR reaction using the flanldng primers of this fragment LMB3 and PCRHLINK. the product of this was gel purified for subsequent assembly of this with the light chain from the clone.



   For both CDR shuffling regimes, a final PCR reaction using the scFV-flanking primers
LMB3 and FDTSEQ1 is performed. The CDR shuffled material is then digested with Notl and Sfil for cloning into pCANTAB5-his 6 (see FIG. 7).



  Method For CDR Shuffling
 The primers FDTSEQ1, LMB3, PCRHLINK and LINKPCRL are described in
Example 4.2. In addition, the following primers are used:
VHCDR3BACK 5'GAC ACG GC (TC) GT (AG) TAT TAC TGT 3'
REV VHCDR3BACK 5'ACA GTA ATA (CT) AG (GA) GC CGT GTC 3'
 (Nucleotides in paraentheses indicate introduced"wobbles"in the primer design to ensure universal amplification.)
 Primary PCR products are prepared in the following reactions:

  
CD 1 and 2 Fragment
LBprimer (10pmoles/1ll) 2.5) il
REV VHCDR3BACK primer (10 pmoles/lll) 2.5 111 10X PCR
Reaction Buffer 5.0 Etl  5 mM each dNTP (S) 2.5  l
Taq polymerase (5U/I) 0.3  l
Water to 50 p1
CDR3 Fragment
PCRHUNK primer (l0pmoles/1ll) 2.5 it
VHCDR3BACK primer (10pmoles l) 2.5  l 1 ( ? X PCR
Reaction Buffer 5.0 tit 5 mM each dNTP (s) 2.5 p1
Taq polymerase (5U/ l) 0. t
Water to 50 gel
VHCDR3-Linker-VL Framnetit
FDTSEQ1 primer (10pmoles/ l) 2.5!
VISCDR3BACK primer (10pmoles l) 2.5 il lot PCR
Reaction Buffer 5.0 tt 5 mM each dNTP (s) 2.5  l
Ta (l polymerase (5U/ l) 0.3 us
Water to 50tl
Linker-VL Fragment
FDTSEQ1 primer (10pmoles/ l) 2.5  l
LINKPCRL primer (10pmoles l) 2.5 il
 10X PCR
Reaction Buffer 5.0 f. U 5 mM each dNTP (s) 2.5 Rl
Taq polymerase (5U/ l) 0.3 pi
Water to 50  l
 Miniprep DNA was prepared from the library described by Marks et al. (l991) as temptate for PCR.

   PCR produced from the clone was prepared by innoculating from a bacterial colony.



   PCR conditions were 25 cycles of 94  C 1 minute, 55  C 1 minute, 72  C 2 minutes with a final 10 minutes at 72'C.



   Primary PCR products were gel purified using the Promega Magic PCR Prep System     5 mM ench dNTP (S) 2.5i I2.5i I
 Taq polymerase
 (5U/ l) 0.3  l
 Water to50fd   
 CDR3 Fragment
 PCRHLINK primer    (10pmoles/ l) 2.5  l
 VHCDR3BACK primer
 (10pmoles l) 2.5 lil
 10X PCR
 Reaction Buffer 5.0  l
 5mMeachdNTP (s) 2.5 ytl
 Taqpolymerase
 (5U/ l) 0. 3 il
 Water to 50 iti   
 VnCDR3-Li) tker-VL Frapment
 FDTSEQI primer    (10pmoles/ l) 2.5 y.

   l
 VIlCDR3I3ACK primer
 (10pmoles l) 2.5 ut
 10X PCR
 Reaction Buffer 5.0 All
 5 mM each dNTP (s)2.5ttt
 Taq polymerase
 (5U/ l) 0.3 ul
 Water to 50 111
 Linker-VL rragmott
 FDTSEQ1 primer
 (10pmoles/ l) 2.5!
 LINKPCRL primer
 (10pmoles l) 2.5 ni
 10X PCR
 Reaction Buffer 5.0  l
 5 mM each dNTP(s) 2.5  l
 Taqpotymerase
 (5U/ l)0.3!
 Water to 50 Iti
 Miniprep DNA was prepared from the library described by Marks et al. (1991) as tcmpiatc for PCR. PCR produced from the clone was prepared by innoculating from a bacteria ! coiony.



   PCR conditions were 25 cycles of 94  C I minute, 55* C I minute, 72  C 2 minutes with a final 10 minutes at 72'C.   



   I'rilllilly PCR products were gel purified using the Promega Magic PCR Prep System  except for the CDR3 fragment which requires Mermaici purification (Bio 101) due to its mater size.



  CDR +2 Shufffling
Assembly Of Library CDR1+2 Fragments With VII CDR3-LinkerVL Fragment From An
Isolated Oone
Purified library CDRl+2 l) NA 20-5 () ng
Purified VIICDR3-Linker-VL Fragment
 from an isolated ctone 20-50 ng t OX PCR Buffer 5.0 Ill 5 mM each dNTP (s) 2.5 ttt
Taq polymerase (5U/Etl) (). 3 tt)
Water to 50 il
PCR conditions as for primary PCRs.



  Secondary PCR Of Assembled CDR1+2 Shuffled DNA
Assembly product 1.0 tint
FDTSEQ ! primer (10pmoles/ l) 2.5  l
LMB3primer (10pmoles/ l) 2.5 lil
 10X PCR
 Reaction Buffer 5.0 l l
 5 mM each dNTP (s) 2.5 ti
Taq yolymerase
 (5U/ l) 0.3 il
 Water to 50  l
 CDR3 Shuffling
 Assenas) Of Library CDR3 Fragments With The CDR 14 (ni Fragment From An Isolated Clone
 Purified library CDR 3 DNA 20-50 ng
 Purified CDR 1+2
 from an isolated clone 20-50 ng
 10X PCR Buffer 5. zip
 5 mM each dNTP (s) 2.5 il
 Taq polymerase
 (5U/ l) 0.3  l
 Water to 50  l
 Second PCR Of Assemblez Library CDR3 - CDR1+2 From An Isolated Clone
 Assembly product 1.0 til
 PCRHLINK primer  
ExampleI I
Derivation Of Human Catalytic Antibodies By"Imprinting"
 The process of"imprinting"involves using an existing antibody with desired binding characteristics, to derive new antibodies, with similar characteristics.

   This is done by recombining original antibody chains, or parts thereof, with a library of complementary parts.



  When new antibody elements are found, which complement the original antibody binding characteristics, these are recombined with a library which replaces the original antibody binding characteristics, these are recombined with a library which replaces the original antibody part, to give an entirely new antibody which mimics the binding of the original antibody (PCT/GB/ 92/01755). This approach might be used to derive human catalytic antibodies from an existing
 mouse catalytic antibody.



   This example describes a"two-step conversion". This, of course, may be done over
 multiple steps or in a single step, if a hybrid molecule consisting of part of the original antibody
 is sufficient.



   This is a useful method for deriving human antibodies with similar binding activities to
 an existing mouse antibody for example.



   The catalytic phage antibody clones 18 and 83 (see Example 5.2) in pCANTAb vectors,
 (cloned in pCANTAB vectors), were used as template for PCR amplification of separate heavy
 and light chains. Heavy chains were amplified with LMB3 and PCRHLINK and light chains
 were amplified with LINKPCRL and FDTSEQ1 as described above. Libraries of human
 heavy and light chains were also amplified by PCR using the samer primers and with DNA
 prepared from the human scFv library described by Marks et al., J. Mol. Biol. 222 (1991): 581
 597 as described above.



   The individual mouse heavy chains from each clone were then recombined with the
 library of human light chains by PCR linkage as described above. Similarly, the individual
 light chains were recombined with the library of heavy chains in the same way.



   The resulting linked products were cleaved with ApaLl (for mouse heavy chains) or  
SFil (for human heavy chains) along with Notl, ligated into the appropriate pCANTAB vector and transformation into E. coli TG1 cells. All steps were as described above.



   Individual populations of TG 1 cells carrying each separate library, were grown for 2-3 hours at 30  C and rescued by infection with VCSM 13 helper phage at 37* C. After overnight growth phage particles were collected and concentrated. Each population was panned several times against RT3-BSA and indivdual binding clones identified by ELISA.



   Binding clones were selected and the human chain of each clone was amplified by PCR as before. This chain was recombined with the PCR product of the human library of complementary chains. PCR linkage, cleavage with Slwil and Nos 1, ligation, transformation, phage rescue, panning and screening were as before.



   In this way, a new population of RT3 binders were derived whose binding profile was directed by the original mouse clone but which were entirely human. The above example covers imprinting by shuffling separate chains but could equally apply to shuffling parts of chains in a single or multiple rounds (as above). The library material was derived from the
 library of Marks et al. J. Mol. Biol. 222 (1991): 581-597 but could equally come from PCR products of human blood, spleen, etc., or could be partially or totally derived from synthetic
DNA.



   The example given above involves shuffling chains within a single chain Fv on a single
 replicon. A similar result can be achieved by using ncm-linkccl VI 1/VL or VH-CHG/VL-CL
 fragments displayed on phage (McCafferty et al. WO 92/01047). These again may be on the
 same replicon or may be on different replicons. For example, the heavy chain of the original
 mouse antibody may be cloned into pUC19 or other plasmid, in frame with appropriate
 promoters, signal peptide and stop codon (s) enabling it to be expressed as a soluble VH or
 VER-CHU fragment in the bacterial periplasm (Better et al., 1989; Skerra et al., 1989).

   A
 growing culture of cells carrying this plasmid could then be infected with helper phage derived
 from a library of human light chains (either VL or VL-CI., as appropriate), cloned as fusions
 with gene III in fd-CATI or fd-DOG 1 (IvIcCafferty et al., supra. 1991) for example. This will
 give rise to a population of phage expressing indivudal human light chain fused to gene III,  with a heavy chain partner derived from the mouse clone. Those human chains, which complement the binding activity of die mouse chain, will be enriched by panning (McCafferty, et al., supra. 1991) and the gene encoding this chain will be present in the phage particle.



   Light chains derived in this way can be recloned into a vector for soluble expression of the single chain in the periplasm, as was done for the original mouse chain. As before, a growing culture of cells expressing these individual human light chains could be infected with helper phage derived from a library of human heavy chains, cloned as fusions with gene III in fd-CAT1 or fd-DOGI (McCafferty et al., supra. 1991) for example. As before panning against antigen with enrich those clones with the appropriate binding activity. This will result in a pair of human clones which mirror the binding of the original mouse clone.



   A similar process can be carried out by shuffling with the human heavy chain first and then the light chain. Alternatively, the enriched population or clones derived from one round of separate shuffling of heavy and light chains can be recombined with each other in the same way as described above for either SCFv (s) or separate chains.
  

Claims

WHAT IS CLAIMED IS 1. A method for producing catalytic antibodies displayed on phage comprising the steps of : (a) generating a gene library of antibody-derived domains; (b) inserting coding for said domains into a phage expression vector; and (c) isolating said catalytic antibodies.

2. A method as recited in Claim 1 wherein said catalytic antibodies are single chain antibodies.

3. A method as recited in Claim l wherein the antibodies isolated in step (c) are produced in quantity by culturing E. coli cells.

4. Catalytic antibodies prepared by the method of Claim 1.

5. A method as recited in Claim 1 wherein said gene library of antibody-derived domains is generated from one or more of the following groups: (a) gene fragments obtained from lymphocytes from an immunized animal; b) gene fragments obtained from lymphocytes from a non-immunized animal; (c) gene fragments obtained by shuffling of VH and VL chains; (d) gene fragments obtained by shuffling of CDR regions; (e) gene fragments obtained by mutagenesis of CDR regions; (f) imprinting; or (g) synthetic antibody genes.

6. A method for isolating catalytic antibodies displayed on phage comprising the following steps: (a) preparing an antigen; (b) immunizing with said antigen; (c) generating a library of VI-I and VL domains from said immunized animal; (ci) cloning said VH and VL domains into a phage expression vector to generate phage display antibodies; (e) selecting phage display antibodies which bind specifically to said antigen; (f) screening said selected phage display antibodies for catalytic activity to substrate; and (g) isolating said catalytic antibodies.

7. A method as recited in Claim 6 wherein said cataltyic antibodies are single chain antibodies.

8. A method as recited in Claim 6 wherein said antigen is a transition state analog.

9. A method as recited in Claim 6 wherein said antigen is a phosphonate.

10. A method as recited on Claim 6 wherein said antigen is EMI79.1 1 1. Catalytic antibodies prepared by the method of Claim 6.

12. A method for isolating catalytic human antibodies displayed on phage comprising the following steps: (a) preparing an antigen; (b) generating a library of VH and VL domains; (c) cloning said Vli and VL domains into a phage expression vector to generate phage display antibodies; (d) selecting plicge display antibcxiies which binei specifically to said antigen; (e) screening said selected phage display antibodies for catalytic activity to substrate; and (f) isolating said catalytic antibodies.

13. A method as recited in Claim 12 wherein said library is mouse-derived.

14. A method as recited in Claim 12 wherein said antigen is a transition state analog. 15. A method as recited in Clam! 2 wherein said antigen is a phosphonate.

16. A method as recited in Claim 12 wherein said antigen is EMI80.1 17. Catalytic antibodies prepared by the method of Claim 12.

18. A method for producing catalytic antibodies displayed on phage through chain shuffling comprising the following steps: (a) combining a library of VL genes with VH genes to form a chain shuffled library; (b) cloning the shuffled chain; (c) expressing said chain shuffled antibody on phage; (d) selecting against an antigen; and (e) screening for catalytic activity.

19. A method for producing catalytic antibodies displayed on phage through CDR shuffling comprising the following steps: (a) isolating VL and VH genes; (b) isolating a library of CDR regions; (c) recombining said VL and VH genes with said library of CDR regions to produce a CDR shuffled library; and (d) cloning the CDR shuffled library; (e) expressing said CDR shuffled library on phage; (f) selecting against an antigen; and (g) screening for catalytic activity.

20. A method for producing catalytic antibodies displayed on phage through imprinting comprising the following steps: (a) selecting a set of antibodies; (b) isolating a set of VH and a set of VL genes from said antibodies; (c) combining said set of VII with a library of VL and combining said set of VL with a library of VIl to form two combination libraries; (d) cloning said combination libraries; (e) expressing said libraries on phage; (f) selecting against an antigen; (g) isolating selected libraries of VH and VL genes; (h) combining said libraries of Vll and VL genes; (i) cloning said combined libraries; (j) expressing said combined libraries on phage; (k) reselecting against an antigen; and (1) screening for catalytic activity.

21. A recombinant cloning vector comprising an antibody, 6 His residues, a portion of a myc gene, and a phage gene III.

22. A recombinant cloning vector of Claim 21 wherein said vector allows the production of a fusion protein consisting of the following elements: an antibody which retains binding activity, a His-6 sequence which retains metal ion binding activity, and a mvc sequence which retains antigenicity.

23. A recombinant cloning vector of Claim 21 wherein said vector allows production of a fusion protein consisting of the following elements: an antiboiy which retains bincling activity, a His-6 sequence, a myc sequence, and a gene III protein which retains biological activity.

24. A method for enhancing the rate of cleavage or formation of a specific bond within a molecule in vivo which comprises introducing into an animal an effective amount of a phage derived catalytic antibody.

25. A method for in vivo activation of a prodrug comprising: (a) introducing a prodrug into a patient, said prodrug having a chemical bond therein which upon cleavage releases the active form of said drug; and (b) introducing into said patient an effective amount of a phage-derived catalytic antibody capable of cleaving said bond in said prodrug.

26. A method for activating or deactivating a biological function in an animal by enhancing the rate of cleavage or formation of a specific bond within a molecule in vivo which comprises introducing into an animal an effective amount of a catalytic antibody, said antibody having been produced by the method of Claim 1.

27. A method for activating or deactivating a biological function in an animal by enhancing the rate of cleavage or formation of a specific bond within a molecule in vivo which comprises introducing into an animal an effective amount of a catalytic antibody, said antibody having been produced by the method of Claim 18.

28. A method for activating or deactivating a biological function in an animal by enhancing the rate of cleavage or formation of a specific bond within a molecule in vivo which comprises introducing into an animal an effective amount of a catalytic antibody, said antibody having been produced by the method of Claim 19.

29. A method for activating or deactivating a biological function in an animal by enhancing the rate of cleavage or formation of a specific bond within a molecule in vivo which comprises introducing into an animal an effective amount of a catalytic antibody, said antibody having been produced by the method of Claim 20.