HK1064708B - Transgenic transchromosomal rodents for making human antibodies - Google Patents

Description

Transgenic transchromosomal rodents for production of human antibodies

Cross Reference to Related Applications

This application claims priority from united states provisional application No. 60/250,340, filed on 30/11/2000, the disclosure of which is incorporated herein by reference in its entirety.

Technical Field

The present invention belongs to the field of transgenic animal, molecular immunology and medical technology.

Background

Antibodies represent a class of therapeutic molecules for a number of different applications including transplantation, cardiovascular disease, infectious disease, cancer and autoimmune disease (Goldenberg, M., 1999, Clin. Ther.21: 309-. The development of hybridoma technology has enabled the isolation of rodent monoclonal antibodies (also known as MAbs) as candidate therapeutic molecules (Kohler, G., and Milstein, C., 1975, Nature 256: 495-. However, early studies involving non-human monoclonal antibodies for in vivo human therapy demonstrated that human anti-mouse antibodies (HA)MA) responses may limit the utility of such drugs (Schroff, r. et al, 1985, cancer res.45, 879-; shawler, d, et al, 1985, j.immunol.135: 1530-1535). Thus, it is recognized that there is a need to reduce the immunogenicity of therapeutic antibodies. Recombinant DNA techniques have been used to reduce the immunogenicity of non-human antibodies (Boulianne, G. et al, 1984, Nature 312, 643. 646; Morrison, S. et al, 1984, Proc. Natl. Acad. Sci. U.S.A.81: 6851. 6855; Riechmann, L. et al, 1988, Nature 332: 323. 327; Jones, P. et al, 1986, Nature 321: 522. 525; Queen, C. et al, 1989; Proc. Natl. Acad. Sci. U.S.A.86: 10029. 10033). However, it is also recognized that fully human monoclonal antibodies are a potential source of low immunogenicity therapeutics for the treatment of human diseases (Little, M. et al, 2000, immunological. today 21: 364-70). The use of transgenic mice bearing a human immunoglobulin (Ig) locus in germline configuration allows for the isolation of high affinity fully human monoclonal antibodies against a variety of targets including human self-antigens normally tolerated by the human immune system (Lonberg, N. et al, 1994, Nature 368: 856-9; Green, L. et al, 1994, Nature Genet.7: 13-21; Green, L. and Jakobovits, 1998, exp. Med.188: 483-95; Lonberg, N and Huszar, D.1995, int.Rev. Immunol.13: 65-93; Bruggemann, M. et al, 1991, Eur. J.Immunol.21: 1323; Fishwild, D.e., Immunol.21, Nat. Biotechnol.14: 845, Mennz et al, 1997, J.Immunol.156: 19; Nature J.35, J.1997, J.Immunol.35; Nature J.35, J.35; Eur. J.35, J.Immunol.11, J.35; C.35, J.J.Immunol.11, J.35; C.11, J.35, J.E.E.E.23, J.E.E.E.J.E.E.23, J.E.J.E.J.E.E.E.E.E.E.J.J.J.E.D.D.11, J.11, J.E.11, J.E.E.E.E.E. Human antibodies are based on light (kappa and lambda) and heavy (IgA) chains₁、IgA₂、IgD、IgE、IgG₁、IgG₂、IgG₃、IgG₄And IgM) into a variety of different classes. These different categories may provide different therapeutic applications. For example, different heavy chain isotypes have different interactions with complement and Fc-based receptors with cells. Some of the heavy chain classes (IgM and IgA) may also form multimers, thus increasing the valency of the Fc and V region interactions. Thus, it is desirable to have a single human monomer that produces all isoformsPlatform for cloning antibody. However, the large size of the human Ig locus (1-2Mb) is a major obstacle to the introduction of the complete locus into transgenic mice to reconstitute a fully diverse human antibody repertoire, since cloning of megabase-sized DNA fragments containing the complete human Ig locus is difficult even when yeast artificial chromosomes are employed. Recently, a new approach using the human chromosome itself as a gene transfer vector has facilitated the transfer of the complete IgH and Ig kappa loci into transgenic mice without the need to clone DNA fragments into artificial DNA vectors (Tomizuka, K.et al 1997, Nature Genet.16: 133-727; Tomizuka, K.et al 2000, Proc. Natl.Acad.Sci.97: 722-727). Tomizuka et al (Tomizuka, K. et al, 2000, Proc. Natl. Acad. Sci. U.S.A.97: 722-727) demonstrated the introduction of two heritable human chromosome fragments (hCF), one containing the immunoglobulin (Ig) heavy chain locus (IgH,. about.1.5 Mb) and the other containing the kappa light chain locus (Ig kappa,. about.2 Mb), into transgenic mouse strains whose endogenous IgH and Ig kappa loci are inactivated. In the resulting double chromosome (Tc)/double knock-out (KO) mice, a substantial percentage of somatic cells retained both hcfs, with high levels of expression of human Ig heavy and kappa light chains in the absence of mouse heavy and kappa light chains, indicating rescue in the absence of Ig production. In addition, the serum expression profiles of the 4 human Ig γ subclasses resemble those observed in humans. The transgenic mice produce antigen-specific human antibody responses upon immunization with Human Serum Albumin (HSA), and HSA-specific human monoclonal antibodies of various isotypes are obtained from these mice. Studies by Tomizuka et al, supra, also demonstrated the instability of hCHr.2-derived hCF (hCF (2-W23)) containing the Ig kappa locus in mice. The observed instability of the kappa transchromosome may be a barrier to optimal expression of human kappa light chains and production of human kappa positive hybridomas. Indeed, the 2/3 anti-HSA hybridoma from the double Tc/KO mouse was mouse lambda positive (m lambda⁺) And most (83%) IgG/m.lambda.hybridomas were found to have lost hCF (2-W23). Thus, there is a need for transgenic animals that retain the properties conferred by the transchromosomes described by Tomizuka et al (supra), particularly those that express essentially the entire repertoire of human heavy chain isotypes and that also exhibit the introduced humanAnimals with improved sequence stability, allowing for increased efficiency in obtaining fully human antibodies.

Summary of The Invention

The present invention provides a transgenic non-human mammal comprising two human immunoglobulin loci, wherein one of the two human immunoglobulin loci is a human heavy chain locus and the other locus is a human light chain locus; and wherein only one of said loci is of a transchromosome (transchromosome). In certain transgenic non-human mammals, the transchromosome is autonomous. In some transgenic non-human mammals, the transchromosome comprises a fragment of human chromosome 14. In certain transgenic non-human mammals, the human light chain locus is linked to an endogenous mammalian chromosome. In certain transgenic non-human mammals, the human heavy chain locus is transchromosomal and the human light chain locus is linked to an endogenous mammalian chromosome. In some such transgenic non-human mammals, at least a portion of the human light chain locus is cloned into a YAC vector. In certain transgenic non-human mammals, the human heavy chain locus is contained in hCF (SC20) and the human light chain locus is contained in the human kappa light chain locus transgene KCo 5. In certain transgenic non-human mammals, the human light chain locus is the locus of a transchromosome, while the human heavy chain locus is linked to an endogenous mammalian chromosome. In certain transgenic non-human mammals, the transgenic non-human mammal is a mouse. In the transgenic non-human mammal, the endogenous mammalian heavy chain locus and at least one mammalian light chain locus are inactivated. In some such transgenic non-human mammals, the endogenous mammalian heavy chain locus and the kappa light chain locus are inactivated.

In another aspect, the transgenic non-human mammal further comprises a genetic mutation, wherein the mutation enhances the immune response to the autoantigen. In certain transgenic non-human mammals, the mutation is an inactivation of the Fc- γ RIIB gene.

The invention also provides a method for generating a plurality of B cells expressing human antibody sequences, the method comprising: providing a transgenic non-human mammal comprising two human immunoglobulin loci, wherein one of the two human immunoglobulin loci is a human heavy chain locus and the other locus is a human light chain locus; and wherein only one of said loci is transchromosomal; and immunizing the transgenic non-human mammal to produce a plurality of B cells expressing human antibody sequences. In some such methods, the transchromosome is a fragment of human chromosome 14. In some such methods, the human transchromosome is the human chromosome fragment SC20(hCF (SC 20)). Some such methods further comprise collecting a plurality of B cells expressing human antibody expression sequences. Some such methods further comprise fusing the plurality of B cells with immortalized cells to form a hybridoma. Other such methods further comprise collecting the human antibody sequences from the hybridoma. In some such methods, the human antibody sequence is purified. Some such methods further comprise collecting sequences encoding human antibodies. In some such methods, the sequence encoding the human antibody is full-length. In certain methods, the sequence encoding the human antibody is expressed in transfected cells. In some such methods, the human light chain locus comprises unrearranged sequences from a native human kappa light chain locus. In some such methods, the human kappa light chain locus is the inserted KCo5 transgene. In some such methods, the plurality of B cells comprises at least a first B cell encoding an antibody having a first isotype selected from the group consisting of: IgA, IgD, IgE, IgG and IgM. In certain methods, the IgA isotype is IgA₁Or IgA₂. In certain methods, the IgG isotype is IgG₁、IgG₂、IgG₃Or IgG₄. In some such methods, the plurality of B cells comprises at least a second B cell encoding an antibody having a second isotype selected from IgA, IgD, IgE, IgG, and IgM that is different from the first isotype. In some methods, the plurality of B is fineThe cells comprise at least 5B cells, each B cell encoding an antibody of a different isotype, wherein the isotypes of antibodies are IgA, IgD, IgE, IgG and IgM, respectively. In another aspect, the transgenic non-human mammal further comprises a genetic mutation, wherein the mutation enhances the immune response to the autoantigen. In some such methods, the mutation is inactivation of the Fc- γ RIIB gene.

The present invention also provides a method for producing a human sequence antibody that binds to a predetermined antigen, the method comprising the steps of: immunizing a transgenic non-human mammal with a predetermined antigen, wherein said transgenic non-human mammal comprises two human immunoglobulin loci, wherein one of said two human immunoglobulin loci is a human heavy chain locus and the other is a human light chain locus; and wherein only one of said loci is transchromosomal; and collecting the human sequence antibody from the immunized non-human mammal. In another aspect, the transgenic non-human mammal further comprises a genetic mutation, wherein the mutation enhances the immune response to the autoantigen. In some such methods, the mutation is inactivation of the Fc- γ RIIB gene.

The human sequence antibodies of the invention may include various antibody isotypes or mixtures thereof, e.g., IgG₁、IgG₂、IgG₃、IgG₄、IgM、IgA₁、IgA₂IgD and IgE. The human sequence antibody may be full-length (e.g., IgG)₁、IgG₄、IgA₁Or IgA₂Antibodies), or may include only antigen-binding portions (e.g., Fab, F (ab') 2, Fv, or Fd fragments). Certain human sequence antibodies are recombinant human sequence antibodies. The human sequence antibodies of the invention can typically be raised to a predetermined antigen of at least 10⁸M^-1、10⁹M^-1、10¹⁰M^-1、10¹¹M^-1And 10¹²M^-1Equilibrium association constant (K) of_a) And (4) combining. Certain human sequence antibodies of the invention are monoclonal antibodies. Certain human sequence antibodies of the invention are antigen-specific.

The present invention also provides a method of producing an antigen-specific hybridoma that secretes a human sequence antibody, the method comprising: immunizing the transgenic non-human mammal with a predetermined antigen, wherein the transgenic non-human mammal comprises two human immunoglobulin loci, wherein one of the two human immunoglobulin loci is a human heavy chain locus and the other locus is a human light chain locus; and wherein only one of said loci is transchromosomal; fusing lymphocytes from the transgenic non-human mammal with immortalized cells to form hybridoma cells; and determining binding of the antibody produced by the hybridoma cells to the predetermined antigen. In some such methods, more than 50% of the antigen-specific hybridoma clones secrete antibodies with human heavy and light chains. In another aspect, the transgenic non-human mammal further comprises a genetic mutation, wherein the mutation enhances the immune response to the autoantigen. In some such methods, the mutation is inactivation of the Fc- γ RIIB gene.

The present invention also provides a method of producing a human sequence antibody that binds to a predetermined antigen, the method comprising the steps of: immunizing a transgenic non-human mammal with a predetermined antigen, wherein the transgenic non-human mammal comprises two human immunoglobulin loci, wherein one of the two human immunoglobulin loci is a human heavy chain locus and the other locus is a human light chain locus, wherein only one locus is transchromosomal; and screening the resulting hybridoma cells for the presence of antigen-reactive antibodies. In some such methods, the hybridoma cells are subcloned with an efficiency of more than 20%. In certain such methods, the antigen-reactive antibody is secreted by the hybridoma in culture. In some such methods, the antigen-reactive antibody is substantially pure. In certain methods, the substantially pure antibody is formulated for therapeutic use. In another aspect, the transgenic non-human mammal further comprises a genetic mutation, wherein the mutation enhances the immune response to the autoantigen. In some such methods, the mutation is inactivation of the Fc- γ RIIB gene.

The present invention also provides a method of producing a rearranged immunoglobulin sequence, the method comprising: providing a transgenic non-human mammal, wherein the transgenic non-human mammal comprises two human immunoglobulin loci, wherein one of the two human immunoglobulin loci is a human heavy chain locus and the other locus is a human light chain locus, wherein only one locus is transchromosomal; and obtaining the rearranged human immunoglobulin sequence from the transgenic non-human mammal. In certain methods, the obtaining step comprises collecting B lymphocytes containing the rearranged human immunoglobulin sequences from the transgenic non-human mammal. In such a method, the obtaining step comprises isolating and amplifying mRNA from the B lymphocytes to produce cDNA. Some such methods further comprise isolating and amplifying heavy and light chain variable region sequences from the cDNA. The invention also provides isolated nucleic acids encoding these amplified heavy chain variable region sequences from the cDNA. The invention also provides an isolated nucleic acid encoding the amplified light chain variable region sequence obtained from the cDNA. In another aspect, the transgenic non-human mammal further comprises a genetic mutation, wherein the mutation enhances the immune response to the autoantigen. In some such methods, the mutation is inactivation of the Fc- γ RIIB gene.

In another aspect, the invention provides a nucleic acid molecule of the invention encoding the human sequence antibody, or antigen-binding portion. Thus, recombinant expression vectors comprising nucleic acids encoding the antibodies of the invention and host cells transfected with such vectors (or progeny of such host cells) are also encompassed by the invention, as are methods for producing the antibodies of the invention by culturing such host cells. Certain such methods comprise culturing the host cell under conditions that cause the nucleic acid to be expressed; and recovering the nucleic acid from the cultured host cell or the culture medium thereof. Some host cells are eukaryotic cells. Some such expression vectors comprise nucleic acids encoding the heavy and light chain variable region sequences of the present invention operably linked to regulatory sequences that control expression of the nucleic acids in a host cell.

The present invention also provides a method of generating a human antibody display library, the method comprising: introducing an immunogen into the transgenic non-human mammal, wherein the transgenic non-human mammal comprises two human immunoglobulin loci, wherein one of the two human immunoglobulin loci is a human heavy chain locus and the other locus is a human light chain locus, wherein only one locus is transchromosomal; isolating a population of nucleic acids encoding human antibody chains from lymphocytes of the transgenic non-human mammal; and generating a display packaging library displaying the antibody chains, wherein one library member comprises nucleic acid encoding an antibody chain displayed by the packaging. In certain such methods, the transgenic non-human mammal lacks a detectable potency against the immunogen when subjected to the isolating step. In some such methods, the immunogen is a nucleic acid. In certain such methods, the nucleic acid encodes a membrane-bound receptor. In another aspect, the transgenic non-human mammal further comprises a genetic mutation, wherein the mutation enhances the immune response to the autoantigen. In some such methods, the mutation is inactivation of the Fc- γ RIIB gene.

The present invention also provides a method of producing a human sequence antibody or fragment thereof that binds to a predetermined antigen, the method comprising the steps of: immunizing a transgenic non-human mammal with a predetermined antigen, wherein the transgenic non-human mammal comprises two human immunoglobulin loci, wherein one of the two human immunoglobulin loci is a human heavy chain locus and the other locus is a human light chain locus, wherein only one locus is transchromosomal; collecting antibody V region sequences from the immunized transgenic non-human mammal; cloning the collected V regions into a DNA vector to generate an expression library; allowing the library to express to identify V region sequences encoding antibodies or fragments thereof that bind to the predetermined antigen. In another aspect, the transgenic non-human mammal further comprises a genetic mutation, wherein the mutation enhances the immune response to the autoantigen. In some such methods, the mutation is inactivation of the Fc- γ RIIB gene.

The present invention also provides a method of producing a human sequence antibody or fragment thereof that binds to a predetermined antigen, the method comprising the steps of: immunizing a transgenic non-human mammal with a predetermined antigen, wherein the transgenic non-human mammal comprises at least two human immunoglobulin loci, wherein one of the human immunoglobulin loci is a human heavy chain locus and the other locus is a human light chain locus; and wherein at least one locus is transchromosomal; isolating cDNA encoding at least one human antibody V region from the B cells of the immunized transgenic non-human mammal or from a hybridoma produced by fusing the B cells with immortalized cells; cloning the cDNA into an expression vector; introducing the vector into a host cell; culturing the host cell; and collecting the human sequence antibody or fragment thereof from the host cell or the culture medium thereof. In some such methods, the isolating step is performed by PCR. In some such methods, the isolating step is performed by screening a cDNA library with at least one DNA probe. In some such methods, the isolating step is performed by phage display library screening. In some such methods, the cDNA encodes a full-length human antibody sequence. In certain methods, the collected human sequence antibody isotypes are different from the isotypes of the antibody producing cells of the immunized transgenic non-human mammal. In another aspect, the transgenic non-human mammal further comprises a genetic mutation, wherein the mutation enhances the immune response to the autoantigen. In some such methods, the mutation is inactivation of the Fc- γ RIIB gene.

The present invention also provides a method of improving the stability of a transgenic mouse hybridoma cell expressing a human antibody reactive with a predetermined antigen, the method comprising: mating together a first mouse comprising a first human immunoglobulin locus on a transchromosome and a second mouse comprising a second human immunoglobulin locus inserted into an endogenous mouse chromosome; obtaining a third mouse from the breeding, the third mouse comprising both the first human immunoglobulin locus and the second human immunoglobulin locus; immunizing the third mouse or progeny thereof with the predetermined antigen; collecting B cells from the immunized mouse; and fusing the B cell with an immortalized cell to obtain a hybridoma cell expressing a human antibody reactive with the predetermined antigen. Some such methods further comprise: culturing the hybridoma cells in a culture medium; testing the culture medium to identify the presence of hybridoma cells expressing a human antibody reactive with the predetermined antigen; diluting the hybridoma cells; and culturing the diluted hybridoma cells to obtain a clonal cell line expressing a human monoclonal antibody reactive with the predetermined antigen. In some such methods, the clonal cell line is obtained from at least 50% of the identified hybridoma cells.

In another aspect, the invention provides a mouse hybridoma cell that secretes a human sequence antibody of the IgA isotype of at least 10¹⁰M^-1Equilibrium association constant (K) of_a) Binding to a specific antigen.

In another aspect, the invention provides an antibody having the IgA isotype human sequence, said antibody having at least 10¹⁰M^-1Equilibrium association constant (K) of_a) Binding to a specific antigen.

Brief Description of Drawings

FIG. 1. design of C.mu.targeting vector. A) Mouse genomic DNA of C.mu.region. B) mu.C targeting vector. C) Mouse genomic DNA homologously recombined with the C.mu.targeting vector.

FIG. 2 serum concentrations of human Ig. mu.g, gamma, kappa and mouse lambda chains in dual TC/KO and hybrid mice.

FIG. 3 serum concentration of anti-CD 4 human Ig gamma at day 34 in immunized twin TC/KO and hybrid mice.

FIG. 4 serum concentration of anti-CD 4 human Ig kappa at day 34 in immunized twin TC/KO and hybrid mice.

Figure 5 time course of anti-CD 4 human Ig γ response at day 34 in the dual TC/KO and hybrid mice showing the highest serum titers in each group (N ═ 5).

Figure 6 time course of anti-CD 4 human Ig κ response at day 34 in the dual TC/KO and hybrid mice showing the highest serum titers in each group (N ═ 5).

FIG. 7 growth curves of KM2-3 hybridoma cells and production of anti-CD 4 human monoclonal antibodies.

FIG. 8 serum concentrations of anti-GCSF human Ig gamma at day 34 in immunized twin TC/KO and hybrid mice.

FIG. 9 serum concentrations of anti-GCSF human Ig kappa at day 34 in immunized twin TC/KO and hybrid mice.

Figure 10 time course of anti-GCSF human Ig γ response at day 34 in the dual TC/KO and hybrid mice showing the highest serum titers in each group (N ═ 5).

Figure 11 time course of anti-GCSF human Ig κ response at day 34 for the dual TC/KO and hybrid mice showing the highest serum titers in each group (N ═ 5).

Figure 12 dose response curves of blocking activity of anti-CTLA-4 human monoclonal antibodies.

Figure 13 inhibition of binding of CTLA-4 (biotin) to B7.2 cells with monoclonal antibodies.

FIG. 14 enhanced response to bovine C-IV in hybrid (Fc) mouse serum.

Detailed Description

The term "transchromosome" refers to a chromosome or fragment thereof that can be transferred into a non-human mammalian cell. An exemplary cell into which the transchromosome is introduced is an ES cell. The transchromosome may comprise a selectable marker and may be derived from a different species of non-human mammal. The transchromosome may comprise a portion of a human chromosome. The term "transchromosomic" or "transchromosome" refers to "maintenance of a transchromosome" or "of a transchromosome".

The human sequence antibodies of the invention can be produced in transgenic non-human mammals, such as transgenic mice, which are capable of producing multiple human (e.g., monoclonal or polyclonal) antibody isotypes (e.g., IgM, IgD, IgG, IgA, and/or IgE) against a variety of antigens by undergoing antibody V-D-J recombination and isotype switching for non-IgM/non-IgD. Accordingly, various aspects of the invention include antibodies and antibody fragments and pharmaceutical compositions thereof, as well as transgenic non-human mammals, B cells and hybridomas for making such monoclonal antibodies.

Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably and refer to mammals such as human patients and non-human primates, and laboratory animals such as rabbits, rats, and mice, among others.

The term "treating" includes administering a compound or drug of the invention to prevent or delay the onset of symptoms, complications, or biochemical indicators of a disease, alleviate symptoms, or arrest or inhibit further development of a disease, condition, or disorder (e.g., an autoimmune disease). Treatment may be prophylactic (to prevent or delay the onset of disease, or to prevent the manifestation of clinical or subclinical symptoms thereof), or therapeutic inhibition or alleviation of symptoms following disease manifestation.

In general, the phrase "well tolerated" means that there are no adverse changes in health conditions that occur as a result of the treatment and that may affect treatment decisions.

The term "lymphocyte" as used herein has the normal meaning in the art and refers to any mononuclear, non-phagocytic leukocyte present in blood, lymph and lymphoid tissues, i.e., B lymphocytes and T lymphocytes.

THE phrase "subpopulation of T lymphocytes" or "T cell subpopulation" refers to T lymphocytes or T cells characterized by expression of a particular cell surface marker (see Barclay, a.n. et al (eds.), 1997, tee lekocyt ANTIGEN FACTS BOOK, 2nd.

The terms "cytotoxic T lymphocyte-associated antigen-4", "CTLA 4", "CTLA-4 antigen" and "CD 152" (see, e.g., Murata, 1999, am. J. Pathol. 155: 453-.

The complete cDNA sequence of human CTLA-4 is Genbank accession number L15006. The region of amino acids 1-37 is a leader peptide; 38-161 are extracellular V-like domains; 162-187 is a transmembrane domain; and 188-223 is a cytoplasmic domain. Variants of the nucleotide sequence have been reported, including the G → A transition at position 49, the C → T transition at position 272 and the A → G transition at position 439. The complete DNA sequence of mouse CTLA-4 has the EMBL accession number X05719(Brunet et al, 1987, Nature 328: 267-270). The region of amino acids 1-35 is a leader peptide.

The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Epitopes usually consist of chemically active surface groups (surface groups) of molecules, such as amino acid or sugar side chains, usually with specific three-dimensional structural characteristics and specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that in the presence of denaturing solvents, binding to the former is lost, but not to the latter.

An intact "antibody" comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains-CH 1, CH2, and CH 3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain-CL. The VH and VL regions may be further subdivided into hypervariable regions (known as Complementarity Determining Regions (CDRs)) interspersed with more conserved regions (known as Framework Regions (FRs), each VH and VL consisting of three CDRs and four FRs, arranged from amino to carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen the constant regions of the antibody may mediate binding of an immunoglobulin to host tissues or factors including various cells of the immune system (e.g., effector cells) via cellular receptors such as Fc receptors (e.g., Fc γ RI, Fc γ RIIa, Fc γ RIIb, Fc γ RIII and FcR η) and the first component of the classical complement system (Clq), examples of antigen binding moieties include (i) Fab fragments, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; (ii) a F (ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) (iv) Fv fragments consisting of the VL and VH domains of one arm of an antibody, (v) dAb fragments (Ward et al, 1989 Nature 341: 544-546) consisting of one VH domain; and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they may be joined recombinantly using a synthetic linker which enables them to be a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al, 1988, Science 242: 423-. Such single chain antibodies are included within the scope of the term "antibody". Fragments may be prepared by recombinant techniques or by enzymatic or chemical cleavage of intact antibodies.

The term "human sequence antibody" includes antibodies having variable and constant regions (if any) derived from human immunoglobulin sequences. The human sequence antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, the term "human sequence antibody" as used herein does not include antibodies in which the complete CDR sequences sufficient to confer antigen specificity and derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human framework sequences (i.e., humanized antibodies).

The term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody having variable and constant regions (if any) derived from human germline immunoglobulin sequences that exhibits a single binding specificity. In one embodiment, the human monoclonal antibodies are produced by a hybridoma comprising a B cell obtained from a transgenic non-human mammal (e.g., a transgenic mouse) having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

The term "monoclonal antibody" refers to a preparation of at least two antibodies directed against one antigen. Typically, different antibodies bind different epitopes.

The term "oligoclonal antibody" refers to a preparation of 3-100 different antibodies to an antigen. Typically, the antibody in such a preparation binds to a number of different epitopes.

The term "polyclonal antibody" refers to preparations of more than one (two or more) different antibodies directed against an antigen. Typically, such a preparation comprises antibodies that bind to a number of different epitopes.

The present invention provides human sequence antibodies against a variety of antigens, including human antibodies against human CTLA-4, human G-CSF, human HSA, human CD4, and human EGFR. The human antibodies of the invention include those that block or antagonize the receptor on the cell surface (e.g.Human CTLA-4 receptor and human CD4 co-receptor). Some of these antibodies can bind to an epitope on human CTLA-4 to inhibit CTLA-4 interaction with the human B7 counter receptor. Also certain of the antibodies can bind to an epitope on human CD4 to inhibit CD4 interaction with human MHC class II. Because the interaction of human CTLA-4 with human B7 transduces a signal that results in the inactivation of T cells bearing human CTLA-4 receptors, antagonism of the interaction is effective to induce, increase or prolong activation of T cells bearing human CTLA-4 receptors, thereby prolonging or enhancing the immune response. By "blocking antibody" is meant an antibody having a ratio of binding sites for human CTLA-4 ligand of greater than 1: 1 and a concentration of antibody of greater than 10^-8M, an antibody that reduces binding of soluble human CTLA-4 to cells expressing human B7 ligand by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 99.9%.

The invention also provides human sequence IgA antibodies to a variety of human antigens. Exemplary IgA antibodies include CD4, G-CSF, CTLA-4, and EGFR. Because IgA antibodies can form dimers, such IgA antibodies can have improved crosslinking properties.

Other antibody preparations, sometimes referred to as multivalent preparations, bind to cell surface receptors such as human CTLA-4 in a manner that allows cross-linking of multiple human CTLA-4 receptors on the same cell.

Cross-linking can also be accomplished by combining soluble bivalent antibodies with different epitope specificities. These polyclonal antibody preparations comprise at least two pairs of heavy and light chains that bind to different epitopes on the antigen, such that signals can be transduced due to antibody-mediated cross-linking.

The term "recombinant human antibody" includes all human sequence antibodies of the invention made, expressed, constructed or isolated by recombinant methods, e.g., antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (described further below); antibodies expressed using recombinant expression vectors transfected into host cells, antibodies isolated from libraries of recombinant combinatorial human antibodies, or antibodies prepared, expressed, constructed, or isolated by any other method involving splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions (if any) derived from human germline immunoglobulin sequences. However, such antibodies can be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when using animals transgenic for human Ig sequences), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not normally exist in human antibody germline repertoires in vivo.

A "heterologous antibody" is defined for a transgenic non-human organism that produces such an antibody. The term refers to antibodies having amino acid sequences or encoding nucleic acid sequences corresponding to those present in organisms not including the transgenic non-human animal, typically obtained from a species other than the transgenic non-human animal.

"Heterohybrid antibody" refers to an antibody having a light chain and a heavy chain derived from different organisms. For example, an antibody to a human heavy chain that binds to a murine light chain is a heterohybrid antibody.

The term "substantially pure" or "isolated" refers to a species of interest (e.g., an antibody of the invention) that has been identified and separated and/or recovered from its natural environmental components to make it the predominant species present (i.e., it is more abundant in molar concentration than any other individual species in the composition); a "substantially pure" or "isolated" composition is also one in which the target species comprises at least about 50% (on a molar basis) of all macromolecular species present. The substantially pure or isolated composition may also comprise more than about 80-90% by weight of all macromolecular species present in the composition. An isolated target species (e.g., an antibody of the invention) can also be purified to substantial homogeneity (contaminant species are not detectable in the composition by conventional assays), wherein the composition consists essentially of derivatives of a single macromolecular species. For example, an isolated anti-human CTLA-4 antibody can be substantially free of other antibodies that do not bind to human CTLA-4 but bind to a different antigen. Furthermore, an isolated antibody that specifically binds to an epitope, isoform or variant of human CTLA-4 may have cross-reactivity with other relevant antigens, such as antigens from other species (e.g., CTLA-4 species homologs). Furthermore, the isolated antibodies of the invention are substantially free of other cellular material (e.g., non-immunoglobulin related proteins) and/or chemicals.

By "specifically binds" is meant that an antibody binds preferentially to a particular antigen relative to other, non-particular antigens. The phrase "specifically (or selectively) binds" to an antibody refers to determining the presence of a binding reaction of the protein in a heterogeneous population of proteins and other biologics. Typically, the antibody is administered in an amount of at least about 1X 10⁶M^-1Or 10⁷M^-1Or about 10⁸M^-1-10⁹M^-1Or about 10¹⁰M^-1-10¹¹M^-1Or higher association constant (K)_a) Binds with at least twice the affinity of said specific antigen than to non-specific antigens other than said specific antigen or closely related antigens (e.g. BSA, casein). The phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen". The predetermined antigen is an antigen that is selected prior to selecting an antibody that binds to the antigen.

The phrase "specifically binds," when referring to a peptide, refers to a peptide molecule having a moderate or high binding affinity that binds only to, or predominantly to, a target molecule. The phrase "specifically binds" refers to a binding reaction that determines the presence of a target protein in a heterogeneous population of proteins and other biological products. Thus, under the specified assay conditions, the specified binding moiety preferentially binds to the particular target protein and does not bind in a significant amount to other components present in the test sample. Specific binding to a target protein under such conditions may require a binding moiety selected for its specificity for a particular target antigen. A wide variety of assay formats can be used to select ligands that specifically react with a particular protein. For example, solid phase ELISA immunoassays, immunoprecipitations, Biacore, and western blots are used to identify peptides that specifically react with the antigen. Typically, a specific or selective reaction will be at least twice the background signal or noise, more typically 10 times above the background.

The term "high affinity" of an antibody refers to the equilibrium association constant (K)_a) At least about 10⁷M^-1At least about 10⁸M^-1At least about 10⁹M^-1At least about 10¹⁰M^-1At least about 10¹¹M^-1Or at least about 10¹²M^-1Or higher, e.g. up to 10¹³M^-1Or 10¹⁴M^-1Or higher. However, "high affinity" binding may vary for other antibody isotypes.

The term "K" as used herein_a"refers to the equilibrium association constant for a particular antibody-antigen interaction. The unit of this constant is 1/M.

The term "K" as used herein_d"refers to the equilibrium dissociation constant for a particular antibody-antigen interaction. The unit of this constant is M.

The term "K" as used herein_a"refers to the dynamic (kinetic) association constant of a particular antibody-antigen interaction. The unit of this constant is 1/Ms.

The term "k" as used herein_d"refers to the dynamic dissociation constant of a particular antibody-antigen interaction. The unit of this constant is 1/s.

"specific antibody-antigen interaction" refers to the test conditions under which equilibrium constants and kinetic constants are measured.

"isotype" refers to the class of antibodies encoded by the heavy chain constant region gene. The heavy chain is divided into gamma, mu, alpha,δ or ε, defining the isotype of the antibody as IgG, IgM, IgA, IgD and IgE, respectively. Additional structural variations identify IgG (e.g., IgG)₁、IgG₂、IgG₃And IgG₄) And IgA (e.g. IgA)₁And IgA₂) Different subtypes of (2).

"isotype switching" refers to the phenomenon whereby the class or isotype of an antibody changes from one Ig class to one of the other Ig classes.

"non-switching isotype" refers to the isotype class of heavy chains that are produced when isotype switching does not occur; the CH gene encoding the non-switching isoform is typically the first CH gene immediately downstream of the functionally rearranged VDJ gene. Isotype switching is classified as either classical or non-classical isotype switching. Classical isotype switching occurs through recombination events involving at least one switching sequence region in the transgene. Non-classical isotype switching may be achieved by, for example, human sigma_μSigma of man_μ(delta-related deletions) between the two. Alternative non-classical switching mechanisms, for example, where inter-transgene and/or inter-chromosome recombination may occur, accomplish isotype switching.

The term "switching sequences" refers to those DNA sequences which are responsible for switching recombination. The "switch donor" sequence is typically the μ switch region, located 5' (i.e., upstream) of the construct region that will be deleted during the switch recombination. The "switch receptor" region is located between the region of the construct to be deleted and the replacement constant region (e.g., γ, ε, etc.). Since there is no specific site at which recombination always occurs, the final gene sequence is often unpredictable depending on the construct.

"glycosylation pattern" is defined as the pattern of sugar units covalently linked to a protein, more specifically to an immunoglobulin. Given that one skilled in the art will recognize that the glycosylation pattern of the heterologous antibody is more similar to that in the transgenic non-human animal species than the species from which the transgenic CH gene is derived, the glycosylation pattern of the heterologous antibody can be described as being similar to that which normally occurs on antibodies produced in the transgenic non-human animal species.

The term "naturally-occurring" when used with respect to an object refers to the fact that the object may be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses), which can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory, is naturally-occurring.

The term "immunoglobulin locus" refers to a genetic element or a set of related genetic elements that contain information that B cells or B cell precursors can use to express immunoglobulin peptides. Such a peptide may be a heavy chain peptide, a light chain peptide, or a fusion of a heavy chain peptide and a light chain peptide. In the case of unrearranged loci, the genetic elements are assembled by B cell precursors to form genes encoding immunoglobulin peptides. For rearranged loci, the gene encoding the immunoglobulin peptide is contained in the locus.

The term "rearranged" refers to a configuration of a heavy or light chain immunoglobulin locus in which V segments are located immediately adjacent to D-J or J segments in a conformation encoding a substantially intact VH or VL domain, respectively. By comparison with germline DNA, rearranged immunoglobulin loci can be identified; the rearranged locus has at least one recombinant heptamer/nonamer homology element.

The term "unrearranged" or "germline configuration" in reference to a V segment refers to a configuration in which the V segment is immediately adjacent to a D segment or a J segment without recombination.

The term "nucleic acid" or "nucleic acid molecule" refers to a deoxyribonucleotide or ribonucleotide polymer in either single-or double-stranded form, and unless otherwise indicated, can include known analogs of natural nucleotides that can function in a manner similar to naturally occurring nucleotides.

The term "isolated nucleic acid" in relation to a nucleic acid encoding an antibody or antibody portion (e.g., VH, VL, CDR3) that binds to the antigen refers to a nucleic acid in which the nucleotide sequence encoding the antibody or antibody portion does not bind other nucleotide sequences encoding antibodies or antibody portions that bind to antigens other than, for example, CTLA-4, which other sequences may naturally flank the nucleic acid in human genomic DNA.

The term "substantially identical" in the context of two nucleic acids or polypeptides refers to two or more sequences or subsequences that have at least about 80%, about 90%, about 95% or more nucleotide or amino acid residue identity, as determined by sequence comparison and/or by visual inspection, when compared and aligned for maximum correspondence. Such "substantially identical" sequences are generally considered homologous. "substantial identity" may exist over a region of at least about 50 residues in length, over a region of at least about 100 residues, or over a region of at least 150 residues in the sequence, or over both full-length sequences to be compared. As described below, any two antibody sequences can be aligned in only one way by employing the numbering scheme of Kabat. Thus, for antibodies, the percent identity has a unique and well-defined meaning.

The amino acids from the mature heavy and light chain variable regions of immunoglobulins are designated Hx and Lx, respectively, where x is the number designating the amino acid position according to the protocol of Kabat, Sequences of Proteins of immunologicals Interest (National Institutes of Health, Bethesda, MD, 1987 and 1991). Kabat lists many amino acid sequences of each subgroup antibody and lists the most commonly occurring amino acid at each residue position in the subgroup, thereby generating a consensus sequence. Kabat uses a method of assigning a residue number to each amino acid in a listed sequence, which has become standard in the art. The Kabat protocol can be extended to other antibodies not included in its schema by aligning the antibody to one of the consensus sequences in Kabat with conserved amino acids as coordinates. Amino acids at equivalent positions in different antibodies can be readily identified using the Kabat numbering system. For example, the amino acid at position L50 of the human antibody occupies a position equivalent to the mouse antibody amino acid position L50. Likewise, nucleic acids encoding antibody chains are aligned when the amino acid sequences encoded by the corresponding nucleic acids are aligned according to the Kabat numbering convention (Kabat numbering convention). An alternative structural definition has been proposed by Chothia et al (Chothia, 1987J. mol. biol. 196: 901-917; Chothia et al, 1989, Nature 342: 878-883; and Chothia et al, J. mol. biol. 186: 651-663(1989), which are incorporated herein by reference for all purposes).

The phrase "selectively (or specifically) hybridizes" refers to the binding, duplexing, or hybridizing of a molecule to a particular nucleotide sequence under stringent hybridization conditions when the sequence is present in a complex mixture (e.g., total cell or library DNA or RNA) wherein at least about 10 times the background of the particular nucleotide sequence is detected. In one embodiment, a nucleic acid can be determined to be within the scope of the invention based on its ability to hybridize under stringent conditions to a nucleic acid that is otherwise determined to be within the scope of the invention (e.g., the exemplary sequences described herein).

The phrase "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but not to other sequences in significant amounts (about 10 times background hybridization of a positive signal (e.g., identification of a nucleic acid of the invention)). The stringency conditions depend on the sequence and differ from case to case. Longer sequences hybridize specifically at higher temperatures. Various guidelines for nucleic acid hybridization are found, for example, in Sambrook, MOLECULAR CLONING, in: a LABORATORY MANUAL (2NDED.), Vols.1-3, Cold Spring Harbor LABORATORY (1989); currentprotocols IN MOLECULAR BIOLOGY, Ausubel, John Wiley & Sons, Inc., New York (1997); laborary TECHNIQUES INBIOCHEMISTRY AND MOLECULAR BIOLOGY: HyBRIDIZATIONWITH NUCLEIC ACID PROBES, PART I.Theory and NUCLEIC ACID preparation, Tijssen, Elsevier, N.Y. (1993).

Generally, stringent conditions are selected to be about 5-10 ℃ below the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence under equilibrium conditions (when the target sequence is present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0M sodium ion, typically about 0.01-1.0M sodium ion concentration (or other salts), pH 7.0-8.3 and the temperature is at least about 30 ℃ for short probes (e.g., 10-50 nucleotides) and at least about 60 ℃ for long probes (e.g., more than 50 nucleotides). Stringent conditions may also be achieved by the addition of destabilizing agents such as formamide as described in Sambrook (described below). For high stringency hybridization, the positive signal is at least twice background, preferably 10 times background hybridization. Exemplary high stringency or stringency hybridization conditions include: 50% formamide, 5 XSSC and 1% SDS at 42 ℃ or 5 XSSC and 1% SDS at 65 ℃; washing was carried out in 0.2 XSSC and 0.1% SDS at 65 ℃. For selective or specific hybridization, a positive signal (e.g., identification of a nucleic acid of the invention) is about 10 times background hybridization. Stringent hybridization conditions used to identify nucleic acids within the scope of the invention include, for example, hybridization at 42 ℃ in a buffer comprising 50% formamide, 5 XSSC and 1% SDS, or hybridization at 65 ℃ in a buffer comprising 5 XSSC and 1% SDS, with wash conditions of 0.2 XSSC and 0.1% SDS, 65 ℃. In the present invention, genomic DNA or cDNA comprising a nucleic acid of the invention can be identified in standard southern blots under stringent conditions using the nucleic acid sequences disclosed herein. Other stringency conditions for such hybridization (to identify nucleic acids within the scope of the invention) are those which include hybridization in a buffer of 40% formamide, 1M NaCl, 1% SDS at 37 ℃.

However, the choice of hybridization format is not critical, and it is the stringency of the washing conditions that establish the conditions for determining whether a nucleic acid is within the scope of the invention. Washing conditions used to identify nucleic acids within the scope of the invention include, for example, a salt concentration of about 0.02M, a pH of 7, and a temperature of at least about 50 ℃ or about 55 ℃ to about 60 ℃; or a salt concentration of about 0.15M NaCl at 72 ℃ for about 15 minutes; or a salt concentration of 0.2 XSSC, a temperature of at least about 50 ℃ or about 55 ℃ to 60 ℃ for about 15 minutes to about 20 minutes; or; the hybridization complex was washed 2 times for 15 minutes at room temperature with a salt concentration of about 2 XSSC in a solution containing 0.1% SDS, and then 2 times for 15 minutes at 68 ℃ with 0.1 XSSC containing 0.1% SDS; or an equivalent condition. See Sambrook, Tijssen and Ausubel for a description of SSC buffers and equivalent conditions.

The term "sequence identity" refers to a measure of similarity between amino acid sequences or nucleotide sequences, which can be measured using methods known in the art, for example, as described below:

the terms "identical" or percent "identity" with respect to two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same, or that are the same, as measured by the following sequence comparison algorithm, or by manual sequence alignment and visual inspection, when compared and aligned for maximum correspondence over a comparison window or designated region, i.e., 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity.

The phrase "substantially identical" in reference to two nucleic acids or polypeptides refers to two or more sequences or subsequences that are at least 60%, typically at least 70%, preferably at least 80%, and most preferably at least 90% or at least 95% identical in nucleotide or amino acid residue, as measured by the following sequence comparison algorithm or by visual inspection, when compared and aligned for maximum correspondence. Preferably, there is substantial identity over a region of the sequence that is at least about 50 bases or residues in length, more preferably over a region of at least about 100 bases or residues, and most preferably the sequences are substantially identical over at least about 150 bases or residues. In a most preferred embodiment, the sequences are substantially identical over the entire coding region.

For sequence comparison, one sequence is typically used as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters may be used or parameters may be specified. Based on the program parameters, a sequence comparison algorithm calculates the percent sequence identity of the test sequence relative to the reference sequence. For sequence comparisons of nucleic acids and proteins, the BLAST and BLAST 2.0 algorithms, as well as the default parameters discussed below, can be used.

As used herein, a "comparison window" includes reference to a segment having any number of contiguous positions selected from 20-600, typically about 50-200, more typically about 100-150, wherein a sequence can be compared to a reference sequence having the same number of contiguous positions after optimal alignment of the two sequences. Methods of aligning sequences for comparison are well known in the art. An optimal sequence alignment for comparison can be performed, for example, by the following algorithm: the local homology algorithm by Smith and Waterman, 1981, adv.appl.math.2: 482) (ii) a Homology alignment algorithm by Needleman and Wunsch, 1970, j.mol.biol.48: 443; similarity search method of Pearson and Lipman, 1988, proc.natl.acad.sci.u.s.a.85: 2444; computer tools for these algorithms (FASTDB (intelligentics), BLAST (national Center for biological information), GAP in Wisconsin genetics software Package, BESTFIT, FASTA and TFASTA, genetics computer Group, 575 Science Dr., Madison, Wis.); or manual sequence alignment and visual inspection (see, e.g., Ausubel et al, 1987(1999Suppl.), Current Protocols in molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y.).

One preferred example of an algorithm suitable for determining sequence identity and percent sequence similarity is the FASTA algorithm, which is described in Pearson, w.r., and Lipman, d.j., 1988, proc.natl.acad.sci.u.s.a.85: 2444 as described herein. See also w.r. pearson, 1996, Methods enzymol.266: 227-258. Preferred parameters for calculating percent identity in the FASTA algorithm for DNA sequences are preferred, BL50 Matrix 15: -5, k-tuple 2; joining dependency is 40, optimization is 28; gap penalty-12, gap length penalty ═ 2; and width 16.

Another example of a preferred algorithm suitable for determining sequence identity and percent sequence similarity is the BLAST and BLAST 2.0 algorithms, respectively, described in Altschul et al, 1977, nuc. acids res.25: 3389 3402 and Altschul et al, 1990, J.mol.biol.215: 403-410. Percent sequence identity for nucleic acids and proteins of the invention is determined using BLAST and BLAST 2.0 and the parameters described herein. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http:// www.ncbi.nlm.nih.gov /). The algorithm involves first identifying high scoring sequence pairings (HSPs) by identifying short strings of length W that either match or satisfy some positive numerical minimum score T when aligned in a query sequence with a string of the same length in a database sequence. T is called the neighborhood word lowest score (Altschul et al, supra). These primary neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. String hits extend in both directions along each sequence, as long as the cumulative sequence alignment score can be increased. For nucleotide sequences, cumulative scores were calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is used to calculate cumulative scores. Extension of string hits in each direction terminates when one of the following conditions is encountered: the cumulative sequence alignment score is lower by an amount X than its maximum value obtained; the cumulative score value is zero or below zero due to the accumulation of one or more negative-scoring residue alignments; or to the end of either sequence. BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a default word length (W) of 11, an Expectation (E) of 10, M-5, N-4, and compares the two strands. For amino acid sequences, the BLASTP program uses a default word size (W) of 3, an expectation (E) of 10, a BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, proc. natl. acad. sci. u.s.a.89: 10915) sequence alignment (B) of 50, an expectation (E) of 10, M-5, N-4, and comparing the two strands.

The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A.90: 5873-. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P (N)), which provides an indication of the probability by which two nucleotide or amino acid sequences may be matched by chance. For example, a test nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

Another example of a useful algorithm is PILEUP. PILEUP creates a multiple sequence alignment from a group of related sequences using asymptotically paired sequence alignments to show the correlation and percent sequence identity. It also draws a tree of evolution or branching (dendogram) to show the clustering relationships used to create the sequence alignments. PILEUP was performed using Feng and Doolittle, 1987, j.mol.evol.35: 351-360 is a simplified method of the asymptotic sequence alignment method. The method used was similar to Higgins and Sharp, 1989, cabaos 5: 151-153. The program can align up to 300 sequences, each with a maximum length of 5,000 nucleotides or amino acids. The multiple sequence alignment begins with a pairwise alignment of the two most similar sequences, resulting in a set of two aligned sequences. The set of sequences is then aligned with the next most related sequence or the next set of aligned sequences. Two sets of sequences are aligned by a simple extension of the pairwise alignment of the two individual sequences. The final sequence alignment is accomplished by a series of asymptotically paired sequence alignments. The program is run by specifying the amino acid or nucleotide coordinates of the specific sequence and its regions of sequence comparison and specifying the program parameters. For example, using PILEUP, the following parameters are used: default gap weight (3.00), default gap length weight (0.10), and weighted end gap (weighted end gap) the reference sequence can be compared to other test sequences to determine percent sequence identity relationships. PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al, 1984, Nuc. acids Res.12: 387- > 395).

Another preferred example of an algorithm suitable for multiple DNA and amino acid sequence alignments is the CLUSTALW program (Thompson, J.D. et al, 1994, Nucl. acids. Res.22: 4673-4680). ClustalW performs multiple pairwise comparisons between sets of sequences and then assembles them into a multiple sequence alignment based on homology. Gap open and Gap extension penalties are 10 and 0.05, respectively. For amino acid sequence alignment, the BLOSUM algorithm can be used as a protein molecular weight matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. U.S.A.89: 10915-10919).

The nucleic acids of the invention are present in whole cells, in cell lysates, or in partially purified or substantially pure form. Nucleic acids are "isolated" or "rendered substantially pure" when they are purified from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, by standard techniques, including alkali/SDS treatment, CsCl banding (CsCl banding), column chromatography, agarose gel electrophoresis, and other methods well known in the art (see, for example, the methods of Sambrook, Tijssen, and Ausubel, discussed herein, incorporated by reference for all purposes). The nucleic acid sequences of the present invention and other nucleic acids, whether RNA, cDNA, genomic DNA, or hybrids thereof, used in the practice of the present invention can be isolated from a variety of sources, genetically engineered, amplified, and/or recombinantly expressed. Any recombinant expression system may be used, including, for example, yeast, insect or mammalian systems, in addition to bacteria. Alternatively, these nucleic acids can be chemically synthesized in vitro. Techniques for manipulation of nucleic acids, such as subcloning into expression vectors, labeling probes, sequencing, and hybridization, are described extensively in the scientific and patent literature, see, e.g., Sambrook, Tijssen, and Ausubel. Nucleic acids can be analyzed and quantified using any of a variety of general methods well known to those skilled in the art. These include, for example, analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, High Performance Liquid Chromatography (HPLC), Thin Layer Chromatography (TLC), and ultra-diffusion chromatography; various immunological methods, such as fluid or gel precipitin reactions, immunodiffusion (one-way or two-way), immunoelectrophoresis, Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assays, southern blot analysis, northern blot analysis, dot blot analysis, gel electrophoresis (e.g., SDS-PAGE), RT-PCR, quantitative PCR, amplification of other nucleic acids or targets or signals, radiolabelling, scintillation counting and affinity chromatography.

The nucleic acid compositions of the invention, although typically in the native sequence (except for modified restriction sites, etc.), can be made by mutating a nucleic acid of the invention from either a cDNA, a genome or a mixture to provide a gene sequence, in accordance with standard techniques. For coding sequences, these mutations may affect the desired amino acid sequence. In particular, DNA sequences substantially homologous to or derived from native V, D, J, constant regions, switching sequences, and other such sequences described herein are contemplated (wherein "derived from" means that one sequence is identical to or modified from another sequence).

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence. With respect to transcriptional regulatory sequences, operably linked means that the DNA sequences being linked are contiguous and, if necessary, the two protein coding sequences are contiguous and linked in frame. For a switching sequence, operably linked means that the sequence is capable of effecting switching recombination.

The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors useful in recombinant DNA techniques are typically in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably, as plasmids are the most commonly used form of vector. However, the invention includes such other forms of expression vectors, e.g., viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), for equivalent function.

The term "recombinant host cell" (or simply "host cell") refers to a cell into which a recombinant expression vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

A "label" is a composition that is detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful markers include³²P, a fluorescent dye, an electron-dense reagent, an enzyme (e.g., an enzyme commonly used in ELISA), biotin, digoxigenin, or a hapten, and a protein from which antisera or monoclonal antibodies are available (e.g., a polypeptide of the invention can be made detectable by incorporating a radioactive label into the peptide of the invention, which is used to detect antibodies specifically reactive with the peptide).

The term "sorting" as used herein in reference to cells refers to both physical sorting of cells (which can be achieved using, for example, a fluorescence activated cell sorter) and cell analysis based on expression of cell surface markers (e.g., FACS analysis in the absence of sorting).

The phrase "immune cell response" refers to the response of cells of the immune system to external or endogenous stimuli (e.g., antigens, cytokines, chemokines, and other cells) to produce biochemical changes in the immune cells that result in migration of the immune cells, killing of target cells, phagocytosis, production of antibodies, other soluble effectors of the immune response, and the like.

The terms "T lymphocyte response" and "T lymphocyte activity" are used interchangeably herein and refer to components of an immune response that are dependent on T lymphocytes (i.e., T lymphocytes proliferate and/or differentiate into helper T lymphocytes, cytotoxic killer T lymphocytes, or suppressive T lymphocytes, helper T lymphocytes provide signals to B lymphocytes, elicit or prevent antibody production, cytotoxic T lymphocytes kill specific target cells, and release soluble factors such as cytokines that regulate other immune cell functions).

The term "immune response" refers to the synergistic effect of lymphocytes, antigen presenting cells, phagocytes, granulocytes and soluble macromolecules produced by the above cells or liver (including antibodies, cytokines and complement) leading to the selective damage, destruction of invading pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in the case of autoimmune or pathological inflammation, or their elimination from the human body.

The components of the immune response can be detected in vitro using a variety of methods well known to those skilled in the art. For example, (1) cytotoxic T lymphocytes can be incubated with radiolabeled target cells, lysis of these target cells is then detected on the basis of the release of radioactivity, (2) helper T lymphocytes can be incubated with antigen and antigen presenting cells, cytokine synthesis and secretion is then measured by standard methods (Windhagen, A. et al, 1995, Immunity 2: 373-80), (3) antigen presenting cells can be incubated with intact protein antigen and then either assayed by T lymphocyte activation, or the presentation of the antigen on the MHC by biophysical methods (Harding et al, 1989, Proc. Natl. Acad. Sci. U.S.A., 86: 4230-4), (4) mast cells can be incubated with reagents that crosslink their Fc-epsilon receptors and then histamine release measured by enzyme immunoassay (Siraganian et al, 1983, TIPS 4: 432-.

Likewise, the detection of immune response products in a model organism (e.g., a mouse) or a human patient can also be carried out by various methods well known to those skilled in the art. For example, (1) antibody production in response to vaccination can be readily detected using standard methods currently used in clinical laboratories, such as ELISA; (2) migration of immune cells to inflammatory sites can be detected by scratching the skin surface, placing a sterile container to capture the migrating cells at the scratched site (Peters et al, 1988, Blood 72: 1310-5); (3) peripheral blood mononuclear cell proliferation or mixed lymphocyte response to mitogens can be measured using 3H-thymidine; (4) phagocytic capacity of granulocytes, macrophages and other phagocytic cells in PBMCs can be measured by seeding PBMCs into wells together with labeled particles (Peters et al, 1988); and (5) by labeling PBMCs with anti-CD molecule (e.g., CD4 and CD8) antibodies and measuring the proportion of PBMCs expressing these markers, the differentiation of immune system cells can be measured.

The phrase "signal transduction pathway" or "signal transduction event" as used herein refers to at least one biochemical reaction, but more typically a series of biochemical reactions, resulting from the interaction of a cell with a stimulatory compound or factor. Thus, the interaction of the stimulatory compound with the cell produces a "signal" that is transmitted through the signal transduction pathway, ultimately leading to a cellular response (e.g., the immune response described above).

Signal transduction pathways refer to biochemical relationships between a variety of signal transduction molecules that play a role in the transmission of signals from one part of a cell to another. The phrase "cell surface receptor" as used herein includes molecules and complexes of molecules that are capable of receiving a signal and transmitting such a signal across the plasma membrane of a cell. An example of a "cell surface receptor" of the present invention is the B7 ligand of the T Cell Receptor (TCR) or CTLA-4.

Signal transduction pathways in cells are initiated by the interaction of the cell with a stimulus either inside or outside the cell. If an exogenous (i.e., outside the cell) stimulus (e.g., an MHC-antigen complex on an antigen presenting cell) interacts with a cell surface receptor (e.g., a T cell receptor), the signal transduction pathway can transmit a signal across the cell membrane of the cell to the cytoplasm of the cell, and in some cases, into the nucleus. If an internal (e.g., within the cell) stimulus interacts with an intracellular signaling molecule, the signaling pathway may result in the transmission of a signal through the cytoplasm of the cell, and in some cases into the nucleus of the cell.

Signal transduction may occur, for example, through the following pathways: phosphorylation of the molecule; non-covalent allosteric interactions; complexing of molecules; a conformational change in the molecule; release of calcium; production of inositol phosphates; carrying out proteolysis; cyclic nucleotide production and diacylglycerol ester production. Typically, signal transduction occurs by phosphorylating a signaling molecule.

The term "non-specific T cell activation" refers to the stimulation of T cells independent of their antigen specificity.

General procedure

To achieve improved stability of the human kappa light chain locus, transgenic animals were generated by combining transchromosome technology with early prokaryotic microinjection technology. The human kappa light chain locus transgene KCo5(Fishwild, D. et al, 1996, nat. Biotechnol. 14: 845-. This transgene was combined with stable hCF (SC20) transchromosomes and functionally inactivating mutations in the mouse endogenous heavy and kappa light chain loci to produce animals expressing a large repertoire of human antibodies including multiple human heavy chain isotypes. Thus, the improved stability of the light chain transgene relative to the bis-TC/KO mice (Tomizuka, K. et al, 2000, Proc. Natl. Acad. Sci U.S.A.97: 722-one 727) achieved the recovery of large numbers of hybridomas from each fusion.

The invention provides for the isolation of fully human antibodies of any desired heavy chain isotype, including IgA₁、IgA₂、IgD、IgE、IgG₁、IgG₂、IgG₃、IgG₄And IgM. In particular, several different antibodies with high affinity for a predetermined antigen can be isolated from a transgenic non-human mammal.

Transgenic non-human mammals of the invention (preferably mice or other rodents) can also be produced using the deposited material. Chicken DT40 cells retaining hCF (SC20) have been deposited under the Budapest treaty at the International Patent organization for Integrated Industrial Science and Technology, national institute of Advanced Industrial Science and Technology, Tsukuba Central6, 1-1, Higashi 1-Chome Tsukuba-shi, Ibaraki-ken, 305-. The deposit number is FERM BP-7583. The name of the cell line is designated SC 20.

Chicken DT40 cells retaining hCF (SC20) have also been deposited at taiwan at 18.8.1999 at the institute for research and development of food industry (FIRDI). Accession number CCRC 960099. The name of the cell line was designated SC20(D) in taiwan collection. hCF (SC20) retained in chicken DT-40 cells can be transferred to mouse ES cells as described in WO00/10383(EP 1106061). Briefly, minicells were generated from chicken DT-40 cells and fused to CHO cells. CHO cells retaining hCF (SC20) can then be selected on the basis of G418 resistance. Retention of hCF (SC20) can be confirmed by PCR or FISH analysis using commercially available human COT1 DNA probe or human chromosome 14 specific probe. Thereafter, minicells were generated from the hCF (SC20) -retaining CHO cells and fused to mouse ES cells. In the same manner as for CHO cells, ES cells retaining hCF (SC20) were selected.

Cells retaining the KCo5 transgenic DNA have been deposited under the Budapest treaty at the American Type Culture Collection (ATCC) at 11/8.2001, 10801 University Boulevard, Manassas, VA 20110-: 17E1(YAC y17, MetarexKCo 5) in yeast was designated ATCC No. PTA-3842 (the genetic material was a yeast artificial chromosome containing an insert of the human immunoglobulin kappa variable region locus); pKV4 (Metarex KCo5) in Escherichia coli (E.coli) was designated ATCC No. PTA-3843 (plasmid containing human immunoglobulin variable region gene); pKCIB (Metarx KCo5) in E.coli was designated ATCC No. PTA-3844 (a plasmid containing the human immunoglobulin J κ and κ constant region genes).

Cells retaining KCo5 transgenic DNA have also been deposited at the institute for research and development of food industry (FIRDI) in taiwan at 11/22/2001. pKV4, YACy17 and pKCIB accession numbers are: CCRC 940383, CCRC 940385, and CCRC 940386.

Transgenic KCo5 can be transferred into mouse cells as previously described (Fishwild, D. supra, see also example 38 in U.S. Pat. No. 5,770,429; see also example 2 below).

General characteristics of immunoglobulins

The basic antibody structural unit is known to comprise a tetramer of subunits. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region, primarily responsible for effector function.

Light chains are classified either as κ or λ. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, and antibody isotypes are identified as IgG, IgM, IgA, IgD, and IgE, respectively. In both the light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 1 to 10 more amino acids. (see generally fundamentals immunology (Paul, W. eds., 2nd ed. raven Press, N.Y., 1989), Ch.7 (incorporated herein by reference in its entirety for all purposes).

The variable regions of each light/heavy chain pair constitute antibody binding sites. Thus, an intact antibody has two binding sites. The two binding sites are identical except in a bifunctional or bispecific antibody. The chains all exhibit the same general structure of relatively conserved Framework Regions (FR) connected by three hypervariable regions, also known as complementarity determining regions or CDRs. The CDRs from both chains of each pair are arranged by the framework regions to enable binding to a particular epitope. Both the light and heavy chains comprise, from N-terminus to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. According to Kabat, Sequences of Proteins of immunologicals interest (National Institutes of Health, Bethesda, MD, 1987 and 1991) or Chothia and Lesk, J.mol.biol.196: 901-917 (1987); chothia et al, Nature 342: 878-883(1989), each domain is assigned an amino acid number.

Natural human immunoglobulin loci

Human immunoglobulins are naturally encoded by three distinct loci: heavy chain, kappa light chain and lambda light chain. These natural loci are located on chromosomes 14, 2 and 22, respectively. The natural human heavy chain locus is located at 14q32.3, near the telomere of the long arm of human chromosome 14, and extends over approximately 1.3 megabases, encoding approximately 45 expressed V gene segments, 27D gene segments, 6J gene segments, and 9 constant (C) region gene segments. The native human kappa light chain locus is located on chromosome 2 at 2p11.12, extends over about 1.8 megabases, and comprises about 34 expressed V gene segments, 5J gene segments, and one C region gene segment. The native human λ locus is located at 22q11.2, extends over about 0.9 megabases, encodes about 30V gene segments and 4 functional J-C pairs, each pair comprising one J gene segment and one C gene segment. Each of these native loci can also comprise deletions, insertions, and single nucleotide polymorphisms.

Production of monoclonal antibodies by hybridoma fusion

The production of monoclonal antibodies can be achieved, for example, by immunizing an animal with an antigen (e.g., a human protein antigen, such as CD4, G-CSF, HSA, EGFR, or CTLA-4, pathogen-encoded antigen, toxin, or other antigen). Longer polypeptides comprising said antigen or an immunogenic fragment of said antigen or an anti-idiotypic antibody directed against an antibody to said antigen may also be used. See Harlow and Lane, Antibodies, A Laboratory Manual (CSHP New York, NY, 1988) and Mishell and Shiigi, Selected Methods in cellular immunology, (W.H.Freeman and Co.New York, NY 1980) (both of which are incorporated herein by reference for all purposes). Such an immunogen may be obtained from natural sources, by peptide synthesis or by recombinant expression. Optionally, the immunogen is administered linked to or complexed with a carrier protein, as described below. Optionally, the immunogen is administered with an adjuvant. Several types of adjuvants may be used as described below. It is preferable to immunize experimental animals with complete Freund's adjuvant and then with incomplete adjuvant. Polyclonal antibodies are typically produced in rabbits and guinea pigs. Rodents (e.g., mice, rats, and hamsters) are commonly used to produce monoclonal antibodies. These mice may be transgenic and may contain human immunoglobulin gene sequences, as described below. After immunization, the immunized animal will develop a serum response to the introduced immunogen. This serum response can be measured by determining the titer of the collected serum using a variety of different assays. One example of a commonly used assay is ELISA. The magnitude of the serum response is commonly referred to as titer. For example, a titer of 1,000 indicates that the presence of reactive antibodies can be detected by measuring 1,000-fold dilutions of serum. Typically, immunization will elicit a serum response several orders of magnitude greater than that present in an unimmunized animal. Only one or two orders of magnitude of serum response is considered a weak response, often indicating the presence of a small number of B cells expressing antigen-reactive antibodies. Monoclonal antibodies are obtained by fusing lymphocytes with immortalized cells (e.g., myeloma cells) to form hybrid cells called hybridoma cells. The newly formed hybridoma cells acquire antibody expression characteristics from the parent lymphocytes and growth characteristics from the parent immortalized cells. The newly formed hybridoma cells are grown in culture dishes (e.g., 96-well plates) containing culture medium. The culture supernatants are tested (typically between 1 and 2 weeks after fusion) for the presence of antigen-reactive antibodies of the desired heavy and light chain isotype. The cells in this primary culture are then diluted and reseeded to isolate individual clones of hybridoma cells that secrete a monoclonal antibody. Such secondary cultures may be further subcloned, a third culture obtained, and so forth. The ratio of antigen-reactive primary cultures from which hybridoma clones can be obtained by this subcloning method provides a measure of subcloning efficiency. If all of the antigen-positive primary hybridoma cultures can be used to obtain a clonal cell line, the subcloning efficiency is 100%. If the immunoglobulin locus encoding the expressed antibody is unstable, e.g., is susceptible to loss during cell division-either by loss of chromosomes, chromosome fragments or transchromosomes, or by deletion recombination of inserted sequences, or by some other mechanism-then the efficiency of subcloning will be reduced (i.e., less than 100%). Platforms with high subcloning efficiencies (e.g., greater than 20%, preferably greater than 50%, more preferably greater than 80%, most preferably greater than 90% or 95%) that yield monoclonal antibodies are particularly useful. Antibodies are screened for specific binding to the antigen. Antibodies are optionally further screened for binding to specific regions of the antigen. For protein antigens, this latter screening can be accomplished by determining the binding of an antibody to a panel of deletion mutants of the antigenic peptide, and then determining which deletion mutants bind to the antibody. Binding can be assessed, for example, by western blotting or ELISA. The smallest fragment that shows specific binding to the antibody defines the epitope of the antibody. However, some epitopes contain non-contiguous structural elements that may be lost due to the deletion of elements located outside the exact epitope. Alternatively, epitope specificity can be determined using a competition assay in which the test antibody and the reference antibody compete for binding to the antigen. If the test antibody and the reference antibody compete, they bind to the same epitope, or an epitope sufficiently adjacent to such binding of one of the antibodies will interfere with the binding of the other antibody.

Cloning of nucleic acids encoding antibodies from B cell hybridomas

Nucleic acids encoding at least the heavy and light chain variable regions can be cloned from transgenic animals, either immunized or not tested. The nucleic acid can be cloned as genomic DNA or cDNA from lymphocytes of such animals. No immortalization of such cells is required prior to cloning of the immunoglobulin sequences. Typically, mRNA is isolated and amplified by reverse transcription using oligo-dT primers. The cDNA of the conserved region of human immunoglobulin is then amplified with primers. Non- μ isotypes (e.g., IgG or IgA) can be easily enriched in the library using 3' primers specific for non- μ sequences. Typically, the amplified population of light chains comprises at least 100, 1000, 10,000, 100,000, or 1,000,000 different light chains. Likewise, an amplified population of heavy chains comprises at least 100, 1000, 10,000, 100,000, or 1,000,000 different heavy chains. For example, using IgG primers, typically at least 90%, 95%, or 99% of the amplified heavy chains are of the IgG isotype. Nucleic acids encoding at least the variable regions of the heavy and light chains can also be cloned from the above hybridomas by screening cDNA libraries with various well-known methods, such as PCR or with DNA probes specific for the conserved regions of human antibodies. The nucleic acid encoding the antibody chain to be subcloned may be excised by restriction digestion of the flanking sequences, or the nucleic acid may be amplified by PCR using primers directed to sites adjacent to the coding sequence. See generally PCR Technology: principles and Applications for DNA Amplification (h.a. erlich, Freeman Press, NY, 1992); PCR Protocols: a guides to Methods and Applications (Innis et al, Academic Press, San Diego, Calif., 1990); mattila et al, 1991, Nucleic Acids Res.19: 967; eckert et al, 1991, PCR Methods and applications 1: 17; PCR (McPherson et al, IRL Press, Oxford). These references and the references cited therein are incorporated by reference herein for all purposes.

Recombinant expression of antibodies

Nucleic acids encoding the light and heavy chain variable regions operably linked to the constant regions are inserted into an expression vector. The light and heavy chains may be cloned in the same or different expression vectors. The DNA segment encoding the antibody chain is operably linked to control sequences in the expression vector that ensure expression of the antibody chain. Such control sequences include signal sequences, promoters, enhancers, and transcription termination sequences. Expression vectors are generally replicable in host organisms either as episomes or as an integral part of the host chromosome.

Coli is a prokaryotic host particularly useful for expression of the antibodies of the invention. Other microbial hosts suitable for use include Bacillus species such as Bacillus subtilis and other Enterobacteriaceae species such as Salmonella (Salmonella), Serratia (Serratia) and various Pseudomonas species. In these prokaryotic hosts, one can also prepare expression vectors, which typically contain expression control sequences (e.g., origins of replication) and regulatory sequences such as a lactose promoter system, a tryptophan (trp) promoter system, a beta-lactamase promoter system, or a promoter system derived from bacteriophage lambda, compatible with the host cell.

Other microorganisms, such as yeast, may also be used for expression. Saccharomyces (Saccharomyces) is a preferred host, and if desired, has expression control sequences such as promoters, including promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, and origins of replication, termination sequences, and the like.

Mammalian tissue cell cultures may also be used to express and produce the antibodies of the invention (see Winnacker, From Genes to Clones (VCH Publishers, N.Y., 1987.) preferred eukaryotic cells since many suitable host cell lines capable of secreting intact antibodies have been developed preferred suitable host cells for expressing nucleic acids encoding immunoglobulins of the invention include monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL 1651), human embryonic kidney line (293) (Graham et al, 1977, J.Gen.Virol.36: 59), baby hamster kidney cells (Graham et al, ATCC CCL 10), Chinese hamster ovary cells-DHFR (CHO, Urlaub and Chasin, 1980, Proc.Natl.Acad.Sci.U.S.A.77: 4216), mouse support cells (TM4, Mather, 1980, biol.23: Henry. Acad. Sci.Sci.U.S.A.77: 4276), mouse support cells (TM. 4, Mather, 1980, Biol.Reverd.23: BHLA; ATCC # WO 19; ATCC # WO-76, kidney cells; ATCC # 1587, ATCC # 3676, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); and TRI cells (Mather et al, 1982, Annals N.Y Acad.Sci.383: 44-46); a baculovirus cell.

Vectors containing the polynucleotide sequences of interest (e.g., heavy and light chain coding sequences and expression control sequences) can be transferred into host cells. For prokaryotic cells calcium chloride transfection is commonly used, while for other cellular hosts calcium phosphate treatment or electroporation may be used (see generally Sambrook et al, Molecular Cloning: A Laboratory Manual (Cold spring harbor Press, 2nd ed., 1989), which is incorporated herein by reference in its entirety for all purposes.

Once expressed, the intact antibodies of the invention, dimers, single light and heavy chains thereof, or other immunoglobulin forms may be purified according to standard methods in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like (see generally Scopes, protein purification (Springer-Verlag, n.y., 1982), preferably substantially pure immunoglobulins of at least about 90-95% homogeneity, most preferably 98-99% or greater homogeneity.

Chimeric and humanized antibodies

Chimeric and humanized antibodies have the same or similar binding specificity and affinity as mouse or other non-human antibodies that provide the starting material for constructing the chimeric or humanized antibody. Chimeric antibodies are antibodies whose light and heavy chain genes have been constructed, usually by genetic engineering, from immunoglobulin gene segments belonging to different species. For example, variable region (V) segments from a mouse monoclonal derived gene can be combined with human constant region (C) segments such as IgG₁And IgG₄And (4) connecting. Preferably human isotype IgG₁. A typical chimeric antibody is thus a hybrid protein consisting of a V region or antigen-binding region derived from a mouse antibody and a C region or effector region derived from a human antibody.

Humanized antibodies have variable region framework residues substantially from human antibodies (referred to as acceptor antibodies) and complementarity determining regions substantially from mouse antibodies (referred to as donor immunoglobulins). See Queen et al, 1989, proc.natl.acad.sci.u.s.a.86: 10029-. The constant region, if present, is also substantially or entirely derived from a human immunoglobulin. The human variable regions are typically selected from human antibodies in which the framework sequences exhibit a high degree of sequence identity to the murine variable region domains from which the CDRs are derived. The heavy and light chain variable region framework residues may be derived from the same or different human antibody sequences. The human antibody sequences may be naturally occurring human antibody sequences, or may be a consensus sequence of several human antibodies. See Carter et al, WO 92/22653. Certain amino acids from human variable region framework residues are selected for substitution based on their possible effect on CDR conformation and/or antigen binding. Such possible effects are studied by modeling, examining the properties of amino acids at specific positions, or empirically observing the effects of substitution or mutagenesis of specific amino acids.

For example, when an amino acid differs between murine variable region framework residues and selected human variable region framework residues, the human framework amino acid should generally be replaced with an equivalent framework amino acid from a mouse antibody when one of the following is reasonably predictable:

(1) the amino acid is non-covalently bound directly to the antigen,

(2) the amino acids are adjacent to the CDR regions,

(3) the amino acids otherwise interact with the CDR regions (i.e., within about 6A of the CDR regions), or

(4) The amino acids are involved in the VL-VH linkage.

Other candidates for substitution are the rare acceptor human framework amino acids at this position for human immunoglobulins. These amino acids may be substituted with amino acids from equivalent positions in a mouse donor antibody or from equivalent positions in a more general human immunoglobulin. Other candidates for substitution are the rare acceptor human framework amino acids at this position for human immunoglobulins. The variable region framework of humanized immunoglobulins typically exhibit at least 85% sequence identity to the human variable region framework sequence or to a consensus sequence of such sequences.

Human antibodies

Human antibodies to a particular antigen can be provided by a variety of methods as described below. Certain human antibodies are selected by competitive binding experiments, or certain human antibodies are selected that have the same epitope specificity as a particular mouse antibody (e.g., one of the mouse monoclonal antibodies described in the examples). It is also possible to screen human antibodies for a particular epitope specificity using only fragments of the antigen as immunogens and/or, in the case of protein antigens, by screening antibodies against a panel of deletion mutants of said antigen.

Trioma method

The basic method and exemplary cell fusion components (fusion partner) -SPAZ-4 for this method have been described by the following references: oestberg et al, 1983, Hybridoma 2: 361-; oestberg, U.S. Pat. No. 4,634,664; and Engleman et al, U.S. patent 4,634,666 (each incorporated by reference herein in its entirety for all purposes). The antibody-producing cell lines obtained by this method are called trioma because they are transformed from three cells-two human cells and one mouse cell. Initially, a mouse myeloma line is fused with human B lymphocytes to obtain non-antibody producing xenogeneic hybrid cells, such as the SPAZ-4 cell line described by Oestberg (see above). Then fusing the xenogeneic cells with immortalized human B lymphocytes to obtain the antibody-producing trioma cell line. It has been found that antibodies are produced in trioma which are more stable than normal hybridomas prepared from human cells.

While triomas are genetically stable, they do not produce very high levels of antibodies. Expression levels can be increased by cloning antibody genes from the trioma into one or more expression vectors, transforming the vectors into standard mammalian, bacterial or yeast cell lines.

Transgenic non-human mammals

Transgenic non-human mammals containing human immunoglobulin loci can also be used to produce human antibodies against a variety of antigens. Generally, these immunoglobulin loci can encode antibodies that are substantially human sequences, preferably 95% or greater identity, more preferably 98-99% or greater identity, and most preferably 100% identical to human sequences. Immunoglobulin loci may be rearranged or unrearranged, and may contain deletions or insertions relative to the native human immunoglobulin locus. The locus may include genetic elements (e.g., non-coding elements such as enhancers, promoters and switching sequences, or coding elements such as mu constant region gene segments) from other species and from non-immunoglobulin loci that produce substantially no secondary component (non-IgM) antibody-encoding portions. Preferred human immunoglobulin loci undergo DNA sequence changes, including v (d) J-junctions, heavy chain class switching, and somatic mutations, in lymphoid cells and/or lymphoid cell precursors in the transgenic non-human mammal, resulting in high affinity human antibodies to a predetermined antigen. The human immunoglobulin loci contained in these transgenic mammals preferably include the unreleased sequences of the native human heavy and human light chain loci. In general, the endogenous immunoglobulin locus of such transgenic mammals is functionally inactivated (U.S. Pat. No. 5,589,369; Takeda, S. et al, 1993, EMBO J.12: 2329-2366; Jakobovits, A. et al, 1993, Proc. Natl.Acad. Sci. U.S.A.90: 2551-2555; Kitamura, D. and Rajewsky, K.1992, Nature 356: 154-156; Gu, H. et al, 1991, 65: 47-54; Chen, J. et al, EMBO J12: 821-830; Sun, W. et al, 1994, J. Immunol 152: 695-za-704; Chen, J. et al, 1993, Intl. Immunol 5: 647-656; Zz, X. et al, 1995, Eur. J. 92: 21525; Euro J. 92-90; Euro J. 1165: Thr. J. 1993, Intl. Immunol. J. 22-90; Chen. J. 22, H. J. 055; Chen. J. 35; Chen. J. 0520; Chen. J. K. K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.K.1165; 35; Chen, s. et al, 1993, Science 262: 1268-71; kitamura, d. et al, 1991, Nature 350: 423-6; lutz, C. et al, 1998, Nature 393: 797-801; zuo, y, et al, 1994, Current Biology 4: 1099-; chen, J. et al, 1993, EMBO J.12: 4635-4645; serwe, m. and sablitizky, f., 1993, EMBO j.12: 2321-2327; sanchez, p. et al, 1994, intl.immunology 6: 711-; zou, y, et al, 1993, EMBO j.12: 811-820). Inactivation of endogenous immunoglobulin genes can preferably be achieved, for example, by targeted homologous recombination. The exogenous human immunoglobulin locus may be bound to the endogenous mouse chromosome or may be (e.g., part of, inserted into, or linked to) the introduced transchromosome. Transchromosomes are introduced into cells as non-endogenous chromosomes or chromosome fragments with one centromere and two telomeres. These transchromosomes typically contain telomere and centromere sequences and may contain deletions relative to the parent intact chromosome. The transchromosomes may also contain additional insertion sequences. For example, two human immunoglobulin loci can be bound on a single transchromosome by inserting the sequence of a first immunoglobulin locus (e.g., from a YAC clone, a transchromosome, or an entire chromosome) into a transchromosome comprising a second immunoglobulin locus. This process can also be repeated to bind all three human immunoglobulin loci to one transchromosome. A single transchromosome containing two or three different immunoglobulin loci provides genetic linkage of these loci, which increases the rate of transgenic progeny that can be used to produce human antibodies. Preferred forms of transchromosomes are those detailed in the following references: tomizuka, k, et al, 2000, proc.natl.acad.sci.u.s.a.97: 722-727, Tomizuka, K. et al, 1997, Nature Genetics 16: 133-143, and WO 97/07671, WO98/37757, and WO00/10383, each of which is incorporated herein by reference in its entirety for all purposes. The transchromosome may also include an integrated selectable marker (e.g., a neomycin resistance gene) and other sequences not present in the parent intact chromosome. In the event of recombination between a transchromosome and an endogenous mouse chromosome, sequences from the transchromosome are inserted or added to the endogenous mouse chromosome. The transchromosomes may be modified by deletion, translocation, substitution, etc. as described in WO98/37757, EP 0972445 and WO00/10383, which are incorporated herein by reference for all purposes. For example, transchromosomes may spontaneously fragment during introduction into mouse Embryonic Stem (ES) cells, fragment by telomere-directed truncation and/or translocate by Cre/loxP site-directed recombination or similar methods. Specific insertion recombination sites (e.g., loxP sequences and other sequences; see, e.g., Abuin, A. and Bradley, A., 1996, mol. cell. biol. 16: 1851-1856; Mitani, K. et al, 1995, Somat. cell. mol. Genet. 21: 221-231; Li, Z. W. et al, 1996, Proc. Natl. Acad. Sci. U.S.A.93: 6158-6162; Smith, AJ. et al, 1995, nat. Genet. 9: 376-385; Trinh, K. and Morrison, S.L., 2000, J. Immunol. Metds 244: 185-42; Sunaga, S. et al, 1997, mol. Dev. 46: 109-113; Dymei, S. M. Actic, 6191: 185-11; Sunaga, S. 1997, Mol. Ser. No. 31, No. Ser. 31, No. 32, No. 10, No. 5, No. 103, No. 3, No. 7, No. 5, No. 7, No. 3, No. 7. In case loxP sites are introduced, expression of a transgene encoding cre recombinase will promote recombination between two loxP sites. The transchromosome may also be a fused chromosome composed of different chromosome fragments due to the translocation described above. The transchromosome may be autonomous. The autosomal transgene is distinct from, non-contiguous with, and insertional into, a mouse endogenous chromosome. These autonomous transchromosomes contain telomeres and centromere sequences that enable autonomous replication. Alternatively, the transchromosome sequence can be translocated to the mouse chromosome after it is introduced into the mouse nucleus. The mouse endogenous chromosomes include 19 pairs of autosomes as well as the X and Y chromosomes.

Introduction of exogenous human immunoglobulin loci can be accomplished by a variety of methods including, for example, microinjection of half day old embryos, transfection of embryonic stem cells, or fusion of embryonic stem cells with yeast spheroplasts or minicores containing transchromosomes. The transgenic mammals produced by the above method are capable of functionally rearranging the introduced exogenous immunoglobulin component sequences and expressing the antibody repertoire of the various isotypes encoded by the human immunoglobulin genes, without expressing endogenous immunoglobulin genes. The production and characterization of mammals with these properties is described in detail, for example, in Lonberg et al, WO 93/12227 (1993); U.S. patent nos. 5,877,397, 5,874,299, 5,814,318, 5,789,650, 5,770,429, 5,661,016, 5,633,425, 5,625,126, 5,569,825, 5,545,806, Nature 48: 1547 (1994), Nature Biotechnology 14, 826(1996), Kucherlapati, WO 91/10741(1991), WO94/02602(1993), WO 96/34096(1995), WO 96/33735(1996), WO98/24893(1997), U.S. Pat. Nos. 5,939,598, 6,075,181, 6,114,598, Tomizuka, K. et al, 2000, Proc. Natl.Acad.Sci.U.S.A.97: 722-727, Tomizuka, K. et al, 1997, Nature Genetics 16: 133-. Transgenic non-human mammals such as rodents are particularly suitable. Monoclonal antibodies can be prepared, for example, by fusing B cells obtained from such mammals with a suitable immortalized cell line using conventional Kohler-Milstein technology. Monoclonal antibodies can also be obtained directly from individual B cells using PCR to amplify the V region (Schrader et al, 1997, U.S. Pat. No. 5,627,052), and isolated from the culture medium. Alternatively, a preparation of B cells sorted by FACs, or enriched, can be used as a source of RNA or DNA for PCR amplification of V region sequences. Phage display methods (described below) can also be used to obtain human antibody sequences from immunized transgenic mice containing human immunoglobulin loci. The human antibody V region sequences obtained by these methods can then be used to generate a complete antibody that retains the binding properties of the original parent antibody. This method is described below.

Phage display method

Another method for obtaining human antibodies is according to Huse et al, 1989, Science 246: 1275-1281 screening of cDNA libraries from B cells was performed using the general protocol outlined above. Such B cells may be obtained from a human immunized with the desired antigen, fragment, longer polypeptide containing said antigen or fragment, or anti-idiotypic antibody. Such B cells are optionally obtained from individuals not contacted with the antigen. B cells may also be obtained from transgenic non-human mammals expressing human immunoglobulin sequences. The transgenic non-human mammal may be immunized with an antigen or a set of antigens. The animal may also be non-immunized. cDNA was generated using reverse transcriptase using B cell mRNA sequences encoding human antibodies. The V region encoding segment of the cDNA sequence is then cloned into a DNA vector that directs expression of the antibody V region. Typically, the V region sequences are specifically amplified by PCR prior to cloning. The V region sequences are also typically cloned into the DNA vector in a site that is constructed such that the V region is expressed as a fusion protein. Examples of such fusion proteins include the m13 E.coli phage gene 3 and gene 8 fusion proteins. This set of cloned V region sequences is then used to generate an expression library of antibody V regions. To generate an expression library, eukaryotic or prokaryotic host cells are transformed with a DNA vector comprising the cloned V region sequences. In addition to the V region, the vector may optionally encode all or part of a viral locus, and may comprise viral packaging sequences. In some cases, the vector does not contain the entire viral genome, and the vector is then used with a helper virus or helper virus DNA sequence. The expressed antibody V regions are present within or on the surface of the transformed cells or viral particles derived from the transformed cells. Such an expression library comprising said cells or viral particles is then used to identify V region sequences encoding antibodies or antibody fragments reactive with a predetermined antigen. To identify these V region sequences, the expression library is screened or selected for reactivity with the predetermined antigen. Cells or viral particles comprising the cloned V-region sequences and having expressed V-regions are screened or selected using methods that identify or enrich for cells or viral particles having V-regions reactive (e.g., binding association or catalytic activity) with a predetermined antigen. For example, a radioactively or fluorescently labeled antigen bound to an expressed V region can be detected and used to identify or sort cells or viral particles. Antigens bound to solid phase matrices or microbeads can also be used to select for cells or virus particles having reactive V-regions on the surface. The V region sequences so identified from the expression library can then be used to direct the expression of antibodies or fragments thereof reactive with the predetermined antigen in transformed host cells. The protocol described by Huse has become more efficient in combination with phage display technology. See, e.g., Dower et al, WO91/17271, and McCafferty et al, WO 92/01047, U.S. patent nos. 5,871,907, 5,858,657, 5,837,242, 5,733,743, and 5,565,332 (each of which is incorporated herein by reference in its entirety for all purposes). In these methods, a phage library is generated in which the members (display packages) display different antibodies on their outer surface. Antibodies are typically displayed as Fv or Fab fragments. Phage displaying antibodies with the desired specificity can be selected by affinity enrichment with the antigen or fragment thereof. Phage display in combination with an immune transgenic non-human animal expressing human immunoglobulin genes can be used to obtain antigen-specific antibodies even when the immune response to the antigen is weak.

In one variant of phage display, human antibodies can be generated with the binding specificity of a selected murine antibody. See, for example, Winter, WO 92/20791. In this method, either the heavy chain variable region or the light chain variable region of the selected murine antibody is used as the starting material. If, for example, a light chain variable region is selected as the starting material, a phage library is constructed whose members display the same light chain variable region (i.e., murine starting material) and different heavy chain variable regions. The heavy chain variable region is obtained from a library of rearranged human heavy chain variable regions. The selection shows strong specific binding to CTLA-4 (e.g., at least 10)⁸M^-1Preferably at least 10⁹M^-1) The bacteriophage of (1). The human heavy chain variable region from this phage was then used as starting material for the construction of another phage library. In this library, each phage displays the same heavy chain variable region (i.e., the region identified from the first display library) and a different light chain variable region. The light chain variable region is obtained from a library of rearranged human light chain variable regions. Again, phage showing strong specific binding to the selected antigen are selected. Artificial antibodies similar to human antibodies can be obtained, for example, from phage display libraries incorporating random or synthetic sequences in the CDR regions.

Selection of constant regions

The heavy and light chain variable regions of a chimeric, humanized or human antibody can be linked to at least a portion of a human constant region using a variety of well-known methods (see, e.g., Queen et al, 1989, Proc. Natl. Acad. Sci. U.S.A.86: 10029-10033 and WO 90/07861; these references and the references cited therein are incorporated herein by reference for all purposes). The choice of constant region depends in part on whether antibody-dependent complement and/or cell-mediated toxicity is desired. For example, isotype IgG₁And IgG₃IgG isotype in general₂Or IgG₄Has higher complement fixation activity. The choice of isotype can also affect antibody entry in the brain. The light chain constant region can be a lambda or a kappa constant region. The antibody may be a tetramer comprising two light chains and two heavy chains,Expressed as isolated heavy and light chains, as Fab, Fab ', F (ab') 2 and Fv, or as single chain antibodies with heavy and light chain variable regions linked by a spacer.

For certain applications, non-IgG antibodies may be useful. For example, when multivalent antibody complexes are desired, IgM and IgA antibodies can be used.

Expression of intact antibodies using partial antibody sequences

Antibodies interact with a target antigen primarily through amino acid residues located in the 6 heavy and light chain Complementarity Determining Regions (CDRs). For this reason, the amino acid sequences within the CDRs are more diverse than sequences outside the CDRs among the individual antibodies. Because CDR sequences are responsible for most of the antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular naturally occurring antibody by constructing expression vectors that include CDR sequences from the particular naturally occurring antibody grafted onto framework sequences from different antibodies having different properties (see, e.g., Riechmann, L. et al, 1988, Nature 332: 323-. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences. These germline sequences will differ from the mature antibody gene sequences in that they will not include fully assembled variable region genes formed by v (d) J junctions during B cell maturation. Germline gene sequences will also differ from the sequences of high affinity subcomponent antibodies by somatic mutations at individual nucleotides. However, somatic mutations are not evenly distributed in the variable region. For example, the frequency of somatic mutations in the amino-terminal portion of framework region 1 and the carboxy-terminal portion of framework region 4 is relatively low. In addition, many somatic mutations do not significantly alter the binding properties of antibodies. Thus, it is not necessary to obtain the complete DNA sequence of a particular antibody to reconstitute a complete recombinant antibody with similar binding properties as the original antibody (see PCT/US99/05535, filed 3/12 of 1999, which is incorporated herein by reference for all purposes). Portions of the heavy and light chain sequences spanning the CDR regions are generally sufficient for this purpose. The partial sequences are used to determine which germline variable region gene segments and linker gene segments contribute to the recombinant antibody variable region gene. The germline sequence is then used to complement the deleted portion of the variable region. The heavy and light chain leader sequences are cleaved off during protein maturation and do not affect the properties of the final antibody. Thus, the expression construct does not have to use the corresponding germline leader sequence. To add the deleted sequences, the cloned cDNA sequences can be combined with synthetic oligonucleotides by ligation or PCR amplification. Alternatively, the entire variable region can be synthesized as a set of short overlapping oligonucleotides, which are then ligated by PCR amplification to construct an entire synthetic variable region clone. This approach has several advantages, such as the elimination or inclusion of specific restriction sites or optimization of specific codons.

The nucleotide sequences of the heavy and light chain transcripts from a hybridoma can be used to design a set of overlapping synthetic oligonucleotides to construct synthetic V sequences having the same amino acid coding ability as the native sequence. The synthetic heavy and kappa light chain sequences may differ from the natural sequence in three ways: the repeated nucleotide base sequence segment is interrupted to facilitate oligonucleotide synthesis and PCR amplification; the addition of an optimal translation initiation site according to the Kozak principle (Kozak, 1991, J.biol.chem.266: 19867-19870); and engineering a HindIII site upstream of the translation initiation site.

For the heavy and light chain variable regions, the optimized coding strand sequence and the corresponding non-coding strand sequence are broken into segments of 30-50 nucleotides such that a break between the coding strand sequence nucleotides occurs at about the midpoint of the corresponding non-coding oligonucleotide. Thus, for each strand, the oligonucleotides can be assembled as a set of overlapping double strands that completely span the desired sequence. These oligonucleotides were mixed into pools spanning segments of 150400 nucleotides. The library was then used as a template to generate a PCR amplification product of 150-400 nucleotides. Typically, one variable region oligonucleotide set is divided into two pools, which are amplified separately, producing two overlapping PCR products. These overlapping products are then combined by PCR amplification to form the complete variable region. It may also be desirable to include overlapping fragments of the heavy or light chain constant regions (including the BbsI site of the kappa light chain or the AgeI site of the gamma heavy chain) in PCR amplification to produce fragments that can be readily cloned into the expression vector construct.

The heavy chain and light chain variable regions are then combined with a cloning promoter sequence, a translation initiation sequence, a constant region sequence, a 3' untranslated sequence, a polyadenylation sequence, and a transcription termination sequence to form an expression vector construct. The heavy and light chain expression constructs may be incorporated into one vector, co-transfected, sequentially transfected or transfected separately into a host cell and then fused to form a host cell expressing both chains.

Plasmids used for constructing the human IgG kappa expression vector are described below. The plasmid was constructed so that PCR amplified V heavy and V κ light chain cDNA sequences could be used to reconstruct the complete heavy and light chain minigenes. These plasmids can be used to express intact human or chimeric IgG₁Kappa or IgG₄Kappa antibodies. Similar plasmids can be constructed for expression of other heavy chain isotypes or for expression of antibodies comprising lambda light chains.

The kappa light chain plasmid pCK7-96(SEQ ID NO: 1) shown below includes a kappa constant region and a polyadenylation site, such that kappa sequences amplified with a 5' primer that includes a HindIII site upstream of the initiator methionine can be digested with HindIII and BbsI, then cloned into HindIII and BbsI digested pCK7-96, and the entire light chain coding sequence with polyadenylation site is reconstructed. This cassette can be isolated as a HindIII/NotI fragment and ligated with a transcriptional promoter sequence to construct a functional minigene for transfection into cells.

The gamma 1 heavy chain plasmid pCG7-96(SEQ ID NO: 2) includes a human gamma 1 constant region and a polyadenylation site, such that the gamma sequence amplified with a 5' primer including a HindIII site upstream of the initiating methionine can be digested with HindIII and AgeI, then cloned into HindIII and AgeI digested pCG7-96, and the complete gamma 1 heavy chain coding sequence with a polyadenylation site is reconstructed. This cassette can be isolated as a HindIII/SalI fragment and ligated with a transcriptional promoter sequence to construct a functional minigene for transfection into cells.

The gamma 4 heavy chain plasmid pG4HE (SEQ ID NO: 3) included a human gamma 4 constant region and a polyadenylation site, such that the gamma sequence amplified with a 5' primer including a HindIII site upstream of the initiating methionine could be digested with HindIII and AgeI, then cloned into pG4HE digested with HindIII and AgeI, and the entire gamma 4 heavy chain coding sequence with polyadenylation site reconstructed. This cassette can be isolated as a HindIII/EcoRI fragment and ligated with the transcription promoter sequence to construct a functional minigene for transfection into cells.

The reconstructed heavy and light chain genes can be expressed using a variety of different promoters, including but not limited to the CMV promoter, the ubiquitin promoter, the SR α promoter, and the β -actin promoter. For example, vector pCDNA3.1+ (Invitrogen, Carlsbad, Calif.) can be cleaved with HindIII and either NotI, XhoI or EcoRI for ligation with either the kappa, gamma 1 or gamma 4 cassettes described above to construct an expression vector that can be directly transfected into mammalian cells.

[150]pCK7-96(SEQ ID NO：1)

TCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATC

AGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACA

TGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTC

CATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAA

CCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTG

TTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTT

TCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTG

TGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGT

CCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGA

GCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAG

AAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTA

GCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAG

ATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGC

TCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCA

CCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACT

TGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCG

TTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCAT

CTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCA

ATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCAT

CCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCA

ACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTC

AGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGT

TAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGG

TTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACT

GGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCC

GGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAA

AACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAA

CCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGC

AAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATAC

TCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGA

TACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAA

AGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTA

TCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAG

CTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGG

CGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTG

TACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGC

ATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTC

TTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGC

CAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGCCAAGCTAGCGGCCGCGGTC

CAACCACCAATCTCAAAGCTTGGTACCCGGGAGCCTGTTATCCCAGCACAGTCCTGGAAGAG

GCACAGGGGAAATAAAAGCGGACGGAGGCTTTCCTTGACTCAGCCGCTGCCTGGTCTTCTTC

AGACCTGTTCTGAATTCTAAACTCTGAGGGGGTCGGATGACGTGGCCATTCTTTGCCTAAAG

CATTGAGTTTACTGCAAGGTCAGAAAAGCATGCAAAGCCCTCAGAATGGCTGCAAAGAGCTC

CAACAAAACAATTTAGAACTTTATTAAGGAATAGGGGGAAGCTAGGAAGAAACTCAAAACAT

CAAGATTTTAAATACGCTTCTTGGTCTCCTTGCTATAATTATCTGGGATAAGCATGCTGTTT

TCTGTCTGTCCCTAACATGCCCTGTGATTATCCGCAAACAACACACCCAAGGGCAGAACTTT

GTTACTTAAACACCATCCTGTTTGCTTCTTTCCTCAGGAACTGTGGCTGCACCATCTGTCTT

CATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGA

ATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGT

AACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCAC

CCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATC

AGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGAGGGAGAAGTGC

CCCCACCTGCTCCTCAGTTCCAGCCTGACCCCCTCCCATCCTTTGGCCTCTGACCCTTTTTC

CACAGGGGACCTACCCCTATTGCGGTCCTCCAGCTCATCTTTCACCTCACCCCCCTCCTCCT

CCTTGGCTTTAATTATGCTAATGTTGGAGGAGAATGAATAAATAAAGTGAATCTTTGCACCT

GTGGTTTCTCTCTTTCCTCAATTTAATAATTATTATCTGTTGTTTACCAACTACTCAATTTC

TCTTATAAGGGACTAAATATGTAGTCATCCTAAGGCGCATAACCATTTATAAAAATCATCCT

TCATTCTATTTTACCCTATCATCCTCTGCAAGACACGCCTCCCTCAAACCCACAAGCCTTCT

GTCCTCACAGTCCCCTGGGCCATGGATCCTCACATCCCAATCCGCGGCCGCAATTCGTAATC

ATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAG

CCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCG

TTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGG

CCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGC

[151]pCG7-96(SEQ ID NO：2)

GAACTCGAGCAGCTGAAGCTTTCTGGGGCAGGCCAGGCCTGACCTTGGCTTTGGGGCAGGGA

GGGGGCTAAGGTGAGGCAGGTGGCGCCAGCCAGGTGCACACCCAATGCCCATGAGCCCAGAC

ACTGGACGCTGAACCTCGCGGACAGTTAAGAACCCAGGGGCCTCTGCGCCCTGGGCCCAGCT

CTGTCCCACACCGCGGTCACATGGCACCACCTCTCTTGCAGCCTCCACCAAGGGCCCATCGG

TCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTG

GTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGG

CGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGA

CCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGC

AACACCAAGGTGGACAAGAAAGTTGGTGAGAGGCCAGCACAGGGAGGGAGGGTGTCTGCTGG

AAGCCAGGCTCAGCGCTCCTGCCTGGACGCATCCCGGCTATGCAGCCCCAGTCCAGGGCAGC

AAGGCAGGCCCCGTCTGCCTCTTCACCCGGAGGCCTCTGCCCGCCCCACTCATGCTCAGGGA

GAGGGTCTTCTGGCTTTTTCCCCAGGCTCTGGGCAGGCACAGGCTAGGTGCCCCTAACCCAG

GCCCTGCACACAAAGGGGCAGGTGCTGGGCTCAGACCTGCCAAGAGCCATATCCGGGAGGAC

CCTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCACTCCCTCAGCTCGGACACCT

TCTCTCCTCCCAGATTCCAGTAACTCCCAATCTTCTCTCTGCAGAGCCCAAATCTTGTGACA

AAACTCACACATGCCCACCGTGCCCAGGTAAGCCAGCCCAGGCCTCGCCCTCCAGCTCAAGG

CGGGACAGGTGCCCTAGAGTAGCCTGCATCCAGGGACAGGCCCCAGCCGGGTGCTGACACGT

CCACCTCCATCTCTTCCTCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCC

CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGA

CGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATA

ATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTC

ACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGC

CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGGGACCCGTGGGGTGCGAG

GGCCACATGGACAGAGGCCGGCTCGGCCCACCCTCTGCCCTGAGAGTGACCGCTGTACCAAC

CTCTGTCCCTACAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATG

AGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATC

GCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCT

GGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGC

AGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAG

AGCCTCTCCCTGTCTCCGGGTAAATGAGTGCGACGGCCGGCAAGCCCCCGCTCCCCGGGCTC

TCGCGGTCGCACGAGGATGCTTGGCACGTACCCCCTGTACATACTTCCCGGGCGCCCAGCAT

GGAAATAAAGCACCCAGCGCTGCCCTGGGCCCCTGCGAGACTGTGATGGTTCTTTCCACGGG

TCAGGCCGAGTCTGAGGCCTGAGTGGCATGAGGGAGGCAGAGCGGGTCCCACTGTCCCCACA

CTGGCCCAGGCTGTGCAGGTGTGCCTGGGCCCCCTAGGGTGGGGCTCAGCCAGGGGCTGCCC

TCGGCAGGGTGGGGGATTTGCCAGCGTGGCCCTCCCTCCAGCAGCACCTGCCCTGGGCTGGG

CCACGGGAAGCCCTAGGAGCCCCTGGGGACAGACACACAGCCCCTGCCTCTGTAGGAGACTG

TCCTGTTCTGTGAGCGCCCCTGTCCTCCCGACCTCCATGCCCACTCGGGGGCATGCCTGCAG

GTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCATCGATGATATCAGATCTGCCGG

TCTCCCTATAGTGAGTCGTATTAATTTCGATAAGCCAGGTTAACCTGCATTAATGAATCGGC

CAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTC

GCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGT

TATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCC

AGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCA

TCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGG

CGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATAC

CTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCT

CAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCG

ACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCG

CCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGA

GTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTC

TGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACC

GCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCA

AGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAG

GGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA

AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAAT

CAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCG

TCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCG

CGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGA

GCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAG

CTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATC

GTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCG

AGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTG

TCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTT

ACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTG

AGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGC

CACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCA

AGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTC

AGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAA

AAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTAT

TGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAA

TAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCA

TTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGT

TTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCT

GTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTC

GGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGGACATA

TTGTCGTTAGAACGCGGCTACAATTAATACATAACCTTATGTATCATACACATACGATTTAG

GTGACACTATA

[152]pG4HE(SEQ ID NO：3)

GAACTCGAGCAGCTGAAGCTTTCTGGGGCAGGCCGGGCCTGACTTTGGCTGGGGGCAGGGAG

GGGGCTAAGGTGACGCAGGTGGCGCCAGCCAGGTGCACACCCAATGCCCATGAGCCCAGACA

CTGGACCCTGCATGGACCATCGCGGATAGACAAGAACCGAGGGGCCTCTGCGCCCTGGGCCC

AGCTCTGTCCCACACCGCGGTCACATGGCACCACCTCTCTTGCAGCTTCCACCAAGGGCCCA

TCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTG

CCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCA

GCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTG

GTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCC

CAGCAACACCAAGGTGGACAAGAGAGTTGGTGAGAGGCCAGCACAGGGAGGGAGGGTGTCTG

CTGGAAGCCAGGCTCAGCCCTCCTGCCTGGACGCACCCCGGCTGTGCAGCCCCAGCCCAGGG

CAGCAAGGCATGCCCCATCTGTCTCCTCACCCGGAGGCCTCTGACCACCCCACTCATGCTCA

GGGAGAGGGTCTTCTGGATTTTTCCACCAGGCTCCGGGCAGCCACAGGCTGGATGCCCCTAC

CCCAGGCCCTGCGCATACAGGGGCAGGTGCTGCGCTCAGACCTGCCAAGAGCCATATCCGGG

AGGACCCTGCCCCTGACCTAAGCCCACCCCAAAGGCCAAACTCTCCACTCCCTCAGCTCAGA

CACCTTCTCTCCTCCCAGATCTGAGTAACTCCCAATCTTCTCTCTGCAGAGTCCAAATATGG

TCCCCCATGCCCATCATGCCCAGGTAAGCCAACCCAGGCCTCGCCCTCCAGCTCAAGGCGGG

ACAGGTGCCCTAGAGTAGCCTGCATCCAGGGACAGGCCCCAGCCGGGTGCTGACGCATCCAC

CTCCATCTCTTCCTCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAA

AACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTG

AGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGC

CAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCG

TCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTC

CCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGTGGGACCCACGGGGTGCGAGGGCC

ACATGGACAGAGGTCAGCTCGGCCCACCCTCTGCCCTGGGAGTGACCGCTGTGCCAACCTCT

GTCCCTACAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGAT

GACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCG

TGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC

TCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGG

GAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCC

TCTCCCTGTCTCTGGGTAAATGAGTGCCAGGGCCGGCAAGCCCCCGCTCCCCGGGCTCTCGG

GGTCGCGCGAGGATGCTTGGCACGTACCCCGTCTACATACTTCCCAGGCACCCAGCATGGAA

ATAAAGCACCCACCACTGCCCTGGGCCCCTGTGAGACTGTGATGGTTCTTTCCACGGGTCAG

GCCGAGTCTGAGGCCTGAGTGACATGAGGGAGGCAGAGCGGGTCCCACTGTCCCCACACTGG

CCCAGGCTGTGCAGGTGTGCCTGGGCCACCTAGGGTGGGGCTCAGCCAGGGGCTGCCCTCGG

CAGGGTGGGGGATTTGCCAGCGTGGCCCTCCCTCCAGCAGCAGCTGCCCTGGGCTGGGCCAC

GGGAAGCCCTAGGAGCCCCTGGGGACAGACACACAGCCCCTGCCTCTGTAGGAGACTGTCCT

GTCCTGTGAGCGCCCTGTCCTCCGACCCCCCATGCCCACTCGGGGGGATCCCCGGGTACCGA

GCTCGAATTCATCGATGATATCAGATCTGCCGGTCTCCCTATAGTGAGTCGTATTAATTTCG

ATAAGCCAGGTTAACCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTAT

TGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAG

CGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGA

AAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGC

GTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGT

GGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGC

TCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGT

GGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGC

TGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGT

CTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGAT

TAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCT

ACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGA

GTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAA

GCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGT

CTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGG

ATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGA

GTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTC

TATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGC

TTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTT

ATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCG

CCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGT

TTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTGTGGTATGGC

TTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAA

AAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCA

CTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTC

TGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCT

CTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATC

ATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTC

GATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTG

GGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGT

TGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCAT

GAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTC

CCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAAT

AGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACA

CATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCC

GTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAG

CAGATTGTACTGAGAGTGCACCATATGGACATATTGTCGTTAGAACGCGGCTACAATTAATA

CATAACCTTATGTATCATACACATACGATTTAGGTGACACTATA

Expression of recombinant antibodies

Chimeric, humanized and human antibodies are typically produced by recombinant expression. Recombinant polynucleotide constructs typically include expression control sequences, including naturally associated promoter regions or heterologous promoter regions, operably linked to antibody chain coding sequences. Preferably, the expression control sequence is a eukaryotic promoter system in a vector capable of transforming or transfecting a eukaryotic host cell. Once the vector is incorporated into a suitable host, the host is maintained under conditions suitable for high level expression of the nucleotide sequence and collection and purification of the cross-reactive antibodies.

These expression vectors are typically replicable in the host organism either as episomes or as an integral part of the host chromosomal DNA. Expression vectors typically contain a selectable marker, such as ampicillin resistance or hygromycin resistance, to allow detection of those cells transformed with the desired DNA sequence.

Coli is a prokaryotic host particularly useful for cloning the DNA sequences of the present invention. Microorganisms such as yeast may also be used for expression. Saccharomyces is a preferred yeast host, and according to the need with expression control sequences, replication origin, termination sequence suitable vectors. Commonly used promoters include the 3-phosphoglycerate kinase promoter and other glycolytic enzyme promoters. Inducible yeast promoters include, among others, promoters derived from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization.

Mammalian cells are preferred hosts for expression of nucleotide segments encoding immunoglobulins or fragments thereof. See Winnacker, FROM GENES TO CLONES, (VCHPublishers, NY, 1987). Many suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, including CHO cell lines, various COS cell lines, HeLa cells, L cells, and myeloma cell lines. The cell is preferably non-human. Expression vectors for use in these cells may include expression control sequences such as origins of replication, promoters, enhancers (Queen et al, 1986, Immunol. Rev.89: 49); and essential processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcription termination sequences. Preferred expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adenovirus, bovine papilloma virus, and the like. See Co et al, 1992, j. immunol.148: 1149.

alternatively, antibody coding sequences can be incorporated into a transgene for introduction into the genome of a transgenic animal and subsequent expression in the milk of the transgenic animal (see, e.g., U.S. patent nos. 5,741,957, 5,304,489, and 5,849,992). Suitable transgenes include coding sequences for light and/or heavy chains operably linked to promoters and enhancers from mammary gland-specific genes (e.g., casein gene or β -lactoglobulin).

The vector containing the DNA segment of interest may be transferred into the host cell by well-known methods, depending on the type of cellular host. For example, for prokaryotic cells, calcium chloride transfection is commonly utilized, while for other cellular hosts, calcium phosphate treatment, electroporation, lipofection, biolistics (biolistics), or viral-based transfection may be used. Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see generally, Sambrook et al, supra). For the production of transgenic animals, the transgene may be microinjected into fertilized eggs, or may be incorporated into the genome of embryonic stem cells, and the nuclei of such cells are then transferred into enucleated oocytes.

Once expressed, the antibody can be purified according to standard procedures in the art, including HPLC purification, column chromatography, gel electrophoresis, and the like (see generally Scopes, protein purification (Springer-Verlag, NY, 1982)).

Modified antibodies

The invention also includes modified antibodies. The term "modified antibody" includes antibodies that have been modified, e.g., by the deletion, addition or substitution of portions of the antibody, such as monoclonal antibodies, chimeric antibodies and humanized antibodies. For example, an antibody may be modified by deleting its constant regions and substituting constant regions that may result in an increase in the half-life of the antibody, such as serum half-life, stability or affinity.

The antibody conjugates of the invention can be used to alter a given biological response or to elicit a biological response (e.g., recruitment of effector cells). The drug moiety should not be construed as limited to classical chemotherapeutic drugs. For example, the drug moiety may be a protein or polypeptide having a desired biological activity. Such proteins may include, for example, enzymatically active toxins or active fragments thereof, such as abrin, ricin a, pseudomonas exotoxin, or diphtheria toxin; proteins, such as tumor necrosis factor or interferon- α; or biological response modifiers, such as lymphokines, interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte macrophage colony stimulating factor ("GM-CSF"), granulocyte colony stimulating factor ("G-CSF"), or other growth factors.

Techniques For conjugating such therapeutic moieties to Antibodies are well known, see, e.g., Arnon et al, "Monoclonal Antibodies For Immunotargeting Of Drugs Incancer Therapy", Monoclonal Antibodies And Cancer Therapy, Reisfeld et al (eds.), pp.243-56(Alan R.Liss, Inc.1985); hellstrom et al, "Antibodiesfor Drug Delivery", Controlled Drug Delivery (2nd Ed.), Robinson et al (eds.), pp.623-53(Marcel Dekker, Inc.1987); thorpe, "Antibody Carriers of Cytotoxin Agents In Cancer Therapy: a Review ", Monoclonal Antibodies' 84: biological And Clinical Applications, Pinchera et al (eds.), pp.475-506 (1985); "Analysis, Results, And" Analysis, applied Of The Therapeutic Using Of Radiolabed anti In Cancer Therapy ", Monoclonal Antibodies for Cancer Detection And Therapy, Baldwin et al (eds.), pp.303-16(academic Press 1985), And Thorpe et al," The Preparation Of anti cytological Properties Of anti-body-Toxin Conjugates ", Immunol.Rev., 62: 119-58(1982).

Treatment regimens

The present invention provides pharmaceutical compositions comprising one or a combination of monoclonal antibodies (intact or binding fragments thereof) formulated with a pharmaceutically acceptable carrier. Certain compositions comprise a plurality (e.g., two or more) of the monoclonal antibodies of the invention, or antigen-binding portions thereof, in a combination. In certain compositions, each antibody or antigen-binding portion thereof of the composition is a monoclonal antibody or human sequence antibody that binds to a different preselected epitope of the antigen.

In prophylactic applications, a subject susceptible to or at risk of a disease or condition (i.e., an immune disorder) is administered a pharmaceutical composition or drug in an amount sufficient to eliminate or reduce this risk, to alleviate the disease (including biochemical, histological, and/or behavioral symptoms of the disease), its complications, and the severity of or delay the onset of intermediate pathological phenotypes that exist during the onset of the disease. In therapeutic applications, a patient suspected of having or having such a disease is administered a sufficient amount of a composition or drug to cure or at least partially arrest the symptoms of the disease (biochemical, histological and/or behavioral), including its complications and intermediate pathological phenotypes during pathogenesis. An amount suitable to effect therapeutic or prophylactic treatment is defined as a therapeutically effective amount or a prophylactically effective amount. In both prophylactic and therapeutic regimens, the drug is usually administered in divided doses until a sufficient immune response is achieved. The immune response is typically monitored and if the immune response begins to resolve, repeated doses are administered.

An effective amount

The effective amount of the compositions of the present invention for treating the immune-related disorders and diseases described herein will vary depending upon a number of different factors, including the method of administration, the target site, the physiological condition of the patient, whether the patient is a human or an animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. The patient is typically a human, but non-human mammals, including transgenic mammals, may also be treated. The therapeutic dose needs to be gradually increased to optimize safety and efficacy.

For administration of the antibody, the dosage range is about 0.0001-100mg/kg of host body weight, more usually 0.01-5mg/kg of host body weight. For example, the dose may be 1mg/kg body weight or 10mg/kg body weight, or in the range of 1-10 mg/kg. An exemplary treatment regimen entails administration 1 time every two weeks or 1 time per month, or 1 time every 3-6 months. In certain methods, two or more monoclonal antibodies with different binding specificities are administered simultaneously, in which case the dose of each antibody administered is within the specified range. The antibody is typically administered multiple times. The interval between single administrations can be weekly, monthly or yearly. The intervals may also be irregular as indicated by measuring blood antibody levels in the patient. In some methods, the dose is adjusted to achieve a plasma antibody concentration of 1-1000. mu.g/ml, and in some methods 25-300. mu.g/ml. On the other hand, the antibody may be administered as a sustained release formulation, in which case less frequent administration is required. The dose and frequency will vary depending on the half-life of the antibody in the patient. In general, human antibodies exhibit the longest half-life, followed by humanized, chimeric, and non-human antibodies. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at relatively low frequency intervals over a prolonged period of time. Some patients will receive treatment for life thereafter. In therapeutic applications, it is sometimes desirable to administer relatively high doses at relatively short intervals until disease progression is diminished or terminated, preferably until the patient exhibits partial or complete remission of the condition. Thereafter, the patient may be given a prophylactic regimen.

For nucleic acids encoding immunogens, the dosage range is about 10ng-1g, 100ng-100mg, 1 μ g-10mg, or 30-300 μ g DNA per patient. The dose for infectious viral vectors varies from 10-100 or more virions per dose.

Route of administration

The agent for inducing an immune response may be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular routes for prophylactic and/or therapeutic treatment. The most common route of administration of immunogenic drugs is subcutaneously, although other routes may be equally effective. The next most common route is intramuscular injection. This type of injection is most commonly performed in the arm or leg muscles. In certain methods, the drug is injected directly into a specific tissue where the precipitate (precipitate) has accumulated, e.g., intracranial injection. For administration of the antibody, intramuscular injection is preferred over intravenous infusion. In certain methods, a particular therapeutic antibody is injected directly into the cranium. In certain methods, the antibodies are administered as a slow release composition or device, such as Medipad^TMThe device is administered.

The agents of the invention may optionally be administered in combination with other agents that are at least partially effective in the treatment of various diseases, including immune-related diseases. In the case of Alzheimer's disease and Down's syndrome, where amyloid deposits occur in the brain, the agents of the invention may also be administered in combination with other agents that increase the crossing of the Blood Brain Barrier (BBB) by the agents of the invention.

Preparation

The agents of the invention are typically administered as pharmaceutical compositions comprising the active therapeutic agent, i.e., and a wide variety of other pharmaceutically acceptable components. See Remington's pharmaceutical Science (15th ed., Mack Publishing Company, Easton, Pennsylvania, 1980). The preferred form depends on the intended mode of administration and therapeutic application. Depending on the desired formulation, the composition may also include a pharmaceutically acceptable non-toxic carrier or diluent, which is defined as a carrier commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate buffered saline, Ringer's solution, dextrose solution and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, non-therapeutic, non-immunogenic stabilizers, and the like.

The pharmaceutical composition may also include large slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid, and copolymers (e.g., latex functionalized sepharose)^TMAgarose, cellulose, etc.), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes). In addition, these carriers can be used as immunostimulants (i.e., adjuvants).

For parenteral administration, the medicaments of the invention may be administered as a solution or suspension of an injectable dose of the substance in a physiologically acceptable diluent with a pharmaceutically acceptable carrier, which may be a sterile liquid such as water, oil, saline, glycerol or ethanol. In addition, adjuvants may be present in the composition, such as wetting or emulsifying agents, surfactants, pH buffering substances and the like. Other ingredients of the pharmaceutical compositions are those derived from petroleum, animal, vegetable or synthetic sources, such as peanut oil, soybean oil and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, especially for injectable solutions. The antibody may be administered in the form of a long acting injection (depot) or implant formulation which may be formulated in a manner which allows sustained release of the active ingredient. One exemplary composition comprises 5mg/ml of monoclonal antibody formulated in an aqueous buffer consisting of 50mM L-histidine, 150mM NaCl adjusted to pH 6.0 with HCl.

Typically, the compositions are formulated as injections, either as liquid solutions or suspensions; solid forms suitable for solution or suspension in a liquid vehicle prior to injection can also be prepared. The formulations may also be emulsified as described above or encapsulated in liposomes or microparticles such as polylactides, polyglycolides or copolymers to enhance the adjuvant effect (see Langer, 1990, Science 249: 1527 and Hanes, 1997, Advanced Drug Delivery Reviews 28: 97-119). The medicament of the present invention may be administered in the form of a long-acting injection or an implant formulation, which is formulated in such a manner as to allow sustained or pulsed release of the active ingredient.

Other formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications.

For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1% to 2%. Oral formulations include excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. These compositions are in the form of solutions, suspensions, tablets, pills, capsules, sustained release preparations or powders, containing 10% to 95% of active principle, preferably 25% to 70% of active principle.

Topical administration can result in transdermal or intradermal delivery. Topical administration may be facilitated by co-administration of the drug with cholera toxin or detoxified derivatives or subunits thereof, or other similar bacterial toxins (see Glenn et al, 1998, Nature 391: 851). Co-administration can be achieved using linker molecules obtained as a mixture or by chemical cross-linking or as expression of fusion proteins.

On the other hand, transdermal delivery can be achieved with skin patches or transferosomes (Paul et al, 1995, Eur.J.Immunol.25, 3521-24; Cevc et al, 1998, biochem.Biophys.acta 1368, 201-15).

The pharmaceutical compositions are generally formulated to be sterile, substantially isotonic, and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. food and drug administration.

Toxicity

Preferably, a therapeutically effective amount of a protein described herein will provide a therapeutic benefit without causing significant toxicity

Toxicity of the proteins described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining LD₅₀(dose lethal to 50% of the population) or LD₁₀₀(the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used to formulate a non-toxic dosage range for human use. The dosage of the proteins described herein is preferably within a range of circulating concentrations that include an effective amount that is low or non-toxic. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be selected by The individual physician according to The condition of The patient (see, e.g., Fingl et al, 1975, in: The Pharmacological Basis of Therapeutics, Chapter 1, page 1).

Reagent kit

Kits comprising the compositions of the invention (e.g., monoclonal antibodies, human sequence antibodies, human antibodies, multispecific molecules, and bispecific molecules) and instructions for use are also within the scope of the invention. The kit may further comprise at least one additional agent, or one or more additional antibodies of the present invention (e.g., a human antibody having complementary activity to bind to an epitope in the antigen that is different from the first human antibody). The kit typically includes a label indicating the intended use of the kit contents. The term label includes any writing or recording material supplied on, with or accompanying the kit.

Examples

Example 1

Generation of C.mu.M-primed mice

And constructing a CMD targeting vector. Plasmid pICE. mu. contains an EcoRI/XhoI fragment spanning the murine Ig heavy chain locus of the. mu. gene, obtained from the Balb/C genomic lambda phage library (Marcu et al, 1980, Cell 22: 187). This genomic fragment was subcloned into the XhoI/EcoRI site of the plasmid pCEMI 9H (Marsh et al, 1984, Gene 32: 481-485). The heavy chain sequence included in pICE μ extends from just downstream of the EcoRI site located 3' to the μ intron enhancer to an XhoI site located about 1kb downstream of the last transmembrane exon of the μ gene; however, most of the μ -switching repeat region has been deleted due to passage in E.coli.

The targeting vector was constructed as follows (see FIG. 1). A1.3 kb HindIII/SmaI fragment was excised from pICE. mu.and subcloned into HindIII/SmaI digested pBluescript (Stratagene, La Jolla, Calif.). This pICE μ fragment extends from a HindIII site located at about 1kb of C μ 15' to a SmaI site within C μ 1. The resulting plasmid was digested with SmaI/SpeI and the approximately 4kb SmaI/XbaI fragment from pICI μ, extending from the SmaI site in C μ 13' to the XbaI site just downstream of the last C μ exon, was inserted. The resulting plasmid pTAR1 was linearized at the SmaI site and inserted into a neo expression cassette. The cassette consists of the neo Gene under the transcriptional control of the mouse phosphoglycerate kinase (pgk) promoter (XbaI/TaqI fragment; Adra et al, 1987, Gene 60: 65-74) and containing a pgk polyadenylation site (PvuII/HindIII fragment; Boer et al, 1990, Biochemical Genetics 28: 299-308). This cassette was obtained from plasmid pKJ1 (described by Tybuliwicz et al, 1991, Cell 65: 1153-1163), from which the neo cassette was excised as an EcoRI/HindIII fragment and subcloned into EcoRI/HindIII digested pGEM-7Zf (+) to yield pGEM-7(KJ 1). The neo cassette was excised from pGEM-7(KJ1) by EcoRI/SalI digestion, blunted and subcloned into the SmaI site in plasmid pTAR1 in the opposite direction to the genomic C.mu.sequence. The resulting plasmid was linearized with NotI and inserted into the herpes simplex virus thymidine kinase (tk) cassette to facilitate enrichment of ES clones with homologous recombination, as described by Mansour et al, 1988, Nature 336: 348 while 352. The cassette consists of the coding sequence of the tk gene, with the mouse pgk promoter and polyadenylation site as a scaffold, as described by tybuliwicz et al, 1991, Cell 65: 1153 and 1163. The resulting CMD targeting vector shares a total of approximately 5.3kb homology with the heavy chain genome and was designed to generate a mutant μ gene in which the neo expression cassette was inserted into the unique SmaI site of the first C μ exon. The targeting vector is linearized with PvuI cleaved within the plasmid sequence prior to electroporation into ES cells.

Production and analysis of the ES cells being hit. AB-1ES Cells (McMahon, A.P. and Bradley, A.1990, Cell 62: 1073-1085) were grown on a mitotically inactive SNL76/7 Cell feeder layer (supra) essentially as described (Robertson, E.J. (1987) Teratoocci and Embryonic Stem Cells: APracial apparatus (E.J.Robertson), Oxford, IRL Press, p.71-112). The linearized CMD targeting vector was electroporated into AB-1 cells using the method described by Hasty et al (Hasty, P.R., et al, 1991, Nature 350: 243-. Electroporated cells were plated at 1-2X 10⁶The density of cells/dish was seeded into 100mm culture dishes. After 24 hours, G418 (200. mu.g/ml active principle) and FIAU (5X 10)^-7M), allowing resistant clones to develop within 8-9 days. The clones were picked, treated with trypsin, divided into two parts, and further expanded for culture. Half of the cells from each clone were then frozen and the other half analyzed for homologous recombination between the vector and the target sequence.

DNA analysis was performed by southern blot hybridization. DNA was isolated from the clones as described by Laird et al (Laird, P.W. et al, 1991, Nucleic Acids Res.19: 4293). The isolated genomic DNA was digested with SpeI and probed with a 915bp SacI fragment, Probe A (FIG. 1), which hybridizes to the sequence between the μ intron enhancer and the μ transition region. Probe A detected a 9.9kb SpeI fragment from the wild-type locus and a 7.6kb discriminatory band from the mu locus that had been homologously recombined with the CMD targeting vector (neo expression cassette contains a SpeI site). Of the 1132G 418 and FIAU resistant clones screened by southern analysis, 3 clones showed the 7.6kb SpeI band indicative of homologous recombination at the μ locus. These 3 clones were further digested with the enzymes BglI, BstXI and EcoRI to confirm the homologous integration of the vector into the μ gene. Southern blots of wild-type DNA digested with BglI, BstXI or EcoRI produced 15.7, 7.3 and 12.5kb fragments, respectively, while 7.7, 6.6 and 14.3kb fragments indicate the presence of the hit mu allele, respectively, when hybridized with probe a. All 3 positive clones detected by SpeI digestion showed the expected restriction fragments identifying BglI, BstXI and EcoRI for the insertion of the neo cassette into the C.mu.1 exon.

Generation of mice with mutant μ genes. The 3 hit ES clones designated 264, 272 and 408 were thawed and recovered as described by Bradley (Bradley, A., 1987, by Teratocaccinomas and Embrying Stem Cells: a Practical apparatus, (E.J. Robertson eds.) Oxford: IRL Press, p.113-151) and injected into C57BL/6J blastocysts. The injected blastocysts were transferred into the uterus of pseudopregnant female mice, resulting in chimeric mice representing a mixture of cells derived from the input ES cells and host blastocysts. The degree of contribution of ES cells to the chimera can be visually estimated based on the amount of ES cell line derived from the guinea pig hair color on a black background of C57 BL/6J. Clones 272 and 408 produced only a low percentage of chimeras (i.e., a low percentage of guinea pig staining), while clone 264 produced a high percentage of male chimeras. These chimeras were bred to C57BL/6J females to produce guinea pig offspring indicative of germline inheritance of the ES cell genome. The knockdown μ genes were screened by southern blot analysis of DNA from BglI-digested tail biopsy samples (as described above for ES cell DNA analysis). About 50% of the guinea pig progeny showed a 7.7kb BglI hybridizing band in addition to the 15.7kb wild-type band, demonstrating germline inheritance of the targeted μ gene.

Analysis of functional inactivation of the mu gene of transgenic mice. To determine whether insertion of the neo cassette into C μ 1 inactivated the Ig heavy chain gene, clone 264 chimeras were bred to mice homozygous for the JHD mutation, which inactivated heavy chain expression due to deletion of the JH gene segment (Chen et al, 1993, Immunol.5: 647-656). 4 guinea pig offspring were generated. Sera were obtained from 1 month old animals and analyzed for the presence of murine IgM by ELISA. 2 of the 4 progeny completely lacked IgM (table 1). Tail biopsy sample DNA was genotyped in the 4 animals by digestion at BlI and hybridization to probe a (fig. 1), and by StuI digestion and hybridization to a 475bp EcoRI/StuI fragment (supra), demonstrating that animals that were unable to express serum IgM were animals in which one allele in the heavy chain locus carried the JHD mutation and the other allele carried the C μ 1 mutation. Mice heterozygous for the JHD mutation show wild-type levels of serum Ig. These data demonstrate that the C μ 1 mutation inactivates the expression of the μ gene.

TABLE 1

Mouse	Serum IgM (microgram/ml)	IgH chain genotype
			42	＜0.002	CMD/JHD
43	196	+/JHD
			44	＜0.002	CMD/JHD
45	174	+/JHD
			129×BL6F1	153	+/+
JHD	＜0.002	JHD/JHD

Table 1 shows serum IgM levels of mice carrying the CMD mutation and the JHD mutation (CMD/JHD), JHD mutation heterozygous mice (+/JHD), wild type mice (129Sv × C57BL/6J) F1 mice (+/+) and B cell deficient JHD mutation homozygous mice (JHD/JHD) detected by ELISA.

Example 2

Human kappa light chain transgene KCo5

The generation of the human kappa light chain transgenic mouse line KCo5-9272 has been previously described as KCo5(Fishwild, D. et al, 1996, nat. Biotechnol.14, 845-. This line was generated by co-injection of the human worker kappa light chain locus and YAC clones containing multiple human vk segments. YAC clone DNA was isolated from yeast strains containing 450kb Yeast Artificial Chromosomes (YACs) comprising a portion of the human vk locus (ICRF YAC library designation 4x17E 1). DNA sequence analysis of the V gene segments amplified from the YAC DNA demonstrated that this clone contained a substantial portion of the human distal vk region, including approximately 32 different vk segments. Analysis of the different isolates of this clone (Brensing-Kuppers, J.et al 1997, Gene 191: 173-181) confirmed this result, and also demonstrated that this clone represents an example of a C haplotype of the human kappa locus, in which the 5' portion of the distal V Gene cluster resembles the homologous region of the proximal V Gene cluster. Thus, the 5' O family V gene segment is in sequence proximity to the homologous proximal Op family V segment.

To obtain purified YAC DNA for microinjection into embryonic pronuclei, total genomic DNA was size fractionated on agarose gels. Yeast cells containing YAC 4x17E1 were embedded in agarose prior to lysis, YAC DNA was separated from yeast chromosomal DNA by pulsed field gel electrophoresis, YAC DNA was isolated and microinjected into half-day-old embryo pronuclei.

Southern analysis of genomic DNA demonstrated that the human VkA10 gene (Cox, J. et al, 1994, Eur. J. Immunol.24: 827-. PCR analysis (Brensing-Kuppers, J. et al, 1997, Gene 191: 173-181) using a probe specific for the V.kappa.O 15 ' region (m217-1, Genbank X76071; AB129, ccaccccataaacactgattc (SEQ ID NO: 4); AB130, ttgatgcatcctacccagggc (SEQ ID NO: 5)) and a probe specific for the intergenic region between V.kappa.L 24 and L25 (m138-13, Genbank X72824; AB127, cctgccttacagtgctgtag (SEQ ID NO: 6); AB128, ggacagcaacaggacatggg (SEQ ID NO: 7)) showed that the 5 ' and 3 ' regions of the V.kappa.gene cluster from YAC clone 4X17E1 were included in the KCo5-9272 transgene integrants. Mice of line KCo5-9272 were then bred with mice with mutated human heavy chain transgenic endogenous immunoglobulin loci to obtain mice homozygous for disruption of endogenous heavy chain and kappa light chain loci, as well as hemizygous or homozygous for human heavy chain transgenic HC2 or HCo7 (U.S. Pat. No. 5,770,429) and human kappa light chain transgenic KCo 5. Animals homozygous for disruption of endogenous heavy and kappa light chain loci, and hemizygous or homozygous for the human heavy chain transgene and the human kappa light chain transgene were designated double transgenic/double deleted mice.

DNA sequence analysis of cDNA clones either directly from KCo5 double transgenic/double deleted mice or from hybridomas produced by these animals revealed the following vk genes expressing L6, a27, O12, O4/O14, a10, L15, L18, L19 and L24.

Example 3

Crossbreeding

The human heavy chain locus containing chromosome 14 fragment hCF (SC20) and the human kappa light chain transgene were combined into one line by cross breeding. The hCF (SC20) transgenic mouse strain was homozygous for the endogenous heavy chain locus (CM2D) and the endogenous kappa light Chain (CKD) inactivating mutation. This line is also homozygous for the λ 1(low) mutation (Tomizuka, K. et al, 2000, Proc. Natl. Acad. Sci. U.S.A.97: 722-one 727). The CM2D mutation contained a portion covering C μ 2, C μ 3-C μ 4, and a deletion of the 3.7kb BamHI-XhoI segment of M μ 1 and M μ 2. The CKD mutation comprises a deletion of the 2kb SacII-BglII segment covering the C.kappa.exon. These two mutations have been previously reported (Tomizuka, K. et al, 2000, Proc. Natl. Acad. Sci. U.S.A.97: 722-one 727). These mice were bred with KCo5-9272 human kappa transgene insertion homozygous mice and CMD and JKD disruption of endogenous heavy and kappa chain loci, respectively. The CMD mutations are described in example 1 above. JKD mutations are described in U.S. patent No. 5,770,429 and Chen et al, 1993, EMBO j.12: 821-830). hCF (SC20) transfectant positive progeny (SC20/KCo5 mice, or hybrid mice) obtained from these matings were hemizygous for the following 6 different genetic modifications: SC20, KCo5, CMD, CM2D, JKD and CKD. However, these SC20/KCo5 mice were homozygous for disruption of each of the two loci, since CMD and CM2D mutations of the endogenous heavy chain locus prevented expression of the mouse μ gene, while JKD and CKD mutations of the endogenous kappa locus prevented expression of mouse κ. Thus, the expression of kappa light chain-containing antibodies in the mice was dependent on SC20 and KCo5 transgenes. They can also form hybrid human/mouse antibodies because the endogenous mouse lambda light chain locus is still functional. The mouse may also express a chimeric human/mouse antibody comprising human heavy chain V region and mouse non-mu heavy chain isotype constant region sequences. These chimeric antibodies can be formed by class switching-mediated chromosomal translocation of the human SC20IgH locus into the mouse IgH locus. Such a "switching" event was previously found to occur in mice containing a small locus heavy chain transgene (Taylor, L. et al, 1994, int. Immunol.6: 579-. Cross breeding between 40 male Kco5/CMD/JKD mice and 98 female hCF (SC20)/CM2D/CKD mice yielded 305 pups. Of serum samples prepared from these young animals

ELISA analysis (Tomizuka, K.et al, 2000, Proc. Natl.Acad.Sci.U.S.A.97: 722-727) showed that 125 (41%) of 305 pups were positive for human Ig. mu. chain expression further analysis detecting human Ig. kappa. chains showed that all h. mu. positive individuals were also h. kappa positive, indicating KCo5 transgene retention (see example 2). The primer pairs (Tomizuka, K.et al, 2000, Proc. Natl.Acad.Sci.U.S.A.97, Acassa.722 727) used to detect hCF (SC20) showed that all h. mu. DNA positive PCR analysis retained, all hCbCk.82, and all negative PCR analysis (hCmax. Natl.Sci.S.A.727) showed that all H. mu. positive PCR (hCzoc) retained the DNA tail DNA, PCR (Sci.32) and all negative PCR (Sci.S.32) were reported from previous rC. Natl.S.S.S.S.S.46727) 2: SC 39727).

Example 4

Expression of human Ig in serum of hybrid mice

Serum samples prepared from 6-12 week old hybrid mice were examined by ELISA to determine the concentrations of human Ig μ, γ, κ and mouse λ chains (fig. 2). In contrast to endogenous C.mu.deleted hemizygous mice maintained under similar conditions, the average levels of human Ig. mu.and Ig γ were higher than the mouse μ chain level (273mg/l), which was 1/3 of the mouse γ chain level (590mg/l), respectively. These heavy chain expression levels were similar to those of the double Tc/double KO mice (hCF (SC20)/hCF (2-W23)/CM2D/CKD Tomizuka, K. et al, 2000, Proc. Natl. Acad. Sci. U.S.A.97: 722-727). The F2 progeny of 1/4 produced by mating between male and female hybrid mice are expected to be homozygous for the m λ C1(λ low) mutation, since the first generation of hybrid mice is heterozygous for the mutation. The serum concentrations of human Ig kappa light chain and mouse Ig lambda light chain were determined by ELISA in 21F 2 hybrid mice (Tomizuka, K. et al, 2000, Proc. Natl. Acad. Sci. U.S.A.97: 722-727) previously described in the report. Of the 21 mice examined, 6 showed a low (< 0.1) mouse λ/human κ ratio, which is characteristic of λ low mutant homozygous mice (Tomizuka, K. et al, 2000, Proc. Natl. Acad. Sci. U.S.A.97: 722-727). Thus, these 6 hybrid mice are likely homozygous for the λ (low) mutation, which can be used to efficiently produce hybridomas secreting antibodies comprising human Ig heavy and κ light chains.

Example 5

Generation of human monoclonal antibodies to human CD4

Immunization of antigens. Hybrid and double Tc/KO mice (n ═ 5) were immunized subcutaneously by injection of 100 μ g of soluble human CD4(sCD4) in freund's complete adjuvant (Sigma) on day 0, and then immunized with 100 μ g of soluble human CD4 in freund's complete adjuvant (Sigma) on days 9, 19, and 27. PBS containing 40. mu.g sCD4 was injected intravenously last on day 37.

Humoral responses in mice. Sera were collected on days 0, 16, 26, 34 and 40. Antigen-specific human Ig γ and Ig κ were measured by enzyme-linked immunosorbent assay (ELISA) based on the production of monoclonal antibodies (MAb) against sCD 4. The detailed protocol for ELISA is described in example 4. Antigen specific plates were coated overnight with bicarbonate buffer (Sigma) containing 1. mu.l/ml antigen. Antigen-specific Ig γ and Ig κ were quantitatively determined using a human monoclonal IgG specific to the antigen as a standard. The results are shown in FIGS. 3,4, 5 and 6. Human gamma and kappa responses were observed in hybrid and double Tc/double KO mice 34 days after primary immunization.

Generation of hybridomas. On day 40, splenocytes from immunized mice were fused with Sp2/0-Ag14 cells. The cell suspension was seeded into 384-well plates at 20,000 splenocytes/well. The resulting hybridomas were screened for production of anti-sCD 4 monoclonal antibodies (MAb). The results are shown in table 1 below.

TABLE 1

Production of CD4 monoclonal antibody

	Hybrid seed	Bis Tc/KO
			Number of wells with colonies	1265	720

	Hybrid seed	Bis Tc/KO
			Antigen-specific hgamma/hkappa positive well number	18	4
Number of positive wells for antigen specificity h gamma/m lambda	0	0
			Number of subcloned parent wells	14	1
Subcloning efficiency (%)	88	21

The parental hybridomas from the hybrid mice were subcloned efficiently by two rounds of limiting dilution. All hybridomas from the hybrid mice secreted human γ/human κ anti-CD 4MAb, and none secreted human γ/mouse λ anti-CD 4 MAb. These data indicate that hybrid mice are superior to the dual TC/KO strain for the production of antigen-specific human monoclonal antibodies. The isoforms of MAb secreted by these subcloned hybridomas were further examined by multiple ELISA. 7 holes are h.gamma.1⁺And 7 holes are h gamma 4⁺。

Growth curves and anti-CD 4 human IgG in Small Scale cultures₁Secretion level of monoclonal antibody. Generation of anti-CD 4 human IgG with one of them₁Kappa hybridoma clone (KM2-3) to determine growth curves and secretion levels of human monoclonal antibodies in small-scale cultures. Day 0, KM2-3 hybridoma cells were cultured at 1X 10⁵Cells/ml were seeded into 4 liter spinner flasks (Bellco). Culture was performed using 1 liter of ERDF medium supplemented with ITS-X (Gibco BRL) and 1% low IgG serum (Hyclone). 1ml of medium was collected daily, cell number was measured and IgG was measured by ELISA as described in previous reports (Tomizuka, K. et al, 2000, Proc. Natl. Acad. Sci. U.S.A.97: 722-727)₁Kappa concentration. The results are shown in FIG. 7. The estimated productivity was 24.6 pg/cell/day, which is in a range similar to that expected for murine hybridomas that were superior under these conditions.

Example 6

Generation of human monoclonal antibodies to human G-CSF

Immunization of antigens. Hybrid and dual Tc/KO mice (n-5) were immunized by subcutaneous injection of 100 μ G of soluble human G-CSF in TiterMaxGold adjuvant (CytRx) on days 0, 9, 19, 27. On day 37, hybrid and double Tc/KO mice were given a final intravenous injection of 20 μ g G-CSF in PBS.

Humoral responses in each mouse strain. Sera were collected on days 0, 16, 26, 34 and 40. The concentration of antigen-specific human Ig was determined quantitatively by ELISA. Antigen specific plates were coated overnight with bicarbonate buffer (Sigma) containing 1. mu.g/ml antigen. Antigen-specific Ig gamma and Ig kappa were quantitatively determined using a human monoclonal IgG specific for G-CSF as a standard. The results are shown in fig. 8, 9, 10 and 11. The antigen-specific h γ and h κ concentrations in the sera of the hybrid mice were about 10 times those of the double Tc/KO mice.

Generation of hybridomas. On day 40, splenocytes from immunized mice were fused with Sp2/0-Ag14 cells, and the resulting hybridomas were screened by ELISA for the production of anti-G-CSF monoclonal antibodies (MAb). The results are shown in table 2 below.

TABLE 2

Production of G-CSF monoclonal antibodies

	Hybrid seed	Bis Tc-KO
			Number of wells with colonies	3880	1580
Antigen-specific hgamma/hkappa positive well number	13	3
			Number of positive wells for antigen specificity h gamma/m lambda	13	0
Number of subcloned parent wells	11	2
			Subcloning efficiency (%)	83	64

Half of the hybridomas producing anti-G-CSF IgG secrete human γ/human κ anti-G-CSFMAb, and the remaining hybridomas secrete human γ/murine λ anti-G-CSF MAb. Hybridomas producing h γ/h κ antibodies were subcloned by two rounds of limiting dilution. Further ELISA experiments demonstrated that 5,3 and 3 wells, respectively, were h γ 1⁺、hγ2⁺And h γ 4⁺。

Example 7

Production of human monoclonal antibodies to human serum albumin

The hybrid mice were immunized by intraperitoneal injection of 50 μ g of human serum albumin in Freund's complete adjuvant (Sigma) on day 0, and then immunized with 50 μ g of human serum albumin in Freund's incomplete adjuvant (Sigma) on days 7, 14, and 21.

Generation of hybridomas. On day 24, splenocytes from immunized mice were fused with Sp2/0-Ag14 cells, and the resulting hybridomas were screened by ELISA for the production of monoclonal antibodies (MAb) against the antigen. 10-well hybridomas were randomly selected from the albumin h γ resistant hybridomas and subcloned. All hybridomas secrete human gamma/human kappa anti-albumin. This data indicates that hybrid mice are superior to dual TC/dual KO mice for the generation of antigen-specific fully human monoclonal antibodies, since the 2/3 anti-albumin IgG hybridoma from a dual TC/dual KO mouse is m λ⁺(Tomizuka, K. et al, 2000, Proc. Natl. Acad. Sci. U.S.A.97: 722-one 727).

Example 8

Production of anti-human CTLA-4 monoclonal antibodies

An antigen. DNA-encoding segments of fusion proteins comprising sequences from the human CTLA-4 and murine CD3 ζ genes were constructed by PCR amplification of cDNA clones and bridging synthetic oligonucleotides. The encoded fusion protein contains the following sequence: i.) human CTLA-4 encoding amino acids 1-190 (containing the signal peptide, extracellular domain and entire human CTLA-4 putative transmembrane sequence of human CTLA-4); and ii.) murine CD3 ζ from amino acid 52 to the carboxy terminus. The PCR amplification product was cloned into a plasmid vector and the DNA sequence was determined. The cloned insert was then subcloned into the vector pBABE containing the gene coding for puromycin resistance (Morgansten, JP and Land, H, 1990 Nucl. acids Res.18: 3587-96) to produce pBABE-huCTLA-4/CD3 ζ, pBABE-huCTLA4/CD3 ζ was transfected into the retroviral packaging line ψ -2, a puromycin resistant cell bank was selected, these cells were co-cultured with the murine T cell hybridoma BW5147(ATCC # TIB-47), after 2 days of co-culture, nonadherent BW5147 cells were removed, selection was made for puromycin resistance by limiting dilution, the puromycin resistant cell bank was subcloned, and a clone expressing high levels of human CTLA-4 on the cell surface (BW-huCTLA-4CD 3-3 #12) was selected by FACS-4 testing based on surface expression of human CTLA-4 From R & D Systems (Cat. # 325-CT-200).

And (4) immunization. 3 SC20/KCo5 hybrid mice (ID #22227, 22230 and 22231) were immunized by intraperitoneal (i.p.) injection of 10e7 whole cells of BW-huCTLA-4CD3 ζ -3#12, which express the extracellular domain of human CTLA-4 by washing, respectively. This immunization step was repeated 2 or more times for mice #22227 and 22230 at approximately 1 month intervals. At month 3, mouse #22231 was injected intraperitoneally with the washed whole cells for a third time, while mice #22227 and 22230 were injected intraperitoneally and subcutaneously (s.c.) with 20 micrograms of soluble recombinant antigen in MPL + TDM adjuvant (Sigma Cat. # M6536), respectively. The mice were then allowed to rest for 10 days, and mice #22227 and 22230 were injected in tail vein (i.v.) with 20 micrograms of soluble recombinant antigen and intraperitoneally with 20 micrograms of soluble recombinant antigen in MPL + TDM adjuvant 2 days prior to harvesting spleen cells for hybridoma fusion, respectively. One day prior to harvesting splenocytes, these mice were given an additional intravenous injection of 20 micrograms of soluble recombinant antigen. 3 days before harvesting splenocytes, 10e7 washed BW-huCTLA-4CD3 ζ -3#12 cells in mouse #22231MPL + TDM adjuvant were administered intraperitoneally, followed by 2 days before fusion, intraperitoneally, with 10 without adjuvant⁷Washed BW-huCTLA-4CD3 ζ -3#12 cells.

And (4) fusing. IN 3 independent experiments, cells were obtained from mice, such as mouse myeloma cells, mouse hybridoma cells, mouse myeloma cells, mouse clone cells, mouse myeloma cells, mouse cells. The cells were cultured in DMEM, 10% FBS, OPI (Sigma O-5003), BME (Gibco 21985-023) and 3% origenHybroma Cloning Factor (Igen IG 50-0615). During initial growth and selection, HAT or HT supplements were added to the medium.

And (4) screening the hybridoma. To identify hybridomas secreting antigen-reactive human IgG antibodies, ELISA plates (Nunc MaxiSorp) were coated overnight at 4 ℃ with 100. mu.l/well of PBS containing 0.2. mu.g/ml of human CD152Mu-Ig fusion (Ancel # 501-820). Plates were washed and blocked with 100. mu.l/well PBS-Tween containing 1% BSA. 50 μ l of cell culture supernatant was added, followed by incubation for 1-2 hours. Plates were washed and then incubated for 1 hour with 100. mu.l/well of goat anti-human gamma heavy chain conjugated with alkaline phosphatase (anti-human gamma (fc) AP Jackson # 109-056-098). Plates were washed 3 times with PBS-Tween between each step. 76 hybridomas secreting gamma positive antigen reactive antibodies were identified. These clones were then further analyzed to determine the presence or absence of gamma heavy or light chain isotype and contaminating IgM secreting cells (table 3).

TABLE 3

Analysis of heavy chain isotypes from 1 ° hybridoma wells containing antigen-reactive human IgG antibodies

Mouse ID #	IgM	IgG	IgG	IgG	IgG	Igκ	Igλ	All IgG' s
									22227	0	4	1	0	3	7	0	8
22230	9	25	8	5	7	48	6	45

Mouse ID #	IgM	IgG	IgG	IgG	IgG	Igκ	Igλ	All IgG' s
									22231	1	11	2	3	7	23	1	23
Total of	10	40	11	8	17	75	7	76

Hybridoma supernatants were first tested for the presence of antigen-reactive human IgG. 76 positive supernatants were then tested for antigen reactive human IgM, IgG₁、IgG₂、IgG₃、IgG₄Ig κ and mouse Ig λ. Capture reagent: human CD 152. mu. Ig fusion (Ancel # 501-820). Detection reagent: anti-human gamma (fc) HRP (Jackson # 109-; anti-human kappa HRP (Bethyyl # A80-115P); anti-human gamma 1 HRP (Southern Biotech # 9050-05); anti-human gamma 2HRP (Southern Biotech # 9070-05); anti-human gamma 3HRP (southern Biotech # 9210-05); anti-human gamma 4 HRP (Southern Biotech # 9200-05); anti-human μ HRP (Southern Biotech # 1060-05).

75 of the 76 IgG antigen-positive wells were also positive for human kappa light chain antigen-reactive antibodies, while 7 of the wells were positive for hybrid antibodies containing mouse lambda. However, 6 of the 7 λ -positive wells also contained kappa light chains, 3 of these 3 wells were positive for contaminating IgM antigen-reactive antibodies. Since these contaminating IgM antibodies may have an impact, including lambda light chains, there are 3-7 IgG lambda clones out of a total of 76 IgG clones. Thus, endogenous mouse λ appears to produce only 4-9% of the IgG-positive antigen-reactive hybridomas. Cells from 22 of the 76 positive hybridoma wells were then reseeded by limiting dilution to subclone hybridomas secreting individual monoclonal antibodies. From 19 out of 22 of the 1 ° hybridomas, stable antigen-reactive human IgG subclones were obtained (see table 4 below).

TABLE 4

Subcloning of anti-CTLA-4 hybridomas

Cloning	OD	Number of clones tested	Number of positive	Percentage of positivity
					4C1	0.44	24	5	21％
2E4	1.48	24	9	38％
					1H5	1.39	24	14	58％
9C4	1.30	24	5	21％
					6D11	3.24	16	10	63％
10H3	1.59	16	2	13％
					8H4	3.14	16	7	44％
8G5	1.38	8	3	38％
					4A9	1.35	24	20	83％

Cloning	OD	Number of clones tested	Number of positive	Percentage of positivity
					10E1	1.17	24	3	13％
9F6	1.08	24	0	0％
					689	1.16	16	5	31％
9B10	2.70	32	9	28％
					10D1	0.90	48	6	13％
1B6	1.34	24	9	38％
					4C7	1.34	8	2	25％
1D11	0.97	8	0	0％
					1B5	2.75	8	3	38％
4E9	1.36	24	1	4％
					11H7	0.40	16	0	0％
2D8	1.31	24	10	42％
					8F2	1.28	16	5	31％

Thus, a subcloning efficiency of 86% was obtained. When subcloned, one of the 1 ° hybridomas was found to contain 2 different clones with different IgG isotypes (see table 5 below).

TABLE 5

Isotype analysis of human IgG kappa anti-CTLA-4 subclones

Mouse	Cloning	Parent well	IgGκ	IgGκ	IgGκ	IgGκ
							22227	8G5	IgGκ	+	-	-	-
22227	6B9	IgGκ	+	-	-	-
							22230	1B5	IgGκ	-	-	+	-
22230	2D8	IgGκ	+	-	-	-

Mouse	Cloning	Parent well	IgGκ	IgGκ	IgGκ	IgGκ
							22230	6D11	IgGκ	-	-	-	+
22230	8H4	IgGκ	-	-	-	+
							22230	9C4	IgGκ	-	-	+	-
22230	10H3	IgGκ	-	-	+	-
							22231	1B6	IgGκ	+	-	-	-
22231	1H5	IgGκ	+	-	-	-
							22231	2E4	IgGκ	+	-	-	-
22231	4A9	IgGκ	+	-	-	-
							22231	4C1.1	IgGκ	-	-	-	+
22231	9B10	IgGκ	+	-	-	-
							22231	4C7	IgGκ	-	-	+	-
22231	10D1.1	IgGκ，IgGκ	+	-	-	-
							22231	10D1.4	IgGκ，IgGκ	-	-	-	+
22231	10E1	IgGκ	-	-	-	+
							22231	8F2	IgGκ	+	-	-	-
22231	4E9	IgGκ	+	-	-	-

Thus, 20 different subclones were obtained. All 20 clones used human kappa light chains and were fully human clones.

Monoclonal antibodies were isolated from 5 of the subcloned hybridomas (1H5, 4a9, 4C1, 8H4, and 10E1) and tested for their ability to block CTLA-4 binding to B7.2 (fig. 12 and 13).

Briefly, ELISA plates were coated with 0.7. mu.g/ml (100. mu.l/well) of B7.2Ig fusion protein (see WO 01/14424, which is incorporated herein by reference in its entirety for all purposes). Plates were washed and blocked with PBS-T + 1% BSA for 30 min. The antibody was mixed with an equal volume of 0.2. mu.g/ml biotin-labeled CTLA-4Ig (Ancell #501-030), pre-incubated at room temperature for 1 hour, then transferred to a B7.2-coated ELISA plate and incubated for 1 hour. Plates were washed and 100. mu.l/well streptavidin alkaline phosphatase (Kirkegaard and Perry Labs15-30-0) was added and incubated for 1 hour. Plates were developed with pnp substrate. Inhibition of binding of biotin-labeled CTLA-4 to B7.2 was plotted as antibody concentration versus absorbance at 405 nm. Antibody 10D1 is a CTLA-4 specific human IgG₁(see WO 01/14424). Antibody isotype is 1H5.1(γ)₁)、4A9.(γ₁)、4C1.1(γ₄)、8H4.4(γ₄)、10E1.1(γ₄) And 10D1(γ)₁)。

Of these, 2 antibodies (1H5 and 4a9) were found to be blocking antibodies, and 3 antibodies (4C1, 8H4 and 10E1) were found to be non-blocking antibodies (fig. 12 and 13).

Administration of anti-CTLA-4 can enhance T cell mediated immune responses (Krummel, 1995, J.Exp.Med.182: 459-523; Krummel et al, 1996, Int' l Immunol.8: 519-523). Thus, CTLA-4 antibodies can be used as an adjuvant to enhance the immunogenicity of another factor. When an anti-CTLA-4 antibody is administered with another agent, the two may be administered in either order or simultaneously. The methods can be used in a variety of vaccines and treatments for which an enhanced immune response is beneficial. For example, infectious diseases and cancers, including melanoma, colon, prostate, and renal cancers.

CTLA-4 antibodies may also be used to down-regulate T cell mediated immune responses. This activity can be achieved with multivalent formulations of anti-CTLA-4 antibodies. For example, latex microspheres coated with anti-CTLA-4 (to increase the valency of the antibody) can inhibit T cell proliferation and activation. Factors with the same binding site for antibodies can act as CTLA-4 antagonists when presented as Fab or soluble IgG, and as CTLA-4 agonists when highly cross-linked. Thus, multivalent forms of anti-CTLA-4 antibodies are useful therapeutic agents for immunosuppression.

In addition to being linked to latex microspheres or other insoluble particles, the antibodies can be cross-linked to each other or genetically engineered to form multimers. Crosslinking may be performed by direct chemical linkage, or by indirect linkage such as an antibody-biotin-avidin complex. The cross-linking may be covalent, where chemical linking groups are utilized, or non-covalent, where protein-protein or other protein-ligand interactions are utilized. Genetic engineering methods of ligation include, for example, re-expression of the variable region or any protein portion (e.g., polylysine, etc.) of high affinity IgG antibodies in an IgM expression vector. By converting high-affinity IgG antibodies into IgM antibodies, a ten-valent complex with extremely high affinity can be constructed. IgA for use in therapy₂Expression vectors may also be used to produce multivalent antibody complexes. IgA for use in therapy₂Polymers may be formed with the J chain and secretory component. IgA for use in therapy₂May have the additional advantage that it may additionally be neutrophil-, megakaryocyte-depletedIgA receptor CD89 expressed on phagocytic and monocyte cells was cross-linked. Alternatively, since approximately 2% of hybridomas produced from C20/KCo5 hybrid mice are IgA, these animals can be used to produce human IgA isotype anti-CTLA-4 antibodies directly.

Agonism may also be achieved using certain preparations of anti-CTLA-4 polyclonal antibodies comprising at least two non-overlapping epitopes on CTLA-4. An antibody in this formulation, which contains two binding sites, can bind to two molecules of CTLA-4 to form a small cluster. A second antibody with a different binding site can then link (aggregate) these small clusters to form large clusters, forming CTLA-4 complexes (on the cell surface) that can transduce signals to the T cells to inhibit, attenuate, or prevent activation of CTLA-4 bearing T cells. Thus, certain polyclonal antibody preparations exhibit agonistic effects similar to the multivalent preparations described above.

Thus, multivalent or polyclonal formulations of anti-CTLA-4 antibodies can be used to agonize the CTLA-4 receptor, thereby inhibiting immune responses that would otherwise be mediated by CTLA-4 receptor-bearing T cells. Some examples of diseases that can be treated with such multivalent or polyclonal antibody preparations include autoimmune diseases, transplant rejection and inflammation.

Example 9

Production of anti-human EGFR antibodies

An antigen. Purified soluble Epithelial Growth Factor Receptor (EGFR) from human cancer a431 cells was obtained from Sigma Chemical Co (E3641). The human cancer A431 cell line was obtained from the American Type Culture Collection (ATCC CRL-1555). RibimPL + TDM adjuvant was obtained from Sigma Chemical Co (M-6536).

And (4) immunization. Two SC20/KCo5 hybrid mice (ID #22232 and 22239) were injected intraperitoneally (i.p.) with 10 injections, respectively⁷Washed human carcinoma a431 whole cells were immunized. This immunization procedure was repeated in two mice after 1 month. At month 4, mice 22239 were immunized intraperitoneally with 25 μ g of soluble EGFR in MPL + TDM adjuvant; rest on the tableDay 11, then immunization was performed by intravenous injection of 10 μ g EGFR in PBS and intraperitoneal injection of 10 μ g EGFR in MPL + TDM adjuvant. After 2 days, mouse 22239 again received 10 μ g egfr intravenously in PBS, and next day splenocytes were harvested from mouse 22239 for fusion. After the first two injections of A431 cells, mice 22232 were allowed to rest for 3 months, and then injected intraperitoneally with 10 mixed with MPL + TDM adjuvant⁷A431 cells. After 4 days, splenocytes were harvested from mouse 22232 for fusion.

And (4) fusing. In two independent experiments, splenocytes from mice #22232 and 22239 were fused with either the P3X 63 Ag8.6.53(ATCC CRL 1580; mouse #22239) or SP2/0-Ag14(ATCC CRL 1581; mouse #22232) myeloma cell lines. Fusion was performed using the standard method outlined in example 8.

And (4) screening the hybridoma. The EGFR hybridoma was screened in a manner similar to that used for CTLA-4 in example 8. ELISA plates (Nunc MaxiSorp) were coated overnight with 100. mu.l/well of 1. mu.g/ml PBS solution of soluble EGFR antigen. Plates were washed and blocked with 100. mu.l/well PBS-Tween containing 1% BSA. Add 50. mu.l of cell culture supernatant and incubate for 1-2 hours. Plates were washed and then incubated for 1 hour with 100. mu.l/well of goat anti-human gamma heavy chain conjugated with alkaline phosphatase (anti-human gamma (fc) AP Jackson # 109-. Plates were washed 3 times with PBS-Tween between each step. 5 and 2 hybridomas secreting human IgG kappa anti-EGFR specific antibodies were subcloned from the mouse 22232 and mouse 22239 fusions, respectively. Isotype analysis of the heavy and light chains of the EGFR-specific antibody included 4 IgGs₁Kappa, 1 IgG₂Kappa and 1 IgG₄Kappa antibodies.

Example 10

Rate constants and equilibrium constants for purification of human IgG kappa monoclonal antibodies

The hybridomas were cultured in eRDF containing 1% fetal bovine serum (low IgG). Human MAb was purified using a protein G column. The rate equilibrium association constants of purified G-CSF MAb and soluble CD4MAb were determined using a BIAcore2000 instrument. Human G-CSF (120RU) or CD 4: fc (1600 RU)) Immobilized to the sensor chip surface of BIAcore2000(BIAcore) by amino covalent coupling. Flowing the monoclonal antibody over the antigen. The chip was washed with glycine-HCl buffer (pH 1.5) or 4M MgCl₂And (c) regenerating to remove any residual anti-human G-CSF or anti-CD 4 MAb. This cycle was repeated with different concentrations of MAb. Binding to and dissociation from the antigen was determined using BIAevaluation 3.0 software. Will k_{Association of}Divided by k_{Solution (II) Separation device}To obtain K_a. These values are compared to the mouse anti-human G-CSF MAb, clone 3316.111 (R), as shown in Table 6 below&D) Or the values obtained for mouse anti-CD 4MAb, Leu3a (Pharmingen).

TABLE 6

Rate constants and equilibrium constants for purified human IgG kappa MAb

MAb	Subclass of	Mouse	A antigen	k(Ms)	K(s)	K(M)
							#4	IgG	Bis Tc/KO	G-CSF	4.1x10	3.1x10	1.3x10
#5	IgG	Bis Tc/KO	G-CSF	5.9x10	5.8x10	1.0x10

MAb	Subclass of	Mouse	A antigen	k(Ms)	K(s)	K(M)
							#11	IgG	Hybrid seed	G-CSF	4.0x10	1.5x10	2.8x10
#21	IgG	Hybrid seed	G-CSF	1.1x10	2.0x10	5.4x10
							#27	IgG	Hybrid seed	G-CSF	1.3x10	1.9x10	6.5x10
#23	IgG	Hybrid seed	CD4	7.6x10	5.7x10	1.3x10
							3316.111	Mouse	Wild type	G-CSF	1.5x10	2.3x10	6.3x10
Leu3a	Mouse	Wild type	CD4	2.2x10	7.1x10	3.1x10

Example 11

Hybrid (Fc) mouse production

It is well known that immune tolerance avoids reactivity to self-antigens, and generally prevents the production of mouse monoclonal antibodies against foreign antigens whose amino acid sequences are similar or identical to those of the murine counterparts. Mouse monoclonal antibodies that bind to a common epitope between human antigens and their murine counterparts can be useful because the amino acid sequences in the active site of protein antigens are often well conserved. In addition, any effects due to in vivo administration of these antibodies can be readily studied using a mouse model. However, it has also been difficult to obtain mouse monoclonal antibodies directed against such common epitopes. As described above, the hybrid mouse of the present invention can be used to obtain human monoclonal antibodies against various human antigens. However, in some cases, it may be difficult to obtain human monoclonal antibodies that have the ability to bind well conserved human antigens or that cross-react with murine counterparts. Thus, in another aspect of the invention, there is provided a further hybrid mouse of the invention in which Fc γ receptor IIB has been inactivated. These mice, referred to herein as hybrid (Fc) mice, are available for the production of monoclonal antibodies that bind to well-conserved antigens or cross-react with their murine counterparts. Biochemical and genetic studies have shown that the low affinity receptor type IIB of immunoglobulin (Ig) G (Fc γ RIIB), inhibits cell activation triggered by antibodies or immune complexes, and may be an important component in preventing the development of autoimmunity (Takai, t. et al, 1996, Nature 379: 346-. Animals deficient in Fc γ RIIB (i.e., inhibitory Fc receptor) have a generalized enhanced antibody response and increased inflammation in all antibody-mediated classes of allergic reactions (Takai, T. et al, 1996, Nature 379: 346-349). For example, instead of wild-type mice, mutant mice immunized with bovine type IV collagen (C-IV) exhibited increased autoantibody responses to mouse C-IV (Nakamura, A. et al, 2000, J.Exp.Med.191: 899-. However, there has been no report on the study of whether Fc γ RIIB mutant mice can be used to efficiently produce autoreactive monoclonal antibodies. Furthermore, there is no report demonstrating efficient production of human monoclonal antibodies that bind to both human antigens and murine counterparts in mice.

Fc γ RIIB mutations were grown into the hybrid mice of the invention as described below. Immunization of the resulting hybrid (Fc) mice with bovine C-IV elicits human antibody responses against both bovine and murine C-IV. Hybridomas secreting human monoclonal antibodies that bind to both bovine C-IV and murine C-IV can also be produced. Thus, the hybrid (Fc) mice are available for the production of human monoclonal antibodies that bind both to immunizing foreign antigens and to their murine counterparts. The hybrid (Fc) mice can also be used to obtain human monoclonal antibodies against well-conserved antigens. Fc γ RIIB knock-out homozygous (Fc (-/-)) mice (Takai, T. et al, 1996, Nature 379: 346-. Fc (-/-) male mice were mated with female hybrid mice (as described in example 3). The retention of KCo5 transgene and hCF (SC20) in each individual F1 was examined by ELISA and PCR as described in example 3. The genotype of the Fc γ RIIB knockout was determined by PCR analysis using the following three primers:

neo，5’-CTCGTGCTTTACGGTATCGCC(SEQ ID NO：8)；

5 'EC 1, 5' -AAACTCGACCCCCCGTGGATC (SEQ ID NO: 9); and

3’EC1，5’-TTGACTGTGGCCTTAAACGTGTAG(SEQ ID NO：10).

genomic DNA samples prepared from tail biopsies were subjected to PCR using AmpliTaq DNA polymerase (Perkin Elmer). Samples were amplified for 35 cycles in a standard reaction mixture containing the three primers described above (0.5 pM each): 30 seconds at 94 ℃,30 seconds at 62 ℃ and 30 seconds at 72 ℃ (Gene Amp PCR system 9600, Perkin Elmer). The wild type allele and the homozygous allele gave band sizes of 161bp and 232bp, respectively. F1 males with genotype KCo5/CMD or CM2D (-/+)/CKD or JKD (-/+)/Fc (-/+) and F1 females with genotype hCF (SC20)/KCo5/CMD or CM2D (-/+)/CKD or JKD (-/+)/Fc (-/+) were selected and used for further breeding. Finally, mice of genotypes hCF (SC20)/KCo5/CMD or CM2D (-/-)/CKD or JKD (-/-)/Fc (-/-) (hybrids (Fc)) were obtained. It was confirmed that the serum expression levels of human Ig μ and κ in the hybrid (Fc) mice were comparable to those in the hybrid mice (see example 4).

Example 12

Generation of human monoclonal antibodies against mouse type IV collagen

Immunization of antigens. Bovine C-IV (Cellmatrix IV) was obtained from Nitta Gellan, Inc. The C-IV solution (3.0mg/ml in 1mM HCl, pH 3.0) was neutralized by the addition of 1mM NaOH (final concentration) prior to emulsification with Freund's adjuvant. Hybrid mice and hybrid (FC) mice were used at the base of the tail in mice containing Mycobacterium tuberculosis (Mycobacterium tuberculosis) strain H₃₇Rv in CFA (Wako Pure Chemical Industries, Ltd.) emulsified 150. mu. g C-IV. After 26 days and 48 days, mice were boosted at the same site with 150. mu. g C-IV + IFA (Wako pure chemical Industries, Ltd.) (Nakamura, A. et al, 2000, J.Exp.Med.191: 899-905).

Humoral responses in mice. Serum was collected on day 58. Antigen-reactive human Ig. gamma.in serum was measured by ELISA as described and modified (Nakamura, A. et al, 2000, J.Exp.Med.191: 899-905). Anti-bovine C-IV antibodies were detected in a 96-well microplate assay (Nunc, MaxiSorp) in which each well was coated overnight at 4 ℃ with 50. mu.l/well of 20. mu.g/ml bovine C-IV in PBS. Anti-mouse C-IV antibodies were detected using a BIOCOAT cellware mouse C-IV 96 well plate assay (Becton Dickinson Labware). Diluted serum (1: 20-1280) was added at 50. mu.l/well and allowed to react overnight at 4 ℃. The wells were washed, incubated with goat anti-human IgG (Fc) (Sigma, A0170) conjugated with horseradish peroxidase for 2 hours at 4 ℃, washed, and then developed with 50. mu.l of TMB substrate (Sumitomo Bakelite, ML-1120T) for 30 minutes at room temperature. OD 450nm was read with a small plate reader (Arvo, Wallac Berthold Japan). Specific human gamma autoantibody responses against mouse C-IV were observed in the serum of hybrids (Fc) but not in the hybrid mice. Enhanced anti-bovine C-IV responses were observed in the serum of hybrid (Fc) mice (fig. 14).

Fusion and hybridoma screening. After 66 days, the mice were additionally injected intraperitoneally (KM # 1: hybrid, FC # 1: hybrid (Fc)) or intravenously (KM # 2: hybrid, FC # 2: hybrid (Fc)) with 150. mu.g of antigen, and splenocytes were harvested after 69 days. Spleen cells from mice were fused with mouse myeloma cells (Sp2/0-Ag14) using standard methods. The cell suspension was seeded into 96-well plates at 200,000 splenocytes/well. Cells were cultured in DMEM, 10% FBS, insulin, IL-6. During initial growth and selection, HAT or HT supplements were added to the medium. Hybridomas were screened by ELISA. To identify hybridomas secreting mouse C-IV, ELISA plates (Nunc MaxiSorp) were coated overnight at 4 ℃ with 50. mu.l/well of 40. mu.g/ml mouse C-IV (Sigma, C0534) in PBS. 50 μ l of cell culture supernatant was added. Two hybridomas secreting h γ positive mouse C-IV reactive antibodies were obtained from hybrid (Fc) mice and successfully subcloned by limiting dilution (see table 7 below).

TABLE 7

Production of anti-type IV collagen monoclonal antibodies

This data indicates that the hybrid (Fc) mice can be used to produce human monoclonal antibodies against well-conserved antigens or epitopes.

***

The scope of the invention is not limited to the illustrated embodiments used to illustrate various aspects of the invention, and any clone, DNA or amino acid sequence that is functionally equivalent is within the scope of the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. It also goes without saying that the size of all base pairs of the nucleotides given is approximate for illustration.

All publications and patent documents cited above are incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication or patent document were individually indicated to be incorporated by reference.

Sequence listing

<110>Tomizuka，Kazuma

Ishida，Isao

Lonberg，Nils

Halk，Ed

<120> transgenic transchromosomal rodents for producing human antibodies

<130>014643-012110US

<140> is to be determined

<141> is to be determined

<150>US 60/250,340

<151>2000-11-30

<160>10

<170>PatentIn Ver.2.1

<210>1

<211>3881

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: kappa light chain plasmid

<220>

<223>pCK7-96

<400>1

tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta 60

tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag 120

aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg 180

tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg 240

tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg 300

cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga 360

agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc 420

tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt 480

aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact 540

ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg 600

cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt 660

accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt 720

ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct 780

ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg 840

gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt 900

aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt 960

gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc 1020

gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg 1080

cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc 1140

gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg 1200

gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca 1260

ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga 1320

tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct 1380

ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg 1440

cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca 1500

accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata 1560

cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct 1620

tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact 1680

cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa 1740

acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc 1800

atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga 1860

tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga 1920

aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg 1980

cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac 2040

atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 2100

cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca 2160

gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg 2220

agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga 2280

tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga 2340

ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgcc 2400

aagctagcgg ccgcggtcca accaccaatc tcaaagcttg gtacccggga gcctgttatc 2460

ccagcacagt cctggaagag gcacagggga aataaaagcg gacggaggct ttccttgact 2520

cagccgctgc ctggtcttct tcagacctgt tctgaattct aaactctgag ggggtcggat 2580

gacgtggcca ttctttgcct aaagcattga gtttactgca aggtcagaaa agcatgcaaa 2640

gccctcagaa tggctgcaaa gagctccaac aaaacaattt agaactttat taaggaatag 2700

ggggaagcta ggaagaaact caaaacatca agattttaaa tacgcttctt ggtctccttg 2760

ctataattat ctgggataag catgctgttt tctgtctgtc cctaacatgc cctgtgatta 2820

tccgcaaaca acacacccaa gggcagaact ttgttactta aacaccatcc tgtttgcttc 2880

tttcctcagg aactgtggct gcaccatctg tcttcatctt cccgccatct gatgagcagt 2940

tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctatccc agagaggcca 3000

aagtacagtg gaaggtggat aacgccctcc aatcgggtaa ctcccaggag agtgtcacag 3060

agcaggacag caaggacagc acctacagcc tcagcagcac cctgacgctg agcaaagcag 3120

actacgagaa acacaaagtc tacgcctgcg aagtcaccca tcagggcctg agctcgcccg 3180

tcacaaagag cttcaacagg ggagagtgtt agagggagaa gtgcccccac ctgctcctca 3240

gttccagcct gaccccctcc catcctttgg cctctgaccc tttttccaca ggggacctac 3300

ccctattgcg gtcctccagc tcatctttca cctcaccccc ctcctcctcc ttggctttaa 3360

ttatgctaat gttggaggag aatgaataaa taaagtgaat ctttgcacct gtggtttctc 3420

tctttcctca atttaataat tattatctgt tgtttaccaa ctactcaatt tctcttataa 3480

gggactaaat atgtagtcat cctaaggcgc ataaccattt ataaaaatca tccttcattc 3540

tattttaccc tatcatcctc tgcaagacag tcctccctca aacccacaag ccttctgtcc 3600

tcacagtccc ctgggccatg gatcctcaca tcccaatccg cggccgcaat tcgtaatcat 3660

ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 3720

ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 3780

cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 3840

tcggccaacg cgcggggaga ggcggtttgc gtattgggcg c 3881

<210>2

<211>4723

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: gamma 1 heavy chain plasmid

<220>

<223>pCG7-96

<400>2

gaactcgagc agctgaagct ttctggggca ggccaggcct gaccttggct ttggggcagg 60

gagggggcta aggtgaggca ggtggcgcca gccaggtgca cacccaatgc ccatgagccc 120

agacactgga cgctgaacct cgcggacagt taagaaccca ggggcctctg cgccctgggc 180

ccagctctgt cccacaccgc ggtcacatgg caccacctct cttgcagcct ccaccaaggg 240

cccatcggtc ttccccctgg caccctcctc caagagcacc tctgggggca cagcggccct 300

gggctgcctg gtcaaggact acttccccga accggtgacg gtgtcgtgga actcaggcgc 360

cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag tcctcaggac tctactccct 420

cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc cagacctaca tctgcaacgt 480

gaatcacaag cccagcaaca ccaaggtgga caagaaagtt ggtgagaggc cagcacaggg 540

agggagggtg tctgctggaa gccaggctca gcgctcctgc ctggacgcat cccggctatg 600

cagccccagt ccagggcagc aaggcaggcc ccgtctgcct cttcacccgg aggcctctgc 660

ccgccccact catgctcagg gagagggtct tctggctttt tccccaggct ctgggcaggc 720

acaggctagg tgcccctaac ccaggccctg cacacaaagg ggcaggtgct gggctcagac 780

ctgccaagag ccatatccgg gaggaccctg cccctgacct aagcccaccc caaaggccaa 840

actctccact ccctcagctc ggacaccttc tctcctccca gattccagta actcccaatc 900

ttctctctgc agagcccaaa tcttgtgaca aaactcacac atgcccaccg tgcccaggta 960

agccagccca ggcctcgccc tccagctcaa ggcgggacag gtgccctaga gtagcctgca 1020

tccagggaca ggccccagcc gggtgctgac acgtccacct ccatctcttc ctcagcacct 1080

gaactcctgg ggggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 1140

atctcccgga cccctgaggt cacatgcgtg gtggtggacg tgagccacga agaccctgag 1200

gtcaagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccgcgg 1260

gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc tcaccgtcct gcaccaggac 1320

tggctgaatg gcaaggagta caagtgcaag gtctccaaca aagccctccc agcccccatc 1380

gagaaaacca tctccaaagc caaaggtggg acccgtgggg tgcgagggcc acatggacag 1440

aggccggctc ggcccaccct ctgccctgag agtgaccgct gtaccaacct ctgtccctac 1500

agggcagccc cgagaaccac aggtgtacac cctgccccca tcccgggatg agctgaccaa 1560

gaaccaggtc agcctgacct gcctggtcaa aggcttctat cccagcgaca tcgccgtgga 1620

gtgggagagc aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc 1680

cgacggctcc ttcttcctct acagcaagct caccgtggac aagagcaggt ggcagcaggg 1740

gaacgtcttc tcatgctccg tgatgcatga ggctctgcac aaccactaca cgcagaagag 1800

cctctccctg tctccgggta aatgagtgcg acggccggca agcccccgct ccccgggctc 1860

tcgcggtcgc acgaggatgc ttggcacgta ccccctgtac atacttcccg ggcgcccagc 1920

atggaaataa agcacccagc gctgccctgg gcccctgcga gactgtgatg gttctttcca 1980

cgggtcaggc cgagtctgag gcctgagtgg catgagggag gcagagcggg tcccactgtc 2040

cccacactgg cccaggctgt gcaggtgtgc ctgggccccc tagggtgggg ctcagccagg 2100

ggctgccctc ggcagggtgg gggatttgcc agcgtggccc tccctccagc agcacctgcc 2160

ctgggctggg ccacgggaag ccctaggagc ccctggggac agacacacag cccctgcctc 2220

tgtaggagac tgtcctgttc tgtgagcgcc cctgtcctcc cgacctccat gcccactcgg 2280

gggcatgcct gcaggtcgac tctagaggat ccccgggtac cgagctcgaa ttcatcgatg 2340

atatcagatc tgccggtctc cctatagtga gtcgtattaa tttcgataag ccaggttaac 2400

ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 2460

gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 2520

cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 2580

tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 2640

cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 2700

aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 2760

cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 2820

gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 2880

ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 2940

cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 3000

aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 3060

tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 3120

ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 3180

tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 3240

ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 3300

agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 3360

atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 3420

cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 3480

ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 3540

ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc 3600

agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct 3660

agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc 3720

gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg 3780

cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc 3840

gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat 3900

tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag 3960

tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat 4020

aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg 4080

cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca 4140

cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga 4200

aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc 4260

ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata 4320

tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg 4380

ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc 4440

acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag 4500

ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag 4560

ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc atcagagcag 4620

attgtactga gagtgcacca tatggacata ttgtcgttag aacgcggcta caattaatac 4680

ataaccttat gtatcataca catacgattt aggtgacact ata 4723

<210>3

<211>4694

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: gamma 4 heavy chain plasmid

<220>

<223>pG4HE

<400>3

gaactcgagc agctgaagct ttctggggca ggccgggcct gactttggct gggggcaggg 60

agggggctaa ggtgacgcag gtggcgccag ccaggtgcac acccaatgcc catgagccca 120

gacactggac cctgcatgga ccatcgcgga tagacaagaa ccgaggggcc tctgcgccct 180

gggcccagct ctgtcccaca ccgcggtcac atggcaccac ctctcttgca gcttccacca 240

agggcccatc cgtcttcccc ctggcgccct gctccaggag cacctccgag agcacagccg 300

ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg tggaactcag 360

gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca ggactctact 420

ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc tacacctgca 480

acgtagatca caagcccagc aacaccaagg tggacaagag agttggtgag aggccagcac 540

agggagggag ggtgtctgct ggaagccagg ctcagccctc ctgcctggac gcaccccggc 600

tgtgcagccc cagcccaggg cagcaaggca tgccccatct gtctcctcac ccggaggcct 660

ctgaccaccc cactcatgct cagggagagg gtcttctgga tttttccacc aggctccggg 720

cagccacagg ctggatgccc ctaccccagg ccctgcgcat acaggggcag gtgctgcgct 780

cagacctgcc aagagccata tccgggagga ccctgcccct gacctaagcc caccccaaag 840

gccaaactct ccactccctc agctcagaca ccttctctcc tcccagatct gagtaactcc 900

caatcttctc tctgcagagt ccaaatatgg tcccccatgc ccatcatgcc caggtaagcc 960

aacccaggcc tcgccctcca gctcaaggcg ggacaggtgc cctagagtag cctgcatcca 1020

gggacaggcc ccagccgggt gctgacgcat ccacctccat ctcttcctca gcacctgagt 1080

tcctgggggg accatcagtc ttcctgttcc ccccaaaacc caaggacact ctcatgatct 1140

cccggacccc tgaggtcacg tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc 1200

agttcaactg gtacgtggat ggcgtggagg tgcataatgc caagacaaag ccgcgggagg 1260

agcagttcaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac caggactggc 1320

tgaacggcaa ggagtacaag tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga 1380

aaaccatctc caaagccaaa ggtgggaccc acggggtgcg agggccacat ggacagaggt 1440

cagctcggcc caccctctgc cctgggagtg accgctgtgc caacctctgt ccctacaggg 1500

cagccccgag agccacaggt gtacaccctg cccccatccc aggaggagat gaccaagaac 1560

caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 1620

gagagcaatg ggcagccgga gaacaactac aagaccacgc ctcccgtgct ggactccgac 1680

ggctccttct tcctctacag caggctaacc gtggacaaga gcaggtggca ggaggggaat 1740

gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacaca gaagagcctc 1800

tccctgtctc tgggtaaatg agtgccaggg ccggcaagcc cccgctcccc gggctctcgg 1860

ggtcgcgcga ggatgcttgg cacgtacccc gtctacatac ttcccaggca cccagcatgg 1920

aaataaagca cccaccactg ccctgggccc ctgtgagact gtgatggttc tttccacggg 1980

tcaggccgag tctgaggcct gagtgacatg agggaggcag agcgggtccc actgtcccca 2040

cactggccca ggctgtgcag gtgtgcctgg gccacctagg gtggggctca gccaggggct 2100

gccctcggca gggtggggga tttgccagcg tggccctccc tccagcagca gctgccctgg 2160

gctgggccac gggaagccct aggagcccct ggggacagac acacagcccc tgcctctgta 2220

ggagactgtc ctgtcctgtg agcgccctgt cctccgaccc cccatgccca ctcgggggga 2280

tccccgggta ccgagctcga attcatcgat gatatcagat ctgccggtct ccctatagtg 2340

agtcgtatta atttcgataa gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg 2400

gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 2460

ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 2520

agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 2580

ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 2640

caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 2700

gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 2760

cctgtccgcc tttctccctt cgggaagcgt ggcgctttct caatgctcac gctgtaggta 2820

tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 2880

gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 2940

cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 3000

tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 3060

tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 3120

caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 3180

aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 3240

cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 3300

ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 3360

tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 3420

atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 3480

tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 3540

aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 3600

catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 3660

gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 3720

ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 3780

aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 3840

atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 3900

cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 3960

gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 4020

agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 4080

gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 4140

caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 4200

ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 4260

tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 4320

aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat 4380

catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg 4440

tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta 4500

agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg 4560

gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatggacat 4620

attgtcgtta gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt 4680

taggtgacac tata 4694

<210>4

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: probes for the vk 015' region

<400>4

ccaccccata aacactgatt c 21

<210>5

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: probes for the vk 015' region

<400>5

ttgatgcatc ctacccaggg c 21

<210>6

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: probes for the intergenic region between V.kappa.L 24 and L25

<400>6

cctgccttac agtgctgtag 20

<210>7

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: probes for the intergenic region between V.kappa.L 24 and L25

<400>7

ggacagcaac aggacatggg 20

<210>8

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer neo

<400>8

ctcgtgcttt acggtatcgc c 21

<210>9

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer 5' EC1

<400>9

aaactcgacc ccccgtggat c 21

<210>10

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PCR primer 3' EC1

<400>10

ttgactgtgg ccttaaacgt gtag 24

Claims (10)

1. A method of making a human antibody-producing mouse carrying a transchromosome comprising a fragment of human chromosome 14 including a human antibody heavy chain locus and a transgene that has been inserted into a mouse chromosome and has an unrearranged human antibody light chain locus, the method comprising crossing a mouse carrying a transchromosome comprising a fragment of human chromosome 14 including a human antibody heavy chain locus with a mouse carrying a transgene inserted into a mouse chromosome and having an unrearranged human antibody light chain locus, wherein an endogenous mouse antibody heavy chain locus and an endogenous mouse antibody kappa light chain locus are inactivated.

2. The method of claim 1, wherein the transchromosome is a fragment of human chromosome 14 containing the human heavy chain locus.

3. The method of claim 1, wherein the transchromosome is hCF (SC 20).

4. The method of claim 1 wherein at least a portion of the transgene having unrearranged human antibody light chain loci is introduced into the mouse with a YAC vector.

5. The method of claim 1, wherein the unrearranged human antibody light chain locus is a human antibody light chain kappa locus.

6. The method of claim 1, wherein the transgene having an unrearranged human antibody light chain locus is the KCo5 transgene.

7. The method of claim 1, said mouse carrying human chromosome fragment SC20 and human antibody kappa light chain locus transgene KCo5, the method comprising crossing a mouse carrying human chromosome fragment SC20 with a mouse carrying human antibody kappa light chain locus transgene KCo5, wherein the endogenous mouse antibody heavy chain locus and the endogenous mouse antibody kappa light chain locus are inactivated.

8. The method of claim 1, wherein the transchromosome is a fused chromosome formed by translocation, consisting of a fragment of human chromosome 14 and a different chromosome fragment.

9. The method of claim 1, wherein the transchromosome further comprises an unrearranged human antibody light chain locus.

10. The method of claim 1, further comprising a genetic mutation that enhances an immune response to the autoantigen, wherein the genetic mutation that enhances an immune response to the autoantigen is inactivation of the fcyriib gene.