Patent Application
20220369609
The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 23, 2020, is named 0133-0007WO1_SL.txt and is 119,740 bytes in size.
This invention relates to production of immunoglobulin molecules, including methods for generating transgenic mammals capable of producing bovine antigen-specific antibody-secreting cells for the generation of monoclonal antibodies.
In the following discussion certain articles and methods are described for background and introductory purposes. Nothing contained herein is to be construed as an “admission” of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the articles and methods referenced herein do not constitute prior art under the applicable statutory provisions.
Antibodies have emerged as important biological pharmaceuticals because they (i) exhibit exquisite binding properties that can target antigens of diverse molecular forms, (ii) are physiological molecules with desirable pharmacokinetics that make them well tolerated in treated humans and animals, and (iii) are associated with powerful immunological properties that naturally ward off infectious agents. Furthermore, established technologies exist for the rapid isolation of antibodies from laboratory animals, which can readily mount a specific antibody response against virtually any foreign substance not present natively in the body.
In their most elemental form, antibodies are composed of two identical heavy (H) chains that are each paired with an identical light (L) chain. The N-termini of both H and L chains includes a variable domain (VH and VL, respectively) that together provide the paired H-L chains with a unique antigen-binding specificity.
The exons that encode the antibody VH and VL domains do not exist in the germline DNA. Instead, each VH exon is generated by the recombination of randomly selected VH, D, and JH gene segments present in the immunoglobulin H chain locus (IGH); likewise, individual VL exons are produced by the chromosomal rearrangements of randomly selected VL and JL gene segments in a light chain locus.
The bovine genome contains two alleles that can express the H chain (one allele from each parent), two alleles that can express the kappa (κ) L chain, and two alleles that can express the lambda (λ) L chain. There are multiple VH, D, and JH gene segments at the H chain locus as well as multiple VL and JL gene segments at both the immunoglobulin κ (IGK) and immunoglobulin λ (IGL) L chain loci (Collins and Watson (2018) Immunoglobulin Light Chain Gene Rearrangements, Receptor Editing and the Development of a Self-Tolerant Antibody Repertoire. Front. Immunol. 9:2249. (doi: 10.3389/fimmu.2018.02249)).
In a typical immunoglobulin heavy chain variable region locus, VH gene segments lie upstream (5′) of JH gene segments, with D gene segments located between the VH and JH gene segments. Downstream (3′) of the JH gene segments of the IGH locus are clusters of exons that encode the constant region (CH) of the antibody. Each cluster of CH exons encodes a different antibody class (isotype). Eight classes of antibody exist in mouse: IgM, IgD, IgG3, IgG1, IgG2a (or IgG2c), IgG2b, IgE, and IgA (at the nucleic acid level, they are respectively referred to as: μ, δ, γ3, γ1, γ2a/c, γ2b, ε, and α). In bovine animals, the putative isotypes are IgM, IgD, IgG, IgE, and IgA (
The question of whether there is bovine IgD has been controversial. Early studies found no evidence for a Cδ gene or IgD protein in cows (Butler, et al. (1996) The swine Ig heavy chain locus has a single JH and no identifiable IgD. Int. Immunol. 8:1897-1094 (DOI: 10.1093/intimm/8.12.1897); Naessens J (1997) Surface Ig on B lymphocytes from cattle and sheep. Int. Immunol. 9:349-354 (DOI:10.1093/intimm/9.3.349). Subsequently, evidence for the existence of bovine Cδ genes (Zhao, et al. (2002) J. Immunol. Artiodactyl IgD: The missing link. 160:4408-4416 (DOI:10.4049/jimmunol.169.8.4408) and expression of bovine IgD (Xu, et al. (2012) PLoS ONE Expressional Analysis of Immunoglobulin D in Cattle (Bos taurus), a Large Domesticated Ungulate. 7:e44719. (doi.org/10.1371/journal.pone.0044719) have been reported, although the frequency of IgD+ cells was much lower than in mice. The current annotation of the bovine IGH locus lists five Cδ genes, IGHDD1P, IGHDD2P, IGHDD39, and IGHD, none of which are functional. There are two IGHD alleles, one is a pseudogene due to a frameshift in M1 and the other is an ORF due to a non-canonical splice donor, NGC instead of NGT. Perhaps the low frequency of IgD+ cells observed by Xu et al. is due to occasional leaky splicing from the ORF allele.
At the IGK locus of most mammalian species, a cluster of Vκ gene segments are located upstream of a small number of Jκ gene segments, with the Jκ gene segment cluster located upstream of a single Cκ gene. This organization of the κ locus can be represented as (Vκ)a . . . (Jκ)b . . . Cκ, wherein a and b, independently, are an integer of 1 or more. The bovine IGK locus is small, ˜400 Kb.
The IGL locus of most species includes a set of Vλ gene segments that are located 5′ to a variable number of J-C tandem cassettes, each made up of a Jλ gene segment and a Cλ gene segment (see schematic of the bovine IGL locus in
During B cell development, gene rearrangements occur first on one of the two homologous chromosomes that contain the H chain variable gene segments. The resultant VH exon is then spliced at the RNA level to the Cμ exons for IgM H chain expression. Subsequently, the VL-JL rearrangements occur on one L chain allele at a time until a functional L chain is produced, after which the L chain polypeptides can associate with the IgM H chain homodimers to form a fully functional B cell receptor (BCR) for antigen. In mouse and humans, as B cells continue to mature, IgD is co-expressed with IgM, with IgD being expressed at a level 10 times higher than IgM in the main B cell population.
It is widely accepted by experts in the field that in mouse and human, VL-JL rearrangements first occur at the IGK locus on both chromosomes before the IGL light chain locus on either chromosome becomes receptive for VL-JL recombination. This is supported by the observation that in mouse B cells that express κ light chains, the λ locus on both chromosomes is usually inactivated by non-productive rearrangements. This may explain the predominant κ L chain usage in mouse, which is >90% κ and <10% λ.
However, the ratio of κ to λ immunoglobulins varies between species. Many livestock and companion animals are highly λ dominant, with cattle producing approximately 91% λ chains. Arun et al. (1996) Immunohistochemical examination of light chain expression (lambda/kappa ratio) in canine, feline, equine, bovine and porcine plasma cells. Zentralblatt für Veterinärmedizin Reihe A. 43:573-576.
Upon encountering an antigen, the B cell then undergoes another round of DNA recombination at the IGH locus to remove the Cμ and Cδ exons, effectively switching the CH region to one of the downstream isotypes (this process is called class switching).
The genes encoding various bovine (e.g., domestic cattle) and mouse immunoglobulins have been characterized, although the sequence and annotation of the bovine Ig loci in the genome databases is not yet complete. For example, Sinclair, et al., describe the bovine IgG repertoire as being dominated by a single diversified VH gene segment family in J. Immunol., 159(8):3883-89 (1997); Lopez, et al., describe a single VH family and long CDR3 as being the targets for hypermutation in bovine IgG heavy chains in Immunol. Rev. 162(1):55-66 (1998); Hosseini, et al., demonstrate that duplicated copies of the bovine JH locus contribute to the Ig repertoire in Int. Immunol. 16(6):355-63 (1998); Wang, et al., describe antigen-binding sites in certain bovine antibodies as ultralong CDR3 loops, each with a stalk with a projecting knob that can be further somatically diversified by changing the number of Cys residues, as well as the patterns and connectivities of the somatically generated disulfide bonds in Cell, 153(6):1379-1393 (2013). Blankenstein and Krawinkel describe the mouse variable heavy chain region in Eur. J. Immunol. 17:1351-1357 (1987).
The generation of transgenic animals—such as mice having varied immunoglobulin loci—has allowed the use of such transgenic animals in various research and development applications, e.g., in drug discovery and basic research into various biological systems. For example, the generation of transgenic mice bearing human immunoglobulin genes is described in International Application WO 90/10077 and WO 90/04036. WO 90/04036 describes a transgenic mouse with an integrated human immunoglobulin “mini” locus. WO 90/10077 describes a vector containing the immunoglobulin dominant control region for use in generating transgenic animals.
Numerous methods have been developed for modifying the mouse endogenous immunoglobulin variable region gene locus with, e.g., human immunoglobulin sequences to create partly or fully human antibodies for drug discovery purposes. Examples of such mice include those described in, e.g., U.S. Pat. Nos. 7,145,056; 7,064,244; 7,041,871; 6,673,986; 6,596,541; 6,570,061; 6,162,963; 6,130,364; 6,091,001; 6,023,010; 5,593,598; 5,877,397; 5,874,299; 5,814,318; 5,789,650; 5,661,016; 5,612,205; and 5,591,669. However, many of the fully humanized immunoglobulin transgenic mice exhibit suboptimal antibody production because B cell development in these mice is severely hampered by inefficient V(D)J recombination, and by inability of the fully human antibodies/BCRs to function optimally with mouse signaling proteins. Other humanized immunoglobulin transgenic mice, in which the mouse coding sequences have been “swapped” with human sequences, are very time consuming and expensive to create due to the approach of replacing individual mouse exons with the syntenic human counterpart.
The use of antibodies that function as drugs is not necessarily limited to the prevention or therapy of human disease. The high population density of modern intensively managed livestock operations results in sharing of both commensal flora and pathogens, which results in rapid dissemination of infectious agents. As a result, livestock commonly require aggressive infection management strategies, which often include the use of antibiotics. The massive use of antibiotics favors the outgrowth of resistant microbes, endangering both farm animals and the humans who consume them. As such, there is increasing pressure to reduce or eliminate the use of antibiotics in domestic livestock, such as cattle.
Based on the foregoing, a clear need exists for efficient and cost-effective methods to produce bovine antibodies for the treatment of diseases in domestic cattle. More particularly, there is a need for small, rapidly breeding mammals capable of producing antigen-specific bovine immunoglobulins that can be used to generate hybridomas capable of large-scale production of bovine monoclonal antibodies.
This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written Detailed Description including those aspects illustrated in the accompanying drawings and defined in the appended claims.
Described herein is a non-bovine mammalian cell and a non-bovine mammal having a genome comprising an exogenously introduced, partly bovine immunoglobulin locus, where the introduced locus comprises coding sequences of the bovine immunoglobulin variable region gene segments and non-coding sequences based on the endogenous immunoglobulin variable region locus of the non-bovine mammalian host. Thus, the non-bovine mammalian cell or mammal is capable of expressing a chimeric B cell receptor (BCR) or antibody comprising H and L chain variable regions that are fully bovine in conjunction with the respective constant regions that are native to the non-bovine mammalian host cell or mammal. In one aspect, the transgenic cells and animals have genomes in which part or all of the endogenous immunoglobulin variable region gene locus is removed.
At a minimum, the production of chimeric bovine monoclonal antibodies in a non-bovine mammalian host requires the host to have at least one locus that expresses a chimeric bovine immunoglobulin H or L chain. In most aspects, there are one heavy chain locus and two light chain loci that, respectively, express chimeric bovine immunoglobulin H and L chains.
In some aspects, the partly bovine immunoglobulin locus comprises bovine VH coding sequences and non-coding regulatory or scaffold sequences present in the endogenous VH gene locus of the non-bovine mammalian host. In these aspects, the partly bovine immunoglobulin locus further comprises bovine D and JH gene segment coding sequences in conjunction with the non-coding regulatory or scaffold sequences present in the vicinity of the endogenous D and JH gene segments of the non-bovine mammalian host cell genome.
In one aspect, the partly bovine immunoglobulin locus comprises bovine VH, D and JH gene segment coding sequences embedded in non-coding regulatory or scaffold sequences present in an endogenous immunoglobulin heavy chain locus of the non-bovine mammalian host. In one aspect, the partly bovine immunoglobulin locus comprises bovine VH, D and JH gene segment coding sequences embedded in non-coding regulatory or scaffold sequences present in an endogenous immunoglobulin heavy chain locus of a rodent, such as a mouse. In other aspects, the partly bovine immunoglobulin locus comprises bovine VL coding sequences and non-coding regulatory or scaffold sequences present in the endogenous VL gene locus of the non-bovine mammalian host. In one aspect, the exogenously introduced, partly bovine immunoglobulin locus comprising bovine VL coding sequences further comprises bovine L-chain J gene segment coding sequences and non-coding regulatory or scaffold sequences present in the vicinity of the endogenous L-chain J gene segments of the non-bovine mammalian host cell genome. In one aspect, the partly bovine immunoglobulin locus comprises bovine Vλ and Jλ gene segment coding sequences embedded in non-coding regulatory or scaffold sequences present in an immunoglobulin light chain locus of the non-bovine mammalian host cell. In one aspect, the partly bovine immunoglobulin locus comprises bovine Vκ and Jκ gene segment coding sequences embedded in non-coding regulatory or scaffold sequences present in an immunoglobulin locus of the non-bovine mammalian host. In one aspect, the endogenous κ locus of the non-bovine mammalian host is inactivated or replaced by sequences encoding bovine λ chain, to increase production of bovine λ immunoglobulin light chain over bovine κ chain. In one aspect, the endogenous κ locus of the non-bovine mammalian host is inactivated but not replaced by sequences encoding bovine λ chain.
In certain aspects, the non-bovine mammal is a rodent, for example, a mouse or rat.
In one aspect, the engineered immunoglobulin locus includes a partly bovine immunoglobulin light chain locus that includes one or more bovine λ variable region gene segment coding sequences. In one aspect, the engineered immunoglobulin locus is a partly bovine immunoglobulin light chain locus that includes one or more bovine κ variable region gene segment coding sequences.
In one aspect, a transgenic rodent or rodent cell is provided that has a genome comprising an engineered partly bovine immunoglobulin locus. In one aspect, a transgenic rodent or rodent cell is provided that has a genome comprising an engineered partly bovine immunoglobulin light chain locus. In one aspect, the partly bovine immunoglobulin light chain locus of the rodent or rodent cell includes one or more bovine immunoglobulin variable region gene segment coding sequences. In one aspect, the partly bovine immunoglobulin light chain locus of the rodent or rodent cell includes one or more bovine immunoglobulin κ variable region gene segment coding sequences. In one aspect, the engineered immunoglobulin locus is capable of expressing immunoglobulin comprising bovine variable domains.
In one aspect, a transgenic rodent that produces more immunoglobulin comprising λ light chain than immunoglobulin comprising κ light chain is provided. In one aspect, the transgenic rodent produces at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% and up to about 100% λ light chain immunoglobulin. In one aspect, the transgenic rodent produces at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% and up to about 100% λ light chain immunoglobulin comprising a bovine variable domain. In one aspect, more λ light chain-producing cells than κ light chain-producing cells are likely to be isolated from the transgenic rodent. In one aspect, more cells producing λ light chain with a bovine variable domain are likely to be isolated from the transgenic rodent than cells producing κ light chain with a bovine variable domain.
In one aspect, a transgenic rodent cell is provided that is more likely to produce immunoglobulin comprising λ light chain than immunoglobulin comprising κ light chain. In one aspect, the rodent cell is isolated from a transgenic rodent described herein. In one aspect, the rodent cell is recombinantly produced as described herein. In one aspect, the transgenic rodent cell or its progeny, has at least about a 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% and up to about 100%, probability of producing λ light chain immunoglobulin. In one aspect, the transgenic rodent cell or its progeny, has at least about about a 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, and up to about 100%, probability of producing λ light chain immunoglobulin with a bovine variable domain.
In one aspect, the engineered partly bovine immunoglobulin locus comprises bovine Vλ gene segment coding sequences and Jλ gene segment coding sequences and non-coding sequences such as regulatory or scaffold sequences of a rodent immunoglobulin light chain variable region gene locus.
In one aspect, the engineered immunoglobulin locus comprises bovine Vλ and Jλ gene segment coding sequences embedded in rodent non-coding regulatory or scaffold sequences of a rodent immunoglobulin λ light chain variable region gene locus. In one aspect, the engineered immunoglobulin locus comprises bovine Vλ and Jλ gene segment coding sequences embedded in non-coding regulatory or scaffold sequences of the rodent immunoglobulin κ light chain variable region gene locus. In one aspect, the partly bovine immunoglobulin locus comprises one or more bovine Vλ gene segment coding sequences and Jλ gene segment coding sequences and one or more rodent immunoglobulin λ constant region coding sequences.
In one aspect, the engineered immunoglobulin variable region locus comprises one or more bovine Vλ gene segment coding sequences and one or more J-C units wherein each J-C unit comprises a bovine Jλ gene segment coding sequence and rodent region Cλ coding sequence. In one aspect, the engineered immunoglobulin variable region locus comprises one or more bovine Vλ gene segment coding sequences and one or more J-C units wherein each J-C unit comprises a bovine Jλ gene segment coding sequence and rodent Cλ region coding and non-coding sequences. In one aspect, the rodent Cλ region coding sequence is selected from a rodent Cλ1, Cλ2 or Cλ3 coding sequence. In one aspect, one or more bovine Vλ gene segment coding sequences are located upstream of one or more J-C units, wherein each J-C unit comprises a bovine Jλ gene segment coding sequence and a rodent Cλ gene segment coding sequence. In one aspect, one or more bovine Vλ gene segment coding sequences are located upstream of one or more J-C units, wherein each J-C unit comprises a bovine Jλ gene segment coding sequence and a rodent Cλ gene segment coding sequence and rodent Cλ non-coding sequences. In one aspect, the J-C units comprise bovine Jλ gene segment coding sequences and rodent Cλ region coding sequences embedded in non-coding regulatory or scaffold sequences of a rodent immunoglobulin κ light chain locus.
In one aspect, a transgenic rodent or rodent cell is provided with an engineered immunoglobulin locus that includes a rodent immunoglobulin κ locus in which one or more rodent Vκ gene segment coding sequences and one or more rodent Jκ gene segment coding sequences have been deleted and replaced with one or more bovine Vλ gene segment coding sequences and one or more Jλ gene segment coding sequences, respectively, and in which rodent Cκ coding sequence in the locus has been replaced by rodent Cλ1, Cλ2, or Cλ3 coding sequence(s).
In one aspect, the engineered immunoglobulin locus includes one or more bovine Vλ gene segment coding sequences upstream and in the same transcriptional orientation as one or more bovine Jλ gene segment coding sequences which are upstream of one or more rodent Cλ coding sequences.
In one aspect, the engineered immunoglobulin locus includes one or more bovine Vλ gene segment coding sequences upstream and in the opposite transcriptional orientation as one or more bovine Jλ gene segment coding sequences which are upstream of one or more rodent Cλ coding sequences.
In one aspect, a transgenic rodent or rodent cell is provided in which an endogenous rodent immunoglobulin κ light chain locus is deleted, inactivated, or made nonfunctional by one or more of:
In one aspect, a transgenic rodent or rodent cell is provided in which expression of an endogenous rodent immunoglobulin λ light chain variable domain is suppressed or inactivated by one or more of:
In one aspect, a transgenic rodent or rodent cell is provided in which the engineered immunoglobulin locus expresses immunoglobulin light chains comprising a bovine variable domain and a rodent constant domain. In one aspect, a transgenic rodent or rodent cell is provided in which the engineered immunoglobulin locus expresses immunoglobulin light chains comprising a bovine λ variable domain and rodent λ constant domain. In one aspect, a transgenic rodent or rodent cell is provided in which the engineered immunoglobulin locus expresses immunoglobulin light chains comprising a bovine κ variable domain and rodent κ constant domain.
In one aspect, a transgenic rodent or rodent cell is provided in which the genome of the transgenic rodent or rodent cell comprises an engineered immunoglobulin locus comprising bovine Vκ and Jκ gene segment coding sequences. In one aspect, the bovine Vκ and Jκ gene segment coding sequences are inserted into a rodent immunoglobulin κ light chain locus. In one aspect, the bovine Vκ and Jκ gene segment coding sequences are embedded in rodent non-coding regulatory or scaffold sequences of the rodent immunoglobulin κ light chain variable region gene locus. In one aspect, the bovine Vκ and Jκ coding sequences are inserted upstream of a rodent immunoglobulin κ light chain constant region coding sequence.
In one aspect, a transgenic rodent or rodent cell is provided in which the genome of the transgenic rodent or rodent cell comprises an engineered immunoglobulin locus comprising bovine Vκ and Jκ gene segment coding sequences inserted into a rodent immunoglobulin λ light chain locus. In one aspect, the bovine Vκ and Jκ gene segment coding sequences are embedded in rodent non-coding regulatory or scaffold sequences of the rodent immunoglobulin λ light chain variable region gene locus. In one aspect, the genome of the transgenic rodent or rodent cell includes a rodent immunoglobulin κ light chain constant region coding sequence inserted downstream of the bovine Vκ and Jκ gene segment coding sequences. In one aspect, the rodent immunoglobulin κ light chain constant region is inserted upstream of an endogenous rodent Cλ coding sequence. In one aspect, the rodent immunoglobulin κ light chain constant region is inserted upstream of an endogenous rodent Cλ2 coding sequence. In one aspect, expression of an endogenous rodent immunoglobulin λ light chain variable domain is suppressed or inactivated by one or more of:
In one aspect, the engineered partly bovine immunoglobulin light chain locus comprises a rodent intronic κ enhancer (iEκ) and 3′ κ enhancer (3′Eκ) regulatory sequences.
In one aspect, the transgenic rodent or rodent cell further comprises an engineered partly bovine immunoglobulin heavy chain locus comprising bovine immunoglobulin heavy chain variable region gene segment coding sequences and non-coding regulatory and scaffold sequences of the rodent immunoglobulin heavy chain locus. In one aspect, the engineered bovine immunoglobulin heavy chain locus comprises bovine VH, D and JH gene segment coding sequences. In one aspect, each bovine/rodent chimeric VH, D or JH gene segment comprises VH, D or JH coding sequence embedded in non-coding regulatory and scaffold sequences of the rodent immunoglobulin heavy chain locus. In one aspect, the heavy chain scaffold sequences are interspersed by one or both functional ADAM6 genes.
In one aspect, the rodent regulatory and scaffold sequences comprise one or more enhancers, promoters, splice sites, introns, recombination signal sequences, or a combination thereof.
In one aspect, an endogenous rodent immunoglobulin locus of the transgenic rodent or rodent cell has been inactivated. In one aspect, an endogenous rodent immunoglobulin locus of the transgenic rodent or rodent cell has been deleted and replaced with the engineered partly bovine immunoglobulin locus.
In one aspect, the rodent is a mouse or a rat. In one aspect, the rodent cell is an embryonic stem (ES) cell or a cell of an early stage embryo. In one aspect, the rodent cell is a mouse or rat embryonic stem (ES) cell, or mouse or rat cell of an early stage embryo.
In one aspect, a cell of B lymphocyte lineage is provided that is obtained from a transgenic rodent described herein, wherein the B cell expresses or is capable of expressing a chimeric immunoglobulin heavy chain or light chain comprising a bovine variable region and a rodent immunoglobulin constant region. In one aspect, a hybridoma cell or immortalized cell line is provided that is derived from a cell of B lymphocyte lineage obtained from a transgenic rodent or rodent cell described herein.
In one aspect, antibodies or antigen binding portions thereof are provided that are produced by a cell from a transgenic rodent or rodent cell described herein.
In one aspect, a nucleic acid sequence of a VH, D, or JH, or a VL or JL gene segment coding sequence is provided that is derived from an immunoglobulin produced by a transgenic rodent or rodent cell described herein. In one aspect, a method for generating a non-bovine mammalian cell comprising a partly bovine immunoglobulin locus is provided, said method comprising: a) introducing two or more recombinase targeting sites into the genome of a non-bovine mammalian host cell and integrating at least one site upstream and at least one site downstream of a genomic region comprising endogenous immunoglobulin variable region genes wherein the endogenous immunoglobulin variable genes comprise VH, D and JH gene segments, or Vκ and Jκ gene segments, or Vλ and Jλ gene segments, or VΔ, Jλ and Cλ gene segments; and b) introducing into the non-bovine mammalian host cell via recombinase-mediated cassette exchange (RMCE) an engineered partly bovine immunoglobulin variable gene locus comprising bovine immunoglobulin variable region gene coding sequences and non-coding regulatory or scaffold sequences corresponding to the non-coding regulatory or scaffold sequences present in the endogenous immunoglobulin variable region gene locus of the non-bovine mammalian host.
In another aspect, the method further comprises deleting the genomic region flanked by the two exogenously introduced recombinase targeting sites prior to step b.
In a specific aspect, the exogenously introduced, engineered partly bovine immunoglobulin heavy chain locus is provided that comprises bovine VH gene segment coding sequences, and further comprises i) bovine D and JH gene segment coding sequences and ii) non-coding regulatory or scaffold sequences upstream of the bovine D gene segments (pre-D sequences,
In other aspects, the non-coding regulatory or scaffold sequences derive (at least partially) from other sources, e.g., they could be rationally designed artificial sequences or otherwise conserved sequences of unknown functions, sequences that are a combination of bovine and artificial or other designed sequences, or sequences from other species. As used herein, “artificial sequence” refers to a sequence of a nucleic acid not derived from a sequence naturally occurring at a genetic locus. In one aspect, the non-coding regulatory or scaffold sequences are derived from non-coding regulatory or scaffold sequences of a rodent immunoglobulin heavy chain variable region locus. In one aspect, the non-coding regulatory or scaffold sequences have at least about 75%, 80%, 85%, 90%, 95% or 100% sequence identity to non-coding regulatory or scaffold sequences of a rodent immunoglobulin heavy chain variable region locus. In another aspect, the non-coding regulatory or scaffold sequences are rodent immunoglobulin heavy chain variable region non-coding or scaffold sequences.
In yet another specific aspect of the method, the introduced engineered partly bovine immunoglobulin locus comprises bovine immunoglobulin VL gene segment coding sequences, and further comprises i) bovine L-chain J gene segment coding sequences and ii) non-coding regulatory or scaffold sequences corresponding to the non-coding regulatory or scaffold sequences present in the endogenous L chain locus of the non-bovine mammalian host cell genome. In one aspect, the engineered partly bovine immunoglobulin locus is introduced into the host cell using recombinase targeting sites that have been previously introduced upstream of the endogenous immunoglobulin VL gene locus and downstream of the endogenous J gene locus on the same chromosome.
In a more particular aspect, an exogenously introduced, engineered partly bovine immunoglobulin light chain locus is provided that comprises bovine Vλ gene segment coding sequences and bovine Jλ gene segment coding sequences. In one aspect, the partly bovine immunoglobulin light chain locus is introduced into the host cell using recombinase targeting sites that have been previously introduced upstream of the endogenous immunoglobulin Vλ gene locus and downstream of the endogenous Jλ gene locus on the same chromosome.
In one aspect, the exogenously introduced, engineered partly bovine immunoglobulin light chain locus comprises bovine Vκ gene segment coding sequences and bovine Jκ gene segment coding sequences. In one aspect, the partly bovine immunoglobulin light chain locus is introduced into the host cell using recombinase targeting sites that have been previously introduced upstream of the endogenous immunoglobulin Vκ gene locus and downstream of the endogenous Jκ gene locus on the same chromosome.
In one aspect, the non-coding regulatory or scaffold sequences are derived from non-coding regulatory or scaffold sequences of a rodent λ immunoglobulin light chain variable region locus. In one aspect, the non-coding regulatory or scaffold sequences have at least about 75%, 80%, 85%, 90%, 95% or 100% sequence identity to non-coding regulatory or scaffold sequences of a rodent immunoglobulin λ light chain variable region locus. In another aspect, the non-coding regulatory or scaffold sequences are rodent immunoglobulin λ light chain variable region non-coding or scaffold sequences.
In one aspect, the non-coding regulatory or scaffold sequences are derived from non-coding regulatory or scaffold sequences of a rodent immunoglobulin κ light chain variable region locus. In one aspect, the non-coding regulatory or scaffold sequences have at least about 75%, 80%, 85%, 90%, 95% or 100% sequence identity to non-coding regulatory or scaffold sequences of a rodent immunoglobulin κ light chain variable region locus. In another aspect, the non-coding regulatory or scaffold sequences are rodent immunoglobulin κ light chain variable region non-coding or scaffold sequences.
In one aspect, the engineered partly bovine immunoglobulin locus is synthesized as a single nucleic acid, and introduced into the non-bovine mammalian host cell as a single nucleic acid region. In one aspect, the engineered partly bovine immunoglobulin locus is synthesized in two or more contiguous segments, and introduced to the mammalian host cell as discrete segments. In another aspect, the engineered partly bovine immunoglobulin locus is produced using recombinant methods and isolated prior to being introduced into the non-bovine mammalian host cell.
In another aspect, methods for generating a non-bovine mammalian cell comprising an engineered partly bovine immunoglobulin locus are provided, said method comprising: a) introducing into the genome of a non-bovine mammalian host cell two or more sequence-specific recombination sites that are not capable of recombining with one another, wherein at least one recombination site is introduced upstream of an endogenous immunoglobulin variable region gene locus while at least one recombination site is introduced downstream of the endogenous immunoglobulin variable region gene locus on the same chromosome; b) providing a vector comprising an engineered partly bovine immunoglobulin locus having i) bovine immunoglobulin variable region gene coding sequences and ii) non-coding regulatory or scaffold sequences based on an endogenous immunoglobulin variable region gene locus of the host cell genome, wherein the partly bovine immunoglobulin locus is flanked by the same two sequence-specific recombination sites that flank the endogenous immunoglobulin variable region gene locus of the host cell of a); c) introducing into the host cell the vector of step b) and a site specific recombinase capable of recognizing the two recombinase sites; d) allowing a recombination event to occur between the genome of the cell of a) and the engineered partly bovine immunoglobulin locus, resulting in a replacement of the endogenous immunoglobulin variable region gene locus with the engineered partly bovine immunoglobulin variable region gene locus.
In one aspect, the partly bovine immunoglobulin locus comprises VH immunoglobulin gene segment coding sequences, and further comprises i) bovine D and JH gene segment coding sequences, ii) non-coding regulatory or scaffold sequences surrounding the codons of individual VH, D, and JH gene segments present endogenously in the genome of the non-bovine mammalian host, and iii) pre-D sequences based on the endogenous genome of the non-bovine mammalian host cell. The recombinase targeting sites are introduced upstream of the endogenous immunoglobulin VH gene locus and downstream of the endogenous D and JH gene locus.
In one aspect, there is provided a transgenic rodent with a genome deleted of a rodent endogenous immunoglobulin variable gene locus and in which the deleted rodent endogenous immunoglobulin variable gene locus has been replaced with an engineered partly bovine immunoglobulin locus comprising bovine immunoglobulin variable gene coding sequences and non-coding regulatory or scaffold sequences based on the rodent endogenous immunoglobulin variable gene locus, wherein the engineered partly bovine immunoglobulin locus of the transgenic rodent is functional and expresses immunoglobulin chains with bovine variable domains and rodent constant domains. In some aspects, the engineered partly bovine immunoglobulin locus comprises bovine VH, D, and JH coding sequences, and in some aspects, the engineered partly bovine immunoglobulin locus comprises bovine VL and JL coding sequences. In one aspect, the partly bovine immunoglobulin locus comprises bovine Vλ and Jλ coding sequences. In another aspect, the partly bovine immunoglobulin locus comprises bovine Vκ and Jκ coding sequences.
Some aspects provide a cell of B lymphocyte lineage from the transgenic rodent, a part or whole immunoglobulin molecule comprising bovine variable domains and rodent constant domains obtained from the cell of B lymphocyte lineage, a hybridoma cell derived from the cell of B lymphocyte lineage, a part or whole immunoglobulin molecule comprising bovine variable domains and rodent constant domains obtained from the hybridoma cell, a part or whole immunoglobulin molecule comprising bovine variable domains derived from an immunoglobulin molecule obtained from the hybridoma cell, an immortalized cell derived from the cell of B lymphocyte lineage, a part or whole immunoglobulin molecule comprising bovine variable domains and rodent constant domains obtained from the immortalized cell, a part or whole immunoglobulin molecule comprising bovine variable domains derived from an immunoglobulin molecule obtained from the immortalized cell.
In one aspect, a transgenic rodent is provided, wherein the engineered partly bovine immunoglobulin locus comprises bovine VL and JL coding sequences, and a transgenic rodent, wherein the engineered partly bovine immunoglobulin loci comprise bovine VH, D, and JH or VL and JL coding sequences. In some aspects, the rodent is a mouse. In some aspects, the non-coding regulatory sequences comprise the following sequences of endogenous host origin: promoters preceding each V gene segment coding sequence, introns, splice sites, and recombination signal sequences for V(D)J recombination; in other aspects, the engineered partly bovine immunoglobulin locus further comprises one or more of the following sequences of endogenous host origin: ADAM6A or ADAM6B gene, a Pax-5-Activated Intergenic Repeat (PAIR) elements, or CTCF binding sites from a heavy chain intergenic control region 1.
In one aspect, the non-bovine mammalian cell for use in each of the above methods is a mammalian cell, for example, a mammalian embryonic stem (ES) cell. In one aspect, the mammalian cell is a cell of an early stage embryo. In one aspect, the non-bovine mammalian cell is a rodent cell. In one aspect, the non-bovine mammalian cell is a mouse cell.
Once the cells have been subjected to the replacement of the endogenous immunoglobulin variable region gene locus by the introduced partly bovine immunoglobulin variable region gene locus, the cells can be selected and isolated. In one aspect, the cells are non-bovine mammalian ES cells, for example, rodent ES cells, and at least one isolated ES cell clone is then utilized to create a transgenic non-bovine mammal expressing the engineered partly bovine immunoglobulin variable region gene locus.
In one aspect, a method for generating the transgenic rodent is provided, said method comprising: a) integrating at least one target site for a site-specific recombinase in a rodent cell's genome upstream of an endogenous immunoglobulin variable gene locus and at least one target site for a site-specific recombinase downstream of the endogenous immunoglobulin variable gene locus, wherein the endogenous immunoglobulin variable locus comprises VH, D and JH gene segments, or Vκ and Jκ gene segments, or Vλ and Jλ gene segments, or Vλ, Jλ and Cλ gene segments; b) providing a vector comprising an engineered partly bovine immunoglobulin locus, said engineered partly bovine immunoglobulin locus comprising chimeric bovine immunoglobulin gene segments, wherein each of the partly bovine immunoglobulin gene segment comprises bovine immunoglobulin variable gene coding sequences and rodent non-coding regulatory or scaffold sequences, with the partly bovine immunoglobulin variable gene locus being flanked by target sites for a site-specific recombinase wherein the target sites are capable of recombining with the target sites introduced into the rodent cell; c) introducing into the cell the vector and a site-specific recombinase capable of recognizing the target sites; d) allowing a recombination event to occur between the genome of the cell and the engineered partly bovine immunoglobulin locus resulting in a replacement of the endogenous immunoglobulin variable gene locus with the engineered partly bovine immunoglobulin locus; e) selecting a cell that comprises the engineered partly bovine immunoglobulin variable locus generated in step d); and utilizing the cell to create a transgenic rodent comprising partly bovine the engineered partly bovine immunoglobulin variable locus. In some aspects, the cell is a rodent embryonic stem (ES) cell, and in some aspects the cell is a mouse embryonic stem (ES) cell. Some aspects of this method further comprise after, after step a) and before step b), a step of deleting the endogenous immunoglobulin variable gene locus by introduction of a recombinase that recognizes a first set of target sites, wherein the deleting step leaves in place at least one set of target sites that are not capable of recombining with one another in the rodent cell's genome. In some aspects, the vector comprises bovine VH, D, and JH, coding sequences, and in some aspects the vector comprises bovine VL and JL coding sequences. In some aspects, the vector further comprises rodent promoters, introns, splice sites, and recombination signal sequences of variable region gene segments.
In another aspect, a method for generating a transgenic non-bovine mammal comprising an exogenously introduced, engineered, partly bovine immunoglobulin variable region gene locus is provided, said method comprising: a) introducing into the genome of a non-bovine mammalian host cell one or more sequence-specific recombination sites that flank an endogenous immunoglobulin variable region gene locus and are not capable of recombining with one another; b) providing a vector comprising a partly bovine immunoglobulin locus having i) bovine variable region gene coding sequences and ii) non-coding regulatory or scaffold sequences based on the endogenous host immunoglobulin variable region gene locus, wherein the coding and non-coding regulatory or scaffold sequences are flanked by the same sequence-specific recombination sites as those introduced to the genome of the host cell of a); c) introducing into the cell the vector of step b) and a site-specific recombinase capable of recognizing one set of recombinase sites; d) allowing a recombination event to occur between the genome of the cell of a) and the engineered partly bovine immunoglobulin variable region gene locus, resulting in a replacement of the endogenous immunoglobulin variable region gene locus with the partly bovine immunoglobulin locus; e) selecting a cell which comprises the partly bovine immunoglobulin locus; and f) utilizing the cell to create a transgenic animal comprising the partly bovine immunoglobulin locus.
In a specific aspect, the engineered partly bovine immunoglobulin locus comprises bovine VH, D, and JH gene segment coding sequences, and non-coding regulatory and scaffold pre-D sequences (including a fertility-enabling gene) present in the endogenous genome of the non-bovine mammalian host. In one aspect, the sequence-specific recombination sites are then introduced upstream of the endogenous immunoglobulin VH gene segments and downstream of the endogenous JH gene segments.
In one aspect, a method for generating a transgenic non-bovine animal comprising an engineered partly bovine immunoglobulin locus is provided, said method comprising: a) providing a non-bovine mammalian cell having a genome that comprises two sets of sequence-specific recombination sites that are not capable of recombining with one another, and which flank a portion of an endogenous immunoglobulin variable region gene locus of the host genome; b) deleting the portion of the endogenous immunoglobulin locus of the host genome by introduction of a recombinase that recognizes a first set of sequence-specific recombination sites, wherein such deletion in the genome retains a second set of sequence-specific recombination sites; c) providing a vector comprising an engineered partly bovine immunoglobulin variable region gene locus having bovine coding sequences and non-coding regulatory or scaffold sequences based on the endogenous immunoglobulin variable region gene locus, where the coding and non-coding regulatory or scaffold sequences are flanked by the second set of sequence-specific recombination sites; d) introducing the vector of step c) and a site-specific recombinase capable of recognizing the second set of sequence-specific recombination sites into the cell; e) allowing a recombination event to occur between the genome of the cell and the partly bovine immunoglobulin locus, resulting in a replacement of the endogenous immunoglobulin locus with the engineered partly bovine immunoglobulin variable locus; f) selecting a cell that comprises the partly bovine immunoglobulin variable region gene locus; and g) utilizing the cell to create a transgenic animal comprising the engineered partly bovine immunoglobulin variable region gene locus.
In one aspect, a method for generating a transgenic non-bovine mammal comprising an engineered partly bovine immunoglobulin locus is provided, said method comprising: a) providing a non-bovine mammalian embryonic stem ES cell having a genome that contains two sequence-specific recombination sites that are not capable of recombining with each other, and which flank the endogenous immunoglobulin variable region gene locus; b) providing a vector comprising an engineered partly bovine immunoglobulin locus comprising bovine immunoglobulin variable gene coding sequences and non-coding regulatory or scaffold sequences based on the endogenous immunoglobulin variable region gene locus, where the partly bovine immunoglobulin locus is flanked by the same two sequence-specific recombination sites that flank the endogenous immunoglobulin variable region gene locus in the ES cell; c) bringing the ES cell and the vector into contact with a site-specific recombinase capable of recognizing the two recombinase sites under appropriate conditions to promote a recombination event resulting in the replacement of the endogenous immunoglobulin variable region gene locus with the engineered partly bovine immunoglobulin variable region gene locus in the ES cell; d) selecting an ES cell that comprises the engineered partly bovine immunoglobulin locus; and e) utilizing the cell to create a transgenic animal comprising the engineered, partly bovine immunoglobulin locus.
In one aspect, the transgenic non-bovine mammal is a rodent, e.g., a mouse or a rat.
In one aspect, a non-bovine mammalian cell and a non-bovine transgenic mammal are provide that express an introduced immunoglobulin variable region gene locus having bovine variable region gene coding sequences and non-coding regulatory or scaffold sequences based on the endogenous non-bovine immunoglobulin locus of the host genome, where the non-bovine mammalian cell and transgenic animal express chimeric antibodies with fully bovine H or L chain variable domains in conjunction with their respective constant regions that are native to the non-bovine mammalian cell or animal.
Further, B cells from transgenic animals are provided that are capable of expressing partly bovine antibodies having fully bovine variable sequences, wherein such B cells are immortalized to provide a source of a monoclonal antibody specific for a particular antigen. In one aspect, a cell of B lymphocyte lineage from a transgenic animal is provided that is capable of expressing partly bovine heavy or light chain antibodies comprising a bovine variable region and a rodent constant region.
In one aspect, bovine immunoglobulin variable region gene sequences cloned from B cells are provided for use in the production or optimization of antibodies for diagnostic, preventative and therapeutic uses.
In one aspect, hybridoma cells are provided that are capable of producing partly bovine monoclonal antibodies having fully bovine immunoglobulin variable region sequences. In one aspect, a hybridoma or immortalized cell line of B lymphocyte lineage is provided.
In another aspect, antibodies or antigen binding portions thereof produced by a transgenic animal or cell described herein are provided. In another aspect, antibodies or antigen binding portions thereof comprising variable heavy chain or variable light chain sequences derived from antibodies produced by a transgenic animal or cell described herein are provided.
In one aspect, methods for determining the sequences of the H and L chain immunoglobulin variable domains from the monoclonal antibody-producing hybridomas or primary plasma cells or B cells and combining the VH and VL sequences with bovine constant regions are provided for creating a fully bovine antibody that is not immunogenic when injected into cattle.
These and other aspects, objects and features are described in more detail below.
The terms used herein are intended to have the plain and ordinary meaning as understood by those of ordinary skill in the art. The following definitions are intended to aid the reader in understanding the present invention but are not intended to vary or otherwise limit the meaning of such terms unless specifically indicated.
The term “locus” as used herein refers to a chromosomal segment or nucleic acid sequence that, respectively, is present endogenously in the genome or is (or about to be) exogenously introduced into the genome. For example, an immunoglobulin locus may include part or all of the genes (i.e., V, D, J gene segments as well as constant region genes) and intervening sequences (i.e., introns, enhancers, etc.) supporting the expression of immunoglobulin H or L chain polypeptides. Thus, a locus (e.g., immunoglobulin heavy chain variable region gene locus) may refer to a specific portion of a larger locus (e.g., a portion of the immunoglobulin H chain locus that includes the VH, D and JH gene segments). Similarly, an immunoglobulin light chain variable region gene locus may refer to a specific portion of a larger locus (e.g., a portion of the immunoglobulin L chain locus that includes the VL and JL gene segments). The term “immunoglobulin variable region gene” as used herein refers to a V, D, or J gene segment that encodes a portion of an immunoglobulin H or L chain variable domain. The term “immunoglobulin variable region gene locus” as used herein refers to part of, or the entire, chromosomal segment or nucleic acid strand containing clusters of the V, D, or J gene segments and may include the non-coding regulatory or scaffold sequences.
The term “gene segment” as used herein, refers to a nucleic acid sequence that encodes a part of the heavy chain or light chain variable domain of an immunoglobulin molecule. A gene segment can include coding and non-coding sequences. The coding sequence of a gene segment is a nucleic acid sequence that can be translated into a polypeptide, such the leader peptide and the N-terminal portion of a heavy chain or light chain variable domain. The non-coding sequences of a gene segment are sequences flanking the coding sequence, which may include the promoter, 5′ untranslated sequence, intron intervening the coding sequences of the leader peptide, recombination signal sequence(s) (RSS), and splice sites. The gene segments in the immunoglobulin heavy chain (IGH) locus comprise the VH, D and JH gene segments (also referred to as IGHV, IGHD and IGHJ, respectively). The light chain variable region gene segments in the immunoglobulin κ and λ light loci can be referred to as VL and JL gene segments. In the κ light chain, the VL and JL gene segments can be referred to as Vκ and Jκ gene segments or IGKV and IGKJ. Similarly, in the λ light chain, the VL and JL gene segments can be referred to as Vλ and Jλ gene segments or IGLV and IGLJ.
The heavy chain constant region can be referred to as CH or IGHC. The CH region exons that encode IgM, IgD, IgG1-4, IgE, or IgA can be referred to as, respectively, Cμ, Cδ, Cγ1-4, Cε or Cα. Similarly, the immunoglobulin κ or λ constant region can be referred to as Cκ or Cλ as well as IGKC or IGLC, respectively.
“Partly bovine” as used herein refers to a strand of nucleic acids, or their expressed protein and RNA products, comprising sequences corresponding to the sequences found in a given locus of both a bovine and a non-bovine mammalian host. “Partly bovine” as used herein also refers to an animal comprising nucleic acid sequences from both a bovine and a non-bovine mammal, for example, a rodent. In one aspect, the partly bovine nucleic acids have coding sequences of bovine immunoglobulin H or L chain variable region gene segments and sequences based on the non-coding regulatory or scaffold sequences of the endogenous immunoglobulin locus of the non-bovine mammal.
The term “based on” when used with reference to endogenous non-coding regulatory or scaffold sequences from a non-bovine mammalian host cell genome refers to the non-coding regulatory or scaffold sequences that are present in the corresponding endogenous locus of the mammalian host cell genome. In one aspect, the term “based on” means that the non-coding regulatory or scaffold sequences that are present in the partly bovine immunoglobulin locus share a relatively high degree of homology with the non-coding regulatory or scaffold sequences of the endogenous locus of the host mammal. In one aspect, the non-coding sequences in the partly bovine immunoglobulin locus share at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% homology with the corresponding non-coding sequences found in the endogenous locus of the host mammal. In one aspect, the non-coding sequences in the partly bovine immunoglobulin locus are retained from an immunoglobulin locus of the host mammal. In one aspect, the bovine coding sequences are embedded in the non-regulatory or scaffold sequences of the immunoglobulin locus of the host mammal. In one aspect, the host mammal is a rodent, such as a rat or mouse.
“Non-coding regulatory sequences” refer to sequences that are known to be essential for (i) V(D)J recombination, (ii) isotype switching, (iii) proper expression of the full-length immunoglobulin H or L chains following V(D)J recombination, and (iv) alternate splicing to generate, e.g., membrane and secreted forms of the immunoglobulin H chain. “Non-coding regulatory sequences” may further include the following sequences of endogenous origin: enhancer and locus control elements such as the CTCF and PAIR sequences (Proudhon, et al., Adv. Immunol. 128:123-182 (2015)); promoters preceding each endogenous V gene segment; splice sites; introns; recombination signal sequences flanking each V, D, or J gene segment. In one aspect, the “non-coding regulatory sequences” of the partly bovine immunoglobulin locus share at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% and up to 100% homology with the corresponding non-coding sequences found in the targeted endogenous immunoglobulin locus of the non-bovine mammalian host cell.
“Scaffold sequences” refer to sequences intervening the gene segments present in the endogenous immunoglobulin locus of the host cell genome. In certain aspects, the scaffold sequences are interspersed by sequences essential for the expression of a functional non-immunoglobulin gene, for example, ADAM6A or ADAM6B. In certain aspects, the scaffold sequences are derived (at least partially) from other sources—e.g., they could be rationally designed or artificial sequences, sequences present in the immunoglobulin locus of the bovine genome, sequences present in the immunoglobulin locus of another species, or combinations thereof. It is to be understood that the phrase “non-coding regulatory or scaffold sequence” is inclusive in meaning (i.e., referring to both the non-coding regulatory sequence and the scaffold sequence existing in a given locus).
The term “homology targeting vector” refers to a nucleic acid sequence used to modify the endogenous genome of a mammalian host cell by homologous recombination; such nucleic acid sequence may comprise (i) targeting sequences with significant homologies to the corresponding endogenous sequences flanking a locus to be modified that is present in the genome of the non-bovine mammalian host, (ii) at least one sequence-specific recombination site, (iii) non-coding regulatory or scaffold sequences, and (iv) optionally one or more selectable marker genes. As such, a homology targeting vector can be used to introduce a sequence-specific recombination site into particular region of a host cell genome.
“Site-specific recombination” or “sequence-specific recombination” refers to a process of DNA rearrangement between two compatible recombination sequences (also referred to as “sequence-specific recombination sites” or “site-specific recombination sequences”) including any of the following three events: a) deletion of a preselected nucleic acid flanked by the recombination sites; b) inversion of the nucleotide sequence of a preselected nucleic acid flanked by the recombination sites, and c) reciprocal exchange of nucleic acid sequences proximate to recombination sites located on different nucleic acid strands. It is to be understood that this reciprocal exchange of nucleic acid segments can be exploited as a targeting strategy to introduce an exogenous nucleic acid sequence into the genome of a host cell.
The term “targeting sequence” refers to a sequence homologous to DNA sequences in the genome of a cell that flank or are adjacent to the region of an immunoglobulin locus to be modified. The flanking or adjacent sequence may be within the locus itself or upstream or downstream of coding sequences in the genome of the host cell. Targeting sequences are inserted into recombinant DNA vectors which are used to transfect, e.g., ES cells, such that sequences to be inserted into the host cell genome, such as the sequence of a recombination site, are flanked by the targeting sequences of the vector.
The term “site-specific targeting vector” as used herein refers to a vector comprising a nucleic acid encoding a sequence-specific recombination site, an engineered partly bovine locus, and optionally a selectable marker gene, which is used to modify an endogenous immunoglobulin locus in a host using recombinase-mediated site-specific recombination. The recombination site of the targeting vector is suitable for site-specific recombination with another corresponding recombination site that has been inserted into a genomic sequence of the host cell (e.g., via a homology targeting vector), adjacent to an immunoglobulin locus that is to be modified. Integration of an engineered partly bovine sequence into a recombination site in an immunoglobulin locus results in replacement of the endogenous locus by the exogenously introduced partly bovine region.
The term “transgene” is used herein to describe genetic material that has been or is about to be artificially inserted into the genome of a cell, and particularly a cell of a mammalian host animal. The term “transgene” as used herein refers to a partly bovine nucleic acid, e.g., a partly bovine nucleic acid in the form of an engineered expression construct or a targeting vector.
“Transgenic animal” refers to a non-bovine animal, usually a mammal, having an exogenous nucleic acid sequence present as an extrachromosomal element in a portion of its cells or stably integrated into its germ line DNA (i.e., in the genomic sequence of most or all of its cells). In one aspect, a partly bovine nucleic acid is introduced into the germ line of such transgenic animals by genetic manipulation of, for example, embryos or embryonic stem cells of the host animal according to methods well known in the art.
A “vector” includes plasmids and viruses and any DNA or RNA molecule, whether self-replicating or not, which can be used to transform or transfect a cell.
Note that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a locus” refers to one or more loci, and reference to “the method” includes reference to equivalent steps and methods known to those skilled in the art, and so forth.
As used herein, the term “or” can mean “and/or”, unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive. The terms “including,” “includes” and “included”, are not limiting.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for the purpose of describing and disclosing devices, formulations and methodologies that may be used in connection with the presently described invention.
Where a range of values is provided, it is understood that each intervening value, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either both of those included limits are also included in the invention.
The practice of the techniques described herein may employ, unless otherwise indicated, conventional techniques and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and sequencing technology, which are within the skill of those who practice in the art. Such conventional techniques include polymer array synthesis, hybridization and ligation of polynucleotides, polymerase chain reaction, and detection of hybridization using a label. Specific illustrations of suitable techniques can be had by reference to the examples herein. However, other equivalent conventional procedures can, of course, also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Green, et al., Eds. (1999), Genome Analysis: A Laboratory Manual Series (Vols. I-IV); Weiner, Gabriel, Stephens, Eds. (2007), Genetic Variation: A Laboratory Manual; Dieffenbach and Veksler, Eds. (2007), PCR Primer: A Laboratory Manual; Bowtell and Sambrook (2003), DNA Microarrays: A Molecular Cloning Manual; Mount (2004), Bioinformatics: Sequence and Genome Analysis; Sambrook and Russell (2006), Condensed Protocols from Molecular Cloning: A Laboratory Manual; and Sambrook and Russell (2002), Molecular Cloning: A Laboratory Manual (all from Cold Spring Harbor Laboratory Press); Stryer, L. (1995) Biochemistry (4th Ed.) W.H. Freeman, New York N.Y.; Gait, “Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press, London; Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3.sup.rd Ed., W. H. Freeman Pub., New York, N.Y.; and Berg et al. (2002) Biochemistry, 5.sup.th Ed., W.H. Freeman Pub., New York, N.Y., all of which are herein incorporated in their entirety by reference for all purposes.
In the following description, numerous specific details are set forth to provide a more thorough understanding of the present invention. However, it will be apparent to one of skill in the art that the present invention may be practiced without one or more of these specific details. In other instances, well-known features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the invention.
Described herein is a transgenic rodent or rodent cell having a genome comprising an engineered partly bovine immunoglobulin heavy chain or light chain locus. In one aspect, the partly bovine immunoglobulin heavy chain locus comprises one or more bovine immunoglobulin heavy chain variable region gene segments. In one aspect, the partly bovine immunoglobulin light chain locus comprises one or more bovine immunoglobulin λ light chain variable region gene segments. In one aspect, the partly bovine immunoglobulin light chain locus comprises one or more bovine immunoglobulin κ light chain variable region gene segments. In one aspect, the partly bovine immunoglobulin heavy chain or light chain locus comprise immunoglobulin region gene segments from Bos Taurus.
In one aspect, non-bovine mammalian cells are provided that comprise an exogenously introduced, engineered partly bovine nucleic acid sequence comprising coding sequences for bovine variable regions and non-coding regulatory or scaffold sequences present in the immunoglobulin locus of the mammalian host genome, e.g., mouse genomic non-coding sequences when the host mammal is a mouse. In one aspect, one or more coding sequences for bovine variable region gene segments are embedded in non-coding regulatory or scaffold sequences corresponding to those of an immunoglobulin locus in a mammalian host genome. In one aspect, the coding sequences for bovine variable region gene segments are embedded in non-coding regulatory or scaffold sequences of a rodent or mouse immunoglobulin locus.
In one aspect, the partly bovine immunoglobulin locus is synthetic and comprises bovine VH, D, or JH or VL or JL gene segment coding sequences that are under the control of regulatory elements of the endogenous host. In one aspect, the partly bovine immunoglobulin locus comprises bovine VH, D, or JH or VL or JL gene segment coding sequences embedded in non-coding regulatory or scaffold sequences corresponding to those of an immunoglobulin locus in a mammalian host genome.
Methods are also provided for generating a transgenic rodent or rodent ES cell comprising exogenously introduced, engineered partly bovine immunoglobulin loci, wherein the resultant transgenic rodent is capable of producing more immunoglobulin comprising λ light chain than immunoglobulin comprising κ light chain.
In the humoral immune system, a diverse antibody repertoire is produced by combinatorial and junctional diversity of IGH and IGL chain gene loci by a process termed V(D)J recombination. In the developing B cell, the first recombination event to occur is between one D and one JH gene segment of the heavy chain locus, and the DNA between these two gene segments is deleted. This D-JH recombination is followed by the joining of one VH gene segment from a region upstream of the newly formed DJH complex, forming a rearranged VHDJH exon. All other sequences between the recombined VH and D gene segments of the newly generated VHDJH exon are deleted from the genome of the individual B cell. This rearranged exon is ultimately expressed on the B cell surface as the variable region of the H-chain polypeptide, which is associated with an L-chain polypeptide to form the B cell receptor (BCR).
The light chain repertoire in the mouse is believed to be shaped by the order of gene rearrangements. The IGK light chain locus on both chromosomes is believed to undergo Vκ-Jκ rearrangements first before the IGL light chain locus on either chromosome becomes receptive for Vλ-Jλ recombination. If an initial κ rearrangement is unproductive, additional rounds of secondary rearrangement can proceed, in a process known as receptor editing (Collins and Watson. (2018) Immunoglobulin light chain gene rearrangements, receptor editing and the development of a self-tolerant antibody repertoire. Front. 9:2249.) A process of serial rearrangement of the κ chain locus may continue on one chromosome until all possibilities of recombination are exhausted. Recombination will then proceed on the second κ chromosome. A failure to produce a productive rearrangement on the second chromosome after multiple rounds of rearrangement will be followed by rearrangement on the λ loci (Collins and Watson (2018) Immunoglobulin light chain gene rearrangements, receptor editing and the development of a self-tolerant antibody repertoire. Front. Immunol. 9:2249.) This preferential order of light chain rearrangements is believed to give rise to a light chain repertoire in mouse that is >90% κ and <10% λ.
However, immunoglobulins in the bovine immune system are dominated by λ light chain usage, which has been estimated to be at least 90% λ to <10% κ (Arun et al. (1996) Immunohistochemical examination of light-chain expression (λ/κ ratio) in canine, feline, equine, bovine and porcine plasma cells. Zentralbl Veterinarmed A. 43(9):573-6).
The murine and bovine Ig loci are highly complex in the numbers of features they contain and in how their coding regions are diversified by V(D)J rearrangement; however, this complexity does not extend to the basic details of the structure of each variable region gene segment. The V, D and J gene segments are highly uniform in their compositions and organizations. For example, V gene segments have the following features that are arranged in essentially invariant sequential fashion in immunoglobulin loci: a short transcriptional promoter region (<600 bp in length), an exon encoding the 5′ UTR and the majority of the signal peptide for the antibody chain; an intron; an exon encoding a small part of the signal peptide of the antibody chain and the majority of the antibody variable domain, and a 3′ recombination signal sequence necessary for V(D)J rearrangement. Similarly, D gene segments have the following necessary and invariant features: a 5′ recombination signal sequence, a coding region and a 3′ recombination signal sequence. The J gene segments have the following necessary and invariant features: a 5′ recombination signal sequence, a coding region and a 3′ splice donor sequence.
Non-bovine mammalian cells are provided that comprise an exogenously introduced, engineered, partly bovine nucleic acid sequence comprising coding sequences for bovine variable regions and non-coding sequences (e.g., promoter, 5′ and 3′ recombination sequences, and splice acceptor site).
Compared with humans and mice, cattle have fewer germline heavy chain V, D and J segments. Only a single VH family, designated as BoVH1, encoded on chromosome 21, is expressed at the cDNA level.
The sequences of the bovine IGH are in Table 1.
Because of the limited number of germline heavy chain V, D and J segments, bovine immunoglobulin diversity relies on somatic hypermutation of ultralong CDR H3, rather than germline combinatorial diversity. (Stanfield et al. (2018) The unusual genetics and biochemistry of bovine immunoglobulins. Adv. Immunol. 137:135-164). Cattle of different breeds all appear to have unusually long CDR H3 s, which are encoded by long bovine D segments. One bovine D (termed D2) contains 149 nucleotides, accounting for 49 amino acid codons. This long D contributes significantly to the length of the complementarity-determining region 3 (CDR3) of the bovine immunoglobulin H chain. By comparison, the average H chain CDR3 length in mice is ˜11 amino acids and in humans is ˜15 amino acids. Bovine antibodies are of two types, conventional antibodies, albeit with comparatively longer CDR3s (˜25 amino acids), and those with ultralong CDR3s, which range from 40-67 residues, with an average of ˜58 amino acids. The exceptionally long CDR3 has a unique structure that includes a supporting stalk with a projecting knob. In conventional antibodies the CDR loops of both H and L chain variable regions can contribute to antigen binding. However, in the ultralong (UL) CDR3 antibodies the CDR H1, H2, L1, and L2 loops only form the supporting stalk, and the UL CDR H3 loop forms the knob structure, which binds antigen and can be enormously diversified by changing the number of Cys residues as well as the resulting patterns and connectivities of the somatically generated disulfide bonds. The heavy chains of all UL bovine antibodies analyzed to date are encoded by a single VH gene segment, VHBUL, a single D gene segment, D2, and a single JH gene segment, JH1.
Similar to humans and mice, two types of Ig light chains (κ and λ) are expressed in cattle, though the λ to κ ratio differs significantly among these animals. In mice, approximately 96% of light chains in the serum antibodies are the κ type, while the κ type in humans accounts for only 66% of the total population of IGL chains. In contrast, the L chain repertoire in cattle is dominated by λ chains. Cattle have 20 functional Vλ and 8 functional Vκ genes. The UL bovine antibodies use a single Vλ, Vλ1x.
The bovine κ locus (1240) is located on chromosome 11 and is approximately 280 kb in size. The κ locus contains 22 Vκ genes (1225), 3 Jκ genes (1226) and one Cκ gene (1227).
The bovine λ locus (1216) is located on chromosome 17 and is larger than the κ locus, and contains approximately 63 Vλ genes (1218), 8 JΔ genes and 9 Cλ genes arrayed in Jλ-Cλ tandem clusters (Cλ6 lacks a Jλ segment).
The mouse immunoglobulin κ locus is located on chromosome 6.
The mouse immunoglobulin λ locus is located on chromosome 16.
The partly bovine nucleic acid sequence described herein allows the transgenic animal to produce a heavy chain or light chain repertoire comprising bovine VH or VL regions, while retaining the regulatory sequences and other elements that can be found within the intervening sequences of the host genome (e.g., rodent) that help to promote efficient antibody production and antigen recognition in the host.
In one aspect, synthetic, or recombinantly produced, partly bovine nucleic acids are engineered to comprise both bovine coding sequences and non-bovine non-coding regulatory or scaffold sequences from an immunoglobulin VH, Vλ or Vκ locus, or, in some aspects, a combination thereof.
In one aspect, a transgenic rodent or rodent cell that expresses immunoglobulin with a bovine variable region can be generated by inserting one or more bovine VH gene segment coding sequences into a VH locus of a rodent heavy chain immunoglobulin locus. In another aspect, a transgenic rodent or rodent cell that expresses immunoglobulin with bovine a variable region can be generated by inserting one or more bovine VL gene segment coding sequences into a VL locus of a rodent light chain immunoglobulin locus.
The existence of two light chain loci—κ and λ—means that a variety of light chain insertion combinations are possible for generating a transgenic rodent or rodent cell that expresses immunoglobulin with bovine a variable region, including but not limited to: inserting one or more bovine Vλ or Jλ gene segment coding sequences into a rodent Vλ locus, inserting one or more bovine Vκ or Jκ gene segment coding sequences into a rodent Vκ locus, inserting one or more bovine Vλ or Jλ gene segment coding sequences into a rodent Vκ locus and inserting one or more bovine Vκ or Jκ gene segment coding sequences into a rodent Vλ locus.
The selection and development of a transgenic rodent or rodent cell that expresses partly bovine immunoglobulin is complicated by the fact that more than 90% of light chains produced by mice are κ and less than 10% are λ, whereas more than 90% of light chains produced by cattle are λ and less than 10% K.
Since mice produce mainly κ LC-containing antibodies, one reasonable method to increase production of λ LC-containing partly bovine immunoglobulin by the transgenic rodent would be to insert one or more bovine Vλ or Jλ gene segment coding sequences into a rodent κ locus. However, as shown in the Example 9 below, coupling bovine Vλ region exon with rodent Cκ region exon results in sub-optimal expression of the partly bovine immunoglobulin in vitro.
Provided herein is a transgenic rodent or rodent cell that is capable of expressing immunoglobulin comprising bovine variable domains, wherein the transgenic rodent produces more or is more likely to produce immunoglobulin comprising λ light chain than immunoglobulin comprising κ light chain. While not wishing to be bound by theory, it is believed that a transgenic rodent or rodent cell that produces more, or is more likely to produce, immunoglobulin comprising λ light chain will result in a fuller antibody repertoire for the development of therapeutics.
A transgenic rodent or rodent cell having a genome comprising an engineered partly bovine immunoglobulin light chain locus is provided herein. In one aspect, the partly bovine immunoglobulin light chain locus comprises bovine immunoglobulin λ light chain variable region gene segments. In one aspect, the engineered immunoglobulin locus is capable of expressing immunoglobulin comprising a bovine variable domain. In one aspect, the engineered immunoglobulin locus is capable of expressing immunoglobulin comprising a bovine λ variable domain. In one aspect, the engineered immunoglobulin locus is capable of expressing immunoglobulin comprising a bovine κ variable domain. In one aspect, the engineered immunoglobulin locus expresses immunoglobulin light chains comprising a bovine variable domain and a rodent constant domain. In one aspect, the engineered immunoglobulin locus expresses immunoglobulin light chains comprising a bovine λ variable domain and a rodent λ constant domain. In one aspect, the engineered immunoglobulin locus expresses immunoglobulin light chains comprising a bovine κ variable domain and a rodent κ constant domain.
In one aspect, the transgenic rodent or rodent cell produces more, or is more likely to produce, immunoglobulin comprising λ light chain than immunoglobulin comprising κ light chain. In one aspect, a transgenic rodent is provided in which more λ light chain producing cells than κ light chain producing cells are likely to be isolated from the rodent. In one aspect, a transgenic rodent is provided that produces at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% and up to about 100% immunoglobulin comprising λ light chain. In one aspect, a transgenic rodent cell, or its progeny, is provided that is more likely to produce immunoglobulin with λ light chain than immunoglobulin with κ light chain. In one aspect, the transgenic rodent cell, or its progeny, has at least about a 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% and up to about 100%, probability of producing immunoglobulin comprising λ light chain. In one aspect, a transgenic rodent or rodent cell is provided in which an endogenous rodent light chain immunoglobulin locus has been deleted and replaced with an engineered partly bovine light chain immunoglobulin locus. In one aspect, the transgenic rodent is a mouse.
In one aspect, a transgenic rodent or rodent cell is provided that has a genome comprising a recombinantly produced partly bovine immunoglobulin variable region locus. In one aspect, the partly bovine immunoglobulin variable region locus is a light chain variable region (VL) locus. In one aspect, the partly bovine immunoglobulin variable region locus comprises one or more bovine Vλ gene segment coding sequences or one or more bovine Jλ gene segment coding sequences. In one aspect, the partly bovine immunoglobulin variable region locus comprises one or more bovine Vκ gene segment coding sequences or one or more bovine Jκ gene segment coding sequences. In one aspect, the partly bovine immunoglobulin variable region locus comprises one or more rodent constant domain genes or coding sequences. In one aspect, the partly bovine immunoglobulin variable region locus comprises one or more rodent Cλ genes or coding sequences. In one aspect, the partly bovine immunoglobulin variable region locus comprises one or more rodent Cκ genes or coding sequences. In one aspect, an endogenous rodent light chain immunoglobulin locus has been inactivated. In one aspect, an endogenous rodent light chain immunoglobulin locus has been deleted and replaced with an engineered partly bovine light chain immunoglobulin locus.
In one aspect, the engineered immunoglobulin locus expresses immunoglobulin light chains comprising a bovine λ variable domain and rodent λ constant domain. In one aspect, the engineered immunoglobulin locus expresses immunoglobulin light chains comprising a bovine κ variable domain and rodent κ constant domain.
In one aspect, the engineered partly bovine immunoglobulin variable region locus comprises a VL locus comprising most or all of the Vλ gene segments coding sequences from a bovine genome. In one aspect, the engineered partly bovine immunoglobulin locus variable region comprises a VL locus comprising at least 20, 30, 40, 50 and up to 63 bovine Vλ gene segment coding sequences. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VL locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the Vλ gene segment coding sequences from a bovine genome. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VL locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the Vλ gene segment coding sequences from Bos taurus.
In one aspect, the engineered partly bovine immunoglobulin locus variable region comprises a VL locus comprising most or all of the Jλ gene segment coding sequences found in the bovine genome. In one aspect, the engineered partly bovine immunoglobulin locus variable region comprises a VL locus comprising at least 1, 2 or, 3 bovine Jλ gene segment coding sequences. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VL locus comprising at least about 50%, 75%, and up to 100% of the Jλ gene segment coding sequences found in the bovine genome. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VL locus comprising at least about 50%, 75%, and up to 100% of the Jλ gene segment coding sequences from Bos taurus.
In one aspect, the engineered partly bovine immunoglobulin locus variable region comprises a VL locus comprising most or all of the Vλ and Jλ gene segment coding sequences from the bovine genome. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VL locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the Vλ and Jλ gene segment coding sequences from the bovine genome. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VL locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the Vλ and Jλ gene segment coding sequences from the Bos taurus.
In one aspect, the engineered partly bovine immunoglobulin locus variable region comprises a VL locus comprising most or all of the Vκ gene segment coding sequences from the bovine genome. In one aspect, the engineered partly bovine immunoglobulin locus variable region comprises a VL locus comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and up to 22 bovine Vκ gene segment coding sequences. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VL locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the Vκ gene segment coding sequences from the bovine genome. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VL locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the Vκ gene segment coding sequences from Bos taurus.
In one aspect, the engineered partly bovine immunoglobulin locus variable region comprises a VL locus comprising most or all of the Jκ gene segment coding sequences found in the bovine genome. In one aspect, the engineered partly bovine immunoglobulin locus variable region comprises a VL locus comprising at least 1, 2, or 3 bovine Jκ gene segment coding sequences. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VL locus comprising at least about 50%, 75%, and up to 100% of the Jκ gene segment coding sequences found in the bovine genome. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VL locus comprising at least about 50%, 75%, and up to 100% of the Jκ gene segment coding sequences from Bos taurus.
In one aspect, the engineered partly bovine immunoglobulin locus variable region comprises a VL locus comprising most or all of the Vκ and Jκ gene segment coding sequences from the bovine genome. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VL locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the Vκ and Jκ gene segment coding sequences from the bovine genome. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VL locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the Vκ and Jκ gene segment coding sequences from Bos taurus.
In one aspect, the engineered immunoglobulin locus comprises bovine VL gene segment coding sequences and rodent non-coding regulatory or scaffold sequences from a rodent immunoglobulin light chain variable region gene locus. In one aspect, the engineered immunoglobulin locus comprises bovine Vλ or Jλ gene segment coding sequences and rodent non-coding regulatory or scaffold sequences from a rodent immunoglobulin light chain variable region gene locus. In one aspect, the rodent non-coding regulatory or scaffold sequences are from a rodent immunoglobulin λ light chain variable region gene locus. In one aspect, the rodent non-coding regulatory or scaffold sequences are from a rodent immunoglobulin κ light chain variable region locus. In one aspect, the engineered immunoglobulin locus comprises bovine Vλ and Jλ gene segment coding sequences and rodent non-coding regulatory or scaffold sequences from a rodent immunoglobulin λ light chain variable region gene locus. In one aspect, the partly bovine immunoglobulin locus comprises one or more rodent immunoglobulin λ constant region (Cλ) coding sequences. In one aspect, the partly bovine immunoglobulin locus comprises one or more bovine Vλ and Jλ gene segment coding sequences and one or more rodent immunoglobulin Cλ coding sequences. In one aspect, the engineered immunoglobulin locus comprises bovine Vλ and Jλ gene segment coding sequences and one or more rodent Cλ coding sequences embedded in rodent non-coding regulatory or scaffold sequences of a rodent immunoglobulin λ light chain variable region gene locus.
In one aspect, the engineered immunoglobulin locus comprises bovine Vλ or Jλ gene segment coding sequences and rodent non-coding regulatory or scaffold sequences from a rodent immunoglobulin κ light chain variable region gene locus. In one aspect, the engineered immunoglobulin locus comprises bovine Vλ or Jλ gene segment coding sequences embedded in rodent non-coding regulatory or scaffold sequences of a rodent immunoglobulin κ light chain variable region gene locus. In one aspect, the engineered immunoglobulin locus comprises bovine Vλ and Jλ gene segment coding sequences and one or more rodent immunoglobulin Cλ coding sequences and rodent non-coding regulatory or scaffold sequences from a rodent immunoglobulin κ light chain variable region gene locus. In one aspect, the engineered immunoglobulin locus comprises bovine Vλ and Jλ gene segment coding sequences and one or more rodent immunoglobulin Ca coding sequences embedded in rodent non-coding regulatory or scaffold sequences of a rodent immunoglobulin κ light chain variable region gene locus.
In one aspect, one or more bovine Vλ gene segment coding sequences are located upstream of one or more Jλ gene segment coding sequences, which are located upstream of one or more rodent Cλ genes. In one aspect, one or more bovine Vλ gene segment coding sequences are located upstream and in the same transcriptional orientation as one or more Jλ gene segment coding sequences, which are located upstream of one or more rodent lambda Cλ genes.
In one aspect, the engineered immunoglobulin variable region locus comprises one or more bovine Vλ gene segment coding sequences, one or more bovine Jλ gene segment coding sequences and one or more rodent Cλ genes. In one aspect, the engineered immunoglobulin variable region locus comprises one or more bovine Vλ gene segment coding sequences, one or more bovine Jλ gene segment coding sequence and one or more rodent Cλ region genes, wherein the Vλ and Jλ gene segment coding sequences and the rodent Cλ region genes are inserted into a rodent immunoglobulin κ light chain locus. In one aspect, the engineered immunoglobulin variable region locus comprises one or more bovine Vλ gene segment coding sequences, one or more bovine Jλ gene segment coding sequence and one or more rodent Cλ genes, wherein the Vλ and Jλ gene segment coding sequences and the rodent (Ca) region genes are embedded in non-coding regulatory or scaffold sequences of a rodent immunoglobulin κ light chain locus.
In one aspect, one or more bovine Vλ gene segment coding sequences are located upstream of one or more Jλ gene segment coding sequences, which are located upstream of one or more rodent Cλ genes, wherein the Vλ and Jλ gene segment coding sequences and rodent Cλ genes are inserted into a rodent immunoglobulin κ light chain locus. In one aspect, one or more bovine Vλ gene segment coding sequences are located upstream of one or more Jλ gene segment coding sequences, which are located upstream of one or more rodent Cλ genes, wherein the Vλ and Jλ gene segment coding sequences and rodent Cλ genes are embedded in non-coding regulatory or scaffold sequences of a rodent immunoglobulin κ light chain locus.
In one aspect, the rodent Cλ coding sequence is selected from a rodent Cλ1, Cλ2, or Cλ3 coding sequence.
In one aspect, a transgenic rodent or rodent cell is provided, wherein the engineered immunoglobulin locus comprises a rodent immunoglobulin κ locus in which one or more rodent Vκ gene segment coding sequences and one or more rodent Jκ gene segment coding sequences have been deleted and replaced by one or more bovine Vλ gene segment coding sequences and one or more Jλ gene segment coding sequences, respectively, and in which rodent Cκ coding sequences in the locus have been replaced by rodent Cλ1, Cλ2, or Cλ3 coding sequence.
In one aspect, the engineered immunoglobulin variable region locus comprises one or more bovine Vλ gene segment coding sequences and one or more J-C units wherein each J-C unit comprises a bovine Jλ gene segment coding sequence and a rodent Cλ gene. In one aspect, the engineered immunoglobulin variable region locus comprises one or more bovine Vλ gene segment coding sequences and one or more J-C units wherein each J-C unit comprises a bovine Jλ gene segment coding sequence and rodent Cλ region coding sequence, wherein the Vλ gene segment coding sequences and the J-C units are inserted into a rodent immunoglobulin κ light chain locus. In one aspect, the engineered immunoglobulin variable region locus comprises one or more bovine Vλ gene segment coding sequences and one or more J-C units wherein each J-C unit comprises a bovine Jλ gene segment coding sequence and rodent Cλ coding sequence, wherein the Vλ gene segment coding sequences and the J-C units are embedded in non-coding regulatory or scaffold sequences of a rodent immunoglobulin κ light chain locus.
In one aspect, one or more bovine Vλ gene segment coding sequences are located upstream and in the same transcriptional orientation as one or more J-C units, wherein each J-C unit comprises a bovine Jλ gene segment coding sequence and a rodent Cλ gene. In one aspect, one or more bovine Vλ gene segment coding sequences are located upstream and in the same transcriptional orientation as one or more J-C units, wherein each J-C unit comprises a bovine Jλ gene segment coding sequence and a rodent Cλ coding sequence. In one aspect, the engineered immunoglobulin variable region locus comprises one or more bovine Vλ gene segment coding sequences located upstream of one or more J-C units wherein each J-C unit comprises a bovine Jλ gene segment coding sequence and rodent CX coding sequence, wherein the Vλ gene segment coding sequences and the J-C units are inserted into a rodent immunoglobulin κ light chain locus. In one aspect, the engineered immunoglobulin variable region locus comprises one or more bovine Vλ gene segment coding sequences upstream and in the same transcriptional orientation as one or more J-C units wherein each J-C unit comprises a bovine Jλ gene segment coding sequence and rodent CX coding sequence, wherein the Vλ gene segment coding sequences and the J-C units are embedded in non-coding regulatory or scaffold sequences of a rodent immunoglobulin κ light chain locus. In one aspect, the rodent Cλ coding sequence is selected from a rodent Cλ1, Cλ2, or Cλ3 coding sequence.
In one aspect, the engineered immunoglobulin locus comprises bovine Vκ coding sequences and rodent non-coding regulatory or scaffold sequences from a rodent immunoglobulin light chain variable region gene locus. In one aspect, the engineered immunoglobulin locus comprises bovine Vκ or Jκ gene segment coding sequences and rodent non-coding regulatory or scaffold sequences from a rodent immunoglobulin light chain variable region gene locus. In one aspect, the rodent non-coding regulatory or scaffold sequences are from a rodent immunoglobulin λ light chain variable region gene locus. In one aspect, the rodent non-coding regulatory or scaffold sequences are from a rodent immunoglobulin κ light chain variable region locus. In one aspect, the engineered immunoglobulin locus comprises bovine Vκ and Jκ gene segment coding sequences and rodent non-coding regulatory or scaffold sequences from a rodent immunoglobulin κ light chain variable region gene locus. In one aspect, the engineered immunoglobulin locus comprises bovine Vκ and Jκ gene segment coding sequences and rodent non-coding regulatory or scaffold sequences from a rodent immunoglobulin λ light chain variable region gene locus. In one aspect, the partly bovine immunoglobulin locus comprises one rodent immunoglobulin Cκ coding sequences. In one aspect, the partly bovine immunoglobulin locus comprises one or more rodent immunoglobulin Cλ coding sequences. In one aspect, the partly bovine immunoglobulin locus comprises one or more bovine Vκ and Jκ gene segment coding sequences and one rodent immunoglobulin Cκ coding sequences. In one aspect, the engineered immunoglobulin locus comprises bovine Vκ and Jκ gene segment coding sequences and one rodent immunoglobulin Cκ coding sequences embedded in rodent non-coding regulatory or scaffold sequences of a rodent κ light chain variable region gene locus. In one aspect, the engineered immunoglobulin locus comprises bovine Vκ and Jκ gene segment coding sequences and one rodent immunoglobulin Cκ coding sequences embedded in rodent non-coding regulatory or scaffold sequences of a rodent immunoglobulin λ light chain variable region gene locus.
While not wishing to be bound by theory, it is believed that inactivating or rendering nonfunctional an endogenous rodent κ light chain locus may increase expression of λ light chain immunoglobulin from the partly bovine immunoglobulin locus. This has been shown to be the case in otherwise conventional mice in which the κ light chain locus has been inactivated in the germline (Zon, et al. (1995) Subtle differences in antibody responses and hypermutation of λ light chains in mice with a disrupted κ constant region. Eur. J. Immunol. 25:2154-2162). In one aspect, inactivating or rendering nonfunctional an endogenous rodent κ light chain locus may increase the relative amount of immunoglobulin comprising λ light chain relative to the amount of immunoglobulin comprising κ light chain produced by the transgenic rodent or rodent cell.
In one aspect, a transgenic rodent or rodent cell is provided in which an endogenous rodent immunoglobulin κ light chain locus is deleted, inactivated, or made nonfunctional. In one aspect, the endogenous rodent immunoglobulin κ light chain locus is inactivated or made nonfunctional by one or more of the following deleting or mutating all endogenous rodent Vκ gene segment coding sequences; deleting or mutating all endogenous rodent Jκ gene segment coding sequences; deleting or mutating the endogenous rodent Cκ coding sequence; deleting, mutating, or disrupting the endogenous intronic κ enhancer (iEκ) and 3′ enhancer sequence (3′Eκ); or a combination thereof.
In one aspect, a transgenic rodent or rodent cell is provided in which an endogenous rodent immunoglobulin λ light chain variable domain is deleted, inactivated, or made nonfunctional. In one aspect, the endogenous rodent immunoglobulin λ light chain variable domain is inactivated or made nonfunctional by one or more of the following: deleting or mutating all endogenous rodent Vκ gene segments; deleting or mutating all endogenous rodent Jλ gene segments; deleting or mutating all endogenous rodent Cλ coding sequences; or a combination thereof.
In one aspect, the partly bovine immunoglobulin locus comprises rodent regulatory or scaffold sequences, including, but not limited to enhancers, promoters, splice sites, introns, recombination signal sequences, and combinations thereof. In one aspect, the partly bovine immunoglobulin locus comprises rodent λ regulatory or scaffold sequences. In one aspect, the partly bovine immunoglobulin locus comprises rodent κ regulatory or scaffold sequences.
In one aspect, the partly bovine immunoglobulin locus includes a promoter to drive gene expression. In one aspect, the partly bovine immunoglobulin locus includes a κ V-region promoter. In one aspect, the partly bovine immunoglobulin locus includes a λ V-region promoter. In one aspect, the partly bovine immunoglobulin locus includes a λ V-region promoter to drive expression of one or more λ LC gene coding sequences created after Vλ to Jλ gene segment rearrangement. In one aspect, the partly bovine immunoglobulin locus includes a λ V-region promoter to drive expression of one or more κ LC gene coding sequences created after Vκ to Jκ gene segment rearrangement. In one aspect, the partly bovine immunoglobulin locus includes a κ V-region promoter to drive expression of one or more λ LC gene coding sequences created after Vλ to Jλ gene segment rearrangement. In one aspect, the partly bovine immunoglobulin locus includes a κ V-region promoter to drive expression of one or more κ LC gene coding sequences created after Vκ to Jκ gene segment rearrangement.
In one aspect, the partly bovine immunoglobulin locus includes one or more enhancers. In one aspect, the partly bovine immunoglobulin locus includes a mouse κ iEκ or 3′Eκ enhancer. In one aspect, the partly bovine immunoglobulin locus includes one or more Vλ or Jλ gene segment coding sequences and a moue κ iEκ or 3′Eκ enhancer. In one aspect, the partly bovine immunoglobulin locus includes one or more Vκ or Jκ gene segment coding sequences and a κ iEκ or 3′Eκ enhancer.
In one aspect, a transgenic rodent or rodent cell has a genome comprising a recombinantly produced partly bovine immunoglobulin heavy chain variable region (VH) locus. In one aspect, the partly bovine immunoglobulin variable region locus comprises one or more bovine VH, D or JH gene segment coding sequences. In one aspect, the partly bovine immunoglobulin heavy chain variable region locus comprises one or more rodent constant domain (CH) genes or coding sequences. In one aspect, an endogenous rodent heavy chain immunoglobulin locus has been inactivated. In one aspect, an endogenous rodent heavy chain immunoglobulin locus has been deleted and replaced with an engineered partly bovine heavy chain immunoglobulin locus.
In one aspect, the synthetic H chain DNA segment contains the ADAM6A or ADAM6B gene needed for male fertility, Pax-5-Activated Intergenic Repeats (PAIR) elements involved in IGH locus contraction and CTCF binding sites from the heavy chain intergenic control region 1, involved in regulating normal VDJ rearrangement ((Proudhon, et al., Adv. Immunol., 128:123-182 (2015)), or various combinations thereof. The locations of these endogenous non-coding regulatory and scaffold sequences in the mouse IGH locus are depicted in
In one aspect, the engineered partly bovine region to be integrated into a mammalian host cell comprises all or a substantial number of the known bovine VH gene segments. In some instances, however, it may be desirable to use a subset of such VH gene segments, and in specific instances even as few as one bovine VH coding sequence may be introduced into the cell or the animal.
In one aspect, the engineered partly bovine immunoglobulin locus variable region comprises a VH locus comprising most or all of the VH gene segment coding sequences from the bovine genome. In one aspect, the engineered partly bovine immunoglobulin locus variable region comprises a VH locus comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and up to 12 functional bovine VH gene segment coding sequences. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VH locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the VH gene segment coding sequences from the bovine genome. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VH locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the VH gene segment coding sequences from Bos taurus.
In one aspect, the engineered partly bovine immunoglobulin locus variable region comprises a VH locus comprising most or all of the VH gene segment coding sequences from the bovine genome. In one aspect, the engineered partly bovine immunoglobulin locus variable region comprises a VH locus comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and up to 12 bovine VH gene segment coding sequences. In this aspect, the VH gene segment pseudogenes are reverted to restore their functionality, e.g., by mutating an in-frame stop codon into a functional codon, using methods well known in the art. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VH locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the VH gene segment coding sequences from the bovine genome.
In one aspect, the engineered partly bovine immunoglobulin locus variable region comprises a VH locus comprising most or all of the D gene segment coding sequences found in the bovine genome. In one aspect, the engineered partly bovine immunoglobulin locus variable region comprises a VH locus comprising at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 and up to 23 bovine D gene segment coding sequences. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VH locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the D gene segment coding sequences found in the bovine genome. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VH locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the D gene segment coding sequences from Bos taurus.
In one aspect, the engineered partly bovine immunoglobulin locus variable region comprises a VH locus comprising most or all of the JH gene segment coding sequences found in the bovine genome. In one aspect, the engineered partly bovine immunoglobulin locus variable region comprises a VH locus comprising at least 1, 2, or 3 and up to 4 functional bovine JH gene segment coding sequences. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VH locus comprising at least about 50%, 75%, and up to 100% of JH gene segment coding sequences found in the bovine genome. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VH locus comprising at least about 50%, 75%, and up to 100% of JH gene segment coding sequences from Bos taurus.
In one aspect, the engineered partly bovine immunoglobulin locus variable region comprises a VH locus comprising most or all of the VH, D and JH gene segment coding sequences from the bovine genome. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VH locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the VH, D and JH gene segment coding sequences from the bovine genome. In one aspect the engineered partly bovine immunoglobulin variable region locus comprises a VH locus comprising at least about 50%, 60%, 70%, 80%, 90% and up to 100% of the VH, D and JH gene segment coding sequences from Bos taurus.
In one aspect, a transgenic rodent or rodent cell is provided that includes an engineered partly bovine immunoglobulin heavy chain locus comprising bovine immunoglobulin heavy chain variable region gene coding sequences and non-coding regulatory or scaffold sequences of the rodent immunoglobulin heavy chain locus. In one aspect, the engineered bovine immunoglobulin heavy chain locus comprises bovine VH, D or JH gene segment coding sequences. In one aspect, the engineered bovine immunoglobulin heavy chain locus comprises bovine VH, D or JH gene segment coding sequences embedded in non-coding regulatory or scaffold sequences of a rodent immunoglobulin heavy chain locus.
In one aspect, non-bovine mammals and mammalian cells comprising an engineered partly bovine immunoglobulin locus that comprises coding sequences of bovine VH, bovine D, and bovine JH genes are provided that further comprise non-coding regulatory and scaffold sequences, including pre-D sequences, based on the endogenous IGH locus of the non-bovine mammalian host. In certain aspects, the exogenously introduced, engineered partly bovine region can comprise a fully recombined V(D)J exon.
In one aspect, the transgenic non-bovine mammal is a rodent, for example, a mouse, comprising an exogenously introduced, engineered partly bovine immunoglobulin locus comprising codons for multiple bovine VH, bovine D, and bovine JH genes with intervening sequences, including a pre-D region, based on the intervening (non-coding regulatory or scaffold) sequences in the rodent. In one aspect, the transgenic rodent further comprises partly bovine IGL loci comprising coding sequences of bovine Vκ or Vλ genes and Jκ or Jλ genes, respectively, in conjunction with their intervening (non-coding regulatory or scaffold) sequences corresponding to the immunoglobulin intervening sequences present in the IGL loci of the rodent.
In an exemplary embodiment, as set forth in more detail in the Examples section, the entire endogenous VH immunoglobulin locus of the mouse genome is deleted and subsequently replaced with a partly bovine immunoglobulin locus comprising 20 bovine VH gene segments containing interspersed non-coding sequences corresponding to the non-coding sequences of the J558 VH locus of the mouse genome. The complete, exogenously introduced, engineered immunoglobulin locus further comprises bovine D and JH gene segments, as well as the mouse pre-D region. Thus, the bovine VH, D and JH codon sequences are embedded in the rodent intergenic and intronic sequences.
In one aspect, an endogenous immunoglobulin locus variable region of a non-bovine mammal, such as a rodent, for example a rat or mouse, which contains VH, D and JH or VL and JL gene segments, is deleted using site-specific recombinases and replaced with an engineered partly bovine immunoglobulin locus. In one aspect, the partly bovine immunoglobulin locus is inserted into the genome of the host animal as a single nucleic acid or cassette. Because a cassette that includes the partly bovine immunoglobulin locus is used to replace the endogenous immunoglobulin locus variable region, the bovine coding sequences can be inserted into the host genome in a single insertion step, thus providing a rapid and straightforward process for obtaining a transgenic animal.
In one aspect, the engineered partly bovine immunoglobulin locus variable region is prepared by deleting murine VH, D and JH or VL and JL coding sequences from a mouse immunoglobulin locus variable region and replacing the murine coding sequences with bovine coding sequences. In one aspect, the non-coding flanking sequences of the murine immunoglobulin locus, which include regulatory sequences and other elements, are left intact.
In one aspect, the nucleotide sequence for the engineered partly bovine immunoglobulin locus is prepared in silico and the locus is synthesized using known techniques for gene synthesis. In one aspect, coding sequences from a bovine immunoglobulin variable region locus and sequences of the host animal immunoglobulin locus are identified using a search tool such as BLAST (Basic Local Alignment Search Tool). After obtaining the genomic sequences of the host immunoglobulin locus and the coding sequences of the bovine immunoglobulin variable region locus, the host coding sequences can be replaced in silico with the bovine coding sequences using known computational approaches to locate and delete the endogenous host animal immunoglobulin coding segments and replace the coding sequences with bovine coding sequences, leaving the endogenous regulatory and flanking sequences intact.
In one aspect, a combination of homologous recombination and site-specific recombination is used to create the cells and animals described herein. In some embodiments, a homology targeting vector is first used to introduce the sequence-specific recombination sites into the mammalian host cell genome at a desired location in the endogenous immunoglobulin loci. In one aspect, in the absence of a recombinase protein, the sequence-specific recombination site inserted into the genome of a mammalian host cell by homologous recombination does not affect expression and amino acid codons of any genes in the mammalian host cell. This approach maintains the proper transcription and translation of the immunoglobulin genes which produce the desired antibody after insertion of recombination sites and, optionally, any additional sequence such as a selectable marker gene. However, in some cases it is possible to insert a recombinase site and other sequences into an immunoglobulin locus sequence such that an amino acid sequence of the antibody molecule is altered by the insertion, but the antibody still retains sufficient functionality for the desired purpose. Examples of such codon-altering homologous recombination may include the introduction of polymorphisms into the endogenous locus and changing the constant region exons so that a different isotype is expressed from the endogenous locus. In one aspect, the immunoglobulin locus includes one or more of such insertions.
In one aspect, the homology targeting vector can be utilized to replace certain sequences within the endogenous genome as well as to insert certain sequence-specific recombination sites and one or more selectable marker genes into the host cell genome. It is understood by those of ordinary skill in the art that a selectable marker gene as used herein can be exploited to weed out individual cells that have not undergone homologous recombination and cells that harbor random integration of the targeting vector.
Exemplary methodologies for homologous recombination are described in U.S. Pat. Nos. 6,689,610; 6,204,061; 5,631,153; 5,627,059; 5,487,992; and 5,464,764, each of which is incorporated by reference in its entirety.
Site/sequence-specific recombination differs from general homologous recombination in that short specific DNA sequences, which are required for recognition by a recombinase, are the only sites at which recombination occurs. Depending on the orientations of these sites on a particular DNA strand or chromosome, the specialized recombinases that recognize these specific sequences can catalyze i) DNA excision or ii) DNA inversion or rotation. Site-specific recombination can also occur between two DNA strands if these sites are not present on the same chromosome. A number of bacteriophage- and yeast-derived site-specific recombination systems, each comprising a recombinase and specific cognate sites, have been shown to work in eukaryotic cells and are therefore applicable for use in connection with the methods described herein, and these include the bacteriophage P1 Cre/lox, yeast FLP-FRT system, and the Dre system of the tyrosine family of site-specific recombinases. Such systems and methods of use are described, e.g., in U.S. Pat. Nos. 7,422,889; 7,112,715; 6,956,146; 6,774,279; 5,677,177; 5,885,836; 5,654,182; and 4,959,317, each of which is incorporated herein by reference to teach methods of using such recombinases.
Other systems of the tyrosine family of site-specific recombinases such as bacteriophage lambda integrase, HK2022 integrase, and in addition systems belonging to the separate serine family of recombinases such as bacteriophage phiC31, R4Tp901 integrases are known to work in mammalian cells using their respective recombination sites, and are also applicable for use in the methods described herein.
Since site-specific recombination can occur between two different DNA strands, site-specific recombination occurrence can be utilized as a mechanism to introduce an exogenous locus into a host cell genome by a process called recombinase-mediated cassette exchange (RMCE). The RMCE process can be exploited by the combined usage of wild-type and mutant sequence-specific recombination sites for the same recombinase protein together with negative selection. For example, a chromosomal locus to be targeted may be flanked by a wild-type LoxP site on one end and by a mutant LoxP site on the other. Likewise, an exogenous vector containing a sequence to be inserted into the host cell genome may be similarly flanked by a wild-type LoxP site on one end and by a mutant LoxP site on the other. When this exogenous vector is transfected into the host cell in the presence of Cre recombinase, Cre recombinase will catalyze RMCE between the two DNA strands, rather than the excision reaction on the same DNA strands, because the wild-type LoxP and mutant LoxP sites on each DNA strand are incompatible for recombination with each other. Thus, the LoxP site on one DNA strand will recombine with a LoxP site on the other DNA strand; similarly, the mutated LoxP site on one DNA strand will only recombine with a likewise mutated LoxP site on the other DNA strand.
In one aspect, combined variants of the sequence-specific recombination sites are used that are recognized by the same recombinase for RMCE. Examples of such sequence-specific recombination site variants include those that contain a combination of inverted repeats or those which comprise recombination sites having mutant spacer sequences. For example, two classes of variant recombinase sites are available to engineer stable Cre-loxP integrative recombination. Both exploit sequence mutations in the Cre recognition sequence, either within the 8 bp spacer region or the 13-bp inverted repeats. Spacer mutants such as lox511 (Hoess, et al., Nucleic Acids Res, 14:2287-2300 (1986)), lox5171 and lox2272 (Lee and Saito, Gene, 216:55-65 (1998)), m2, m3, m7, and m11 (Langer, et al., Nucleic Acids Res, 30:3067-3077 (2002)) recombine readily with themselves but have a markedly reduced rate of recombination with the wild-type site. This class of mutants has been exploited for DNA insertion by RMCE using non-interacting Cre-Lox recombination sites and non-interacting FLP recombination sites (Baer and Bode, Curr Opin Biotechnol, 12:473-480 (2001); Albert, et al., Plant J, 7:649-659 (1995); Seibler and Bode, Biochemistry, 36:1740-1747 (1997); Schlake and Bode, Biochemistry, 33:12746-12751 (1994)).
Inverted repeat mutants represent the second class of variant recombinase sites. For example, LoxP sites can contain altered bases in the left inverted repeat (LE mutant) or the right inverted repeat (RE mutant). An LE mutant, lox71, has 5 bp on the 5′ end of the left inverted repeat that is changed from the wild type sequence to TACCG (Araki, et al, Nucleic Acids Res, 25:868-872 (1997)). Similarly, the RE mutant, lox66, has the five 3′-most bases changed to CGGTA. Inverted repeat mutants are used for integrating plasmid inserts into chromosomal DNA with the LE mutant designated as the “target” chromosomal loxP site into which the “donor” RE mutant recombines. Post-recombination, loxP sites are located in cis, flanking the inserted segment. The mechanism of recombination is such that post-recombination one loxP site is a double mutant (containing both the LE and RE inverted repeat mutations) and the other is wild type (Lee and Sadowski, Prog Nucleic Acid Res Mol Biol, 80:1-42 (2005); Lee and Sadowski, J Mol Biol, 326:397-412 (2003)). The double mutant is sufficiently different from the wild-type site that it is unrecognized by Cre recombinase and the inserted segment is not excised.
In certain aspects, sequence-specific recombination sites can be introduced into introns, as opposed to coding nucleic acid regions or regulatory sequences. This avoids inadvertently disrupting any regulatory sequences or coding regions necessary for proper antibody expression upon insertion of sequence-specific recombination sites into the genome of the animal cell.
Introduction of the sequence-specific recombination sites may be achieved by conventional homologous recombination techniques. Such techniques are described in references such as e.g., Sambrook and Russell (2001) (Molecular cloning: a laboratory manual 3rd ed. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press) and Nagy, A. (2003). (Manipulating the mouse embryo: a laboratory manual, 3rd ed. (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press). Renault and Duchateau, Eds. (2013) (Site-directed insertion of transgenes. Topics in Current Genetics 23. Springer). Tsubouchi, H. Ed. (2011) (DNA recombination, Methods and Protocols. Humana Press).
Specific recombination into the genome can be facilitated using vectors designed for positive or negative selection as known in the art. In order to facilitate identification of cells that have undergone the replacement reaction, an appropriate genetic marker system may be employed and cells selected by, for example, use of a selection tissue culture medium. However, in order to ensure that the genome sequence is substantially free of extraneous nucleic acid sequences at or adjacent to the two end points of the replacement interval, desirably the marker system/gene can be removed following selection of the cells containing the replaced nucleic acid.
In one aspect, cells in which the replacement of all or part of the endogenous immunoglobulin locus has taken place are negatively selected against upon exposure to a toxin or drug. For example, cells that retain expression of HSV-TK can be selected against by using nucleoside analogues such as ganciclovir. In another aspect, cells comprising the deletion of the endogenous immunoglobulin locus may be positively selected for by use of a marker gene, which can optionally be removed from the cells following or as a result of the recombination event. A positive selection system that may be used is based on the use of two non-functional portions of a marker gene, such as HPRT, that are brought together through the recombination event. These two portions are brought into functional association upon a successful replacement reaction being carried out and wherein the functionally reconstituted marker gene is flanked on either side by further sequence-specific recombination sites (which are different from the sequence-specific recombination sites used for the replacement reaction), such that the marker gene can be excised from the genome, using an appropriate site-specific recombinase.
The recombinase may be provided as a purified protein, or as a protein expressed from a vector construct transiently transfected into the host cell or stably integrated into the host cell genome. Alternatively, the cell may be used first to generate a transgenic animal, which then may be crossed with an animal that expresses said recombinase.
Because the methods described herein can take advantage of two or more sets of sequence-specific recombination sites within the engineered genome, multiple rounds of RMCE can be exploited to insert the partly bovine immunoglobulin variable region genes into a non-bovine mammalian host cell genome.
Although not yet routine for the insertion of large DNA segments, CRISPR-Cas technology is another method to introduce the chimeric bovine Ig locus.
In one aspect, methods for the creation of transgenic animals, for example rodents, such as mice, are provided that comprise the introduced partly bovine immunoglobulin locus.
In one aspect, the host cell utilized for replacement of the endogenous immunoglobulin genes is an embryonic stem (ES) cell, which can then be utilized to create a transgenic mammal. In one aspect, the host cell is a cell of an early stage embryo. In one aspect, the host cell is a pronuclear stage embryo or zygote. Thus, in accordance with one aspect, the methods described herein further comprise: isolating an embryonic stem cell or a cell of an early stage embryo such as a pronuclear stage embryo or zygote, which comprises the introduced partly bovine immunoglobulin locus and using said ES cell to generate a transgenic animal that contains the replaced partly bovine immunoglobulin locus.
In one aspect, a method of producing antibodies comprising bovine variable regions is provided. In one aspect, the method includes providing a transgenic rodent or rodent cell described herein and isolating antibodies comprising bovine variable regions expressed by the transgenic rodent. In one aspect, a method of producing monoclonal antibodies comprising bovine variable regions is provided. In one aspect, the method includes providing B-cells from a transgenic rodent or cell described herein, immortalizing the B-cells; and isolating antibodies comprising bovine variable domains expressed by the immortalized B-cells.
In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise bovine HC variable domains. In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise mouse HC constant domains. These can be of any isotype, IgM, IgD, IgG1, IgG2a/c, IgG2b, IgG3, IgE or IgA.
In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise bovine HC variable domains and mouse HC constant domains. In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise bovine LC variable domains and mouse LC constant domains. In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise bovine HC variable domains and bovine LC variable domains and mouse HC constant domains and mouse LC constant domains.
In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise bovine λ LC variable domains. In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise mouse λ constant domains. In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise bovine λ LC variable domains and mouse λ constant domains. In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise bovine κ LC variable domains. In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise mouse κ constant domains. In one aspect, the antibodies expressed by the transgenic rodent or rodent cell comprise bovine κ LC variable domains and mouse κ constant domains.
In one aspect, a method of producing antibodies or antigen binding fragments comprising bovine variable regions is provided. In one aspect, the method includes providing a transgenic rodent or cell described herein and isolating antibodies comprising bovine variable regions expressed by the transgenic rodent or rodent cell. In one aspect, the variable regions of the antibody expressed by the transgenic rodent or rodent cell are sequenced. Antibodies comprising bovine variable regions obtained from the antibodies expressed by the transgenic rodent or rodent cell can be recombinantly produced using known methods.
In one aspect, a method of producing an immunoglobulin specific to an antigen of interest is provided. In one aspect, the method includes immunizing a transgenic rodent as described herein with the antigen and isolating immunoglobulin specific to the antigen expressed by the transgenic rodent or rodent cell. In one aspect, the variable domains of the antibody expressed by the rodent or rodent cell are sequenced and antibodies comprising bovine variable regions that specifically bind the antigen of interest are recombinantly produced using known methods. In one aspect, the recombinantly produced antibody or antigen binding fragment comprises bovine HC and LC, κ or λ, constant domains.
All references cited herein, including patents, patent applications, papers, textbooks and the like, and the references cited therein, to the extent that they are not already, are hereby incorporated herein by reference in their entirety for all purposes.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent or imply that the experiments below are all of or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.
Efforts have been made to ensure accuracy with respect to terms and numbers used (e.g., vectors, amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees centigrade, and pressure is at or near atmospheric.
The examples illustrate targeting by both a 5′ vector and a 3′ vector that flank a site of recombination and introduction of synthetic DNA. It will be apparent to one skilled in the art upon reading the specification that the 5′ vector targeting can take place first followed by the 3′, or the 3′ vector targeting can take place first followed by the 5′ vector. In some circumstances, targeting can be carried out simultaneously with dual detection mechanisms.
An exemplary method illustrating the introduction of an engineered partly bovine immunoglobulin locus into the genomic locus of a non-mammalian ES cell is illustrated in more detail in
As illustrated in
Once the recombination sites are integrated into the mammalian host cell genome, the endogenous region of the immunoglobulin domain is then subjected to recombination by introducing one of the recombinases corresponding to the sequence-specific recombination sites integrated into the genome, e.g., either Flp or Cre. Illustrated in
ES cells that are insensitive to diphtheria toxin are then screened for the deletion of the endogenous variable region gene loci. The primary screening method for the deleted endogenous immunoglobulin locus can be carried out by Southern blotting, or by polymerase chain reaction (PCR) followed by confirmation with a secondary screening technique such as Southern blotting.
The sequences of the bovine VH, D and JH gene segment coding regions are in Table 1.
Primary screening procedure for the introduction of the partly bovine immunoglobulin locus can be carried out by Southern blotting, or by PCR followed by confirmation with a secondary screening method such as Southern blotting. The screening methods are designed to detect the presence of the inserted VH, D and JH gene loci, as well as all the intervening sequences.
In certain aspects, the partly bovine immunoglobulin locus comprises the elements as described in Example 1, but with additional non-coding regulatory or scaffold sequences e.g., sequences strategically added to introduce additional regulatory sequences, to ensure the desired spacing within the introduced immunoglobulin locus, to ensure that certain coding sequences are in adequate juxtaposition with other sequences adjacent to the replaced immunoglobulin locus, and the like.
The primary screening procedure for the introduction of the engineered partly bovine immunoglobulin region can be carried out by Southern blotting, or by PCR with confirmation by a secondary screening method such as Southern blotting. The screening methods are designed to detect the presence of the inserted PAIR elements, the VH, D and JH gene loci, as well as all the intervening sequences.
A method for replacing a portion of a mouse genome with an engineered partly bovine immunoglobulin locus is illustrated in
The targeting vectors (803, 805) employed for introducing the site-specific recombinase sequences on either side of the VH (815), D (817) and JH (819) gene segment clusters and upstream of the constant region genes (821) in the wild-type mouse immunoglobulin locus (801) include an additional site-specific recombination sequence that has been modified so that it is still recognized efficiently by the recombinase, but does not recombine with unmodified sites. This mutant modified site (e.g., lox5171) is positioned in the targeting vector such that after deletion of the endogenous VH, D and JH gene segments (802) it can be used for a second site-specific recombination event in which a non-native piece of DNA is moved into the modified IGH locus by RMCE. In this example, the non-native DNA is a synthetic nucleic acid comprising both bovine and non-bovine sequences (809).
Two gene targeting vectors are constructed to accomplish the process just outlined. One of the vectors (803) comprises mouse genomic DNA taken from the 5′ end of the IGH locus, upstream of the most distal VH gene segment. The other vector (805) comprises mouse genomic DNA taken from within the locus downstream of the JH gene segments.
The key features of the 5′ vector (803) in order from 5′ to 3′ are as follows: a gene encoding the diphtheria toxin A (DTA) subunit under transcriptional control of a modified herpes simplex virus type I thymidine kinase gene promoter coupled to two mutant transcriptional enhancers from the polyoma virus (823); 4.5 Kb of mouse genomic DNA mapping upstream of the most distal VH gene segment in the IGH locus (825); a FRT recognition sequence for the Flp recombinase (827); a piece of genomic DNA containing the mouse Polr2a gene promoter (829); a translation initiation sequence (methionine codon embedded in a “Kozak” consensus sequence, 835)); a mutated loxP recognition sequence (lox5171) for the Cre recombinase (831); a transcription termination/polyadenylation sequence (pA. 833); a loxP recognition sequence for the Cre recombinase (837); a gene encoding a fusion protein with a protein conferring resistance to puromycin fused to a truncated form of the thymidine kinase (pu-TK) under transcriptional control of the promoter from the mouse phosphoglycerate kinase 1 gene (839); and 3 Kb of mouse genomic DNA (841) mapping close to the 4.5 Kb mouse genomic DNA sequence present near the 5′ end of the vector and arranged in the native relative orientation.
The key features of the 3′ vector (805) in order from 5′ to 3′ are as follows; 3.7 Kb of mouse genomic DNA mapping within the intron between the JH and CH gene loci (843); an HPRT gene under transcriptional control of the mouse Polr2a gene promoter (845); a neomycin resistance gene under the control of the mouse phosphoglycerate kinase 1 gene promoter (847); a loxP recognition sequence for the Cre recombinase (837); 2.1 Kb of mouse genomic DNA (849) that maps immediately downstream of the 3.7 Kb mouse genomic DNA fragment present near the 5′ end of the vector and arranged in the native relative orientation; and a gene encoding the DTA subunit under transcriptional control of a modified herpes simplex virus type I thymidine kinase gene promoter coupled to two mutant transcriptional enhancers from the polyoma virus (823).
Mouse embryonic stem (ES) cells (derived from C57Bl/6NTac mice) are transfected by electroporation with the 3′ vector (805) according to widely used procedures. Prior to electroporation, the vector DNA is linearized with a rare-cutting restriction enzyme that cuts only in the prokaryotic plasmid sequence or the polylinker associated with it. The transfected cells are plated and after ˜24 hours they are placed under positive selection for cells that have integrated the 3′ vector into their DNA by using the neomycin analogue drug G418. There is also negative selection for cells that have integrated the vector into their DNA but not by homologous recombination. Non-homologous recombination results in retention of the DTA gene (823), which kills the cells when the gene is expressed, whereas the DTA gene is deleted by homologous recombination since it lies outside of the region of vector homology with the mouse IGH locus. Colonies of drug-resistant ES cells are physically extracted from their plates after they became visible to the naked eye about a week later. These picked colonies are disaggregated, re-plated in micro-well plates, and cultured for several days. Thereafter, each of the clones of cells is divided such that some of the cells can be frozen as an archive, and the rest used for isolation of DNA for analytical purposes.
DNA from the ES cell clones is screened by PCR using a widely practiced gene-targeting assay design. For this assay, one of the PCR oligonucleotide primer sequences maps outside the region of identity shared between the 3′ vector (805) and the genomic DNA, while the other maps within the novel DNA between the two arms of genomic identity in the vector, i.e., in the HPRT (845) or neomycin resistance (847) genes. According to the standard design, these assays detect pieces of DNA that would only be present in clones of ES cells derived from transfected cells that undergo fully legitimate homologous recombination between the 3′ targeting vector and the endogenous mouse IGH locus. Two separate transfections are performed with the 3′ vector (805). PCR-positive clones from the two transfections are selected for expansion followed by further analysis using Southern blot assays.
The Southern blot assays are performed according to widely used procedures using three probes and genomic DNA digested with multiple restriction enzymes chosen so that the combination of probes and digests allow the structure of the targeted locus in the clones to be identified as properly modified by homologous recombination. One of the probes maps to DNA sequence flanking the 5′ side of the region of identity shared between the 3′ targeting vector and the genomic DNA; a second probe maps outside the region of identity but on the 3′ side; and the third probe maps within the novel DNA between the two arms of genomic identity in the vector, i.e., in the HPRT (845) or neomycin resistance (847) genes. The Southern blot identifies the presence of the expected restriction enzyme-generated fragment of DNA corresponding to the correctly mutated, i.e., by homologous recombination with the 3′ IGH targeting vector, part of the IGH locus as detected by one of the external probes and by the neomycin or HPRT probe. The external probe detects the mutant fragment and also a wild-type fragment from the non-mutant copy of the immunoglobulin IGH locus on the homologous chromosome.
Karyotypes of PCR- and Southern blot-positive clones of ES cells are analyzed using an in situ fluorescence hybridization procedure designed to distinguish the most commonly arising chromosomal aberrations that arise in mouse ES cells. Clones with such aberrations are excluded from further use. ES cell clones that are judged to have the expected correct genomic structure based on the Southern blot data—and that also do not have detectable chromosomal aberrations based on the karyotype analysis—are selected for further use.
Acceptable clones are then modified with the 5′ vector (803) using procedures and screening assays that are similar in design to those used with the 3′ vector (805) except that puromycin selection is used instead of G418/neomycin for selection. The PCR assays, probes and digests are also tailored to match the genomic region being modified by the 5′ vector (805).
Clones of ES cells that have been mutated in the expected fashion by both the 3′ and the 5′ vectors, i.e., doubly targeted cells carrying both engineered mutations, are isolated following vector targeting and analysis. The clones must have undergone gene targeting on the same chromosome, as opposed to homologous chromosomes (i.e., the engineered mutations created by the targeting vectors must be in cis on the same DNA strand rather than in trans on separate homologous DNA strands). Clones with the cis arrangement are distinguished from those with the trans arrangement by analytical procedures such as fluorescence in situ hybridization of metaphase spreads using probes that hybridize to the novel DNA present in the two gene targeting vectors (803 and 805) between their arms of genomic identity. The two types of clones can also be distinguished from one another by transfecting them with a vector expressing the Cre recombinase, which deletes the pu-TK (839), HPRT (845) and neomycin resistance (847) genes if the targeting vectors have been integrated in cis, and then comparing the number of colonies that survive ganciclovir selection against the thymidine kinase gene introduced by the 5′ vector (803) and by analyzing the drug resistance phenotype of the surviving clones by a “sibling selection” screening procedure in which some of the cells from the clone are tested for resistance to puromycin or G418/neomycin. Cells with the cis arrangement of mutations are expected to yield approximately 103 more ganciclovir-resistant clones than cells with the trans arrangement. The majority of the resulting cis-derived ganciclovir-resistant clones are also sensitive to both puromycin and G418/neomycin, in contrast to the trans-derived ganciclovir-resistant clones, which should retain resistance to both drugs. Doubly targeted clones of cells with the cis-arrangement of engineered mutations in the heavy chain locus are selected for further use.
The doubly targeted clones of cells are transiently transfected with a vector expressing the Cre recombinase and the transfected cells subsequently are placed under ganciclovir selection, as in the analytical experiment summarized above. Ganciclovir-resistant clones of cells are isolated and analyzed by PCR and Southern blot for the presence of the expected deletion between the two engineered mutations created by the 5′ (803) and the 3′ (805) targeting vectors. In these clones, the Cre recombinase causes a recombination (802) to occur between the loxP sites (837) introduced into the heavy chain locus by the two vectors to create the genomic DNA configuration shown at 807. Because the loxP sites are arranged in the same relative orientations in the two vectors, recombination results in excision of a circle of DNA comprising the entire genomic interval between the two loxP sites. The circle does not contain an origin of replication and thus is not replicated during mitosis and therefore is lost from the cells as they undergo proliferation. The resulting clones carry a deletion of the DNA that was originally between the two loxP sites. Clones that have the expected deletion are selected for further use.
ES cell clones carrying the deletion of sequence in one of the two homologous copies of their immunoglobulin heavy chain locus are retransfected (804) with a Cre recombinase expression vector together with a piece of DNA (809) comprising a partly bovine immunoglobulin heavy chain locus containing bovine VH, D and JH region gene coding region sequences flanked by mouse regulatory and flanking sequences. The key features of this piece of synthetic DNA (809) are the following: a lox5171 site (831); a neomycin resistance gene open reading frame (847) lacking the initiator methionine codon, but in-frame and contiguous with an uninterrupted open reading frame in the lox5171 site a FRT site (827); an array of 20 bovine VH heavy chain variable region genes (851), each with bovine coding sequences embedded in mouse noncoding sequences; optionally a 10 kb pre-D region from the mouse heavy chain locus (not shown); a 58 Kb piece of DNA containing the 10 bovine D gene segments (853) and 4 bovine JH gene segments (855) where the bovine VH, D and JH coding sequences are embedded in mouse noncoding sequences; a loxP site (837) in opposite relative orientation to the lox5171 site (831).
The transfected clones are placed under G418 selection, which enriches for clones of cells that have undergone RMCE in which the engineered partly bovine donor immunoglobulin locus (809) is integrated in its entirety into the deleted endogenous immunoglobulin heavy chain locus between the lox5171 (831) and loxP (837) sites to create the DNA region illustrated at 811. Only cells that have properly undergone RMCE have the capability to express the neomycin resistance gene (847) because the promoter (829) as well as the initiator methionine codon (835) required for its expression are not present in the vector (809) but are already pre-existing in the host cell IGH locus (807). The remaining elements from the 5′ vector (803) are removed via Flp-mediated recombination (806) in vitro or in vivo, resulting in the final bovine-based locus as shown at 813.
G418-resistant ES cell clones are analyzed by PCR and Southern blot to determine if they have undergone the expected RMCE process without unwanted rearrangements or deletions. Clones that have the expected genomic structure are selected for further use.
ES cell clones carrying the partly bovine immunoglobulin heavy chain DNA (813) in the mouse heavy chain locus are microinjected into mouse blastocysts from strain DBA/2 to create partially ES cell-derived chimeric mice according to standard procedures. Male chimeric mice with the highest levels of ES cell-derived contribution to their coats are selected for mating to female mice. The female mice of choice here are of C57Bl/6NTac strain, and also carry a transgene encoding the Flp recombinase that is expressed in their germline. Offspring from these matings are analyzed for the presence of the partly bovine immunoglobulin heavy chain locus, and for loss of the FRT-flanked neomycin resistance gene that was created in the RMCE step. Mice that carry the partly bovine locus are used to establish a colony of mice.
Another method for replacing a portion of a mouse genome with partly bovine immunoglobulin locus is illustrated in
The targeting vectors employed for introducing the site-specific recombination sequences on either side of the Vκ (915) and Jκ (919) gene segments also include an additional site-specific recombination sequence that has been modified so that it is still recognized efficiently by the recombinase, but does not recombine with unmodified sites. This site is positioned in the targeting vector such that after deletion of the Vκ and Jκ gene segment clusters it can be used for a second site specific recombination event in which a non-native piece of DNA is moved into the modified Vκ locus via RMCE. In this example, the non-native DNA is a synthetic nucleic acid comprising bovine Vκ and Jκ gene segment coding sequences embedded in mouse regulatory and flanking sequences.
Two gene targeting vectors are constructed to accomplish the process just outlined. One of the vectors (903) comprises mouse genomic DNA taken from the 5′ end of the locus, upstream of the most distal Vκ gene segment. The other vector (905) comprises mouse genomic DNA taken from within the locus downstream (3′) of the Jκ gene segments (919) and upstream of the constant region genes (921).
The key features of the 5′ vector (903) are as follows: a gene encoding the diphtheria toxin A (DTA) subunit under transcriptional control of a modified herpes simplex virus type I thymidine kinase gene promoter coupled to two mutant transcriptional enhancers from the polyoma virus (923); 6 Kb of mouse genomic DNA (925) mapping upstream of the most distal variable region gene in the κ chain locus; a FRT recognition sequence for the Flp recombinase (927); a piece of genomic DNA containing the mouse Polr2a gene promoter (929); a translation initiation sequence (935, methionine codon embedded in a “Kozak” consensus sequence); a mutated loxP recognition sequence (lox5171) for the Cre recombinase (931); a transcription termination/polyadenylation sequence (933); a loxP recognition sequence for the Cre recombinase (937); a gene encoding a fusion protein with a protein conferring resistance to puromycin fused to a truncated form of the thymidine kinase (pu-TK) under transcriptional control of the promoter from the mouse phosphoglycerate kinase 1 gene (939); 2.5 Kb of mouse genomic DNA (941) mapping close to the 6 Kb sequence at the 5′ end in the vector and arranged in the native relative orientation.
The key features of the 3′ vector (905) are as follows: 6 Kb of mouse genomic DNA (943) mapping within the intron between the Jκ (919) and Cκ (921) gene loci; a gene encoding the human hypoxanthine-guanine phosphoribosyl transferase (HPRT) under transcriptional control of the mouse Polr2a gene promoter (945); a neomycin resistance gene under the control of the mouse phosphoglycerate kinase 1 gene promoter (947); a loxP recognition sequence for the Cre recombinase (937); 3.6 Kb of mouse genomic DNA (949) that maps immediately downstream in the genome of the 6 Kb DNA fragment included at the 5′ end in the vector, with the two fragments oriented in the same transcriptional orientation as in the mouse genome; a gene encoding the diphtheria toxin A (DTA) subunit under transcriptional control of a modified herpes simplex virus type I thymidine kinase gene promoter coupled to two mutant transcriptional enhancers from the polyoma virus (923).
Mouse embryonic stem (ES) cells derived from C57Bl/6NTac mice are transfected by electroporation with the 3′ vector (905) according to widely used procedures. Prior to electroporation, the vector DNA is linearized with a rare-cutting restriction enzyme that cuts only in the prokaryotic plasmid sequence or the polylinker associated with it. The transfected cells are plated and after ˜24 hours they are placed under positive selection for cells that have integrated the 3′ vector into their DNA by using the neomycin analogue drug G418. There is also negative selection for cells that have integrated the vector into their DNA but not by homologous recombination. Non-homologous recombination results in retention of the DTA gene, which kills the cells when the gene is expressed, whereas the DTA gene is deleted by homologous recombination since it lies outside of the region of vector homology with the mouse IGK locus. Colonies of drug-resistant ES cells are physically extracted from their plates after they became visible to the naked eye about a week later. These picked colonies are disaggregated, re-plated in micro-well plates, and cultured for several days. Thereafter, each of the clones of cells is divided such that some of the cells could be frozen as an archive, and the rest used for isolation of DNA for analytical purposes.
DNA from the ES cell clones is screened by PCR using a widely used gene-targeting assay design. For this assay, one of the PCR oligonucleotide primer sequences maps outside the region of identity shared between the 3′ vector (905) and the genomic DNA (901), while the other maps within the novel DNA between the two arms of genomic identity in the vector, i.e., in the HPRT (945) or neomycin resistance (947) genes. According to the standard design, these assays detect pieces of DNA that are only present in clones of ES cells derived from transfected cells that had undergone fully legitimate homologous recombination between the 3′ vector (905) and the endogenous mouse IGK locus. Two separate transfections are performed with the 3′ vector (905). PCR-positive clones from the two transfections are selected for expansion followed by further analysis using Southern blot assays.
The Southern blot assays are performed according to widely used procedures; they involve three probes and genomic DNA digested with multiple restriction enzymes chosen so that the combination of probes and digests allowed for conclusions to be drawn about the structure of the targeted locus in the clones and whether it is properly modified by homologous recombination. One of the probes maps to DNA sequence flanking the 5′ side of the region of identity shared between the 3′ κ targeting vector (905) and the genomic DNA; a second probe also maps outside the region of identity but on the 3′ side; the third probe maps within the novel DNA between the two arms of genomic identity in the vector, i.e., in the HPRT (945) or neomycin resistance (947) genes. The Southern blot identifies the presence of the expected restriction enzyme-generated fragment of DNA corresponding to the correctly mutated, i.e., by homologous recombination with the 3′ κ targeting vector (905) part of the κ locus, as detected by one of the external probes and by the neomycin resistance or HPRT gene probe. The external probe detects the mutant fragment and also a wild-type fragment from the non-mutant copy of the immunoglobulin κ locus on the homologous chromosome.
Karyotypes of PCR- and Southern blot-positive clones of ES cells are analyzed using an in situ fluorescence hybridization procedure designed to distinguish the most commonly arising chromosomal aberrations that arise in mouse ES cells. Clones with such aberrations are excluded from further use. Karyotypically normal clones that are judged to have the expected correct genomic structure based on the Southern blot data are selected for further use.
Acceptable clones are then modified with the 5′ vector (903) using procedures and screening assays that are similar in design to those used with the 3′ vector (905), except that puromycin selection is used instead of G418/neomycin selection, and the protocols are tailored to match the genomic region modified by the 5′ vector (903). The goal of the 5′ vector (903) transfection experiments is to isolate clones of ES cells that have been mutated in the expected fashion by both the 3′ vector (905) and the 5′ vector (903), i.e., doubly targeted cells carrying both engineered mutations. In these clones, the Cre recombinase causes a recombination (902) to occur between the loxP sites introduced into the κ locus by the two vectors, resulting in the genomic DNA configuration shown at 907.
Further, the clones must have undergone gene targeting on the same chromosome, as opposed to homologous chromosomes; i.e., the engineered mutations created by the targeting vectors must be in cis on the same DNA strand rather than in trans on separate homologous DNA strands. Clones with the cis arrangement are distinguished from those with the trans arrangement by analytical procedures such as fluorescence in situ hybridization of metaphase spreads using probes that hybridize to the novel DNA present in the two gene targeting vectors (903 and 905) between their arms of genomic identity. The two types of clones can also be distinguished from one another by transfecting them with a vector expressing the Cre recombinase, which deletes the pu-Tk (939), HPRT (945) and neomycin resistance (947) genes if the targeting vectors have been integrated in cis, and comparing the number of colonies that survive ganciclovir selection against the thymidine kinase gene introduced by the 5′ vector (903) and by analyzing the drug resistance phenotype of the surviving clones by a “sibling selection” screening procedure in which some of the cells from the clone are tested for resistance to puromycin or G418/neomycin. Cells with the cis arrangement of mutations are expected to yield approximately 103 more ganciclovir-resistant clones than cells with the trans arrangement. The majority of the resulting cis-derived ganciclovir-resistant clones should also be sensitive to both puromycin and G418/neomycin, in contrast to the trans-derived ganciclovir-resistant clones, which should retain resistance to both drugs. Clones of cells with the cis-arrangement of engineered mutations in the κ chain locus are selected for further use.
The doubly targeted clones of cells are transiently transfected with a vector expressing the Cre recombinase (902) and the transfected cells are subsequently placed under ganciclovir selection, as in the analytical experiment summarized above. Ganciclovir-resistant clones of cells are isolated and analyzed by PCR and Southern blot for the presence of the expected deletion (907) between the two engineered mutations created by the 5′ vector (903) and the 3′ vector (905). In these clones, the Cre recombinase has caused a recombination to occur between the loxP sites (937) introduced into the κ chain locus by the two vectors. Because the loxP sites are arranged in the same relative orientations in the two vectors, recombination results in excision of a circle of DNA comprising the entire genomic interval between the two loxP sites. The circle does not contain an origin of replication and thus is not replicated during mitosis and is therefore lost from the clones of cells as they undergo clonal expansion. The resulting clones carry a deletion of the DNA that was originally between the two loxP sites. Clones that have the expected deletion are selected for further use.
The ES cell clones carrying the deletion of sequence in one of the two homologous copies of their immunoglobulin κ chain locus are retransfected (904) with a Cre recombinase expression vector together with a piece of DNA (909) comprising a partly bovine immunoglobulin κ chain locus containing Vκ (951) and Jκ (955) gene segment coding sequences. The key features of this piece of DNA (referred to as “K-K”) are the following: a lox5171 site (931); a neomycin resistance gene open reading frame (947, lacking the initiator methionine codon, but in-frame and contiguous with an uninterrupted open reading frame in the lox5171 site (931)); a FRT site (927); an array of 8 bovine Vκ gene segments (951), each with bovine coding sequences embedded in mouse noncoding sequences; optionally a 13.5 Kb piece of genomic DNA from immediately upstream of the cluster of Jκ region gene segments in the mouse κ chain locus (not shown); a 2 Kb piece of DNA containing the 3 bovine Jκ region gene segments (955) embedded in mouse noncoding DNA; a loxP site (937) in opposite relative orientation to the lox5171 site (931).
The sequences of the bovine Vκ and Jκ gene coding regions are in Table 2.
In a second independent experiment, an alternative piece of partly bovine DNA (909) is used in place of the K-K DNA. The key features of this DNA (referred to as “L-K”) are the following: a lox5171 site (931); a neomycin resistance gene open reading frame (947) lacking the initiator methionine codon, but in-frame and contiguous with an uninterrupted open reading frame in the lox5171 site (931); a FRT site (927); an array of 25 functional bovine Vλ variable region gene segments (951), each with bovine coding sequences embedded in mouse noncoding regulatory or scaffold sequences; optionally, a 13.5 Kb piece of genomic DNA from immediately upstream of the cluster of the Jκ region gene segments in the mouse κ chain locus (not shown); a 2 Kb piece of DNA containing bovine Jλ region gene segments embedded in mouse noncoding DNA (955); a loxP site (937) in opposite relative orientation to the lox5171 site (931).
The transfected clones from the K-K and L-K transfection experiments are placed under G418 selection, which enriches for clones of cells that have undergone RMCE, in which the partly bovine donor DNA (909) is integrated in its entirety into the deleted immunoglobulin κ chain locus between the lox5171 (931) and loxP (937) sites that were placed there by 5′ (903) and 3′ (905) vectors, respectively. Only cells that have properly undergone RMCE have the capability to express the neomycin resistance gene (947) because the promoter (929) as well as the initiator methionine codon (935) required for its expression are not present in the vector (909) and are already pre-existing in the host cell IGH locus (907). The DNA region created using the K-K sequence is illustrated at 911. The remaining elements from the 5′ vector (903) are removed via Flp-mediated recombination (906) in vitro or in vivo, resulting in the final bovine-based light chain locus as shown at 913.
G418-resistant ES cell clones are analyzed by PCR and Southern blotting to determine if they have undergone the expected RMCE process without unwanted rearrangements or deletions. Both K-K and L-K clones that have the expected genomic structure are selected for further use.
The K-K ES cell clones and the L-K ES cell clones carrying the partly bovine immunoglobulin DNA in the mouse κ chain locus (913) are microinjected into mouse blastocysts from strain DBA/2 to create partly ES cell-derived chimeric mice according to standard procedures. Male chimeric mice with the highest levels of ES cell-derived contribution to their coats are selected for mating to female mice. The female mice of choice for use in the mating are of the C57Bl/6NTac strain, and also carry a transgene encoding the Flp recombinase that is expressed in their germline. Offspring from these matings are analyzed for the presence of the partly bovine immunoglobulin κ or λ light chain locus, and for loss of the FRT-flanked neomycin resistance gene that was created in the RMCE step. Mice that carry the partly bovine locus are used to establish colonies of K-K and L-K mice.
Mice carrying the partly bovine heavy chain locus, produced as described in Example 3, can be bred with mice carrying a bovine-based κ chain locus. Their offspring are in turn bred together in a scheme that ultimately produces mice that are homozygous for both bovine-based loci, i.e., bovine-based for heavy chain and κ. Such mice produce partly bovine heavy chains with bovine variable domains and mouse constant domains. They also produce partly bovine κ proteins with bovine κ variable domains and the mouse κ constant domain from their κ loci. Monoclonal antibodies recovered from these mice have bovine heavy chain variable domains paired with bovine κ variable domains.
A variation on the breeding scheme involves generating mice that are homozygous for the bovine-based heavy chain locus, but heterozygous at the κ locus such that on one chromosome they have the K-K bovine-based locus and on the other chromosome they have the L-K bovine-based locus. Such mice produce partly bovine heavy chains with bovine variable domains and mouse constant domains. They also produce partly bovine κ proteins with bovine κ variable domains and the mouse κ constant domain from one of their κ loci. From the other κ locus, they produce partly bovine λ proteins with bovine λ variable domains the mouse κ constant domain. Monoclonal antibodies recovered from these mice have bovine variable domains paired in some cases with bovine κ variable domains and in other cases with bovine λ variable domains.
Another method for replacing a portion of a mouse genome with an engineered partly bovine immunoglobulin locus is illustrated in
The key features of the gene targeting vector (1003) for accomplishing the 194 Kb deletion are as follows: a negative selection gene such as a gene encoding the A subunit of the diphtheria toxin (DTA, 1059) or a herpes simplex virus thymidine kinase gene (not shown); 4 Kb of genomic DNA from 5′ of the mouse Vλx/Vλ2 variable region gene segments in the λ locus (1025); a FRT site (1027); a piece of genomic DNA containing the mouse Polr2a gene promoter (1029); a translation initiation sequence (methionine codon embedded in a “Kozak” consensus sequence) (1035); a mutated loxP recognition sequence (lox5171) for the Cre recombinase (1031); a transcription termination/polyadenylation sequence (1033); an open reading frame encoding a protein that confers resistance to puromycin (1037), whereas this open reading frame is on the antisense strand relative to the Polr2a promoter and the translation initiation sequence next to it and is followed by its own transcription termination/polyadenylation sequence (1033); a loxP recognition sequence for the Cre recombinase (1039); a translation initiation sequence (a methionine codon embedded in a “Kozak” consensus sequence) (1035) on the same, antisense strand as the puromycin resistance gene open reading frame; a chicken beta actin promoter and cytomegalovirus early enhancer element (1041) oriented such that it directs transcription of the puromycin resistance open reading frame, with translation initiating at the initiation codon downstream of the loxP site and continuing back through the loxP site into the puromycin open reading frame all on the antisense strand relative to the Polr2a promoter and the translation initiation sequence next to it; a mutated recognition site for the Flp recombinase known as an “F3” site (1043); a piece of genomic DNA upstream of the Jλ3, Cλ3, Jλ1 and Cλ1 gene segments (1045).
Mouse embryonic stem (ES) cells derived from C57Bl/6NTac mice are transfected (1002) by electroporation with the targeting vector (1003) according to widely used procedures. Homologous recombination replaces the native DNA with the sequences from the targeting vector (1003) in the 196 Kb region resulting in the genomic DNA configuration depicted at 1005.
Prior to electroporation, the vector DNA is linearized with a rare-cutting restriction enzyme that cuts only in the prokaryotic plasmid sequence or the polylinker associated with it. The transfected cells are plated and after ˜24 hours placed under positive drug selection using puromycin. There is also negative selection for cells that have integrated the vector into their DNA but not by homologous recombination. Non-homologous recombination results in retention of the DTA gene, which kills the cells when the gene is expressed, whereas the DTA gene is deleted by homologous recombination since it lies outside of the region of vector homology with the mouse IGL locus. Colonies of drug-resistant ES cells are physically extracted from their plates after they became visible to the naked eye approximately a week later. These picked colonies are disaggregated, re-plated in micro-well plates, and cultured for several days. Thereafter, each of the clones of cells are divided such that some of the cells are frozen as an archive, and the rest used for isolation of DNA for analytical purposes.
DNA from the ES cell clones is screened by PCR using a widely used gene-targeting assay design. For these assays, one of the PCR oligonucleotide primer sequences maps outside the regions of identity shared between the targeting vector and the genomic DNA, while the other maps within the novel DNA between the two arms of genomic identity in the vector, e.g., in the puro gene (1037). According to the standard design, these assays detect pieces of DNA that would only be present in clones of cells derived from transfected cells that had undergone fully legitimate homologous recombination between the targeting vector (1003) and the native DNA (1001).
Six PCR-positive clones from the transfection (1002) are selected for expansion followed by further analysis using Southern blot assays. The Southern blots involve three probes and genomic DNA from the clones that has been digested with multiple restriction enzymes chosen so that the combination of probes and digests allow identification of whether the ES cell DNA has been properly modified by homologous recombination.
Karyotypes of the six PCR- and Southern blot-positive clones of ES cells are analyzed using an in situ fluorescence hybridization procedure designed to distinguish the most common chromosomal aberrations that arise in mouse ES cells. Clones that show evidence of aberrations are excluded from further use. Karyotypically normal clones that are judged to have the expected correct genomic structure based on the Southern blot data are selected for further use.
The ES cell clones carrying the deletion in one of the two homologous copies of their immunoglobulin λ chain locus are retransfected (1004) with a Cre recombinase expression vector together with a piece of DNA (1007) comprising a partly bovine immunoglobulin λ chain locus containing Vλ, Jλ and Cλ region gene segments. The key features of this piece of DNA (1007) are as follows: a lox5171 site (1031); a neomycin resistance gene open reading frame lacking the initiator methionine codon, but in-frame and contiguous with an uninterrupted open reading frame in the lox5171 site (1047); a FRT site 1027); an array of 25 functional bovine λ region gene segments, each with bovine λ coding sequences embedded in mouse λ noncoding sequences (1051); an array of J-C units where each unit has a bovine Jκ gene segment and a mouse λ constant domain gene segment embedded within noncoding sequences from the mouse λ locus (1055) (the bovine Jκ gene segments are those encoding Jλ1, Jλ2, Jλ3, Jλ4, Jλ5, Jλ6, and Jλ7, while the mouse λ constant domain gene segments are Cλ1 or Cλ2 or Cλ3); a mutated recognition site for the Flp recombinase known as an “F3” site (1043); an open reading frame conferring hygromycin resistance (1057), which is located on the antisense strand relative to the immunoglobulin gene segment coding information in the construct; a loxP site (1039) in opposite relative orientation to the lox5171 site.
The sequences of the bovine Vλ and Jλ gene coding regions are in Table 3.
The transfected clones are placed under G418 or hygromycin selection, which enriches for clones of cells that have undergone a RMCE process, in which the partly bovine donor DNA is integrated in its entirety into the deleted immunoglobulin λ chain locus between the lox5171 and loxP sites that were placed there by the gene targeting vector. The remaining elements from the targeting vector (1003) are removed via FLP-mediated recombination (1006) in vitro or in vivo resulting in the final bovinized locus as shown at 1011.
G418/hygromycin-resistant ES cell clones are analyzed by PCR and Southern blotting to determine if they have undergone the expected recombinase-mediated cassette exchange process without unwanted rearrangements or deletions. Clones that have the expected genomic structure are selected for further use.
The ES cell clones carrying the partly bovine immunoglobulin DNA (1011) in the mouse λ chain locus are microinjected into mouse blastocysts from strain DBA/2 to create partially ES cell-derived chimeric mice according to standard procedures. Male chimeric mice with the highest levels of ES cell-derived contribution to their coats are selected for mating to female mice. The female mice of choice here are of the C57Bl/6NTac strain, which carry a transgene encoding the Flp recombinase expressed in their germline. Offspring from these matings are analyzed for the presence of the partly bovine immunoglobulin λ chain locus, and for loss of the FRT-flanked neomycin resistance gene and the F3-flanked hygromycin resistance gene that were created in the RMCE step. Mice that carry the partly bovine locus are used to establish a colony of mice.
In some aspects, the mice comprising the bovine-based heavy chain and κ locus (as described in Examples 3 and 4) are bred to mice that carry the bovine-based λ locus. Mice generated from this type of breeding scheme are homozygous for the bovine-based heavy chain locus and can be homozygous for the K-K bovine-based locus or the L-K bovine-based locus. Alternatively, they can be heterozygous at the κ locus carrying the K-K locus on one chromosome and the L-K locus on the other chromosome. Each of these mouse strains is homozygous for the bovine-based λ locus. Monoclonal antibodies recovered from these mice has bovine heavy chain variable domains paired in some cases with bovine κ variable domains and in other cases with bovine λ variable domains. The λ variable domains are derived from either the bovine-based L-K locus or the bovine-based λ locus.
In certain other aspects, the partly bovine immunoglobulin locus comprises a bovine variable domain minilocus such as the one illustrated in
A site-specific targeting vector (1131) comprising the partly bovine immunoglobulin locus to be integrated into the mammalian host genome is introduced (1102) into the genomic region (1101) with the deleted endogenous immunoglobulin locus comprising the puro-TK gene (1105) and the following flanking sequence-specific recombination sites: mutant FRT site (1109), mutant LoxP site (1111), wild-type FRT site (1107), and wild-type LoxP site (1105). The site-specific targeting vector comprises i) an array of optional PAIR elements (1141); ii) a VH locus (1119) comprising, e.g., 1-20 functional bovine VH coding regions and intervening sequences based on the mouse genome endogenous sequences; iii) a 21.6 kb pre-D region (1121) comprising mouse sequence; iv) a D locus (1123) and a JH locus (1125) comprising 10 D and JH bovine coding sequences and intervening sequences based on the mouse genome endogenous sequences. The partly bovine immunoglobulin locus is flanked by recombination sites—mutant FRT (1109), mutant LoxP (1111), wild-type FRT (1107), and wild-type LoxP (1105)—that allow recombination with the modified endogenous locus. Upon introduction of the appropriate recombinase, e.g., Cre) (1104), the partly bovine immunoglobulin locus is integrated into the genome upstream of the constant gene region (1127) as shown at 1129.
As described in Example 1, the primary screening for introduction of the partly bovine immunoglobulin variable region locus is carried out by primary PCR screens supported by secondary Southern blotting assays. The deletion of the puro-TK gene (1105) as part of the recombination event allows identification of the cells that did not undergo the recombination event using ganciclovir negative selection.
Cattle antibodies mostly contain λ light chains, whereas mouse antibodies mostly contain κ light chains. To increase production of antibodies containing a λ LC, the endogenous mouse Vκ and Jκ are replaced with a partly bovine locus containing Vλ and Jλ gene segment coding sequences embedded in mouse Vκ region flanking and regulatory sequences, the L-K mouse of Example 4. In such a mouse, the endogenous regulatory sequences promoting high level κ locus rearrangement and expression are predicted to have an equivalent effect on the ectopic λ locus. However, in vitro studies demonstrated that bovine Vλ domains do not function well with mouse Cκ (see Example 9). Thus, the expected increase in λ LC-containing antibodies in the L-K mouse might not occur. As an alternate strategy, the endogenous mouse Vκ and Jκ are replaced with a partly bovine locus containing Vλ and Jλ gene segment coding sequences embedded in mouse Vκ region flanking and regulatory sequences and mouse Cκ is replaced with mouse Cλ.
The method for replacing a portion of a mouse genome with a partly bovine immunoglobulin locus is illustrated in
The targeting vectors employed for introducing the site-specific recombination sequences on either side of the Vκ (1315) gene segments and the Cκ exon (1321) also include an additional site-specific recombination sequence that has been modified so that it is still recognized efficiently by the recombinase, but does not recombine with unmodified sites. This site is positioned in the targeting vector such that after deletion of the Vκ and Jκ gene segment clusters and the Cκ exon it can be used for a second site specific recombination event in which a non-native piece of DNA is moved into the modified Vκ locus via RMCE. In this example, the non-native DNA is a synthetic nucleic acid comprises bovine Vλ and Jλ gene segment coding sequences and mouse Cλ exon(s) embedded in mouse IGK regulatory and flanking sequences.
Two gene targeting vectors are constructed to accomplish the process just outlined. One of the vectors (1303) comprises mouse genomic DNA taken from the 5′ end of the locus, upstream of the most distal Vκ gene segment. The other vector (1305) comprises mouse genomic DNA taken from within the locus in a region spanning upstream (5′) and downstream (3′) of the Cκ exon (1321).
The key features of the 5′ vector (1303) are as follows: a gene encoding the diphtheria toxin A (DTA) subunit under transcriptional control of a modified herpes simplex virus type I thymidine kinase gene promoter coupled to two mutant transcriptional enhancers from the polyoma virus (1323); 6 Kb of mouse genomic DNA (1325) mapping upstream of the most distal variable region gene in the κ chain locus; a FRT recognition sequence for the Flp recombinase (1327); a piece of genomic DNA containing the mouse Polr2a gene promoter (1329); a translation initiation sequence (1335, methionine codon embedded in a “Kozak” consensus sequence); a mutated loxP recognition sequence (lox5171) for the Cre recombinase (1331); a transcription termination/polyadenylation sequence (1333); a loxP recognition sequence for the Cre recombinase (1337); a gene encoding a fusion protein with a protein conferring resistance to puromycin fused to a truncated form of the thymidine kinase (pu-TK) under transcriptional control of the promoter from the mouse phosphoglycerate kinase 1 gene (1339); 2.5 Kb of mouse genomic DNA (1341) mapping close to the 6 Kb sequence at the 5′ end in the vector and arranged in the native relative orientation.
The key features of the 3′ vector (1305) are as follows: 6 Kb of mouse genomic DNA (1343) mapping within the locus in a region spanning upstream (5′) and downstream (3′) of the Cκ exon (1321); a gene encoding the human hypoxanthine-guanine phosphoribosyl transferase (HPRT) under transcriptional control of the mouse Polr2a gene promoter (1345); a neomycin resistance gene under the control of the mouse phosphoglycerate kinase 1 gene promoter (1347); a loxP recognition sequence for the Cre recombinase (1337); 3.6 Kb of mouse genomic DNA (1349) that maps immediately downstream in the genome of the 6 Kb DNA fragment included at the 5′ end in the vector, with the two fragments oriented in the same transcriptional orientation as in the mouse genome; a gene encoding the diphtheria toxin A (DTA) subunit under transcriptional control of a modified herpes simplex virus type I thymidine kinase gene promoter coupled to two mutant transcriptional enhancers from the polyoma virus (1323).
One strategy to delete the endogenous mouse IGK locus is to insert the 3′ vector (1305) in the flanking region downstream of the mouse Cκ exon (1321). However, the 3′κ enhancer, which needs to be retained in the modified locus, is located 9.1 Kb downstream of the Cκ exon, which is too short to accommodate the upstream and downstream homology arms of the 3′ vector, which total 9.6 Kb. Therefore, the upstream region of homology was extended.
Mouse embryonic stem (ES) cells derived from C57Bl/6NTac mice are transfected by electroporation with the 3′ vector (1305) according to widely used procedures. Prior to electroporation, the vector DNA is linearized with a rare-cutting restriction enzyme that cuts only in the prokaryotic plasmid sequence or the polylinker associated with it. The transfected cells are plated and after ˜24 hours they are placed under positive selection for cells that have integrated the 3′ vector into their DNA using the neomycin analogue drug G418. There is also negative selection for cells that have integrated the vector into their DNA but not by homologous recombination. Non-homologous recombination retains the DTA gene, which kills the cells when the gene is expressed, but the DTA gene is deleted by homologous recombination since it lies outside of the region of vector homology with the mouse IGK locus. Colonies of drug-resistant ES cells are physically extracted from their plates after they are visible to the naked eye about a week later. These colonies are disaggregated, re-plated in micro-well plates, and cultured for several days. Thereafter, each of the clones of cells is divided—some of the cells are frozen as an archive, and the rest are used to isolate DNA for analytical purposes.
DNA from the ES cell clones is screened by PCR using a widely used gene-targeting assay design. For this assay, one of the PCR oligonucleotide primer sequences maps outside the region of identity shared between the 3′ vector (1305) and the genomic DNA (1301), while the other maps within the novel DNA between the two arms of genomic identity in the vector, i.e., in the HPRT (1345) or neomycin resistance (1347) genes. According to the standard design, these assays detect pieces of DNA that are only present in clones of ES cells derived from transfected cells that had undergone fully legitimate homologous recombination between the 3′ vector (1305) and the endogenous mouse IGK locus. Two separate transfections are performed with the 3′ vector (1305). PCR-positive clones from the two transfections are selected for expansion followed by further analysis using Southern blot assays.
Southern blot assays are performed according to widely used procedures using three probes and genomic DNA digested with multiple restriction enzymes chosen so that the combination of probes and digests allowed for conclusions to be drawn about the structure of the targeted locus in the clones and whether it is properly modified by homologous recombination. A first probe maps to DNA sequence flanking the 5′ side of the region of identity shared between the 3′ κ targeting vector (1305) and the genomic DNA; a second probe also maps outside the region of identity but on the 3′ side; a third probe maps within the novel DNA between the two arms of genomic identity in the vector, i.e., in the HPRT (1345) or neomycin resistance (1347) genes. The Southern blot identifies the presence of the expected restriction enzyme-generated fragment of DNA corresponding to the correctly mutated, i.e., by homologous recombination with the 3′ κ targeting vector (1305) part of the κ locus, as detected by one of the external probes and by the neomycin resistance or HPRT gene probe. The external probe detects the mutant fragment and also a wild-type fragment from the non-mutant copy of the immunoglobulin κ locus on the homologous chromosome.
Karyotypes of PCR- and Southern blot-positive clones of ES cells are analyzed using an in situ fluorescence hybridization procedure designed to distinguish the most commonly arising chromosomal aberrations that arise in mouse ES cells. Clones with such aberrations are excluded from further use. Karyotypically normal clones that are judged to have the expected correct genomic structure based on the Southern blot data are selected for further use.
Acceptable clones are then modified with the 5′ vector (1303) using procedures and screening assays that are similar in design to those used with the 3′ vector (1305), except that puromycin selection is used instead of G418/neomycin selection, and the protocols are tailored to match the genomic region modified by the 5′ vector (1303). The goal of the 5′ vector (1303) transfection experiments is to isolate clones of ES cells that have been mutated in the expected fashion by both the 3′ vector (1305) and the 5′ vector (1303), i.e., doubly targeted cells carrying both engineered mutations. In these clones, the Cre recombinase causes a recombination (1302) to occur between the loxP sites introduced into the κ locus by the two vectors, resulting in the genomic DNA configuration shown at 1307.
Further, the clones must have undergone gene targeting on the same chromosome, as opposed to homologous chromosomes; i.e., the engineered mutations created by the targeting vectors must be in cis on the same DNA strand rather than in trans on separate homologous DNA strands. Clones with the cis arrangement are distinguished from those with the trans arrangement by analytical procedures such as fluorescence in situ hybridization of metaphase spreads using probes that hybridize to the novel DNA present in the two gene targeting vectors (1303 and 1305) between their arms of genomic identity. The two types of clones can also be distinguished from one another by transfecting them with a vector expressing the Cre recombinase, which deletes the pu-Tk (1339), HPRT (1345) and neomycin resistance (1347) genes if the targeting vectors have been integrated in cis, and comparing the number of colonies that survive ganciclovir selection against the thymidine kinase gene introduced by the 5′ vector (1303) and by analyzing the drug resistance phenotype of the surviving clones by a “sibling selection” screening procedure in which some of the cells from the clone are tested for resistance to puromycin or G418/neomycin. Cells with the cis arrangement of mutations are expected to yield approximately 103 more ganciclovir-resistant clones than cells with the trans arrangement. The majority of the resulting cis-derived ganciclovir-resistant clones should also be sensitive to both puromycin and G418/neomycin, in contrast to the trans-derived ganciclovir-resistant clones, which should retain resistance to both drugs. Clones of cells with the cis-arrangement of engineered mutations in the κ chain locus are selected for further use.
The doubly targeted clones of cells are transiently transfected with a vector expressing the Cre recombinase (1302) and the transfected cells are subsequently placed under ganciclovir selection, as in the analytical experiment summarized above. Ganciclovir-resistant clones of cells are isolated and analyzed by PCR and Southern blot for the presence of the expected deletion (1307) between the two engineered mutations created by the 5′ vector (1303) and the 3′ vector (1305). In these clones, the Cre recombinase causes a recombination to occur between the loxP sites (1337) introduced into the κ chain locus by the two vectors. Because the loxP sites are arranged in the same relative orientations in the two vectors, recombination results in excision of a circle of DNA comprising the entire genomic interval between the two loxP sites. The circle does not contain an origin of replication and thus is not replicated during mitosis and is therefore lost from the clones of cells as they undergo clonal expansion. The resulting clones carry a deletion of the DNA that was originally between the two loxP sites and have the genomic structure show at 1307. Clones that have the expected deletion are selected for further use.
The ES cell clones carrying the sequence deletion in one of the two homologous copies of their immunoglobulin κ chain locus are retransfected (1304) with a Cre recombinase expression vector together with a piece of DNA (1309) comprising a partly bovine immunoglobulin λ chain locus containing Vλ (1351) and Jλ (1355) gene segment coding sequences and mouse Cλ exon(s) (1357). The key features of this piece of DNA are the following: a lox5171 site (1331); a neomycin resistance gene open reading frame (1347, lacking the initiator methionine codon, but in-frame and contiguous with an uninterrupted open reading frame in the lox5171 site (1331); a FRT site (1327); an array of 1-24 functional bovine Vλ variable region gene segments (1351), each with bovine coding sequences embedded in mouse noncoding regulatory or scaffold sequences; optionally, a 13.5 Kb piece of genomic DNA from immediately upstream of the cluster of the Jκ region gene segments in the mouse κ chain locus (not shown); a 2 Kb piece of DNA containing 1-5 functional bovine Jλ region gene segments embedded in mouse noncoding DNA (1355) and mouse Cλ exon(s) (1357); a loxP site (1337) in opposite relative orientation to the lox5171 site (1331). The piece of DNA also contains the deleted iEκ (not shown).
The sequences of the bovine Vλ and Jλ gene coding regions are in Table 3.
The transfected cells are placed under G418 selection, which enriches for clones of cells that have undergone RMCE, in which the partly bovine donor DNA (1309) is integrated in its entirety into the deleted immunoglobulin κ chain locus between the lox5171 (1331) and loxP (1337) sites that were placed there by 5′ (1303) and 3′ (1305) vectors, respectively. Only cells that have properly undergone RMCE have the capability to express the neomycin resistance gene (1347) because the promoter (1329) as well as the initiator methionine codon (1335) required for its expression are not present in the vector (1309) and are already pre-existing in the host cell IGK locus (1307). The DNA region created by RMCE is illustrated at 1311. The remaining elements from the 5′ vector (1303) are removed via Flp-mediated recombination (1306) in vitro or in vivo, resulting in the final bovine-based light chain locus as shown at 1313.
G418-resistant ES cell clones are analyzed by PCR and Southern blotting to determine if they have undergone the expected RMCE process without unwanted rearrangements or deletions. Clones that have the expected genomic structure are selected for further use.
Clones carrying the partly bovine immunoglobulin DNA in the mouse κ chain locus (1313) are microinjected into mouse blastocysts from strain DBA/2 to create partly ES cell-derived chimeric mice according to standard procedures. Male chimeric mice with the highest levels of ES cell-derived contribution to their coats are selected for mating to female mice. The female mice of choice for use in the mating are of the C57Bl/6NTac strain, and also carry a transgene encoding the Flp recombinase that is expressed in their germline. Offspring from these matings are analyzed for the presence of the partly bovine immunoglobulin λ light chain locus, and for loss of the FRT-flanked neomycin resistance gene that was created in the RMCE step. Mice that carry the partly bovine locus are used to establish colonies of mice.
Mice carrying the partly bovine heavy chain locus, produced as described in Example 3, can be bred with mice carrying a bovine λ-based κ chain locus. Their offspring are in turn bred together in a scheme that ultimately produces mice that are homozygous for both bovine-based loci, i.e., bovine-based for heavy chain and λ-based λ. Such mice produce partly bovine heavy chains with bovine variable domains and mouse constant domains. They also produce partly bovine λ proteins with bovine λ variable domains and the mouse λ constant domain from their κ loci. Monoclonal antibodies recovered from these mice have bovine heavy chain variable domains paired with bovine λ variable domains.
A variation on the breeding scheme involves generating mice that are homozygous for the bovine-based heavy chain locus, but heterozygous at the κ locus such that on one chromosome they have the K-K bovine-based locus described in Example 4 and on the other chromosome they have the partly bovine λ-based κ locus described in this example. Such mice produce partly bovine heavy chains with bovine variable domains and mouse constant domains. They also produce partly bovine κ proteins with bovine κ variable domains and the mouse κ constant domain from one of their κ loci. From the other κ locus, partly bovine λ proteins comprising bovine λ variable domains and the mouse λ constant domain is produced. Monoclonal antibodies recovered from these mice include bovine variable domains paired in some cases with bovine κ variable domains and in other cases with bovine λ variable domains.
This example describes an alternate strategy to Example 7 in which the endogenous mouse Vκ and Jκ are replaced with a partly bovine locus containing bovine Vλ and Jλ gene segment coding sequences embedded in mouse Vκ region flanking and regulatory sequences and mouse Cκ is replaced with mouse Cλ. However, in this example the structure of the targeting vector containing the partly bovine locus is different. The bovine V gene locus coding sequences include an array of anywhere from 1 to 24 functional Vλ gene segment coding sequences, followed by an array of Jλ-Cλ tandem cassettes in which the Jλ is of bovine origin and the Cλ is of mouse origin, for example, Cλ1, Cλ2 or Cλ3. The number of cassettes ranges from one to five, the number of unique functional bovine Jλ gene segments. The overall structure of the partly bovine λ locus in this example is similar to the endogenous mouse λ locus, whereas the structure of the locus in Example 7 is similar to the endogenous mouse κ locus, which is being replaced by the partly bovine λ locus in that example.
The method for replacing a portion of a mouse genome with a partly bovine immunoglobulin locus is illustrated in
The targeting vectors employed for introducing the site-specific recombination sequences on either side of the Vκ (1415) gene segments and the Cκ exon (1421) also include an additional site-specific recombination sequence that has been modified so that it is still recognized efficiently by the recombinase, but does not recombine with unmodified sites. This site is positioned in the targeting vector such that after deletion of the Vλ and Jκ gene segment clusters and the Cκ exon it can be used for a second site specific recombination event in which a non-native piece of DNA is moved into the modified Vκ locus via RMCE. In this example, the non-native DNA is a synthetic nucleic acid comprising an array of bovine Vλ gene segment coding sequences and an array of Jλ-Cλ tandem cassettes in which the Jλ is of bovine origin and the Cλ is of mouse origin, for example, Cλ1, Cλ2 or Cλ3 embedded in mouse IGK regulatory and flanking sequences.
Two gene targeting vectors are constructed to accomplish the process just outlined. One of the vectors (1403) comprises mouse genomic DNA taken from the 5′ end of the locus, upstream of the most distal Vλ gene segment. The other vector (1405) comprises mouse genomic DNA taken from within the locus in a region spanning upstream (5′) and downstream (3′) of the Cκ exon (1321).
The key features of the 5′ vector (1403) and the 3′ vector (1405) are described in Example 7.
Mouse embryonic stem (ES) cells derived from C57Bl/6NTac mice are transfected by electroporation with the 3′ vector (1405) according to widely used procedures as described in Example 7. DNA from the ES cell clones is screened by PCR using a widely used gene-targeting assay as described in Example 7. The Southern blot assays are performed according to widely used procedures as described in Example 7.
Karyotypes of PCR- and Southern blot-positive clones of ES cells are analyzed using an in situ fluorescence hybridization procedure designed to distinguish the most commonly arising chromosomal aberrations that arise in mouse ES cells. Clones with such aberrations are excluded from further use. Karyotypically normal clones that are judged to have the expected correct genomic structure based on the Southern blot data are selected for further use.
Acceptable clones are modified with the 5′ vector (1403) using procedures and screening assays as described in Example 7. The resulting correctly targeted ES clones have the genomic DNA configuration of the endogenous κ locus in which the 5′ vector (1403) is inserted upstream of endogenous Vκ gene segments and the 3′ vector (1405) is inserted downstream of the endogenous Cκ. In these clones, the Cre recombinase causes recombination (1402) to occur between the loxP sites introduced into the κ locus by the two vectors, resulting in the genomic DNA configuration shown at 1407.
Acceptable clones undergo gene targeting on the same chromosome, as opposed to homologous chromosomes; such that the engineered mutations created by the targeting vectors are in cis on the same DNA strand rather than in trans on separate homologous DNA strands. Clones with the cis arrangement are distinguished from those with the trans arrangement by analytical procedures as described in Example 7.
The doubly targeted clones of cells are transiently transfected with a vector expressing the Cre recombinase (1402) and the transfected cells are subsequently placed under ganciclovir selection and analyses using procedures described in Example 7. In selected clones, the Cre recombinase has caused a recombination to occur between the loxP sites (1437) introduced into the κ chain locus by the two vectors. Because the loxP sites are arranged in the same relative orientations in the two vectors, recombination results in excision of a circle of DNA comprising the entire genomic interval between the two loxP sites. The circle does not contain an origin of replication and thus is not replicated during mitosis and is therefore lost from the clones of cells as they undergo clonal expansion. The resulting clones carry a deletion of the DNA that was originally between the two loxP sites and have the genomic structure show at 1407. Clones that have the expected deletion are selected for further use.
The ES cell clones carrying the deletion of sequence in one of the two homologous copies of their immunoglobulin κ chain locus are retransfected (1404) with a Cre recombinase expression vector together with a piece of DNA (1409) comprising a partly bovine immunoglobulin λ chain locus containing Vλ (1451) segment coding sequences and a tandem array of cassettes containing bovine Jλ gene segment coding sequences and mouse Ca exon(s) embedded in mouse IGK flanking and regulatory DNA sequences (1457). The key features of this piece of DNA are the following: a lox5171 site (1431); a neomycin resistance gene open reading frame (1447, lacking the initiator methionine codon, but in-frame and contiguous with an uninterrupted open reading frame in the lox5171 site (1431); a FRT site (1427); an array of 1-24 functional bovine Vλ variable region gene segments (1451), each containing bovine coding sequences embedded in mouse noncoding regulatory or scaffold sequences; optionally, a 13.5 Kb piece of genomic DNA from immediately upstream of the cluster of the Jκ region gene segments in the mouse κ chain locus (not shown); DNA containing a tandem array of cassettes containing bovine Jλ gene segment coding sequences and mouse Cλ exon(s) embedded in mouse IGK flanking and regulatory DNA sequences (1457); a loxP site (1437) in opposite relative orientation to the lox5171 site (1431).
The sequences of the bovine Vλ and Jλ gene coding regions are in Table 3.
The transfected cells are placed under G418 selection, which enriches for clones of cells that have undergone RMCE, in which the partly bovine donor DNA (1409) is integrated in its entirety into the deleted immunoglobulin κ chain locus between the lox5171 (1431) and loxP (1437) sites placed there by the 5′ (1403) and 3′ (1405) vectors, respectively. Only cells that properly undergo RMCE have the capability to express the neomycin resistance gene (1447) because the promoter (1429) as well as the initiator methionine codon (1435) required for its expression are not present in the vector (1409) and are already pre-existing in the host cell IGK locus (1407). The DNA region created by RMCE is illustrated at 1411. The remaining elements from the 5′ vector (1403) are removed via Flp-mediated recombination (1406) in vitro or in vivo, resulting in the final bovine-based light chain locus as shown at 1413.
G418-resistant ES cell clones are analyzed by PCR and Southern blotting to determine if they have undergone the expected RMCE process without unwanted rearrangements or deletions. Clones that have the expected genomic structure are selected for further use.
Clones carrying the partly bovine immunoglobulin DNA in the mouse κ chain locus (1413) are microinjected into mouse blastocysts from strain DBA/2 to create partly ES cell-derived chimeric mice according to standard procedures. Male chimeric mice with the highest levels of ES cell-derived contribution to their coats are selected for mating to female mice. The female mice of choice for use in the mating are of the C57Bl/6NTac strain, and also carry a transgene encoding the Flp recombinase that is expressed in their germline. Offspring from these matings are analyzed for the presence of the partly bovine immunoglobulin λ light chain locus, and for loss of the FRT-flanked neomycin resistance gene that was created in the RMCE step. Mice that carry the partly bovine locus are used to establish colonies of mice.
Mice carrying the partly bovine heavy chain locus, produced as described in Example 3, can be bred with mice carrying a bovine λ-based κ chain locus. Their offspring are in turn bred together in a scheme that ultimately produces mice that are homozygous for both bovine-based loci, i.e., bovine-based for heavy chain and λ-based κ. Such mice produce partly bovine heavy chains with bovine variable domains and mouse constant domains. They also produce partly bovine λ proteins with bovine λ variable domains and the mouse λ constant domain from their κ loci. Monoclonal antibodies recovered from these mice have bovine heavy chain variable domains paired with bovine λ variable domains.
A variation on the breeding scheme involves generating mice that are homozygous for the bovine-based heavy chain locus, but heterozygous at the κ locus such that on one chromosome they have the K-K bovine-based locus described in Example 4 and on the other chromosome they have the partly bovine λ-based κ locus described in this example. Such mice produce partly bovine heavy chains with bovine variable domains and mouse constant domains. They also produce partly bovine κ proteins with bovine κ variable domains and the mouse κ constant domain from one of their κ loci. From the other κ locus, they produce partly bovine λ proteins with bovine λ variable domains and the mouse λ constant domain. Monoclonal antibodies recovered from these mice have bovine variable domains paired in some cases with bovine κ variable domains and in other cases with bovine λ variable domains.
The method described above for introducing an engineered partly bovine immunoglobulin locus with bovine λ variable region coding sequences and mouse λ constant region sequences embedded in mouse κ immunoglobulin non-coding sequences involve deletion of the mouse Cκ exon. An alternate method involves inactivating the Cκ exon by mutating its splice acceptor site. Introns must be removed from primary mRNA transcripts by a process known as RNA splicing in which the spliceosome, a large molecular machine located in the nucleus, recognizes sequences at the 5′ (splice donor) and 3′ (splice acceptor) ends of the intron, as well as other features of the intron including a polypyrimidine tract located just upstream of the splice acceptor. The splice donor sequence in the DNA is NGT, where “N” is any deoxynucleotide and the splice acceptor is AGN (Cech T R, Seitz J A and Atkins J F Eds. (2019) (RNA Worlds: New Tools for Deep Exploration, CSHL Press) ISBN 978-1-621822-24-0).
The mouse Cκ exon is inactivated by mutating its splice acceptor sequence and the polypyrimidine tract. The wild type sequence upstream of the Cκ exon is CTTCCTTCCTCAG (SEQ ID NO: 294) (the splice acceptor site is underlined). It is mutated to AAATTAATTAACC (SEQ ID NO: 295), resulting in a non-functional splice acceptor site and thus a non-functional Cκ exon. The mutant sequence also introduces a PacI restriction enzyme site (underlined). As an eight base pair recognition sequence, this restriction site is expected to be present only rarely in the mouse genome (˜every 65,000 bp), making it simple to detect whether the mutant sequence has been inserted into the IGK locus by Southern blot analysis of the ES cell DNA that has been digested with PacI and another, more frequently cutting restriction enzyme. The wild type sequence is replaced with the mutant sequence by homologous recombination, a technique widely known in the art, as to insert the 3′ RMCE vector. The key features of the homologous recombination vector (MSA, 1457) to mutate the Cκ exon splice acceptor sequence and the polypyrimidine tract are as follows: 6 Kb of mouse genomic DNA (1443) mapping within the κ locus in a region spanning upstream (5′) and downstream (3′) of the Cκ exon (1421) and containing the mutant AAATTAATTAACC (SEQ ID NO: 295) (1459) sequence instead of the wild type CTTCCTTCCTCAG (SEQ ID NO: 294) sequence in its natural position just upstream of the Cκ exon; a neomycin resistance gene under the control of the mouse phosphoglycerate kinase 1 gene promoter (1447) and flanked by mutant FRT sites (1461); 3.6 Kb of mouse genomic DNA (1449) that maps immediately downstream in the genome of the 6 Kb DNA fragment included at the 5′ end in the vector, with the two fragments oriented in the same transcriptional orientation as in the mouse genome; a gene encoding the diphtheria toxin A (DTA) subunit under transcriptional control of a modified herpes simplex virus type I thymidine kinase gene promoter coupled to two mutant transcriptional enhancers from the polyoma virus (1423). Mutant FRT sites (1461), e.g., FRT F3 or FRT F5 (Schlake and Bode (1994) Use of mutated FLP recognition target (FRT) sites for the exchange of expression cassettes at defined chromosomal loci. Biochemistry 33:12746-12751 PMID: 7947678 DOI: 10.1021/bi00209a003), are being used here because, once the spicing mutation is introduced and the Neo gene is deleted by transient transfection of a FLP recombinase expression vector (1406), the ES cells are subjected to further genetic manipulation. This process requires wild type FRT sites to delete another Neo selection gene (1447 at 1403). If the FRT site (1461) remaining in the IGK locus (1469) after introduction of the splicing mutation is wild type, attempted FRT-mediated deletion of this second Neo gene (1406 at 1413) may inadvertently result in deletion of the entire newly-introduced partly bovine locus and the inactivated mouse Cκ exon.
Mouse embryonic stem (ES) cells derived from C57Bl/6NTac mice are transfected by electroporation with the MSA vector (1457) according to widely used procedures. Prior to electroporation, the vector DNA is linearized with a rare-cutting restriction enzyme that cuts only in the prokaryotic plasmid sequence or the polylinker associated with it. The transfected cells are plated and after ˜24 hours they are placed under positive selection for cells that have integrated the MSA vector into their DNA by using the neomycin analogue drug G418. There is also negative selection for cells that have integrated the vector into their DNA but not by homologous recombination. Non-homologous recombination results in retention of the DTA gene, which kills the cells when the gene is expressed, whereas the DTA gene is deleted by homologous recombination since it lies outside of the region of vector homology with the mouse IGK locus. Colonies of drug-resistant ES cells are physically extracted from their plates after they became visible to the naked eye about a week later. These picked colonies are disaggregated, re-plated in micro-well plates, and cultured for several days. Thereafter, each of the clones of cells is divided such that some of the cells are frozen as an archive, and the rest used to isolate DNA for analytical purposes.
The IGK locus in ES cells that are correctly targeted by homologous recombination has the configuration depicted at 1463.
DNA from the ES cell clones is screened by PCR using a widely used gene-targeting assay design. For this assay, one of the PCR oligonucleotide primer sequences maps outside the region of identity shared between the MSA vector (1457) and the genomic DNA (1401), while the other maps within the novel DNA between the two arms of genomic identity in the vector, i.e., the neomycin resistance (1447) gene. According to the standard design, these assays detect pieces of DNA that are only present in clones of ES cells derived from transfected cells that had undergone fully legitimate homologous recombination between the MSA vector (1457) and the endogenous mouse IGK locus. Two separate transfections are performed with the MSA vector (1457). PCR-positive clones from the two transfections are selected for expansion followed by further analysis using Southern blot assays.
The Southern blot assays are performed according to widely used procedure using three probes and genomic DNA digested with multiple restriction enzymes chosen so that the combination of probes and digests allowed for conclusions to be drawn about the structure of the targeted locus in the clones and whether it is properly modified by homologous recombination. In in this particular example, the DNA is double digested with Pac1 and another restriction enzyme such as EcoRI or HindIII, as only cells with the integrated MSA vector contains the PacI site. A first probe maps to DNA sequence flanking the 5′ side of the region of identity shared between the MSA vector (1457) and the genomic DNA; a second probe also maps outside the region of identity but on the 3′ side; a third probe maps within the novel DNA between the two arms of genomic identity in the vector, i.e., in the neomycin resistance (1447) gene. The Southern blot identifies the presence of the expected restriction enzyme-generated fragment of DNA corresponding to the correctly mutated, i.e., by homologous recombination with the MSA κ targeting vector (1457) part of the κ locus, as detected by one of the external probes and by the neomycin resistance gene probe. The external probe detects the mutant fragment and also a wild-type fragment from the non-mutant copy of the immunoglobulin κ locus on the homologous chromosome. The Southern blot assays are performed according to widely used procedures described in Example 7.
Karyotypes of PCR- and Southern blot-positive clones of ES cells are analyzed using an in situ fluorescence hybridization procedure designed to distinguish the most commonly arising chromosomal aberrations that arise in mouse ES cells. Clones with such aberrations are excluded from further use. Karyotypically normal clones that are judged to have the expected correct genomic structure based on the Southern blot data are selected for further use.
Although the ability of the ES cell DNA to be digested by PacI in the mutated IGK allele confirms the presence of the TTAATTAA sequence, DNA sequencing focusing on the region upstream of the Cκ exon is performed to confirm the presence of the complete expected splicing mutation. The region is amplified by genomic PCR using primers that flank the mutation [1465 and 1467 (Table 5: SEQ ID NO: 275 and SEQ ID NO:276)]. An alternate primer pair is shown in SEQ ID NO: 277 and SEQ ID NO: 278. These primers are designed using NCBI Primer-Blast and verified in silico to lack any predicted off-target binding sites in the mouse genome.
Sequence-verified ES cell clones are transiently transfected (1406) with a FLP recombinase expression vector to delete the neomycin resistance gene (1427). The cells are then subcloned and the deletion is confirmed by PCR. The IGK locus in the ES cells have the genomic configuration depicted at 1469.
The ES cells are electroporated with the 5′ and 3′ RMCE vectors, as described above. The only differences are that the 3′ vector (1405) is inserted upstream of the mutant Cκ exon at the position shown in
For the proposed L-K mouse (Example 4), bovine Vλ and Jλ gene segment coding sequences flanked by mouse non-coding and regulatory sequences are embedded in the mouse IGK locus from which endogenous Vκ and Jκ gene segments have been deleted. After productive VλJλ gene rearrangement, the resulting Ig gene encodes a LC with a bovine λ variable domain and a mouse κ constant domain. To test whether such a hybrid LC was properly expressed and forms an intact Ig molecule, a series of transient transfection assays were performed with different combinations of Vs, both Vκ and Vλ, and C light chain exons, both Cκ and Cλ, together with an Ig HC and tested for cell surface and intracellular expression and secretion of the encoded Ig.
For these experiments, bovine sequences encoding two bovine IGH variable region domains were tested, both of which were cloned from heterohybridomas and are somatically hypermutated. One contained an ultra-long HCDR3 (clone BLV1H12, Accession #AF015506.1) and the other contained a normal-length HCDR3 (clone B4, Accession #U11628.1) linked to a mouse IgMb allotype HC. Each VH-encoding DNA contains the endogenous bovine L1-intron-L2. Each was individually cloned into a pCMV vector. Cow Vλ (AF023843.1) or Vκ (BC122795) VL exon was linked to the constant region of mouse Cκ, Cλ1, Cκ2, or Cλ3 and cloned into a pFUSE vector. The L1-intron-L2 sequences in each VL are all of cow origin. These sequences contain somatically hypermutated residues that were reverted back to germline sequence.
To examine cell surface IgM expression, 293T/17 cells were co-transfected with a human CD4 (hCD4) expression vector as a transfection control plus one of the HC and LC constructs and a CD79a/b expression vector. The CD79a/b heterodimer was required for cell surface expression of the IgM. Approximately 24 hr later, the transfected cells were subjected to cell surface or intracellular staining by flow cytometry. For analysis of Ig secretion, the same VH genes as above were cloned into a pFUSE vector containing mouse IgG2a Fc. 293T/17 cells were co-transfected with a human CD4 (hCD4) expression vector as a transfection control plus one of the HC and LC constructs described above. Approximately 48 hr later, the transfected cells and their corresponding supernatants were harvested and analyzed for HC/LC expression/secretion by western blotting.
To summarize the data obtained from these experiments, when bovine VH domains containing either ultra-long or normal-length HCDR3 and linked to a mouse IgM backbone were co-expressed with a LC having Vκ or Vλ with a mismatched CL region, i.e., Vλ with Cκ or Vκ with Cλ, IgM expression on the cell surface as assessed by flow cytometry was at least two times less than when the same bovine VH was co-expressed with a LC having a matched VL/CL, i.e., when Vλ was linked to Cλ1, Cλ2 or Cλ3, or when Vκ was linked to Cκ. These results were confirmed by staining for cell surface CD79b, an obligate component of the BCR. The extent of the expression defect was dependent of the particular VH gene being used; some VH genes allowed for some cell surface expression of the hybrid light chains, but others were more stringent. The same trends were seen with Ig secretion.
The results of this analysis indicate that hybrid light chains composed of bovine Vλ and mouse Cκ or bovine Vκ and mouse Cλ1, Cλ2 or Cλ3 were relatively poorly expressed on the cell surface with μHC. Since B cell survival depends on IgM BCR expression, it is clear that pairing of bovine Vλ and mouse Cκ would result in a major reduction in the development of λLC-expressing B cells. Similarly, pairing of bovine Vκ with mouse Cλ1, Cλ2 or Cλ3 would reduce the development of κ-LC expressing B cells.
Expression and secretion of the Ig with hybrid or homologous LC. Supernatants and cell lysates of the transiently transfected cells were analyzed by western blotting.
Molecular weight standards are in lane 1711. This particular bVκ/MCL combination was not particularly well secreted, no matter what the LC, but clearly bVκ/mCκ (1703) was secreted better than bVκ/mCλ2 (1705) or bVκ/mCλ3 (1706). On the other hand, bVλ was secreted very poorly with mCκ (1707) but was secreted well with all three mouse Cλ subclasses (1708-1710). The intracellular HC levels were similar with all eight constructs (blot 1702, lanes 1703-1710), indicating that differential intracellular protein stability was not responsible for the observed differences in secretion.
IgD is co-expressed with IgM on mature B cells in most mammals.
However, the issue of whether cows have a functional constant region gene to encode the δHC has been quite controversial. Early studies found no evidence for a Cδ gene or IgD protein in cows (Butler, et al. ibid; Naessens J ibid). Subsequently, evidence for the existence of bovine Cδ genes (Zhao, et al. ibid) and expression of bovine IgD (Xu, et al. ibid) have been reported, although the reported frequency of bovine IgD+ cells was much lower than in mice. The current annotation of the bovine IGH locus by the International ImMunoGeneTics Information System® http://www.imgt.org, (IMGT) lists four Cδ genes, IGHDD1P, IGHDD2P, IGHDD39, and IGHD, none of which is functional. There are two IGHD alleles, one is a pseudogene due to a frameshift in M1 and the other is an ORF due to a non-canonical splice donor, NGC instead of NGT. The low frequency of IgD+ cells observed by Xu et al. (ibid) may be due to occasional leaky splicing from the ORF allele. In any case, one concern was that the bovine VH domains might not fold properly when linked to mouse Cδ, since the bovine VH gene region has apparently been evolving with a partial or completely non-functional Cδ gene. A problem with partial or absent assembly of the partly bovine IgD could disturb normal B cell development.
To test whether bovine VH domains with a Cδ backbone can assemble into an IgD molecule expressible on the cell membrane, transient transfection and flow cytometry analysis were conducted using methods similar to Example 9.
293T/17 cells were co-transfected with a human CD4 (hCD4) expression vector as a transfection control plus one of the HC constructs from Example 9 (IGHV BLV1H12 or B4), except that Cμ was replaced with Cδ, as well as an additional IGHV BL5B8, also linked to Cδ, Both IGHV BLV1H12 B4 have ultra-long HCDR3 regions, 61 and 56 amino acids, respectively. Each heavy chain construct was co-transfected one of the λ LC constructs described in Example 9, along with a CD79a/b expression vector. As can be seen if
Thus, the three tested bovine VH domains were expressed with a mouse IgD backbone, although there was some variability in the level of cell surface expression depending on the particular HC/LC combination. It is believed that HC/LC combinations that can be expressed as IgD on the cell surface are selected into the follicular B cell compartment during B cell development, generating a diverse BCR repertoire
The preceding merely illustrates the principles of the methods described herein. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims that follow, unless the term “means” is used, none of the features or elements recited therein should be construed as means-plus-function limitations pursuant to 35 U.S.C. § 112 ¶6. All references cited herein are incorporated by reference in their entirety for all purposes.
(NB, the sequence and annotation of the bovine genome is still incomplete. These tables do not necessarily describe the complete bovine VH, D and JH, Vκ AND Jκ, or Vλ and Jλ gene segment repertoire.)
(F=Functional, ORF=open reading frame, P=pseudogene, *01 indicates the IMGT allele number)
This application claims priority to U.S. Provisional Application No. 62/869,415, filed Jul. 1, 2019, the disclosure of which is incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/040290 | 6/30/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62869415 | Jul 2019 | US |
