Human antibodies derived from transgenic rodents possessing multiple heavy chain immunoglobulin gene loci.

Abstract

The present invention relates to transgenic animals useful for the optimal production of functional immunoglobulins with human idiotypes. [Selected Figure] Figure 1A

Description

[Technical Field]

[0001] This invention relates to transgenic animals useful for producing immunoglobulins with a human idiotype in rodents, and to methods for producing such animals. The invention further relates to compositions and methods for producing humanized antibodies and fully human antibodies using polynucleotides derived from modified broad regions on bacterial artificial chromosomes and their combined tandem incorporations. Crossbreeding of independently obtained transgenic animals has enabled the expression of a highly diverse repertoire of human antibodies using many different, and sometimes all, human VH, D, and JH segments. Expression is controlled in vivo by simultaneously regulating separate incorporation sites to obtain diversity and selection of VH genes without interference. [Background technology]

[0002] Human monoclonal antibodies have proven useful for therapeutic applications as either normal-sized, single-stranded, or domain-module IgG (Chan & Carter Nature reviews. Immunology 10, 301-316 (2010); Enever et al. Current opinion in biotechnology 20, 405-411 (2009)). Despite this success, their production still faces significant drawbacks, which depend either on the specific selection of available human material and subsequent modification of individual products, or on the immunization of transgenic animals with limited availability (Bruggemann et al. Part I: Selecting and shaping the antibody molecule, Selection Strategies III: Transgenic mice, in Handbook of Therapeutic Antibodies. Ed. Dubel, S. Wiley-VHC, 69-93 (2007)).

[0003] DNA rearrangement and expression of the human immunoglobulin (Ig) gene in transgenic animals by stably inserting heavy chain genes into the germline structure was developed more than 20 years ago (Bruggemann, M. et al. PNAS 86, 6709-6713 (1989)). One problem associated with the therapeutic use of non-human immunoglobulins is their potential immunogenicity in human patients. To reduce the immunogenicity of such formulations, various strategies have been developed to produce chimeric antibodies, partially human (humanized) antibodies, and fully human antibodies. Chimeric antibodies contain a human constant region and a binding region encoded by the non-human V gene. The ability to produce transgenic antibodies with a human idiotype in non-human animals is particularly desirable because the antigen-binding determinant is located within the idiotype region, and the non-human idiotype is thought to be a factor in the immunogenicity of current antibody therapeutics. Human idiotype is a particularly important issue with regard to monoclonal antibody therapeutics, which consist of a single idiotype delivered at relatively high concentrations, in contrast to the diverse idiotypes delivered at low concentrations by polyclonal antibody mixtures.

[0004] Two new strategies—gene knockout in embryonic stem (ES) cells (Kitamura et al. Nature 350, 423-426 (1991)) and gene locus extension on artificial chromosomes (Davies et al. Nucleic acids research 21, 767-768 (1993))—have been combined to bring about significant improvements, higher expression levels, and exclusive production of human Ig. Silencing endogenous Ig genes by gene targeting in ES cells has produced several inactive mouse lines without the ability to rearrange their IgH and IgL loci, or without producing fully functional IgH, IgK, or IgL products. More recently, zinc finger nucleases (ZFNs) have been designed to induce site-specific double-strand breaks in Ig genes, enabling gene disruption via deletion and non-homologous DNA repair. Ig-silencing rats and rabbits were created by injecting ZFN plasmids into fertilized eggs, resulting in disruption of IgH and IgL (Geurts, A. et al. Science 325, 433 (2009); Menoret, S. et al. European journal of immunology 40, 2932-2941 (2010); Flisikowska, T. et al. PloS one 6, e21045 (2011)).

[0005] A key technical challenge encountered in many prior art approaches for producing humanized transgenic antibodies in non-human animals concerns the apparent competition between dual Ig loci in the same animal, e.g., between an existing or endogenous Ig locus introduced into the transgenic animal and an exogenous or artificial locus. Historically, in the absence of effective knockout, the endogenous locus has outperformed the exogenous locus in terms of antibody production, thus effectively silencing the duplicate locus (Lonberg et al., Nat Bio, 23, 1117, 2005; Nicholson et al., J Immunol, 163, 6898, 1999; Bruggemann et al., AITE 63, 101, 2015). Therefore, in this regard, the prior art has neither addressed nor resolved whether dual Ig loci integrated into different chromosomal regions can act cooperatively in the production of transgenic antibodies in the same host animal; in fact, it would be reasonably suggested to those skilled in the art that the opposite is true.

[0006] Another technical challenge encountered in the production of transgenic antibodies with human idiotypes in non-human animals is the difficulty in providing complete complements to the human immunoglobulin VDJ or VJ gene segments used to produce human antibodies. Some have attempted to address this problem by introducing megabase-sized fragments from human heavy chain and kappa light chain loci. However, this approach has only proven successful for approximately 80% of human immunoglobulin genes contained in germline composition, and has relied on the use of protoplasts to deliver large fragments of the relevant chromosomes via a yeast artificial chromosome (YAC) system (U.S. Patent No. 5,939,598).

[0007] To maximize antibody diversity, extensive duplication V is required to maintain the full functionality of the IgH locus and to ensure that DNA rearrangement is essential. H DJ HWhile region incorporation has been utilized in transgenic animals, duplicate incorporations have primarily been reported as occurring in much smaller regions (less than 100kb) (Wagner et al. Genomics 35,405-414 (1996); Bruggemann et al. European Journal of Immunology 21,1323-1326 (1991)) or in larger regions, but still limited to a single incorporation site (WO2014 / 093908; Bruggemann et al.). At the time of filing, the common understanding in the art was that diffusion or multiple incorporation of BAC or YAC mixtures was rare and could be detrimental to homozygous mating. Furthermore, the challenging integration of large YACs into stem cells, followed by the induction of animals from these stem cells, has been commonly performed (Mendez et al. Nature Genetics 15,146-156 (1997); Davies et al. Biotechnology (NY) 11,911-914 (1993)).

[0008] Maximizing the diversity of antibodies with human idiotypes using transgenic animals possessing complete complements of the human V gene, and optimizing the production of immunoglobulins or antibodies, remains a challenge in generating novel specificity for therapeutic applications across a wide range of disease areas. [Overview of the project] [Problems that the invention aims to solve]

[0009] The present invention resolves the aforementioned uncertainties in the art by providing transgenic animals that contain multiple artificial Ig heavy chain gene loci, each containing a double / duplication human immunoglobulin VDJ or VJ gene segment integrated into different chromosomal sites, and that lack the ability to produce endogenous immunoglobulins. The method used to create these transgenic animals, which involves inserting two different gene loci at two different locations on two different chromosomes, surprisingly produced functional B cells that favorably avoid allele exclusion and result in increased antibody diversity due to the complete complementarity of the human immunoglobulin VDJ heavy chain gene segment integrated into the genome of the transgenic animals. [Means for solving the problem]

[0010] In one aspect of the present invention, a novel polynucleotide comprising a nucleic acid sequence encoding a chimeric immunoglobulin chain, particularly a chimeric heavy chain for use in the production of transgenic animals, is disclosed. The polynucleotide of the present invention benefits from optimal expression, at least in part, by the inclusion of a 3' enhancer, because a trans locus lacking this 3' enhancer results in reduced isotype switching and low IgG expression. Accordingly, in a preferred embodiment, the present invention provides a chimeric polynucleotide comprising a rat 3' enhancer sequence, an Ig constant region gene, and at least one human immunoglobulin (Ig) ligation (J) region gene. In a preferred embodiment, the rat 3' enhancer sequence comprises the sequence described as Sequence ID No. 1, or a portion thereof.

[0011] In one embodiment, the chimeric polynucleotide described herein may further include at least one human variable (V) gene, at least one diversity (D) gene, or a combination thereof. In one embodiment, the constant region gene of the chimeric polynucleotide is selected from the group consisting of human constant region genes and rat constant region genes. In a preferred embodiment, the constant region gene includes a rat constant region gene. In another preferred embodiment, the constant region gene is selected from the group consisting of Cμ and Cγ.

[0012] In one embodiment, the chimeric polynucleotide comprises a nucleic acid sequence (e.g., SEQ ID NO: 10, or a part thereof) that is substantially homologous to the bacterial artificial chromosome (BAC) Annabel disclosed herein, optionally BAC6-V H 3-11 and BAC3 constructs and / or at least one human variable Ig gene isolatable from BAC9 and BAC14 / 5 constructs may further be included. In a preferred embodiment, the chimeric polynucleotides contemplated herein have nucleic acid sequences (a) and (b) in the 5' to 3' order: (a) a human Ig variable region containing a human V gene in its native configuration isolatable from BAC6-V H 3-11 and BAC3 constructs and / or BAC9 and BAC14 / 5 constructs, and (b) a human Ig joining region containing a human J gene in its native configuration isolatable from BAC Annabel. In another embodiment, each of the human Ig variable region, human Ig diversity region, human Ig joining region, Ig constant region, and rat 3' enhancer region of the chimeric polynucleotides disclosed herein is in the relative position shown in FIG. 1a. In another embodiment, the disclosed chimeric polynucleotide comprises the sequence described as SEQ ID NO: 2 or a part thereof, or has a sequence substantially homologous thereto. In another embodiment, the disclosed chimeric polynucleotide comprises the sequence described as SEQ ID NO: 11 or a part thereof, or has a sequence substantially homologous thereto. In a further embodiment, the chimeric polynucleotide disclosed herein comprises a rearranged V-D-J region encoding a heavy chain variable domain exon.

[0013] In one embodiment, the transgenic animal further comprises a chimeric polynucleotide in which the human Ig V region contains at least one human V region gene that can be isolated from BAC9 and / or BAC14 / 5. In a preferred embodiment, the chimeric polynucleotide comprises nucleic acid sequences in 5' to 3' order (a) and (b): (a) a human Ig variable region containing a human V region gene in a natural composition used (or rearranged) from BAC9 and / or BAC14 / 5; and (b) a human Ig ligation region containing a human J region gene in a natural composition used (or rearranged) from bacterial artificial chromosome (BAC) Annabel. In another embodiment, each of the human immunoglobulin variable region (gene), human immunoglobulin diversity region (segment), human immunoglobulin ligation region (segment), immunoglobulin constant region gene, and rat 3' enhancer are located in the positions shown in Figure 1b. In another embodiment, the disclosed chimeric polynucleotide contains or has substantially homologous sequences to those shown in Figure 6. In another embodiment, the disclosed chimeric polynucleotide includes the sequence or a portion thereof shown in Figure 7, or has a substantially homologous sequence. In a further embodiment, the chimeric polynucleotide disclosed herein may include a rearranged VDJ, the rearranged gene segment derived from the above sequence number and figure.

[0014] Polynucleotides encoding the human kappa light chain gene are also disclosed herein. In one embodiment, the polynucleotides disclosed herein include a nucleic acid sequence selected from the group consisting of RP11-1156D9 (described as SEQ ID NO: 3) and RP11-1134E24 (described as SEQ ID NO: 4), or have a nucleic acid sequence substantially homologous thereto. In another embodiment, the isolated polynucleotide has nucleic acid sequences (a) and (b) in order from 5' to 3': (a) a human Ig variable region containing a human V gene in a native configuration isolatable from bacterial artificial chromosome (BAC) RP11-156D9 and / or RP11-1134E24, and (b) a human Ig joining region containing a human J gene in a native configuration isolatable from bacterial artificial chromosome (BAC) RP11-1134E24 and / or RP11-344F17 (described as SEQ ID NO: 5). In a preferred embodiment, each of the human Ig variable region, the human Ig joining region, and the human Ig constant region is in the relative position shown in FIG. 2. In another embodiment, the disclosed chimeric polynucleotide includes the sequence described as SEQ ID NO: 6 or a portion thereof, or has a sequence substantially homologous thereto.

[0015] Rodent cells containing one or more polynucleotides of the present invention are also provided herein. For example, a polynucleotide disclosed herein, preferably comprising a nucleic acid sequence encoding a chimeric heavy chain (e.g., a nucleic acid sequence encoding a rat 3' enhancer sequence, an Ig constant region gene, and at least one human J region gene), and optionally a nucleic acid sequence substantially homologous to a nucleic acid sequence selected from the group consisting of RP11-1156D9, RP11-1134E24, and portions thereof, are provided herein. Rodent cells contemplated herein may further include a polynucleotide encoding a functional light chain, for example, a nucleic acid sequence selected from the group consisting of the sequence shown in FIG. 2a (described as SEQ ID NO: 6), the sequence shown in FIG. 2b (described as SEQ ID NO: 7), and portions thereof, or having a sequence substantially homologous thereto. In one embodiment, the one or more polynucleotides are integrated into the rodent cell genome.

[0016] In another embodiment of the present invention, a transgenic animal is provided comprising at least one inactivated endogenous Ig locus and a plurality of artificial transgenic Ig heavy chain loci, integrated into different chromosomal regions of the animal's genome. In one embodiment, the transgenic animal having a plurality of artificial Ig heavy chain loci comprises (i) a V region having at least one human V gene segment encoding a germline or hypermutant human V region amino acid sequence, (ii) one or more J gene segments, and (iii) one or more constant region gene segments, wherein the artificial Ig heavy chain locus is functional, capable of undergoing gene rearrangement, and acts cooperatively to produce a repertoire of artificial immunoglobulins. In another embodiment, the transgenic animal comprises a complete complement of the human variable heavy chain region. In various other embodiments, the transgenic animal has i) an artificial heavy chain locus comprising overlapping heavy chain gene segments, ii) lacks a functional endogenous Ig light chain locus, and / or iii) lacks a functional endogenous Ig heavy chain locus. In yet another embodiment, the transgenic animal expresses a diverse repertoire of antibodies encoded by the V gene at a transgenic immunoglobulin locus located in a different chromosomal region.

[0017] In some embodiments, transgenic animals lack a functional Ig light chain locus and can produce antibodies consisting only of the heavy chain.

[0018] In another embodiment, a transgenic animal having at least two artificial Ig heavy chain loci has at least one artificial Ig heavy chain locus comprising at least one human immunoglobulin (Ig) linkage (J) region gene, an Ig constant region gene, and a rat 3' enhancer. In these transgenic animals, the rat 3' enhancer may comprise the sequence described as Sequence ID No. 1. The transgenic animals described in the above embodiments may further comprise at least one human Ig variable (V) region gene and / or a human Ig diversity (D) region gene. In other embodiments of the present invention, the constant region gene is selected from the group consisting of human constant region genes and rat constant region genes. In certain embodiments, the constant region gene comprises a constant region gene selected from the group consisting of Cμ and Cγ. In various embodiments, the transgenic animal comprises a nucleic acid sequence substantially homologous to the bacterial artificial chromosome (BAC) Annabel or a portion thereof.

[0019] In a particular embodiment, the human IgV region of a transgenic animal is BAC6-V H The transgenic animal comprises at least one human V region gene that can be isolated from 3-11 and / or BAC3. In certain embodiments, the transgenic animal has (a)BAC6-V in the order of 5' to 3'. HThe transgenic animal comprises (b) a human Ig variable region containing a human V region gene in a native structure that can be isolated from 3-11 and / or BAC3, and (b) a human Ig ligation region containing a human J region gene in a native structure that can be isolated from bacterial artificial chromosome (BAC) Annabel. In one embodiment, the human immunoglobulin variable region, human immunoglobulin diversity region, human immunoglobulin ligation region, immunoglobulin constant region, and rat 3' enhancer are each in the relative positions shown in Figure 1a. In another embodiment, the transgenic animal has a nucleic acid sequence substantially homologous to the nucleic acid sequence described as SEQ ID NO: 2. In yet another embodiment, the transgenic animal has a nucleic acid sequence substantially homologous to the nucleic acid sequence described as SEQ ID NO: 11. In one embodiment, the transgenic animal has a rearranged VDJ region that forms a complete exon encoding a heavy chain variable domain.

[0020] In other specific embodiments, the transgenic animal is BAC9-V H They have a human Ig V region containing at least one human V region gene that can be isolated from 3-53 and / or BAC14 / 5. In certain embodiments, these transgenic animals have (a)BAC9-V in the order of 5' to 3'. H (b) A nucleic acid comprising a human Ig variable region containing a human V region gene in a natural composition that can be isolated from 3-53 and / or BAC14 / 5, and (b) a human Ig ligation region containing a human J region gene in a natural composition that can be isolated from bacterial artificial chromosome (BAC) Annabel. In one embodiment, the human immunoglobulin variable region, human immunoglobulin diversity region, human immunoglobulin ligation region, immunoglobulin constant region, and rat 3' enhancer are each in the relative positions shown in Figure 1b. In another embodiment, the transgenic animal has a nucleic acid sequence substantially homologous to the nucleic acid sequence shown in Figure 6. In yet another embodiment, the transgenic animal has a nucleic acid sequence substantially homologous to the nucleic acid sequence shown in Figure 7.

[0021] Another aspect of the present invention provides a method for producing an antibody, comprising immunizing the transgenic animal described above with an immunogen. In one embodiment, a polyclonal antiserum composition is produced, the antiserum comprising an antigen-specific antibody encoded by a V gene encoded by a transgenic immunoglobulin locus located at a different chromosomal site. In another embodiment, a method for producing a monoclonal antibody comprises (i) immunizing the transgenic animal described above with an immunogen; (ii) isolating monoclonal antibody-producing cells from the transgenic animal that produce a monoclonal antibody that specifically binds to the immunogen; and (iii) using the monoclonal antibody-producing cells to produce the monoclonal antibody that specifically binds to the immunogen, or using the monoclonal antibody-producing cells to produce hybridoma cells that produce the monoclonal antibody, and using the hybridoma cells to produce the monoclonal antibody.

[0022] In another embodiment, a method for producing a monoclonal antibody includes (i) immunizing the transgenic animal with an immunogen; (ii) isolating monoclonal antibody-producing cells from the transgenic animal that produce a monoclonal antibody that specifically binds to the immunogen; (iii) isolating a monoclonal antibody nucleic acid encoding the monoclonal antibody that specifically binds to the immunogen from the monoclonal antibody-producing cells; and (iv) producing the monoclonal antibody that specifically binds to the immunogen using the monoclonal antibody nucleic acid. In a particular embodiment, the monoclonal antibody has a human idiotype.

[0023] In yet another embodiment, a method for producing a fully human monoclonal antibody includes (i) immunizing the transgenic animal with an immunogen; (ii) isolating monoclonal antibody-producing cells from the transgenic animal that produce a monoclonal antibody that specifically binds to the immunogen; (iii) isolating a monoclonal antibody nucleic acid encoding the monoclonal antibody that specifically binds to the immunogen from the monoclonal antibody-producing cells; (iv) modifying the monoclonal antibody nucleic acid to produce a recombinant nucleic acid encoding a fully human monoclonal antibody; and (v) producing the encoded fully human monoclonal antibody using the recombinant nucleic acid encoding the fully human monoclonal antibody.

[0024] Another aspect of the present invention is a monoclonal antibody produced by the method described above.

[0025] In yet another embodiment, a method is provided for neutralizing an antigenic element in a human body component, comprising contacting the body component with the above-mentioned polyclonal antiserum composition comprising an immunoglobulin molecule that specifically binds to and neutralizes the antigenic element. In one embodiment, the method for neutralizing an antigenic element in a human body component comprises contacting the body component with the above-mentioned monoclonal antibody that specifically binds to and neutralizes the antigenic element. [Brief explanation of the drawing]

[0026] [Figure 1A]This is an overview of the incorporated chimeric (human, rat) and complete human Ig loci. The two chimeric human-rat IgH regions (HC14 and HC30) each contain three overlapping BACs with more than 22 different and potentially functional human VH segments. In HC14, BAC6-3 extends at VH3-11, resulting in a 10.6kb overlap with BAC3, which then overlaps 11.3kb with the C region BAC Hu-rat Annabel via VH6-1 (A). In HC30, BAC9 extends at BAC14 / 5, which then extends by adding a portion of BAC5 following VH3-43, resulting in a 6.1kb overlap with Hu-rat Annabel (B). The latter is a chimeric region containing all human D and JH segments followed by a rat C region with a complete enhancer sequence. The arrows indicate the use of the VH gene when HC14, HC30, and HC14 / HC30 are combined. Thinner bands indicate the VH gene that is less frequently expressed. Sequences were obtained by unbiased RT-PCR and NGS. [Figure 1B]This is an overview of the incorporated chimeric (human, rat) and complete human Ig loci. The two chimeric human-rat IgH regions (HC14 and HC30) each contain three overlapping BACs with more than 22 different and potentially functional human VH segments. In HC14, BAC6-3 extends at VH3-11, resulting in a 10.6kb overlap with BAC3, which then overlaps 11.3kb with the C region BAC Hu-rat Annabel via VH6-1 (A). In HC30, BAC9 extends at BAC14 / 5, which then extends by adding a portion of BAC5 following VH3-43, resulting in a 6.1kb overlap with Hu-rat Annabel (B). The latter is a chimeric region containing all human D and JH segments followed by a rat C region with a complete enhancer sequence. The arrows indicate the use of the VH gene when HC14, HC30, and HC14 / HC30 are combined. Thinner bands indicate the VH gene that is less frequently expressed. Sequences were obtained by unbiased RT-PCR and NGS. [Figure 2] (A) Human Igk BACs with 12 Vk regions and all Jk regions result in approximately 14kb of overlap in the Vk region and approximately 40kb of overlap in the Ck region, and include a KDE. (B) Human Igl regions with 17 Vl regions and all J-Cl regions including the 3' enhancer are derived from YACs (Vincent-Fabert, C. et al. Blood 116, 1895-1898 (2010)). [Figure 3] This shows the integration of the HC14 locus into chromosome 6 and the HC30 locus into chromosome 15. [Figure 4] This is an ELISA analysis of IgM and IgG concentrations in the serum of HC30 and HC14 / HC30 animals. Each dot (HC30) or square (HC14 / HC30) represents the titer (μg / ml) of one animal. IgG is further analyzed for the content of IgG1 and IgG2b. [Figure 5]This is an ELISA analysis of anti-β-gal specific antibodies derived from HC30 and HC14 / HC30. Each dot (HC30) or square (HC14 / HC30) represents the serum titer (comparative dilution) of a single animal. [Figure 6-1] This is a BAC9 sequence. [Figure 6-2] This is a BAC9 sequence. [Figure 6-3] This is a BAC9 sequence. [Figure 6-4] This is a BAC9 sequence. [Figure 6-5] This is a BAC9 sequence. [Figure 6-6] This is a BAC9 sequence. [Figure 6-7] This is a BAC9 sequence. [Figure 6-8] This is a BAC9 sequence. [Figure 6-9] This is a BAC9 sequence. [Figure 6-10] This is a BAC9 sequence. [Figure 6-11] This is a BAC9 sequence. [Figure 6-12] This is a BAC9 sequence. [Figure 6-13] This is a BAC9 sequence. [Figure 6-14] This is a BAC9 sequence. [Figure 6-15] This is a BAC9 sequence. [Figure 6-16] This is a BAC9 sequence. [Figure 6-17] This is a BAC9 sequence. [Figure 6-18] This is a BAC9 sequence. [Figure 6-19] This is a BAC9 sequence. [Figure 6-20] This is a BAC9 sequence. [Figure 6-21] This is a BAC9 sequence. [Figure 6-22] This is a BAC9 sequence. [Figure 6-23] This is a BAC9 sequence. [Figure 6-24] This is a BAC9 sequence. [Figure 6-25] This is a BAC9 sequence. [Figure 6-26] This is a BAC9 sequence. [Figure 6-27] This is a BAC9 sequence. [Figure 6-28] This is a BAC9 sequence. [Figure 6-29] This is a BAC9 sequence. [Figure 6-30] This is a BAC9 sequence. [Figure 6-31] This is a BAC9 sequence. [Figure 6-32] This is a BAC9 sequence. [Figure 6-33] This is a BAC9 sequence. [Figure 6-34] This is a BAC9 sequence. [Figure 6-35] This is a BAC9 sequence. [Figure 6-36] This is a BAC9 sequence. [Figure 7-1] This is the BAC14 / 5 sequence. [Figure 7-2] This is the BAC14 / 5 sequence. [Figure 7-3] This is the BAC14 / 5 sequence. [Figure 7-4] This is the BAC14 / 5 sequence. [Figure 7-5] This is the BAC14 / 5 sequence. [Figure 7-6] This is the BAC14 / 5 sequence. [Figure 7-7] This is the BAC14 / 5 sequence. [Figure 7-8] This is the BAC14 / 5 sequence. [Figure 7-9] This is the BAC14 / 5 sequence. [Figure 7-10] This is the BAC14 / 5 sequence. [Figure 7-11] This is the BAC14 / 5 sequence. [Figure 7-12] This is the BAC14 / 5 sequence. [Figure 7-13] This is the BAC14 / 5 sequence. [Figure 7-14] This is the BAC14 / 5 sequence. [Figure 7-15] This is the BAC14 / 5 sequence. [Figure 7-16]This is the BAC14 / 5 sequence. [Figure 7-17] This is the BAC14 / 5 sequence. [Figure 7-18] This is the BAC14 / 5 sequence. [Figure 7-19] This is the BAC14 / 5 sequence. [Figure 7-20] This is the BAC14 / 5 sequence. [Figure 7-21] This is the BAC14 / 5 sequence. [Figure 7-22] This is the BAC14 / 5 sequence. [Figure 7-23] This is the BAC14 / 5 sequence. [Figure 7-24] This is the BAC14 / 5 sequence. [Figure 7-25] This is the BAC14 / 5 sequence. [Figure 7-26] This is the BAC14 / 5 sequence. [Figure 7-27] This is the BAC14 / 5 sequence. [Figure 7-28] This is the BAC14 / 5 sequence. [Figure 7-29] This is the BAC14 / 5 sequence. [Figure 7-30] This is the BAC14 / 5 sequence. [Figure 7-31] This is the BAC14 / 5 sequence. [Figure 7-32] This is the BAC14 / 5 sequence. [Figure 7-33] This is the BAC14 / 5 sequence. [Figure 7-34] This is the BAC14 / 5 sequence. [Figure 7-35] This is the BAC14 / 5 sequence. [Figure 7-36] This is the BAC14 / 5 sequence. [Figure 7-37] This is the BAC14 / 5 sequence. [Figure 7-38] This is the BAC14 / 5 sequence. [Figure 7-39] This is the BAC14 / 5 sequence. [Figure 7-40] This is the BAC14 / 5 sequence. [Figure 7-41] This is the BAC14 / 5 sequence. [Modes for carrying out the invention]

[0027] Chimeric polynucleotides encoding recombinant or artificial immunoglobulin chains or loci are provided herein. As described above, the chimeric polynucleotides disclosed herein are useful for transforming rodents to contain the human Ig gene and for using such rodents to produce immunoglobulins or antibodies having a human idiotype. As further provided herein, transgenic animals have been constructed comprising at least three different transgene constructs having the complete complement of human immunoglobulin VDJ heavy chain gene segments tandemly incorporated into the genome of the transgenic animals, thereby ensuring the availability of the full human immunoglobulin gene in germline configurations under conditions of complete inactivation of the endogenous immunoglobulin gene or locus. Surprisingly, as demonstrated for the first time herein, multiple transgenic loci containing different V genes may act synergistically in the expression of humanized transgenic antibodies and full human transgenic antibodies.

[0028] definition Immunoglobulins refer to proteins consisting of one or more polypeptides substantially encoded by immunoglobulin genes. Recognized human immunoglobulin genes include kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. A full-length immunoglobulin "light chain" (approximately 25 kd, or 214 amino acids) typically contains a variable domain (approximately 110 amino acids) encoded by an exon containing one or more variable region genes and one or more linking region genes at the NH2 terminus, and a constant domain encoded by a kappa or lambda constant region gene at the COOH terminus. A full-length immunoglobulin "heavy chain" (approximately 50 kd, or 446 amino acids) similarly comprises (1) a variable domain (approximately 116 amino acids) encoded by exons including one or more variable region genes, one or more diversity region genes, and one or more linking region genes, and (2) one of the aforementioned constant domains (encoding approximately 330 amino acids) including one or more constant region genes such as alpha, gamma, delta, epsilon, or mu. The immunoglobulin heavy chain constant region genes encode an antibody class, i.e., isotype (e.g., IgM or IgG1).

[0029] As used herein, the term “antibody” refers to a protein comprising at least one, and preferably two, heavy (H) chain variable domains (hereinafter abbreviated as VH) and at least one, and preferably two, light (L) chain variable domains (hereinafter abbreviated as VL). Those skilled in the art will recognize that the variable domains of an immune chain are encoded in a gene segment that must first undergo somatic recombination to form a complete exon encoding the variable domain. There are three types of regions or gene segments that undergo rearrangement to form a variable domain: a variable region containing a variable gene, a diversity region containing a diversity gene (in the case of an immunoglobulin heavy chain), and a ligation region containing a ligation gene. The VH and VL domains may be further subdivided into hypervariable regions called “complementarity-determining regions” (“CDR”), which are interspersed with more conserved regions called “framework regions” (“FR”). The degrees of FR and CDR are precisely defined (see Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242; and Chothia et al. (1987) J.Mol.Biol.196:901-17, which are incorporated herein by reference). Each VH and VL domain generally consists of three CDRs and four FRs arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. An antigen-binding fragment of an antibody (or simply “antibody portion” or “fragment”) refers, as used herein, to one or more fragments of a full-length antibody that retain the ability to specifically bind to an antigen (e.g., CD3).

[0030] Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include: (i) Fab fragments, i.e., monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, i.e., bivalent fragments containing two Fab fragments linked at a hinge region by disulfide crosslinking; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of a VH domain (Ward et al. (1989) Nature 341:544-46); and (vi) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker that allows them to be produced using recombination methods as a single protein chain (known as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-26; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-83) where the VL and VH region pair forms a monovalent molecule. Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of antibodies. These antibody fragments are obtained using prior art known to those skilled in the art, and the fragments are screened for utility, as are intact antibodies.

[0031] Antibodies may further comprise heavy chain and / or light chain constant domains, thereby forming an immunoglobulin heavy chain and an immunoglobulin light chain, respectively. In one embodiment, the antibody is a tetramer of two immunoglobulin heavy chains and two immunoglobulin light chains, where the immunoglobulin heavy chains and immunoglobulin light chains are interlinked, for example, by disulfide bonds. The heavy chain constant domain consists of three gene segments, CH1, CH2, and CH3. The light chain constant domain consists of one gene, CL. The variable domains of the heavy chain and / or light chain include binding domains that interact with antigens. The constant domains of the antibody typically mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complementarity system (C1q).

[0032] A polynucleotide encoding an artificial immunoglobulin locus or artificial immunoglobulin chain means a recombinant polynucleotide comprising multiple immunoglobulin regions, for example, a variable (V) region or gene segment comprising a V gene, a linked (J) gene region or gene segment comprising a J gene, a diversity (D) region or gene segment comprising a D gene in the case of a heavy chain locus, and / or at least one constant (C) region comprising at least one C gene. Preferably, each region of the variable domain, for example, the V, D, or J region comprises or extends to at least two genes of the same type. For example, a variable region, as used herein, comprises a linked region comprising at least two variable genes, at least two linked genes, and a diversity region comprising two diversity genes. A constant region may comprise just one constant gene, for example, a κ gene or a λ gene, or multiple genes, for example, CH1, CH2, and CH3.

[0033] As used herein, "enhancer sequence" or "enhancer" refers to a sequence identified near many active genes due to hypersensitivity to nuclease digestion and degradation. Hypersensitivity sites may precede promoter sequences, and the intensity of their activity correlated with the DNA sequence. Linking to reporter genes showed increased transcription in the presence of enhancer function (Mundt et al., J.Immunol., 166, 3315

[2001] ). At the IgH locus, two important transcription or expression regulators, Eμ and 3'E, have been identified at the locus terminals (Pettersson et al., Nature, 344, 165

[1990] ). In mice, removal of the entire 3' regulatory region (including hs3a, hs1,2, hs3b, and hs4) allows for normal early B cell development but disrupts class switch recombination (Vincent-Fabert et al., Blood, 116, 1895

[2010] ) and may hinder the optimization of somatic hypermutation (Pruzina et al., Protein Engineering, Design and Selection, 1,

[2011] ). Regulatory function to achieve optimal isotype expression is particularly desirable when transgenic human IgH genes are used. Transgene constructs with an incomplete 3'E region that typically provides only the hs1,2 elements resulted in disappointing expression levels in transgenic mice, even when the endogenous IgH locus was knocked out. As a result, only a small amount of antigen-specific complete human IgG has been isolated from constructs produced over the past 20 years (Lonberg et al., Nature 368, 856

[1994] ; Nicholson et al., J.Immunol., 163, 6898

[1999] ; Davis et al., Cancer Metastasis Rev.18, 421

[1999] ; Pruzina et al., Protein Engineering, Design and Selection, 1,

[2011] ). In the rat IgH locus, the 3'E region has not been adequately analyzed.Comparison of mouse and rat sequences failed to identify hs4, a crucial fourth element further downstream containing additional important regulatory sequences (Chatterjee et al., J. Biol. Chem., 286, 29303

[2011] ). The polynucleotides of the present invention, at least partially, favorably result in optimal expression through the inclusion of the rat 3' enhancer, because chimeric polynucleotides lacking this 3' enhancer result in reduced isotype switching and low IgG expression. In one embodiment, the rat 3' enhancer includes, or has substantially homologous to, the sequence described as Sequence ID No. 1.

[0034] As used herein, a polynucleotide containing a portion of a second sequence (e.g., SEQ ID NO: 1, SEQ ID NO: 2, etc.), for example, less than the whole, or having a substantially homologous sequence, preferably retains the biological activity of the second sequence (e.g., retains the biological activity of a 3' enhancer to provide optimal immunoglobulin expression and / or isotype switching, can be rearranged to provide a humanized chimeric heavy chain, etc.). In one embodiment, a nucleic acid containing a portion of SEQ ID NO: 1, or a substantially homologous sequence, comprises a continuous nucleic acid substantially homologous to SEQ ID NO: 1, at least 8 kB, preferably at least 10 kB. In another embodiment, a second nucleic acid containing a portion of SEQ ID NO: 59 or 60, or a substantially homologous sequence, comprises a continuous nucleic acid substantially homologous to SEQ ID NO: 59 or 60, at least 8 kB, preferably at least 10 kB.

[0035] When used herein, “artificial Ig locus” may refer to a non-reorganized, partially reorganized, or reorganized polynucleotide (e.g., a sequence including the V, D, and / or J regions in the case of a heavy chain, or the V and / or J regions in the case of a light chain, and optionally the constant regions of either or both of the heavy and light chains). An artificial Ig locus includes an artificial Ig light chain locus and an artificial Ig heavy chain locus. In one embodiment, the artificial immunoglobulin locus of the present invention is functional and capable of reorganization and production of a repertoire of immunoglobulin chains. In a preferred embodiment, the variable domain or portion of the polynucleotide disclosed herein includes a gene of a synthetic sequence encoding a polypeptide sequence substantially identical to the natural composition, i.e., the natural sequence of the human Ig gene segment, a degenerate form of the natural sequence of the human Ig gene segment, and the polypeptide encoded by the natural sequence of the human Ig gene segment. In another preferred embodiment, the polynucleotide includes a variable domain or portion of the natural composition found in humans. For example, the polynucleotides encoding the artificial Ig heavy chain disclosed herein may, in their natural composition, include at least two human V genes, at least two D genes, at least two J genes, or a combination thereof.

[0036] In a preferred embodiment, the artificial Ig locus includes a non-human C region gene, making it possible to create a repertoire of immunoglobulins, including chimeric immunoglobulins having a non-human C region. In one embodiment, the artificial Ig locus includes a human C region gene, making it possible to create a repertoire of immunoglobulins, including immunoglobulins having a human C region. In one embodiment, the artificial Ig locus includes an "artificial constant region gene," thereby meaning a constant region gene containing nucleotide sequences derived from human and non-human constant region genes. For example, exemplary artificial C constant region genes are constant region genes encoding the human IgG CH1 domain, as well as the rat IgG CH2 and CH3 domains.

[0037] In some embodiments, the artificial Ig heavy chain locus lacks the CH1 or equivalent sequence, which allows the resulting immunoglobulin to avoid typical immunoglobulin:chaperone association. Such an artificial locus lacks the functional Ig light chain locus, resulting in the production of heavy chain-only antibodies in transgenic animals that do not express the functional Ig light chain. Such an artificial Ig heavy chain locus is used in the method intended herein to produce transgenic animals that lack the functional Ig light chain locus, contain the artificial Ig heavy chain locus, and are capable of producing heavy chain-only antibodies. Alternatively, the artificial Ig locus may be manipulated in situ to disrupt the CH1 or equivalent region, thereby generating an artificial Ig heavy chain locus that results in the production of heavy chain-only antibodies. For more information on heavy chain-only antibody production in light chain-deficient mice, see, for example, Zou et al., JEM, 204:3271-3283, 2007.

[0038] "Human idiotype" refers to the polypeptide sequence present on a human antibody encoded by an immunoglobulin V gene segment. As used herein, the term "human idiotype" includes both the natural sequence of a human antibody and a synthetic sequence substantially identical to the polypeptide found in natural human antibodies. "Substantially" means that the degree of amino acid sequence identity is at least about 85% to 95%. Preferably, the degree of amino acid sequence identity is greater than 90%, or more preferably greater than 95%.

[0039] "Chimera antibody" or "chimeric immunoglobulin" means an immunoglobulin molecule comprising a portion of a human immunoglobulin polypeptide sequence (or a polypeptide sequence encoded by a human Ig gene segment) and a portion of a non-human immunoglobulin polypeptide sequence. The chimeric immunoglobulin molecules of the present invention are immunoglobulins having a non-human Fc region or an artificial Fc region and a human idiotype. Such immunoglobulins can be isolated from animals of the present invention that have been engineered to produce chimeric immunoglobulin molecules.

[0040] "Artificial Fc region" refers to the Fc region encoded by an artificial constant region gene.

[0041] The term "Ig gene segment," as used herein, refers to a region of DNA that encodes various parts of the Ig molecule, present in the germline cells of non-human animals and humans, and assembled within B cells to form a rearranged Ig gene. Thus, as used herein, an Ig gene segment includes the V gene segment, D gene segment, J gene segment, and C gene segment.

[0042] As used herein, the term “human Ig gene segment” includes any of the following: the natural sequence of a human Ig gene segment, a degenerate form of the natural sequence of a human Ig gene segment, and a synthetic sequence encoding a polypeptide sequence substantially identical to the polypeptide encoded by the natural sequence of a human Ig gene segment. “Substantially” means that the degree of amino acid sequence identity is at least about 85% to 95%. Preferably, the degree of amino acid sequence identity is greater than 90%, and more preferably greater than 95%.

[0043] The polynucleotides relating to the present invention may include DNA or RNA and may be entirely or partially synthesized. References to nucleotide sequences expressed herein, unless otherwise specified by context, encompass DNA molecules having a particular sequence and RNA molecules having a particular sequence in which U is substituted with T.

[0044] The calculation of “homology” or “sequence identity” (these terms are used interchangeably herein) between two sequences is carried out as follows: The sequences are aligned for the purpose of best comparison (for example, gaps may be introduced in one or both of the first and second amino acid or nucleic acid sequences of best alignment, and non-homologous sequences may be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90%, or 100% of the length of the reference sequence. Then, amino acid residues or nucleotides are compared at the corresponding amino acid or nucleotide positions. The molecules are identical at the position when the position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence (wherein used herein, “identity” of an amino acid or nucleic acid is equivalent to “homology” of an amino acid or nucleic acid). The identity percentage between two arrays is a function of the number of identical positions shared by those arrays, taking into account the number of gaps and the length of each gap, which must be introduced for optimal alignment of the two arrays.

[0045] The comparison of sequences between two sequences and the determination of the sequence identity percentage can be achieved using mathematical algorithms. In a preferred embodiment, the identity percentage between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J.Mol.Biol.48:444-53) algorithm, which is incorporated within the GAP program in the GCG software package (available online at gcg.com), using either the Blossum62 matrix or the PAM250 matrix, and gap weightings of 16, 14, 12, 10, 8, 6, or 4 and length weightings of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the identity percentage between two nucleotide sequences is determined using the NWSgapdna.CMP matrix, with gap weightings of 40, 50, 60, 60, 70, or 80 and length weightings of 1, 2, 3, 4, 5, or 6, using the GAP program in the GCG software package (available at www.gcg.com). A particularly preferred set of parameters (and one to be used when practitioners are uncertain which parameters should be applied to determine whether a molecule falls within the limits of sequence identity or homology of the present invention) is a Blossum62 score matrix with a 12-gap penalty, a 4-gap elongation penalty, and a 5-frameshift gap penalty. The percentage of identity between two amino acid or nucleotide sequences can also be determined using the Meyers and Miller ((1989) CABIOS 4:11-17) algorithm, incorporated into the ALIGN program (version 2.0), using a PAM120 weighted residue table, a 12-gap length penalty, and a 4-gap penalty.

[0046] Artificial Ig locus The present invention further relates to artificial Ig loci and their use in the creation of transgenic animals capable of producing immunoglobulins having a human idiotype. Each artificial Ig locus comprises multiple immunoglobulin gene segments, each comprising at least one V region gene segment, one or more J gene segments, one or more D gene segments in the case of a heavy chain locus, and one or more constant region genes. In the present invention, at least one of the V gene segments encodes a germline or hypermutant human V region amino acid sequence. Thus, such transgenic animals have the ability to produce a diversified repertoire of immunoglobulin molecules, including antibodies having a human idiotype. In heavy chain loci, human or non-human derived D gene segments may be included in the artificial Ig locus. The gene segments in such loci are juxtaposed with one another in a non-reorganized configuration (or "germline configuration") or in a partially or completely reorganized configuration. The artificial Ig locus has the ability to undergo gene rearrangement in the target animal (if the gene segment is not completely rearranged), thereby producing a diversified repertoire of immunoglobulins with human idiotypes.

[0047] Regulatory elements such as promoters, enhancers, switch regions, recombinant signals, and similar entities may be of human or non-human origin. These elements are required to be operable in the relevant animal species in order to make the artificial locus functional. Suitable regulatory elements are described in detail herein.

[0048] In one embodiment, the present invention provides a transgenic construct comprising an artificial heavy chain locus that can undergo gene rearrangement in a host animal, thereby producing a diversified repertoire of heavy chains having a human idiotype. The transgene artificial heavy chain locus comprises a V region having at least one human V gene segment. Preferably, the V region comprises at least about 5 to 100 human heavy chain V (or "VH") gene segments. In preferred embodiments, the V region comprises more than 20, more than 25, more than 30, more than 35, or more than 40 VH gene segments. As described above, the human VH segment comprises the native sequence of the human VH gene segment, a degenerate form of the native sequence of the human VH gene segment, and a synthetic sequence encoding a polypeptide sequence substantially (i.e., at least about 85% to 95%) identical to the human heavy chain V domain polypeptide.

[0049] In a preferred embodiment, the artificial heavy chain locus comprises at least one or more rat constant region genes, e.g., Cδ, Cμ, and Cγ (including any Cγ subclass).

[0050] In another preferred embodiment, the artificial heavy chain locus includes an artificial constant region gene. In a preferred embodiment, such an artificial constant region gene encodes a human CH1 domain and a rat CH2CH3 domain, or a human CH1 and a rat CH2, CH3, and CH4 domain. A hybrid heavy chain having a human CH1 domain effectively pairs with a complete human light chain.

[0051] In a preferred embodiment, the artificial Ig locus includes hs1,2, hs3a, hs3b, and a 3' enhancer sequence comprising the sequence between rat C-alpha and 3'hs3b.

[0052] In another preferred embodiment, the artificial heavy chain locus comprises an artificial constant region gene lacking a CH1 domain. In a preferred embodiment, such an artificial constant region gene encodes cleaved IgM and / or IgG lacking a CH1 domain but containing CH2 and CH3, or CH1, CH2, CH3, and CH4 domains. The heavy chain lacking a CH1 domain cannot effectively pair with the Ig light chain and forms an antibody consisting only of the heavy chain.

[0053] In another embodiment, the present invention provides a transgenic construct comprising an artificial light chain locus that can undergo gene rearrangement in a host animal, thereby producing a diversified repertoire of light chains having a human idiotype. The transgene artificial light chain locus comprises a V region having at least one human V gene segment, for example, a V region having at least one human VL gene and / or at least one rearranged human VJ segment. Preferably, the V region comprises at least about 5 to 100 human light chain V (or "VL") gene segments. Consistently, the human VL segment comprises the native sequence of the human VL gene segment, a degenerate form of the native sequence of the human VL gene segment, and a synthetic sequence encoding a polypeptide sequence substantially (i.e., at least about 85% to 95%) identical to the human light chain V domain polypeptide. In one embodiment, the artificial light chain Ig locus has a C region having at least one rat C gene (e.g., rat Cλ or Cκ).

[0054] Another aspect of the present invention relates to a method for constructing a transgenic vector containing an artificial Ig locus. Such a method involves isolating an Ig locus or a fragment thereof and conjugating it with one or more DNA fragments containing sequences encoding human V region elements. The Ig gene segment(s) is inserted into the artificial Ig locus or a portion thereof by ligation or homologous recombination in a manner that preserves the ability of the locus to undergo effective gene rearrangement in the animal of interest.

[0055] Preferably, non-human Ig loci are isolated by screening libraries of plasmids, cosmids, YACs, or BACs, and similar products, prepared from their genomic DNA. Since YAC clones can hold DNA fragments up to 2 megabase pairs in size, an entire or large portion of an animal heavy chain locus may be isolated within a single YAC clone or reconstructed to be contained within a single YAC clone. BAC clones can hold smaller DNA fragments (approximately 50–500 kb). However, multiple BAC clones containing duplicate fragments of an Ig locus may be individually modified and then injected together into animal recipient cells, where the duplicate fragments recombine to generate a continuous Ig locus.

[0056] Human Ig gene segments can be incorporated into the Ig locus of a vector (e.g., a BAC clone) by various methods, including ligation of DNA fragments or insertion of DNA fragments by homologous recombination. The incorporation of a human Ig gene segment is carried out in such a manner that the human Ig gene segment operably ligates to the host animal sequence within the transgene to produce a functional humanized Ig locus, i.e., an Ig locus that enables gene rearrangement resulting in the production of a diversified repertoire of antibodies possessing the human idiotype. Homologous recombination can be performed in bacteria, yeast, and other cells with high-frequency homologous recombination events. Manipulated YACs and BACs can be readily isolated from these cells and used in the production of transgenic animals.

[0057] Transgenic animals capable of producing antibodies containing an artificial Ig gene locus and possessing a human idiotype. In one embodiment, the present invention provides a transgenic animal capable of producing immunoglobulins having a human idiotype, and a method for producing the same. The transgenic animal used is selected from rodents (e.g., rats, hamsters, mice, and guinea pigs).

[0058] The transgenic animals used for humanized antibody production in the present invention harbor germline mutations at endogenous Ig loci. In preferred embodiments, the transgenic animals are homozygous for mutated endogenous Ig heavy chain and / or endogenous Ig light chain genes. Furthermore, these animals harbor at least two artificial heavy chain Ig loci that are functional and capable of producing a repertoire of immunoglobulin molecules in the transgenic animals. The artificial Ig loci used herein include at least one human V gene segment.

[0059] In a preferred embodiment, the transgenic animal possesses at least two artificial Ig heavy chain loci and at least one artificial Ig light chain loci, each capable of producing a repertoire of immunoglobulin molecules, each functional and containing antibodies having a human idiotype in the transgenic animal. In one embodiment, an artificial locus containing at least one non-human C gene is used, and an animal is provided capable of producing a chimeric antibody having a human idiotype and a non-human constant region. In another embodiment, an artificial locus containing at least one human C gene is used, and an animal is provided capable of producing an antibody having a human idiotype and a human constant region.

[0060] In another preferred embodiment, the transgenic animal possesses at least two artificial Ig heavy chain loci and lacks a functional Ig light chain locus. Such animals find applications in the production of heavy chain-only antibodies.

[0061] The production of such transgenic animals involves the integration into the genome of a transgenic animal having at least one endogenous Ig locus, in which at least two artificial heavy chain Ig loci and one or more artificial light chain Ig loci have been or will be deactivated by the action of one or more meganucleases. Preferably, the transgenic animal is null-conjugated with respect to the endogenous Ig heavy chain and / or endogenous Ig light chain and therefore cannot produce endogenous immunoglobulins. Regardless of chromosomal location, the artificial Ig loci of the present invention are capable of undergoing gene rearrangement, thereby producing a diversified repertoire of immunoglobulin molecules. Ig loci capable of undergoing gene rearrangement are also referred to herein as “functional” Ig loci, and antibodies having diversity produced by functional Ig loci are also referred herein as “functional” antibodies or a “functional” repertoire of antibodies.

[0062] The artificial loci used to generate such transgenic animals each comprise multiple immunoglobulin gene segments, each containing at least one V-region gene segment, one or more J-region segments, one or more D-region segments in the case of a heavy chain locus, and one or more constant-region genes. In this invention, at least one of the V-region segments encodes a germline or hypermutant human V-region amino acid sequence. Thus, such transgenic animals have the ability to produce a diversified repertoire of immunoglobulin molecules, including antibodies having a human idiotype.

[0063] In one embodiment, the artificial locus used includes at least one non-human C-region gene segment. Thus, such a transgenic animal has the ability to produce a diversified repertoire of immunoglobulin molecules, including chimeric antibodies having a human idiotype.

[0064] In one embodiment, the artificial locus used includes at least one human C-region gene segment. Thus, such a transgenic animal has the ability to produce a diversified repertoire of immunoglobulin molecules, including antibodies having human idiotypes and human constant regions.

[0065] In one embodiment, the artificial locus used includes at least one artificial constant region gene. For example, an exemplary artificial C constant region gene is a constant region gene encoding the human IgG CH1 domain, as well as the rat IgG CH2 and CH3 domains. Thus, such a transgenic animal has the ability to produce a diversified repertoire of immunoglobulin molecules, including antibodies having human idiotypes and artificial constant regions containing both human and non-human components.

[0066] A transgenic vector containing an artificial Ig gene locus is introduced into recipient cells(s), and subsequently integrated into the genome of the recipient cells(s) by random or targeted integration.

[0067] In the case of random insertion, a transgenic vector containing an artificial Ig locus can be introduced into recipient cells by standard transgenic techniques. For example, the transgenic vector can be directly injected into the pronucleus of a fertilized oocyte. The transgenic vector can also be introduced by co-incubating sperm with the transgenic vector before fertilization of the oocyte. The transgenic animal can develop from the fertilized oocyte. Another method for introducing the transgenic vector is to transfect embryonic stem cells or other pluripotent cells (e.g., primordial germ cells) and then inject the genetically modified cells into a developing embryo. Alternatively, the transgenic vector (as is or in combination with an accelerating agent) can be directly injected into a developing embryo. Finally, a chimeric transgenic animal is produced from an embryo containing an artificial Ig transgene integrated into the genome of at least some somatic cells of the transgenic animal. In another embodiment, the transgenic vector is introduced into the genome of cells, and the animal is obtained from transfected cells by nuclear transfer cloning.

[0068] In a preferred embodiment, the transgene containing the artificial Ig locus is randomly integrated into the genome of a recipient cell (such as a fertilized oocyte or developing embryo). In a preferred embodiment, offspring are obtained that are null-zygous for endogenous Ig heavy chains and / or Ig light chains and therefore unable to produce endogenous immunoglobulins but capable of producing transgenic immunoglobulins.

[0069] In the case of targeted integration, the transgenic vector can be introduced into suitable recipient cells, such as embryonic stem cells, other pluripotent cells, or already differentiated somatic cells. Cells into which the transgene has been integrated into the animal genome can then be selected by standard methods. The selected cells can then be fused with enucleated nuclear transfer unit cells, such as oocytes or embryonic stem cells (cells that are totipotent and capable of forming functional neonates). Fusion is performed according to well-established conventional techniques. See, for example, Cibelli et al., Science (1998) 280:1256; Zhou et al., Science (2003) 301:1179. Oocyte enucleation and nuclear transfer can also be performed by microsurgery using an injection pipette (see, for example, Wakayama et al., Nature (1998) 394:369). The resulting cells are then cultured in appropriate media and transferred to synchronized recipients for the production of transgenic animals. Alternatively, selected genetically modified cells can be injected into developing embryos that will subsequently develop into chimeric animals.

[0070] In one embodiment, a meganuclease is used to increase the frequency of homologous recombination at a target site during double-strand DNA cleavage. For integration into a specific site, a site-specific meganuclease may be used. In one embodiment, a meganuclease targeting an endogenous Ig locus is used to increase the frequency of homologous recombination and substitution of an artificial Ig locus or a portion thereof with an endogenous Ig locus or a portion thereof. In one embodiment, the transgenic animal lacks a functional Ig light chain locus and contains an artificial Ig heavy chain locus.

[0071] Preferred embodiments for the incorporation of human Ig gene segments using YACs and BACs, and for their mutual exchange, offer advantages in both speed and the ability to verify the completeness of constructs of larger regions due to overlapping homology. Tandem incorporation of constructs with overlapping regions has the ability to maintain full functionality, which is essential for DNA rearrangement. Preferred embodiments of the present invention not only have the desired incorporation by homology but also result in tandem incorporation as a frequent event. This substantially facilitates transgenic techniques as it eliminates the need for the difficult incorporation of large YACs into stem cells and subsequent induction of animals from those stem cells. In addition, ZFN techniques, similarly implemented via DNA injection (Geurts et al. Science 325,433 (2009); Menoret et al. European journal of immunology 40,2932-2941 (2010)), facilitate the production of Ig KO lines and may be a future technique of choice for gene disruption and substitution. Silencing endogenous Ig gene expression in OmniRat®, including the human-rat IgH and human IgL loci, has the advantage of preventing the generation of interfering or undesirable rat Ig in the mixed product.

[0072] In mice, the enhancer region downstream of Cα plays an essential role in class switch recombination (Vincent-Fabert et al. Blood 116, 1895-1898 (2010)), and elements within this region may promote hypermutation (Pruzina et al. Protein engineering, design & selection: PEDS 24, 791-799 (2011)). This may be the reason why high-frequency immune responses and the generation of diverse hybridomas can be difficult even in mice carrying large complete human loci (Davis et al. Cancer metastasis reviews 18, 421-425 (1999); Lonberg Current opinion in immunology 20, 450-459 (2008)). Since the chimeric human-rat IgH locus promotes differentiation and expression levels similar to wild type in OmniRat, the endogenous rat C region, and actually approximately 30 kb enhancer sequence 3’ of Cα, provide optimal locus control for expressing and maturing human V H genes. Another region, Cδ with its 3’ regulatory motif cluster (Mundt et al. J Immunol 166, 3315-3323 (2001)), has been removed from the chimeric C region BAC because silencing or absence of IgD did not seem to reduce immune function (Chen Immunol Rev 237, 160-179 (2010)). Normally, mature IgM + IgD + B cells downregulate IgD upon antigen contact, which initiates class switch recombination (Id). Therefore, switching can increase without IgD control, which is supported by the inventors' finding that IgG transcripts and serum levels are significantly lower when the Cδ region is retained within the transgenic construct (data not shown).

[0073] The inventors found that, under various immunization conditions, mAbs with sequence and epitope diversity equivalent to those produced in wild-type controls could be isolated via spleen and lymph node fusion, making the production of specific IgG in OmniRat® particularly promising. The V, D, and J gene diversity was as expected, and almost all segments were found to be productively utilized as anticipated (Lefranc & Lefranc The immunoglobulin factsbook. FactsBook Series, Academic Press, GB, 45-68 (2001)). This was in striking contrast to mice with a fully human trans locus, where clonal proliferation from a small number of precursor B cells yielded little diversity (Pruzina et al. Protein engineering, design & selection: PEDS 24, 791-799 (2011)). Because the number of transplanted V genes is only about half that used in humans, the inventors predicted that limited immune responses and limited diversity would be found when comparing OmniRat to wild-type animals. However, this was not the case, and a comparison of CDR3 diversity in over 1000 clones (sequences may be provided) revealed that OmniRat exhibited extensive conjugation differences similar to those in wild-type animals. A small number of identical gene segment combinations were found to be V H From D and / or from D to J H Further diversification occurred through the addition or deletion of the N sequence at conjugation, as well as through hypermutation. Therefore, the rat C region sequence is similar to that of human V. H DJ HIt has been shown to be highly efficient in regulating DNA rearrangement and expression. Extensive diversity was also observed for the introduced human Igκ and Igλ loci, similar to that already shown in mice (Nicholson et al. J Immunol 163,6898-6906(1999);Pruzina et al. Protein engineering, design & selection:PEDS 24,791-799(2011);Popov et al. The Journal of experimental medicine 189,1611-1620(1999)). Therefore, the significantly reduced efficiency in the production of human antibodies from mice (Lonberg,N. Nature biotechnology 23,1117-1125(2005)) has been overcome in OmniRat™, which reliably and extensively diversifies the rearranged H chains through class switching and hypermutation, producing high-affinity antibodies not occasionally but in large quantities. The yield and hypermutation levels of transgenic IgG, which are impressively utilized in antigen-specific mAbs, showed similar levels of clonal diversification and production between OmniRat® and wild-type animals. Regular generation of high affinity specificity in the sub-nanomolar range was achieved even by different monoimmunizations, and here again, it was preferable compared to wild-type animals (results have not been shown in transgenic mice that produce a human antibody repertoire exclusively from human loci) (Mendez et al. Nature Genetics 15, 146-156 (1997)).

[0074] In summary, to maximize human antibody production, the IgH locus should be considered essential, utilizing human genes for antibody specificity but rodent genes for differentiation and high expression control. The flexibility of the light chain is an additional advantage, as it allows for highly efficient assembly of human IgH / IgL even in the presence of wild-type Ig. For therapeutic applications, chimeric heavy chains can be readily converted to full human abs by C gene substitution without compromising specificity.

[0075] Immunoglobulins with human idiotypes Once transgenic animals capable of producing immunoglobulins having a human idiotype are created, immunoglobulins and antibody preparations against antigens can be easily obtained by immunizing the animals with the antigen. When used herein, "polyclonal antiserum composition" includes affinity-purified polyclonal antibody preparations.

[0076] Various antigens can be used to immunize transgenic animals. Such antigens include, but are not limited to, microorganisms, such as viruses and single-celled organisms (bacteria and fungi, etc.), fragments of live, attenuated, or dead microorganisms, or antigenic molecules isolated from microorganisms.

[0077] Suitable bacterial antigens for use in animal immunization include purified antigens from Staphylococcus aureus, such as capsular polysaccharides types 5 and 8; recombinant virulence factors such as alpha toxins; adhesion junction proteins; collagen-binding proteins; and fibronectin-binding proteins. Suitable bacterial antigens also include attenuated forms of S. aureus, Pseudomonas aeruginosa, enterococcus, enterobacter, and Klebsiella pneumoniae, or culture supernatants from these bacterial cells. Other bacterial antigens that may be used in immunization include purified lipopolysaccharide (LPS), capsular antigens, capsular polysaccharides, and / or recombinant outer membrane proteins; fibronectin-binding proteins; and endotoxins and exotoxins of Pseudomonas aeruginosa, enterococcus, enterobacter, and Klebsiella pneumoniae.

[0078] Preferred antigens for the production of antibodies against fungi include attenuated fungi or their outer membrane proteins, and fungi include, but are not limited to, Candida albicans, Candida parapsilosis, Candida tropicalis, and Cryptococcus neoformans.

[0079] Preferred antigens for use in immunization to produce antibodies against viruses include, but are not limited to, viral envelope proteins and attenuated bodies, viral syncytial virus (RSV) (especially the F protein), hepatitis C virus (HCV), hepatitis B virus (HBV), cytomegalovirus (CMV), EBV, and HSV.

[0080] Cancer-specific antibodies can be produced by immunizing transgenic animals with isolated tumor cells or tumor cell lines, as well as tumor-associated antigens, including but not limited to Her-2-neu antigen (an antibody useful for treating breast cancer), CD20, CD22, and CD53 antigens (antibodies useful for treating B-cell lymphoma), prostate-specific membrane antigen (PMSA) (an antibody useful for treating prostate cancer), and the 17-1A molecule (an antibody useful for treating colon cancer).

[0081] The antigen may be administered to the transgenic animal by any conventional method, with or without adjuvant, and may be administered according to a predetermined plan.

[0082] To produce monoclonal antibodies, spleen cells are isolated from immunized transgenic animals and used in cell fusion with transformed cell lines for hybridoma production, or the cDNA encoding the antibody is cloned using standard molecular biology techniques and expressed in transfected cells. Procedures for producing monoclonal antibodies are well established in the art. See, for example, European Patent Application No. 0 583 980 A1 ("Method For Generating Monoclonal Antibodies From Rabbits"), U.S. Patent No. 4,977,081 ("Stable Rabbit-Mouse Hybridomas And Secretion Products Thereof"), WO97 / 16537 ("Stable Chicken B-cell Line And Method of Use Thereof"), and European Patent No. 0 491 057 B1 ("Hybridoma Which Produces Avian Specific Immunoglobulin G"), whose disclosures are incorporated herein by reference. In vitro production of monoclonal antibodies from clonal cDNA molecules has been described by Andris-Widhopf et al. J Immunol Methods 242:159 (2000) and by Burton Immunotechnology 1:87 (1995).

[0083] When chimeric monoclonal antibodies with a human idiotype are generated, such chimeric antibodies can be readily converted to fully human antibodies using standard molecular biology techniques. Fully human monoclonal antibodies are not immunogenic in humans and are suitable for use in therapeutic treatments for human subjects.

[0084] The antibodies of the present invention include antibodies consisting solely of heavy chains. In one embodiment, a transgenic animal lacking a functional Ig light chain locus and containing at least two artificial heavy chain loci is immunized with an antigen to produce an antibody consisting only of the heavy chain that specifically binds to the antigen.

[0085] In one embodiment, the present invention provides monoclonal antibody-producing cells of such animal origin, and nucleic acids obtained therefrom. Hybridomas derived therefrom are also provided. Antibodies consisting solely of fully human heavy chains, and coding nucleic acids derived therefrom are also provided.

[0086] Instructions regarding antibodies consisting solely of heavy chains can be found in the art. See, for example, PCT International Publications WO02085944, WO02085945, WO2006008548, and WO2007096779. See also U.S. Patents 5,840,526, 5,874,541, 6,005,079, 6,765,087, 5,800,988, European Patent No. 1589107, WO9734103, and U.S. Patent No. 6,015,695.

[0087] Pharmaceutical composition In further embodiments of the present invention, purified monoclonal or polyclonal antibodies are mixed with a suitable pharmaceutical carrier suitable for administration to a patient in order to provide a pharmaceutical composition.

[0088] Patients treated with the pharmaceutical compositions of the present invention are preferably mammals, more preferably humans, but veterinary use is also intended.

[0089] Any pharmaceutically acceptable carrier that may be used in the present pharmaceutical composition may be any and all solvents, dispersions, isotonic agents, and the like. Any conventional medium, agent, diluent, or carrier is suitable for use in the pharmaceutical composition of the present invention unless it is detrimental to the therapeutic efficacy of the recipient or the antibodies contained therein.

[0090] The carrier may be a liquid, semi-solid, such as a paste or solid carrier. Examples of carriers include oils, water, saline solution, alcohols, sugars, gels, lipids, liposomes, resins, porous matrices, binders, fillers, coatings, preservatives, and the like, or combinations thereof.

[0091] Treatment method In a further aspect of the present invention, a method is provided for treating a disease in a vertebrate, preferably a mammal, preferably a primate (a human subject is a particularly preferred embodiment), by administering a purified antibody composition of the present invention that is desirable for the treatment of such disease.

[0092] Antibody compositions may be used to bind to, neutralize, or modulate antigenic elements in human tissues that cause or contribute to disease, or that induce an undesirable or abnormal immune response. “Antigenic element” is defined herein to include any soluble or cell surface-bound molecules, including proteins, and cells or organisms or active substances that cause infection, which can at least bind to an antibody and preferably stimulate an immune response.

[0093] Administration of antibody compositions against infectious agents, either as monotherapy or in combination with chemotherapy, leads to the elimination of infectious particles. A single dose of antibody generally reduces the number of infectious particles by 10 to 100 times, and more commonly by more than 1000 times. Similarly, antibody therapy in patients with malignant diseases, used either as monotherapy or in combination with chemotherapy, generally reduces the number of malignant cells by 10 to 100 times, or more than 1000 times. Treatment may be repeated for extended periods to ensure the complete elimination of infectious particles, malignant cells, etc. In some cases, antibody therapy may be continued for extended periods in the absence of detectable levels of infectious particles or unwanted cells.

[0094] Similarly, the use of antibody therapy to modulate the immune response may consist of a single or multiple doses of therapeutic antibodies. The treatment may be continued for an extended period in the absence of any disease symptoms.

[0095] The targeted therapy may be used in conjunction with chemotherapy at doses sufficient to suppress infection or malignancy. In patients with autoimmune diseases or transplant recipients, antibody therapy may be used in conjunction with immunosuppressive therapy at doses sufficient to suppress the immune response. [Examples]

[0096] In mice transgenic to the human immunoglobulin (Ig) gene locus, the suboptimal efficiency of full-human antibody delivery is attributed to incomplete interaction between the constant region of the human membrane IgH chain and the mouse cell signaling mechanism. To prevent this problem, we hereby introduce, in this specification, human V at the chimeric human / rat IgH gene locus

[22] . H , has germline gene space but rat C H All human D and J gene loci linked to the locus H This section describes a humanized rat strain (OmniRat®) that possesses a complete human light chain locus [12 Vκs linked to Jκ-Cκ and 16 Vλs linked to Jλ-Cλ] along with the segment [including the segment]. The endogenous rat Ig locus was silenced with a designer zinc finger nuclease. After immunization, OmniRat functions as efficiently as normal rats in producing high-affinity serum IgG. Monoclonal antibodies containing a complete human variable region with sub-nanomolecular antigen affinity and carrying a wide range of somatic mutations can be easily obtained, similar to the production of conventional antibodies from normal rats.

[0097] Materials and methods Construction of modified human Ig gene loci on YAC and BAC a) IgH gene locus The human IgH V gene was encompassed by two BACs: BAC6-VH3-11 (modified from commercially available BAC clone 3054M17 CITB), which contained a true region extending from VH4-39 to VH3-23 and then to VH3-11, and BAC3 (811L16 RPCI-11), which contained a true region extending from VH3-11 to VH6-1. A BAC called Annabel was constructed by ligating a rat CH region gene directly downstream of the human VH6-1-D-JH region (Figure 1). All BAC clones containing portions of the human or rat IgH gene locus were purchased from Invitrogen.

[0098] Both BAC6-VH3-11 and Annabel were first constructed as cyclic YACs (cYACs) within S. cerevisiae, and then confirmed and maintained as BACs within E. coli.

[0099] Unlike YACs, BAC plasmid preparations produce large quantities of the desired DNA. To convert linear YACs to cYACs, or to assemble DNA fragments with overlapping ends into a single cYAC within S. cerevisiae (which can also be maintained as a BAC within E. coli), we constructed two self-replicating S. cerevisiae / E. coli shuttle vectors, pBelo-CEN-URA and pBelo-CEN-HYG. Briefly, S. cerevisiae CEN4 was excised from pYAC-RC as an AvrII fragment (Marchuk & Collins Nucleic Acids Research 16,7743 (1988)) and ligated to SpeI-linearized pAP599 (Kaur & Cormack PNAS 104,7628-7633 (2007)). The resulting plasmid contains CEN4 cloned between S. cerevisiae URA3 and a hygromycin resistance expression cassette (HygR). From this plasmid, an ApaLI-BamHI fragment containing URA3 is excised, followed by a CEN4 or a PmlI-SphI fragment containing CEN4, and then the HygR is ligated into pBACBelo11 (New England Biolabs) double-digested with ApaLI and BamHI, or HpaI and SphI, to produce pBelo-CEN-URA and pBelo-CEN-HYG.

[0100] To construct BAC6-VH3-11, two fragments, a 115kb NotI-PmeI and a 110kb RsrII-SgrAI, were first excised from BAC clone 3054M17 CITB. The 3' end of the former fragment overlapped with the 5' end of the latter by 22kb. The NotI-PmeI fragment was ligated to a NotI-BamHI YAC arm containing S. cerevisiae CEN4 and TRP1 / ARS1 from pYAC-RC, and the RsrII-SgrAI fragment was similarly ligated to a SgrAI-BamHI YAC arm containing S. cerevisiae URA3 from pYAC-RC. Subsequently, the ligation mixtures were transformed into spheroplasts. 41S. cerevisiae AB1380 cells were transformed via [method / method], and a URA+TRP+ yeast clone was selected. A clone called YAC6, containing a linear region from human VH4-39 to VH3-23, was identified by Southern blot analysis. YAC6 was further extended by adding a 10.6kb VH3-23 fragment to the 3' end, converting it to cYAC. The 10.6kb extension includes human VH3-11, which also occurs at the 5' end of BAC3. For modification of YAC6, pBeloHYG-YAC6+BAC3(5') was constructed. Briefly, three fragments with overlapping ends were prepared by PCR: 1) S. cerevisiae adjacent to the HpaI site in YAC6, with a 5' end matching the upstream sequence of VH4-39 and a 3' end matching the downstream sequence of VH3-23. 1) A "stuff" fragment containing TRP1-ARS1 (using long oligos 561 and 562, and pYAC-RC as a template), 2) a 10.6kb elongated fragment having a 5' end matching the downstream sequence of VH3-23, as described above, and a unique AscI site at its 3' end (using long oligos 570 and 412, and human genomic DNA as a template), 3) a pBelo-CEN-HYG vector having a CEN4 downstream ligated to a homologous end matching the 3' end of the 10.6kb elongated fragment, as described above, and a HygR upstream ligated to an end matching the upstream sequence of VH4-39 (using long oligos 414 and 566, and pBelo-CEN-HYG as a template). Next, the three PCR fragments were assembled into a small cYAC conferring HYGR and TRP+ in S. cerevisiae via homologous recombination associated with spheroplast transformation, and this cYAC was further converted to BACpBeloHYG-YAC6+BAC3(5'). Finally, using HpaI-digested pBeloHYG-YAC6+BAC3(5'), yeast cells retaining YAC6 were transformed, and via homologous recombination, a cYAC BAC6-VH3-11 conferring only HYGR was generated. This cYAC was introduced into E. coli as BAC via transformation (see below).The human VH gene within BAC6-VH3-11 was excised as an approximately 182kb AsiSI (spontaneously occurring within HygR)-AscI fragment, and the VH gene within BAC3 was excised as an approximately 173kb NotI fragment (Figure 1, top).

[0101] A self-replicating shuttle vector called pCAU, which efficiently acts in both Saccharomyces cerevisiae and E. coli, was constructed based on the previously published pBelo-CEN-URA (Osborn et al. J Immunol 2013;190:1481-1490). Briefly, ARSH4 was amplified from S. cerevisiae genomic DNA using primers 878 and 879 (all primer sequences are listed below), and AsiSI and SexAl were introduced at both ends following the ApaLI site. The fragment was digested with ApaLI and SexAI and ligated with pBelo-CEN-URA digested with the same restriction enzymes to obtain pCAU. This vector contains S. cerevisiae CEN4, URA3, and ARSH4 in a pBeloBAC11 backbone (New England BioLabs).

[0102] Three BACs derived from human chromosome 14, CTD-2011A5 (BAC9), CTD-3148C6 (BAC14), and CTD-2548B8 (BAC5), were purchased from Invitrogen / Thermo Fisher. A human genomic region encompassing IgHV3-74 to IgHV1-58 was isolated from BAC9 as a 185kb NotI-fragment. BAC(14+5) was constructed from BAC14 and BAC5. As a 210kb BsiwI-fragment, we isolated a combined genomic region of this BAC that included a 90.6kb region from BAC14 containing a 4.6kb sequence from 5' to 3' that overlaps with the 3' of the NotI fragment from BAC9, followed by an 86kb region containing IgHV5-51 to IgHV1-45, a 1.7kb synthetic region centrally located and linking IgHV3-43 with BAC14 and BAC5, a 111.7kb region from BAC5 containing IgHV3-21 to IgHV3-13, and a 6.1kb region overlapping with Anabel's 5' (BAC that holds the human Ig constant region).

[0103] BAC (also known as 14+5 or 14 / 5) was constructed in three steps, including the generation of cyclic YAC (cYAC) by homologous recombination within yeast as described above, and the conversion of cYAC to BAC. Firstly, the BAC vector-pCAU+GAP-BAC14,5 was generated in yeast by assembling the following three duplicate fragments: from 5' to 3': a 116 bp sequence overlapping the 5' and 3' ends of the desired region of BAC14 having a centrally specific RsrII site, a 1.6 kb IgHV3-43 gene [including a 1.0 kb 5' untranslated region (UTR) and a 0.2 kb 3' UTR], a 106 bp sequence overlapping the 5' and 3' ends of the desired region of BAC5 having a centrally specific PmeI site, and a 38 bp sequence overlapping the 5' end of Anabel, a 6.1 kb PCR fragment corresponding to Anabel's 5' using primers 383 and 384, and a pCAU vector amplified using primers 1066 and 1088. Secondly, the pCAU+GAP-BAC14,5 vector was linearized with PmeI and co-transformed with a 154kb NotI-fragment isolated from BAC5 into the yeast strain AB1380. The resulting BAC (approximately 128kb in length) possessed the desired region of BAC5 incorporated into the BAC vector via homologous recombination mediated by homologous ends to BAC5 exposed in the PmeI linearized vector. Thirdly, the BAC holding the second-stage BAC5 was linearized with RsrII, exposing the homologous ends to the desired region of BAC14, and co-transformed with a 114kb SnaBI-fragment isolated from BAC14 to obtain BAC(14+5).

[0104] For the assembly of C regions with VH duplication, to obtain cYAC / BAC, the human VH6-1-D-JH region had to be ligated to the rat genome sequence immediately downstream of the last JH, followed by rat C. To achieve this, five overlapping restriction and PCR fragments were used: a 6.1kb fragment 5' of human VH6-1 (using oligos 383 and 384, and human genomic DNA as a template), an approximately 78kb PvuI-PacI fragment containing the human VH6-1-D-JH region excised from BAC1 (RP11645E6), an 8.7kb fragment containing a portion of the rat μ-coding sequence (using oligos 488 and 346, and rat genomic DNA as a template), and BAC. A NotI-PmeI fragment of approximately 52kb containing the true rat μ, δ, and γ2c regions excised from M5 (CH230-408M5) was prepared, along with a pBelo-CEN-URA vector (using long-chain oligos 385 and 550, and pBelo-CEN-URA as a template) having URA3 ligated downstream with a homologous end corresponding to the 3' end of the rat γ2c region, and CEN4 ligated upstream with a end corresponding to the 5' region of human VH6-1, as described above. Accurate assembly via homologous recombination within S. cerevisiae was analyzed by PCR, and purified cYAC from accurate clones was converted to BAC in E. coli.

[0105] The assembly of the Annabel portion described above used human VH6-1-D-JH, followed by cYAC / BAC containing the true rat μ, δ, and γ2c regions, as well as PCR fragments. The five duplicate fragments included, as described above, a 6.1kb fragment at the 5' end of human VH6-1, an approximately 83kb SpeI fragment containing human VH6-1-D-JH, immediately followed by the rat genome sequence downstream of the last JH, and a portion of rat Cμ, a 5.2kb fragment (using oligos 490 and 534, and rat genomic DNA as a template) ligated at the 5' end of rat γ1 to the 3' end of rat μ, a NotI-SgrAI fragment of approximately 118kb containing the true rat γ1, γ2b, ε, α, and 3'E IgH enhancer regions excised from BAC I8 (CH230-162I08), and a pBelo-CEN-URA vector having URA3 ligated downstream with a homologous end matching the 3' end of rat 3'E as described above, and CEN4 ligated upstream with an end matching the 5' end of human VH6-1. There is a 10.3kb overlap between the human VH6-1 regions in both BAC3 and Annabel. In Annabel, the human VH6-1-D-JH, which follows the rat CH region and S. cerevisiae URA3, can be excised as a single NotI-fragment of approximately 183kb (see Figure 1).

[0106] BAC6-VH3-11, BAC3, BAC9, and BAC(14+5), as well as Annabel, were thoroughly verified for their authenticity through restriction analysis and partial sequencing.

[0107] b) IgL gene locus The human Igλ locus on a YAC of approximately 410kb was obtained by recombinant assembly of cosmid-containing 3Cλ and Vλ YACs (Popov et al. Gene 177,195-201 (1996)). Rearrangement and expression were validated in transgenic mice derived from ES cells containing one copy of the complete human Igλ YAC (Popov et al. The Journal of Experimental Medicine 189,1611-1620 (1999)). This Igλ YAC was shortened by generating a circular YAC by removing approximately 100kb of the 5' region of Vλ3-27. The vector pYAC-RC was digested with ClaI and BspEI to remove URA3 and ligated with a ClaI / NgoMIV fragment from pAP599 containing HYG. PCR of the yeast centromere and hygromycin marker gene region from a novel vector (pYAC-RC-HYG) was performed using a primer with a 5' end homologous to the 5' region of Vλ3-27 (primer 276) and a primer within the ADE2 marker gene in the YAC arm (primer 275). PCR fragments (3.8kb) were incorporated into Igλ YAC using a highly efficient lithium acetate transformation method (Gietz & Woods Methods in Microbiology 26, 53-66 (1998)) and hygromycin selection including YPD plates. DNA was prepared from clones (Epicentre MasterPure Yeast DNA Purification Kit), and accurate conjugation was analyzed by PCR using the following oligos: 243+278 and Hyg-terminal R+238. Plugs were prepared (Peterson Nature protocols 2, 3009-3015 (2007)), yeast chromosomes were removed by PFGE (0.8% agarose (PFC) (Biorad) gel [6V / cm, pulse times of 60 seconds and 10 seconds per 10 hours, 8°C]), and circular yeast artificial chromosomes were trapped in the agarose block (Beverly, Nucleic acids research 16, 925-939 (1988)). The block was removed and digested with NruI.In short, the block was pre-incubated on ice for 1 hour with excess restriction enzyme buffer at a final concentration of 1×. The excess buffer was removed, leaving just enough to cover the plug, and the restriction enzyme was added to a final concentration of 100 U / ml. The tube was incubated at 37°C for 4–5 hours. Linearized YAC was eluted from the block by PFGE, cut off as a fragment from the gel, and purified as described below.

[0108] Three BACs (RP11-344F17, RP11-1134E24, and RP11-156D9, Invitrogen) were selected for the human Igκ locus, covering a region of over 300kb from 5'Vκ1-17 to 3'KDE (Kawasaki et al. European Journal of Immunology 31, 1017-1028 (2001)). Digestion and sequence analysis identified three duplicate fragments: from Vκ1-17 to Vκ3-7 (150kb NotI with approximately 14kb of duplication), from Vκ3-7 to 3' of Cκ (158kb NotI with approximately 40kb of duplication), and from Cκ to 3' of KDE (55kb PacI with 40kb of duplication). When overlapping regions are co-injected into oocytes, ligation and integration are generally preferred (Wagner et al. Genomics 35, 405-414 (1996)).

[0109] Gel analysis and DNA purification Purified YAC and BAC DNA were analyzed by restriction digestion and separation on a conventional 0.7% agarose gel (Sambrook & Russell Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY (2001)). Larger fragments of 50–200 kb were separated by PFGE (Biorad Chef Mapper®) at 80°C using 0.8% PFC agarose in 0.5% TBE, with a switch time of 2–20 seconds over 16 hours, at 6 V / cm, 10 mA. Purification allowed for direct comparison of the obtained fragments with the expected sizes obtained from sequence analysis. Modifications were analyzed by PCR and sequencing.

[0110] The digested linear YAC, circular YAC, and BAC fragments were purified by electroelution using Elutrap™ (Schleicher and Schuell) from sections cut from conventional 0.8% agarose gel electrophoresis or pulsed-field gel electrophoresis (PFGE) (Gu et al. Journal of biochemical and biophysical methods 24,45-50 (1992)). The DNA concentration was typically several ng / μl in a volume of approximately 100 μl. For fragments up to approximately 200 kb, the DNA was precipitated and redissolved to the desired concentration in microinjection buffer (10 mM Tris-HCl pH 7.5, 100 mM EDTA pH 8, and 100 mM NaCl, but without spermine / spermidine).

[0111] Circular YACs were purified from yeast using Nucleobond AX silica-based anion exchange resin (Macherey-Nagel, Germany). Briefly, spheroplasts were prepared using zymolyase or liticase and pelletized (Davies et al. Human antibody repertoires in transgenic mice: Manipulation and transfer of YACs. IRL Oxford, 59-76 (1996)). The cells were then lysed with alkali, conjugated to an AX100 column, and eluted as described in the Nucleobond method for low-copy plasmids. Contaminating yeast chromosomal DNA was hydrolyzed using Plamid-Safe™ ATP-Dependent DNase (Epicentre Biotechnologies), followed by a final purification step using SureClean (Bioline). Subsequently, aliquots of DH10 electrocompetent cells (Invitrogen) were transformed with circular YACs to obtain BAC colonies. For microinjection, the insertion DNA (150-200kb) was separated from the BAC vector DNA (approximately 10kb) using a filtration step with Sepharose 4B-CL (Yang et al. Nature biotechnology 15, 859-865 (1997)).

[0112] Rats and the induction of mating Purified DNA encoding recombinant immunoglobulin loci was resuspended in microinjection buffer with 10 mM spermine and 10 mM spemidine. The DNA was injected into oocytes at various concentrations ranging from 0.5 to 3 ng / μl.

[0113] Plasmid DNA or mRNA encoding a rat immunoglobulin gene-specific ZFN was injected into oocytes at various concentrations ranging from 0.5 to 10 ng / μl.

[0114] Microinjection was performed at the Caliper Life Sciences facility. Uninbred SD / Hsd (wild-type) strain animals were housed in standard micro-isolator cages under approved animal care protocols in an animal facility certified by the International Association of Accredited Laboratory Animal Care (AAALAC). Rats were maintained with free access to feed and water on a 14-10 hour light / dark cycle. Before mating with uninbred SD / Hsd males, 4-5 week old SD / Hsd female rats were injected with 20-25 IU of PMSG (Sigma-Aldrich), followed by 20-25 IU of hCG (Sigma-Aldrich) 48 hours later. Single-cell stage fertilized embryos were collected for subsequent microinjection. The manipulated embryos were transferred to pseudopregnant SD / Hsd female rats and allowed to give birth.

[0115] Human Ig rats with multiple traits (human IgH, Igκ, and Igλ combined with rat J KO, κKO, and λKO), as well as wild-type rats as controls, were analyzed at 10–18 weeks of age. These animals were mated in the Charles River under specific pathogen-free conditions.

[0116] Multiple different Vs at separate gene loci H The procedure for introducing the regions can be carried out by inserting these different loci into separate transgenic rats (preferably rats with the knockout IgH locus), as described in the examples above. Separate transgenic rat lines are created using these separate loci, and then they are crossed to obtain double transgenic rats in which all the VH regions used can be made available for the recombination process. Crossing these rats to be homozygous for both loci can double the number of VH regions available for recombination (see the karyotype in Figure 3, where one locus is integrated on chromosome 6 and the other on chromosome 15). Having multiple copies of the integrated locus can further increase this number.

[0117] Multiple different Vs along with one constant-region array HThe procedure of introducing different gene loci separately by transposing regions enabled the incorporation of unlinked and multiple trans loci. Following this, animals expressing antibodies from both separately incorporated loci were produced through mating.

[0118] The same procedure was applied to the light chain. One lineage of animals was produced at the kappa locus, while another lineage was produced at the lambda locus. These loci are combined within animals through crossbreeding.

[0119] PCR and RT-PCR Transgenic rats were identified by PCR from tail or ear clip DNA using Genomic DNA Mini Kid (Bioline). For IgH PCR, GoTaq Green Master mix (Promega) less than 1kb was used under the following conditions: 2 minutes at 94°C, 32× (30 seconds at 94°C, 30 seconds at 54-67°C (see Table 1 for primers and specific annealing temperatures), 1 minute at 72°C), 2 minutes at 72°C. For IgH PCR, KOD polymerase (Novagen) greater than 1kb was used under the following conditions: 2 minutes at 95°C, 32× (20 seconds at 95°C, 20 seconds at 56-62°C, Table 1), 90 seconds at 70°C), 2 minutes at 70°C. For Igκ and Igλ PCR, all were less than 1kb, and the above conditions were used except for a 50-second extension at 72°C.

[0120] RNA was extracted from blood using the RiboPure Blood Kit (Ambion), and RNA was extracted from the spleen, bone marrow, or lymph nodes using the RNASpin mini kit (GE Healthcare). cDNA was prepared using oligo-dT and Promega reverse transcriptase at 42°C for 1 hour. The concentration was determined by GAPDH PCR reaction (oligo-429-430).

[0121] RT-PCR was set up using VH reader primers with rat μCH2 or rat γCH2 primers (Table 2). Amplification in GoTaq Green Master mix was performed at 94°C for 2 minutes, 34× (94°C for 30 seconds, 55–65°C for 30 seconds, 72°C for 50–60 seconds), and 72°C for 2 minutes. PCR products of expected size were purified by either gel or QuickClean (Bioline) and either directly sequenced or cloned into pGemT (Promega).

[0122] The primer sequences used in PCR and RT-PCR assays for detecting the integration and expression of human IgH and IgL are provided in Table 3.

[0123] Next-generation sequencing for characterizing antibodies in immunized OmniRat animals Six OmniRat2 animals were immunized with β-gal, and B cells were isolated from the lymph nodes in the influx region. The B cells were pelleted, and after removing the supernatant, total RNA was prepared from the lymph node-derived B cells. The RNA was reverse transcribed, and the resulting cDNA was used as a template to amplify the entire variable region (VH region) of the Ig heavy chain rearrangement locus. The amplified products were then prepared for next-generation sequencing (NGS), and the complete VH repertoire of each animal was determined by NGS.

[0124] After post-processing and quality control of raw NGS reads, the V gene usage frequency for each animal was determined by aligning each unique VH sequence with a germline V gene reference sequence. The V gene usage rate was calculated by dividing the number of VH sequences using a particular V gene by the total number of VH sequences in that animal.

[0125] Protein purification IgM was purified on an anti-IgM affinity matrix (BAC BV, Netherlands, CaptureSelect #2890.05) as described in the protocol. Similarly, human Igκ and Igλ were purified on anti-L chain affinity matrices (CaptureSelect anti-Igκ #0833 and anti-Igλ #0849) according to the protocol.

[0126] For rat IgG purification (Bruggemann et al. J Immunol 142, 3145-3150 (1989)), protein A and protein G agarose were used (Innova, Cambridge, UK, #851-0024 and #895-0024). Serum was incubated with resin, and binding was promoted with 0.1 M sodium phosphate at pH 7 for protein G and at pH 8 for protein A under gentle mixing. A polyprep column (Bio-Rad) was packed with the mixture and thoroughly washed with PBS at pH 7.4. The elution buffer was 0.1 M sodium citrate at pH 2.5, and the neutralization buffer was 1 M Tris-HCl at pH 9.

[0127] Electrophoresis was performed on 4-15% SDS-PAGE, using Coomassie Brilliant Blue for staining. The MW standard was a HyperPage-stained protein marker (#BIO-33066, Bioline).

[0128] Flow cytometry analysis and FISH The cell suspension was washed and prepared at 5 × 10⁵ cells / 100 μl in PBS-1% BSA-0.1% azide. Different B cell subsets were identified using mouse anti-rat IgM FITC-labeled mAb (MARM4, Jackson Immunoresearch Laboratories) along with anti-B cell CD45R (rat B220)-PE-conjugate mAb (His24, BD Biosciences) or anti-IgD-PE-conjugate mAb (MARD-3, Abd Serotec). A FACS Canto II flow cytometer and FlowJo software (Becton Dickinson, Pont de Claix, France) were used for analysis.

[0129] As described, fluorescence in situ hybridization was performed on fixed blood lymphocytes using purified IgH and IgL C-region BACs. (Meisner & Johnson Methods 45, 133-141 (2008))

[0130] Immunization, cell fusion, and affinity measurement Immunization was performed in the ridge region with 125 μg of PG, 150 μg of hGHR, 200 μg of Tau / KLH, 150 μg of HEL, and 150 μg of OVA in CFA, and as described, medial iliac lymph node cells were fused with mouse P3X63Ag8.653 myeloma cells after 22 days (Kishiro et al. Cell structure and function 20, 151-156 (1995)). For multiple immunizations, protein, 125 μg of PG or HEL, or 100 μg of hGHR or CD14 in GERBU adjuvant (www.Gerbu.com) was administered intraperitoneally as follows: spleen cell fusion with P3X63Ag8.653 cells without adjuvant on days 0, 14, 28, and 41, followed by 4 days later (Meisner & Johnson Methods 45, 133-141 (2008)).

[0131] As described, the binding kinetics were analyzed by surface plasmon resonance using Biacore2000 with the antigen directly immobilized. (Pruzina et al. Protein engineering, design & selection: PEDS 24, 791-799 (2011)).

[0132] Detection of antigen-specific antibodies by ELISA Rat serum samples were analyzed for B-Gal IgG and IgM antibodies and antigenic titers using antigen-coated, anti-IgG, or IgM reporter ELISA. 96-well plates were coated with B-Gal overnight at 2–6°C, blocked with PBS casein blocker / diluent 1×, washed with ELISA wash buffer, incubated with serum, washed with ELISA wash buffer, incubated with either a mixture of goat anti-rat IgG1-HRP, goat anti-rat IgG2a-HRP, and goat anti-rat IgG2b-HRP (1 / 5,000 dilution each) or goat anti-rat IgM (1 / 5,000 dilution), washed with ELISA wash buffer, incubated with TMB substrate solution for 30 minutes, and ELISA stop solution was added to the wells. Absorbance in the plate wells was measured at 450 nm. Otherwise, incubation was 1.5–2 hours at ambient temperature.

[0133] Measurement of IgM and IgG concentrations in rat serum Rat serum samples were analyzed for total rat IgG1, rat IgG2b, and rat IgM concentrations using a dual antibody ELISA sandwich assay format. Total rat IgG1, rat IgG2b, and rat IgM concentrations were calculated using standard curves prepared individually for each isotype. 96-well plates were coated overnight at 2–6°C with each isotype-specific capture antibody (either mouse anti-rat IgG1, mouse anti-rat IgG2b, or goat anti-rat IgM), blocked with PBS casein blocker / diluent 1×, washed with ELISA wash buffer, incubated with serum, washed with ELISA wash buffer, incubated with each detection antibody (either mouse anti-rat IgG or goat anti-rat IgM), washed with ELISA wash buffer, incubated with TMB substrate solution for 30 minutes, and ELISA stop solution was added to the wells. Absorbance in the plate wells was measured at 450 nm. Otherwise, incubation was 1.5–2 hours at ambient temperature. [Table 1] [Table 2] [Table 3-1] [Table 3-2] [Table 3-3]

[0134] result Human IgH and IgL gene loci The construction of the human Ig locus utilized established techniques for assembling large DNA segments using YACs and BACs (Davies et al. Nucleic acids research 20,2693-2698(1992); Davies et al. Biotechnology(NY)11,911-914(1993); Wagner et al. Genomics 35,405-414(1996); Popov et al. Gene 177,195-201(1996); Mundt et al. J Immunol 166,3315-3323(2001)). Multiple BAC modifications in E. coli frequently resulted in deletions of repeating regions such as switch sequences and enhancers. Therefore, a method was developed to assemble sequences with overlapping ends as circular YACs (cYACs) within S. cerevisiae and then convert such cYACs to BACs. The advantages of YACs include their large size, ease of homologous modification in the yeast host, and sequence stability, while BACs, which grow within E. coli, offer the advantages of easy preparation and high yield. In addition, detailed restriction mapping and sequence analysis can be achieved better with BACs than with YACs.

[0135] Sequence analysis and digestion identified the target gene cluster and ensured the integrity and functionality of the locus to stabilize broad-area DNA rearrangement and switching, as shown in Figure 1. As previously shown, overlapping regions are generally preferred to be ligated and incorporated when co-injected into oocytes (Wagner et al. Genomics 35, 405-414 (1996)). Thus, insertion of the 173kb NotI fragment BAC3, the 193kb NotI fragment BAC3-1N12M5I8 (Hu-rat Annabel), and the 182kb AsiSI-AscI fragment BAC6-VH3-11 resulted in the rearrangement of the fully functional transgenic IgH locus (HC14) in the rat genome. Similarly, injection of BAC9, BAC(14+5), and BAC3-1N12M5I8 resulted in the rearrangement of the fully functional transgenic IgH locus (HC30) in the rat genome.

[0136] Similarly, the human Igκ locus was incorporated by homologous duplication. The human Igλ locus was isolated intact as a YAC of approximately 300kb and then completely inserted into the rat chromosome. Successful incorporation was confirmed by transcriptional analysis, which showed V(D)JC recombination from the 5' end to the 3' end of the injected locus. Multiple copies were identified by qPCR (not shown), suggesting that incorporation occurred from the head to the tail. In all cases, transgenic animals with single-site incorporation were generated by mating.

[0137] Homozygous mating Induction of transgenic rats by DNA microinjection into oocytes, their mating, and immunization are comparable to those in mice. However, ZFN technology, which results in gene knockout, has only recently been reported (Geurts et al. Science 325,433 (2009); Flisikowska et al. PloS one 6,e21045 (2011)). HSilencing of the rat IgH locus by deletion has been described (Menoret et al. European Journal of Immunology 40, 2932-2941 (2010)), and documentation describing silencing of the rat IgL locus, targeting of Cκ, and deletion of the J-Cλ gene is in preparation. We derived several founders in which the human Ig locus was incorporated and endogenous Ig production was silenced, and all were analyzed by PCR and FISH after incorporating complete trans loci through selection and heterocrossing (Table 4). Several founder rats retained low trans locus copy numbers, and it is believed that the rat C gene BAC in OmniRat® is completely incorporated within 5 copies, as determined by qPCR of Cμ and Cα products (not shown). FISH identification of single-position insertions in many lineages confirmed that diffusion of BAC mixtures or multiple incorporations is rare, and that the advantage of homozygous crossing was achieved. [Table 4]

[0138] Rats carrying individual human trans loci, IgH, Igκ, and Igλ, were successfully crossbred with Ig locus KO rats, resulting in homozygous crosses. This resulted in 22 functional V H Human V files of 400kb or more, including H -DJ H We produced a novel, highly efficient, multi-characteristic lineage (OmniRat®) containing the HC14 heavy chain locus and a rat C region of approximately 116kb. DNA rearrangements, expression levels, class switching, and hypermutation were remarkably similar between different founders and equivalent to wild-type rats. This is likely due to the authentic configuration of the relevant rat constant region containing several Cs and the 3'E (enhancer-controlled) region. OmniRat2 was created by crossing OmniRat animals holding the HC14 heavy chain locus with OmniRat animals holding the HC30 locus. OmniRat2 animals contain two heavy chain loci, each containing 43 functional VHs.

[0139] B cell development in a knockout background Flow cytometry analysis was performed to evaluate whether the introduced human Ig locus could reconstitute normal B cell development. Specific differentiation stages were analyzed in spleen and bone marrow lymphocytes (Osborn et al. J Immunol 2013;190:1481-1490), and the lack of B cell development in JKO / JKO rats (Menoret et al. European journal of immunology 40,2932-2941(2010)), as well as the absence of corresponding IgL expression in κKO / κKO and λKO / λKO animals, were already shown (data not shown). Most notably, similar numbers of B220 (CD45R) were observed in bone marrow and spleen. + Compared to wild-type animals with lymphocytes, B cell development was completely restored in OmniRat. CD45R + IgM expression in the majority of B cells indicated a fully reconstituted immune system. Since the size and shape segregation of spleen cells was indistinguishable between OmniRat® and wild-type animals, reconstruction was successful in transgenic rats expressing the human idiotype and rat C region. Furthermore, small sIgG + A lymphocyte population was found within the omniRat (Osborn et al. J Immunol 2013;190:1481-1490).

[0140] Analysis of other OmniRat lymphocyte tissues has shown that they are indistinguishable from wild-type controls, for example, that the T cell subset is completely retained (data not shown), which further supports the view that optimal immune function is fully restored.

[0141] Serum Ig levels To obtain clear information about antibody production, the inventors compared the quality and quantity of serum Ig from HC30 and HC14 / HC30 animals (Figure 4). The results demonstrated that animals with one Ig gene locus (HC30) expressed the same amount of IgM and IgG in their serum compared to animals with two heavy chain gene loci (HC14 and HC30).

[0142] ELISA analysis of serum from immunized OmniRat animals with one HC locus (HC30) or two HC loci (HC14 and HC30) revealed similar titers of anti-β-gal IgM and IgG in such animals (Figure 5).

[0143] Diverse human H chain and L chain transcripts Extensive transcriptional analysis was performed using blood lymphocytes or spleen cells from transgenic rats possessing functional endogenous Ig loci. Specific human V was identified prior to Cμ or Cγ reverse primers. H RT-PCR from the group showed human V H DJ H The frequency of use is indicated. For the light chain analysis group, specific human Vκ or Vλ forward primers were used together with Cκ or Cγ reverse primers.

[0144] In addition, animal B cells were collected, RNA was prepared, reverse transcribed, and the resulting cDNA was used as a template to amplify the entire variable region (VH region) of the Ig heavy chain rearrangement locus. The amplified products were then prepared for next-generation sequencing (NGS), and the complete VH repertoire of each animal was determined by NGS. After post-processing and quality control of the raw NGS reads, the V gene usage frequency of each animal was determined by aligning each unique VH sequence with a germline V gene reference sequence. The V gene usage rate was calculated by dividing the number of VH sequences using a particular V gene by the total number of VH sequences in that animal. Of the 43 total human V genes introduced into the OmniRat2 transgene, the inventors detected 33 V genes expressed at levels exceeding 0.1% in the rearranged IgG transcript.

[0145] The results of RT-PCR VH gene expression analysis and NGS repertory analysis are summarized in Figure 1. These results show that the D segment and all J segments are affected. H All integrated human V segments that are considered functional in combination with the diverse use of segments H The use of the gene was demonstrated (Lefranc & Lefranc The immunoglobulin factsbook. FactsBook Series, Academic Press, GB, 45-68 (2001)).

[0146] The results clearly demonstrate that the addition of more variable regions provided by the two loci (HC14+HC30) results in a broader antibody repertoire. In conclusion, we have demonstrated that potentially any class of antigen-specific high-affinity antibodies can be produced in transgenic animals having one or two Ig heavy chain loci. This technique would enable the production of any class of full human antibodies or fragments thereof in response to antigen administration for use as therapeutic or diagnostic agents in humans. By using different loci, our technique also enables the production of high-affinity mature antibodies from rodents for use as reagents, diagnostic agents, or for use in human treatment.

[0147] Consideration Combinations of human and rat genes to assemble a novel IgH locus resulted in highly efficient, near-normal expression of antibodies possessing the human idiotype. Furthermore, the incorporation of human Igκ and Igγ loci revealed that fully human-specific chimeric Ig antibodies are readily produced, and that the association of the human light chain with the rat C region is not harmful. The advantage of using a portion of the rat IgH locus is that it essentially allows for the expression of diverse human V H DJ HThe key is that only the region is transplanted, while the species-specific C region and enhancer regulatory elements are retained in their native configuration. Furthermore, expression of antibodies containing the rat Fc region enables the assembly of normal B cell receptors and the optimal activation of downstream signaling pathways essential for the initiation of a highly efficient immune response. In particular, the quality of the immune response to antigen administration depends on many receptor-related signaling pathways and the binding of the modified components (see www.biocarta.com / pathfiles / h bcrpathway.asp).

[0148] Approaches using YAC and BAC, as well as mutual exchange between the two, offer advantages in both speed and the ability to verify the completeness of constructing larger regions due to duplication homology. Several founder rats retain low trans-locus copy numbers, suggesting that the rat C gene BAC in OmniRat is fully integrated within five copies, as determined by qPCR of Cμ and Cα products (not shown). FISH identification of single-position insertions in many lineages confirmed that diffusion or multiple integration of BAC mixtures is rare, thus achieving the advantage of homozygous mating. Little was known about whether extensive duplication regions could be integrated to maintain the complete functionality essential for DNA rearrangement. While overlapping integrations have been reported previously, they were for much smaller regions (less than 100kb) (Wagner et al. Genomics 35,405-414 (1996); Bruggemann et al. European Journal of Immunology 21,1323-1326 (1991)). Our results indicate that desired integration due to homology or in tandem is a frequent occurrence. This substantially facilitates transgenic techniques because it eliminates the need for the difficult integration of large YACs into stem cells and subsequent induction of animals from those stem cells. In addition to (Mendez et al. Nature Genetics 15,146-156 (1997); Davies et al. Biotechnology (NY) 11,911-914 (1993)), ZFN technology, similarly implemented via DNA injection (Geurts et al. Science 325,433 (2009); Menoret et al. European Journal of Immunology 40,2932-2941 (2010)), can easily produce Ig KO lines and may become a future technology of choice for gene disruption and substitution. Silencing endogenous Ig gene expression in OmniRat, including the human-rat IgH and human IgL loci, has the advantage that the mixed product may not produce interfering or undesirable rat Ig.Interestingly, immunization and hybridoma formation in OmniRats still producing wild-type Ig revealed that many products were fully human, human-rat IgH, and human IgL, despite incomplete Ig knockout. It was noteworthy that, despite numerous wild-type V genes, the introduced human genes were readily amplified, thereby demonstrating their efficient competitiveness. This is consistent with our view that all incorporated transgenes exhibit generally favorable expression levels, preferably competing with endogenous loci. Previously, Ig knockout was essential in mice expressing human antibody repertoires because human product expression was minimal when wild-type Ig was released (Bruggemann et al. PNAS 86,6709-6713 (1989); Mendez et al. Nature Genetics 15,146-156 (1997)).

[0149] Because strain-specific cis-acting sequences are required for high-level expression, the production of a fully human Ig locus may not be optimal even within Ig KO mice. In mice, the enhancer region downstream of Cα plays an essential role in class switch recombination (Vincent-Fabert et al. Blood 116, 1895-1898 (2010)), and elements within that region may promote hypermutation (Pruzina et al. Protein engineering, design & selection: PEDS 24, 791-799 (2011)). This may explain why high-frequency immune responses and the generation of diverse hybridomas may make it difficult to maintain a larger fully human locus in mice (Davis et al. Cancer metastasis reviews 18, 421-425 (1999); Lonberg Current opinion in immunology 20, 450-459 (2008)). The chimeric human-rat IgH locus promotes differentiation and expression levels close to wild-type in OmniRat, because the endogenous rat C region and, in fact, the 3' of the 30kb Cα enhancer sequence, are related to human V HIt can be concluded that this provides optimal locus control for gene expression and maturation. Another region, Cδ (Mundt et al. J Immunol 166, 3315-3323 (2001)), which has its 3' regulatory motif cluster, has been removed from the chimeric C region BAC because silencing or absence of IgD did not appear to reduce immune function.37 Normally, mature IgM + IgD + B cells downregulate IgD upon antigen contact, which initiates class switch recombination (Chen Immunol Rev 237, 160-179 (2010)). Therefore, switching can increase without IgD regulation, supported by our findings that IgG transcript and serum levels are significantly lower when the Cδ region is retained within the transgenic construct (data not shown).

[0150] The inventors found that, under various immunization conditions, mAbs with sequence and epitope diversity equivalent to those produced in wild-type controls could be isolated via spleen and lymph node fusion, making the production of specific IgG in OmniRat particularly promising. The V, D, and J gene diversity was as expected, and almost all segments were found to be productively utilized as anticipated (Lefranc & Lefranc The immunoglobulin factsbook. FactsBook Series, Academic Press, GB, 45-68 (2001)). This was in striking contrast to mice with a fully human trans locus, where clonal proliferation from a small number of precursor B cells yielded little diversity (Pruzina et al. Protein engineering, design & selection: PEDS 24, 791-799 (2011)). Because the number of transplanted V genes is only about half that used in humans, the inventors predicted that limited immune responses and limited diversity would be found when comparing OmniRat to wild-type animals. However, this was not the case, and a comparison of CDR3 diversity in over 1000 clones revealed that OmniRat exhibited extensive conjugation differences similar to those in wild-type animals. A small number of identical gene segment combinations were found to be related to V H From D and / or from D to J H Further diversification occurred through the addition or deletion of the N sequence at conjugation, as well as through hypermutation. Therefore, the rat C region sequence is similar to that of human V. H DJ HIt is clear that it is highly efficient in controlling DNA rearrangement and expression. Extensive diversity was also observed for introduced human Igκ and Igλ loci, similar to that already shown in mice (Nicholson et al. J Immunol 163,6898-6906(1999);Pruzina et al. Protein engineering, design & selection:PEDS 24,791-799(2011);Popov et al. The Journal of experimental medicine 189,1611-1620(1999)). Therefore, the significantly reduced efficiency in the production of human antibodies from mice (Lonberg Nature biotechnology 23,1117-1125(2005)) has been overcome in OmniRat, which reliably and extensively diversifies the rearranged H chains through class switching and hypermutation, producing high-affinity antibodies not occasionally but in large quantities. The yield and hypermutation levels of transgenic IgG, which are impressively utilized in antigen-specific mAbs, showed similar levels of clonal diversification and production between OmniRat and wild-type animals. Regular generation of high affinity specificity in the sub-nanomole range was achieved even by different monoimmunizations, and again, this was preferable compared to wild-type animals (results have not been shown in transgenic mice that produce a human antibody repertoire exclusively from human loci). (Mendez et al. Nature Genetics 15, 146-156 (1997))

[0151] In summary, to maximize human antibody production, the IgH locus, which uses human genes for antibody specificity but rodent genes for differentiation and high expression control, should be considered essential. The flexibility of the light chain is an additional advantage, as it allows for highly efficient human IgH / IgL assembly even in the presence of wild-type Ig. For therapeutic applications, chimeric heavy chains can be readily converted to full human antibodies by C gene substitution without compromising specificity.

[0152] All patents and patent publications referenced herein are incorporated herein by reference.

[0153] Those skilled in the art will likely consider certain modifications and improvements based on the above description. For the sake of brevity and readability, all such modifications and improvements have been omitted herein, but it should be understood that they are appropriately covered by the following claims.

Claims

1. A transgenic rodent comprising at least one inactivated endogenous Ig locus and a plurality of artificial transgenic Ig heavy chain loci integrated into different chromosomal regions of the rodent genome; wherein the artificial transgenic Ig heavy chain loci are functional, capable of gene rearrangement, and act cooperatively to produce a repertoire of artificial immunoglobulins. The first of the multiple artificial transgenic Ig heavy chain loci is HC30, as shown in Figure 1B. 【Chemistry 1】 As shown, the HC30 is: (i) Human heavy chain V gene segments IgHV3-74, IgHV3-73, IgHV3-72, IgHV2-70, IgHV1-69, IgHV3-66, IgHV3-64, IgHV4-61, IgHV4-59, IgHV1-58, IgHV3-53, IgHV5-51, IgHV3-49, IgHV3-48, IgHV1-46, IgHV1-45, IgHV3-43, IgHV3-21, IgHV3-20, IgHV1-18, IgHV3-15, IgHV3-13, and IgHV6-1; (ii) Human heavy chain D segment; (iii) Human heavy chain JH1-6; and (iv) Rat constant region extending from Eμ to the rat 3' enhancer sequence; It includes, and the second artificial transgenic Ig heavy chain locus is double / duplication: (i) Human heavy chain V gene segment; (ii) Human heavy chain D segment; (iii) Human heavy chain JH1-6; and (iv) Rat constant region extending from Eμ to the rat 3' enhancer sequence; Including; and The second artificial transgenic Ig heavy chain locus of the plurality of artificial transgenic Ig heavy chain loci is HC14, and HC14 is shown in Figure 1A 【Chemistry 2】 As shown below: (i) Human heavy chain V gene segments IgHV4-39, IgHV3-38, IgHV3-35, IgHV4-34, IgHV3-33, IgHV4-31, IgHV3-30, IgHV4-28, IgHV2-26, IgHV1-24, IgHV3-23, IgHV3-22-2, IgHV3-11, IgHV3-9, IgHV1-8, IgHV3-7, IgHV2-5, IgHV7-4, IgHV4-4, IgHV1-3, IgHV1-2, and IgHV6-1; (ii) Human heavy chain D segment; (iii) Human heavy chain JH1-6; and (iv) Rat constant region extending from Eμ to the rat 3' enhancer sequence; including; Transgenic rodents.

2. The transgenic rodent according to claim 1, wherein the transgenic rodent comprises a complete complement of the human Ig heavy chain gene segment.

3. The first artificial transgenic Ig heavy chain gene locus mentioned above contains the following three bacterial artificial chromosomes (BACs): i) A first BAC containing a 185kb NotI-fragment of BAC9 encompassing IgHV3-74 to IgHV1-58, ii) A second BAC containing the rat CH region gene immediately downstream of the human VH6-1-Ds-JHs region, and iii) A BsiwI fragment containing 90.6kb of BAC14 including a 4.6kb sequence overlapping with BAC9, a 1.7kb synthetic fragment containing 86kb of IgHV5-51 to IgHV1-45 and centrally located IgHV3-43, a 111.7kb fragment of BAC5 containing IgHV3-21 to IgHV3-13, and a third BAC containing a 6.1kb region overlapping with the BAC of ii), A transgenic rodent according to claim 1 or 2, which is reconstructed in the rodent genome by overlapping regions.

4. The second artificial transgenic Ig heavy chain locus mentioned above contains the following three BACs: i) The first BAC, which includes the true region extending from VH4-39 to VH3-23 and then to VH3-11, ii) A second BAC containing the rat CH region gene immediately downstream of the human VH6-1-Ds-JHs region, and iii) The third BAC, which includes the true region extending from VH3-11 to VH6-1, The transgenic rodent according to claim 1, which is reconstructed in the rodent genome by the overlapping regions of the transgenic rodent according to claim 1.

5. The transgenic rodent according to claim 4, wherein the transgenic rodent comprises a nucleic acid sequence substantially homologous to the nucleic acid sequence of Sequence ID No.

2.

6. The transgenic rodent according to claim 5, wherein the transgenic rodent comprises a nucleic acid sequence substantially homologous to the nucleic acid sequence of Sequence ID No.

11.

7. The transgenic rodent according to any one of claims 1 to 6, wherein the plurality of artificial Ig heavy chain gene loci include complete complements of human variable heavy chain regions.

8. The transgenic rodent according to any one of claims 1 to 7, wherein the transgenic rodent lacks a functional endogenous Ig light chain locus.

9. The transgenic rodent according to any one of claims 1 to 8, wherein the transgenic rodent lacks a functional endogenous Ig heavy chain locus.

10. The transgenic rodent according to any one of claims 1 to 9, wherein at least one of the artificial Ig heavy chain loci comprises a rat 3' enhancer.

11. The transgenic rodent according to claim 10, wherein the rat 3' enhancer comprises the sequence described as Sequence ID No.

1.

12. The transgenic rodent according to any one of claims 1 to 11, wherein the constant region gene includes a constant region gene selected from the group consisting of Cμ and Cγ.

13. A method for producing a polyclonal antiserum composition, comprising immunizing a transgenic rodent according to any one of claims 1 to 12 with an immunogen.

14. A method for producing a monoclonal antibody, comprising: (i) immunizing a transgenic rodent according to any one of claims 1 to 12 with an immunogen; (ii) isolating monoclonal antibody-producing cells from the transgenic rodent that produce a monoclonal antibody that specifically binds to the immunogen; and (iii) producing the monoclonal antibody that specifically binds to the immunogen using the monoclonal antibody-producing cells, or producing hybridoma cells that produce the monoclonal antibody using the monoclonal antibody-producing cells, and then producing the monoclonal antibody using the hybridoma cells.

15. A method for producing a monoclonal antibody, comprising: (i) immunizing a transgenic rodent according to any one of claims 1 to 12 with an immunogen; (ii) isolating monoclonal antibody-producing cells from the transgenic rodent that produce a monoclonal antibody that specifically binds to the immunogen; (iii) isolating a monoclonal antibody nucleic acid encoding the monoclonal antibody that specifically binds to the immunogen from the monoclonal antibody-producing cells; and (iv) producing the monoclonal antibody that specifically binds to the immunogen using the monoclonal antibody nucleic acid.

16. The method according to either claim 14 or 15, wherein the monoclonal antibody has a human idiotype.

17. A method for producing a fully human monoclonal antibody, comprising: (i) immunizing a transgenic rodent according to any one of claims 1 to 12 with an immunogen; (ii) isolating monoclonal antibody-producing cells from the transgenic rodent that produce a monoclonal antibody that specifically binds to the immunogen; (iii) isolating a monoclonal antibody nucleic acid encoding the monoclonal antibody that specifically binds to the immunogen from the monoclonal antibody-producing cells; (iv) modifying the monoclonal antibody nucleic acid to produce a recombinant nucleic acid encoding a fully human monoclonal antibody; and (v) producing the encoded fully human monoclonal antibody using the recombinant nucleic acid encoding the fully human monoclonal antibody.

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