WO1994025586A1

WO1994025586A1 - Transgenic animals having an engineered immune response

Info

Publication number: WO1994025586A1
Application number: PCT/US1994/004708
Authority: WO
Inventors: Nora Sarvetnick; Richard A. Lerner; Peter Schultz
Original assignee: The Scripps Research Institute
Priority date: 1993-04-30
Filing date: 1994-04-29
Publication date: 1994-11-10
Also published as: AU6904794A

Abstract

The invention describes a transgenic animal having somatic and germ cells that comprise an exogenous exon expressable in antibody-producing cells of the animal, wherein the exon codes for an immunoglobulin V region capable of forming a coordination complex with a metal cation. Also described are methods of producing and using the transgenic animal for the production of antibody molecules that have a metal binding site.

Description

TRANSGENIC ANIMALS HAVING AN ENGINEERED IMMUNE RESPONSE

Description Technical Field

The present invention relates to transgenic animals that are capable of producing an engineered immune response comprising antibody molecules encoded by a transgene defining a protein having a preselected biological activity, particularly antibody molecules that have metal cation binding activity. More specifically, this invention relates to the production of transgenic non-human animals carrying exogenous DNA sequences that encode antibody light and/or heavy chains having preselected activity.

Background

The humoral immune response provides a mechanism for evolving receptors specific either for stable molecules or for highly energetic transition states. The latter antibodies have been shown to act as selective chemical catalysts and share many properties with enzymes. Lerner et al. Science. 252: 659-667 (1991) . Catalytic antibodies were shown to accelerate reactions by many of the same mechanisms used by enzymes. However, to extend the chemical potential of the immune system, one would like to introduce into the immunological repertoire catalytic functionality beyond the side chains provided by the twenty natural amino acids.

Catalytic cofactors have been introduced into an antibody by directed mutagenesis of an existing antibody to create a cofactor binding site. See for example, Iverson et al, Science. 249:659-662 (1990), and Roberts et al, Proc. Natl. Acad. Sci. USA. 87:6654-6658 (1990). However, such engineering has been conducted in vitro. Antibody and enzyme technology has not advanced to the degree that cofactor binding sites can be incorporated into an immune response in an animal.

Previous work with transgenic mice demonstrated that exogenous immunoglobulin light chain genes can be expressed in the B cell compartment of the animal, that the expression of the transgene can dominate the antibody repertoire of the transgenic animal, (Richie et al, Nature. 312:517-520, 1984; Brinster et al. Nature. 306:332-336, 1983; and Carmack et al, J. Immunol.. 147:2024-2032, 1991), and that the transgene can undergo somatic hypermutation. O'Brien et al, Nature, 326:405-409 (1987). However, there continues to be a need for systems for the in vivo production of an immunological repertoire incorporating the catalytic features of a cofactor binding site.

Brief Summary of the Invention

It has now been discovered that transgenic animals can be prepared in which the immune response of the animal is engineered to include the production of preselected heavy or light chain immunoglobulin variable region polypeptides having cofactor binding sites, and other interesting biological activities. Thus, the transgenic animal can produce antibody molecules of preselected activity. In particular, the specificity of a diverse immune response in a transgenic animal of this invention can be influenced by the presence of a preselected and custom-engineered activity in the heavy or light chain region of antibody molecules in the immune response. By the present methods, one can introduce new chemistries into the germline antibody repertoire of a transgenic animal .

The transgenic non-human animals described herein are useful as a source of an engineered immune response, ie. , for the preparation of antibody molecules in the transgenic animal in which the antibody molecules have preselected biological activity. In particular, the antibody molecules contain antigen binding specificity and also contain a biological activity engineered into the variable region of the immunoglobulin heavy or light chain. In one embodiment, the invention describes a transgenic animal having somatic and germ cells that comprise an exogenous exon expressable in antibody- producing cells of the transgenic animal, wherein the exon codes for an immunoglobulin V region capable of forming a coordination complex with a metal cation. Preferably, the transgenic animal is characterized by a phenotype of enhanced immune-responsiveness, such as is provided by a deficiency in the Fas antigen gene as where there is lymphoproliferation (lpr) gene mutation, or where the transgenic animal further contains a Jcl-2 gene that promotes lymphoproliferation. A preferred animal is a transgenic mouse. The invention also provides a method of producing a transgenic mouse which comprises: a) providing an exon expressable in antibody-producing cells of a mouse wherein the exon codes for an immunoglobulin V region capable of forming a coordination complex with a metal cation, b) introducing the exon into an embryo of a mouse, c) transplanting the embryo into a pseudopregnant mouse, and d) allowing the embryo to develop to term. The invention additionally describes methods for producing an antibody molecule having a preselected activity which comprises the steps of: a) providing an exon expressable in antibody-producing cells of a mouse wherein the exon codes for an immunoglobulin V region capable of forming a coordination complex with a metal cation; b) introducing the exon into an embryo of a mouse; c) transplanting the embryo into a pseudopregnant mouse; d) allowing the embryo to develop to term and producing a transgenic mouse which is capable of expressing the exon; e) immunizing the transgenic mouse with a preselected immunogen to induce an immune response that includes antibody molecules immunoreactive with the immunogen and which include, the immunoglobulin V region and have a metal cation binding site; and f) harvesting the antibody molecules formed in step (e) from the transgenic mouse. In a related embodiment, the a method of producing an antibody molecule having a preselected activity is contemplated which comprises: a) providing a transgenic animal having somatic and germ cells that comprise an exogenous exon expressable in antibody-producing cells of the animal wherein the exon codes for an immunoglobulin V region capable of forming a coordination complex with a metal cation; b) harvesting genes coding for immunoglobulin heavy and/or light chain polypeptides from antibody-producing cells of the animal; c) expressing the harvested genes in an expression vector capable of expressing the harvested genes and producing an antibody molecule; and d) collecting an antibody having the capacity to bind a metal cation from the produced antibody molecules. Other embodiments will be apparent to one skilled in the art in view of the present specification and claims.

Brief Description of the Drawings In the drawings, forming a portion of this disclosure:

Figure 1 illustrates the map of plasmid DNA pB,-14 (also referred to as PB-14) in which the functional MOPC-21 kappa light chain gene was cloned. Figure 2 is a schematic representation of the

14.5 Kb construct used for preparing a transgenic animal. Following site-directed mutagenesis of pB-14, the QM212 coding sequences were inserted into the second exon. The hatched boxes represent the kappa gene exons from pB-14 shown in Figure 1, and the filled box represents the inserted QM212 sequences. Arrows indicate position of PCR primers (ATG/PVU) used in analysis of the transgenic mice.

Figures 3A and 3B illustrate the expression of transgene sequences in tissue in an ethidium bromide stained agarose gel of PCR reactions. PCR analysis of tail genomic DNA with primers (BAM/PVU) flanking the inserted QM212 sequences (see Figure 2) is shown on the left side of the gel labeled as Figure 3A. PCR analysis with primers (ATG/PVU) of cDNA derived from blood RNA from nontransgenic (BALB and 1908) and transgenic (1853 and 1873) mice is shown on the right side of the gel labeled as Figure 3B. DNA from a nontransgenic mouse (1908) is used as a control for the PCR analysis. The results are described in Example 3 .

Figures 4A and 4B illustrate the expression of transgene sequences in hybridomas by PCR analysis of hybridomas H1-H6 derived from spleen cells of FITC-BSA immunized transgenic mice. The cDNAs used in the PCR analysis were prepared from RNA extracted from the hybridomas, H1-H6, or from the liver (L) and spleen (S) from transgenic (2422) and nontransgenic (2424) mice. In Figure 4A, the analysis is shown for hybridomas H1-H4 and the liver and spleen samples from 2422 and 2424 mice. In Figure 4B, the results of PCR is shown for H5 and H6 along with the 2422 and 2424 mice again. The band corresponding to the transgene transcript (arrow) was seen in hybridomas H3 and H6, in addition to the spleen of the transgenic mouse 2422.

Figure 5 is a Western blot of isoelectric focusing gel. Protein G purified ascites from hybridomas H6 (left lane), H5, H4, and H3 (right lane) were reduced, separated on an isoelectric focusing gel, blotted, and reacted with an anti-kappa antibody. Hybridomas 6 and 3 contain a unique band corresponding to an isoelectric focusing point of pH 8.6-9.3.

Detailed Description of the Invention A. Definitions

Animal Cells: Unless specifically stated otherwise, animal cells includes cells in cell cultures, embryos, and differentiated animals.

Antibody Molecule: The phase "antibody molecule" in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule, i.e. molecules that contain an antibody combining site or paratope.

Antibody Combining Site: An "antibody combining site" is that structural portion of an antibody molecule that specifically binds (immunoreacts with) antigen and is comprised of variable regions of both the heavy and light chains. ^• The term "immunoreact" in its various forms means specific binding between an antigenic determinant-containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or a portion thereof.

Antibody: The term antibody in its various grammatical forms refers to a composition containing antibody molecules. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules, portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab', F(ab^,)₂, F(v) , combinations of polypeptides having a variable (V) region of heavy (V_H) and light (V_L) chain, and single chain antigen binding proteins.

Fab and F(ab')₂ portions of antibody molecules are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well known. See for example, U.S. Patent No. 4,342,566 to Theofilopolous and Dixon. Fab' antibody molecule portions are also well known and are produced from F(ab')₂ portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide. Exogenous Gene: An exogenous gene, or transgene, is a gene present in a transgenic animal which originates from outside the animal, i.e., is a gene not originally present in the animal prior to introduction of the transgene into the animal.

Im unoreaction Conditions: Immunoreaction conditions are those that maintain the immunological activity of a antibody of this invention. Those conditions include a temperature range of about 4 degrees C (4C) to about 45C, preferably about 37C, a pH value range of about 5 to about 9, preferably about 7 and an ionic strength varying from that of distilled water to that of about one molar sodium chloride, preferably about that of physiological saline. Methods for optimizing such conditions are well known in the art. Metal: The term "metal" in the context of a metal binding site refers to the metal cations disclosed herein that form a coordination complex with a metal binding protein.

Monoclonal Antibody: The phrase "monoclonal antibody" in its various grammatical forms refers to an antibody containing having only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immunospecific for a different antigen, e.g., a bispecific monoclonal antibody. Nucleic Acid: A term to refer to any of a class of molecules that includes ribonucleic acid (RNA) , deoxynucleic acid (DNA) in its single or double stranded forms, and polynucleotides.

Nucleotide: A monomeric unit of DNA or RNA consisting of a sugar moiety (pentose) , a phosphate. and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1' carbon of the pentose) and that combination of base and sugar is a nucleoside. When the nucleoside contains a phosphate group bonded to the 3' or 5' position of the pentose it is referred to as a nucleotide. A sequence of operatively linked nucleotides is typically referred to herein as a "base sequence" or "nucleotide sequence", and is represented herein by a formula whose left to right orientation is in the conventional direction of 5'-terminus to 3'-terminus.

Polynucleotide: A nucleic acid molecule comprising a polymeric unit of DNA or RNA having a sequence of two or more operatively linked nucleotides that form a single linear strand of nucleotides, also referred to as an oligonucleotide.

Polypeptide: refers to a linear series of amino acid residues connected to one another by peptide bonds between the alpha-amino group and carboxy group of contiguous amino acid residues. Protein: refers to a linear series of greater than 50 amino acid residues connected one to the other as in a polypeptide. Recombinant DNA Molecule (rDNA) : A sequence of nucleotides either DNA or RNA that is used for preparing a non-human transgenic organism.

Transgenic Animal: A non-human animal which contains an exogenous gene introduced into somatic and germ cells of the animal, or into an ancestor of the animal, at an embryonic stage.

B. Transgenic Animals

The present invention provides engineered somatic and germ cells of an animal, animal embryos or differentiated animals, having a genome characterized by the presence of a gene comprising at least an exon useful in the present methods and in a transgenic animal. Overall, the methods described herein produce a transgenic animal having a transgene that is expressable in antibody-producing cells of the animal during an immune response to produce antibody molecules having preselected biological activities engineered into the immunological repertoire of the transgenic animal. This capability is particularly useful because a diversity of antibody molecules is produced in the animal during a typical immune response, and in this case the response includes an engineered V region of predetermined chemical activity. The predetermined activity can include the capacity to bind metal cations, binding sites for enzymic cofactors, and the like biologically active structures engineered into a V region of an antibody molecule.

Theoretically, any animal may be used to produce a transgenic animal of this invention so long as the animal is capable of an immune response. A transgenic animal is typically a mammal, and can be any species of mammal, including agriculturally significant species, such as sheep, cow, lamb, horse and the like. Preferred are animals significant for scientific purposes, including but not limited to rabbits, primates and rodents, such as mice, rats and the like. A transgenic mammal is not human. A preferred and exemplary animal is a mouse. Thus, although the present methods are readily adaptable to other species, the discussion will generally refer to mice as exemplary of the invention. The invention therefore describes a transgenic animal having somatic and germ cells that comprise an exogenous exon that is expressable in antibody- producing cells of the transgenic animal. The exogenous exon codes for an immunoglobulin V region having an engineered biological activity that is capable of being incorporated into antibody molecules during an immune response in the transgenic animal. The engineering of immunoglobulin V regions to contain preselected biological activities has been described earlier for a variety of biological activities. Such engineering is not to be construed as limiting of the invention insofar as it has been discovered that once engineered, the V region can be incorporated into the ger line of an animal so that the modified V region contributes to the immunological repertoire of the animal and can be included in an immune response in the transgenic animal.

An exemplary biological activity engineered into an immunoglobulin V region is the capacity to bind metal cations as described by Iverson et al, Science. 249:659-662 (1990), and by Roberts et al, Proc. Natl. Acad. Sci. USA. 87:6654-6658 (1990). Based on the teachings of these references, immunoglobulin variable (V) regions on both heavy or light chains can be engineered to contain a metal binding site, and such engineered metal cation binding sites are exemplary of the present invention, and represent a preferred embodiment.

Thus, in one embodiment, an engineered V region has the ability to complex with (bind) a metal cation through ligands (contacts) provided by contact amino acid residues in the polypeptide sequence defining the V region. The contact amino acid residues are presented in a geometry that coordinates the complexation of a metal cation. The V region contains a site that binds metal cation and is therefore referred to as a metal cation binding site. A metal cation is bound because the engineered V region is capable of forming a coordination complex with the metal cation in a manner analogous to the coordination complex formations found in other well known metalloproteins, such as carbonic anhydrase, superoxide dismutase and the like.

Immunoglobulins represent a large family of molecules that include several types of molecules, such as IgD, IgG, IgA, IgM and IgE. An immunoglobulin is typically comprised of two heavy (H) and two light (L) chains with both a variable (V) and constant (C) region present on each chain. Several different regions of an immunoglobulin contain conserved sequences on the basis of comparative studies of known sequences of heavy or light chains. Extensive amino acid and nucleic acid sequence data displaying exemplary conserved sequences have been compiled for immunoglobulin molecules by Kabat et al., in Seguences of Proteins of Immunological Interest. 4th Ed., National Institutes of Health, Bethesda, MD (1987) .

Present understanding of the sites of an antibody molecule responsible for antigen-antibody binding indicates that part of the antibody combining site is formed by heavy chain hypervariable regions, and part of the combining site is formed by the light chain hypervariable regions. See for example, Getzoff et al., Adv. Immunol.. 43:1-98 (1988). Six loops of polypeptide comprise the hypervariable regions; three loops from the variable region of the light chain (V_L) and three loops from the variable region of the heavy chain (V_H) , denoted LI, L2, L3 and HI, H2, H3 respectively. See for example, Chothia et al., Nature, 342:877-883 (1989). The hypervariable regions are also known as complementarity determining regions, or CDR's.

Comparative studies of the known three dimensional structure of numerous antibody molecules have identified that each hypervariable region adopts one of a few main chain conformations or canonical structures. Using sequence homologies, the amino acid residue sequence of a heavy or light chain are aligned with the sequence of a known immunoglobulin heavy or light chain structure, respectively, the hypervariable region loops and specific amino acid residue positions within the loops, of the heavy or light chain can be reproducibly identified.

Thus, for example, a LI, L2 or L3 loop structure (CDR LI, CDR L2 or CDR L3) can be reproducibly identified solely on the basis of sequence homologies to other immunoglobulin light chains, thereby locating the position of critical residue positions within the loop structure. Due to the existence of variation in the chain length for a particular immunoglobulin light chain, amino acid residue position numbers are referred to herein by a numbering scheme that is based on alignments using homologous sequences as described by Kabat et al., in Sequences of Proteins of Immunological Interest. 4th Ed., U.S. Department of Health and Human Services, National Institute of Health, Bethesda, MD (1987) . Specific residue number reference to the amino acid residue positions for particular residues, therefore, will be cited herein as "Kabat position number" or "Kabat amino acid residue position number" to connote a reproducibly identifiable residue position on a recognized CDR loop structure. Wherever position numbers are given, they refer to Kabat positions. The V region of any immunoglobulin heavy or light chain molecule having the identifiable loop structures are useful in the present invention to produce a metal cation binding site.

A metal cation binding site is formed by the reproducible folding of a V region into its characteristic folded structure engineered to contain specific contact amino acid residues. The metal binding site is formed by the geometric positioning of three metal ligands (contact sites) provided by the side chain residues of three contact amino acid residues to form coordinating ligands for complexing a metal cation. Thus the positioning of three contact amino acid residues in the amino acid sequence of an immunoglobulin V region defines a metal binding site. The structure and stereochemistry of protein-metal interactions in metalloproteins is generally well understood. See for example. Freeman et al.. Adv. Protein Chem.. 22:257-424 (1967); Kannan et al.. Annals. NY Acad. Sci.. 429:49 (1984); and Tainer et al., J. Mol. Biol.. 160:181-217 (1982).

The metal ligands (contact sites) for binding a metal cation to a metal binding site in a V region used in this invention are positioned at three locations to provide three ligand contact points typically required for a metal cation coordination complex. Representative coordination complex geometries for the metal ligands can potentially be tetrahedral, square planar or trigonal depending upon the metal cation. However, tetrahedral geometries are preferred. Representative coordination metal complexes of the preferred tetrahedral coordinating geometry are shown by the structure of the zinc(II) complex in the enzyme superoxide dismutase, or the copper(II) complex in carbonic anhydrase. See Tainer et al., J. Mol. Biol.. 160:181-217 (1982); and Kannan et al., Annals. NY Acad. Sci.. 429:49 (1984). In these examples, although a metal cation presents four potential contact sites, typically three participate in the metal-ligand contact, and the fourth site on the metal cation is free to participate in electron exchanges or sharing with solvent or solute in solution having access to the co plexed metal. Similarly, in the present metal binding protein, three contact amino acid residues (ligand contact sites) participate in complexing the metal cation and the fourth is available for contributing to catalysis mediated by the engineered V region.

An amino acid residue that occupies one of the three amino acid residue positions to provide a metal ligand contact site in a metal binding site is referred to as a contact amino acid residue. Amino acid residues suitable for use as contact amino acid residues are known in the art of metalloprotein biochemistry and include histidine, cysteine, methionine, aspartic acid, glutamic acid and the like residues known to provide a ligand for metal cations in metalloproteins. In one preferred embodiment, the three contact amino acid residues are two histidines and a third residue selected from the group of histidine, aspartic acid, cysteine and glutamic acid. Particularly preferred is the use of histidine residues for all three contact amino acid residues. Immunoglobulin heavy and light chain molecules each contain in their V regions potential sites for positioning three contact amino acid residues suitable to produce a metal ligand binding site. See Roberts et al, Proc. Natl. Acad. Sci. USA. 87:6654-6658 (1990) . These sites can be reproducibly located in a V region as to present metal cation ligand contacts at the proper coordinates for forming a metal binding site because the location of the critical amino acid residue position for forming a metal binding hypervariable regions of either a heavy or light chain can be reproducibly identified in the CDR loop structure due to their conserved features. See Getzoff et al., Adv. Immunol.. 43:1-98 (1988); and Chothia et al.,. Nature, 342:877-883 (1989). Therefore, any immunoglobulin heavy or light chain molecule can be modified to contain a metal binding site by first identifying contact amino acid residue positions using the Kabat position number at a position in a V region as summarized in TABLE 1.

TABLE 1 Metal Binding Sites in Antibody CDRs

Metal Binding Site¹ Cation H-Bond³ Other location² ligands θ-Strand

LI (34), L3 (89,91) Center 34-89 LI (32)

L3 (90,92,97) LI and L3 90-97 L3 (95)

L3 (89,91,96) Center 91:96

HI (33,35), H3 (95) Center 33-95 HI (33,35) , H2 (50) Center 35:50 H2 (52)

HI (31,33) , H2 (52) Center 33:52

H2 (61,52,58) H2 and L3 50-58 H2 (56)

H2 (50,58,60) H2 and L3 50-58

H3 (95,101X, 101X-2)⁴ Center 95:101X fl-Turn

L2 (50,53,55) L2 and H3 50...53 ce-Helix

LI (27d,29) , L3 (93)⁵ Above site 27d...29

ⁱ Numbering in parenthesis is for the amino acid residue position for a contact amino acid residue that contributes to a metal binding site. The numbers refer to the Kabat position number. The CDR in which the contact residue is located is also indicated.

² The cation was either positioned in the center of the binding pocket between L3 and H3 (center) , or between the two CDRs indicated.

³ Pairs of residues having hydrogen bonds conserved in all antibodies are indicated by "-" and those having N-0 distances greater than 3.5 A in some antibodies by "...". Nonhydrogen-bonded pairs of residues that occur in _-strands are indicated by

II . • II • ⁴ "lOlx" indicates either residue 101 or the preceding residue of H3, and "10lx-2" indicates the residue located two amino acid residue positions away from lOlx towards the amino terminus of the heavy chain, as described further herein.

⁵ The site is found in V lambda only. The amino acid residue position 27d in CDR LI is located four amino acid residue positions away from position 29 towards the amino terminus of the light chain as described further herein.

Thus, in one embodiment the amino acid residue positions used to provide three metal ligand contact sites are located in specific positions of the amino acid sequence that defines the V region of the immunoglobulin light chain molecule.

In one light chain embodiment, the V region includes a LI region and a L3 region and the three contact amino acid residues are located at any three of the four amino acid residue positions 32, 34, 89 and 91 .

Thus, one preferred metal binding site includes three contact amino acid residues occupying amino acid residue positions 32, 34 and 89; positions 32, 34 and 91; positions 32, 89 and 91; or 34, 89 and 91 of an immunoglobulin light chain variable region. A preferred and exemplary metal binding site is formed by using histidine as the contact amino acid residue at positions 34, 89 and 91, and is described in more detail in the Examples.

Several features of the CDR LI and CDR L3-containing V region of the light chain provide an optimum environment for positioning a metal binding site on the claimed protein as disclosed herein. The LI and L3 regions form anti-parallel strand main-chain structures hydrogen bond between strands at several points. This feature provides a beta sheet structure that is reproducible and readily identifiable in all light chain V regions. It also provides stabilization to facilitate proper atomic distances so that the preselected sites for contact amino acid residues are properly located to form a tetrahedral array for presenting ligands to complex the metal cation.

Hydrogen bonds are typically found between main- chain atoms of residue pair 89 and 34, or pair 91 and 32, that stabilize the L3 and LI main chain structures, respectively.

In the embodiment utilizing contact amino acid residues at positions 34, 89 and 91 of the light chain, a metal binding V region of the present invention preferably includes either glycine or a non-polar amino acid residue at the amino acid residue position 36 of the light chain V region sequence, where the non-polar residue is an amino acid residue having fewer side chain atoms than tyrosine, and is not proline. Preferred residues are leucine, valine and alanine. In particularly preferred embodiments, a metal binding protein has a leucine residue at position 36 in the light chain V region. In another light chain embodiment, the V region includes a L3 region and the three contact amino acid residues are located at amino acid residue positions 90, 92 and 97. Hydrogen bonds are preferably located between the main-chain atoms of residue pair 90 and 99 to stabilize the L3 main-chain structure.

Another light chain embodiment includes a L3 region in the V region containing the three contact amino acid residues at amino acid residue positions 89, 91 and 96. A related light chain embodiment includes an L2 region in the V region containing the three contact amino acid residues at amino acid residue positions 50, 53 and 55.

Another light chain embodiment includes a LI region and a L3 region of a light chain V region and contains three contact amino acid residues at amino acid residue positions 27d, 29 and 93. Positions 27d and 29 are located in the LI region. The Kabat position 27d connotes an amino acid residue position that is four amino acid residues away from position 29 in the direction towards the amino terminus of the light chain.

In another embodiment the amino acid residue positions used to provide three metal ligand contact sites are located in specific positions of the amino acid sequence that defines the V region of the immunoglobulin heavy chain molecule.

In one heavy chain embodiment, the V region includes a HI region and a H3 region and the three contact amino acid residues are located at amino acid residue positions 33, 35 and 95. Hydrogen bonds are preferably located between the main-chain atoms of the residue pair 33 and 95 to stabilize the HI and H3 main-chain structures. In another heavy chain embodiment, the V region includes a HI region and a H2 region and the three contact amino acid residues are located at any three of the four amino acid residue positions 33, 35, 50 and 52. Another heavy chain embodiment includes a HI region and a H2 region in the V region containing the three contact amino acid residues at amino acid residue positions 31, 33 and 52.

A related heavy chain embodiment includes a H2 region in the V region containing three contact amino acid residues at any three of the four amino acid residue positions 50, 52, 56 and 58. Hydrogen bond pairs are preferably located between the main-chain atoms of residue pair 50 and 58 to stabilize the H2 main-chain structure.

In another heavy chain embodiment, the V region includes a H2 region, and the three contact amino acid residues are located at the amino acid residue positions 50, 58 and 60. Hydrogen bond pairs are preferably located between the main-chain atoms of residue pair 50 and 58 to stabilize the H2 main-chain structure.

Another heavy chain embodiment includes a H3 region in the V region containing the three contact amino acid residues at amino acid residue positions

95, lOlx and lOlx-2. The position designation "lOlx" connotes alternative positions of either Kabat position number 101 or the amino acid residue position preceding position 101, i.e., the position one residue away from position 101 and in the direction towards the amino terminus of the heavy chain. The position designation "101x-2" connotes alternative positions that depend on the position indicated by the term "lOlx". If lOlx is position 101, then lOlx-2 is a position two residues away from position 101 in the direction towards the amino terminus of the heavy chain. If lOlx is the position preceding 101 by one residue, then 101x-2 is a position three residues away from position 101 in the direction towards the amino terminus of the heavy chain.

Additional engineered metal binding sites have been prepared in V regions using randomized oligonucleotide sequences to mutate immunoglobulin CDR regions, followed by selection of the resulting mutated antibody populations for binding to preselected metal cations, as described herein. Those additional engineered V regions are described in the Examples.

Transgenic animals can be prepared as described herein incorporating a transgene of this invention.

Preferred transgenic animals contain a transgene that codes for a V region having a metal binding site.

A particularly preferred transgenic mammal exemplary of the present methods is the transgenic mouse described herein and designated met-mouse that contains a transgene coding a metal binding site in a V region of an immunoglobulin light chain. An embryo of the preferred transgenic mouse line met-mouse was deposited with the American Type Culture Collection (ATCC; Rockville, MD) no later that April 30, 1993 and assigned the ATCC accession number 72015.

A transgenic animal of the present invention is useful for producing antibodies of preselected biological activity. Additionally, the animal can be a source of an immunological repertoire for the purpose of producing antibody molecule libraries which contain the preselected biological activity. The preparation of such antibodies or antibody libraries are described herein. Under normal circumstances, self-reactive B cells are eliminated or inactivated from the repertoire of circulating B lymphocytes by mechanisms including programmed cell death. An expanded B cell repertoire that includes anti-self specificities would be useful for eliciting humoral responses to antigens that are the same or similar in sequence to self protein. Thus, a transgenic animal of this invention having the additional trait of producing an expanded repertoire would be useful in facilitating the isolation of a greater diversity of antibodies, including antibodies that recognize self-related antigens.

In this regard, a transgenic animal of this invention may further contain an expressed genetic characteristic (phenotype) of increased, altered or misregulated immune responsiveness to facilitate the production of an immune response of increased diversity. Examples of animals having these traits include mice with lymphoproliferative disorder produced by defects in Fas antigen, or mice having a bcl-2 gene expressed in their bone marrow cells.

A representative Fas antigen defect is produced by the presence of mutations in the lymphoproliferation (lpr) gene described by Watanabe- Fukunaga et al, Nature, 356:314-317 (1992). Mice having lpr gene mutations include the MRL/MpJ strain (Murphy et al, in "Genetic Control of Autoimmune Disease", Rose et al, eds., pp.207-221, Elsevier, New York, 1978) , and the lpr⁰⁸ mutation in the CBA/KlJms strain (Matεuzawa et al, J. EXP. Med.. 171:519-531, 1990) . Mice having the lpr mutation are available from a variety of commercial sources that carry mice strains for research purposes, including MRL/MpJ-lpr and C3H.MRL-lpr strains from Jackson Labs (Bar Harbor, Maine) and the MRL/SCR/lpr/lpr strain from the mouse colony at The Scripps Research Institute (La Jolla, California) .

A representative transgenic mouse having a bcl-2 gene in bone marrow cells can be prepared as described by Vaux et al. Nature. 335:440-442 (1988).

C. Methods for Preparing a Transgenic Animal The preparation of a transgenic animal containing a transgene according to the present invention is prepared using standard technologies for producing a transgenic animal. The critical factor is providing a recombinant DNA molecule that contains the transgene of interest that is capable of expressing the exon contained therein in antibody-producing cells of the animal.

1. Recombinant DNA Molecules Useful as a Transgene

A transgene of the present invention is a recombinant DNA (rDNA) molecule that includes an endogenous exon expressable in an antibody-producing cell of the transgenic animal. The exon in a transgene codes for an immunoglobulin V region comprising an exogenous biological function, such as the metal cation binding site that is exemplary of the invention. The exon can be prepared by the general methods of molecular cloning of rDNA molecules, manipulation of those cloned molecules and regulated protein expression of the exon in the transgenic animal's antibody-producing cells using the manipulated DNA molecules. An overview of the method for preparing a transgene useful in this invention involves the following steps:

1. Obtaining a nucleic acid that includes an exon coding for a sequence of amino acid residues that define a variable (V) region of an immunoglobulin molecule.

2. Determining the sequence of the gene to locate the regions that code for amino acid residue positions disclosed herein that are suitable for introducing a nucleotide mutation to encode a domain in the immunoglobulin V region having a desired exogenous biological function. As an example, the biological function is the ability to bind a metal cation, and the mutations introduce contact amino acid residues and produce a metal cation binding site.

3. Modifying the nucleotide base sequence of the gene to encode metal ligand contact residues at three of the four metal ligand contact site residue positions within the V region.

4. Inserting the modified nucleotide base sequence into an expression vector that is expressable in antibody-producing cells of the animal.

A nucleic acid useful in preparing a transgene for the present invention may be obtained by a variety of means. A DNA segment containing a preselected nucleotide sequence may be prepared by chemical synthesis using, for example, the phosphotriester method of Matteucci et al., J. Am. Chem. Soc.. 103:3185 (1981). Once prepared, the DNA segment is included into a recombinant DNA vector that is useful to manipulate and express the transgene.

A DNA segment containing a preselected nucleotide sequence may also be assembled from a number of chemically synthesized polynucleotides designed to form a particular DNA segment when hybridized together to form a double stranded DNA segment. The double stranded DNA segment is then ligated together using T4DNA ligase and inserted into an appropriate expression vector.

Alternatively, nucleic acids used in a transgene can be obtained by molecular cloning of messenger RNA (mRNA) or genomic DNA.

Methods for isolating and manipulating nucleic acids and genes are well known in the art. See, for example, "Guide To Molecular Cloning Techniques", in Methods In Enzvmology. Volume 152, Berger and Kimmel, eds. (1987) ; and Current Protocols in Molecular Biology. Ausubel et al., eds., John Wiley and Sons, NY (1987) , whose disclosures are herein incorporated by reference.

Genes useful in practicing this invention include genes coding for a sequence of. amino acid residues defining a variable (V) region contained in immunoglobulin products, immunoglobulin molecules, Fab fragments, F_v fragments and abzymes, and the like. Particularly preferred is a gene coding for an intact immunoglobulin V_L or V_H region.

In one embodiment described herein as exemplary, a gene coding for an immunoglobulin V_L region of an immunoglobulin capable of binding a preselected antigen is used, and an exogenous biological function is introduced into the V_L region prior to its introduction into the animal host as a transgene. Immunoglobulin V region exons or whole genes are isolated from cells obtained from a vertebrate, preferably a mammal, which has been immunized with an antigenic ligand (antigen) against which activity is sought, i.e., a preselected antigen. The immunization can be carried out conventionally and antibody titer in the animal can be monitored to determine the stage of immunization desired, which corresponds to the affinity or avidity desired. Partially immunized animals typically receive only one immunization and cells are collected therefrom shortly after a response is detected. Fully immunized animals display a peak titer which is. achieved with one or more repeated injections of the antigen into the host mammal, normally at two to three week intervals. Usually three to five days after the last challenge, the spleen is removed and the genes coding for immunoglobulin heavy and immunoglobulin light chain are isolated from the rearranged B cells present in the spleen using standard procedures. See Current Protocols in Molecular Biology. Ausubel et al., eds., John Wiley and Sons, NY (1987) ; and Antibodies: A Laboratory Manual. Harlowe and Lane, eds.. Cold Spring Harbor, NY (1988) .

Genes coding for V_H and V_L polypeptides can be derived from cells producing IgA, IgD, IgE, IgG or IgM, most preferably from IgM and IgG, producing cells. Methods for preparing fragments of genomic DNA from which immunoglobulin variable region genes can be cloned are well known in the art. See for example, Herrmann et al.. Methods in Enzymol. _f 152:180-183

(1987); Frischauf, Methods in Enzymol.. 152:183-190 (1987); Frischauf, Methods in Enzymol.. 152:199-212 (1987); and DiLella et al.. Methods in Enzymol.. 152:199-212 (1987). Genes coding for a V region of an immunoglobulin can be isolated from either the genomic DNA containing the gene as described above or from the mRNA which codes for the variable region. The difficulty in using genomic DNA is in juxtaposing the sequences coding for a polypeptide where the sequences are separated by introns. The DNA fragment(s) containing the proper exons must be isolated, the introns excised, and the exons spliced together in the proper order and orientation. Therefore, techniques employing mRNA will be the method of choice because the sequence is contiguous (free of introns) for the entire polypeptide obviating the need to splice exons. Methods for isolating mRNA coding for peptides or proteins are well known in the art. See, for example, Current Protocols in Molecular Biology. Ausubel et al., John Wiley and Sons, NY (1987); Guide to Molecular Cloning Technigues. in Methods In Enzymology. Volume 152, Berger and Kimmel, eds. (1987) , and Molecular Cloning; A Laboratory Manual. Second Edition. Sambrook et al., eds.. Cold Spring Harbor, NY (1989) .

Genes coding for both heavy and light chain V regions can be obtained from mRNA and/or genomic DNA using the polymerase chain reaction and various primers to form antibody gene repertoires. See, for example, Orlandi et al., Proc. Natl. Acad. Sci. USA. 86:3833-3837 (1989); Sastry et al., Proc. Natl. Acad. Sci.. USA. 86:5728-5732 (1989); Ward et al.. Nature, 341:544 (1989); Huse et al., Science. 246:1275-1281 (1989) and PCR Protocol: A Guide to Methods and Applications. Innis et al., eds., Academic Press, London (1990) .

Modifications to the V region coding nucleotide sequence may be introduced using any of the well known methods of random or site-directed mutagenesis. See, for example, Smith, Ann. Rev. Genet.. 19:423-463 (1985); Kunkel et al., Proc. Natl. Acad. Sci.. USA, 82:488-492 (1985); and Kunkel et al., Meth. Enzymol.. 154:367-382 (1987). The nucleotide sequence (exon) coding a V region that includes an exogenous biological function, such as a metal cation binding site, is linked into a transcriptional unit for expression of the exon. A transcriptional unit compatible with the host animal is used to express the transgene including the exon in antibody-producing cells of the transgenic animal. The transcriptional unit includes a promoter capable of directing the expression (transcription and translation) of the exon in the transgene in antibody- producing cells of the host animal. A promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur. Promoter sequences compatible with expression in antibody-producing cells are generally well known eukaryotic promoter sequences and contain a TATA box, an RNA polymerase binding site, enhancer elements that provide tissue specificity for expression, and the like transcription initiation control components. Particularly important is the enhancer element which promotes transcription in antibody-producing cells, i.e., immunoglobulin specific enhancer sequences.

Immunoglobulin specific enhancer sequences that promote expression in antibody-producing cells are well known in the art, and any of a variety can be used in the present invention, so long as the enhancer is compatible with expression in an antibody-producing cell of the species of the transgenic animal. Preferably, the enhancer element and the promoter are derived from immunoglobulin genes derived from the transgenic animal species. More preferably the configuration of the promoter and enhancer elements of the transcriptional unit are the same as is found in an immunoglobulin gene of the transgenic animal species, such as is described in the Examples. The transcriptional unit contains at least the exon coding an immunoglobulin V region. In addition, the transcriptional unit preferably also contains other immunoglobulin region exons such as the J and C regions to facilitate immunoglobulin gene rearrangement during clonal expansion of immune- responding B-cells (antibody producing cells) . However, particularly preferred are transcriptional units having a "pre"-rearranged immunoglobulin gene locus, i.e., where the V and C regions have already been joined, so as to provide an advantage in clonal expansion of the antibody-producing cells over the native V regions present in the animal. An exemplary construction is shown in Figure 2. A transcriptional unit also preferably contains a transcriptional control element to accommodate somatic hypermutation of the immunoglobulin V region in the responding antibody-producing cells. The presence of this element allows the production of increased diversity of produced antibody molecules that derive from the exon-containing transgene. The construct shown in Figure 2 and described in the Examples combines the features described above, including a mouse immunoglobulin promoter, a mouse immunoglobulin enhancer, pre-rearranged V-C region, and at the 3' end of the construct a somatic hypermutation element.

Thus, the transgene contains expression control elements including the promoter to drive expression of the exon when present in antibody-producing cells of the transgenic animal. The engineered V region-coding nucleotide sequence of the exon is operatively linked in the transgene to the expression control elements to allow the promoter sequence to direct RNA polymerase binding and synthesis of the desired polypeptide coding gene. The choice of which expression control elements to which an engineered V region-coding nucleotide sequence (exon) is operatively linked depends directly, as is well known in the art, on the functional properties desired, e.g. the location and timing of protein expression, and the particular antibody-producing host cell in which expression is to occur, these being limitations inherent in the art of constructing recombinant DNA molecules. 2. Preparation of a Structurally Defined

Metal Cation Binding Site in an Immunoglobulin V Region A preferred transgenic animal contains a transgene of this invention in which the engineered V region has the capacity to bind a metal cation.

Antibodies which contain a metal cation binding site, particularly repertoires of antibody molecules produced in an immune response, provide a source for the development of antibody molecules having complexed metal cations. Such antibodies are useful in cases where the complexed metal cation participates in antigen binding, but more importantly, are useful for the development of catalytic antibodies, i.e., antibodies which contribute as a catalyst to a chemical reaction, where the complexed metal cation contributes to the catalysis.

The preparation of an engineered V region which binds to a metal binding site has been described previously by Iverson et al, Science. 249:659-662 (1990), and by Roberts et al, Proc. Natl. Acad. Sci. USA, 87:6654-6658 (1990), the teachings of which are hereby incorporated by reference.

In this embodiment, a gene coding for an antibody V region from a heavy or light chain of interest is first cloned as described herein. Thereafter, amino acid residues in the V region at contact amino acid residue positions are identified by sequencing and mutated to substitute a contact amino acid residue for the native residue at the designated positions. The resulting constructed gene codes for a V region with a metal binding site introduced therein.

The contact amino acid residue positions suitable for introducing a metal binding site into a V region are identified as described earlier herein, and preferred residue positions are described in TABLE 1. Because the identification of the contact positions are defined structurally, the binding site can be referred to as a structurally defined metal binding site. A preferred metal binding site in which the contacts are located at Kabat positions 34, 89 and 91 was engineered into a V region of an immunoglobulin light chain, and the resulting gene was introduced into a mouse to form the met-mouse described in the Examples. A preferred rDNA molecule containing a transgene that was used to prepare the transgenic animal designated met-mouse is prepared as described in Example 1, and is designated pSKPl.

Applying the methods described herein, a metal binding site can be engineered into any heavy or light chain immunoglobulin V region at any of the positions identified in TABLE 1.

3. Preparation of a Metal Cation Binding Site in an Immunoglobulin V Region by Random Mutagenesis

In addition to preparing engineered V regions for use in a transgene of this invention using the contact amino acid residue positions described above, one can use random mutagenesis of CDR domains in a V region and screening methods such as is described by Barbas et al, Proc. Natl. Acad. Sci. USA. 89:4457-4461, (1992).

The mutagenesis approach for preparing a metal cation binding site in a V region involves the use of phage display vectors for their particular advantage of providing a means to screen a very large population of expressed display proteins and thereby locate one or more specific clones that code for a desired binding reactivity. The use of phage display vectors derives from the previously described use of combinatorial libraries of antibody molecules based on phagemids. The combinatorial library production and manipulation methods have been extensively described in the literature, and will not be reviewed in detail herein, except for those feature required to make and use unique embodiments of the present invention. However, the methods generally involve the use of a filamentous phage (phagemid) surface expression vector system for cloning and expressing antibody species from a library of antibodies.

Various phagemid cloning systems to produce combinatorial libraries have been described by others. See, for example the preparation of combinatorial antibody libraries on phagemids as described by Kang et al., Proc. Natl. Acad. Sci.. USA. 88:4363-4366 (1991); Barbas et al., Proc. Natl. Acad. Sci.. USA. 88:7978-7982 (1991); Zebedee et al., Proc. Natl. Acad. Sci.. USA. 89:3175-3179 (1992); Kang et al. , Proc. Natl. Acad. Sci.. USA. 88:11120-11123 (1991); Barbas et al., Proc. Natl. Acad. Sci.. USA. 89:4457-4461 (1992); and Gram et al., Proc. Natl. Acad. Sci.. USA. 89:3576-3580 (1992), which references are hereby incorporated by reference. A phagemid vector for use herein is a recombinant DNA (rDNA) molecule containing a nucleotide sequence that codes for and is capable of expressing an antibody-derived heterodimeric protein on the surface of the phagemid in the form of a phagemid display protein. An exemplary and preferred phagemid vector is plasmid pC3muFab described in Example 5.

The method for producing a metal binding site in a phagemid display protein generally involves (1) introducing a heavy or light chain V region-coding gene of interest into the phagemid display vector; (2) introducing a metal binding site into the phagemid display protein vector by primer extension with an oligonucleotide containing regions of homology to a CDR domain of the antibody V region gene and containing regions of degeneracy for producing randomized coding sequences as described herein, to form a large population of display vectors each capable of expressing different putative binding sites displayed on a phagemid surface display protein, (3) expressing the display protein and binding site on the surface of a filamentous phage particle, and (3) isolating the surface-expressed phage particle using affinity techniques such as panning of phage particles against a preselected metal cation, thereby isolating one or more species of phagemid containing a display protein containing a binding site that binds a preselected metal cation.

As a further characterization of the produced metal binding site, the nucleotide and corresponding amino acid residue sequence of the gene coding the engineered V region is determined by nucleic acid sequencing. The primary amino acid residue sequence information provides essential information regarding the binding site's reactivity. An exemplary preparation of a metal binding site in the CDR3 region of a light chain of an immunoglobulin is described in the Examples. The isolation of a particular vector capable of expressing a metal binding site of interest involves the introduction of the dicistronic expression vector able to express the phagemid display protein into a host cell permissive for expression of filamentous phage genes and the assembly of phage particles. Typically, the host is E. coli. Thereafter, a helper phage genome is introduced into the host cell containing the phagemid expression vector to provide the genetic complementation necessary to allow phage particles to be assembled. The resulting host cell is cultured to allow the introduced phage genes and display protein genes to be expressed, and for phage particles to be assembled and shed from the host cell. The shed phage particles are then harvested (collected) from the host cell culture media and screened for desirable metal cation binding properties. Typically, the harvested particles are "panned" for binding with a preselected metal cation. The strongly binding particles are then collected, and individual species of particles are clonally isolated and further screened for binding to the metal cation. Phage which produce a binding site of desired metal binding specificity are selected. Using that procedure numerous different engineered V regions were produced as described in Example 5 that are suitable for preparing a transgenic animal of the present invention. 4. Production of a Transgenic Animal

The present invention includes a method of introducing an exogenous transgene into an animal to produce a transgenic animal, i.e., genetically programming a cell within an animal by introducing an exogenous exon of the present invention into the genome of a zygote to produce a genetically altered zygote, or into the genome of individual somatic cells in the organism. The genetically altered zygote is then maintained under appropriate biological conditions for a time period equal to a gestation period or a substantial portion of a gestation period • that is sufficient for the genetically altered zygote to develop into a transgenic animal containing at least 1 copy of the rDNA. The term "genetically programming" or

"engineering" of the immune response as used herein means to permanently alter the DNA content of a cell within an animal so that an exon capable of expressing an immunoglobulin V region in an antibody-producing cell of the animal has been introduced into the somatic and germ cells of the animal.

Methods for producing a transgenic animal containing a transgene of the present invention include standard transgenic technology: introduction of a rDNA into an embryonic stem cell of a mammal followed by appropriate manipulation of the embryonic stem cell to produce a transgenic animal.

The technology for producing a transgenic animal is described by Hogan et al, Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor, NY (1987) ; and Palmiter et al, Ann. Rev. Genet.. 20:465-499 (1986); which methods are described further herein. Production of transgenic mammals is also possible using the homologous recombination transgenic systems described by Capecchi, Science. 244:288-292 (1989) . Furthermore, the preparation of transgenic mammals has also been described in U.S. Patent No. 4,736,866, No. 4,870,009, No. 4,873,191, No. 4,873,316, No. 5,073,490, No. 5,174,986, No. 5,175,383, No. 5,175,384, and No. 5,175,385, the disclosures of which are hereby incorporated by reference.

Further, in accordance with the invention, the transgene is introduced into the host mammal, preferably (but not necessarily) at the single-cell embryo stage, so as to provide the stable presence of the transgene throughout somatic and germ cells of the differentiated animal. The use of chimeric animals is also contemplated herein. Typically, this involves the integration of the transgene into the animal host genome, although methods that allow the transgene to be stably and heritably present through the use of autonomously replicating vectors will also be useful. Elbrecht et al, Mol. Cell. Biol.. 7:1276-1279 (1987). At the cellular level, this may be accomplished using the techniques of microinjection, electroporation, dielectrophoresis or various chemically mediated transformation techniques, all. of which are well known in the art. Following the introduction of the transgene and integration into the genome or cell, the transgenic cell or cells must be allowed to differentiate into a whole organism. This may be accomplished, for example, by embryo implantation into pseudopregnant females, or by other techniques allowing maturation of transgenic embryos. Once such maturation and differentiation has occurred, the animal is assayed for the presence of the transgene. Typically this involves removing small portions of tissue from the animal and using standard DNA hybridization assay techniques to detect the presence of the transgene. Suitable tissue for a mouse is a tail section or a blood sample from a tail bleed.

Transgenic animals carrying the transgene are thereafter bred and offspring carrying the transgene may be selected for use in the present methods.

Thus, one technique for transgenically altering a mammal is to microinject a transgene into the male pronucleus of the fertilized mammalian egg to cause one or more copies of the transgene to be retained in the somatic and germ cells of the developing mammal. Usually 10 to 40 percent of the mammals developing from the injected eggs contain at least 1 copy of the transgene in their tissues. These transgenic mammals usually transmit the gene through the germ line to the next generation. The progeny of the transgenically manipulated embryos may be tested for the presence of the construct by Southern blot analysis of nucleic acids in a tissue of the progeny mammal. The stable integration of the transgene into the genome of the transgenic embryos allows permanent transgenic mammal lines carrying the transgene to be established.

Alternative methods for producing a non-human mammal containing a transgene of the present invention include infection of fertilized eggs, embryo-derived stem cells, totipotent embryonal carcinoma (Ec) cells, or early cleavage embryos with viral expression vectors containing the transgene. See for example, Palmiter et al, Ann. Rev. Genet.. 20:465-499 (1986) and Capecchi, Science. 244:1288-1292 (1989).

In preferred embodiments the transgenic mammal of the present invention is produced by:

1) providing a transgene of this invention, typically in the form of a rDNA containing the exon of interest;

2) introducing, as by microinjecting, the rDNA into a fertilized mammalian egg to produce a genetically altered mammalian egg;

3) implanting (transplanting) the genetically altered mammalian egg (embryo) into a host female mammal, preferably a pseudopregnant animal;

4) allowing the embryo to develop to term by maintaining the host female mammal for a time period equal to a substantial portion of a gestation period of said mammal, to form a transgenic animal.

A fertilized mammalian egg may be obtained from a suitable female mammal (i.e., pseudopregnant) by inducing superovulation with gonadotropins. Typically, pregnant mare's serum is used to mimic the follicle-stimulating hormone (FSH) in combination with human chorionic gonadotropin (hCG) to mimic luteinizing hormone (LH) . The efficient induction of superovulation in mice depends as is well known on several variables including the age and weight of the females, the dose and timing of the gonadotropin administration, and the particular strain of mice used. In addition, the number of superovulated eggs that become fertilized depends on the reproductive performance of the stud males. See, for example, Manipulating the Embryo: A Laboratory Manual. Hogan et al, eds., Cold Spring Harbor, NY (1986).

The transgene (rDNA) may be microinjected into the mammalian egg to produce a genetically altered mammalian egg using well known techniques. Typically, the rDNA is microinjected directly into the pronuclei of the fertilized mouse eggs as has been described by Gordon et al, Proc. Natl. Acad. Sci.. USA. 77:7380-7384 (1980). This leads to the stable chromosomal integration of the rDNA in approximately 10 to 40 percent of the surviving embryos. See for example, Brinster et al, Proc. Natl. Acad. Sci.. USA. 82:4438-4442 (1985). Typically, the integration appears to occur at the 1 cell stage, as a result the rDNA is present in every cell of the transgenic animal, including all of the primordial germ cells. The number of copies of the foreign rDNA that are retained in each cell can range from 1 to several hundred and does not appear to depend on the number of rDNA injected into the egg as is well known. An alternative method for introducing genes into the mouse germ line is the infection of embryos with virus vectors. The embryos can be infected by either wild-type or recombinant viruses leading to the stable of integration of viral genomes into the host chromosomes. See, for example, Jaenisch et al, Cell. 24:519-529 (1981). One particularly useful class of viral vectors are virus vectors derived from retro-viruses. Retroviral integration occurs through a precise mechanism, leading to the insertion of single copies of the virus on the host chromosome. The frequency of obtaining transgenic animals by retroviral infection of embryos can be as high as that obtained by microinjection of the rDNA and appears to depend greatly on the titre of virus used. See, for example, van der Putten et al, Proc. Natl. Acad. Sci.. USA. 82:6148-6152 (1985).

Another method of transferring new genetic information into the mouse embryo involves the introduction of the rDNA into embryonic stem cells and then introducing the embryonic stem cells into the embryo. The embryonic stem cells can be derived from normal blastocysts and these cells have been shown to colonize the germ line regularly and the somatic tissues when introduced into the embryo. See, for example, Bradley et al, Nature, 309:255-256 (1984). Typically, the embryo-derived stem cells are transfected with the rDNA and the embryo-derived stem cells further cultured for a time period sufficient to allow the rDNA to integrate into the genome of the cell. In some situations this integration may occur by homologous recombination with a gene that is present in the genome of the embryo-derived stem cell. See, for example, Capecchi, Science. 244:1288-1292 (1989). The embryo stem cells that have incorporated the rDNA into their genome may be selected and used to produce a purified genetically altered embryo derived stem cell population. See, for example, Mansour et al, Nature, 336:348 (1988). The embryo derived stem cell is then injected into the blastocoel cavity of a preimplantation mouse embryo and the blastocyst is surgically transferred to the uterus of a foster mother where development is allowed to progress to term. The resulting animal is chimeric in that it is composed from cells derived of both the donor embryo derived stem cells and the host blastocyst.

Heterozygous siblings are interbred to produce animals that are homozygous for the rDNA. See for example, Capecchi, Science. 244:1288-1292 (1989).

The genetically altered mammalian egg is implanted into host female mammals. Methods for implanting genetically altered mammalian eggs into host females are well known. See, for example, Hogan et al, Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1986) . Pseudopregnant recipient females may be produced by mating females in natural estrus with vasectomized or genetically sterile males. After mating with a sterile male, the female reproduction tract becomes receptive for transferred embryos even though her own unfertilized eggs degenerate. The genetically altered mammalian eggs are then transferred to the ampullae or the uterine horns of the pseudopregnant recipient. If the genetically altered mammalian egg is transferred into the ampullae it must be enclosed in a zona pellucida membrane. If it is transferred into the uterine horns the genetically altered mammalian egg does not require a zona pellucida membrane.

The host female mammals containing the implanted genetically altered mammalian eggs are maintained for a sufficient time period to give birth to a transgenic mammal having at least 1 cell containing a rDNA of the present invention that has developed from the genetically altered mammalian egg. Typically this gestation period is between 19 to 20 days depending on the particular mouse strain. The breeding and care of mice is well known. See for example, Manipulating the Mouse Embryo: A Laboratory Manual. Hogan et al, eds., Cold Spring Harbor, New York, (1986) . The infection of cells within an animal using a replication incompetent retroviral vector has been described by Luskin et al, Neuron, 1:635-647 (1988). A particular embodiment of a transgenic animal has the genetic phenotype of an enhanced diversity of immune-responsiveness, as described earlier. The preparation of a transgenic animal according to the present invention and having enhanced immune- responsiveness can be prepared by several methods. An animal already possessing the phenotype of enhanced immune-responsiveness may be used as the source of founder stock in producing a transgenic animal according to the present methods. Alternatively, a transgenic animal of this invention may be "back- crossed" with an animal possessing the immune- responsiveness phenotype, whereby the progeny is screened according to Mendelian genetics for that progeny who contains both genes, i.e., both the transgene of this invention and the gene(s) for producing the phenotype of enhanced immune- responsiveness. Further back-crossing can be conducted to produce a transgenic animal homozygous for the transgene of this invention, homozygous for the gene(s) that confer the subject phenotype, or homozygous for both. A transgenic animal capable of expressing an immune response that includes antibodies having a metal cation binding site in the V region of antibody was produced as described herein. In addition, the preparation of a transgenic mouse having the additional phenotype of enhanced immune-responsiveness is described herein using back-crossing to a MRL/SCR/lpr/lpr mouse.

Confirmation that a transgenic animal was successfully prepared can be accomplished by a variety of methods, but generally involves (1) a DNA hybridization assay of nucleic acids from a tissue of the animal to confirm that the transgene was successfully introduced; (2) characterization of an mRNA transcript from the transgene in a tissue of the animal to confirm that the introduction resulted in an intact transgene capable of expression; and (3) identification of antibody molecules in an immune response of the animal that contain the engineered V region to confirm that the transgene can be expressed in amounts sufficient to significantly participate in the immune response.

All these analyses were conducted as described in the Examples. In particular, it was observed that of the four transgenic animals isolated that contain the transgene out of an original set of about twenty progeny mice, two (or 50%) were observed to exhibit transcription of the transgene. Therefore, the Examples demonstrate that a transgenic animal of the present invention can be readily produced. D. Method for Producing a Preselected Antibody from a Transgenic Animal

A transgenic animal of the present invention is used to produce an immune response containing antibodies of preselected activity, i.e., having an engineered immune response. This ability is particularly useful in the context of preparing antibody libraries from which to identify desirable or improved antibody specificities and/or activities. The method for generating useful antibodies in a transgenic animal generally involves immunizing the mouse with an immunogen and harvesting the antibodies in the resulting induced immune response. The immunospecificity of an antibody or monoclonal antibody generated in response to the immunogen is then typically screened for immunoreactivity with a target antigen or the presence of the biological activity engineered into the V.region, and desirable antibodies are selected. The use of a transgenic animal of this invention provides the option of preparing polyclonal antibodies from the sera of the animal, of preparing monoclonal antibodies from the spleen or B cells responding in the animal to the immunogen by preparing a hybridoma, or using the animal as a source of an immunological repertoire for the preparation of a library of responding antibodies according to the combinatorial antibody library methods described herein.

The preparation of hybridoma cell lines for producing monoclonal antibodies is well known in the art. Furthermore, the use of combinatorial antibody library methods and the use of phagemid display technology for producing and screening for a desired antibody in an immune response is described herein and generally well known. Finally, the preparation and use of immunogens for producing an immune response in an animal is extremely well characterized and will not be repeated here. However, where the engineered V region in the transgene of the animal includes an antigen binding site of interest to be boosted, it is preferred to use a corresponding antigen so that the immune response will contain antibodies of interest. It is to be understood that immunization is not necessarily required, particularly where an antibody heavy or light chain in the repertoire is to be mutagenized, shuffled or combined in order to increase the diversity of antibodies in the library.

In a preferred embodiment, the transgenic animal of this invention is used for producing a repertoire of diverse antibody molecules which share the property of having an engineered V region containing a preselected biological activity.

In another embodiment, the invention provides for the use of a transgenic animal having a phenotype of enhanced diversity of immune-responsiveness as described herein. Thus the use of the present methods may include the use of transgenic animals having the enhanced immune-responsiveness phenotype as an additional component increase the diversity of the immunological repertoire to be screened.

A particularly preferred embodiment involves using a transgenic animal of the present invention to produce catalytic antibodies, i.e., antibodies having a metal binding site which can participate in promoting catalysis. Thus, the invention contemplates producing a metalloantibody which has the capacity of promoting a predetermined chemical reaction, i.e., a catalytic metalloantibody. Catalytic antibodies have been described by Tramontano et al., Science. 234:1566-1570 (1986); Pollack et al. , Science. 234:1570-1573 (1986); Janda et al., Science. 241:1188-1191 (1988); Janda et al., Science. 244: 437-440 (1989), and in United States Patent No. 4,659,567, No. 4,900,674, No. 5,030,717, No. 5,079,152 and No. 5,126,258, the disclosures of which are hereby incorporated by reference. In this embodiment, a • preselected antigen with which the antibody combining site immunoreacts is also a substrate for a reaction that is promoted by the metalloantibody. An engineered V region-containing antibody becomes a metalloantibody when it is complexed with a metal cation. Metal cations (cofactor cations) suitable for complexing with a metal binding protein of this invention are any of the transition state metals of the periodic table, and the non-transition state metals calcium (Ca) , zinc (Zn) , cadmium (Cd) , mercury (Hg) , strontium (Sr) , and barium (Ba) , which metals have the capacity to occupy a tetrahedral oxidation state and thereby complex with said protein through coordinated ligands provided by the three contact amino acid residues on the metal binding protein. Preferred metal cations for use in a metalloprotein of this invention are divalent cations, preferably Cu(II) , Zn(II), Ni(II), Co(II), Fe(II), Ag(II), Mn(II) or Cd(II) , and more preferably Cu(II) . In preferred embodiments, the metal cation of a catalytic metalloantibody is Zn(II) , and a preferred chemical reaction is the hydrolysis of a peptide bond. Metalloantibodies are formed by preparing an antibody having an engineered V region and having a metal binding site of the present invention, and then exposing the metal binding protein to a selected metal cation, preferably in a buffered aqueous medium, for a time sufficient to allow a metal-protein coordination complex to form. Preferred is the metalloantibody prepared and described in the Examples.

In another embodiment, the invention provides for the preparation of antibody molecules of preselected activity using well known methods of cloning immunoglobulin genes from antibody-producing cells to produce a repertoire of immunoglobulin genes from which to isolate a desired antibody specificity. The method uses a transgenic animal of the present invention as the source of the cloned immunoglobulin genes.

The method comprises the steps of: a) providing a transgenic animal of the present invention having a transgene that codes for an engineered V region containing a metal binding site; b) harvesting genes coding for immunoglobulin heavy and/or light chain polypeptides from antibody-producing cells of the transgenic animal; c) expressing the harvested genes in an expression vector capable of expressing the harvested genes and producing an antibody molecule; and d) collecting an antibody having the capacity to bind a metal cation from the produced antibody molecules. The methods for harvesting genes that code for immunoglobulin heavy or light chains are extensively described in the literature described herein, and the method generally involves using PCR to selectively amplify and thereby clone populations of heavy and light chain genes from the nucleic acids of antibody- producing cells using PCR primers having homology to conserved domains of the immunoglobulin genes. Thereafter, the cloned genes are expressed in expression vectors designed for producing both heavy and light chains in E.coli. and allowing their assembly into heterodimeric Fab fragments. The methods are described in detail in published International PCT Application No. WO 92/18619, the disclosures of which are hereby incorporated by reference. Alternatively, the cloned genes can be expressed in phagemid display vectors as described herein to form assembled antibody molecules.

The expressed antibody molecules include antibodies having engineered V regions that bind metal cations, and can be identified and collected on that basis to yield antibody molecules that bind metal cations.

In addition, one may optionally immunize the transgenic animal prior to harvesting the immunoglobulin genes so as to induce an immune response directed to the immunogen, using immunization methods described herein, or by methods well known.

Examples The following examples relating to this invention are illustrative and should not, of course, be construed as specifically limiting the invention.

Moreover, such variations of the invention, now known or later developed, which would be within the purview of one skilled in the art are to be considered to fall within the scope of the present invention hereinafter claimed.

1. Construction of a Plasmid That Encodes an Antibody Light Chain Containing a Metal Binding Site

In order to augment the chemical potential of the immunological repertoire, a metal ion binding light chain has been introduced into the murine genome. This was accomplished through the microinjection of a DNA construct that encoded an antibody light chain containing a metal ion binding site. The construction of the fragment containing the requisite genetic information is described herein. Transgenic mice were then prepared as described in Example 2. Confirmation of the presence of the transgene was performed as described in Example 3. The resulting mice containing the transgene of this invention were subsequently immunized with a fluorescein conjugate as described in Example 4. The results, as described in Example 4, show that the transgenic light chain was found at a high frequency in the anti-fluorescein memory B cell compartment.

An alternative approach for obtaining a metal ion binding site light chain of this invention is described in Example 5. The methods of this invention are applicable to other cofactors and small molecules and should lead to the generation of antibodies with novel catalytic activities. The design of the construct for use in creating a light chain containing metal ion binding sites was based on Iverson et al., Science. 249:696-661 (1990). Iverson described a three histidine metal ion coordination site with specificity for Cu(ll) and Zn(ll) that had previously been introduced into the light chain of the fluorescein-specific antibody, designated 4-4-20. In order to express the metal ion binding sites in the light chain in vivo and create a transgenic antibody, the variable and constant coding sequences were embedded into the second exon of the myeloma MOPC-21 kappa gene since this gene had been demonstrated to elicit allelic exclusion in transgenic mice as described by Ritchie et al., Nature, 312:517-520 (1984) and Brinster et al., Nature. 306:332-336 (1983). The starting plasmid pB-14 contained the functional MOPC-21 kappa gene and has been previously described by Ritchie et al., Nature. 312:517-520 (1984) and Brinster et al., Nature. 306:332-336 (1983) , the disclosures of which are hereby incorporated by reference. The plasmid was obtained from U. Storb, University of Chicago. The final plasmid construct for use in this invention was derived from portions of pB-14 as described herein. The variable region of the kappa gene, V_KM.21, was rearranged next to the joining gene segment, J_κ2. The V_KM.21 portion of the gene provided a means to distinguish the expression of the introduced gene from that of the endogenous kappa genes, both on the RNA and protein levels. The MOPC-21 kappa protein has a distinct molecular weight and isoelectric point allowing it to be distinguished from other kappa chains in mouse serum. Brinster et al., supra. have used the pB-14 plasmid in producing transgenic mice that express the MOPC-21 kappa protein.

To derive the plasmid used for microinjection into mice to form transgenic animals of this invention, pB-14 was first digested with EcoR I(blunt) and Pvu I (blunt) to obtain a 1.8 kilobase (kb) fragment corresponding to the 5' regulatory region and variable domain of the MOPC-21 kappa gene. The resultant kappa gene fragment was then subcloned into a Bluescript expression vector, pBS-II SK (Stratagene, La Jolla, CA, Catalog No. 212205) previously digested with Sac I (blunt) and Hinc II. A natural Sac I site within the subcloned kappa sequences was then removed by digestion with Sac I, followed by blunt-ending and self-ligation.

In order to insert the metal-binding light chain sequences into the MOPC-21 kappa gene fragment, a Sac I site was then introduced into the second exon of the MOPC-21 kappa gene in the pBS-II vector by site-directed mutagenesis with the oligonucleotide 5'AGATTGGGTCATTACGGCCGTCGACGGATGAGCTCAATGTTCCCATCAGC3' (SEQ ID NO 1), containing the restriction sites Eag I, Hinc II, and Sac I. Site-directed mutagenesis was performed according to manufacturer's instructions supplied with a kit (Code RPN 1523) commercially available from Amersham, Arlington Heights, IL. The resulting pBS II SK plasmid vector containing a Sac I site in the second exon of the kappa gene was then digested with Sac I and Hinc II to linearize the vector allowing for the ligation of the donor plasmid light chain nucleotide sequence, designated QM 212, that encodes a light chain with metal ion binding sites.

The QM212 donor plasmid that allows for the expression of a light chain variable domain containing metal ion binding sites in preselected coordinates has been previously described by Iverson et al., Science. 249:659-661 (1990), the disclosure of which is hereby incorporated by reference. Iverson et al., supra. describes the construction of an metalloantibody containing a coordination site for metals in the antigen binding pocket. The sites were constructed in a single-chain fluorescein-binding protein previously described by Bird et al., Science. 242:423-426 (1988), the disclosure of which is hereby incorporated by reference. The single-chain fluorescein-binding protein was expressed by a bacterial expression vector as described by Bird et al., supra.

The Bird et al. vector containing sequences for encoding a single-chain fluorescein-binding protein that was subsequently modified by Iverson et al., supra. to incorporate metal ion binding sites was further modified for use in this invention as described herein. The modifications to the Iverson et al. QM212 vector were directed to the light chain variable domain. The modifications resulted in the addition of nucleotide sequences in the 5' end of the variable domain to encode 2 more amino acid residues. In addition, a murine light chain constant domain was also incorporated 3' to the variable domain of the QM212 vector. A Sac I site was engineered in the 5' end of the light chain domain to remove the leader sequence of the QM212 vector and to provide for directional ligation into the PBS II SK vector containing the MOPC-21 gene. This restriction site also provided for the operational insertion of the QM212-derived light chain domain 3' to the first 2 amino' acid residues of the MOPC-21 light chain variable domain. These sites are shown in Figure 2 in the schematic representation of.the final derived light chain construct. In order to accomplish these modifications, two separate polymerase chain reaction (PCR) amplifications were performed followed by a third overlapping PCR amplification resulting in the combining to the first two PCR products into a single chain for insertion into the pBS II SK linearized vector. PCR reactions were performed as described in Example 3 and overlap PCR was performed as described in Example 6. QM212 variable light chain domain containing nucleotides encoding the metal ion binding site were first amplified with the primer pair, AK1, the 5' primer, and AK2, the 3' primer, having the respective nucleotide sequences,

5'GAGGCCGAGCTCGTTATGACTCAGACACCA3' (SEQ ID NO 2) and 5'GGATACAGTTGGTGCAGCATCAGCCCGTTTGGTGCCTCCACCGAACGTCCAC GG3' (SEQ ID NO 3). AK1 contains nucleotide sequences for the Sac I restriction site.

The primer pair, AK3 and AK4, respectively the 5' and 3' primers, were used to amplify the murine kappa constant domain of a Fab antibody-encoding clone, designated 2b, that was previously described by Kang et al., Proc. Natl. Acad. Sci.. USA. 88:4363-4366 (1991) , the disclosure of which is hereby incorporated by reference. AK3 and AK4 had the respective nucleotide sequences 5'CCGTGGACGTTCGGTGGAGGCACCAAACGGGCTGATGCTGCACCAACTGTAT CC3' (SEQ ID NO 4) AND

5'GCGCCGTCTAGAATTAACACTCATTCCTGTTGA3' (SEQ ID NO 5). AK4 contains nucleotide sequences for the Xba I restriction site. The PCR products from both the first and second PCR amplifications were gel purified as described in Example 6 then pooled and subjected to a third PCR amplification with the primer pair AK1 and AK4 to create a combined modified QM212 light chain having both a variable and constant domain. The primers AK2 and AK3 created an overhang region to provide for the PCR recombination event accomplished in the third PCR amplification. The resultant combined PCR light chain was then gel purified and digested with Xba I and Sac I to form a fragment for insertion into the linearized MOPC-21 kappa gene in the pBS II SK vector.

The resulting Xba I (blunt) /Sac I-digested QM212 light chain variable domain fragment was then ligated into the Sac I/Hinc II linearized pBS II SK vector containing the MOPC-21 gene. The resulting circularized pBS II SK plasmid containing the QM212 light chain domain within the MOPC-21 kappa gene sequence was then digested with Eag I (blunt) and Xho I to linearize the vector. A 12 kb Pvu I (blunt) and Xho I fragment from the pB-14 starting vector that contained the enhancer and terminator regions was ligated to the linearized pBS II SK vector to form the final derived plasmid of this invention, designated pSKPl. The plasmid pSKPl was then digested with Bss HII to remove the plasmid sequences and release a 14.5 kb fragment that was purified for microinjection into fertilized oocytes as described in Example 2. The 14.5 kb construct contains the MOPC-21 leader peptide fused to the metal ion binding light chain variable and constant domains as shown in Figure 2. The hatched boxes represent the kappa gene exons from pB- 14, and the filled box represents the inserted QM212 sequences. Arrows indicate position of PCR primers (ATG/PVU) used in analysis of the transgenic mice as described in the Examples below.

The pSKPl plasmid, containing the 14.5 kb insert, was deposited with the American Type Culture Collection (ATCC) , Rockville, MD, on or before April 30, 1993, and has been assigned the Accession Number 75463.

2. Production of Transgenic Mice Containing the DNA Fragment Encoding an Antibody Light Chain Containing a Metal Ion Binding Site

Transgenic mice were produced by standard techniques using mice on the C57BU6 x BALB/c F2 genetic background. Mice were used as the test animal. See, Hogan et al. Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory, (1986) . Also see US Issued Patents 5,073,490, 5,175,383, 5,175,384 and 5,175,385 for methods of producing transgenic animals, the disclosures of which are hereby incorporated by reference. The procedure is briefly summarized herein .

Single cell mouse embryos were harvested from female mice that were impregnated the evening before. The embryos were treated with hyaluronidase and briefly cultured in M16 medium. The embryos were then transferred to M2 medium on a microscope glass 'depression slide. The embryos were observed with a 40X objective and a 10X eyepiece using a Nikon Diaphot microscope equipped with Hoffman optics. The embryos were held in place with a holding pipet that had been rounded with a microfuge. The positions of both the holding pipets and the injection pipets were controlled with micromanipulators. The 14.5 Kb DNA construct, prepared as described above in Example 1, was loaded in the injection pipet at a concentration of 1 to 10 micrograms per milliliter (ug/ml) . Approximately one picoliter, as judged by a refractile change (Hogan et al, supra) of the pronucleus, of DNA solution was injected into the male pronucleus of each of 120 fertilized oocytes.

After DNA injection, the injected embryos were transferred to M16 medium and incubated at 37 degrees Celsius (37C) in a 5% C0₂ atmosphere for one to two hours. The embryos that appeared normal were transferred to one of the fallopian tubes of pseudopregnant foster mothers. The transfers were performed under a dissecting microscope using general anesthesia (avertin) .

From the above procedure, 20 to 70 progeny were obtained. After birth, newborn mice were kept with their foster mothers for 2 weeks, at which point they were then weaned and screened for DNA integration. A 2 centimeter portion of the tail was removed and homogenized in 400 ul of a tail mix solution containing 0.08 mg proteinase K. Tail mix solution was prepared by mixing 25 ml 10% SDS, 10 ml 5 M NaCl, 25 ml 1 M Tris-HCl at pH 8.0, 2 ml 0.5 M EDTA at pH 8.0 and 438 ml distilled water. Before using tail mix, 400 ml of the solution was incubated with proteinase K at 56C overnight. After the tail was maintained with tail mix, 75 ul of 8 M potassium acetate solution was added to each tube and mixed. Four hundred ul of chloroform was then added to each tube and rotated for at least 15 minutes at 4C. The microfuge tubes were then centrifuged at 14,000 rpm for 10 minutes at 4C. The resultant supernatant was removed to a new tube. The DNA in the supernatant was precipitated with ethanol with centrifugation at 14,000 rpm for 5 minutes. The resultant DNA-containing pellet was dried and resuspended in 10 mM Tris-HCl at pH 8.0, 0.5 mM EDTA (TE) solution.

After resuspending the precipitated DNA, some of it was digested with Bam HI endonuclease and electrophoresed through an 0.8% agarose gel. The DNA was denatured by soaking the gel in 1.5 M NaCl and 0.5 M NaOH for one hour and then neutralizing the DNA by soaking it in 1.5 M NaCl, 0.5 M Tris-HCl at pH 7.4 for 30 minutes. The gel was then soaked in 10X SSC for 1 hour. The DNA was then transferred from the gel into a nitrocellulose filter by the method of Southern, as described in Sambrook et al.. Molecular Cloning: A Laboratory Manual. 2nd ed. , New York (1989) . For the Southern, genomic DNA from 21 founder generation progeny were screened for integration of the transgene by hybridization overnight with the ³²P-labeled QM212 polynucleotide insert prepared in Example 1. Following this overnight hybridization, the filter was washed in 0.1 x SSC, 0.1% SDS at 50C and Kodak XAR film was exposed to it in order to identify the kappa light chain containing the metal binding site within the mouse genome. By comparing the transgenic genomic DNA hybridization patterns against the control DNA, it was determined that approximately 5 copies of the transgenic construct integrated into the chromosomal DNA of the transgenic progeny. The potential for a higher copy number is possible ranging from zero up to 200 copies.

Four lines of transgenic animals were produced and identified by this technique. Three of these lines were propagated for further analysis as described in Examples 3 and 4. One line later died in a laboratory mishap. The remaining transgenic mice were maintained by backcrossing to the BALB/c strain. Frozen transgenic embryos, designated met-mouse, were deposited with the American Type Culture Collection (ATCC) , Rockville, MD, on or before April 30, 1993 and have been assigned, the Accession Number 72015.

³- Transcriptional Analysis of Transgenic Mice DNA Containing the DNA Fragment Encoding an Antibody Light Chain Containing a Metal Ion Binding Site In order to determine whether the transgene was expressed in lymphoid tissue, sets of PCR primers were designed that distinguish between the transgene and endogenous light chain mRNAs as indicated by the primer pair positions in Figure 2. The primer specificities were confirmed by PCR analysis of both the injected plasmid and cDNA from non-transgenic mice. Since the transgene consisted of metal ion binding site-containing light chain (also referred to as metallo-light) sequences inserted into kappa genomic sequences, the transgenic transcript was expected to be approximately 400 base pairs longer than the endogenous transcript.

PCR analysis was performed on both genomic DNA samples and RNA samples. Genomic DNA was obtained from tails of mice as described in Example 2. To obtain RNA used in the PCR analysis, flash frozen tissue was homogenized in 7.6 M guanidine hydrochloride, 50 mM potassium acetate, and ethanol precipitated overnight at -20C. RNA was recovered by centrifugation, resuspended in guanidine hydrochloride, and ethanol precipitated for 3 hours.

RNA was again recovered by centrifugation, resuspended in a small volume of guanidine hydrochloride, extracted with phenol:chloroform (1:1), ethanol was added, the RNA was precipitated again, washed with 70% ethanol, resuspended in Tris-EDTA solution (TE) , and stored at -70C. Gibco BRL Reverse Transcriptase Kit (Gibco, Gaithersberg, MD) was used for cDNA production from the purified RNA.

All PCR reactions were performed in an Ericomp Twin Block Cycler (San Diego,CA) . For the transcriptional analysis of the transgenic mice, the oligonucleotide primers used were the 5' primer designated ATG (5ΑTGCATCAGACCAGCATGGGC3' SEQ ID NO 6) and PVU (5'CACTCTGACCATCAGCAGTGTGCA3' SEQ ID NO 7). Each 25 ul PCR reaction contained 2.5 ul of DNA, either genomic or cDNA, 2m5 ul 2 mM dNTP's, 2.5 ul 10X buffer, 2.0 ul 25 mM MgCl₂, 3.0 ul of a 10 micromolar (mM) concentration of primers, 12.3 ul water and 0.2 ul of Taq polymerase. Genomic DNA was amplified with the PVU primer above and a different 5' primer designated BAM (5'GCGATGGTGACTGCGTTGGAGGC3' SEQ ID NO 8) . Plasmid and genomic DNA were amplified by the following program: 94C for 1 minute and 30 seconds (1'30"), 25 cycles of 94C for 30", 60C for 45", 72C for 45", and 72C for 45". For cDNA, the annealing temperature was 66C and the extension time was 1'15". Final extension time was extended to five minutes at the same temperature.

The results of the PCR reactions are shown in an ethidiu bromide stained agarose gel in Figure 3. PCR analysis of tail genomic DNA with primers (BAM/PVU) flanking the inserted QM212 sequences (see Figure 2) is shown on the left side of the gel labeled as Figure 3A. DNA from a nontransgenic mouse (1908) is used as a control for the PCR analysis. DNA from this nontransgenic mouse (1908) contains a single band, whereas DNA from transgenic mice (1853 and 1873) contain a second band (arrow) corresponding to the QM212 containing transgene. PCR analysis of cDNA derived from blood RNA from nontransgenic (BALB and 1908) and transgenic (1853 and 1873) mice is shown on the right side of the gel labeled as Figure 3B. Equivalent bands are seen with primers for actin. Using the ATG/PVU primers, all mice contain the band corresponding to the spliced transcript from the endogenous kappa gene, and the transgenic mice contain an additional band (arrow) corresponding to spliced transcripts from the transgene. Figure 3, thus, shows that amplification of an 850 bp fragment from the cDNA from two transgenic mice, 1853 and 1873, while in the nontransgenic mouse 1908 and BALB/c mice a 400 bp fragment was amplified (Figure 3B) . The size of the PCR product in the transgenic mice corresponded to the predicted length of the transgenic transcript. PCR analysis of genomic DNA from the same mice yielded a fragment of approximately 1150 bp (Figure 3A) . The increase in size of DNA as compared with RNA (cDNA) corresponded to the length of the first intron, indicating that the transgenic transcript was correctly spliced. The structure of the transgenic transcript in this region was confirmed with additional primer sets. Further studies demonstrated that all the remaining transgenic lines expressed the transgene, although the level was somewhat higher in one line when assessed in comparison with actin primer standards.

Non-lymphoid tissue was also evaluated for expression of the transgene and only faint bands were detected on the PCR gels shown in Figure 3, which probably owe their origin to the presence of blood lymphocytes in all tissues. These results provide evidence for tissue specific expression of the metallo-light chain construct in the transgenic mice.

4. Expression and Characterization of Antibodies

Containing a Light Chain Containing a Metal Ion

Binding Site in Transgenic Mice

A. Immunization for the Production of Transgenic Antibodies

In order to determine whether the transgenic metallo-light chain of this invention could participate in an antigen driven immune response in vivo, immunized mice were studied. Since the metallo-light chain was derived from a single chain anti-fluorescein antibody as described in Example 1, the light chain was surmised to be utilized with reasonable frequency to produce anti-fluorescein antibodies. The murine anti-fluorescein response was previously known to exhibit significant affinity maturation and is quite diverse in that there appears to be no dominant idiotype as described by Reinitz et al., J. Immunol.. 135:3365 (1985). The participation of the transgene in the anti-fluorescein response would require selection into the memory B cell compartment, resulting in incorporation of the metallo-light chain into high affinity antibody molecules.

Thus, transgenic and non-transgenic mice from the 1465 line prepared in Example 2 were immunized with fluorescein.

Mice were immunized with fluorescein conjugated to BSA (FITC-BSA) in RIBI adjuvant. Fluorescein was conjugated to BSA according to the methods described in "Antibodies: A Laboratory Manual", eds Harlow et al.. Cold Spring Harbor Laboratory, 1988. The mice initially received 160 ug FITC-BSA subcutaneously. This dose was repeated after three weeks, and followed in two weeks by boosting with 100 ug FITC-BSA intraperitoneally. One month later, a final boost of 50 ug FITC-BSA was given, without adjuvant, by tail vein injection. On the third day following the final boost, spleen cells were used for fusion to produce anti-fluorescein hybridomas as described below in Example 4B. By ELISA performed as described in

Example 4C, both transgenic mice and non-transgenic mice produced comparable high titre anti-fluorescein responses.

B. Production of Transgenic Hybridomas

Following the immunization protocol as described above, the spleens of the immunized mice were separately used to produce hybridomas by fusion with a immortalized myeloma cell line in a procedure well known to one of ordinary skill in the art and described originally by Kohler et al., Nature, 256:495-497 (1975), the disclosure of which is hereby incorporated by reference. Surviving clones were evaluated as described below. C. Screening of Transgenic Hybridoma Supernatants 1) ELISA Assay

In order to determine if functional transgenic antibodies were secreted from hybridomas prepared from fluorescein-immunized transgenic mice, ELISA assays were performed with supernatants from the transgenic hybridoma cultures prepared in Example 4B. For the ELISA, microtiter plates were coated with a 50 g/ml fluorescein-BSA (also referred to as FITC-BSA) conjugate diluted in 0.1 M bicarbonate, pH 8.6. The wells were then washed twice with water and blocked by completely filling the well with 100 ul solution of 1% BSA diluted in PBS to block nonspecific sites on the wells. Afterwards, the plates were inverted and shaken to remove the BSA solution. One hundred ul of hybridoma supernatants obtained from the cultures were then admixed to each well and maintained at 37C for 1 hour to form immunoreaction products. Following the maintenance period, the wells were washed ten times with water to remove unbound soluble antibody and then maintained with a 25 ul of a 1:1000 dilution of secondary goat anti-mouse IgG F(ab')₂ conjugated to alkaline phosphatase diluted in PBS containing 1% BSA. The wells were maintained at 37C for one hour after which the wells were washed 10 times with water followed by development with 50 ul of p-nitrophenyl phosphate (PNPP) . Color development was monitored at 405 nm. Positive clones gave A405 values of >1 (mostly >1.5) after 10 minutes, whereas negative clones gave values of 0.1 to 0.2.

From this initial screening, six hybridoma lines that bound to fluorescein were generated from the immunized transgenic mice (designated H1-H6) . 2) PCR Analysis

In order to determine if any of the fluorescein-reactive hybridomas, H1-H6, expressed the transgene, RNA was extracted and cDNA was prepared and analyzed by PCR methods performed as described in

Example 3. The cDNAs were prepared from RNA extracted from the hybridomas, or from the liver (L) and spleen (S) from transgenic (2422) and nontransgenic (2424) mice. The resultant cDNAs were amplified with the ATG/PVU primers for analysis as described in Example 3.

Out of six hybridomas examined, two (H3 and H6) contained the transgenic transcript as shown in Figure 4, in two panels 4A and 4B. The other four hybridomas also contained the transgene genomic DNA but harbored no corresponding transcript. The band corresponding to the transgene transcript (arrow) was seen in hybridomas H3 and H6, in addition to the spleen of the transgenic mouse 2422. In addition, due to the significant affinity maturation observed in the anti-fluorescein response, DNA sequence analysis was performed to determine whether the metal coordinating histidines were altered by somatic mutation in our hybridomas. The results of the sequencing indicated that the kappa chains of the hybridomas retained the metal coordinating histidine residues.

3) Isoelectric Focusing and Western Blot The transgene RNA expression studies were confirmed by analyzing the corresponding immunoglobulins by Western blot on an isoelectric focusing gel. Isoelectric focusing of the transgenic antibodies, H1-H6, prepared above was accomplished using Novex IEF gels pH 3-10 and the accompanying buffers. Transblotting was done on Millipore Immobilon PVDF-P transfer membrane. Ascites fluid, obtained from primed mice that had previously received injections of 5 X 10⁵ cells of particular transgenic hybridomas, was first purified with protein G then diluted to achieve a lOng/ul concentration before ^•reducing with 2τmercaptoethanol. The diluted ascites fluid was then combined with an equal volume (10 ul each) Novex 2x sample buffer and the admixture was maintained at room temp for 15 minutes. The samples were then loaded on the gel and run in a Novex vertical electrophoresis apparatus at 2W/gel for 2 hours.

After the run, gels were soaked in carbonate transfer buffer before transblotting for 1.5 hours at 150 mAmps. Blocking of the nonspecific sites on the resultant blot was accomplished with 10% nonfat dried milk in 1% TBS containing 0.05%. Tween-20. The blocking step was maintained overnight. Alkaline phosphatase conjugated mouse anti-kappa specific antibody (Caltag, San Francisco, CA) was used at 1:1000 concentration to immunoreact with kappa chains. Color development followed the immunoreaction step with Bio-Rad's alkaline phosphatase color development kit (NBT and BCIP, Bio-Rad, Richmond, CA) . Washes between blocking and maintenance steps were done with 1 X TBS containing 0.05% Tween-20.

The results from Western blot analysis following isoelectric focusing of hybridomas H6 (left lane), H5, H4, and H3 (right lane) are shown in Figure 5. The gels showed that both hybridomas, H3 and H6, contained kappa chains, confirming the RNA PCR analysis. Hybridomas 3 and 6 also demonstrated a unique band corresponding to an 8.6-9.3 isoelectric focusing point that is quite near the 8.8 theoretical pi of the transgenic kappa chain. This result indicates that the kappa chain transcript is translated into the metal ion binding light chain and that it is the only light chain present in these hybridomas.

D. Analysis of the Transgenic Antibodies

Containing a Metal Ion Binding Light Chain This invention demonstrates the power of transgenic technology for in vivo generation of antibodies which contain cofactors for catalysis. In essence, the chemical potential of the antibody repertoire has been vastly increased. The transgenic light chain was found at a high frequency in the anti-fluorescein memory B cell compartment. The methods of this invention are contemplated to be extended to other antigens and other light chains. For production of catalytic antibodies to antigens with multiple epitopes, such as. viral antigens, the in vivo approach would select for responsiveness to epitopes of highest accessibility and immunogenicity and, thus, may be useful for deriving therapeutic antibodies.

Although previous studies have shown that transgenic light chains can participate in the humoral response to multiple antigens as described by Carmack et al., J. Immunol.. 147:2024-2032 (1991), the response to some antigens may be diminished due to the elimination or inactivation of self reactive B cells. See, for example, Hartely et al.. Nature. 353:765-769 (1991), Okamoto et al., J. EXP. Med.. 175:71-79

(1992), Brombacher et al., J. EXP. Med.. 174:1335-1346 (1991) and Erikson et al., Nature. 349:331-334 (1991). Several genetic approaches can be employed to broaden the available B cell repertoire. The transgene of this invention was introduced into Lpr mice as described in Example 6 which produce autoantibodies due to a defect in the Fas gene, which is involved in programmed cell death. See Itoh et al.. Cell. 66:233-243 (1991) and Watanabe-Fukunaga et al., Nature, 356:314-317 (1992). Similarly, also contemplated for use in this invention is the ectopic expression of the Bcl-2 gene which promotes B cell survival. See, Vaux et al.. Nature, 335:440-442 (1988), McDonnell et al., Cell. 57:79-88 (1989) and Strasser et al., Proc. Natl. Acad. Sci.. USA.

88:8661-8665 (1991). Bcl-2 would result in the increase the available B cell repertoire allowing for an enhancement of the immunological response. Utilization of mice deficient in endogenous kappa chains for transgenic experiments should eliminate background and facilitate the screening of a greater number of catalytic antibody molecules.

The ability to augment the murine immunological repertoire with cofactors or other small molecules significantly expands the chemical capacity of the immune system. The methods of this invention can be extended to produce transgenic antibodies that contain alternative metal ion coordination sites such as those described by Roberts et al. , Proc. Natl. Acad. Sci.. USA, 87:6654-6658 (1992), other cofactors such as flavins (Shokat et al., Angew. Chem. Int. Ed. Engl.. 27:1172-1175 (1988) and perhaps even cytotoxic or imaging agents. Finally, the ability to recombine a given metallo light chain in vitro with a large number of heavy chains using combinatorial strategies should expand the potential of this approach even further as described by Barbas et al., Proc. Natl. Acad. Sci.. USA. 89:4457-4461 (1992). 5. Preparation of Synthetic Binding Sites Within the Light Chain CDR3 Domain of a Phagemid Fab Display Protein Produced by a Dicistronic Expression Vector A. Preparation of Nucleotide Sequences Encoding

Synthetic Binding Sites that Bind Metals The immunoglobulin gene phagemid expression vector, pC3muFab, containing the heavy and light chain sequences for expressing both a phagemid displayed and the soluble form of a murine Fab antibody, was used to prepare the synthetic binding site proteins containing metal ion binding sites.

Plasmids, designated p3CmuFab, used in this invention were deposited on or before April 30, 1993, with the American Type Culture Collection, 1301 Parklawn Drive, Rockville, MD, USA (ATCC) . The deposit of the plasmids is listed under the name p3CmuFab and has been assigned the ATCC Accession number 75464. All of the deposits described herein were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty) . The deposit pursuant to the Budapest Treaty assures maintenance of the deposited material for 30 years from the date of deposit. The deposited material will be made available by ATCC under the terms of the Budapest Treaty which assures permanent and unrestricted availability of the progeny of the deposit to the public upon issuance of the pertinent U.S. patent or upon laying open to the public of any U.S. or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto according to 35 U.S.C. §122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.14 with particular reference to 886 OG 638) . The assignee of the present application has agreed that if the deposited material should die, become non-viable or be lost or destroyed when cultivated under suitable conditions, it will be promptly replaced on notification with a viable specimen of the same culture. Availability of the deposited strain is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

The expression vector, p3CmuFab, prepared as described in Example 5B and being 5272 bp in length, was used in PCR amplifications to create unique metal ion binding sites within the CDR3 domain of the Fab light chain in the phagemid. The CDR3 region of a light chain Fab was randomized by overlap PCR amplification and Fabs were subsequently selected for the ability to bind to metals as described in Example 5C.

For preparing PCR products that encode binding sites that exhibited specificity for metals, two separate PCR reactions were performed followed by overlap PCR. In the first PCR amplification reaction, the 5' end of the light chain, beginning at framework 1 and extending to the 3' end of framework 3, was amplified. In the second PCR amplification reaction, the CDR3 region was mutagenized to produce sequences that encode metal binding sites. This was accomplished through the use of a pool of oligonucleotide primers synthesized with a degenerate region sandwiched between and contiguous with conserved framework 3 and 4 region sequences. Degenerate oligonucleotide primers were designed for encoding amino acid residue sequences of 9 amino acid residues in length within the CDR3 domain.

The amplification products resulting from the second PCR, each having a randomized CDR3 region, began at the 3' end of framework 3 and extended downstream of the CDR3. The pool of degenerate oligonucleotide primers were designed to result in the amplification of products having a 5' end that was complementary to and overlapped with the 3' end of the products of the first PCR reaction product.

Thereafter, the two separate PCR reaction products were pooled and subjected to a third PCR reaction in which the overlapping region between the two products was extended to result in a complete light chain having a randomized CDR3 region.

The PCR reactions were performed in a 100 ul reaction containing the following reagents: 1 ug of each of oligonucleotide primer pairs as described below that were synthesized by Operon Technologies Alameda, CA. ; 8 ul 2.5 mM dNTP's (dATP, dCTP, dGTP, dTTP) ; 1 ul Taq polymerase (Perkin-Elmer Corp., Norwalk, CT) ; 10 ng of template pC3muFab; and 10 ul of 10X PCR buffer purchased commercially (Perkin-Elmer Corp.). Thirty-five rounds of PCR amplification in an Ericomp thermocycler (Ericomp) were then performed.

The amplification cycle consisted of denaturing at 94C for one minute, annealing at 47C for one minute, followed by extension at 72C for two minutes. To obtain sufficient quantities of amplification product, 15 identical PCR reactions were performed and thereafter pooled.

In the first PCR amplification reaction, the 5' end of the light chain beginning at framework 1 and extending to the 3' end of framework 3 was amplified. To accomplish this, the 5' coding primer, designated 1-5', having the nucleotide sequence

5'GAGCTCCAGATGACCCAGTCT3' (SEQ ID NO 9) was used in an amplification reaction with the 3' noncoding primer, designated 1-3' , 5'ATAAATCCCAAAATCTTCAGACTGCAGGC3' (SEQ ID NO 10) .

The resultant PCR amplification products were then gel purified on a 1.5% agarose gel using standard electroelution techniques as described in "Molecular Cloning: A Laboratory Manual", Sambrook et al., eds., Cold Spring Harbor, NY (1989). Briefly, after gel electrophoresis of the digested PCR amplified Fab-display encoding synthetic binding sites, the region of the gel containing the DNA fragments of approximately_.258 bp was excised, electroeluted into a dialysis membrane, ethanol precipitated and resuspended in buffer containing 10 mM Tris-HCl at pH 7.5 and 1 mM EDTA to a final concentration of 50 ng/ml.

The purified products were then used in an overlap extension PCR reaction with the products of the second PCR reaction, both as described below, to recombine the two products into reconstructed light chains containing mutagenized CDR3 regions. The second PCR reaction resulted in the amplification of the light chain from the 3' end of framework region 3 extending to the end of CL1 region which was approximately 409 bp in length. To amplify this region for encoding 9 random amino acid residues comprising the CDR3 domain, the following primer pairs were used. The 5' coding oligonucleotide primer pool, designated 2-5', had the nucleotide sequence represented by the formula,

5'GAAGATTTTGGGATTTATNNSNNSNNSNNSNNSNNSNNSNNSNNSACGTTCG GTACTGGGACC3' (SEQ ID NO 11), where N is A, C, G, or T and S is either C or G. The 5' end of the primer pool is complementary to the 3' end of framework 3 represented by the complementary nucleotide sequence of the oligonucleotide primer, 1-3', used in the first PCR reaction and the 3' end of the primer pool after the NNS repeat is complementary to the 5' end of framework 4. The region between the two specified ends of the primer pool is represented by a 27-mer NNS degeneracy which ultimately encodes a diverse population of mutagenized CDR3 regions of 9 amino acid residues in length. The 3' noncoding oligonucleotide primer, designated 2-3', had the nucleotide sequence 5'TCTAGAATTAACACTCATTCC3' (SEQ ID NO 12), and hybridized to the 3' end of framework 4. The second PCR reaction was performed on the PC3muFab in a 100 ul reaction as described above containing 1 ug of each of 2-5' and 2-3' oligonucleotide primers. The resultant PCR amplification products were then gel purified as described above.

One hundred nanograms of gel purified products from the first and second PCR reactions were then admixed with 1 ug each of the 1-5' and 2-3' oligonucleotide primers as a primer pair in a final PCR reaction to form a complete light chain fragment by overlap extension. The PCR reaction admixture also contained 10 ul 10X PCR buffer, 1 ul Taq polymerase and 8 ul 2.5 mM dNTP's as described above. To obtain sufficient quantities of amplification product, 15 identical PCR reactions were performed.

The resulting light chain fragments, beginning at framework 1 and extending downstream of the mutagenized CDR3 domain encoding 9 amino acid residues, were approximately 667 base pairs in length. The light chain fragment amplification products from the 15 reactions were first pooled and then gel purified as described above prior to their incorporation into the pC3muFab surface display phagemid expression vector to form a library. Since the PCR primers 1-5' and 2-3' respectively encoded the restriction sites Sac I and Xba I, the directional ligation into the pC3muFab was easily accomplished. The pooled and purified PCR products were first digested with Sac I and Xba I and ligated into a similarly digested pC3muFab expression vector, resulting in the in-frame positioning of the mutagenized light chains into which heavy chain domains are located upstream.

B. Production of Phagemid Fab-Displayed Synthetic Binding Sites In practicing this invention to obtain expression of Fab-display proteins containing a synthetic binding site on a phage surface, the heavy (Fd consisting of V_H and C_H1) and light (kappa) chains (^V _L, ^C _L) of antibodies were first targeted to the periplasm of E. coli for the assembly of heterodimeric Fab molecules.

In this system, the first cistron encoded a periplasmic secretion signal (pelB leader) operatively linked to the fusion protein, Fd-cpIII. The second cistron encoded a second pelB leader operatively linked to a kappa light chain. The presence of the pelB leader facilitated the coordinated but separate secretion of both the fusion protein containing the synthetic binding site and light chain from the bacterial cytoplasm into the periplasmic space.

In this process, each chain was delivered to the periplasmic space by the pelB leader sequence, which was subsequently cleaved. The heavy chain was anchored in the membrane by the cpIII membrane anchor domain while the light chain containing the synthetic binding site was secreted into the periplasm. Fab molecules were formed from the binding of the heavy chain with the soluble light chains.

1) Preparation of a Dicistronic Expression

Vector. pComb3. Capable of Expressing a Phagemid Fab Display Protein In the phagemid expression vector used in this invention, the antibody Fd chain comprising variable (V_H) and constant (C_H1) domains of the heavy chain were fused with the C-terminal domain of bacteriophage gene III (3) coat protein. Gene III of filamentous phage encodes a 406-residue minor phage coat protein, cpIII (cp3) , which is expressed prior to extrusion in the phage assembly process on a bacterial membrane and accumulates on the inner membrane facing into the periplasm of E. coli.

The phagemid vector, designated pComb3, allowed for both surface display and soluble forms of Fabs. The vector was designed for the cloning of combinatorial Fab libraries. The Xho I and Spe I site were provided for cloning complete PCR-amplified heavy chain (Fd) sequences consisting of the region beginning with framework 1 and extending through framework 4. The Sac I and Xba I sites were provided for cloning PCR amplified antibody light chains. The cloning sites were compatible with previously reported mouse and human PCR primers as described by Huse et al., Science. 246:1275-1281 (1989) and Persson et al., Proc. Natl. Acad. Sci.. USA. 88:2432-2436 (1991). The nucleotide sequence of the pelB, a leader sequence for directing the expressed protein to the periplasmic space, was as reported by Huse et al., supra.

The vector also contained a ribosome binding site as described by Shine et al., Nature, 254:34 (1975). The sequence of the phagemid vector, pBluescript, which includes ColEl and FI origins and a beta-lactamase gene, has been previously described by Short et al., Nuc. Acids Res.. 16:7583-7600 (1988) and has the GenBank Accession Number 52330 for the complete sequence. Additional restriction sites, Sal I, Ace I, Hinc II, Cla I, Hind III, Eco RV, Pst I and Sma I, located between the Xho I and Spe I sites of the empty vector were derived from a 51 base pair stuffer fragment of pBluescript as described by Short et al., supra. A nucleotide sequence that encodes a flexible 5 amino acid residue tether sequence which lacks an ordered secondary structure was juxtaposed between the Fab and cp3 nucleotide domains so that interaction in the expressed fusion protein was minimized.

Thus, the resultant combinatorial vector, pComb3, consisted of a DNA molecule having two cassettes to express one fusion protein, Fd/cp3, and one soluble protein, the light chain, into the periplasmic space. The vector also contained nucleotide residue sequences for the following operatively linked elements listed in a 5' to 3' direction: a first cassette consisting of LacZ promoter/operator sequences; a Not I restriction site; a ribosome binding site; a pelB leader; a spacer region; a cloning region bordered by 5' Xho and 3' Spe I restriction sites; the tether sequence; the sequences encoding bacteriophage cp3 followed by a stop codon; a Nhe I restriction site located between the two cassettes; a second lacZ promoter/operator sequence followed by an expression control ribosome binding site; a pelB leader; a spacer region; a cloning region bordered by 5' Sac I and a 3' Xba I restriction sites followed by expression control stop sequences and a second Not I restriction site. In the above expression vector, the Fd/cp3 fusion and light chain proteins were placed under the control of separate lac promoter/operator sequences and directed to the periplasmic space by pelB leader sequences for functional assembly on the membrane. Inclusion of the phage FI intergenic region in the vector allowed for the packaging of single-stranded phagemid with the aid of helper phage. The use of helper phage superinfection allowed for the expression of two forms of cp3. Consequently, normal phage morphogenesis was perturbed by competition between the Fd/cp3 fusion and the native cp3 of the helper phage for incorporation into the virion. The resulting packaged phagemid carried native cp3, which is necessary for infection, and the encoded Fab fusion protein, which is displayed for selection. Fusion with the C-terminal domain was necessitated by the phagemid approach because fusion with the infective N-terminal domain would render the host cell resistant to infection.

The pComb3 expression vector described above forms the basic construct of pC3muFab antibody display phagemid expression vectors used in this invention for the production of mouse Fab antibodies containing synthetic metal ion binding sites.

2) Preparation of Expression Vector

Libraries for the Expression of the Phagemid Fab-Display Proteins a. Phagemid Library Construction

In order to obtain expressed human Fab antibodies having both heavy and light chain fragments, phagemid libraries were constructed. The libraries provided for the expression of recombinant human Fab antibodies having heavy and light chains where the synthetic binding sites of this invention are displayed in the light chain CDR3 domain. The PCR products resulting from each of the amplification reactions prepared in Example 5A were separately inserted into the pC3muFab phagemid expression vector to prepare phagemid libraries.

For preparation of phagemid libraries for expressing the PCR products prepared in Examples 5A, the PCR products were digested with Sac I and Xba I and separately ligated with a similarly digested pC3muFab phagemid expression vector. The ligation resulted in operatively linking the mutagenized light chains into the phagemid vector.

Phagemid libraries for expressing each of the Fab display synthetic binding sites of this invention were prepared in the following procedure. To form circularized vectors containing the PCR product insert, 640 ng of the digested PCR products were admixed with 2 ug of the linearized pC3muFab phagemid vector and ligation was allowed to proceed overnight at room temperature using 10 units of BRL ligase (Gaithersburg, MD) in BRL ligase buffer in a reaction volume of 150 microliters (ul) . Five separate ligation reactions were performed to increase the size of the phage library having synthetic binding site CDR3 regions in the light chain. Following the ligation reactions, the circularized DNA was precipitated at -20C for 2 hours by the admixture of 2 ul of 20 mg/ml glycogen, 15 ul of 3 M sodium acetate at pH 5.2 and 300 ul of ethanol. DNA was then pelleted by microcentrifugation at 4C for 15 minutes. The DNA pellet was washed with cold 70% ethanol and dried under vacuum. The pellet was resuspended in 10 ul of water and transformed by electroporation into 300 ul of E. coli XLl-Blue cells to form a phage library. The total yield from the PCR amplification and transformation procedure described herein was approximately 5 X 10⁷ transformants.

To isolate phage on which Fabs for selection on metal resins as described below in Example 5C, 3 ml of SOC medium (SOC was prepared by admixture of 20 grams (g) bacto-tryptone, 5 g yeast extract and 0.5 g NaCl in 1 liter of water, adjusting the pH to 7.5 and admixing 20 ml of glucose just before use to induce the expression of the Fd-cpIII and light chain heterodimer) were admixed to the transformed cultures and the culture was shaken at 220 rpm for 1 hour at 37C, after which 10 ml of SB (SB was prepared by admixing 30 g tryptone, 20 g yeast extract, and 10 g Mops buffer per liter with pH adjusted to 7) containing 20 ug/ml carbenicillin and 10 ug/ml tetracycline and the admixture was shaken at 300 rpm for an additional hour. This resultant admixture was admixed to 100 ml SB containing 50 ug/ml carbenicillin and 10 ug/ml tetracycline and shaken for 1 hour, after which helper phage VCSM13 (10¹² pfu) were admixed and the admixture was shaken for an additional 2 hours. After this time, 70 ug/ml kanamycin was admixed and maintained at 30C overnight. The lower temperature resulted in better heterodimer incorporation on the surface of the phage. The supernatant was cleared by centrifugation (4000 rpm for 15 minutes in a JA10 rotor at 4C) . Phage were precipitated by admixture of 4% (w/v) polyethylene glycol 8000 and 3% (w/v) NaCl and maintained on ice for 30 minutes, followed by centrifugation (9000 rpm for 20 minutes in a JA10 rotor at 4C) . Phage pellets were resuspended in 2 ml of PBS and microcentrifuged for 3 minutes to pellet debris, transferred to fresh tubes and stored at -20C for subsequent screening as described below. For determining the titering colony forming units (cfu) , phage (packaged phagemid) were diluted in SB and 1 ul was used to infect 50 ul of fresh (AOD600 = 1) E. coli XLl-Blue cells grown in SB containing 10 ug/ml tetracycline. Phage and cells were maintained at room temperature for 15 minutes and then directly plated on LB/carbenicillin plates.

C. Metal Chelate Affinity Chromatography of the Phage Library for Phagemid Fab-Displaved

Synthetic Metal Binding Site Proteins For selecting Fab displayed synthetic metal binding site proteins of this invention, the phage preparations prepared above were applied to affinity columns on which selected metals had been immobilized. For this selection procedure, metal chelate affinity chromatography was performed using Pharmacia HiTrap columns according to manufacturer's instructions (Pharmacia) . HiTrap columns are packed with 1 or 5 ml chelating Sepharose High Performance, a newly developed matrix. The columns are made of polyethylene which is biocompatible and non-interactive with biomolecules. The amino acids histidine, cysteine and tryptophan, present in almost every protein, allow for the formation of complexes with many transition metal ions. Thus, the chelating Sepharose High Performance, charged with selected metal ions, selectively retains proteins if the complex-forming amino acid residues are exposed on the protein surface. The Sepharose used in the columns consists of highly cross-linked agarose beads coupled by stable ether groups to iminodiacetic acid via 7-atom spacer arms. The coupling technique ensures both high capacity and performance while minimizing leakage of the iminodiacetic groups. For selecting Fab-displaying synthetic binding sites that bind to copper, the metal complex, copper sulfate, (Sigma Chemical Co., St. Louis, MO) was coupled to the chelating Sepharose High Performance following the manufacturer's instructions. The phage preparations from the phage libraries from the 5 separate PCR amplifications were applied to the column. To 100 ul of suspended metal complexed resin 500 ul of 10¹¹ pfu/ml of phage were added. After allowing the phage-anchored Fabs to immobilize to the metal, the column was washed 5 times with TBS containing 0.1% Tween-20. The metal-specific immobilized phage-anchored Fabs were then eluted with 20 ul of 1 mM copper sulfate. Alternatively, the elution was performed with 200 ul of 50 mM EDTA. Thereafter, the eluted phage were infected into bacteria in order to prepare soluble Fab-displayed metal ion binding site proteins..

D. Preparation of Soluble Fab-Displayed Metal

Ion Binding Site Proteins In order to further characterize the specificity of the Fab-displayed synthetic binding site proteins expressed on the surface of phage as described above, soluble heterodimers are prepared. To prepare soluble Fabs consisting of heavy and light chains (i.e., heterodimers) , phagemid DNA from positive clones selected by metal affinity chromatography above is isolated and digested with Spe I and Nhe I. Digestion with these enzymes produces compatible cohesive ends. The 4.7-kb DNA fragment lacking the gill portion is gel-purified (0.6% agarose) and self-ligated. Transformation of E. coli XLl-Blue affords the isolation of recombinants lacking the gill fragment. Clones are then examined for removal of the gill fragment by Xho I/Xba I digestion. Clones are grown in 100 ml SB containing 50 ug/ml carbenicillin and 20 mM MgCl₂ at 37C until an OD^ of 0.2 is achieved. IPTG (1 mM) is added and the culture is grown overnight at 30C (growth at 37C provides only a light reduction in heterodimer yield) . Cells are pelleted by centrifugation at 4000 rpm for 15 minutes in a JA10 rotor at 4C. Cells are resuspended in 4 ml PBS containing 34 ug/ml phenylmethylsulfonyl fluoride (PMSF) and lysed by sonication on ice (2-4 minutes at 50% duty) . Debris is pelleted by centrifugation at 14,000 rpm in a JA20 rotor at 4C for 15 minutes. The supernatant is stored at -20C. For the study of a large number of clones, 10 ml cultures provide a sufficient amount of Fab-displayed synthetic binding site proteins for analysis.

E. Sequence Determination of the Binding Site Proteins Nucleic acid sequencing was performed using

Sequenase 1.0 (USB, Cleveland, OH) on double-stranded DNA encoding the specific Fab-displayed synthetic binding site proteins that bound to copper characterized above. The amino acid residue sequences of the light chain CDR3 domains were derived from the nucleotide sequences and are presented below.

Table 2 Amino Acid Residue Sequence of Metal-Binding CDR3 Light Chain Domains

Amino Acid SeguenceSEO ID NO

Pro-Asn-Ser-Leu-Arg-Trp-Pro-Ser-Cys 13 Ser-Phe-Thr-Phe-Asp-His-Ala-Gly-Leu-Met* 14 His-Cys-Trp-Val-Leu-Gly-Pro-Ser-Leu 15 Arg-Val-Asp-Arg-Thr-His-Ala-Pro-Asp 16 Gly-Asp-Asn-Leu-Tyr-Gly-Ser-Leu-Leu 17 Ala-Gly-Ala-Glu-His-Ala-Asn-Gln-Ser 18 Leu-Trp-Thr-Arg-Leu-Met-Val-Ala-Leu 19 Ser-Gly-Lys-Ile-Tyr-Phe-Arg-Met-Glu 20

* The Met residue comprised the metal binding site of this particular Fab

In addition to incorporating synthetic metal ion binding sites into the light chain, the same procedure can also used to mutagenize the remaining CDR domains of both the light and heavy chains. Mutagenized regions can be used independently or in concert with similar binding site sequences created in the separate chain CDR domains. The use of the two synthetic binding site protein-encoding sequences enhances the ability of obtaining synthetic binding site proteins displayed on human Fabs that exhibit unexpected affinities and avidities to preselected target moleculeε. Thus, the synthetic binding site compositions in the heavy and light chain CDR3 domains of this invention allow for the production of reactive binding molecules not normally attainable that have therapeutic and diagnostic uses. 6. Preparation of a Transgenic Mouse Having an Lpr Mutation

The transgenic mouse produced in Example 2 that expresses in its antibody-producing cells an exogenous engineered V region having a metal binding site (met- mouse) was used to prepare a transgenic mouse with the additional trait of possessing enhanced diversity of immune-responsiveness.

To that end, a male from the met-mouse transgenic line was crossed with two female MRL/SCR/lpr/lpr mice (breeding colony of The Scripps Research Institute, La Jolla, CA) having a mutation in the lpr gene. The progeny of this first cross were screened for the presence of the engineered V region transgene by genomic DNA hybridization as described herein, and approximately 50% of the progeny contained the transgene. A male progeny from the first cross was then crossed again with two female MRL/SCR/lpr/lpr mice because lpr is a recessive trait. The progeny from the second cross were screened for the presence of the engineered V region transgene, and approximately 50% of the progeny contained the transgene. Progeny positive for the engineered transgene were then screened for the presence of anti- DNA animals, which phenotypic trait is indicative of the lpr genotype. Progeny having the highest levels of anti-DNA antibodies were selected as having lpr homozygous genotype in addition to the met-mouse genotype, and were designated as met-lpr mice. The assay for anti-DNA antibodies was conducted by ELISA. Briefly, mouse genomic DNA was adsorbed onto the plastic in the wells of a microtiter plate, and thereafter, sera from a test mouse was added to the wells and allowed to immunoreact with the DNA in the wells. Thereafter, the immunoreacted anti-DNA antibodies, if any, were detected by using a labelled anti-mouse IgG antibody in ELISA format. Of eight mice tested, three had high titres of anti-DNA antibodies and were selected as et-lpr mice of this invention.

The met-lpr mice were maintained by backcrossing to a MRL/SCR/lpr./lpr mouse, and screening the progeny for the presence of the engineered V region transgene. Typically 50% of the progeny were the desired met-lpr mice, and were selected to continue the line.

The met-lpr mouse prepared above is used as a source for immunological repertoires as described herein, and yields a larger diversity of antibody molecules that includes anti-self antigens when compared to the diversity obtained from met-mouse. Thus, the met-lpr mouse is useful to produce an immunological repertoire having an enhanced diversity of antibodies and antibody genes.

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by the cell lines and plasmids deposited, since the deposited embodiment is intended as a single illustration of one aspect of the invention and any cell lines or plasmid vectors that are functionally equivalent are within the scope of this invention. The deposit of material does not constitute an admission that the written description herein contained is inadequate to enable the practice of any aspect of the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustration that it represents. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: The Scripps Research Institute

(B) STREET: 10666 North Torrey Pines Road

(C) CITY: La Jolla

(D) STATE: CA

(E) COUNTRY: USA

(F) POSTAL CODE (ZIP) : 92037

(G) TELEPHONE: 619-554-2937 (H) TELEFAX: 619-554-6312

(ii) TITLE OF INVENTION: TRANSGENIC ANIMALS HAVING AN ENGINEERED IMMUNE RESPONSE

(iii) NUMBER OF SEQUENCES: 20

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO)

(v) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT US 94/

(B) FILING DATE: 29-APR-1994

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/056,365

(B) FILING DATE: 30-APR-1993

(vii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Fitting, Thomas

(B) REGISTRATION NUMBER: 34,163

(C) REFERENCE/DOCKET NUMBER: TSRI331.0PC

(viii) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (619) 554-2937

(B) TELEFAX: (619) 554-6312

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 50 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: AGATTGGGTC ATTACGGCCG TCGACGGATG AGCTCAATGT TCCCATCAGC 50

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 30 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: GAGGCCGAGC TCGTTATGAC TCAGACACCA 30

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 54 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GGATACAGTT GGTGCAGCAT CAGCCCGTTT GGTGCCTCCA CCGAACGTCC ACGG 54 (2) INFORMATION FOR SEQ ID N0:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 54 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4: CCGTGGACGT TCGGTGGAGG CACCAAACGG GCTGATGCTG CACCAACTGT ATCC 54

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: GCGCCGTCTA GAATTAACAC TCATTCCTGT TGA 33

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO ( iv) ANTI - SENSE : NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: ATGCATCAGA CCAGCATGGG C 21

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CACTCTGACC ATCAGCAGTG TGCA 24

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: GCGATGGTGA CTGCGTTGGA GGC 23

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GAGCTCCAGA TGACCCAGTC T 21

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: ATAAATCCCA AAATCTTCAG ACTGCAGGC 29

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 63 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GAAGATTTTG GGATTTATNN SNNSNNSNNS NNSNNSNNSN NSNNSACGTT CGGTACTGGG 60 ACC 63

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE-: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: TCTAGAATTA ACACTCATTC C 21

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY linear

(ii) MOLECULE TYPE protein

(v) FRAGMENT TYPE internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Pro Asn Ser Leu Arg Trp Pro Ser Cys 1 5

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Ser Phe Thr Phe Asp His Ala Gly Leu Met .1 5 10

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY linear

(ii) MOLECULE TYPE protein

(v) FRAGMENT TYPE internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

His Cys Trp Val Leu Gly Pro Ser Leu 1 5

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Arg Val Asp Arg Thr His Ala Pro Asp 1 5

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9 amino acids (B) TYPE: amino acid

(D) TOPOLOGY linear

(ii) MOLECULE TYPE protein

(v) FRAGMENT TYPE internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

Gly Asp Asn Leu Tyr Gly Ser Leu Leu 1 5

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Ala Gly Ala Glu His Ala Asn Gin Ser 1 5

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Leu Trp Thr Arg Leu Met Val Ala Leu 1 5

(2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(v) FRAGMENT TYPE: internal

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

Ser Gly Lys Ile Tyr Phe Arg Met Glu 1 5

Claims

What is claimed is:

1. A transgenic animal having somatic and germ cells that comprise an exogenous exon expressable in antibody-producing cells of said animal, said exon coding for an immunoglobulin V region capable of forming a coordination complex with a metal cation.

2. The transgenic animal of claim 1 wherein said immunoglobulin V region comprises three contact amino acid residues that define a metal cation binding site.

3. The transgenic animal of claim 2 wherein said V region is a light chain V region having a LI region and a L3 region and said three contact amino acid residues are located at any three of the four amino acid residue positions 32, 34, 89, and 91.

4. The transgenic animal of claim 2 wherein said contact amino acid residues are selected from the group of amino acid residues consisting of histidine, cysteine, methionine, aspartic acid, and glutamic acid.

5. The transgenic animal of claim 4 wherein said metal cation is selected from the group consisting of Cu(II) , Zn(II) , Ni(II), Co(II) and Cd(II).

6. The transgenic animal of claim 2 wherein said contact amino acid residues are histidine at positions 34, 89 and 91.

7. The transgenic animal of claim 1 wherein said animal is a mouse.

8. The transgenic animal of claim 1 wherein said animal is characterized by a phenotype of enhanced diversity of immune-responsiveness.

9. The transgenic animal of claim 8 characterized by a deficiency in the Fas antigen gene producing said enhanced immune responsiveness phenotype.

10. The transgenic animal of claim 9 wherein said animal is a mouse having a lymphoproliferation (lpr) gene mutation.

11. The transgenic animal of claim 8 wherein said animal is a mouse further comprising a bcl-2 gene producing said enhanced immune responsiveness phenotype.

12. The method of claim 1 wherein said immunoglobulin V region is present in the form of a pre-rearranged immunoglobulin gene that codes for a V region spliced to a C region.

13. A method of producing a transgenic mouse of claim 7 which comprises: a) providing an exon expressable in antibody-producing cells of a mouse wherein said exon codes for an immunoglobulin V region capable of forming a coordination complex with a metal cation, b) introducing said exon into an embryo of said mouse, c) transplanting said embryo into a pseudopregnant mouse, and d) allowing said embryo to develop to term.

14. A method of producing an antibody molecule having a preselected activity, which method comprises: a) providing an exon expressable in antibody-producing cells of a mouse wherein said exon codes for an immunoglobulin V region capable of forming a coordination complex with a metal cation; b) introducing said exon into an embryo of said mouse; c) transplanting said embryo into a pseudopregnant mouse; d) allowing said embryo to develop to term and producing a transgenic mouse which is capable of expressing said exon; e) immunizing said transgenic mouse with a preselected immunogen to induce an immune response that includes antibody molecules immunoreactive with said immunogen, said antibody molecules comprising said immunoglobulin V region having said metal cation binding site; and f) harvesting said antibody molecules formed in step (e) from said transgenic mouse.

15. A method of producing an antibody molecule having a preselected activity, which method comprises: a) providing a transgenic animal having somatic and germ cells that comprise an exogenous exon expressable in antibody-producing cells of said animal, said exon coding for an immunoglobulin V region capable of forming a coordination complex with a metal cation; b) harvesting genes coding for immunoglobulin heavy and/or light chain polypeptides from antibody-producing cells of said animal; c) expressing said harvested genes in an expression vector capable of expressing said harvested genes and producing an antibody molecule; and d) collecting an antibody having the capacity to bind a metal cation from said produced antibody molecules.

16. The method of claim 15 wherein said transgenic animal is immunized with a immunogen at a time period prior to harvesting said genes sufficient for said immunogen to induce an immune response in said animal.