Human antibodies from transgenic rodents with multiple heavy chain immunoglobulin loci

Abstract

This invention relates to transgenic animals that can be used to optimally produce functional immunoglobulins with human idiotypes.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 201880011903.6, filed on January 19, 2018, entitled "Human Antibody from Transgenic Rodent with Multiple Heavy Chain Immunoglobulin Loci". Technical Field

[0002] This invention relates to transgenic animals that can be used to produce immunoglobulins with human idiotypes in rodents, and methods for preparing said transgenic animals. The invention also relates to compositions and methods for producing humanized and fully human antibodies using the tandem integration of polynucleotides and combinations thereof from large modified regions on bacterial artificial chromosomes. Hybridization of independently obtained transgenic animals allows for the expression of highly diverse human antibody libraries using many different, possibly all, human VH, D, and JH regions. In vivo expression can be controlled by uniformly regulating individual integration sites to, for example, achieve VH gene diversity and selection without interference. Background Technology

[0003] Human monoclonal antibodies (in the form of IgG, single-chain, or domain modules of standard size) have proven extremely useful in therapeutic applications (Chan and Carter, Nature reviews. Immunology 10, 301-316 (2010); Enever et al., Current opinion in biotechnology 20, 405-411 (2009)). Despite these successes, the production of these human monoclonal antibodies remains significantly limited, relying either on specific selection of available human material and subsequent modification of individual products, or on immunization with a limited number of transgenic animals (Brüggemann et al., Part I: Selecting and shaping the antibody molecule, Selection Strategies III: Transgenic mice, in Handbook of Therapeutic Antibodies. Ed. Dübel, S. Wiley-VHC, 69-93 (2007)).

[0004] The rearrangement and expression of human immunoglobulin (Ig) genes in transgenic animals was pioneered more than 20 years ago through the stable insertion of heavy chain genes into germline conformations (Bruggemann, M. PNAS 86, 6709-6713 (1989)). One issue related to the therapeutic applications of non-human immunoglobulins is their potential immunogenicity in human patients. To reduce the immunogenicity of such preparations, various strategies have been developed for generating chimeric antibodies, partially human antibodies (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 generate transgenic antibodies with human idiotypes in non-human animals is particularly desirable because the antigen-binding determinant is located within the idiotype region, and the non-human idiotype is considered to contribute to the immunogenicity of current antibody therapeutics. For monoclonal antibody therapeutics, human idiotype is a particularly important consideration, which consists of a single idiotype delivered at a relatively high concentration, rather than multiple idiotypes delivered at a lower concentration via a mixture of polyclonal antibodies.

[0005] The main improvements in increasing human Ig expression levels and generating exclusivity combine two novel strategies: gene knockout in embryonic stem (ES) cells (Kitamura et al., Nature 350, 423-426 (1991)) and locus extension on artificial chromosomes (Davies et al., Nucleic Acids Research 21, 767-768 (1993)). Silencing endogenous Ig genes in ES cells through gene targeting has produced several inactive mouse strains that are unable to rearrange their IgH and IgL loci or produce fully functional IgH, IgK, or IgL products. More recently, zinc finger nucleases (ZFNs) have been engineered to create site-specific double-strand breaks in Ig genes, allowing for gene disruption via deletion and non-homologous DNA repair. Injecting ZFN plasmids into fertilized eggs produced Ig-silenced rats and rabbits with IgH and IgL disruption (Geurts, AM 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)).

[0006] A major technical challenge encountered by many existing methods for generating humanized transgenic antibodies in nonhuman animals involves significant competition between repetitive Ig loci within the same animal, such as existing or endogenous Ig loci and exogenous or artificial loci introduced into the transgenic animal. Historically, in the absence of effective knockout, endogenous loci have outcompeted exogenous loci for antibody production, resulting in the effective silencing of repetitive loci (Lonberg et al., NatBio, 23, 1117, 2005; Nicholson et al., J Immunol, 163, 6898, 1999; Brüggemann et al., AITE 63, 101, 2015). Therefore, in this respect, the prior art does not explain or resolve whether repetitive Ig loci integrated at different chromosomal sites can function synergistically in the production of transgenic antibodies in the same host animal, and in fact, would reasonably suggest to those skilled in the art that the opposite is true.

[0007] Another technical challenge in generating transgenic antibodies with human idiotypes in non-human animals is the difficulty in providing complete complement for the human immunoglobulin VDJ or VJ gene segments used to generate human antibodies. Some have attempted to address this by introducing megabase-sized fragments from human heavy chain and κ light chain loci. However, this approach has only proven successful in approximately 80% of human immunoglobulin genes contained in germline conformations and relies on the use of protoplasts to deliver large fragments of the relevant chromosome using the yeast artificial chromosome (YAC) system (US 5,939,598).

[0008] Although extensively overlapping V antibodies have been utilized in transgenic animals to maximize antibody diversity, H DJ HIntegration of regions, which enables, for example, the full function of the IgH locus and is essential for DNA rearrangement, is reported primarily in much smaller regions (<100kb) (Wagner et al., Genomics 35, 405-414 (1996); Bruggemann et al., European journal of immunology 21, 1323-1326 (1991)) or larger regions with a limited library at a single integration site (WO2014 / 093908; Bruggemann et al.). At the time of application, it was generally understood in the art that diffusion or multiple integration of BAC or YAC mixtures was rare and would be detrimental to homozygous breeding. In addition, it is more common to painstakingly integrate large YACs into stem cells and then obtain animals from them (Mendez et al., Nature genetics 15, 146-156 (1997); Davies et al., Biotechnology (NY) 11, 911-914 (1993)).

[0009] Optimizing the production of immunoglobulins or antibodies using transgenic animals with the complete complementary sequence of the human V gene to maximize the diversity of antibodies with human idiotypes remains a challenge for generating novel specificity for therapeutic applications across a wide range of disease areas. Summary of the Invention

[0010] This invention addresses the aforementioned uncertainties in the art by providing artificial Ig heavy chain loci comprising multiple repetitive / overlapping human immunoglobulin VDJ or VJ gene segments integrated at different chromosomal sites, and by producing transgenic animals lacking the ability to produce endogenous immunoglobulins. The method for producing these transgenic animals, involving the insertion of two different loci at two different locations on two different chromosomes, surprisingly produces functional B cells. This method advantageously avoids allelic rejection and increases antibody diversity due to the complete complementary sequence of the human immunoglobulin VDJ heavy chain gene segment integrated into the transgenic animal's genome.

[0011] In one aspect of the invention, novel polynucleotides are disclosed comprising nucleic acid sequences encoding chimeric immunoglobulin chains, particularly chimeric heavy chains for the production of transgenic animals. The polynucleotides of the present invention advantageously provide optimal expression, at least in part due to the inclusion of a 3' enhancer, as the absence of this 3' enhancer at the transgenic locus leads to impaired allotype conversion and reduced IgG expression. Therefore, in a preferred embodiment, the present invention provides chimeric polynucleotides comprising a rat 3' enhancer sequence, an Ig constant region gene, and at least one human immunoglobulin (Ig) linker (J) region gene. In a preferred embodiment, the rat 3' enhancer sequence comprises the sequence as described in SEQ ID NO:1 or a portion thereof.

[0012] In one embodiment, the chimeric polynucleotide described herein may further comprise 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 is a rat constant region gene. In another preferred embodiment, the constant region gene is selected from the group consisting of Cμ and Cγ.

[0013] In one embodiment, the chimeric polynucleotide comprises a nucleic acid sequence substantially homologous to the bacterial artificial chromosome (BAC) Annabel (e.g., SEQ ID NO: 10 or a portion thereof) disclosed herein, and optionally also comprises a sequence available from BAC6-V H At least one human variable Ig gene is detachable from BAC6-V and BAC3 constructs and / or BAC9 and BAC14 / 5 constructs. In a preferred embodiment, the chimeric polynucleotides covered herein comprise nucleic acid sequences (a) and (b) in a 5' to 3' sequence: (a) a human Ig variable region comprising a gene detachable from BAC6-V. H(a) The natively configured human V gene isolated from the BAC3 and BAC9 and BAC14 / 5 constructs; and (b) the human Ig linker region comprising the natively configured human J gene, which can be isolated from BAC Annabel. In another embodiment, each of the human Ig variable region, human Ig diversity region, human Ig linker region, Ig constant region, and rat 3' enhancer region of the chimeric polynucleotide disclosed herein is positioned relative to the one shown in Figure 1a. In another embodiment, the disclosed chimeric polynucleotide has a sequence comprising or substantially homologous to the sequence or a portion thereof as described in SEQ ID NO:2. In another embodiment, the disclosed chimeric polynucleotide has a sequence comprising or substantially homologous to the sequence or a portion thereof as described in SEQ ID NO:11. In another embodiment, the chimeric polynucleotide disclosed herein comprises a rearranged VDJ region, wherein the rearranged VDJ region encodes a heavy chain variable domain exon.

[0014] In one embodiment, the transgenic animal further comprises a chimeric polynucleotide, wherein the human Ig V region contains at least one human V region gene separable from BAC9 and / or BAC14 / 5. In a preferred embodiment, the chimeric polynucleotide comprises nucleic acid sequences (a) and (b) in a 5' to 3' sequence: (a) a human Ig variable region containing a human V region gene from BAC9 and / or BAC14 / 5 used in its natural configuration (or rearranged); and (b) a human Ig linker region containing a human J region gene from the bacterial artificial chromosome (BAC) Annabel used in its natural configuration (or rearranged). In another embodiment, each of the human immunoglobulin variable region (gene), human immunoglobulin diversity region (segment), human immunoglobulin linker region (segment), immunoglobulin constant region gene, and rat 3' enhancer is positioned as shown in Figure 1b. In another embodiment, the disclosed chimeric polynucleotide has the following composition: Figure 6 The sequence described herein or a sequence substantially homologous to the described sequence. In another embodiment, the disclosed chimeric polynucleotide has the following composition: Figure 7 The sequence described herein, or a portion thereof, or a sequence substantially homologous to the sequence described herein, or a portion thereof. In another embodiment, the chimeric polynucleotide as disclosed herein may comprise a rearranged VDJ, wherein the rearranged gene segment is derived from SEQ ID NO and Figure above.

[0015] This document also discloses a polynucleotide encoding a human κ light chain gene. In one embodiment, the polynucleotide disclosed herein has a nucleic acid sequence comprising a group consisting of RP11-1156D9 (as described in SEQ ID NO:3) and RP11-1134E24 (as described in SEQ ID NO:4) or a nucleic acid sequence substantially homologous to said nucleic acid sequence. In another embodiment, the isolated polynucleotide comprises nucleic acid sequences (a) and (b) in a 5' to 3' sequence: (a) a human Ig variable region comprising a natively configured human V gene that can be isolated from bacterial artificial chromosome (BAC) RP11-156D9 and / or RP11-1134E24; and (b) a human Ig linker region comprising a natively configured human J gene that can be isolated from bacterial artificial chromosome (BAC) RP11-1134E24 and / or RP11-344F17 (as described in SEQ ID NO:5). In a preferred embodiment, each of the human Ig variable region, the human Ig linker region, and the human Ig constant region is positioned relative to each other as shown in Figure 2. In another embodiment, the disclosed chimeric polynucleotide has a sequence comprising or a portion thereof as described in SEQ ID NO:6, or a sequence substantially homologous to or a portion thereof.

[0016] This document also provides a rodent cell comprising one or more of the polynucleotides of the present invention. For example, this document provides a rodent cell comprising the polynucleotides disclosed herein, wherein the polynucleotides preferably comprise 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 a portion thereof. The rodent cells covered herein may further comprise a polynucleotide encoding a functional light chain, wherein the polynucleotide has, for example, a nucleic acid sequence comprising or substantially homologous to a nucleic acid sequence selected from the group consisting of: the sequence shown in FIG2a (as described in SEQ ID NO:6), the sequence shown in FIG2b (as described in SEQ ID NO:7), and a portion thereof. In one embodiment, one or more polynucleotides are integrated into the rodent cell genome.

[0017] In another aspect of the 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 at different chromosomal sites in the animal genome. In one embodiment, the transgenic animal having a plurality of artificial Ig heavy chain loci comprises (i) a V region of a human V gene segment having at least one amino acid sequence encoding a germline or hypermutant human V region; (ii) one or more J gene segments; and (iii) one or more constant region gene segments, wherein the artificial Ig heavy chain loci are functional and capable of gene rearrangement and synergistically functioning to produce an artificial immunoglobulin library. In another embodiment, the transgenic animal comprises the complete complementary sequence of a human variable heavy chain region. In various other embodiments, the transgenic animal i) has an artificial heavy chain locus comprising an overlapping heavy chain gene segment, 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 library of antibodies encoded by the V gene at transgenic immunoglobulin loci located at different chromosomal sites.

[0018] In some implementations, the transgenic animals lack functional Ig light chain loci and are able to produce heavy chain-only antibodies.

[0019] In another embodiment, at least one artificial Ig heavy chain locus of a transgenic animal having at least two artificial Ig heavy chain loci comprises at least one human immunoglobulin (Ig) linker (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 in SEQ 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 invention, the constant region gene is selected from the group consisting of human constant region genes and rat constant region genes. In some 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.

[0020] In some implementations, the human IgV region of the transgenic animal contains components that can be derived from BAC6-V. H At least one human V region gene is isolated from BAC6-V and / or BAC3. In one particular embodiment, the transgenic animal contains nucleic acids having the following in a 5' to 3' sequence: (a) a human Ig variable region containing a gene that can be isolated from BAC6-V. H(a) A natively configured human V region gene isolated from 3-11 and / or BAC3; and (b) a human Ig linker region containing a natively configured human J region gene that can be isolated from the bacterial artificial chromosome (BAC) Annabel. In one embodiment, each of the human immunoglobulin variable region, human immunoglobulin diversity region, human immunoglobulin linker region, immunoglobulin constant region, and rat 3' enhancer is positioned relative to the location shown in Figure 1a. In another embodiment, the transgenic animal has a nucleic acid sequence substantially homologous to the nucleic acid sequence described in SEQ ID NO:2. In yet another embodiment, the transgenic animal has a nucleic acid sequence substantially homologous to the nucleic acid sequence described in SEQ ID NO:11. In some embodiments, the transgenic animal has a VDJ region that is rearranged to form an intact exon encoding a heavy chain variable domain.

[0021] In some other embodiments, the transgenic animal has a human IgV region, said IgV region containing components that can be derived from BAC9-V. H At least one human V region gene is isolated from BAC9-V and / or BAC14 / 5. In one particular embodiment, these transgenic animals contain nucleic acids having the following in a 5' to 3' sequence: (a) a human Ig variable region containing a gene that can be isolated from BAC9-V. H (a) The natively configured human V region gene isolated from 3-53 and / or BAC14 / 5; (b) the human Ig linker region containing the natively configured human J region gene that can be isolated from the bacterial artificial chromosome (BAC) Annabel. In one embodiment, each of the human immunoglobulin variable region, human immunoglobulin diversity region, human immunoglobulin linker region, immunoglobulin constant region, and rat 3' enhancer is positioned relative to the location shown in Figure 1b. In another embodiment, the transgenic animal has the same... Figure 6 The nucleic acid sequences described herein are substantially homologous to nucleic acid sequences. In yet another embodiment, the transgenic animal possesses [the characteristics of] a nucleic acid sequence that is substantially homologous to [the described sequence]. Figure 7 The nucleic acid sequences described herein are essentially homologous nucleic acid sequences.

[0022] In another aspect of the invention, a method for generating antibodies is provided, the method comprising immunizing a transgenic animal as described above with an immunogen. In one embodiment, a polyclonal antiserum composition is generated, wherein the antiserum comprises an antigen-specific antibody encoded by a V gene encoded by a transgenic immunoglobulin locus located at a different chromosomal locus. In another embodiment, a method for generating monoclonal antibodies comprises (i) immunizing the transgenic animal with an immunogen, (ii) isolating monoclonal antibody-producing cells from the transgenic animal, wherein the monoclonal antibody-producing cells produce monoclonal antibodies that specifically bind to the immunogen; and (iii) using the monoclonal antibody-producing cells to produce the monoclonal antibodies that specifically bind to the immunogen, or using the monoclonal antibody-producing cells to produce hybridoma cells that produce the monoclonal antibodies, and using the hybridoma cells to produce the monoclonal antibodies.

[0023] In another embodiment, the method of generating a monoclonal antibody includes (i) immunizing the transgenic animal with an immunogen, (ii) isolating cells from the transgenic animal that generate monoclonal antibodies, wherein the cells that generate monoclonal antibodies produce monoclonal antibodies that specifically bind to the immunogen; (iii) isolating monoclonal antibody nucleic acid encoding the monoclonal antibody that specifically binds to the immunogen from the cells that generate monoclonal antibodies; and (iv) using the monoclonal antibody nucleic acid to generate the monoclonal antibody that specifically binds to the immunogen. In some embodiments, the monoclonal antibody has a human idiotype.

[0024] In another embodiment, the method for generating a fully human monoclonal antibody includes (i) immunizing the transgenic animal with an immunogen, (ii) isolating cells from the transgenic animal that generate monoclonal antibodies, wherein the cells that generate monoclonal antibodies generate monoclonal antibodies that specifically bind to the immunogen; (iii) isolating monoclonal antibody nucleic acid encoding the monoclonal antibody that specifically binds to the immunogen from the cells that generate monoclonal antibodies; (iv) modifying the monoclonal antibody nucleic acid to generate a recombinant nucleic acid encoding a fully human monoclonal antibody; and (v) using the recombinant nucleic acid encoding the fully human monoclonal antibody to generate a fully human monoclonal antibody.

[0025] Another aspect of the present invention is a monoclonal antibody produced by the above method.

[0026] In another aspect, a method for neutralizing antigenic entities in a human body component is provided, comprising contacting the body component with a polyclonal antiserum composition as described above, wherein the polyclonal antiserum composition comprises immunoglobulin molecules that specifically bind to and neutralize the antigenic entity. In one embodiment, the method for neutralizing antigenic entities in a human body component comprises contacting the body component with a monoclonal antibody according to the above description, wherein the monoclonal antibody specifically binds to and neutralizes the antigenic entity. Brief description of the attached diagram

[0027] Figure 1: Overview of integrated chimeric (human, rat) and fully human Ig loci. The two chimeric human-rat IgH regions (HC14 and HC30) each contain three overlapping BACs, each with ≥22 distinct and potentially functional human V genes. H Section. In HC14, V has already been used. H 3-11 extends BAC6-3 to provide 10.6kb overlap with BAC3, which is achieved through V H 6-1 overlaps 11.3 kb with BAC in region C of Hu-rat Annabel (A), and in HC30, BAC9 provides 4.6 kb overlap with BAC14 / 5, which is extended by adding VH3-43, then adding a portion of BAC5 and equipping it with 6.1 kb overlap with Hu-rat Annabel (B). The latter is chimeric and contains all D and J before and after. H Segments and rat C-regions with complete enhancer sequences. Arrows indicate VH gene usage in HC14, HC30, and the HC14 / HC30 combination. More blurred bands indicate less frequent VH gene expression. Sequences were obtained by unbiased RT-PCR and NGS.

[0028] Figure 2: (A) Human Igk BAC with 12 Vk and all Jk provides about 14 kb of overlap in the Vk region and about 40 kb in the Ck region to include the KDE. (B) Human Ig1 region with 17 Vl and all J-Cl, including the 3' enhancer, from YAC (Vincent-Fabert, C. et al., Blood 116, 1895-1898 (2010)).

[0029] Figure 3 The diagram illustrates the integration of the HC14 locus into chromosome 6 and the integration of the HC30 locus into chromosome 15.

[0030] Figure 4Analysis of IgM and IgG concentrations in serum from HC30 and HC14 / HC30 animals using ELISA. Each dot (HC30) or square (HC14 / HC30) represents the titer (μg / ml) of one animal. Further analysis of IgG levels of IgG1 and IgG2b was performed.

[0031] Figure 5 Analysis of anti-β-gal specific antibodies from HC30 and HC14 / HC30 by ELISA. Each dot (HC30) or square (HC14 / HC30) represents the serum titer of one animal (in comparative dilutions).

[0032] Figure 6 : BAC 9 sequence.

[0033] Figure 7 : BAC 14 / 5 sequence. Implementation

[0034] This paper provides chimeric polynucleotides encoding recombinant or artificial immunoglobulin chains or loci. As described above, the chimeric polynucleotides disclosed herein can be used to transform rodents to include human Ig genes and can be used to generate immunoglobulins or antibodies with human idiotypes using such rodents. As further provided herein, transgenic animals comprising at least three different transgenic constructs having a complete complementary sequence of the human immunoglobulin VDJ heavy chain gene segment tandemly integrated into the transgenic animal genome are generated, thereby ensuring that all human immunoglobulin genes in the germline conformation are available in the context of complete inactivation of endogenous immunoglobulin genes or loci. Surprisingly, as demonstrated for the first time herein, multiple transgenic loci containing different V genes can function synergistically in the expression of humanized and fully human transgenic antibodies.

[0035] definition

[0036] Immunoglobulins are proteins composed of one or more polypeptides essentially encoded by immunoglobulin genes. Recognized human immunoglobulin genes include κ, λ, α (IgA1 and IgA2), γ (IgG1, IgG2, IgG3, IgG4), δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. The full-length immunoglobulin "light chain" (approximately 25 kDa or 214 amino acids) typically contains a variable domain (approximately 110 amino acids) at the NH2 terminus encoded by an exon containing one or more variable region genes and one or more linker region genes, and a constant domain at the COOH terminus encoded by either the κ or λ constant region gene. The full-length immunoglobulin "heavy chain" (approximately 50 kDa or 446 amino acids) similarly comprises (1) a variable domain (approximately 116 amino acids) encoded by an exon containing one or more variable region genes, one or more diversity region genes, and one or more linker region genes; and (2) one of the aforementioned constant domains, containing one or more constant region genes, such as α, γ, δ, ε, or μ (encoding approximately 330 amino acids). The immunoglobulin heavy chain constant region genes encode antibody classes, i.e., isotypes (e.g., IgM or IgG1).

[0037] As used herein, the term "antibody" refers to a protein containing at least one, preferably two, heavy (H) chain variable domains (hereinafter abbreviated as VH) and at least one, preferably two light (L) chain variable domains (hereinafter abbreviated as VL). Those skilled in the art will recognize that the variable domains of the immune chain are encoded in gene segments that must first undergo somatic recombination to form complete exons encoding the variable domains. Three types of regions or gene segments undergo rearrangement to form variable domains: variable regions containing variable genes, diversity regions containing diverse genes (in the case of immunoglobulin heavy chains), and linker regions containing linker genes. VH and VL domains can be further subdivided into hypervariable regions (referred to as "complementarity-determining regions" ("CDRs")) interspersed with more conserved regions (referred to as "framework regions" ("FRs")). The extent of FRs and CDRs has been precisely defined (see, Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., USDapartment 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 typically consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. As used herein, an antigen-binding fragment of an antibody (or simply "antibody portion" or "fragment") refers to one or more fragments of a full-length antibody that retain the ability to specifically bind to an antigen (e.g., CD3).

[0038] Examples of binding fragments covered by the term “antigen-binding fragment” of an antibody include (i) the Fab fragment, a monovalent fragment consisting of VL, VH, CL, and CH1 domains; (ii) the F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) the Fd fragment, consisting of VH and CH1 domains; (iv) the Fv fragment, consisting of the VL and VH domains of a single arm of the antibody; (v) the dAb fragment (Ward et al., (1989) Nature 341:544-46), consisting of the VH domain; and (vi) the separated complementarity-determining region (CDR). Furthermore, although the two domains VL and VH of the Fv fragment are encoded by separate genes, these two domains can be linked together via synthetic linkers using recombination methods, allowing the two domains to be prepared as a single protein chain, where the VL and VH regions pair to form a monovalent molecule (referred to as a single-chain Fv (scFv); see, for example, Bird et al., (1988) Science 242:423-26; and Huston et al., (1988) Proc. Natl. Acad. Sci. USA 85:5879-83). Such single-chain antibodies are also expected to be covered within the term "antigen-binding fragment" in antibody terminology. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and are screened for utility in the same manner as intact antibodies.

[0039] Antibodies may also include heavy chain and / or light chain constant domains, thereby forming heavy chain and light chain immunoglobulin chains, respectively. In one embodiment, the antibody is a tetramer of two immunoglobulin heavy chains and two immunoglobulin light chains, wherein the immunoglobulin heavy chains and light chains are interconnected, for example, by disulfide bonds. The heavy chain constant domain includes three gene segments, namely CH1, CH2, and CH3. The light chain constant domain includes a gene CL. Variable domains of the heavy chain and / or light chain contain 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 (C1q) of the classical complement system.

[0040] The term "polynucleotide encoding an artificial immunoglobulin locus or artificial immunoglobulin chain" refers to a recombinant polynucleotide comprising multiple immunoglobulin regions, such as a variable (V) region or gene segment containing a V gene, a linker (J) gene region or gene segment containing a J gene, a diversity (D) region or gene segment containing a D gene in the case of a heavy chain locus, and / or at least one constant (C) region containing at least one C gene. Preferably, each region of the variable domain (e.g., V, D, or J region) contains or spans at least two genes of the same type. For example, as used herein, a variable region contains at least two variable genes, a linker region contains at least two linker genes, and a diversity region contains two diversity genes. A constant region may contain only one constant gene (e.g., a κ gene or a λ gene) or multiple genes (e.g., CH1, CH2, and CH3).

[0041] As used herein, “enhancer sequence” or “enhancer” refers to a sequence that has been identified near many active genes by nuclease digestion and hypersensitivity to degradation. Hypersensitive sites can be located prior to promoter sequences, and their activity intensity is correlated with the DNA sequence. If enhancer function is present, linkage with a reporter gene shows increased transcription (Mundt et al., J. Immunol, 166, 3315

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

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

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

[2011] ). Achieving optimal allotype expression of the regulatory function is particularly desirable when using transgenic human IgH genes. Transgenic constructs with incomplete 3'E regions (typically providing only hs1,2 elements) result in disappointing expression levels in transgenic mice, even when the endogenous IgH locus is knocked out. Therefore, only a few antigen-specific fully human IgGs have been isolated from constructs produced in 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] ). Analysis of the 3'E region in the rat IgH locus has been inadequate. Comparison of mouse and rat sequences has not allowed for the identification of hs4, a key fourth element with other important regulatory sequences further downstream (Chatterjee et al., J. Biol. Chem., 286, 29303

[2011] ). The polynucleotides of the present invention advantageously provide optimal expression, at least in part due to the inclusion of a rat 3' enhancer, as the chimeric polynucleotide lacking this 3' enhancer results in impaired allotype conversion and reduced IgG expression. In one embodiment, the rat 3' enhancer has a sequence comprising or a portion thereof as described in SEQ ID NO:1 or a sequence substantially homologous to or a portion thereof.

[0042] As used herein, polynucleotides having a portion (e.g., less than all) of a second sequence (e.g., SEQ ID NO:1, SEQ ID NO:2, etc.) or a sequence substantially homologous to said portion preferably retain the biological activity of the second sequence (e.g., retaining the biological activity of the 3' enhancer to provide optimal expression of immunoglobulins and / or isotype conversion, being capable of rearrangement to provide humanized chimeric heavy chains, etc.). In one embodiment, the nucleic acid comprising a portion of SEQ ID NO:1 or a sequence substantially homologous to said portion comprises at least 8 kB, preferably at least 10 kB, of a continuous nucleic acid substantially homologous to SEQ ID NO:1. In another embodiment, the second nucleic acid comprising a portion of SEQ ID NO:59 or 60 or a sequence substantially homologous to said portion comprises at least 8 kB, preferably at least 10 kB, of a continuous nucleic acid substantially homologous to SEQ ID NO:59 or 60.

[0043] As used herein, “artificial Ig locus” can refer to a sequence containing, for example, regions V, D, and / or J (in the case of the heavy chain) or regions V and / or J (in the case of the light chain) and optionally a constant region (in the case of either or both of the heavy and light chains) of an unrearranged, partially rearranged, or rearranged polynucleotide. Artificial Ig loci include artificial Ig light chain loci and artificial Ig heavy chain loci. In one embodiment, the artificial immunoglobulin loci of the present invention are functional and capable of rearrangement and generating a library of immunoglobulin chains. In a preferred embodiment, the variable domain or a portion thereof of the polynucleotide disclosed herein comprises a gene in its natural conformation, i.e., a naturally occurring sequence of a human Ig gene segment, a degenerate form of a naturally occurring sequence of a human Ig gene segment, and a synthetic sequence encoding a polypeptide sequence substantially identical to that encoded by the naturally occurring sequence of the human Ig gene segment. In another preferred embodiment, the polynucleotide comprises a variable domain or a portion thereof in its natural conformation found in humans. For example, the polynucleotides encoding artificial Ig heavy chains disclosed herein may contain at least two human V genes, at least two D genes, at least two J genes, or combinations thereof in their natural configuration.

[0044] In a preferred embodiment, the artificial Ig locus contains a non-human C region gene and is capable of generating an immunoglobulin library including chimeric immunoglobulins with non-human C regions. In one embodiment, the artificial Ig locus contains a human C region gene and is capable of generating an immunoglobulin library including immunoglobulins with human C regions. In one embodiment, the artificial Ig locus contains an "artificial constant region gene," meaning that the constant region gene contains nucleotide sequences from both human and non-human constant region genes. For example, an exemplary artificial C constant region gene is a constant region gene encoding the human IgG CH1 domain and the rat IgG CH2 and CH3 domains.

[0045] In some implementations, the artificial Ig heavy chain locus lacks CH1 or allows the resulting immunoglobulin to bypass the equivalent sequence of typical immunoglobulin:companion protein association. Such artificial loci enable the production of heavy chain-only antibodies in transgenic animals lacking a functional Ig light chain locus and therefore not expressing a functional Ig light chain. The methods envisioned herein utilize such artificial Ig heavy chain loci to generate transgenic animals lacking a functional Ig light chain locus but containing an artificial Ig heavy chain locus, which are capable of producing heavy chain-only antibodies. Alternatively, the artificial Ig locus can be manipulated in situ to disrupt CH1 or an equivalent region, generating an artificial Ig heavy chain locus that provides for the production of heavy chain-only antibodies. For example, regarding the production of heavy chain-only antibodies in light chain-deficient mice, see Zou et al., JEM, 204:3271-3283, 2007.

[0046] "Human idiotype" refers to a polypeptide sequence present on a human antibody encoded by a segment of the immunoglobulin V gene. As used herein, the term "human idiotype" includes both naturally occurring sequences of human antibodies and synthetic sequences substantially identical to polypeptides found in naturally occurring 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%, more preferably greater than 95%.

[0047] "Chimeric antibody" or "chimeric immunoglobulin" refers to an immunoglobulin molecule that contains a portion of a human immunoglobulin polypeptide sequence (or a polypeptide sequence encoded by a segment of the human Ig gene) and a portion of a non-human immunoglobulin polypeptide sequence. The chimeric immunoglobulin molecule of this invention is an immunoglobulin having a non-human Fc region or an artificial Fc region and a human idiotype. Such immunoglobulins can be isolated from the animals of this invention that have been engineered to produce chimeric immunoglobulin molecules.

[0048] "Artificial Fc region" refers to the Fc region encoded by artificial constant region genes.

[0049] As used in this paper, "Ig gene region" refers to the DNA region that encodes the various parts of the Ig molecule, which exists in non-human animal and human lineages and aggregates in B cells to form rearranged Ig genes. Therefore, the Ig gene regions used in this paper include the V gene region, D gene region, J gene region, and C gene region.

[0050] As used herein, the term "human Ig gene segment" includes the naturally occurring sequence of a human Ig gene segment, a degenerate form of the naturally occurring sequence of a human Ig gene segment, and a synthetic sequence encoding a polypeptide sequence substantially identical to that encoded by the naturally occurring sequence of the 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%, more preferably greater than 95%.

[0051] The polynucleotides associated with this invention may comprise DNA or RNA and may be synthesized wholly or partially. Unless the context otherwise requires, references to nucleotide sequences as described herein cover DNA molecules having the specified sequences and RNA molecules having the specified sequences (where T is replaced by U).

[0052] The calculation of “homology” or “sequence consistency” (the terms are used interchangeably herein) between two sequences is performed as follows. The sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences may be ignored for comparison purposes). In a preferred embodiment, the length of the reference sequence to be 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%, 100% of the length of the reference sequence. The amino acid residues or nucleotides at the corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecule at that position is consistent (as used herein, amino acid or nucleic acid “consistency” is equivalent to amino acid or nucleic acid “homology”). The percentage of consistency between the two sequences varies with the number of consistent positions shared by the sequences, taking into account the number of vacancies required for optimal alignment of the two sequences and the length of each vacancy.

[0053] The comparison of two sequences and the determination of the percentage of sequence identity can be accomplished using mathematical algorithms. In a preferred embodiment, the percentage of identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-53) algorithm, which has been incorporated into the GAP program of the GCG software package (available online at gcg.com). This GAP program uses a Blossum 62 matrix or a PAM250 matrix and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, or length weights of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, the percentage of identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at www.gcg.com). This GAP program uses an NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the set of parameters to use if the practitioner is unsure which parameters to apply to determine whether a molecule is within the sequence consistency or homology constraints of this invention) is a Blossum 62 scoring matrix with a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5. The percentage of similarity between two amino acid or nucleotide sequences can also be determined using the algorithm of Meyers and Miller ((1989) CABIOS 4:11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4.

[0054] Artificial Ig loci

[0055] This invention also relates to Ig loci and their use in the preparation of transgenic animals capable of producing immunoglobulins with human idiotypes. Each artificial Ig locus comprises multiple immunoglobulin gene segments, including at least one V region gene segment, one or more J gene segments, one or more D gene segments (in the case of heavy chain loci), and one or more constant region genes. In this invention, at least one V gene segment encodes a germline or hypermutated human V region amino acid sequence. Thus, such transgenic animals are capable of producing a diverse library of immunoglobulin molecules, which include antibodies with human idiotypes. In heavy chain loci, human or non-human D gene segments may be included in the artificial Ig locus. Gene segments in such loci are juxtaposed with each other in an unrearranged configuration (or "germline configuration") or in a partially or completely rearranged configuration. The artificial Ig locus enables gene rearrangement in test animals (if gene segments are not completely rearranged), thereby producing a diverse library of immunoglobulins with human idiotypes.

[0056] Regulatory elements (such as promoters, enhancers, transition regions, recombination signals, etc.) can be of human or non-human origin. It is essential that these elements be operable in the relevant animal species so that the artificial locus can function. Preferred regulatory elements are described in more detail herein.

[0057] In one aspect, the present invention provides a transgenic construct containing an artificial heavy chain locus capable of gene rearrangement in a host animal, thereby generating a diverse heavy chain library with human idiotypes. The transgenic artificial heavy chain locus contains a V region having at least one human V gene segment. Preferably, the V region comprises at least about 5-100 human heavy chain V (or “VH”) gene segments. In a preferred embodiment, 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 encompasses the naturally occurring sequence of the human VH gene segment, a degenerate form of the naturally occurring sequence of the human VH gene segment, and a synthetic sequence encoding a polypeptide sequence substantially (i.e., at least about 85%-95%) identical to the polypeptide sequence of the human heavy chain V domain.

[0058] In a preferred embodiment, the artificial heavy chain locus contains at least one or more rat constant region genes, such as Cδ, Cμ, and Cγ (including any of the Cγ subclasses).

[0059] In another preferred embodiment, the artificial heavy chain locus contains an artificial constant region gene. In one preferred embodiment, such an artificial constant region gene encodes a human CH1 domain and a rat CH2-CH3 domain, or a human CH1 domain and a rat CH2, CH3, and CH4 domain. The hybrid heavy chain containing the human CH1 domain effectively pairs with the fully human light chain.

[0060] In a preferred embodiment, the artificial Ig locus contains a 3' enhancer sequence, including hs1,2, hs3a, hs3b and the sequence between rat Calpha and 3'hs3b.

[0061] In another preferred embodiment, the artificial heavy chain locus contains an artificial constant region gene lacking the CH1 domain. In one preferred embodiment, such an artificial constant region gene encodes a truncated IgM and / or IgG lacking the CH1 domain but containing CH2 and CH3 or CH1, CH2, CH3, and CH4 domains. Heavy chains lacking the CH1 domain cannot effectively pair with Ig light chains and form heavy chain-only antibodies.

[0062] In another aspect, the present invention provides a transgenic construct containing an artificial light chain locus capable of gene rearrangement in a host animal, thereby generating a diverse library of light chains with human idiotypes. The transgenic artificial light chain locus contains a V region having at least one human V gene segment, such as 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 5-100 human light chain V (or “VL”) gene segments. Consistently, the human VL segment encompasses the naturally occurring sequence of the human VL gene segment, a degenerate form of the naturally occurring sequence of the human VL gene segment, and a synthetic sequence encoding a polypeptide sequence substantially (i.e., at least about 85%-95%) identical to the polypeptide sequence of the human light chain V domain. In one embodiment, the artificial light chain Ig locus comprises a C region having at least one rat C gene (e.g., rat Cλ or Cκ).

[0063] Another aspect of the invention relates to a method for preparing a transgenic vector containing an artificial Ig locus. Such a method involves isolating an Ig locus or a fragment thereof, and combining said Ig locus or fragment thereof with one or more DNA fragments containing sequences encoding human V region elements. The Ig gene segment is inserted into the artificial Ig locus or a portion thereof by ligation or homologous recombination to maintain the locus's ability to undergo effective gene rearrangement in test animals.

[0064] Preferably, non-human Ig loci are isolated by screening libraries of plasmids, cosmids, YACs, or BACs, etc., prepared from genomic DNA of the plasmids, cosmids, YACs, or BACs. YAC clones can carry DNA fragments up to 2 megabases, thus allowing the isolation of an entire animal heavy chain locus or a large portion thereof from a single YAC clone, or its reconstruction into a single YAC clone. BAC clones can carry smaller DNA fragments (approximately 50-500 kb). However, multiple BAC clones containing overlapping fragments of Ig loci can be separately modified and then injected together into animal recipient cells, where the overlapping fragments recombine in the recipient animal cells to produce a continuous sequence of Ig loci.

[0065] Human Ig gene segments can be integrated into Ig loci on vectors (e.g., BAC clones) using various methods, including linking DNA fragments or inserting DNA fragments via homologous recombination. The integration of human Ig gene segments allows for operative linking to host animal sequences in transgenic organisms to produce functional humanized Ig loci—Ig loci capable of gene rearrangement leading to a diverse library of antibodies with human idiotypes. Homologous recombination can occur in bacteria, yeast, and other cells with a high frequency of homologous recombination events. Engineered YACs and BACs can be readily isolated from cells and used to prepare transgenic animals.

[0066] Transgenic animals containing artificial Ig loci and capable of producing antibodies with human idiotypes.

[0067] In one aspect, the present invention provides a transgenic animal capable of producing immunoglobulins with human idiotypes, and a method for preparing said transgenic animal. The transgenic animal used is selected from rodents (e.g., rats, hamsters, mice, and guinea pigs).

[0068] The transgenic animals used for the generation of humanized antibodies in this invention carry germline mutations in endogenous Ig loci. In a preferred embodiment, the transgenic animals are homozygous with respect to the mutated endogenous Ig heavy chain and / or endogenous Ig light chain genes. Furthermore, these animals carry at least two artificial heavy chain loc Ig loci, which are functional and capable of generating an immunoglobulin molecule library in the transgenic animals. The artificial Ig loci used in this invention comprise at least one human V gene segment.

[0069] In one preferred embodiment, the transgenic animal carries at least two Ig heavy chain loci and at least one Ig light chain locus, each locus being functional and capable of producing an immunoglobulin library in the transgenic animal, the immunoglobulin library comprising antibodies with human idiotypes. In one embodiment, an artificial locus comprising at least one non-human C gene is used, and an animal capable of producing chimeric antibodies with human idiotypes and non-human constant regions is provided. In one embodiment, an artificial locus comprising at least one human C gene is used, and an animal capable of producing antibodies with both human idiotypes and human constant regions is provided.

[0070] In another preferred embodiment, the transgenic animal carries at least two artificial Ig heavy chain loci and lacks a functional Ig light chain locus. Such animals can be used to produce heavy chain-only antibodies.

[0071] The generation of such transgenic animals involves integrating at least two artificial heavy chain Ig loci and one or more artificial light chain Ig loci into the genome of a transgenic animal having at least one endogenous Ig locus, which has been or will be inactivated by one or more large-scale nucleases. Preferably, the transgenic animal is locus-deficient 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 can undergo gene rearrangement, thereby generating a diverse library of immunoglobulin molecules. Ig loci capable of gene rearrangement are also referred to herein as “functional” Ig loci, and the diverse antibodies generated by functional Ig loci are also referred to herein as “functional” antibodies or a “functional” antibody library.

[0072] The artificial loci used to produce such transgenic animals each comprise multiple immunoglobulin gene segments, including at least one V region gene segment, one or more J gene segments, one or more D gene segments (in the case of heavy chain loci), and one or more constant region genes. In this invention, at least one V gene segment encodes a germline or hypermutated human V region amino acid sequence. Therefore, such transgenic animals are capable of producing a diverse library of immunoglobulin molecules, which include antibodies with human idiotypes.

[0073] In one embodiment, the artificial locus used contains at least one non-human C-region gene segment. Therefore, such transgenic animals are capable of producing a diverse library of immunoglobulin molecules, including chimeric antibodies with human idiotypes.

[0074] In one embodiment, the artificial locus used contains at least one human C-region gene segment. Therefore, such transgenic animals are capable of producing a diverse library of immunoglobulin molecules, including antibodies with human idiotypes and human constant regions.

[0075] In one embodiment, the artificial locus used comprises 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 and the rat IgG CH2 and CH3 domains. Therefore, such transgenic animals are capable of producing a diverse library of immunoglobulin molecules, which include antibodies having human idiotypes and artificial constant regions containing human and non-human components.

[0076] A transgenic vector containing an artificial Ig locus is introduced into one or more recipient cells, and then integrated into the genome of the one or more recipient cells through random or targeted integration.

[0077] For random integration, a transgenic vector containing an artificial Ig locus can be introduced into recipient cells using standard transgenic techniques. For example, the transgenic vector can be injected directly into the pronucleus of a fertilized oocyte. Alternatively, the transgenic vector can 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), followed by injection of the genetically modified cells into a developing embryo. Alternatively, the transgenic vector (naked or bound to a promoter) can be injected directly into the 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 cell genome, and an animal is obtained from the transfected cell via nuclear transfer cloning.

[0078] In a preferred embodiment, a transgene containing an artificial Ig locus is randomly integrated into the genome of a recipient cell (e.g., a fertilized oocyte or a developing embryo). In a preferred embodiment, offspring with a locus deficiency in terms of endogenous Ig heavy chain and / or Ig light chain, and therefore unable to produce endogenous immunoglobulins, and capable of producing transgenic immunoglobulins, are obtained.

[0079] For targeted integration, a transgenic vector can be introduced into suitable recipient cells (such as embryonic stem cells, other pluripotent cells, or differentiated somatic cells). Cells in which the transgene has been integrated into the animal genome can then be selected using standard methods. The selected cells can then be fused with enucleated nuclear transfer unit cells, such as oocytes or embryonic stem cells, which are pluripotent and capable of forming functional newborn animals. Fusion is performed according to recognized 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 via microsurgery using a microinjection needle (see, for example, Wakayama et al., Nature (1998) 394:369). The resulting cells are then cultured in a suitable medium and transferred to concurrently treated recipients for the production of transgenic animals. Alternatively, the selected genetically modified cells can be injected into a developing embryo, which is then allowed to develop into a chimeric animal.

[0080] In one embodiment, a broad range of nucleases is used to increase the frequency of homologous recombination at the target site via double-strand DNA cleavage. For integration into a specific site, site-specific broad range of nucleases can be used. In one embodiment, a broad range of nucleases targeting endogenous Ig loci are used to increase the frequency of homologous recombination, and an artificial Ig locus or a portion thereof replaces a portion of the endogenous Ig locus. In one embodiment, the transgenic animal lacks a functional Ig light chain locus and contains an artificial Ig heavy chain locus.

[0081] The preferred embodiment of integrating human Ig gene segments using YAC and BAC with interleaving between them offers advantages in both speed and the ability to check integrity when preparing large-region constructs via overlapping homology. Tandem integration of constructs with overlapping regions has the ability to integrate (e.g., maintain full function), which is crucial for DNA rearrangement. The preferred embodiment of the invention not only achieves the desired integration via homology but also produces tandem integration as a common event. This greatly simplifies transgenic technology because it eliminates the need for the laborious integration of large YACs into stem cells and subsequent animal derivation from said stem cells. Furthermore, ZFN technology, also performed via DNA injection (Geurts et al., Science 325, 433 (2009); Menoret et al., European journal of immunology 40, 2932-2941 (2010)), readily produces Ig KO strains and is likely to become the preferred technology for future gene disruption and replacement. OmniRats TM The advantage of silencing endogenous Ig gene expression in (containing human-rat IgH and human IgL loci) is that neither interfering nor unwanted rat Ig produces mixed products.

[0082] In mice, the downstream enhancer region of Cα plays a crucial role in class-switching recombination (Vincent-Fabert et al., Blood 116, 1895-1898 (2010)), and elements in this region may promote hypermutation (Pruzina et al., Protein engineering, design & selection: PEDS 24, 791-799 (2011)). This may explain why immune responses and the high frequency of diverse hybridomas are difficult to elicit even in mice carrying large fully 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 in OmniRats favors differentiation and expression levels close to wt%, it can be concluded that the endogenous rat C region, and indeed the approximately 30 kb enhancer sequence of Cα3', provides the enhancement for human V H The optimal locus for gene maturation is controlled by the gene. Another region, Cδ, with a 3' control motif cluster, has been removed from the chimeric C region BAC (Mundt et al., J Immunol 166, 3315-3323 (2001)), because silencing or lack of IgD has not been shown to reduce immune function (Chen Immunol Rev 237, 160-179 (2010)). Normally, mature IgM... + IgD + B cells downregulate IgD upon antigen contact, thereby initiating class-switching recombination (Id). Therefore, in the absence of IgD control, switching may increase, as supported by our following findings: when the Cδ region was retained in the transgenic construct, IgG transcript and serum levels were significantly reduced (data not shown).

[0083] OmniRats TMThe production of medium-specific IgG was particularly encouraging, as we found that mAbs with sequence and epitope diversity comparable to those produced in the wt control could be isolated via spleen and lymph node fusions across various immunizations. The diversity of the V, D, and J genes was as expected, and almost all segments were found to be efficiently utilized as predicted (Lefranc & Lefranc The immunoglobulin factsbook. FactsBook Series, Academic Press, GB, 45-68 (2001)). This contrasts sharply with mice carrying fully human transgenic loci, in which the clonal expansion of a few precursor B cells produced very little diversity (Pruzina et al., Protein engineering, design & selection: PEDS24, 791-799 (2011)). Since only half the number of V genes were transplanted in humans, we expected limited immune responses and limited diversity when comparing OmniRats to wt animals. However, this is not the case, and a comparison of CDR3 diversity in over 1000 clones (sequences available) reveals that the connectivity differences between OmniRats and wt animals are equally extensive. A few consistent gene segment combinations are found via V H -D and / or DJ H The N sequence at the junction can be added or deleted, and further diversified through hypermutation. Therefore, it is clear that the rat C region sequence plays a crucial role in controlling human V. H DJ H It is highly efficient in terms of DNA rearrangement and expression. Extensive diversity was also observed in the introduced human Igκ and Igλ loci, similar to what was previously 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, OmniRats TM The problem of significantly reduced efficiency in generating human antibodies in mice has been overcome (Lonberg, N. Nature biotechnology 23, 1117-1125 (2005)), the OmniRats TMReliable and extensive diversification of rearranged H chains through class switching and hypermutation yields high-affinity antibodies in large quantities rather than occasionally. The impressive yields and hypermutation levels of transgenic IgG for antigen-specific mAbs demonstrate the effectiveness of OmniRats. TM Similar clonal diversification and production levels were observed between wt animals and wt animals. Routine production of high affinity specificity was even achieved in the sub-nanomolar range through different single immunizations, which was advantageous compared to wt animals; however, results have not yet been shown for transgenic mice that produce human antibody libraries from full human loci (Mendez et al., Nature genetics 15, 146-156 (1997)).

[0084] In summary, to maximize human antibody production, it should be considered necessary to use the IgH locus, which uses human genes for antibody specificity but rodent genes for controlling differentiation and high expression. The flexibility of the L chain is an added benefit, as it allows for efficient human IgH / IgL assembly even in the presence of wt Ig. For therapeutic applications, the chimeric H chain can be readily converted into a fully human antibody via C gene substitution without compromising specificity.

[0085] Immunoglobulins with human-specific characteristics

[0086] Once a transgenic animal capable of producing immunoglobulins with a human idiotype is created, immunoglobulins and antibody preparations targeting that antigen can be readily obtained by immunizing the animal with the antigen. As used herein, "polyclonal antiserum compositions" include affinity-purified polyclonal antibody preparations.

[0087] A variety of antigens can be used to immunize transgenic animals. These antigens include, but are not limited to, microorganisms (e.g., live, attenuated, or dead viruses and single-celled organisms such as bacteria and fungi), fragments of microorganisms, or antigenic molecules isolated from microorganisms.

[0088] Preferred bacterial antigens for animal immunization include purified antigens (such as capsular polysaccharides type 5 and 8) from Staphylococcus aureus, recombinant forms of virulence factors (such as alpha-toxin), adhesin-binding proteins, collagen-binding proteins, and fibronectin-binding proteins. Preferred bacterial antigens also include attenuated forms of Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus, Enterobacter, and Klebsiella pneumoniae, or culture supernatants from these bacterial cells. Other bacterial antigens that can be used for immunization include purified lipopolysaccharide (LPS), capsular antigens, capsular polysaccharides, and / or outer membrane proteins, fibronectin-binding proteins, endotoxins, and recombinant forms of exotoxins from Pseudomonas aeruginosa, Enterococcus, Enterobacter, and Klebsiella pneumoniae.

[0089] Preferred antigens for generating antifungal antibodies include attenuated forms of fungi or their outer membrane proteins, including but not limited to Candida albicans, Candida parapsilosis, Candida tropicalis, and Cryptococcus neoformans.

[0090] Preferred antigens for immunization to generate antiviral antibodies include envelope proteins and attenuated forms of viruses, including but not limited to respiratory syncytial virus (RSV) (especially the F protein), hepatitis C virus (HCV), hepatitis B virus (HBV), cytomegalovirus (CMV), EBV, and HSV.

[0091] Cancer-specific antibodies can be generated by immunizing transgenic animals with isolated tumor cells or tumor cell lines and tumor-associated antigens, including but not limited to Her-2-neu antigen (antibodies against said antigen can be used to treat breast cancer); CD20, CD22 and CD53 antigens (antibodies against said antigen can be used to treat B-cell lymphoma), prostate-specific membrane antigen (PMSA) (antibodies against said antigen can be used to treat prostate cancer), and 17-1A molecule (antibodies against said molecule can be used to treat colon cancer).

[0092] The antigen, with or without adjuvant, can be administered to transgenic animals in any convenient manner and can be administered according to a predetermined schedule.

[0093] To prepare monoclonal antibodies, spleen cells are isolated from immunized transgenic animals and used in the form of cell fusions formed together with the transformed cell lines to generate hybridomas, or cDNA encoding the antibody is cloned using standard molecular biology techniques and expressed in transfected cells. Procedures for preparing monoclonal antibodies are well established in the art. See, for example, European Patent Application 0 583 980A1 (“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 EP 0 491 057B1 (“Hybridoma Which Produces Avian Specific Immunoglobulin G”), the disclosures of which are incorporated herein by reference. Methods for producing monoclonal antibodies from cloned cDNA molecules in vitro have been described by Andris-Widhopf et al. in J Immunol Methods 242:159 (2000) and Burton Immunotechnology 1:87 (1995).

[0094] Once chimeric monoclonal antibodies with human idiotypes are developed, they can be readily converted into fully human antibodies using standard molecular biology techniques. Fully human monoclonal antibodies are non-immunogenic in humans and suitable for therapeutic use in human subjects.

[0095] The antibodies of this invention include heavy chain-only antibodies.

[0096] In one implementation, transgenic animals lacking functional Ig light chain loci and containing at least two artificial heavy chain loci are immunized with an antigen to generate heavy chain-only antibodies that specifically bind to the antigen.

[0097] In one embodiment, the present invention provides cells from such animals that produce monoclonal antibodies, and nucleic acids from said cells. Hybridomas from said cells are also provided. Fully human heavy-chain-only antibodies and nucleic acids encoding said fully human heavy-chain antibodies are also provided.

[0098] Teachings regarding heavy chain-only antibodies have been found in the art. See, for example, PCT publications WO02085944, WO02085945, WO2006008548, and WO2007096779. See also US 5,840,526, US 5,874,541, US 6,005,079, US 6,765,087, US 5,800,988, EP 1589107, WO 9734103, and US 6,015,695.

[0099] Pharmaceutical Composition

[0100] In another embodiment of the invention, purified monoclonal or polyclonal antibodies are mixed with a suitable drug carrier for patient administration to provide a pharmaceutical composition.

[0101] Patients treated with the pharmaceutical compositions of the present invention are preferably mammals, more preferably humans, but veterinary use is also included.

[0102] Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention can be any and all solvents, dispersion media, isotonic agents, etc. Their use in the pharmaceutical compositions of the present invention is appropriate unless any conventional medium, reagent, diluent, or carrier is detrimental to the therapeutic effect on the recipient or the antibody contained in said conventional medium, reagent, diluent, or carrier.

[0103] The carrier can be a liquid, a semi-solid (e.g., a paste), or a solid carrier. Examples of carriers include oils, water, saline solutions, alcohols, sugars, gels, lipids, liposomes, resins, porous matrices, adhesives, fillers, coatings, preservatives, or combinations thereof.

[0104] Treatment

[0105] In another aspect of the invention, a method for treating diseases in vertebrates, preferably mammals, preferably primates is provided, wherein a human subject is a particularly preferred embodiment, the method being carried out by administering a purified antibody composition of the invention ideally suited for treating such diseases.

[0106] Antibody compositions can be used to bind to and neutralize or modulate antigenic entities in human tissues that cause or contribute to disease or elicit unwanted or abnormal immune responses. "Antigenic entity" is defined herein as encompassing any soluble or cell-surface-bound molecule (including proteins), and at least capable of binding to an antibody and preferably also capable of stimulating an immune response in cells or organisms that cause infectious diseases.

[0107] Antibody compositions, administered as monotherapy or in combination with chemotherapy, can eliminate infectious particles. Monotherapy with antibodies typically reduces the number of infectious particles by 10 to 100 times, and more commonly by more than 1000 times. Similarly, antibody therapy, used as monotherapy or in combination with chemotherapy in patients with malignant diseases, typically reduces the number of malignant cells by 10 to 100 times, or more than 1000 times. Therapy can be repeated for extended periods to ensure complete elimination of infectious particles, malignant cells, etc. In some cases, where no detectable amounts of infectious particles or malignant cells are present, the duration of antibody therapy will be prolonged.

[0108] Similarly, modulating the immune response using antibody therapy can consist of a single or multiple administrations of therapeutic antibodies. The duration of treatment can be prolonged even in the absence of any disease symptoms.

[0109] Treatment in subjects can be combined with chemotherapy at doses sufficient to suppress infectious or malignant diseases. In patients with autoimmune diseases or transplant recipients, antibody therapy can be combined with immunosuppressive therapy at doses sufficient to suppress the immune response. Example

[0110] In transgenic mice with respect to the human immunoglobulin (Ig) locus, the suboptimal efficacy of fully human antibody delivery is attributed to incomplete interactions between the human membrane IgH chain constant region and mouse cell signaling mechanisms. To avoid this problem, we describe here a humanized rat strain (OmniRat). TM It carries a chimeric human / rat IgH locus [containing 22 human V] H All people D and J H The segment, which has a germline genetic interval but is linked to rat C H [Loci] and fully human light chain loci [12 Vκ linked to Jκ-Cκ and 16 Vλ linked to Jλ-Cλ]. Endogenous rat Ig loci were silenced using a designed zinc finger nuclease. Following immunization, OmniRats performed as efficiently as normal rats in producing high-affinity serum IgG. Monoclonal antibodies containing fully human variable regions with sub-nanomolar antigen affinity and carrying a wide range of somatic mutations were readily available, yielding similar amounts to conventional antibodies from normal rats.

[0111] Materials and methods

[0112] Modified human Ig loci were constructed on YAC and BAC.

[0113] a) IgH locus

[0114] The human IgH V gene is covered by two BACs: BAC6-VH3-11 (modified from commercially available BAC clone 3054M17 CITB) containing the true region spanning from VH4-39 to VH3-23, and subsequently VH3-11, and BAC3 (811L16 RPCI-11) containing the true region spanning from VH3-11 to VH6-1. The BAC known as Annabel was constructed by linking the rat CH region gene downstream of the human VH6-1-D-JH region (Figure 1). All BAC clones containing a portion of the human or rat IgH gene locus were purchased from Invitrogen.

[0115] BAC6-VH3-11 and Annabel were initially constructed as cyclic YACs (cYAC) in Saccharomyces cerevisiae and further examined and maintained as BACs in Escherichia coli.

[0116] Unlike YAC, BAC plasmid preparations produce a large amount of the desired DNA. To convert linear YAC into cYAC or to assemble DNA fragments with overlapping ends into a single cYAC in *Saccharomyces cerevisiae* (which can also be maintained as BAC in *E. coli*), two self-replicating *Saccharomyces cerevisiae* / *E. coli* shuttle vectors, pBelo-CEN-URA and pBelo-CEN-HYG, were constructed. In short, *Saccharomyces cerevisiae* CEN4 was excised from pYAC-RC as an AvrII fragment (Marchuk and Collins, *Nucleic acids research* 16, 7743 (1988)) and ligated into a SpeI-linearized pAP599 (Kaur and Cormack, *PNAS* 104, 7628-7633 (2007)). The resulting plasmid contained CEN4 cloned between *Saccharomyces cerevisiae* URA3 and the hygromycin-resistance expression cassette (HygR). From this plasmid, the ApaLI-BamHI fragment containing URA3 followed by CEN4 or the PmlI-SphI fragment containing CEN4 followed by HygR was excised and ligated into pBACBelo11 (New England Biolabs) that had been double-digested with ApaLI and BamHI or HpaI and SphI to obtain pBelo-CEN-URA and pBelo-CEN-HYG.

[0117] To construct BAC6-VH3-11, two fragments, 115 kb NotI-PmeI and 110 kb 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 fragment by 22 kb. The NotI-PmeI fragment was ligated to the NotI-BamHI YAC arm containing *Saccharomyces cerevisiae* CEN4 from pYAC-RC and TRP1 / ARS1, and the RsrII-SgrAI fragment was ligated to the SgrAI-BamHI YAC arm containing *Saccharomyces cerevisiae* URA3, also from pYAC-RC. Subsequently, the ligation mixture was transformed into *Saccharomyces cerevisiae* AB1380 cells via protoplast transformation. 41We selected a URA+TRP+ yeast clone. The clone named YAC6, containing the linear region from human VH4-39 to VH3-23, was confirmed by Southern blot analysis. YAC6 was further extended by adding a 10.6 kb fragment to the 3' end of VH3-23 and converting it to cYAC. This 10.6 kb extension contained human VH3-11 and also appeared at the 5' end of BAC3. For the modification of YAC6, we constructed pBeloHYG-YAC6+BAC3(5'). In short, three fragments with overlapping ends were prepared by PCR: 1) a 'stuff' fragment containing *Saccharomyces cerevisiae* TRP1-ARS1 side-linked to an HpaI site, wherein the 5' tail in YAC6 matches the upstream sequence of VH4-39 and the 3' tail matches the downstream sequence of VH3-23 (using long oligonucleotides 561 and 562 and pYAC-RC as templates); 2) a 10.6 kb extension fragment, which has a 5' tail matching the downstream sequence of VH3-23 as described above and a unique AscI site at its 3' end (using long oligonucleotides 570 and 412 and human genomic DNA as templates); and 3) the pBelo-CEN-HYG vector, which has a CEN4 downstream homologous to the 3' end of the 10.6 kb extension fragment and a HygR upstream tail matching the upstream sequence of VH4-39 as described above (using long oligonucleotides 414 and 566 and pBelo-CEN-HYG as templates). Subsequently, through homologous recombination associated with protoplast transformation, three PCR fragments were assembled into a small cYAC conferred with HYGR and TRP+ in *Saccharomyces cerevisiae*, and this cYAC was further transformed into a BAC pBeloHYG-YAC6+BAC3 (5'). Finally, pBeloHYG-YAC6+BAC3 (5') digested with HpaI was used to transform yeast cells carrying YAC6, and a cYAC BAC6-VH3-11 conferred with only HYGR was generated through homologous recombination. This cYAC was then introduced into *E. coli* as a BAC through transformation, as described below. The human VH gene in BAC6-VH3-11 was excised as an approximately 182 kb AsiSI (naturally present in HygR)-AscI fragment, and the VH gene in BAC3 was excised as an approximately 173 kb NotI- fragment (Figure 1 top).

[0118] The self-replicating shuttle vector called pCAU, which works efficiently in *Saccharomyces cerevisiae* and *Escherichia coli*, is constructed based on the previously published pBelo-CEN-URA (Osborn et al., *J Immunol* 2013; 190:1481-1490). In short, ARSH4 is amplified from *Saccharomyces cerevisiae* genomic DNA using primers 878 and 879 (all primer sequences are listed below), with an ApaLI site introduced at either end, followed by AsiSI and SexAI. This fragment is digested with ApaLI and SexAI and ligated to pBelo-CEN-URA digested with the same restriction enzymes to produce pCAU. This vector contains *Saccharomyces cerevisiae* CEN4, URA3, and ARSH4 within the pBeloBAC11 backbone (New England BioLabs).

[0119] The three BACs (CTD-2011A5 (BAC9), CTD-3148C6 (BAC14), and CTD-2548B8 (BAC5)) from human chromosome 14 were purchased from Invitrogen / Thermo Fisher. The human genome region covering IgHV3-74 to IgHV1-58 in BAC9 was isolated as a 185kb NotI fragment. BAC(14+5) was constructed from BAC14 and BAC5. The combined genomic region in the BAC was separated into a 210kb BsiwI fragment, from 5' to 3' including: a 90.6kb region from BAC14 containing a 4.6kb sequence overlapping 3' with the NotI fragment of BAC9, followed by an 86kb region covering IgHV5-51 to IgHV1-45, a 1.7kb synthetic region connecting BAC14 and BAC5 with the centrally located IgHV3-43, a 111.7kb region from BAC5 covering IgHV3-21 to IgHV3-13, and a 6.1kb region overlapping 5' with Anabel (a BAC carrying the human Ig constant region).

[0120] BAC (14+5, also known as 14 / 5) is constructed in three steps, all of which involve generating cyclic YAC (cYAC) through homologous recombination in yeast and converting cYAC into BAC as described above. First, synthetic DNA (ordered from ThermoFisher) of the BAC vector pCAU+GAP-BAC14,5 was generated by assembling the following three overlapping fragments in yeast: a 116 bp sequence overlapping the 5' and 3' ends of the desired region in BAC14 with a unique RsrII site at the center; 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 in BAC5 with a unique PmeI site at the center; and a 38 bp sequence overlapping the 5' end of Anabel; a 6.1 kb PCR fragment corresponding to the 5' end of Anabel was synthesized using primers 383 and 384; and the pCAU vector was 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 yeast strain AB1380. The resulting BAC (approximately 128kb in length) incorporated the desired region of BAC5 into the BAC vector via homologous recombination mediated by the exposed homologous ends of BAC5 in the PmeI-linearized vector. Thirdly, the BAC carrying BAC5 from the second step was linearized with RsrII to expose the homologous ends to the desired region in BAC14 and co-transformed with a 114kb SnaBI fragment isolated from BAC14 to produce BAC(14+5).

[0121] For the assembly of the C region overlapping with VH, the human VH6-1-D-JH region must be ligated to the rat genome sequence immediately downstream of the last JH (followed by rat Cs) to obtain cYAC / BAC. To achieve this, five overlap-restricted PCR fragments were prepared: a 6.1 kb fragment of human VH6-1 5' (using oligonucleotides 383 and 384 and human genomic DNA as templates); an approximately 78 kb PvuI-PacI fragment containing the human VH6-1-D-JH region excised from BAC1 (RP11645E6); an 8.7 kb fragment ligating human JH6 to the rat genome sequence immediately downstream of the last JH and containing a portion of the rat μ coding sequence (using oligonucleotides 488 and 346 and rat genomic DNA as templates); and an approximately 52 kb NotI-PmeI fragment containing a portion of the rat μ coding sequence from BAC1 (RP11645E6). The actual rat μ, δ, and γ2c regions excised from M5 (CH230-408M5); and the pBelo-CEN-URA vector, which has URA3 with a homologous tail linked downstream to the 3' end of the matched rat γ2c region and CEN4 with a tail linked upstream to the 5' region of the matched human VH6-1, synthesized using long oligonucleotides 385 and 550 and pBelo-CEN-URA as templates. Correct assembly via homologous recombination in Saccharomyces cerevisiae was analyzed by PCR, and purified cYAC from the correctly cloned strain was converted to BAC in E. coli.

[0122] For the assembly of Annabel, the above cYAC / BAC was used, which contained human VH6-1-D-JH, followed by portions of the real rat μ, δ and γ2c regions, and PCR fragments. The five overlapping fragments include a 6.1 kb fragment at the 5' end of human VH6-1 as described above; an approximately 83 kb SpeI fragment containing the downstream rat genome sequence of human VH6-1-D-JH followed by the last JH and containing a portion of rat Cμ; a 5.2 kb fragment linking the 3' end of rat μ to the 5' end of rat γ1, which was synthesized using oligonucleotides 490 and 534 and rat genomic DNA as templates; an approximately 118 kb NotI-SgrAI fragment containing real rat γ1, γ2b, ε, α, and 3'E IgH enhancer regions excised from BAC I8 (CH230-162I08); and the pBelo-CEN-URA vector having URA3, as described above, with a homologous tail linked downstream to the 3' end of the matched rat 3'E and a tail linked upstream to the 5' end of the matched human VH6-1. There is a 10.3 kb overlap between BAC3 and the human VH6-1 region in Annabel. The human VH6-1-D-JH, which subsequently contains the rat CH region, can be cleaved together with the Saccharomyces cerevisiae URA3 in Annabel into a single NotI fragment of approximately 183 kb (see Figure 1).

[0123] Extensive authenticity checks were performed on BAC6-VH3-11, BAC3, BAC9, BAC(14+5), and Annabel using restriction analysis and partial sequencing.

[0124] b) IgL locus

[0125] Human Igλ locus at approximately 410 kb YAC was obtained by recombinant assembly of VλYAC with three Cλ-containing closomes (Popov et al., Gene 177, 195-201 (1996)). The rearrangement and expression were validated in transgenic mice from ES cells containing a single copy of the fully human IgλYAC (Popov et al., The Journal of Experimental Medicine 189, 1611-1620 (1999)). This IgλYAC was shortened by generating a circular YAC of approximately 100 kb with the Vλ3-27 5' region removed. The vector pYAC-RC was digested with ClaI and BspEI to remove URA3 and ligated to a ClaI / NgoMIV fragment from pAP 599 containing HYG. PCR was performed on regions containing yeast centromeres and hygromycin marker genes from the novel vector (pYAC-RC-HYG) using primers homologous to the 5' region of Vλ3-27 (primer 276) and primers within the ADE2 marker gene in the YAC arm (primer 275). The PCR fragment (3.8 kb) was integrated into IgλYAC using a high-efficiency lithium acetate transformation method (Gietz and Woods, Methods in Microbiology 26, 53-66 (1998)) and selection on YPD plates containing hygromycin. DNA was prepared from the clone (Epicentre MasterPure Yeast DNA Purification Kit) and correctly ligated by PCR using the following oligonucleotides: 243+278 and Hyg-terminal R+238. A stopper was prepared (Peterson Nature protocols 2, 3009-3015 (2007)), and yeast chromosomes were removed by PFGE (0.8% agarose (PFC) (Biorad) gel [6V / cm, 60-second pulse duration for 10 hours, 10-second pulse duration for 10 hours, 8°C], leaving the circular yeast artificial chromosome locked in the agarose block (Beverly, Nucleic acids research 16, 925-939 (1988)). The block was removed and digested with NruI. Briefly, the block was pre-incubated on ice for 1 hour with excess 1X final concentration restriction enzyme buffer. The excess buffer was removed, leaving only enough buffer to cover the stopper, and restriction enzyme was added until its final concentration was 100 U / ml, and the tube was incubated at 37°C for 4-5 hours. Linearized YAC was run from the block by PFGE, excised from the gel as bands, and purified as described below.

[0126] For the human Igκ locus, three BACs (RP11-344F17, RP11-1134E24, and RP11-156D9, Invitrogen) were selected, covering a region greater than 300 kb from 5'Vκ1-17 to 3'KDE (Kawasaki et al., European journal of immunology 31, 1017-1028 (2001)). During digestion and sequence analysis, three overlapping fragments were identified: from Vκ1-17 to Vκ3-7 (approximately 14 kb overlap with 150 kb NotI), from Vκ3-7 to the 3' of Cκ (approximately 40 kb overlap with 158 kb NotI), and from Cκ to the 3' of KDE (40 kb overlap with 55 kb PacI). Overlapping regions can often facilitate integration when co-injected into oocytes (Wagner et al., Genomics 35, 405-414 (1996)).

[0127] Gel analysis and DNA purification

[0128] Purified YAC and BAC DNA were analyzed by restriction digestion and separation on a standard 0.7% agarose gel (Sambrook and 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 transition time of 2–20 seconds (last 16 h), 6 V / cm, and 10 mA. Purification allowed for direct comparison of the resulting fragments with predicted sizes obtained from sequence analysis. Changes were made by PCR and sequencing analysis.

[0129] Linear YAC, circular YAC, and BAC fragments were purified by electroelution from bands excised from conventionally run 0.8% agarose gels or from pulsed-field gel electrophoresis (PFGE) using Elutrap™ (Schleicher and Schuell) (Gu et al., Journal of biochemical and biophysical methods 24, 45-50 (1992)). DNA concentrations were typically a few ng / μl in approximately 100 μl volumes. For fragments up to approximately 200 kb, DNA was precipitated and redissolved in microinjection buffer (10 mM Tris-HCl (pH 7.5), 100 mM EDTA (pH 8), and 100 mM NaCl, but without spermine / spermine) to the desired concentration.

[0130] Cyclic YACs were purified from yeast using a Nucleobond AX silica-based anion exchange resin (Macherey-Nagel, Germany). Briefly, protoplasts were prepared and precipitated using yeast zymolyase or lyticase (Davies et al., Human antibody repertoires in transgenic mice: Manipulation and transfer of YACs.. IRL Oxford, 59-76 (1996)). Cells were then lysed with alkali, bound to an AX100 column, and low-copy-number plasmids were eluted as described in the Nucleobond method. Plamid-Safe was used. TM ATP-Dependent DNase (Epicentre Biotechnologies) hydrolyzed the contaminated yeast chromosome DNA, followed by a final cleanup step using SureClean (Bioline). Aliquots of DH10 electrocompetent cells (Invitrogen) were then transformed with circular YAC to obtain BAC clones. For microinjection, the insert DNA (150–200 kb) was separated from the BAC vector DNA (approximately 10 kb) using a sepharose 4B-CL filtration step (Yang et al., Nature Biotechnology 15, 859–865 (1997)).

[0131] Rat source and breeding

[0132] The purified DNA encoding the recombinant immunoglobulin locus was resuspended in microinjection buffer containing 10 mM spermine and 10 mM spermidine. The DNA was injected into fertilized oocytes at concentrations ranging from 0.5 to 3 ng / μl.

[0133] Plasmid DNA or mRNA encoding rat immunoglobulin gene-specific ZFN was injected into fertilized oocytes at concentrations ranging from 0.5 to 10 ng / µl.

[0134] Microinjection was performed at the Caliper Life Sciences facility. Disrelated hybrid SD / Hsd (WT) rats were housed in standard microisolation cages in an AAALAC-accredited facility under approved animal care protocols. Rats were fed and watered freely according to a 14–10 h light / dark cycle. 4–5 week old female SD / Hsd rats were injected with 20–25 IU PMSG (Sigma-Aldrich), followed by 20–25 IU hCG (Sigma-Aldrich) 48 hours later, and then bred with male disrelated hybrid SD / Hsd rats. Fertilized 1-cell stage embryos were collected for subsequent microinjection. The treated embryos were transferred to pseudopregnant female SD / Hsd rats and carried to parturition.

[0135] Multicharacteristic human Ig rats (a combination of human IgH, Igκ, and Igλ with rat JKO, κKO, and λKO) and WT rats as controls were analyzed at 10–18 weeks of age. The animals were housed at Charles River under specific pathogen-free conditions.

[0136] Introduce multiple different V at separate loci. H The procedure for these regions can be implemented by inserting these different loci into separate transgenic rats (preferably rats with defective IgH loci), as described in the examples above. These separate loci are used to generate separate transgenic rat strains, which are then crossed to obtain double transgenic rats that will possess all VH regions available for recombination. Crossing these rats to make them homozygous for both loci will double the number of VH regions available for recombination (see...). Figure 3 The karyotype has one locus integrated on chromosome 6 and another on chromosome 15. Multiple copies of the integrated locus will further increase this number.

[0137] By combining a constant region array to transfer multiple different V HThe procedure separates the introduction of different loci, allowing for the integration of disconnected multi-loci. Animals expressing antibodies from the two separately integrated loci are then bred.

[0138] The same procedure applies to light chains, in which an animal strain with the κ locus is prepared, and another strain with the λ locus is prepared. These loci are combined in animals through hybridization breeding.

[0139] PCR and RT-PCR

[0140] Transgenic rats were identified using PCR from tail or ear clip DNA using the Genomic DNA Mini Kid (Bioline) kit. For IgH PCR <1kb, the following conditions were met using GoTaq Green Master Mixture (Promega): 94°C for 2 min, 32x (94°C for 30 s, 54-67°C (see Table 1 for primers and specific annealing temperatures) for 30 s, 72°C for 1 min), 72°C for 2 min. For IgH PCR >1kb, the following conditions were met using KOD polymerase (Novagen): 95°C for 2 min, 32x (95°C for 20 s, 56-62°C (Table 1) for 20 s, 70°C for 90 s), 70°C for 2 min. For Igκ and Igλ PCR (both <1kb), the above conditions were met, except for a 50-second extension at 72°C.

[0141] RNA was extracted from blood using the RiboPure Blood Kit (Ambion) and from 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. Concentration was determined by GAPDH PCR reaction (oligonucleotides 429-430).

[0142] RT-PCR was initiated using VH leader primers (Table 2) containing rat μCH2 or rat γCH2 primers. Amplification conditions using the GoTaqGreen Master mixture were 94°C for 2 minutes, 34x (94°C for 30 seconds, 55-65°C for 30 seconds, 72°C for 50-60 seconds), 72°C for 2 minutes. PCR products of the desired size were purified by gel electrophoresis or QuickClean (Bioline) and either directly sequenced or cloned into pGemT (Promega).

[0143] Primer sequences for PCR and RT-PCR assays used to detect the integration and expression of human IgH and IgL are shown in Table 3.

[0144] Antibodies in immunized OmniRat animals were characterized using next-generation sequencing.

[0145] Six OmniRat2 animals were immunized with β-gal, and B cells were isolated from draining lymph nodes. After B cell precipitation and removal of 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. This amplified product was then prepared for next-generation sequencing (NGS), and the complete VH library for each animal was determined by NGS.

[0146] After post-processing and quality control of the raw NGS reads, V gene usage for each animal was determined by comparing each unique VH sequence with a germline V gene reference sequence. The percentage of V gene usage was calculated by dividing the number of VH sequences using a specific V gene by the total number of VH sequences in that animal.

[0147] Protein purification

[0148] IgM was purified on an anti-IgM affinity matrix (BAC BV, Netherlands, CaptureSelect No. 2890.05) as described in the protocol. Similarly, human Igκ and Igλ were purified on anti-L-chain affinity matrices (CaptureSelect anti-Igκ No. 0833 and anti-Igλ No. 0849) according to the same protocol.

[0149] For rat IgG purification (Bruggemann et al., J Immunol 142, 3145-3150 (1989)), protein A and protein G agarose gels were used (Innova, Cambridge, UK, 851-0024 and 895-0024). Serum was incubated with the resin and binding was promoted with gentle mixing in 0.1 M sodium phosphate (pH 7 for protein G and pH 8 for protein A). The mixture was packed into a Poly-prep column (Bio-Rad) and washed thoroughly with PBS (pH 7.4). The elution buffer was 0.1 M sodium citrate (pH 2.5) and the neutralization buffer was 1 M Tris-HCl (pH 9).

[0150] Electrophoresis was performed on 4-15% SDS-PAGE and stained with Coomassie Brilliant Blue. The MW standard was the HyperPage Prestained Protein Marker (BIO-33066, Bioline).

[0151] Flow cytometry analysis and FISH

[0152] Cell suspensions were washed and adjusted to 5 x 10⁵ cells / 100 μl in PBS-1% BSA-0.1% azide. Different B cell subsets were identified using mouse anti-rat IgM FITC-labeled mAbs (MARM4, Jackson Immunoresearch Laboratories) combined with anti-B cell CD45R (rat B220)-PE conjugated mAbs (His 24, BD biosciences) or anti-IgD-PE conjugated mAbs (MARD-3, Abd Serotec). Analysis was performed using FACS CantoII flow cytometry and FlowJo software (Becton Dickinson, Pont de Claix, France).

[0153] Fluorescence in situ hybridization was performed on fixed blood lymphocytes using the purified IgH and IgL C-region BACs described herein. (Meisner and Johnson, Methods 45, 133-141 (2008))

[0154] Immunity, cell fusion and affinity measurements

[0155] Immunization was performed at the tail root with 125 μg PG (in CFA), 150 μg hGHR (in CFA), 200 μg Tau / KLH (in CFA), 150 μg HEL (in CFA), and 150 μg OVA (in CFA), and the medial iliac lymph node cells were fused with mouse P3X63Ag8.653 myeloma cells 22 days later as described (Kishiro et al., Cell structure and function 20, 151-156 (1995)). For multiple immunization, 125 μg of protein PG or HEL or 100 μg of hGHR or CD14 was administered intraperitoneally in GERBU adjuvant (www.Gerbu.com) as follows: no adjuvant on days 0, 14, 28 and 41, followed by fusion of spleen cells with P3x63Ag8.653 cells after 4 days (Meisner and Johnson, Methods 45, 133-141 (2008)).

[0156] Binding kinetics were analyzed using Biacore 2000 via surface plasmon resonance, in which the antigen was directly immobilized as described (Pruzina et al., Protein engineering, design & selection: PEDS 24, 791-799 (2011)).

[0157] Detection of antigen-specific antibodies by ELISA

[0158] Analyze the B-Gal IgG and IgM antibody and antigen titers in rat serum samples using antigen-coated, anti-IgG, or IgM reporter ELISA. Coat 96-well plates overnight with B-Gal at 2–6°C, block with PBS-casein-blocking / diluent 1X, wash with ELISA wash buffer, incubate with serum, wash with ELISA wash buffer, incubate with a mixture of goat anti-rat IgG1-HRP, goat anti-rat IgG2a-HRP, and goat anti-rat IgG2b-HRP (each diluted 1 / 5,000) or goat anti-rat IgM (1 / 5,000 dilution), wash with ELISA wash buffer, incubate with TMB substrate solution for 30 minutes, and add ELISA stop solution to the wells. Measure the absorbance in the wells at 450 nm. Unless otherwise specified, incubation is performed at ambient temperature for 1.5 to 2 hours.

[0159] Determination of IgM and IgG concentrations in rat serum.

[0160] The concentrations of total rat IgG1, rat IgG2b, and rat IgM in rat serum samples were also analyzed using a double-antibody ELISA sandwich assay. The concentrations of total rat IgG1, rat IgG2b, and rat IgM were calculated using standard curves generated separately for each isotype. 96 wells were coated overnight at 2–6 °C with their respective isotype-specific capture antibodies (mouse anti-rat IgG1, mouse anti-rat IgG2b, or goat anti-rat IgM), blocked with PBS-casein-blocking / diluent 1X, washed with ELISA wash buffer, incubated with serum, washed with ELISA wash buffer, incubated with their respective detection antibodies (mouse anti-rat IgG or goat anti-rat IgM), washed with ELISA wash buffer, incubated with TMB substrate solution for 30 min, and ELISA stop solution was added to the wells. The absorbance in the plate wells was measured at 450 nm. Unless otherwise specified, incubation was performed at ambient temperature for 1.5 to 2 hours.

[0161] Table 1

[0162] PCR for detecting the integration and expression of human IgH and IgL * condition

[0163]

[0164]

[0165] KDE 313-314 66℃ Approximately 600bp cκ 307-308 64℃ Approximately 600bp V4-1 333-334 60℃ Approximately 300bp V1-5 329-330 64℃ Approximately 400bp V1-6 331-332 60℃ Approximately 300bp V3-7 309-310 66℃ Approximately 700bp V3-15 311-312 66℃ Approximately 500bp

[0166] V3-27 215-216 67℃ Approximately 400bp V3-19 213-214 67℃ Approximately 700bp V2-14 211-212 67℃ Approximately 400bp V 168-169 65℃ Approximately 500bp Jλ 162-163 67℃ Approximately 800bp cλ 170-171 67℃ Approximately 500bp enhancer 172-173 67℃ Approximately 400bp

[0167] * For DNA extraction from ear and tail clips, use the Genomic DNA Small Kit (Bioline). For PCRs of 1 kb or smaller, use the GoTaq Green Master Mixture (Promega) under the following conditions: 94°C for 2 min, 32x (94°C for 30 s, Tm-5 (below) for 30 s, 72°C for 1 min [50 s for Igκ / λ]), 72°C for 2 min. Set the annealing temperature to the lowest primer Tm-5°C (www.sigmagenosys.com / calc / DNACalc.asp). For PCRs >1 kb, use KOD polymerase (Novagen) under the following conditions: 95°C for 2 min, 32x (95°C for 20 s, Tm-5 for 20 s, 70°C for 90 s), 70°C for 2 min.

[0168] Table 2

[0169] RT-PCR for detecting the integration and expression of human IgH and IgL ** condition

[0170] VH1 leader sequence 390 65℃ ↓ VH2 leader sequence 391 65℃ ↓ VH3 leader sequence 392 65℃ ↓ VH4 leader sequence 393 60℃ ↓ VH6 leader sequence 394 65℃ ↓ VH4-39 leader sequence 761 55℃ ↓ rat μCH2 345 ↑ Approximately 1kb rat γCH2 682 ↑ Approximately 800bp

[0171] HuVK1 leader sequence 400 / 474 63℃ ↓ HuVK3 leader sequence 401 / 475 63℃ ↓ HuVK4 leader sequence 476 63℃ ↓ HuVK5 leader sequence 477 63℃ ↓ HuκC region 402 ↑ Approximately 600bp

[0172]

[0173] ** RNA was extracted from blood using the RiboPure Blood Kit (Ambion). RNA was extracted from 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. PCR settings using the GoTaq Green Master mix were as follows: 94°C for 2 minutes, 34x (94°C for 30 seconds, Tm-5 for 30 seconds, 72°C for 1 minute [50 seconds for Igκ / λ]), 72°C for 2 minutes.

[0174] Table 3

[0175] Primer sequence

[0176]

[0177]

[0178]

[0179] result

[0180] Human IgH and IgL loci

[0181] The construction of human Ig loci utilizes established techniques for assembling large DNA segments using YAC and BAC (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)). Since multiple BAC modifications in *E. coli* often result in the loss of repetitive regions (such as transition sequences and enhancers), a method was developed to assemble sequences with overlapping ends from *Saccharomyces cerevisiae* into circular YACs (cYACs), which were then converted into BACs. YACs offer advantages such as their larger size, ease of homologous alteration in yeast hosts, and sequence stability, while BACs propagated in *E. coli* provide the advantages of ease of preparation and high yield. Furthermore, detailed restriction endonuclease mapping and sequencing analysis can be achieved more effectively in BACs compared to YACs.

[0182] Sequence analysis and digestion identified the gene clusters of interest and ensured the integrity and functionality of the loci, thereby ensuring DNA rearrangement and conversion within broad regions, as shown in Figure 1. As previously mentioned, overlapping regions often facilitate ligation integration when co-injected into oocytes (Wagner et al., Genomics 35, 405-414 (1996)). Therefore, the insertion of BAC6-VH3-11 (a 182 kb AsiSI-AscI fragment) with BAC3 (a 173 kb NotI fragment) and BAC3-1N12M5I8 (Hu-rat Annabel) (a 193 kb NotI fragment) resulted in the reconstruction of a fully functional transgenic IgH locus (HC14) in the rat genome. Similarly, the injection of BAC9, BAC(14+5), and BAC3-1N12M5I8 resulted in the reconstruction of a fully functional transgenic IgH locus (HC30) in the rat genome.

[0183] Similarly, integration of the human Igκ locus was performed via homologous overlap. The human Igλ locus was completely isolated in a YAC format of approximately 300 kb and inserted entirely into the rat chromosome. Successful integration was confirmed by transcriptional analysis, which showed V(D)JC recombination from the 5' to the 3' end of the inserted locus. Multiple copies were identified by qPCR (not shown), and head-to-tail integration was likely observed. In all cases, transgenic animals with single-site integration were bred.

[0184] The breeding is homozygous

[0185] Transgenic rats were produced by microinjecting DNA into oocytes, and the breeding and immunization of these rats were comparable to those of mice. However, the ZFN technique for obtaining gene knockout was only recently reported (Geurts et al., Science 325, 433 (2009); Flisikowska et al., PloS one6, e21045 (2011)). Using ZFN KO technology via J... H Deletion silencing of the rat IgH locus has been described (Menoret et al., European journal of immunology 40, 2932-2941 (2010)), and manuscripts describing silencing of the rat IgL locus, targeting of Cκ, and deletion of the J-Cλ gene are in preparation. We obtained several founders with integrated human Ig loci and silenced endogenous Ig production; all founders were analyzed by PCR and FISH, and selection and interbreed hybridization were performed for complete transgenic locus integration (Table 4). Several founder rats carried low transgenic locus copy numbers; as determined by qPCR of Cμ and Cα products (not shown), OmniRat TM The rat C gene BAC may be fully integrated with 5 copies. FISH identification of single-position insertions in many strains confirmed that the dispersion or multiple integration of BAC mixtures is rare; thus achieving the advantage of breeding homozygosity.

[0186] Table 4: Rat strains resulting from transgenic integration, knockout, and gene use

[0187]

[0188] Rats carrying single human transgenic loci IgH, Igκ, and Igλ were successfully hybridized with KO rats carrying the Ig locus to achieve homozygosity. This resulted in a highly efficient new multi-characteristic strain (OmniRats). TM It contains 22 functional V H People with over 400kb V H -DJ HThe OmniRat region, along with approximately 116 kb of rat C region, exhibits highly similar DNA rearrangements, expression levels, class switching, and hypermutations among different originators, comparable to those in wt rats. This is likely due to the presence of several C regions and the true conformation of the 3'E (enhancer-controlled) region within the relevant rat constant region. OmniRat animals carrying the HC14 heavy chain locus were bred together with OmniRat animals carrying the HC30 locus to produce OmniRat2. OmniRat2 animals contain two heavy chain loci with 43 functional VHs.

[0189] B cell development in a knockout context

[0190] To evaluate whether the introduced human Ig loci could reconstruct normal B cell development, flow cytometry analysis was performed. Specific differentiation stages were analyzed in spleen and bone marrow lymphocytes (Osborn et al., J Immunol 2013; 190:1481-1490), which previously showed a lack of B cell development in JKO / JKO rats (Menoret et al., European Journal of Immunology 40, 2932-2941 (2010)), and no corresponding IgL expression in κKO / κKO and λKO / λKO animals (data not shown). Most notably, B cell development was fully restored in OmniRats compared to wt animals, with similar numbers of B220 (CD45R) in the bone marrow and spleen. + Lymphocytes. A large portion of CD45R + IgM expression in B cells signifies a fully reconstructed immune system. (OmniRats) TM In contrast to wt animals, spleen cell size and shape were indistinguishable, thus successful restoration was achieved in transgenic rats expressing a human idiotype with the rat C region. Furthermore, small sIgG cells were present in OmniRats. + Lymphocyte population (Osborn et al., J Immunol 2013;190:1481-1490).

[0191] Analysis of other OmniRat lymphocyte tissues showed that they were indistinguishable from the wt control; for example, a subset of T-cells was completely preserved (data not shown), which further supports the view that optimal immune function has been fully restored.

[0192] Serum Ig levels

[0193] To obtain definitive information about antibody production, we compared the quality and quantity of serum Ig from HC30 and HC14 / HC30 animals. Figure 4The results showed that the levels of IgM and IgG expressed in the serum of animals with one Ig locus (HC30) were similar to those of animals with two heavy chain loci (HC14 and HC30).

[0194] ELISA analysis of serum from immunized OmniRat animals possessing one HC locus (HC30) or two HC loci (HC14 and HC30) revealed similar titers of anti-βgal IgM and IgG in these animals. Figure 5 ).

[0195] Diverse human H- and L-chain transcripts

[0196] Comprehensive transcriptional analysis was performed using blood lymphocytes or spleen cells from transgenic rats possessing functional endogenous Ig loci. From specific human V... H RT-PCR using forward primers to Cμ or reverse primers indicates that human V H DJ H Use of [specific primers]. For the L-chain analysis group, specific human Vκ or Vλ forward primers are used in conjunction with Cκ or Cλ reverse primers.

[0197] In addition, B cells were collected from animals, RNA was prepared and reverse transcribed, and the resulting cDNA was used as a template to amplify the fully variable region (VH region) of the Ig heavy chain rearrangement locus. This amplified product was then prepared for next-generation sequencing (NGS), and the complete VH library for each animal was determined by NGS. After post-processing and quality control of the raw NGS reads, V gene usage for each animal was determined by comparing each unique VH sequence with a germline V gene reference sequence. The percentage of V gene usage was calculated as the number of VH sequences using a specific V gene divided by the total number of VH sequences in that animal. Of the total 43 human V genes introduced on the OmniRat2 transgene, we detected 33 V genes expressed at levels greater than 0.1% in the rearranged IgG transcript.

[0198] Figure 1 summarizes the results of RT-PCR VH gene expression analysis and NGS library analysis. These results show that all human VH genes considered to be functionally integrated... H Gene usage (Lefranc and Lefranc, The immunoglobulinfactsbook. FactsBook Series, Academic Press, GB, 45-68 (2001)) with D segment and all J H The diverse use of sections is combined.

[0199] The results clearly demonstrate that the addition of more variable regions provided by the two loci (HC14+HC30) results in a broader antibody library. In summary, we have demonstrated that transgenic animals with one or two Ig heavy chain loci can generate potentially any class of antigen-specific high-affinity antibodies. This technology will allow for the production of any class of fully human antibodies or fragments thereof in response to antigen challenge, thus enabling their use as therapeutic or diagnostic agents in humans. By using different loci, our technology also allows for the production of high-affinity mature antibodies from rodents, which can then be used as reagents, diagnostics, or treatments in humans.

[0200] discuss

[0201] Human and rat gene conjugations to assemble novel IgH loci have induced near-normal, highly efficient expression of antibodies with human idiotypes. Furthermore, integration of human Igκ and Igγ loci revealed a readily available chimeric Ig with full human specificity, and association of the rat C region with the human L chain had no adverse effects. The advantage of using a portion of the rat IgH locus is that the species-specific C region and enhancer control elements retain their native conformation, where essentially only diverse human V proteins are transplanted. H DJ H Furthermore, expression of antibodies possessing the rat Fc region allows for normal B cell receptor assembly and optimal activation of downstream signaling pathways necessary to elicit a highly efficient immune response. Specifically, the quality of the immune response to antigen challenge depends on the combined action of numerous receptor-associated signaling and regulatory components (see: www.biocarta.com / pathfiles / hbcrpathway.asp).

[0202] Using YAC and BAC and exchanging them between them offers advantages in both speed and the ability to check integrity when preparing constructs with large regions through overlapping homology. Several founder rats carried low transgenic locus copy numbers; as determined by qPCR of Cμ and Cα products (not shown), the rat C gene BAC in OmniRat may have been fully integrated with 5 copies. FISH identification of single-site insertions in many strains confirmed that the dispersion or multiple integration of BAC mixtures was rare; the advantage of breeding homozygosity was achieved. Little is known about whether extensive overlapping regions can accommodate the integration necessary for DNA rearrangement in order to (e.g.) maintain full function. Previously, overlapping integration has been reported, however, for much smaller regions (<100 kb) (Wagner et al., Genomics 35, 405-414 (1996); Bruggemann et al., European Journal of Immunology 21, 1323-1326 (1991)), and our results indicate that the desired integration via homology or tandem is a frequent event. This greatly simplifies transgenic technology, as it eliminates the need for the laborious integration of large YACs into stem cells and subsequent animal derivation from said stem cells (Mendez et al., Nature Genetics 15, 146-156 (1997); Davies et al., Biotechnology (NY) 11, 911-914 (1993)). Furthermore, ZFN technology, also performed via DNA injection (Geurts et al., Science 325, 433 (2009); Menoret et al., European Journal of Immunology 40, 2932-2941 (2010)), readily produces Ig KO strains and is likely to become the preferred technology for future gene disruption and replacement. The advantage of silenced endogenous Ig gene expression in OmniRats (containing human-rat IgH and human IgL loci) is that neither interfering nor unwanted rat Ig produces mixed products. Interestingly, immunization and hybridoma production in OmniRats that still produce wt Ig revealed that many products were whole human, human-rat IgH, and human IgL, but incomplete Ig KO. Here, despite the presence of numerous wt V genes, it is noteworthy that the introduced human genes are readily amplified, thus indicating efficient expression competitors. This is consistent with the observation that the expression levels of all integrated transgenes are generally good, thus favoring competition with endogenous loci.Previously, Ig KO was required in mice expressing human antibody libraries because a small amount of human product was found to be expressed during the release of wt Ig (Bruggemann et al., PNAS 86, 6709-6713 (1989); Mendez et al., Nature genetics 15, 146-156 (1997)).

[0203] Since strain-specific cis-acting sequences are required for high-level expression, even generating the full human Ig locus in Ig KO mice may be suboptimal. In mice, the downstream enhancer region of Cα plays a crucial role in class-switching recombination (Vincent-Fabert et al., Blood 116, 1895-1898 (2010)), and elements in this region may promote hypermutation (Pruzina et al., Protein engineering, design & selection: PEDS 24, 791-799 (2011)). This may explain why immune responses and the high frequency of diverse hybridomas are difficult to elicit even in mice carrying large full 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 in OmniRats favors differentiation and expression levels close to wt%, it can be concluded that the endogenous rat C region, specifically the approximately 30kb enhancer sequence of Cα3', provides the enhancement for human V expression. H The optimal locus for gene maturation is controlled by the gene. Another region, Cδ, with a 3' control motif cluster, has been removed from the chimeric C region BAC (Mundt et al., J Immunol 166, 3315-3323 (2001)) because IgD silencing or absence has not been shown to reduce immune function.37 Under normal circumstances, mature IgM… + IgD + B cells downregulate IgD upon antigen contact, thereby initiating class switching recombination (ChenImmunol Rev 237, 160-179 (2010)). Therefore, in the absence of IgD control, switching may increase, as supported by our following findings: when the Cδ region was retained in the transgenic construct, IgG transcript and serum levels were significantly reduced (data not shown).

[0204] The production of specific IgG in OmniRats was particularly encouraging, as we found that mAbs with sequence and epitope diversity comparable to those produced in wt controls could be isolated via spleen and lymph node fusions across various immunizations. The diversity of V, D, and J genes was as expected, and almost all segments were found to be efficiently utilized as predicted (Lefranc and Lefran, The immunoglobulin factsbook. FactsBook Series, Academic Press, GB, 45-68 (2001)). This contrasts sharply with mice carrying fully human transgenic loci, where clonal expansion of a small number of precursor B cells resulted in minimal diversity (Pruzina et al., Protein engineering, design & selection: PEDS24, 791-799 (2011)). Since only half the number of V genes were transplanted in humans, we anticipated limited immune responses and diversity when comparing OmniRats to wt animals. However, this is not the case, and a comparison of CDR3 diversity in over 1000 clones reveals that the connectivity differences between OmniRats and wt animals are equally extensive. A few consistent gene segment combinations are found via V H -D and / or DJ H The N sequence at the junction can be added or deleted, and further diversified through hypermutation. Therefore, it is clear that the rat C region sequence plays a crucial role in controlling human V. H DJ HThe OmniRats are highly efficient in terms of DNA rearrangement and expression. Extensive diversity was also observed in the introduced human Igκ and Igγ loci, similar to what was previously 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, OmniRats have overcome the problem of significantly reduced efficiency in generating human antibodies in mice (Lonberg Nature biotechnology 23, 1117-1125 (2005)). The OmniRats reliably and extensively diversify the rearranged H chains through class switching and hypermutation, thereby generating high-affinity antibodies in large quantities rather than occasionally. The yield and hypermutation level of transgenic IgG, used for antigen-specific mAbs, are impressive, indicating similar clonal diversity and production levels between OmniRats and wt animals. Routine production of high affinity specificity in the sub-nanomolar range was even achieved through different single immunizations, and was advantageous compared to wt animals; results have not yet been shown for transgenic mice producing human antibody libraries from full-human loci. (Mendez et al., Nature Genetics 15, 146-156 (1997))

[0205] In summary, to maximize human antibody production, it should be considered necessary to use the IgH locus, which uses human genes for antibody specificity but rodent genes for controlling differentiation and high expression. The flexibility of the L chain is an added benefit, as it allows for efficient human IgH / IgL assembly even in the presence of wt Ig. For therapeutic applications, the chimeric H chain can be readily converted into a fully human antibody via C gene substitution without compromising specificity.

[0206] All patents and patent publications mentioned in this article are incorporated herein by reference.

[0207] Those skilled in the art will conceive of certain modifications and improvements upon reading the above description. It should be understood that, for the sake of brevity and readability, all such modifications and improvements have been removed herein, but these modifications and improvements are reasonably within the scope of the following claims.

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Claims

1. A polyclonal antiserum composition comprising a library of antigen-specific artificial immunoglobulins, each antigen-specific artificial immunoglobulin comprising: Human heavy chain VH segment; and One or more constant regions; The human heavy chain VH region is encoded by the human heavy chain V gene region selected from the following group: 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, 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, The polyclonal antiserum is isolated from a transgenic animal, which contains at least one inactivated endogenous Ig locus and multiple artificial transgenic Ig heavy chain loci. These multiple artificial transgenic Ig heavy chain loci are integrated at different chromosomal sites in the animal genome. The first artificial transgenic Ig heavy chain locus comprises the human heavy chain V gene segment 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, and IgHV3-4...

3. IgHV3-21, IgHV3-20, IgHV1-18, IgHV3-15, IgHV3-13, and IgHV6-1; the second artificially transgenic Ig heavy chain locus contains the human heavy chain V gene segment IgHV4-39, IgHV3-38, IgHV3-35, IgHV4-34, IgHV3-33, IgHV4-31, and IgHV6-1. V3-30, 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, and The transgenic animal is a rodent and is immunized with an antigen bound to an antigen-specific artificial immunoglobulin.

2. The polyclonal antiserum composition of claim 1, wherein the one or more constant region segments comprise human constant region sequences.

3. The polyclonal antiserum composition according to claim 1 or 2, wherein the rodent is a rat.

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